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On the origin of pontocerebellar hypoplasia: Finding genes for a rare disease

Eggens, V.R.C.

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Citation for published version (APA):Eggens, V. R. C. (2016). On the origin of pontocerebellar hypoplasia: Finding genes for a rare disease.

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Download date: 15 Mar 2020

On the rigin of Pontocerebellar Hypoplasia:

finding genes for a rare disease

Uitnodiging

Voor het bijwonen van de openbare verdediging van het proefschrift

On the Origin of Pontocerebellar Hypoplasia: fi nding genes for a rare disease

door Veerle RC Eggens

Vrijdag 17 juni 2016, 10.00 uur

Agnietenkapel, Universiteit van AmsterdamOudezijds Voorburgwal 231, Amsterdam

ParanimfenBrenda Westerhuiswesterhuis.brenda@gmail.com

Bart Appelhofb.appelhof@amc.nl

On the rigin of Pontocerebellar Hypoplasia:

finding genes for a rare disease

On the Origin of Pontocerebellar Hypoplasia: finding genes for a rare disease Veerle RC Eggens

Uitnodiging

Veerle RC Eggens

On the Origin Of POntOcerebellar hyPOPlasia:

finding genes for a rare disease

Veerle r.c. eggens

The work described in this thesis was performed at the department of genome analysis, academic Medi-cal Center amsterdam, University of amsterdam, the netherlands.

Cover design by Veerle eggensLayout and printing by ridderprint B.V.

isBn: 978-94-6299-356-3

On the Origin of

Pontocerebellar hypoplasia:

finding genes for a rare disease

aCadeMisCH ProefsCHrifT

ter verkrijging van de graad van doctor

aan de Universiteit van amsterdam

op gezag van de rector Magnificus

prof. dr. d.C. van den Boom

ten overstaan van een door het College voor Promoties ingestelde commissie,

in het openbaar te verdedigen in de agnietenkapel

op vrijdag 17 juni 2016, te 10:00 uur

door

Veerle rosa Catelijne eggens

geboren te Woerden

Promotiecommissie:

Promotor: prof. dr. f. Baas Universiteit van amsterdamCopromotor: prof. dr. B.T. Poll-The Universiteit van amsterdamoverige leden: dr. g.e.M. abbink VUmc prof. dr. C.B.L.M. Majoie Universiteit van amsterdam prof. dr. e.J. Meijers-Heijboer Universiteit van amsterdam dr. a.s.P. van Trotsenburg Universiteit van amsterdam prof. dr. J.a. Veltman radboud Universiteit nijmegen prof. dr. r.J.a. Wanders Universiteit van amsterdam

faculteit der geneeskunde

Miljoenen jaren wezen werkenHeb ik, Systeem, de weetgrage vlerken

Over mijn doelwitte ratjes gespreidZo spreekt, in vivo, de werkelijkheid

- Jan Lauwereyns

ConTenTs

9 chapter 1 introductionPontocerebellar hypoplasia from a clinical and genetic perspective

27 chapter 2 TOE1 mutations cause pontocerebellar hypoplasia and disorders of sex development

49 chapter 3 EXOSC3 mutations in pontocerebellar hypoplasia type 1: novel mutations and genotype-phenotype correlations

65 chapter 4 CLP1 founder mutation links trna splicing and maturation to cerebellar development and neurodegeneration

105 chapter 5 discussionLost in translation: potential pathomechanisms underlying pontocerebellar hypoplasia

119 summary

129 nederlandse samenvatting

135 List of authors

145 Portfolio

149 dankwoord

1Een goed begin is het halve werkMaar een goed begin is maar de helft- De Jeugd van Tegenwoordig

Based on:

Eggens VRC, Barth PG, Baas F. EXOSC3-Related Pontocerebellar Hypoplasia. In: Pagon

RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong CT, Smith RJH, Stephens K, editors. 

GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2014 (2014)

Eggens VRC, Barth PG, Baas F. Update on pontocerebellar hypoplasia: novel subtypes, genes

and insights. Peadiatric neurological disorders with cerebellar involvement. Diagnosis and

management. Mariani Foundation Peadiatric Neurology: 27 (2014)

Namavar Y, Eggens VRC, Barth PG, Baas F. TSEN54-Related Pontocerebellar Hypoplasia. In:

Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong CT, Smith RJH, Stephens K, editors.

GeneReviews®[Internet]. Seattle (WA): University of Washington, Seattle; 1993-2014. 2009 Sep

08 (updated 2013)

1intrOductiOn

PonToCereBeLLar HyPoPLasia froM a CLiniCaL and geneTiC PersPeCTiVe

Veerle rC eggens

11

Chapter

1

introduction

THe HisTory of PonToCereBeLLar HyPoPLasia

it sounds simple: the main characteristic of pontocerebellar hypoplasia is ponto-cerebellar hypoplasia. in this chapter we will describe the differences between the various types of pontocerebellar hypoplasia (PCH) and we will set out the past and present state of clinical and genetic aspects of PCH research.

The first description of a patient with a hypoplastic pons and cerebellum was published in the beginning of the 20th century [1]. in 1929, Krause associated clinical features to this pathology [2]. He reported a child of 16 months old with swallowing problems, spasticity and microcephaly. Pathological investigation revealed a cerebel-lum severely diminished in size, while gross formation of cortical gyri and sulci was intact.

in the last two decades much progress has been made in research on pontocerebel-lar hypoplasia (PCH). a big step was the identification of the causative gene for PCH in a cluster of related families in Volendam, a genetic isolate in the netherlands. in 1990, Barth described seven children of five related families in this area, presenting micro-cephaly, spastic paresis and extrapyramidal dyskinesia [3]. CT scans revealed severe pontocerebellar hypoplasia and cerebral atrophy. Histologically, loss of neurons was more evident in the pons and the cerebellum compared to other brain regions. PCH was initially classified in two subtypes: PCH with (subtype 1) or without degeneration of the motor neurons in the anterior horn of the spinal cord (subtype 2). nowadays, the number of PCH subtypes is extended to ten, based on clinical and genetic features. shared hallmarks in all subtypes include hypoplasia and/or atrophy of the cerebellum and pons, an early - in most cases fetal - onset of the disease, severe developmental delay and very limited cognitive and motor skills. The small volume of the pons and the cerebellum is mainly due to loss of Purkinje cells, fragmentation of the dentate nucleus and loss of pontine nuclei. impaired foliation of the cerebellar hemispheres is common [4]. Currently, there is no cure for PCH. Treatment is only symptomatic and includes percutaneous endoscopic gastronomy feeding, respiratory support, treatment of dystonia and seizures and physiotherapy [5]. age of death ranges from neonatal to late twenties, though most patients die in childhood.

PCH sUBTyPes – CLiniCs and geneTiCs

The PCH subtypes share common features, but each subtype has distinct clinical and genetic characteristics, which will be briefly discussed below (figure  1.2 and Table 1.1). Pontocerebellar hypoplasia is an autosomal recessive disease. due to the developments in genetics, especially the advantages in next generation sequencing,

12

the identification of new genes involved in rare diseases as PCH became less time consuming. Until a decade ago, linkage analysis in large families was the standard way of identifying a disease locus. nowadays, exome or genome sequencing of “trios” consisting of a patient and his or her parents can be sufficient to identify novel dis-ease genes. This development has boosted PCH research, and currently ten forms of PCH are defined. in the majority of patients the genetic background is known, but still a large cohort remains in which the genetic component of the disease is not yet revealed.

Pch1 (oMiM 607596, 606489, 616081) is characterized by pontocerebellar hypo-plasia plus degeneration of motor neurons in the anterior horn of the spinal cord. initially, PCH1 was associated with death within the first year of life [6]. in recent years, it has become clear that the PCH1 phenotype is much broader, with possible survival into puberty [7]. a subset of patients shows an intact pons, and the level of cerebellar hypoplasia is variable (Chapter 3 of this thesis, [8]). Patients suffer from hypotonia and severe developmental delay. some patients are able to walk or sit independently, but lose this ability as disease progresses [9].

Mutations in the EXOSC3 gene are found in about half of all PCH1 patients [10]. This gene encodes component 3 of the exosome complex, a complex with exoribonuclease activity that is involved in various rna processing and degradation processes [11]. in three families with PCH1, mutations in the vaccinia-related kinase 1 (VRK1) have been found [12,13]. VrK1 is a kinase thought to be involved in cell proliferation, cell cycle and carcinogenesis [14] and regulation of Cajal bodies [15]. recently, evidence for a neuronal function for VrK1 was presented, as Vrk1 knockdown in mice impairs migration of cortical neurons and affects the cell cycle of neuronal progenitors [16]. in one family classified as PCH1, mutations in the TSEN54 gene were identified [17], see below. finally, mutations in EXOSC8, encoding component 8 of the exosome complex, cause a PCH1-like disease, including degeneration of oligodendroglia cells [18].

Pch2 (oMiM 277470, 612389, 612390, 613811) is the most common and therefore best-studied subtype of pontocerebellar hypoplasia. The first reports by Barth [3] describe patients with this subtype. a minority of the patients shows atrophy of the cerebral cortex on Mri [19]. seizures are often reported in this subtype, as well as swallowing problems, dyskinesia, sleep disorders and gastroesophageal reflux. The majority of patients can fix and follow, grasp objects and have social smile. Life expec-tancy ranges from infancy to early puberty [20].

The first gene for PCH was identified in the Volendam PCH2 cohort. a founder muta-tion in the transfer-rna (trna) splicing endonuclease (Tsen) complex was identified [21]. This variant (c.919g>T, p.a307s) lies in one of the subunits of the Tsen complex (TSEN54) and is the most common mutation in PCH2. The p.a307s mutation is highly prevalent in Volendam, with a carrier frequency of 14,2% [22] and is also seen in

13

Chapter

1

introduction

other (Caucasian) populations. Patients with the common mutation have a typical dragonfly-like pattern of the cerebellar hemispheres on coronal brain Mri, where the vermis is relatively intact. Besides the p.a307s mutation, a number of other missense mutations in TSEN54 have been found to underlie PCH2. in a few isolated families mutations in TSEN2 and TSEN34 - other subunits of the Tsen complex - have been identified [21].

Pch3 (oMiM 608027) is described in only a few families [23,24]. Patients show similar symptoms as PCH2 patients, although without extrapyramidal involvement. all but one patient had optic atrophy. PCH3 has also been described in combination with the cardiac malformation syndrome tetralogy of fallot [25] and Vitamin a deficiency [26,27]. recently, a homozygous nonsense mutation in piccolo presynaptic cytomatrix protein (PCLO) was identified in an omani family with PCH3 [28]. PCLo is potentially involved in regulation of presynaptic proteins and vesicles.

for a long time, Pch4 (oMiM 225753) and Pch5 (oMiM 610204) were considered to be distinctive subtypes, based on neuroradiological aspects. in the few families with PCH5 the vermis was supposed to be more affected than the hemispheres, while these structures were equally affected in PCH4 [29]. reconsideration of the phenotypes and genetics of the two subtypes led to the conclusion that separation of the two types is dispensable. Patients with PCH4 or 5 present the same features as PCH2 patients, although with a more severe and earlier onset. Clonus, contractures, hypoventilation and hypoplasia of the cerebrum are more often seen in PCH4 or 5 than in PCH2. also, C-shaped inferior olives indicate a very early prenatal onset of the disease. Most patients with PCH4 or 5 die during infancy [19].

The more severe clinical presentation of PCH4 or 5 in comparison to PCH2 is re-flected in the underlying genetics. Whereas both subtypes can be caused by mutations in TSEN54, PCH2 is due to missense mutations and PCH4 and 5 are due to compound mutations including one heterozygous missense mutation and one functional null allele [19].

Pch6 (oMiM 611523) is rare and combines PCH with mitochondrial respiratory chain defects, manifested in elevated lactate levels. degeneration of the cerebellum and cerebrum is very progressive in patients with PCH6. autopsy on two siblings with PCH6 revealed immature cerebella, similar to a developmental stage of less than 18 weeks, and immature simplified cortical gyri [31]. Most patients survive into childhood [30].

several missense mutations in the RARS2 gene have been identified in patients with PCH6 [32]. rars2 encodes for the nuclear encoded mitochondrial arginyl-trna synthetase, which connects the amino acid arginyl to its corresponding mitochondrial trna.

14

Patients with Pch7 (oMiM 614969) have both brain abnormalities and genital abnormalities. for years it was uncertain whether PCH7 was an isolated disorder or a coincidence of two disorders [33-35]. recently, we described eight families with PCH7, all with mutations in the target of egr1 (TOE1) gene, supporting that PCH7 is an isolated disease (Chapter 2 of this thesis). Brain Mri shows a hypoplastic pons and cerebellum, large ventricles and thin white matter. Patients suffer from axial hypotonia, hypertonic limbs, seizures and severely delayed development. all 46, Xy patients have ambiguous genitals and one 46, XX patient had atrophic ovaries and absent menarche at the age of 20 years. survival range is broad; three patients died at the age of 24 weeks, 2 years and 3 years respectively, while two siblings in their twenties are still alive.

TOE1 is supposed to have various functions, as it is involved in the cell cycle [36], exhibits deadenylation activity [37] and is essential for the maintenance of Cajal bod-ies [38]. Cajal bodies are nuclear entities that include splicing factors, therefore Toe1 might be involved in mrna splicing.

Pch8 (oMiM 614961) is described in three families from Peru and Puerto rico [39] and caused by mutations in charged multivesicular body protein 1a (CHMP1A). it is thought to be a non-progressive form of PCH, including microcephaly, ocular abnor-malities, hypoplasia of pons and cerebellum, reduced cerebral white matter and thin corpus callosum. some patients could sit or walk independently. The developmental rather than degenerative nature of PCH8 is reflected in the function of the affected gene; CHMP1A is involved in cell proliferation and chromatin modelling.

Pch9 (oMiM 615809) is described in five families [40]. distinctive for this subtype is the ‘figure 8’ appearance of the brain stem on axial Mri images and corpus callosum hypoplasia. Patients present progressive microcephaly, impaired swallowing, spastic-ity and absent gross and fine motor skills.

Patients with mutations in the AMPD2 gene have been characterised as PCH9 patients. The gene encodes for adenosine monophosphatase deaminase 2 and is essential in the maintenance of cellular guanine nucleotide levels. a reduction of guanine levels, due to mutations in this gene may lead to a defect in protein transla-tion initiation.

Pch10 (oMiM 615803) is the most recently described subtype of PCH and was initially identified in nine Turkish families (Chapter 4 of this thesis, [41,42]). Patients suffer from both central and peripheral nervous system abnormalities. Brain Mri shows small pons, cerebellum and brainstem, as well as cortical involvement.

Mutations in cleavage and poly-adenylation factor 1 (CLP1) account for PCH10 cases. all nine families harboured a homozygous missense mutation (c.419C>a, p.r140H) in this gene. CLP1 is associated with the Tsen complex and involved in trna processing [43].

hier figuur 1.1hier tabel 1.1

15

Chapter

1

introduction

A B C D E

F G H I J

figure 1.1: Brain Mri of PCH1-10. a control Mri is shown in a. B presents a PCH1 patient with a p.d132a mutation in EXOSC3 [44]. a typically unaffected pons is seen. C shows a PCH2 patient with a homozygous p.a307s mutation in TSEN54 [4]. d is a PCH3 patients with a PCLO mutation [28]. e is an example of a PCH4 patient with cortical atrophy [4]. f presents a PCH6 patient with a RARS2 mutation [32]. g presents a PCH7 patient; characteristically large ventricles and thin corpus callosum are present. Corpus callosum atrophy can also be appreciated in H (PCH8; [39]), i (PCH9; [40]) and J (PCH10; [41]).

table 1.1: Clinical, radiological and genetic features of PCH subtypes.

subtypedistinctive clinical features

anatomic features genetic features Key references

PCH1 anterior horn cell degeneration

Pons not always affected EXOSC3 in majority of cases; TSEN54, RARS2, VRK1 in few families, EXOSC8

[7,8,12,13,17, 18,45,46]

PCH2 extrapyramidal symptoms

dragonfly shaped cerebellar hemispheres in case of p.a307s mutation

TSEN54, TSEN2, TSEN34 [19,21,21,47,48]

PCH3 no extrapyramidal symptoms, optic atrophy

Variable PCLO [23-28]

PCH4 or 5

severe PCH2 often cortical atrophy, immature folia

TSEN54 [19,21]

PCH6 Mitochondrial respiratory chain defects

neocortical atrophy RARS2 [30-32]

PCH7 disorders of sex development

Large ventricles, thin corpus callosum

TOE1 Chapter 2 of this thesis, [33-35]

PCH8 non-progressive Thin corpus callosum CHMP1A [39]

PCH9 ‘figure 8’ appearance of brain stem

‘figure 8’ appearance of brain stem

AMPD2 [40]

PCH10 Both central and peripheral nervous system abnormalities

relatively mild cerebellar hypoplasia

CLP1 [41,42]

16

differenTiaL diagnosis

in the diagnosis of PCH, a small pons and cerebellum on Mri is the main criterion. The degree of hypoplasia varies, making it a broad non-conclusive criterion, which is found in many other diseases. Cerebellar hypoplasia is often not an isolated symptom, but is accompanied by involvement of the cortex, motor neurons and/or white matter. another distinction that can be made within the spectrum of early-onset cerebellar hypoplasias is the origin of the reduced size of the cerebellum; this can be a devel-opmental defect, it can be neurodegenerative or a combination of both. The latter is thought to be the case in most patients: neurodegeneration sets in before the cer-ebellum is fully developed. only PCH8 seems to be a non-progressive developmental form of PCH. since the knowledge of genetics behind these conditions is growing fast, genetic testing can make the final diagnosis of a patient with cerebellar hypoplasia. diseases that present overlapping features with PCH are briefly discussed below. discriminating and shared features of these disease and the various PCH subtypes are displayed in figure 1.2.

a disorder that is very closely related to PCH is mental retardation and microcephaly with pontine and cerebellar hypoplasia (MiCPCH; oMiM 300749), caused by mutations in the X-linked gene CASK. Both males and females can be affected, although the clinical outcome is more severe in males. MiCPCH includes microcephaly, neocortical dysplasia with a simplified gyral pattern in some cases, pontocerebellar hypoplasia, and a thin brain stem. Patients have severe or profound intellectual disability [49].

in progressive cerebello-cerebral atrophy (PCCa, oMiM 613811) biallelic mutations in SEPSECS are found, resulting in the defective synthesis of the seleno-cysteine car-rying trna [50]. Clinical and Mri findings closely resemble those of mild PCH2 [4].

Congenital disorders of glycosylation type 1a  (Cdg1a; oMiM 212065) overlaps with PCH in developmental delay, hypotonia and cerebellar dysfunction. Common additional symptoms in Cdg1a are hepatic dysfunction, hypogonadism, and abnormal subcutaneous fat pads. Cdg1a is caused by biallelic mutations in PMM2 [51].

a critical pathway in neuronal migration in the cortex and cerebellum is the reLn signalling pathway [52]. Two disorders are known in which this pathway is disrupted. Mutations in RELN can cause lissencephaly with cerebellar hypoplasia (oMiM 257320; [53]) and mutations in the VLDLR  gene are associated with cerebellar hypoplasia, ataxia, mental disability and simplified cortical gyri (oMiM 224050). Clinical features are thought to be non-progressive [54].

other non-progressive congenital cerebellar malformations are dandy-Walker syndrome (oMiM 220200) and the various subtypes of Joubert syndrome. Typical for dandy-Walker syndrome is a large posterior fossa with the hypoplastic cerebellum

17

Chapter

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introduction

rotated upwards [55]. a characteristic image on axial Mri in Joubert syndrome is a molar tooth shaped pons and brain stem [56].

in spinocerebellar ataxias (sCas) mental impairment is mild or absent. importantly, cerebellar atrophy rather than hypoplasia is found, which is in line with the often late onset of this disease [57].

also in spinal muscular atrophy type (sMa) cognitive function is usually not impaired. Brain Mri is normal, but eMg reveals denervation and muscle biopsy shows grouped atrophy. sMa type i (oMiM 253300) overlaps with most PCH types considering the early onset (birth – 6 months), muscle weakness and lack of motor development. sMa i is caused by biallelic pathogenic variants in SMN1 [58].

in the majority of cases, pontocerebellar hypoplasia arises due to genetic mutations. However, it is important to realise that prematurity (<32 weeks) is a risk factor for impaired cerebellar development [59,60].

disease MeCHanisM

Many of the genes involved in PCH play a role in rna processing, and trna processing in particular. trnas guide amino acids to the ribosomes for protein synthesis and are therefore essential molecules in all cells of the body. every amino acid is coded by multiple codons and for each codon multiple trna genes exist. Humans have 506 trna genes, of which six percent contains an intron. some trna species are encoded solely by genes without an intron, for example the trna genes coding for trna-ser. other trna species are encoded by almost exclusively intron-containing genes. for

hier figuur 1.2

SMA

VLDLR

MICPCH

PCH8

PCH9

PCH7

PCCA

PCH10

PCH1 PCH4/5

PCH2 PCH3

PCH6

CDG1A SCA

NEURODEGENERATION MOTOR NEURON

INVOLVEMENT

CORTICAL INVOLVEMENT

WHITE MATTER INVOLVEMENT

CEREBELLAR HYPOPLASIA

Joubert syndrome

Dandy-Walker syndrome

RELN

PCH2

figure 1.2: Pathological aspects of PCH subtypes and related disorders. Cerebellar hypoplasia, motor neuron degeneration, cortical atrophy, white matter abnormalities and neurodegeneration play part in the clinical manifestation of PCH and related early-onset brain disorders.

18

instance, thirteen out of fourteen trna-Tyr (gTa) genes contain an intron and all five trna-ile (TaT) genes (Table 1.2).

Unlike mrna, introns of a trnas are spliced out by a specialised splicing machinery, the Tsen complex. as discussed above, this enzyme complex is affected in the major-ity of PCH2 and PCH4 or 5 patients. The complex consists of two structural subunits (Tsen54 and Tsen15) and two catalytic subunits (Tsen2 and Tsen15) [61]. Cleavage of the trna gene results in a 5’ and a 3’ exon, which have to be ligated together. in vertebrates, two pathways of trna ligation exist, the “yeast-like” and the “animal-like” pathway (figure 1.3). in the yeast-like pathway, the oH group on the 5’ end of the 3’

hier tabel 1.2

table 1.2: overview of intron containing trna genes. number and percentage of intron containing trna genes per anticodon. source: http://gtrnadb.ucsc.edu/

trna species - anticodon

number of trna genes with an intron

number of trna genes without an intron

Percentage of trna genes with an intron

Pro-agg 1 9 10.0

arg-TCT 5 1 83.3

Leu-Caa 5 2 71.4

ile-TaT 5 0 100

Tyr-aTa 1 0 100

Tyr-gTa 13 1 92.9

Cys-aCa 1 0 100

Trp-CCa 1 8 11.1

Animal pathway

Yeast-like pathway

TSEN complex CLP1 phosphotransferase and ligase

RTCB

P P

P

P P OH

3’ 3’ 3’ 3’

3’

5’ 5’ 5’ 5’

5’ pre-tRNA

mature tRNA

mature tRNA

PCH10 PCH2 PCH4/5

figure 1.3: simplified representation of trna splicing and ligation pathways in mammals. after splicing out the intron from a pre-trna molecule by the Tsen complex, ligation of the trna exons can occur via either the yeast-like pathway or the animal-pathway. in the yeast-like pathway CLP1 phosphorylates the 5’ end of the 3’ exon. Ligation of the two exons in performed by a yet unidentified enzyme. The animal-like pathway includes direct ligation by rTCB.

19

Chapter

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introduction

exon is phosphorylated by CLP1. a yet unidentified phosphotransferase and ligase join the two exons. in the animal-like pathway, the two exons are ligated in a single gTP-dependent reaction involving the enzyme rTCB [62].

Besides intron removal and ligation, trna molecules undergo various modifications before they can function in protein synthesis. removal of the 5’ and 3’ leader, addition of the CCa tail on the 3’end and various nucleotide modifications as methylations are necessary for a trna to become functional [63]. once all modifications on the trna molecule are done, it can be charged with the corresponding amino acid. This reaction is performed by aminoacyl trna synthetases. for each amino acid a trna synthetase exists, e.g. rars for arginine. some synthetases have a nuclear encoded synthetase functioning in the mitochondria, for example rars2. after charging with an amino acid, the trna can function in protein synthesis in the ribosomes.

since many genes involved in PCH have a function in trna processing, it seems plausible that impairments in this pathway play part in the pathogenesis of the dis-ease. Considering the indispensability of trnas in protein synthesis, it is conceivable that this latter process is affected as well. The first steps are made in elucidating whether this is indeed the case and how defects in these processing give rise to such a specific phenotype remains unclear. This topic will be extensively discussed in Chapter 5.

ConCLUsions

Pontocerebellar hypoplasia is a rare autosomal recessive neurodegenerative disorder. in the vast majority of patients, onset of the disease is prenatal and brain hypoplasia and clinical symptoms are progressive. Brain Mri of PCH patients show pontine and cerebellar hypoplasia, with variable cortical and white matter involvement. in the last decade, the spectrum and classification of pontocerebellar hypoplasia has been expanding. Whereas in the 1990’s two subtypes were distinguished, we now classify ten subtypes of this disease. each subtype has its distinct clinical, radiological and genetic features. implementation and development of next generation sequencing methods gave rise to novel genes associated with PCH. interestingly, the majority of genes associated with PCH can be linked to rna metabolism and protein synthesis. a start is made in elucidating how these mutations influence gene function and how this can lead to PCH. nonetheless, there is still a lot to be discovered.

hier figuur 1.3

20

aiM and oUTLine of THis THesis

in this thesis i investigated the genetics of pontocerebellar hypoplasia on various levels: i have identified novel genes for this disease and i describe novel subtypes and genotype-phenotypes correlations. next, i investigate the pathology of PCH: what cellular and molecular effects do PCH-related mutations have?

in chapter 2 we confirm the presence of PCH subtype 7 as an isolated syndrome with a single genetic locus. exome sequencing revealed two candidate genes for PCH7 – CLK2 and TOE1. We discuss how we found TOE1 as disease-causing gene, and we describe a cohort of eight families with this novel disease. in chapter 3 we zoom in on EXOSC3, a gene that causes PCH1. This subtype turns out to have a much broader phenotype than previously assumed. However, within the broad spectrum of EXOSC3-related PCH, clear genotype-phenotype correlations can be made. We describe a cohort of twelve families with EXOSC3 mutations and show that the specific genetic mutation can predict disease progression. chapter 4 describes our research on the CLP1 gene in various aspects: we identify mutations in CLP1 to be PCH-causing, we model the disease in zebrafish and we investigate the effect of the mutation on cell homeostasis. chapter 5 discusses potential cellular mechanisms underlying PCH and provides directions for further studies.

21

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introduction

referenCes

1. Brun r. Zur Kenntnis der Bildingsfehler des Kleinhirns, epikritische Bermerkungen zur entwick-lungspathologie, Morphologie und Kliniek der umschriebenen entwicklungshemmungen des neozerebellums. Schweiz Arch Neurol Psychiat 1917. 1:61-123.

2. Krause f. Über einen Bildungsfehler des Kleinhirns und einige faseranatomische Beziehungen des organs. Zeitschrift für die gesamte Neurologie und Psychiatrie, 1929. 119: 788-815

3. Barth Pg, Vrensen gf, Uylings HB, oorthuys JW, stam fC: inherited syndrome of microcephaly, dyskinesia and pontocerebellar hypoplasia: a systemic atrophy with early onset. J Neurol Sci 1990, 97: 25-42.

4. namavar y, Barth Pg, Poll-The BT, Baas f: Classification, diagnosis and potential mechanisms in pontocerebellar hypoplasia. Orphanet J Rare Dis 2011, 6: 50.

5. namavar y, eggens VrC, Barth Pg, Baas f. TSEN54-related Pontocerebellar Hypoplasia. in: Pagon ra, adam MP, ardinger HH, Bird Td, dolan Cr, fong CT, smith rJH, stephens K, editors. genereviews®[internet]. seattle (Wa): University of Washington, seattle; 1993-2014. 2009 sep 08 (updated 2013)

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7. rudnik-schoneborn s, senderek J, Jen JC, Houge g, seeman P, Puchmajerova a et al.: Pontocer-ebellar hypoplasia type 1: clinical spectrum and relevance of eXosC3 mutations. Neurology 2013, 80: 438-446.

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Al ben ik meer dan eens de weg kwijtEn rijd ik hele einden omIk ben op weg om je te vindenDus wees gerust, ik kom

- Boudewijn de Groot

2TOE1 mutatiOns cause

POntOcerebellar hyPOPlasia and disOrders Of sex deVelOPment

Veerle rC eggens, david Chitayat, Hülya Kayserili, nicola foulds, Tessa van dijk, Bart

appelhof, Kazuhiro Muramatsu, Kimberly a aldinger, William B dobyns, david Manchester,

Linda de Meirleir, Mary Louise freckmann, Linda Warwick, Chistina fagerberg, Maria

Kibaek, Marie-Cecile nassogne, Justin H davis, Umut altunoglu, Hirotomo saitsu, Masaaki

shiina, Kazuhiro ogata, Kanako Kurata, Peter g Barth, naomichi Matsumoto, frank Baas

Man

uscr

ipt i

n pr

epar

atio

n

28

aBsTraCT

Pontocerebellar hypoplasia type 7 (PCH7) – also described as pontocerebellar hypo-plasia and disorders of sex development (PCH and dsd) - is a rare disease in which both brain and genital development are affected. only four cases have been described in literature displaying this rare combination of symptoms. Until know it was uncer-tain whether the combination of symptoms in these patients occurred coincidentally or not. Here, we show that the presence of PCH and dsd is a single gene syndrome. We collected a cohort of ten patients (eight families) presenting PCH and dsd, and identified recessive mutations in the TOE1 gene in all of them. Brain Mri of the pa-tients revealed a small pons and cerebellum and enlarged ventricles. all eight 46, Xy patients present some degree of undervirilised genitals.

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utations in PCH7

inTrodUCTion

Pontocerebellar hypoplasia (PCH) represents a group of autosomal recessive neu-rodegenerative disorders with prenatal onset. Patients show variable hypoplasia of pons and cerebellum and severe motor and mental impairments. nowadays, ten PCH subtypes have been described (PCH1-10) with each subtype having its characteristic clinical and/or genetic features in addition to a hypoplastic pons and cerebellum.

PCH7 is characterised by PCH plus disorders of sex development (dsd). dsd is a broad group of conditions where defects of gonadal development occur, leading to ambiguous genitals or complete sex reversal. The frequency of 46, Xy dsd is 1:20,000 [1]. in literature, four cases of PCH plus dsd have been described [2-5]. These patients had a small pons and cerebellum, enlarged ventricles and a 46,Xy karyotype with feminizing genitalia. so far, it was unclear whether the combination of PCH and geni-tal anomalies was fortuitous or a distinct syndrome.

in this study we report eight families (ten patients) with PCH and dsd, and present the target of egr1 (TOE1) gene as locus for this disease in all described families. Toe1 is a nuclear protein that can bind rna, has deadenylation activity [6] and can have an inhibitory effect on viral replication [7]. furthermore, it is essential for Cajal body maintenance, and thus potentially involved in mrna splicing [8]. The gene has previously been associated with cerebellar abiotrophy in arabian horses [9]. With the identification of TOE1 mutations in ten patients with PCH and dsd, we confirm the presence of a single gene syndrome causing both brain and genital abnormalities.

MaTeriaLs and MeTHods

exome capture and sequencing

family 1 was analysed at the academic Medical Center, genomics core-facility in am-sterdam and sequenced on a soLid4 platform according to manufacturer’s protocols. fragment libraries were prepared followed by a nimblegens eZexome v2.0 sequence capture. reads were aligned against hg19 with Bioscope1.3. genomic dna for patient 7 and both parents were sequenced by the University of Washington Center for Men-delian genomics. dna was captured using the roche nimblegen seqCap eZ Human exome Library v.2.0 library and sequenced with paired-end 50 bp reads on an illumina Hiseq sequencer. reads were aligned against hg19 using the Burrows-Wheeler aligner v.0.6.2. for patient 8, genomic dna of patient and parental samples was captured using the sureselect Human all exon v5 Kit (agilent Technologies) and sequenced on an illumina Hiseq2000 (illumina) with 101 bp paired-end reads. reads were aligned to grCh37 with novoalign (novocraft Technologies). single nucleotide variants (snVs)

30

were called with the genome analysis Toolkit’s (gaTK) Unifiedgenotyper. rare (<1% in the nHLBi exome sequencing Project exome Variant server), deleterious variants were analysed under de novo and recessive mutation models. snVs in TOE1 were confirmed by sanger sequencing.

sanger sequencing

Variants in TOE1 in families 2,3,4,5 and 6 were identified by sanger sequencing. sanger sequencing of PCr amplified dna was performed using BigdyeTerminator chemistry (applied Biosystems) and analysed on an aBi3730xl sequencer. sequences were analysed using CodonCode aligner software 3.6.1. Primer sequences are outlined in supplementary Table 1.

clK2 knockout mouse

CLK2 whole body knockout mice were bred from Clk2flox/flox mice and zp3-Cre trans-genic mice on a C57BL/6 background. Littermates carrying the floxed allele but not Cre were used as controls. Mice were bred at Puigserver Laboratory (dept. of Can-cer Biology, Harvard Medical school, Boston, Usa) who kindly provided fixed brain samples. standard hematoxylin and eosin staining was performed on sagittal slides.

morpholino injections in zebrafish

Morpholino (Mo) antisense oligonucleotides (gene Tools) were designed to target zebrafish clk2 mrna (nM_001076751.1) either upstream of the aTg startcodon (5’-CCggTgCgTTTgTCCCaCagaaaaT-3’) or at the exon 5 splice donor site (5’-CaTCaaT-gaaCagCTCTTaCTTCTT-3). standard control Mos were provided by gene Tools. Mos were injected in 1-2 cell stage embryos using glass needles and a microinjector. fish injected with splice clk2 Mo were checked for alternative splicing: total rna was ex-tracted with Trizol (Thermofisher)/chloroform, cdna was synthesized using dT-oligos and superscript iii first strand synthesis system (invitrogen) and PCr was performed with primers 5’-agagCCggTCCaTaTCaTCa-3’ and 5’-CCgaaaTCCaCaaTCCTCaC-3’.

mrna transcription and injections in zebrafish

Human CLK2 cdna cloned in a poTB7 vector was obtained from the iMage Consortium. The CLK2 insert was subcloned into a pCs2+ vector using primers with BamHi and Xbai restriction sites (5’- TCgTaCaggCTaCCTggatccgCCaCCaTgCCgCaTCCTCgaa-3’ and 5’- gTgTCaCTgaCTgCaCCgTgtctagaTgggggTCaaaTgaag-3’ respectively, restric-tion sites in bold). The construct was linearized with noti. for zebrafish clk2 cdna (nM_001044879.2), a vector with clk2 insert was synthesised at Life Technologies and cloned into a pcdna3 vector using primers with Hindiii (5’- TaagCaaagcttaTgC-CaCaCTCCaggCggTa -3’) and Xhoi (5’-TgCTTactcgagTCaCCggCTgaTgTCaCggTT-3’)

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restriction sites. The construct was linearized with stui. In vitro transcription was performed using the T7 mMessage mMaCHine Kit (ambion, Warrington, UK).

in situ hybridisation

Plasmids with fgf8 insert were linearised with ecorV and antisense digoxigenin (dig)-rna labelled probes were synthesized using a sP6 polymerase rna labelling kit (roche). Zebrafish embryos were dechorionated, and fixed overnight in formalin. Whole mount in situ hybridisation procedures were adapted from [10]. Preceding isH, fish were bleached (10% H2o2; 5% formamide; 2,5% 20x ssC in water) for a few minutes until pigmentation had vanished. fish were permeabilised using 10 µg/ml proteinase K in PBs, and incubated over night with 1 ng/µl fgf8 dig-rna probe. stain-ing was visualised using a nBT/BCiP solution (roche).

tunel assay

Zebrafish embryos were fixated, bleached and permeabilised as described above. TUneL staining was performed using TdT buffer (roche), TdT enzyme (roche) and dig-UTP (roche). fish were incubated over night with anti-dig-aP antibody (roche). staining was visualised using a nBT/BCiP solution.

recombinant clK2 protein

Murine flag-clk2 cdna in a pcdna3 vector was kindly provided by P. Puigserver (dept. of Cancer Biology, Harvard Medical school, Boston, Usa). Mutant flag-clk2 was gener-ated using site directed Mutagenesis (stratagene) and primers 5’-aTgaCaaCagaga-gCaTCTatCCaTgaTggaaaggaTC-3’ and 5’-gaTCCTTTCCaTCaTggaTagaTgCTCTCTgTT-gTCaT-3, the mutation site indicated in bold. Wild type and mutant flag-clk2 were transfected in HeK293 cells using Xtreme gene HP dna transfection reagent (roche). recombinant protein was purified by immunoprecipitaion with anti-fLag M2 affinity beads (sigma-aldrich) and analysed by Western blotting (1:3000 anti-fLag antibody (stratagene)).

Kinase assay

Western blot band intensities of fLag-CLK2WT and fLag-CLK2a390s were quantified with aida image analyzer v.4.26 to equalize input levels. recombinant protein was incubated with 5ug sK6 substrate (signal Chem) and 33P-aTP (Perkin elkmer) at 30oC for 15 minutes. The reaction mixture was spotted on P81 ion exchange papers (Mil-lipore) and free 33P aTP was removed by extensive washing with 100 mM phosphoric acid. incorporated 33P on the ion exchange paper was measured. recombinant human CLK2 (signalChem) was used as positive control.

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haplotype analysis

exome data from patient 3 was analysed in ingenuity Variant analysis (http://www.ingenuity.com) and snPs with a prevalence <0.5%, 10 mB upstream and downstream of the TOE1 gene were selected. Primers used for sanger sequencing are listed in supplementary Table 2.

cadd analysis

The Cadd score of all identified variants in TOE1 listed in the exome aggregation Consortium (exaC, http://exac.broadinstitute.org) database was determined via http://cadd.gs.washington.edu/. The chance of having two potentially pathogenic variants was calculated as the square of the total of frequencies of all variants with a Cadd score >15.

TOE1 qPcr

Total rna was extracted from fibroblasts. cdna was synthesized using dT-primers and superscript iii first strand synthesis system (invitrogen). qPCr analysis was performed on a LightCycler 480 using primers listed in Table s2 and matching probes (roche).

structural homology modelling

a modelled structure of the dedd domain of human Toe1 was constructed from the crystal structure of mouse poly(a)-specific ribonuclease (Parn) (PdB code; 3d45)[11]. for homology modelling using Phyre2[12], the dedd domain of human Toe1 (amino acid residues 43-265) was used as a query amino acid sequence. images of molecular structures were created using PyMoL.

resULTs

candidate genes for Pch7

in search for a genetic basis for PCH7, we performed exome sequencing in a Turkish consanguineous family with two affected siblings (Table 2.1, family 1). The affected female presented a hypoplastic cerebellum on Mr images and at the age of 20 years, she was spastic and did not have breast development or menarche. The brother - for whom no Mri was available - showed spasticity, hypertonicity and a micropenis, sug-gesting the same disorder as his sister. The siblings were classified as PCH7. exome sequencing was performed on both siblings and their parents and analyzed under an autosomal recessive model. non-synonymous homozygous variants not present in the dbsnP129 were selected. This approach revealed variants in three candidate genes:

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c.307g>a (p.a103T) in target of egr1 (TOE1; nM_025077.3), c.248C>g (p.T83s) in solute carrier family 39 member 1 (SLC39A1; nM_014437.3) and c.1168 g>T (p.a390s) in CdC-like kinase 2 (CLK2; nM_003993.2), all positioned on chromosome 1. The three genes were located in two large shared homozygous regions (2.7Mb around TOE1, 5.9Mb around CLK2 and SLC39A1). TOE1 was initially characterized as a growth suppressor protein acting via egr1 [13]. The protein has deadenylating activity [6], can inhibit viral replication [7] and is possibly fulfilling a role in mrna splicing by maintaining Cajal body integrity [8]. interestingly, TOE1 mutations are found in arabian horses with cerebellar abiotrophy and Purkinje cell degeneration [9]. sLC39a1 is involved in zinc homeostasis [14]. CLK2 is a member of the LaMMer kinase family, which have been shown to phosphorylate proteins of the spliceosomal complex [15].

The majority of genes that are currently known to cause PCH are involved in rna metabolism [16]. Because both TOE1 and CLK2 are potentially involved in mrna splic-ing, we decided to follow up these two candidate genes for PCH7.

CLK2 is an unlikely candidate gene for Pch7

To investigate whether CLK2 is essential for brain development, we knocked down clk2 in zebrafish using antisense morpholinos (Mos). injection of a Mo targeting the translation initiation site of clk2 resulted in embryos with altered expression of mid-hindbrain marker fgf8, and an increased number of TUneL positive cells in the brain,

A

uninjected

control MO

clk2a MO

B

figure 2.1: CLK2 morpholino injections. injections of an antisense morpholino targeted against the start codon of clk2 results in altered formation of the midhindbrain boundary as shown by fgf8 expression (a) and increased cell death in the brain as shown by TUneL staining (B). grey scalebar=200 µm

34

indicative for cell death (fig. 2.1). However, we could not reproduce these results by injecting a Mo targeted against the exon 5 splice donor site in clk2, while partial alternative splicing of clk2 mrna was established (fig. 2.2). in addition, the clk2 aTg Mo induced phenotype could not be rescued by with either human CLK2 mrna or ze-brafish clk2 mrna (fig. 2.3). This could be due to biological issues –mrna is delivered in every cell at the same level, which may not reflect the demand for mrna. However, the observed brain phenotype can also be an off-target effect of the Mo.

importantly, CLK2 Ko mice do not show signs of brain hypoplasia at the age of 8 weeks, again suggesting that CLK2 is not essential in brain development (fig. 2.4). at 3 months of age, the mice still behave normally and can produce offspring (personal communication, M. Hatting, Harvard University, Usa).

hier figuur 2.1hier figuur 2.2hier figuur 2.3

hier figuur 2.4

A B

figure 2.2: CLK2 splice morpholino injections. injection of antisense morpholinos targeting the exon 5 splice donor site induces partial alternative splicing (a) but did not elicit a brain phenotype in zebrafish embryos (B).

A

B A

B figure 2.3: mrna injections in clk2 Mo zebrafish. injection of neither human clk2 mrna (a) nor zebrafish clk2 mrna (B) showed a rescue of the clk2 Mo induced phenotype

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in order to investigate whether the p.a390s variant we identified in the patients has an effect on CLK2 protein function, we performed a kinase assay. recombinant fLag-CLK2WT and fLag-CLK2a390s protein was incubated with s6K substrate and 33P-aTP to measure kinase activity. This revealed that the kinase function of CLK2a390s was not abolished (fig. 2.5). Moreover, sequencing a cohort of over a hundred PCH patients did not reveal any other potentially pathogenic variations in CLK2. from these data we conclude that CLK2 is an unlikely candidate gene for PCH7.

hier figuur 2.5

A B

C D

figure 2.4: Brain morphology CLK2 mouse. gross morphology (a and C) and composition of cell layers (B and d) of the cerebellum in CLK2 knockout mice (cre/cre) is similar to wild types (flox/flox). White scale bar = 500 µm, black scale bar = 100 µm.

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HEK cellsDpm

CLK2 dilution

figure 2.5: CLK2 kinase assay. recombinant murine flag-CLK2 was incubated with s6K substrate and 33P-aTP. CLK2a390s retains its kinase activity. dpm=disintegrations per minute.

36

TOE1 variants in Pch patients

since the p.a390s variant in CLK2 did not seem to have an effect in vitro or in vivo, we proceeded to our second candidate gene: TOE1. Unaffected members of our index family were wild type or heterozygous for the p.a103T variant in Toe1 (fig. 2.6). We sequenced the TOE1 gene in a cohort of over a hundred PCH and PCH-like patients with various PCH subtypes and unknown genetic cause. We identified potentially damaging variants in TOE1 in ten patients (eight families) (fig. 2.7).

hier figuur 2.6hier figuur 2.7

I

II

III Died at 40 days of age

Died at one day of age

Died at 39y of age, moderate mental retardation but self-supporting

1o cousins

1o cousins

IV

figure 2.6: Pedigree of index family. Circle indicates a female, square indicates a male. Black: homozy-gous for p.a103T variant in Toe1; black line, gray filling: heterozygous for p.a103T variant in Toe1; grey line, white filling: wild type at position p.a103 in Toe1; black line, white filling: not tested. diagonal line: deceased.

p.R73S p.A103T

p.F148Y

p.V173G

p.F239S

p.Q314VfsX8

p.H319Q

p.S496F

p.Y231X

p.R253W

p.H319Y

1 510 DEDD ZnF NLS

figure 2.7: Toe1 variants in PCH7 patients. schematic representation of the Toe1 protein with the dna-like exonuclease (dedd) domain, the zinc-finger domain (Znf) and the nuclear localization signal (nLs) indicated. dashed arrows indicate compound heterozygous mutations, solid arrows indicate homozygous mutations. each family is indicated by a different colour.

37

Chapter

2

Toe1 m

utations in PCH7

Three apparently unrelated patients (patients 3, 4 and 5) carried the same missense variant (c.518T>g, p.V173g, allele frequency of 0.00001648 in exaC database). Hap-lotype analysis of the three families revealed a common haplotype of approximately 9Mb, indicative for a common ancestor (supplementary Table 2). all 46,Xy patients in our cohort showed a certain degree of genital undervirilisation in addition to PCH. The two 46,XX cases with cerebellar hypoplasia and variants in TOE1 had normal female external genitals and are sisters of 46, Xy patients with genital abnormalities. The lack of TOE1 variants in 46, Xy PCH patients with unaffected male genitals indicates that variants in this gene are exclusively related to PCH7.

no compound heterozygous TOE1 variants were present in 9300 cases and con-trols in exome sequencing data from a motorneuron disease consortium [17]. The exaC database lists various potentially pathogenic variants in the TOE1 gene (Cadd score > 15), but none of the identified variants in PCH7 patients is reported as a homo-zygous variant. frequencies of compound heterozygous variants cannot be obtained from this database. To estimate the frequency of compound heterozygous cases, we used the reported frequency of variants with a Combined annotation dependent depletion (Cadd) score higher or equal to 15. in this way, we estimated the chance of having two pathogenic variants in TOE1 as 8.0x10-6. This very low probability and the fact that we found TOE1 variants in all eight PCH7 families supports the hypothesis that the identified variants in TOE1 are disease causing.

clinical details of Pch patients with TOE1 mutations

all 46, Xy patients with TOE1 mutations presented both brain and genital abnormali-ties and were therefore diagnosed with PCH7 (Table 2.1).

all index patients were 46, Xy and showed some degree of gonadal undervirilisa-tion. The external genitals ranged in appearance from a nearly normal vagina to a micropenis with hypoplastic scrotum (fig. 2.8). internally, some 46, Xy did not have gonadal tissue at all, some had ovarian or uterine remnants, and some had atrophic undescended testes. The 46, XX siblings had normal female external genitals. in one 46, XX patient the ovaries could not be observed. endocrinological investigations in six patients revealed normal to high fsH and LH levels suggesting intact hypotha-lamic function. in contrast, testosterone levels were low and no response to human chorionic gonadotropin stimulation was observed. This suggests hypergonadotrophic hypogonadism, including a well-functioning hypothalamus and dysfunctional gonads.

The brain Mri scans of the patients with TOE1 mutations show similarities (fig. 2.9). all present pontocerebellar hypoplasia, enlarged ventricles and in most cases de-creased white matter with a thin corpus callosum.

other clinical symptoms included axial hypotonia with increased tone in the limbs and spastic seizures. development is severely delayed with no or very limited sitting,

hier tabel 2.1

hier figuur 2.8

hier figuur 2.9

38

tabl

e 2.

1: C

linic

al fe

atur

es o

f pa

tient

s w

ith T

oe1

mut

atio

ns. y

=yea

rs; w

=wee

ks; m

o=m

onth

s; u

=unk

now

n; U

s: u

ltra

soun

d; L

H=

lute

iniz

ing

horm

one;

fsH

= fo

llicl

e st

imul

atin

g ho

rmon

e; g

nrH

= go

nado

trop

in re

leas

ing

horm

one;

hCg

= hu

man

cho

rioni

c go

nado

trop

in;*

nM

_025

077.

3; **

nP_

0793

53.3

Patie

nt1-

i1-

ii2-

i 22-

ii 2

3 3

45

6 4

78

ethn

icity

Turk

ish

Turk

ish

Mor

occa

nM

oroc

can

Briti

shau

stra

lian

dani

shCa

nadi

anCh

ines

e/Vi

etna

mes

e/Ca

mbo

dian

Japa

nese

age

at la

st

exam

inat

ion

25y

22y

3y (d

eath

)7y

24w

(dea

th)

2y (d

eath

)11

mo

5y16

y14

y

nucl

eotid

e ch

ange

*c.g

307a

, c.g

307a

c.g30

7a,

c.g30

7ac.4

43T>

a,

c.443

T>a

c.443

T>a,

c.4

43T>

ac.5

18T>

g,

c.940

_941

delC

ac.5

18T>

g,

c.940

_941

delC

ac.5

18T>

g,

c.957

C>a

c.219

g>C,

c.6

93T>

ac.7

16T>

C,

c.148

7C>T

c.757

C>T,

c.955

C>T

amin

o ac

id ch

ange

**p.

a103

T, p.

a103

Tp.

a103

T, p.

a103

Tp.

f148

y, p.

f148

yp.

f148

y, p.

f148

yp.

V173

g,

p.Q

314V

fsX8

p. V1

73g,

p.

Q31

4Vfs

X8p.

V173

g,

p.H

319Q

p.r7

3s, p

.y23

1Xp.

f239

s, p.

s496

fp.

r253

W

p.H

319y

Kary

otyp

e46

,Xy

46,X

X46

,Xy

46,X

X46

,Xy

46,X

y46

,Xy

46,X

y46

,Xy

46,X

y

exte

rnal

gen

itals

mic

rope

nis,

hypo

plas

tic

scro

tum

norm

al fe

mal

eno

pen

is, f

used

la

bia

norm

al fe

mal

ere

gres

sing

m

icro

peni

s, la

bios

crot

al

fold

s

peni

s, fu

sed

labi

ano

pen

is, la

bia

maj

ora

pres

ent,

labi

a m

inor

a ab

sent

no p

enis

, no

rmal

labi

ano

pen

is,

norm

al la

bia

no p

enis

, no

rmal

labi

a

inte

rnal

gen

itals

atro

phic

test

esov

arie

s no

t see

n on

Us,

poss

ibly

at

roph

ic

abse

ntno

rmal

ova

ries

abse

ntun

clea

rab

sent

ovar

ies

ovar

ies

unila

tera

l un

desc

ende

d te

stis

uter

usab

sent

pres

ent

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esen

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sent

rem

nant

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ut

erin

e tis

sue

rem

nant

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ut

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sue

pres

ent

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nt

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high

LH

and

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H, lo

w

test

oste

ron

high

LH

an

d fs

H, n

o m

enar

che

at

20y

uu

norm

al

resp

onse

to

gnrH

, no

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onse

to h

Cg

uno

rmal

LH

, hig

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Hno

rmal

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, hig

h fs

Hu

norm

al L

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high

fsH

, low

te

stos

tero

ne

39

Chapter

2

Toe1 m

utations in PCH7

walking, speech and social eye contact. Three patients died at the age of 24 weeks, 2 years and 3 years, the other patients vary in age from 1 year old to late twenties.

functional studies tOe1

Multiple functions have been attributed to TOE1. it was initially identified as gene under control of early growth response 1 (egr1), and therefore named target of egr1. it was assumed to be important in cell cycle and cell growth [13]. furthermore, the Toe1 protein includes a dedd domain, suggesting deadenylating activity [6] and it can interact with tumor suppressor protein p53 [18]. More recent papers about TOE1 describe the protein as essential for Cajal body integrity [8] and as inhibitor of viral replication [7]. all these functions do not give an explanation for the cerebellar hypoplasia. However, TOE1 has been previously linked to Purkinje cell degeneration in

A B C

figure 2.8: genital abnormalities in PCH7 patients. all 46,Xy patients with PCH7 present some degree of genital undervirilisation. Patients 3, 5 and 8 are shown. Patient 3 (a) has a micropenis with labioscrotal folds. Patient 5 (B) is a 46,Xy patient with labia majora and no labia minora. Patient 8 (C) has nearly normal female genitals, and undescended testes.

1-II 2-II 3 4

5 6 7 8

figure 2.9: Mr images of patients with TOE1 mutations. Patients show a hypoplastic pons and cerebel-lum, thin corpus callosum and large ventricles.

40

arabian horses with cerebellar abiotrophy [9]. How TOE1 is functionally linked to brain and genital development is still unknown.

next to a defective function of Toe1 protein, the sequence variants could have a cis-effect on expression of adjacent genes. The TOE1 gene overlaps on the opposite strand with the gene MUTYH on its 5’ end and the gene TESK2 on its 3’ end [19]. TESK2 is involved in spermatogenesis, which could be is of potential interest in view of the genital phenotype we see in PCH7 patients. indications for an increase in MUTYH expression is found in arabian horses with TOE1 mutations [9]. However, we could not confirm this in patients’ fibroblasts, nor did we find alternations in TESK2 expression (fig. 2.10).

To evaluate the impact of the TOE1 mutations at the molecular structural level, we constructed a model structure of the dedd domain of human Toe1 from the crystal structure of mouse Parn protein. five out of the nine missense mutations were mapped onto the obtained model structure, and two other mutations (both at amino acid H319) were mapped onto the nMr structure of the zinc finger domain of Toe1 (fig. 11). The p.a103T and p.V173g mutations are located at a hydrophobic core of the dedd domain and may therefore affect stability and deadenylase activity of Toe1. in case of Parn, the highly conserved p.f148 position is reported to be involved in homodimerization, which is crucial for deadenylase activity [20]. Thus, the p.f148y mutation of Toe1 may impair protein-protein interactions and enzymatic activity. in the zinc finger domain, the p.H319Q and p.H319y mutations could disrupt its folding. The two other mutations identified in the PCH7 patients, p.f239s and p.r253W, are

hier figuur 2.10

0

0,5

1

1,5

2

2,5

3

3,5

TOE1 TESK2 MUTYH

control 1

control 2

het A103T

het p.Q314VfsX8

het p.V173G

p.V173G + p.Q314VfsX8

hom p.A103T

hom p.F148Y

p.R73S + p.Y231X

Fold

chan

ge

figure 2.10: expression levels of genes in the TOE1 locus. displayed expression levels including error bars are means of three independent experiments.

41

Chapter

2

Toe1 m

utations in PCH7

unlikely to affect protein folding substantially. However, since deadenylases often function in a multisubunit complex, it cannot be excluded that these mutations cause disturbances of protein-protein interactions.

disCUssion

in this study, we confirmed the presence of a novel syndrome - pontocerebellar hypo-plasia with disorders of sex development – previously proposed as PCH7. We described eight families with this disorder, all harbouring mutations in the TOE1 gene.

By excluding a functional effect of the p.a390s variant in CLK2 in our index family and identifying TOE1 mutations in all eight families, we show that PCH7 is not a coincident combination of two syndromes, but an isolated disease.

The combination of dsd and neurological anomalies is seen in a few other disorders. for example, patients with smith-Lemli-opitz syndrome (oMiM 270400) can present hypoplasia of the corpus callosum and cerebellum, microcephaly and ambiguous genitals in 46, Xy. The disease is caused by a deficiency in 7-dehydrocholesterol re-ductase [21]. Prader-Willi syndrome (oMiM 176270) includes decreased fetal activity, hypotonia and mental retardation in combination with hypogonadism in 46, Xy and 46, XX. in contrast to PCH7, this hypogonadism is of hypothalamic origin, including low levels of LH and fsH [22]. X-linked alpha-thalassaemia mental retardation syndrome (aTrX, oMiM 301040) is a rare syndrome caused by mutations in the ATRX gene. symptoms include mental retardation, severe developmental delay, microcephaly and genital abnormalities in the majority of patients ranging from hypospadias to Xy sex

hier figuur 2.11

A B

figure 2.11: a modeled structure of the dedd domain of human Toe1. The model is constructed from the crystal structure of mouse poly(a)-specific ribonuclease (Parn) (PdB code; 3d45) and (B) the nMr structure of the C3H-type zinc finger domain of human Toe1 (PdB code; 2fC6) are shown. The residues at the mutation sites are depicted in purple by van der Waals spheres in a and sticks in B, respectively. a gray arrow indicates the rna-binding cleft. Two catalytic residues, d64 and e66 (a), and three cysteine residues of Zf (B) are shown as sticks with oxygen and sulfur atoms colored red and yellow, respectively. The zinc ion is shown as a green sphere.

42

reversal [23]. although PCH7 is not the only disorder that includes both brain and genital abnormalities, in none of previously described disorders brain atrophy is as pronounced as in PCH7.

Up to now, we can only speculate about the exact role of TOE1 in brain and genital development. TOE1 aberrations have shown to cause Purkinje cell degenerations in arabian horses. it was proposed that the TOE1 mutation altered expression of the overlapping MUTYH gene, but we could not confirm this in fibroblasts of PCH7 pa-tients. Perhaps different results would be obtained when investigated mrna levels in brain or gonadal tissue. Taking in account the function of other PCH-genes –  in rna metabolism and protein synthesis - the function of TOE1 in the integrity of Cajal bodies is potentially interesting. Cajal bodies are nuclear organelles that contain high numbers of small nuclear ribonucleoproteins (snrnPs), which are essential for mrna splicing. it is plausible that TOE1 mutations lead to aberrant mrna splicing, subse-quently leading to disturbances in protein synthesis. splicing has also been shown to be important in sex differentiation [24].

This is the first time that TOE1 is linked to genital development. The patients with TOE1 mutations present high LH and fsH, suggesting a functioning pituitary gland. Testosterone is low in 46, Xy patients, indicating malfunctioning gonads. also in a 46, XX patient with TOE1 mutations (patient 1-ii) we see aberrations in gonad develop-ment, as ovaries were not observed and puberty did not initiate. early in gonadal development, a bipotential gonad is formed, which can differentiate in either testes or ovaries [25]. since both 46, Xy and 46, XX present gonadal dysfunction, it is plausible that TOE1 mutations cause a defect in formation of the bipotential gonad.

further functional studies could focus on the role of TOE1 in mrna splicing, dead-enylation and other rna processing events. With the identification of TOE1 as PCH causing gene, yet another link between rna processing and neurodegeneration is made.

43

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utations in PCH7

referenCes

1. ostrer H: disorders of sex development (dsds): an update. J Clin Endocrinol Metab 2014, 99: 1503-1509.

2. Budde Bs, namavar y, Barth Pg, Poll-The BT, nurnberg g, Becker C et al.: trna splicing endo-nuclease mutations cause pontocerebellar hypoplasia. Nat Genet 2008, 40: 1113-1118.

3. anderson C, davies JH, Lamont L, foulds n: early pontocerebellar hypoplasia with vanishing testes: a new syndrome? Am J Med Genet A 2011, 155a: 667-672.

4. siriwardena K, al-Maawali a, guerin a, Blaser s, Chitayat d: Xy sex reversal, pontocerebellar hypoplasia and intellectual disability: confirmation of a new syndrome. Am J Med Genet A 2013, 161a: 1714-1717.

5. Mahbubul Huq aH, nigro Ma: Xy sex reversal and a nonprogressive neurologic disorder: a new syndrome? Pediatr Neurol 2000, 23: 357-360.

6. Wagner e, Clement sL, Lykke-andersen J: an unconventional human Ccr4-Caf1 deadenylase complex in nuclear cajal bodies. Mol Cell Biol 2007, 27: 1686-1695.

7. sperandio s, Barat C, Cabrita Ma, gargaun a, Berezovski MV, Tremblay MJ et al.: Toe1 is an inhibitor of HiV-1 replication with cell-penetrating capability. Proc Natl Acad Sci U S A 2015, 112: e3392-e3401.

8. fong KW, Li y, Wang W, Ma W, Li K, Qi rZ et al.: Whole-genome screening identifies proteins localized to distinct nuclear bodies. J Cell Biol 2013, 203: 149-164.

9. Brault Ls, Cooper Ca, famula Tr, Murray Jd, Penedo MC: Mapping of equine cerebellar abi-otrophy to eCa2 and identification of a potential causative mutation affecting expression of MUTyH. Genomics 2011, 97: 121-129.

10. Thisse C, Thisse B: High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc 2008, 3: 59-69.

11. Wu M, nilsson P, Henriksson n, niedzwiecka a, Lim MK, Cheng Z et al.: structural basis of m(7)gpppg binding to poly(a)-specific ribonuclease. Structure 2009, 17: 276-286.

12. Kelley La, Mezulis s, yates CM, Wass Mn, sternberg MJ: The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 2015, 10: 845-858.

13. de B, i, Wu JX, sperandio s, Mercola d, adamson ed: in vivo cloning and characterization of a new growth suppressor protein Toe1 as a direct target gene of egr1. J Biol Chem 2003, 278: 14306-14312.

14. franklin rB, Ma J, Zou J, guan Z, Kukoyi Bi, feng P et al.: Human ZiP1 is a major zinc uptake transporter for the accumulation of zinc in prostate cells. J Inorg Biochem 2003, 96: 435-442.

15. duncan Pi, stojdl df, Marius rM, scheit KH, Bell JC: The Clk2 and Clk3 dual-specificity protein kinases regulate the intranuclear distribution of sr proteins and influence pre-mrna splicing. Exp Cell Res 1998, 241: 300-308.

16. rudnik-schoneborn s, Barth Pg, Zerres K: Pontocerebellar hypoplasia. Am J Med Genet C Semin Med Genet 2014, 166C: 173-183.

17. Cirulli eT, Lasseigne Bn, Petrovski s, sapp PC, dion Pa, Leblond Cs et al.: exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways. Science 2015.

18. sperandio s, Tardito s, surzycki a, Latterich M, de B, i: Toe1 interacts with p53 to modulate its transactivation potential. FEBS Lett 2009, 583: 2165-2170.

19. Makalowska i: Comparative analysis of an unusual gene arrangement in the human chromo-some 1. Gene 2008, 423: 172-179.

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20. goldstrohm aC, Wickens M: Multifunctional deadenylase complexes diversify mrna control. Nat Rev Mol Cell Biol 2008, 9: 337-344.

21. Bianconi se, Cross JL, Wassif Ca, Porter fd: Pathogenesis, epidemiology, diagnosis and Clinical aspects of smith-Lemli-opitz syndrome. Expert Opin Orphan Drugs 2015, 3: 267-280.

22. angulo Ma, Butler Mg, Cataletto Me: Prader-Willi syndrome: a review of clinical, genetic, and endocrine findings. J Endocrinol Invest 2015.

23. stevenson re: alpha-Thalassemia X-Linked intellectual disability syndrome. in:  Pagon ra, adam MP, ardinger HH, Bird Td, dolan Cr, fong CT, smith rJH, stephens K, editors.  gener-eviews® [internet]. seattle (Wa): University of Washington, seattle; 1993-2014 (updated 2014)

24. ohe K, Lalli e, sassone-Corsi P: a direct role of sry and soX proteins in pre-mrna splicing. Proc Natl Acad Sci U S A 2002, 99: 1146-1151.

25. eggers s, ohnesorg T, sinclair a: genetic regulation of mammalian gonad development. Nat Rev

Endocrinol 2014, 10: 673-683.

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utations in PCH7

supplementary table 1: overview of primers. all primers – except those used for qPCr – were flanked by M13 motives for sequencing; forward: 5’-TgTaaaaCgaCggCCagT-3’; reverse: 5’-CaggaaaCagCTaT-gaCC-3’.

name forward reverse

CLK2_ex1 5’-ggaaaTgaagTgaCCgTgga-3’ 5’-CTaCgaCgCCTCCgCTaga-3’

CLK2_ex2 5’-gTaCCTCCgaggCTCTgaCa-3’ 5’-CCCaaCCCaagCCTaaCC-3’

CLK2_ex3 5’-agCCagTCCTTggaggagaT-3’ 5’-TTaaCaCTgCagCCCaaaTg-3’

CLK2_ex4 5’-TTgTCaCagggTaTggTaggg-3’ 5’-CCaaTgTTCagCTgagaaCaag-3’

CLK2_ex5 5’-gTCCCCCaTCaTCgTCTgT-3’ 5’-CTgTgaCTCaggTTggCTTg-3’

CLK2_ex6and7 5’-CTTCCCCTgTgaCCaTCTgT-3’ 5’-TCTaggaCTCCCCTgTCTgC-3’

CLK2_ex8 5’-CTCCTTgTTggaTggCTgaT-3’ 5’-TgTTTaggggCTTCaCCagT-3’

CLK2_ex9 5’-ggCTTTgCCCTaggTaaCCa-3’ 5’-CagaaggCaaagTggTgCag-3’

CLK2_ex10and11 5’-CCaggCaggagaTCaCagTa-3’ 5’-agTaggCaggTCCTCagCag-3’

CLK2_ex12and13 5’-gCCTggCaTTCTTCTaCCTg-3’ 5’-aCCCTCTgCTTgTgaagagC-3’

CLK2_ex14p1 5’-CTTgaagaaggCagggTTTg-3’ 5’-aggTgagggTggaaaCTgTg-3’

CLK2_ex14p2 5’-CaaCaagTTgTgggaCTCCa-3’ 5’-ggaggaCTCCCTTCaTaCCC-3’

Toe_ex1 5’-aaCggaagTTCgaCCCaTC-3’ 5’-ggaaggagCagTCCTCTgaa-3’

Toe_ex2en3 5’-ggCCTagCTTggTgTCTgTT-3’ 5’-ggTTgCTgTggCTaggagTT-3’

Toe_ex4 5’-gTCaCagaCCTgggTggTTT-3’ 5’-aaaaagaTTTgggagagaaaTgC-3’

Toe_ex5 5’-TTaTggggaaCaggTggTgT-3’ 5’-CCaCTTggaaCgCTCTCTCT-3’

Toe_ex6 5’-ggCagCaTTgggTaaggTag-3’ 5’-gCagggTTTTgTggTCTgTT-3’

Toe_ex7and8a 5’-TggggCCTgaTaCgagTTTa-3’ 5’-ggTTTCTTCaggCTTgaTgC-3’

Toe_ex8b 5’-gaaaaaCggaagagggCTTT-3’ 5’-gaTaaaggaggCggTCCag-3’

sLC39a1 5’-ggagggggTTTgTTTgCTTa-3’ 5’-aaCCaaCaCTCCCagaggTg-3’;

Toe1_qPCr 5’-cctaccataagggcaatgaca-3’ 5’-ggccattgtgtagcaccag-3’

TesK2_qPCr 5’-ccccagattttctgcaactta-3’ 5’-cctgtaggcggctcagaa-3’

MUTyH_qPCr 5’-atgacaccgctcgtctcc-3’ 5’-gcttctgcctcccttcct-3’

rs968322 5’-CCTgagTCTTTgCCCagaag-3’ 5’-aTggTggTgTagggCaTCTg-3’

rs4660360 5’-TgggCTTTCagTgCaaTgC-3’ 5’-TCCTCTTTCTCCCTCTCTgg-3’

rs61784349 5’-agaTTgTgaaaCCTggCTTCa-3’ 5’-CTTCTCaTTgagCaCCTaCCC-3’

rs189962969 5’-TgagagaaggCagTTTgggT-3’ 5’-CTgTaCaaagCTgCTggTCC-3’

rs200725979 5’-CCaaCTTgCCaCaCaTCagg-3’ 5’-gCaCTgCTaTCaaTgaCaagga-3’

rs370490105 5’-aCagaaaCaTTagaagTggagga-3’ 5’-TgTgCCaaCCTCTaCCaCaT-3’

rs75705909 5’-gaaCTCagCCTCTCCTCCag-3’ 5’-CaaaCCTTCTaTCCCTaCCTCCa-3’

rs145119239 5’-gTagCaggTCCTTCTCTggg-3’ 5’-aaCaTCTTgTCCagCCCCTT-3’

rs150795467 5’-TgCaggTTTCCaaaTaCggC-3’ 5’-gaggagCaCTgTCCaCCTag-3’

rs191887693 5’-gTgagTgCTggagggagg-3’ 5’-CaCaTggggTTTTgCTgagC-3’

rs200936475 5’-TTTCagaagagCagCCTgTg-3’ 5’-CTCCTaCaaCTgaCggTCCa-3’

rs202091916 5’-CaagCCgCTgTaTaaCCgTC-3’ 5’-aggCCaTaTaTCTgTgTaCTgCT-3’

46

supplementary table 2: Haplotype analysis of the three patients with a c.518T>g; p.V173g variant in Toe1. The 9Mb homozygous stretch shared amongst the three patients is indicated in red.

snP distance to tOe1 (mb) patient 3 patient 4 patient 5

rs145119239 -9.7 1 0 0

rs75705909 -7.5 1 1 1

rs202091916 -5.8 0 0 0

rs200415763 -5.8 0    

rs150795467 -5-5 1    

rs191887693 -0.3 0 0 0

c.518T>g 0 1 1 1

c.940_941delCa 0 0 0 0

rs200936475 +0.2     0

rs189962969 +0.7 0 0 0

rs4660360 +1.5   1  

rs968322 +1.8   1 0

rs200725979 +7.3 0 0 0

Zalstoe altied bie mie blieven, bie mie blieven, lutje wichtLekker waarm in mien aarms, in dien aarms, ogen dicht

-Ede Staal

3EXOSC3 mutatiOns in

POntOcerebellar hyPOPlasia tyPe 1: nOVel mutatiOns and genOtyPe-

PhenOtyPe cOrrelatiOns

Veerle rC eggens, Peter g Barth, Jikke-Mien f niermeijer, Jonathan n Berg, niklas darin,

abhijit dixit, Joel fluss, nicola foulds, darren fowler, Tibor Hortobágyi, Thomas Jacques,

Mary d King, Periklis Makrythanasis, adrienn Máté, James ar nicoll, declan o’rourke, sue

Price, andrew n Williams, Louise Wilson, Mohnish suri, Laszlo sztriha, Marit B dijns-de

Wissel, Mia T van Meegen, fred van ruissen, eleonora aronica, dirk Troost, Charles BLM

Majoie, Henk a Marquering, Bwee-Tien Poll-Thé, frank Baas

Orp

hane

t Jou

rnal

of R

are

Dis

ease

. 9:2

3 (2

014)

50

aBsTraCT

background: Pontocerebellar hypoplasia (PCH) represents a group of neurodegen-erative disorders with prenatal onset. eight subtypes have been described thus far (PCH1-8) based on clinical and genetic features. Common characteristics include hypoplasia and atrophy of the cerebellum, variable pontine atrophy, and severe men-tal and motor impairments. PCH1 is distinctly characterized by the combination with degeneration of spinal motor neurons. recently, mutations in the exosome component 3 gene (EXOSC3) have been identified in approximately half of the patients with PCH subtype 1.

methods: We selected a cohort of 99 PCH patients (90 families) tested negative for mutations in the TSEN genes, RARS2, VRK1 and CASK. Patients in this cohort were referred with a tentative diagnose PCH type 1, 2, 4, 7 or unclassified PCH. genetic analysis of the EXOSC3 gene was performed using sanger sequencing. Clinical data, Mr images and autopsy reports of patients positive for EXOSC3 mutations were ana-lyzed.

results: EXOSC3 mutations were found in twelve families with PCH subtype 1, and were not found in patients with other PCH subtypes. identified mutations included a large deletion, nonsense and missense mutations. examination of clinical data reveals a prolonged disease course in patients with a homozygous p.d132a mutation. Mri shows variable pontine hypoplasia in EXOSC3 mediated PCH, where the pons is largely preserved in patients with a homozygous p.d132a mutation, but attenuated in patients with other mutations. additionally, bilateral cerebellar cysts were found in patients compound heterozygous for a p.d132a mutation and a nonsense allele.

conclusions: EXOSC3 mediated PCH shows clear genotype-phenotype correlations. a homozygous p.d132a mutation leads to PCH with possible survival into early puberty, and preservation of the pons. Compound heterozygosity for a p.d132a mutation and a nonsense or p.y109n allele, a homozygous p.g31a mutation or a p.g135e mutation causes a more rapidly progressive course leading to death in infancy and attenuation of the ventral pons.

our findings imply a clear correlation between genetic mutation and clinical outcome in EXOSC3 mediated PCH, including variable involvement of the pons.

Keywords: Pontocerebellar hypoplasia, neurodegeneration, eXosC3 gene, genotype-phenotype correlations

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utations in PCH1

BaCKgroUnd

Pontocerebellar hypoplasias represent a group of autosomal recessive neurodegener-ative disorders with prenatal onset. Patients of all subtypes show variable hypoplasia/atrophy of pons and cerebellum and severe motor and cognitive impairments. Based on clinical and genetic criteria, eight subtypes of PCH have been classified (PCH1-8). PCH1 (MiM607596, 614678) is a distinctive subtype of PCH, and is characterized by degeneration of motor neurons in the anterior spinal horn, morphologically similar to spinal muscular atrophy (sMa). PCH1 patients present a broad phenotypic spectrum, ranging from neonatal death [1] to survival into puberty ([2] and patients 7-i and 7-ii in this paper). in few families diagnosed with PCH1, mutations have been found in TSEN54 [3], RARS2 [4] and vaccinia-related kinase 1 (VRK1) [5]. approximately half of PCH1 families carry mutations in EXOSC3, the gene encoding exosome component 3 [2,6-9].

in this study, we report EXOSC3 mutations in twelve families with PCH1. We cat-egorised the patients according to genetic mutation, and correlate this with clinical severity and size of the ventral pons.

MeTHods

Patient cohort

our laboratory is a reference centre for PCH genetic analysis. We received edTa-blood or dna samples from hospitals and institutes worldwide for genetic analysis of genes associated with PCH. for this study, we selected dna material from a cohort of 99 patients (90 families) diagnosed by referring specialists with PCH type 1, 2, 4, 7 or an unknown PCH-like anomaly. all cases were negative for mutations in genes known to cause PCH (TSEN54, TSEN34, TSEN2, RARS2, VRK1 and CASK). informed consent was obtained by referring specialists.

genetic analysis

PCr primer pairs for EXOSC3 [nM_016042.2] were designed for all exons including intron-exon boundaries using Primer3 software (http://frodo.wi.mit.edu/). for primer sequences, see additional file 1. sanger sequencing of PCr amplified dna was per-formed using BigdyeTerminator chemistry (applied Biosystems) and analysed on an aBi3730xl sequencer. sequences were analysed using CodonCode aligner software 3.6.1. analysis of gene mutations was done with the alamut software package (inter-active Biosoftware, version 2.0), which includes the splice site prediction algorithms splicesitefinder, Maxentscan and Human splicing finder. detection of large deletions

52

in EXOSC3 was initially performed with you-MaQ assay (Multiplicon), using primers in the 5’UTr and intron 3 (see additional file 1). detailed analysis of the deletion in patient 8 was performed with the sequalPrep Long PCr Kit (Life Technologies), using primers in the 3’UTr and upstream of EXOSC3 (see additional file 1).

neuroimaging

routine Mr images of EXOSC3 mutation positive patients were re-examined. Coro-nal cerebellar images from patients found to be positive for mutations in EXOSC3 were subclassified as previously described [4]. The main impact of the process that causes pontine hypoplasia/atrophy is on the ventral pons, which leaves the dorsal area (tegmentum) relatively unaffected [10,11]. The ventral pons and tegmentum can be distinguished on routine Mri. on midsagittal Mr scans, surfaces of these areas were determined using iTK-snaP 2.4.0 software [12] and ventral pons/(ventral pons+tegmentum) (VP/(VP+T)) ratios were determined as a measure for pontine hy-poplasia/atrophy. Three-dimensional images were constructed based on a series of Mr scans using the same software. The control group consists of Mr images obtained from children (n=23; neonatal to 11y) referred by paediatric neurologists for diagnos-tic Mri, whose brain Mr image was considered normal.

histological stainings

Paraffinised sections of the pons were stained with Luxol/Pas following standard protocols as described previously [13].

resULTs

EXOSC3 molecular analysis

screening of a cohort of 99 patients (90 families) with various PCH subtypes revealed EXOSC3 mutations in fourteen PCH patients (twelve families, Table 3.1). six patients were homozygous for the c. 92g>C (p.g31a) mutation, three patients were homo-zygous for the c.395a>C (p.d132a) mutation, and one patient was homozygous for the c.404g>a (p.g135e) mutation. We found four patients to be compound hetero-zygous for EXOSC3 mutations (patients 8, 9, 10 and 11). Patient 8 had a hemizygous p.d132a mutation and a heterozygous 6,171 nucleotide large deletion containing the promoter region, the first three exons and a part of intron 3 of the EXOSC3 gene (g.del37781240-37787410), without affecting adjacent genes. Patient 9 had a hetero-zygous p.d132a mutation and a c.743_749delinsa mutation, leading to a premature stop codon (p.L248*). Patient 10 had a p.d132a mutation on the maternal allele. on the paternal allele, this patient had a c.334g>a (p.V112i) variation in addition to a nine

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nucleotide duplication leading to a premature stop codon (c.325-4_329dupgTagTaTgT; p.P111*). The p.V112i amino acid change was predicted to be benign by sifT (score 0,32) and align gVgd (class C0), and possibly damaging by PolyPhen-2 (score 0,949). Patient 11 had the p.d132a mutation plus a c.325T>a (p.y109n) mutation. The c.325 residue is the first nucleotide of exon 2, but the mutation is not predicted to affect splicing by the splicesitefinder, Maxentscan and Human splicing finder software. Unfortunately, tissue was not available for splicing studies. The p.y109n missense mutation was predicted to be deleterious by PolyPhen-2 (score 1,000), sifT (score 0,0) and align-gVgd (class C65). in all cases, we confirmed the mutations by sequencing the parents. in case of expected compound heterozygosity, we could show that the mutations were on different alleles.

in our cohort with different PCH subtypes, all twelve families harbouring EXOSC3 mutations were diagnosed as PCH1. in four other PCH1 families in our cohort we could not detect mutations in coding regions of this gene. no EXOSC3 mutations were found in patients diagnosed with other types of PCH. Therefore, EXOSC3 mutations seem to be exclusively associated with the PCH1 subtype. The patients harbouring the p.g31a mutation are all of romani descent - although living in different countries (sweden and Hungary) - suggesting a common founder as proposed earlier [7,9]. The families with the p.d132a allele are all of Caucasian descent.

clinical data of patients with EXOSC3 mutations

Most EXOSC3 mutation positive patients were previously diagnosed as PCH1 based on clinical features. although patients 3 and 5-i were not tested for lower motor neuron symptoms, they were diagnosed following the diagnosis of patient 5-ii, which is patient 5-iis sister and patient 3s cousin. interestingly, patients 6, 7-i and 7-ii were initially not diagnosed as PCH1, as it was yet unknown that the PCH1 spectrum was broad, and that patients could survive childhood [6]. The finding of depleted motor neurons on autopsy in these patients confirmed PCH1 diagnosis.

Clinical features of EXOSC3 positive patients in our cohort are summarized in Table 3.1. Based on genetic mutations, our cohort can be divided in four subgroups: 1. homozygosity for the p.g31a mutation; 2. homozygosity for the p.d132a mutation; 3. compound heterozygosity for the p.d132a mutation on one allele and a deletion, nonsense or missense mutation on the other allele; 4. homozygosity for the p.g135e mutation.

in the p.g31a group (six patients, five families), hypotonic pareses were present at birth in five patients, and observed at one month in one patient. no patients were born with polyhydramnios, though all suffered from swallowing insufficiency. Tendon reflexes and responses to external stimuli were absent in all patients in this group. at birth, severe contractures were present in two patients: patient 2 presented with

hier tabel 3.1

54

contractures in the knee joints and had equinovarus foot deformities; patient 4 had contractures in hips, ankles, elbows, hands and fingers. dyskinesia or seizures were not observed in this group. Mild facial dysmorphisms were observed in three of the six patients, including low set ears (2/6), broad nasal bridge (2/6) and short palpebral fissures (2/6). Two patients had microcephaly (patient 1: -4 sd at 1.5 months of age; patient 2: -2.5sd at birth), while others had normal or even large ofCs (patient 4: +3sd

table 3.1 Clinical data of 14 patients with EXOSC3 mutation

1 2 3 4 5-I 5-II 6 7-I 7-II 8 9 10 11 12

nucleotide change c.92g > C c.92g > C c.92g > C c.92g > C c.92g > C c.92g > C c.395a > C c.395a > C c.395a > C c.395a > C (he) g.del37781240-37787410 (he)

c.395a > C (he) c.743_749delinsa (he)

c.325-4_329dupgTagTaTgT (he) c.334g > a (he) c.395a > C (he)

c.325 T > a (he) c.395a > C (he)

c.404g > a

amino acid change p.g31a p.g31a p.g31a p.g31a p.g31a p.g31a p.d132a p.d132a p.d132a p.d132a deletion exon 1-3

p.d132a p.L248* p.P111* p.V112i p.d132a p.y109n p.d132a p.g135e

ethnic background roma roma roma roma roma roma Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Pakistan

Pregnancy duration 39w, Cs at term 38w 37w 37w 40w 39w u 35w 39w 38w 42w 41w 40w

hypotonia at birth + + + + + + + + + + + ± + +

Ofc (sd)a (age) −4 (1.5 m) −2.5 (birth) 0 (birth) +3 (4.5mo) 0 (4mo) +2.5 (4mo) +3 (4.5mo) −0.5 (11y) −2 (6.5y) −1 (birth) u −0.5 (10w) −1.5 (6.5mo) −1 (8w)

nystagmus - - u + - - + + + - u - - +

Optic atrophy Pale optic disc - u - - - - u u + u - small optic discs Pale optic disc

seizures - - - - - - - + - + West syndrome at 5 mo - - - -

dyskinesia/dystonia - - - - - - + 1 episode, admitted with high temp and pneunomia

+ + - - - - -

tendon reflexes absent absent absent absent absent absent brisk brisk reduced reduced absent reduced absent absent

response on visual/auditory stimuli

- - u - - - ++ + + ± - ± - -

age at death (cause) 4,5mo(cardiac arrest)

7mo (pneumonia, sepsis)

5d c(respiratory failure)

5mo (u) 6mo (viral infection)

4mo (u) 7y (respiratory failure)

12y (gi failure)

10y (pseudomonas infection)

6mo (respiratory infection) 14w (respiratory failure)

6mo (respiratory infection)

8.5mo (respiratory failure)

8w (respiratory failure)

lower motor neuron signs

neurogenic muscle atrophy

neurogenic muscle atrophy

u, diagnosed following patient 5-ii (cousin)

Tongue fasciculations, denervation (eMg), neurogenic muscle atrophy

u, diagnosed following patient 5-ii (sister)

Muscle denervation (eMg)

u u, diagnosed following patien 7-ii (brother)

denervation (eMg)

neurogenic muscle atrophy

denervation (eMg) denervation (eMg), reduced motor nerve conduction velocity

Tongue fasciculations, neurogenis muscle atrophy

denervation, neurogenic muscle atrophy

he = heterozygous; d = days; w = weeks; mo = months; y = years; u = unknown; ++ = markedly present; + = present; ± = mildly present; - = not present.a sd for head circumference according to WHo standards (http://www.who.int/childgrowth/standards/hc_for_age/en/index.html).

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utations in PCH1

at 4.5 months of age) at the time of presentation. all homozygous p.g31a patients died during infancy (median age of death 4.75 months, range 5 days to 7 months).

in the p.d132a group (three patients, two families), hypotonia was found at birth in two patients, and at 13 weeks in one patient. no polyhydramnios was observed in this group. Tendon reflexes were either brisk (2/3 patients) or reduced (1/3 patients). Patients in this group were able to respond to visual and auditory stimuli. for example, patient 6 recognized familiar faces and voices, made social eye contact and babbled

table 3.1 Clinical data of 14 patients with EXOSC3 mutation

1 2 3 4 5-I 5-II 6 7-I 7-II 8 9 10 11 12

nucleotide change c.92g > C c.92g > C c.92g > C c.92g > C c.92g > C c.92g > C c.395a > C c.395a > C c.395a > C c.395a > C (he) g.del37781240-37787410 (he)

c.395a > C (he) c.743_749delinsa (he)

c.325-4_329dupgTagTaTgT (he) c.334g > a (he) c.395a > C (he)

c.325 T > a (he) c.395a > C (he)

c.404g > a

amino acid change p.g31a p.g31a p.g31a p.g31a p.g31a p.g31a p.d132a p.d132a p.d132a p.d132a deletion exon 1-3

p.d132a p.L248* p.P111* p.V112i p.d132a p.y109n p.d132a p.g135e

ethnic background roma roma roma roma roma roma Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Pakistan

Pregnancy duration 39w, Cs at term 38w 37w 37w 40w 39w u 35w 39w 38w 42w 41w 40w

hypotonia at birth + + + + + + + + + + + ± + +

Ofc (sd)a (age) −4 (1.5 m) −2.5 (birth) 0 (birth) +3 (4.5mo) 0 (4mo) +2.5 (4mo) +3 (4.5mo) −0.5 (11y) −2 (6.5y) −1 (birth) u −0.5 (10w) −1.5 (6.5mo) −1 (8w)

nystagmus - - u + - - + + + - u - - +

Optic atrophy Pale optic disc - u - - - - u u + u - small optic discs Pale optic disc

seizures - - - - - - - + - + West syndrome at 5 mo - - - -

dyskinesia/dystonia - - - - - - + 1 episode, admitted with high temp and pneunomia

+ + - - - - -

tendon reflexes absent absent absent absent absent absent brisk brisk reduced reduced absent reduced absent absent

response on visual/auditory stimuli

- - u - - - ++ + + ± - ± - -

age at death (cause) 4,5mo(cardiac arrest)

7mo (pneumonia, sepsis)

5d c(respiratory failure)

5mo (u) 6mo (viral infection)

4mo (u) 7y (respiratory failure)

12y (gi failure)

10y (pseudomonas infection)

6mo (respiratory infection) 14w (respiratory failure)

6mo (respiratory infection)

8.5mo (respiratory failure)

8w (respiratory failure)

lower motor neuron signs

neurogenic muscle atrophy

neurogenic muscle atrophy

u, diagnosed following patient 5-ii (cousin)

Tongue fasciculations, denervation (eMg), neurogenic muscle atrophy

u, diagnosed following patient 5-ii (sister)

Muscle denervation (eMg)

u u, diagnosed following patien 7-ii (brother)

denervation (eMg)

neurogenic muscle atrophy

denervation (eMg) denervation (eMg), reduced motor nerve conduction velocity

Tongue fasciculations, neurogenis muscle atrophy

denervation, neurogenic muscle atrophy

he = heterozygous; d = days; w = weeks; mo = months; y = years; u = unknown; ++ = markedly present; + = present; ± = mildly present; - = not present.a sd for head circumference according to WHo standards (http://www.who.int/childgrowth/standards/hc_for_age/en/index.html).

56

at the age of six years. Patients 7-i and 7-ii had remarkably good cognitive skills compared to other PCH patients - the boys were able to move a computer mouse to make simple ‘yes’ or ‘no’ choices in a computer game. due to severe motor impairments adequate testing of iQ was not possible, but parents and caretakers considered these brothers’ intellectual skills age appropriate. swallowing insufficiency was present in all three patients. severe contractures at birth were not seen in this group. from the age of 5 and 4 years respectively, patients 7-i and 7-ii developed progressively internally rotated arms and weak, claw-like deformity of the hands. dyskinesia was reported sporadically in patient 6 and regularly in patients 7-i and 7-ii. Patient 7-i had initially few generalised seizures per year, which increased in frequency and severity until death. This patient also developed intense visceral pains and ultimately suc-cumbed to his gastrointestinal tract malfunctioning. all three patients in this group survived into childhood (age of death 7, 12 and 10 years respectively).

The third group consists of patients with a p.d132a mutation in combination with a null allele or missense mutation in the EXOSC3 gene (four patients, four families). all patients were hypotonic at birth, with multiple contractures in three patients. Two patients could be breastfed or bottle fed in the first weeks of life, but developed swallowing difficulties later on. Tendon reflexes were absent (2/4) or reduced (2/4). dyskinesia was not reported in this group. seizures are reported in two patients: patient 8 developed West syndrome at the age of 5 months and in patient 10 seizures were reported by his parents in the days before his death. facial dysmorphisms in two patients included low set ears and large soft ear helices. in addition, patient 10 presented fat distribution as seen in Cdg1a, and patient 11 had a small penis and hypoplastic scrotum. The patients died during infancy (median age of death 6 months, range 3.5 to 8.5 months).

one patient in our cohort was homozygous for the p.g135e (c.404g>a) mutation. Born from consanguineous parents, he presented reduced fetal movements. at birth, he was hypotonic and had contractures of elbows and knees. He had nystagmus, pale optic discs and failed to respond to external stimuli. The patient died at the age of 8 weeks.

brain imaging studies

Mr images of the brain were available for all patients, except for patient 1. Based on coronal images, we categorised the cerebellar hemisphere patterns as described previously [4]: a dragonfly type (flattened hemispheres, relative prominence of ver-mis), a butterfly type (small but normally proportioned cerebellum) and a postnatal atrophy-like type (cerebellar atrophy rather than hypoplasia with the hemispheres reaching the margins of the posterior fossa in at least one spot). as a measure for pontine hypoplasia/atrophy we determined the VP/(VP+T) ratio on midsagittal Mr images (figure 3.1a).

hier figuur 3.1

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in the p.g31a group, four of the five cerebellar hemisphere pathologies resembled a dragonfly pattern (figure 3.2a). Patient 5-i presented a butterfly-like cerebellar pat-tern, albeit hemispheres are smaller than a typical butterfly pattern. in the p.g31a group, the pons is significantly attenuated compared to controls (p<0.01; figure 3.1B, C). supratentorial abnormalities are seen in three of the five patients in this group: these patients had widened extracerebral Csf spaces, and one patient (patient 4, born at 37 weeks gestational age) presented delayed neocortical maturation.

all three patients with a homozygous p.d132a mutation showed a postnatal atrophy-like cerebellar pattern (figure 3.2B). Patient 6 showed supratentorial abnor-malities: she had enlarged ventricles due to atrophy of the basal ganglia. Patient 7-ii underwent a CT scan at the age of one year, which was normal. an Mri performed at the age of five years showed a small cerebellum, suggesting progressive atrophy. The VP/(VP+T) ratio in this group equals that of healthy controls (figure 3.1B, f), indicating a largely unaffected pons in patients with a p.d132a mutation (figure 3.1B, d).

Three of the four patients in the compound heterozygous group presented a drag-onfly cerebellum. one patient in this group (patient 9) presented a butterfly pattern.

hier figuur 3.2

A B

A VPTmedulla

DC

C

p.G31A 3 months

D

p.D132A 11 years

E

p.D132A + deletion 6 days

F

control 2 years

contro

ls

p.G31

A

p.D132A

compound p.D13

2A

p.G13

5E

TSEN54 A30

7S0.0

0.2

0.4

0.6

0.8 p<0.01

VP/(V

P+T)

figure 3.1 Ventral pons/tegmentum ratios in PCH patients. on midsagittal Mr images, surfaces of ventral pons and pontine tegmentum were defined as shown in figure a. Patients with a homozygous p.d132a mutation in EXOSC3 (n=3) present a pons/tegmentum ratio comparable to controls (n=23, age neonatal to 11y). Patients with a homozygous p.g31a mutation (n=5), a p.d132a mutation plus a nonsense or p.y109n allele (n=4) or a homozygous p.g135e mutation (n=1) show a decreased ratio, approaching that seen in patients with a p.a307s mutation in the TSEN54 gene (n=6) (B). Three-dimensional images were constructed of the pons, tegmentum and part of the medulla. The reconstructions show an attenuated pons in a patient with a homozygous p.g31a mutation (C, patient 5-i), compared to patient 7-i (d, homo-zygous p.d132a), patient 8 (e, p.d132a plus large deletion) or a control subject (f). scale bar in C-f=1cm. VP=ventral pons; T=tegmentum.

58

Cerebellar cysts were common in this group (3/4 patients) (figure 3.2C). supratentorial abnormalities are seen in patient 10 (mild atrophy of the cerebral hemispheres and caudate nuclei and widened ventricles) and patient 11 (thin corpus callosum). Midsag-ittal pons measurements showed pontine attenuation in this group (figure 3.1B, e).

The patient homozygous for the p.g135e mutation presents with cerebellar cysts and no supratentorial abnormalities. since no coronal image was available for this patient, it is hard to describe the pattern type of the cerebellar hemispheres. ratio of midsagittal pons/tegmentum surface in this patient is comparable to the p.g31a group (figure 3.1B).

To conclude, the genotype-phenotype correlation of the various EXOSC3 mutations is reflected in the severity of cerebellar and pontine hypoplasia.

neuropathological findings

autopsy was performed in one patient with a p.g31a mutation (patient 1). no Mri was made of this patient, but post mortem results showed atrophy of cerebellar hemispheres, inferior and middle cerebellar peduncles. The cerebellar hemispheres

A B C

figure 3.2 Brain Mri of PCH patients with an EXOSC3 mutation. sagittal and coronal images of a patient with a homozygous p.g31a mutation (a, patient 5-ii, age 2w), a patient with a homozygous p.d132a mutation (B, patient 7-i, age 11y) and a patient with a p.d132a mutation and large deletion (C, patient 8, age 1mo). Cerebellar cysts in this last patient are indicated by arrow heads.

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were poorly developed and presented a smooth surface. Histological analysis showed a reduced number of Purkinje cells and a fragmented dentate nucleus. The number of transverse pontine fibres and pontine neurons was reduced compared to control tissue (figure 3.3).

Post mortem studies were performed on all three patients with a p.d132a mutation. in all, atrophy of cerebellar hemispheres and vermis was noted, including a reduced number of Purkinje cells. Characteristic for PCH1 – the number of motor neurons in the anterior spinal horn was reduced. residual motor neurons showed a shrunken and angulated morphology. in one patient (7-i), depletion of neurons in the dorsal horn was also seen. examination of the pons revealed proportionate reduction of trans-verse pontine fibres, descending fibres and pontine nuclei in patient 6. no pontine abnormalities were found in patient 7-i and 7-ii, in line with the normal appearance of the pons on Mri. Cerebral anomalies were only found in patients 7-i and 7-ii: both presented neuron loss, gliosis and calcifications in the thalamus.

disCUssion

We performed genetic screening on 99 subjects with various types of PCH. among this cohort, we identified fourteen PCH1 patients (twelve families) with mutations in EXOSC3, suggesting that mutations in this gene are restricted to PCH subtype 1. four families with PCH1 that we screened for EXOSC3 mutations did not have mutations in coding regions of this gene. in addition to the EXOSC3 mutations described previously,

hier figuur 3.3

DC

A

VP

TB

VP

T

p.G31Acontrol

figure 3.3 Pons abnormalities in patient with p.g31a mutation. Luxol/Pas staining of the pons shows small pons and reduction of transverse pontine fibres (B) and pontine neurons (d) in a patient with a homozygous p.g31a mutation (patient 1) compared to age matched control (a and C). scale bar a and B=0.5cm, C and d=100µm. VP=ventral pons; T=tegmentum.

60

we identified five novel variants in EXOSC3, leading to an amino acid substitution or a nonsense allele.

after the identification of EXOSC3 as a PCH associated gene [6], many different mutations have been found in this gene (Table  3.2). This genetic heterogeneity is reflected in the broad phenotypic spectrum of EXOSC3 mediated PCH, ranging from relatively mild (e.g. compound heterozygous p.d132a/p.V80f mutation [2]) to severe (e.g. homozygous p.g31a mutation [9]). findings by us and others [6-8], show that EXOSC3 mutations underlie about half of the PCH1 cases. EXOSC3 mediated PCH shows a genotype-phenotype correlation: the p.d132a mutation leads to a relatively prolonged disease course including survival into childhood, limited social interac-tion and preservation of the pons. a homozygous p.g31a mutation, or a p.d132a in combination with a nonsense or p.y109n mutation leads to a more severe type of PCH reflected in clinical symptoms, death during infancy and hypoplasia of the pons. The patient with a homozygous p.g135e mutation shows a similarly severe phenotype.

Previous research suggested that patients with an EXOSC3 mutation show a rela-tively preserved pons in comparison to other types of PCH [4,7,8]. We have quantified this and we demonstrate that the pons/tegmentum ratio on midsagittal Mr images of patients with a homozygous p.d132a mutation indeed resembles that of healthy

hier tabel 3.2

table 3.2 overview of mutations identified in EXOSC3

allele a allele b reference

p.g31a (c.92g > C) p.g31a (c.92g > C) [6,7,9] This paper

p.g31a (c.92g > C) p.W238r (c.712 T > C) [6,7]

p.d132a (c.395a > C) p.d132a (c.395a > C) [6-8] This paper

p.d132a (c.395a > C) start codon affected (c.2 T > C) [7]

p.d132a (c.395a > C) p.P52rfs*2 (c.155delC) [7]

p.d132a (c.395a > C) p.d76gfs*49 (c.226dupg) [7]

p.d132a (c.395a > C) p.V80f (c.238g > T) [2]

p.d132a (c.395a > C) p.V99Wfs*11 (c.294_303del) [6]

p.d132a (c.395a > C) p.y109n (c.325 T > a) This paper

p.d132a (c.395a > C) p.P111*; p.V112i (c.325-4_329dupgTagTaTgT; c. 334g > a) This paper

p.d132a (c.395a > C) p.a139P (c.415g > C) [6]

p.d132a (c.395a > C) exon 3 skipping (c.475-12a > g) [6,7]

p.d132a (c.395a > C) p.C184Lfs*19 (c.551delg) [7]

p.d132a (c.395a > C) p.L248* (c.743_749delinsa) This paper

p.d132a (c.395a > C) deletion exon 1–3 (g.del37781240-37787410) This paper

p.g135e (c.404g > a) p.g135e (c.404g > a) This paper

61

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utations in PCH1

controls. However, in patients with other mutations in EXOSC3, the pons/tegmentum ratio was decreased. The ratio in patients with a homozygous p.g31a mutation does not differ significantly from PCH patients with a p.a307s mutation in the TSEN54 gene, although we should keep in mind that the cohorts are small (p.g31a five pa-tients; p.a307s six patients). interestingly, our control group shows that the ventral pons/tegmentum ratio is rather stable over a large age range (0 to 11 years). Mri analysis showed the presence of cerebellar cysts in 4/14 patients with mutations in EXOSC3. intracerebellar cysts have been reported before in few severe cases of PCH1 [3,14]. Their morphology closely resembles cysts in PCH2 [4]. our results support the occurrence of cysts in severe PCH1.

due to motor handicaps intellectual performance could not be studied in detail, but behavioral study suggests normal cognition in some patients in the p.d132a group ([2], patients 7-i and 7-ii in this paper).

The visceral pain of which patient 7-i suffered from, is a phenomenon not previ-ously reported in PCH1. neuropathological post-mortem examination of this patient revealed degeneration of neurons in both the ventral and dorsal spinal horn. degen-eration of neurons in the dorsal horn may account for the chronic visceral pains in this patient. Patient 7-ii had a normal CT brain scan at 1 year of age, but cerebellar hypo-plasia on Mri at 5 years of age, demonstrating the progressive nature of the disease. additionally, it emphasizes the importance of serial brain imaging in diagnosing PCH.

Motor disorders such as dyskinesia and spasticity were only observed in the p.d132a group which is charactarised by prolonged survival.

To conclude, we show that within the broad spectrum of EXOSC3 mediated PCH, clear genotype-phenotype correlations can be made. a homozygous p.d132a muta-tion leads to a more chronic form of PCH, with survival into childhood, and preserva-tion of the pons. Compound heterozygosity for a p.d132a mutation and a nonsense or p.y109n allele, a homozygous p.g31a mutation or a p.g135e mutation causes a severe disease course including death during infancy and hypoplasia of the pons.

ConCLUsions

We identified new nonsense and missense mutations in the EXOSC3 gene and we show that mutations in this gene are exclusively found in PCH1 patients. There are evident genotype-phenotype correlations in EXOSC3-mediated PCH reflected in clini-cal outcome, age of death and pons hypoplasia: patients with a homozygous p.d132a mutation have a prolonged disease course compared to patients with a p.d132a allele plus a nonsense or p.y109n mutation. Patients with a homozygous p.g31a muta-tion and the patient with a homozygous p.g135e mutation present a similarly severe

62

phenotype with death in infancy. our results refine the current view of an unaffected pons in eXosC3 mediated PCH.

abbreviations

PCH, Pontocerebellar hypoplasia; sMa, spinal muscular atrophy; PCr, Polymerase chain reaction; Mr(i), Magnetic resonance (imaging); VP, Ventral pons; T, Tegmentum.

competing interests

The authors declare to have no competing interests.

authors’ contribution

VrCe collected and analysed the data and wrote the manuscript. PgB had a valu-able contribution in writing and revising the manuscript. fvr, MBd-dW and MTvM performed molecular genetic analysis. PgB, Jfn and BTP-T analysed Mri scans and were closely involved in discussions. JnB, nd, ad, Jf, nf, df, TH, TJ, MK, PM, aM, Jarn, do’r, sP, anW, LW, Ms and Ls provided clinical information. ea, dT, TJ and TH provided post mortem tissue and stainings. CBM and HaM were involved in neuroradiological analysis. fB coordinated the study and the writing of the manuscript. all authors read, revised and approved the final manuscript.

63

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utations in PCH1

referenCes

1. Barth Pg: Pontocerebellar hypoplasias. an overview of a group of inherited neurodegenerative disorders with fetal onset. Brain Dev 1993, 15:411–422.

2. Zanni g, scotton C, Passarelli C, fang M, Barresi s, dallapiccola B, et al: exome sequencing in a family with intellectual disability, early onset spasticity, and cerebellar atrophy detects a novel mutation in eXosC3. Neurogenetics 2013, 14:247–250.

3. simonati a, Cassandrini d, Bazan d, santorelli fM: Tsen54 mutation in a child with pontocer-ebellar hypoplasia type 1. Acta Neuropathol 2011, 121:671–673.

4. namavar y, Barth Pg, Kasher Pr, van rf, Brockmann K, Bernert g, et al: Clinical, neuroradiologi-cal and genetic findings in pontocerebellar hypoplasia. Brain 2011, 134:143–156.

5. renbaum P, Kellerman e, Jaron r, geiger d, segel r, Lee M, et al: spinal muscular atrophy with pontocerebellar hypoplasia is caused by a mutation in the VrK1 gene. Am J Hum Genet 2009, 85:281–289.

6. Wan J, yourshaw M, Mamsa H, rudnik-schoneborn s, Menezes MP, Hong Je, et al: Mutations in the rna exosome component gene eXosC3 cause pontocerebellar hypoplasia and spinal motor neuron degeneration. Nat Genet 2012, 44:704–708.

7. rudnik-schoneborn s, senderek J, Jen JC, Houge g, seeman P, Puchmajerova a, et al: Pontocer-ebellar hypoplasia type 1: clinical spectrum and relevance of eXosC3 mutations. Neurology 2013, 80:438–446.

8. Biancheri r, Cassandrini d, Pinto f, Trovato r, di rM, Mirabelli-Badenier M, et al: eXosC3 muta-tions in isolated cerebellar hypoplasia and spinal anterior horn involvement. J Neurol 2013, 260:1866–1870.

9. schwabova J, Brozkova ds, Petrak B, Mojzisova M, Pavlickova K, Haberlova J, et al: Homozygous eXosC3 mutation c.92g—>C, p.g31a is a founder mutation causing severe Pontocerebellar Hypoplasia Type 1 among the Czech roma. J Neurogenet 2013, 27:163–169.

10. Barth Pg, Blennow g, Lenard Hg, Begeer JH, van der Kley JM, Hanefeld f, et al: The syndrome of autosomal recessive pontocerebellar hypoplasia, microcephaly, and extrapyramidal dyski-nesia (pontocerebellar hypoplasia type 2): compiled data from 10 pedigrees. Neurology 1995, 45:311–317.

11. goutieres f, aicardi J, farkas e: anterior horn cell disease associated with pontocerebellar hypoplasia in infants. J Neurol Neurosurg Psychiatry 1977, 40:370–378.

12. yushkevich Pa, Piven J, Hazlett HC, smith rg, Ho s, gee JC, et al: User-guided 3d active contour segmentation of anatomical structures: significantly improved efficiency and reliability. Neuro-image 2006, 31:1116–1128.

13. goto n: discriminative staining methods for the nervous system: luxol fast blue–peri-odic acid-schiff–hematoxylin triple stain and subsidiary staining methods. Stain Technol 1987, 62:305–315.

14. de Leon ga, grover Wd, d’Cruz Ca: amyotrophic cerebellar hypoplasia: a specific form of infan-tile spinal atrophy. Acta Neuropathol 1984, 63:282–286.

Ik daalde tussen de cellen neerEn sinds ik in het plasma drijfZie ik de bron, dat hele lijfNiet helder meer, niet meer

- Leo Vroman

4CLP1 fOunder mutatiOn linKs

trna sPlicing and maturatiOn tO cerebellar deVelOPment and

neurOdegeneratiOn

ashleigh e schaffer, Veerle rC eggens, ahmet okay Caglayan, Miriam s reuter, eric scott,

nicole g Coufal, Jennifer L silhavy, yuanchao Xue, Hulya Kayserili, Katsuhito yasuno,

rasim ozgur rosti, Mostafa abdellateef, Caner Caglar, Paul r Kasher, J Leonie Cazemier,

Marian a Weterman, Vincent Cantagrel, na Cai, Christiane Zweier, Umut altunoglu, n Bilge

satkin, fesih aktar, Beyhan Tuysuz, Cengiz yalcinkaya, Huseyin Caksen, Kaya Bilguvar,

Xiang-dong fu, Christopher Trotta, stacey gabriel, andré reis, Murat gunel, frank Baas,

Joseph g gleeson

Cell.

157

:651

-63

(201

4)

66

sUMMary

neurodegenerative diseases can occur so early as to affect neurodevelopment. from a cohort of over 2000 consanguineous families with childhood neurological disease, we identified a founder mutation in four independent pedigrees in cleavage and polyad-enylation factor I subunit (CLP1). CLP1 is a multifunctional kinase implicated in trna, mrna and sirna maturation. Kinase activity of the CLP1 mutant protein was defective, and the trna endonuclease complex (Tsen) was destabilized, resulting in impaired pre-trna cleavage. germline clp1 null zebrafish showed cerebellar neurodegeneration that was rescued by wild type but not mutant human CLP1 expression. Patient-derived induced neurons displayed both depletion of mature trnas and accumulation of unspliced pre-trnas. Transfection of partially processed trna fragments into patient cells exacerbated an oxidative stress-induced reduction in cell survival. our data links trna maturation to neuronal development and neurodegeneration through defective CLP1 function in humans.

67

Chapter

4

CLP1 mutations in PCH

10

inTrodUCTion

Transfer rnas (trnas) are abundantly expressed rna molecules required to bring amino acids to the translating ribosome for protein synthesis. of the 506 known hu-man trnas, 32 are encoded as pre-trnas, containing introns that are spliced during maturation (Lowe and eddy, 1997; Phizicky and Hopper, 2010). The nuclear-localized trna splicing endonuclease (Tsen) complex recognizes the secondary structure of pre-trna molecules and cleaves at the exon–intron boundaries to yield a 2’,3’-cyclic phosphate (i.e. phosphodiester) and 5’-oH terminal at the splice site (Peebles et al. , 1983). although still the subject of some debate, there are at least two proposed mechanisms of re-ligating these “half” trnas in mammals (i.e. the 5’- and 3’-exons remaining after excision of the single intron). in the first, HsPC117 directly mediates the ligation of these ends, utilizing the phosphate from the phosphodiester bond in the linkage, and is inhibited by the presence of a 5’-phosphorylation (Popow et al. , 2011). in the second, 5’-phosphorylation by CLP1, a component of the Tsen complex in mammals, is required (Paushkin et al. , 2004; Weitzer and Martinez, 2007; Zillmann et al. , 1991), but the ligase that follows has not yet been discovered. Utilization of both pathways in human cells is documented, but their relative importance and functional redundancy in development and homeostasis remains unclear.

Pontocerebellar hypoplasia represents a group of inherited progressive neurode-generative disorders with prenatal onset, thus intersecting development with degen-eration. all subtypes share common structural defects of the pons and cerebellum, evident upon brain imaging. Targeted therapy is non-existent, and most patients die during infancy (namavar et al. , 2011b). Mutations in any of three subunits of the Tsen complex, in the mitochondrial arginyl-trna synthetase gene, the rna exosome component eXosC3, and the vaccine related kinase are found in some cases (Budde et al. , 2008; edvardson et al. , 2007; renbaum et al. , 2009; Wan et al. , 2012). We recently implicated aMPd2 in PCH, causing a defect in protein translation due to guanosine triphosphate depletion (akizu et al. , 2013). The data implicate rna maturation and protein synthesis defects in PCH, but also suggest further causes are yet to be identi-fied.

Here we demonstrate a requirement for CLP1 in human brain development. We identify four independent families carrying a founder p.r140H which impairs affinity for the Tsen complex, kinase activity in a recombinant assay, as well as function in vivo. Consistent with its role trna splicing, we find depleted mature trnas and excessive pre-trnas accumulating in patient-derived induced neurons (ineurons). We demonstrate sensitivity of patient cells to oxidative-stress induced death exacerbated by the addition of unphosphorylated 3’-trna-exon halves and partially corrected with 5’-phospho- 3’-trna exon addition. in sum, we uncover an evolutionarily conserved

68

requirement for CLP1 during vertebrate neurogenesis, and show CLP1 is necessary for trna maturation, the loss of which leads to stress-induced cell death.

resULTs

Patients harboring CLP1 mutation have progressive brain atrophy

We collaboratively recruited over 2000 families, most with documented parental consanguinity, presenting a child with neurological disease. We performed exome sequencing on at least one affected member per family, then analyzed each for potentially deleterious homozygous mutations. gaTK (dePristo et al. , 2011) was used for variant identification and intersected with identity-by-descent blocks from HomozygosityMapper (seelow et al. , 2009). rare potentially deleterious variants were prioritized against our cumulative in-house 4000 patient exome database and across publically available exome datasets, cumulatively numbering over 10,000 individuals. from this analysis, four independent consanguineous Turkish families with a neu-rodevelopmental/neurodegenerative disorder emerged (figure 1a), all displaying an identical homozygous Chr.11:57427367g>a (hg19) single nucleotide transition in the CLP1 gene, resulting in a p.arg140His (p.r140H) amino acid substitution mutation.

following the identification of the mutation, it became clear that the patients shared many clinical features, but it would have been difficult to separate them clinically from the rest of the cohort. after an unremarkable perinatal history, onset of slow, progressive, neurodegenerative features and/or static encephalopathy ensued by 6 months of age. Clinical features included failure to develop gross or fine motor skills, absent or delayed speech, progressive spasticity and spontaneous epileptic seizures (Table 1 and Table s1). Brain Mri demonstrated mild atrophy of the cerebel-lum, pons and corpus callosum (figure 1B), together with progressive microcephaly. electromyography, while initially normal at a young age, demonstrated age-dependent muscle fibrillations and high amplitude motor unit potentials in one patient, indicat-ing progressive spinal motor neuron loss. extensive testing for known metabolic or degenerative diseases was negative, suggesting a here-to-fore unknown condition.

The mutant allele was observed heterozygous twice in unrelated unaffecteds in our in-house exome database of collectively over 2000 independent exomes (including about 1000 Turkish individuals), and not reported in any public database, suggesting a carrier frequency of 1:1000. in all families, we confirmed that the mutation occurred within a homozygous haplotype block (figure s1a), suggesting a founder mutation. The mutated amino acid residue was highly conserved in all multicellular organisms (figure 1C), and predicted to be damaging (adzhubei et al. , 2010). no other potentially deleterious rare homozygous CLP1 variants were present in the database. Comparison

hier hoofdstuk 4 tabel 1

69

Chapter

4

CLP1 mutations in PCH

10

tabl

e 1.

Clin

ical

phe

noty

pes.

HC:

hea

d ci

rcum

fere

nce;

eeg

: ele

ctro

ence

phal

ogra

m;

eMg:

ele

ctro

myo

gram

; sd

: sta

ndar

d de

viat

ion

belo

w t

he m

ean.

Pro

gres

sive

m

icro

ceph

aly

note

d by

age

-dep

ende

nt d

eclin

e in

hea

d ci

rcum

fere

nce

(sd

, com

pare

sd

at b

irth

to la

st e

xam

). el

ectr

omyo

grap

h w

as n

orm

al in

you

ng p

atie

nts

(<1y

r),

but s

how

ed m

uscl

e fib

rilla

tions

with

incr

ease

d pa

tient

age

, ind

icat

ive

of m

otor

neu

ron

dege

nera

tion

in p

atie

nt 1

810-

Vi-1

. add

ition

al c

linic

al d

ata

foun

d in

Tab

le s

1.

  

1810

-Vi-

113

27-i

V-3

(die

d at

4 y

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27-i

V-4

1327

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572

597-

ii-1

7259

7-ii-

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1337

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eval

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fM

fM

ff

MM

ethn

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rigin

Turk

ish

Turk

ish

Turk

ish

Turk

ish

Turk

ish

Turk

ish

Turk

ish

Turk

ish

Preg

nanc

y du

ratio

n (w

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38

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2,74

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t bir

th

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n/a

n/a

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n/a

n/a

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irth

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d-2

sd

-2 s

dn

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sd0

sd0

sd0

sd

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seiz

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( Lef

t te

mpo

ral)

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brile

(onc

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-

seiz

ure

onse

t3

yrs

50 d

ays

2 m

os2

yrs

3 yr

s-

2 yr

s3

yrs

70

tabl

e 1.

Clin

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phe

noty

pes.

HC:

hea

d ci

rcum

fere

nce;

eeg

: ele

ctro

ence

phal

ogra

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myo

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belo

w t

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ean.

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gres

sive

m

icro

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note

d by

age

-dep

ende

nt d

eclin

e in

hea

d ci

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nce

(sd

, com

pare

sd

at b

irth

to la

st e

xam

). el

ectr

omyo

grap

h w

as n

orm

al in

you

ng p

atie

nts

(<1y

r),

but

show

ed m

uscl

e fib

rilla

tions

with

incr

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d pa

tient

age

, ind

icat

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of m

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dege

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in p

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nt 1

810-

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foun

d in

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le

s1. (

cont

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d)

  

1810

-Vi-

113

27-i

V-3

(die

d at

4 y

o)13

27-i

V-4

1327

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572

597-

ii-1

7259

7-ii-

2 1

337-

ii-1

1337

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Visu

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71

Chapter

4

CLP1 mutations in PCH

10

tabl

e 1.

Clin

ical

phe

noty

pes.

HC:

hea

d ci

rcum

fere

nce;

eeg

: ele

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ence

phal

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m;

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ndar

d de

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belo

w t

he m

ean.

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gres

sive

m

icro

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aly

note

d by

age

-dep

ende

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eclin

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hea

d ci

rcum

fere

nce

(sd

, com

pare

sd

at b

irth

to la

st e

xam

). el

ectr

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h w

as n

orm

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with

incr

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d pa

tient

age

, ind

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of m

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tion

in p

atie

nt 1

810-

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linic

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ata

foun

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le

s1. (

cont

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d)

  

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d at

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27-i

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dia

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14 y

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find

ings

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/aat

roph

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Pons

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opla

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opla

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opla

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opla

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n/a

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/an

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aln

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n/a

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72

of exome allele calls between families suggested a minimal shared haplotype between chr11:57317640-57461472, or 143,832 bp (figure s1B), dated to a common ancestor approximately 16.2 generations in the past (+/- 8.7 generations, see methods), during the height of the ottoman expansion.

direct sanger sequence analysis of all available family members, including deceased member 1810-Vi-2 (from dried umbilical cord) demonstrated segregation according to a strict recessive mode of inheritance (figure s1C), consistent with pathogenesis. obligate carriers were entirely normal. We further found no other CLP1 mutations in any other patient in our collective cohorts with overlapping clinical features, nor any from an additional directly sequenced cohort of 100 cases with familial motor neuron disease.

hier hoofdstuk 4 figuur 1

A

B

d

Control 72597-II-1 72597-II-21327-IV-5 1337-II-11810-VI-1

C

CLP1

1 425

p.R140H

NTD P-loop SwitchI SwitchII Base bindingloop

CTD

R140H

H.s. Y A V R L - G R RM.m. Y A V R L - G R RG.g. Y A V R L - G R RX.l. Y A V R R - G R RD.m. Y A V R V - G R RC.e. Y A V R Q - G R RS.c. Y A L K F N A Y Q

* ***

* * * *

1810I

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1327I

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72597I

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Figure 1

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*

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2 3

figure 1. identification of a homozygous CLP1 p.r140H mutation in families with degeneration/hypo-plasia of the central nervous system. further analysis in figure s1. (a) Pedigrees of four consanguineous Turkish families. filled symbols: affecteds; hash: deceased; double bar: consanguinity; dashed double bar: history of consanguinity but ancestry not established. (B) Midline sagittal (top) and coronal (bottom) brain Mri of control compared with patients from each family, showing ventriculomegaly due to atrophy. red arrowhead: hypoplastic/atrophic pons. White arrowhead: cerebellar folia atrophy. double white ar-rowheads: hypoplastic corpus callosum. red asterisk: fluid cavity as a result of cerebellar atrophy (mega cisterna magna). only axial CT was available for 1337-ii-1. (C) stick figure of CLP1 protein and location of the p.r140H mutation near the aTP-binding P-loop. evolutionary conservation of the p.r140 residue across the animal kingdom. nTd: n-terminal domain; P-loop/Walker a motif; switch loop i; switch loop ii; base-binding loop: involved in nucleotide binding; CTd: C-terminal domain. H.s.: Human; M.m.: Mouse; g.g.: Chicken; X.l.: frog; d.m.: fly; C.e.: Worm; s.c.: yeast.

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clP1r140h is functionally compromised

To determine if the mutation was predicted to disrupt protein function, we modeled human CLP1 using the structure of the partially crystallized yeast nucleotide-bound Clp1 (noble et al. , 2007). in yeast, the p.140arg is substituted for a Lys at the cognate position, which is also a polar basic residue (p.149Lys). structure shows that the yeast p.149Lys is involved in the formation of an inferred hydrogen bond with the highly conserved p.59gLU residue (figure 2a). This polar contact is predicted to be maintained in human, but disrupted in the presence of the mutant p.140His residue, suggesting an alteration in protein structure or function. We found comparable CLP1 protein levels among all genotypes in primary fibroblast lysates derived from skin

AC

ontro

l

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-TUBULIN

48kDa

50kDa

Una

ffect

ed

Affe

cted

R140

Arg 140 His 140 Lys 149

Human CLP1Glu 59Glu 48Yeast Clp1

Figure 2

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rA20

P32-rA20

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prot

ein

T4 P

NK

GST

-CLP

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-CLP

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CLP1 Dapi Merge

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rons

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Cytoplasm Nucleus

CLP1 localization

Perc

ent (

%)

TFIIHCLP1

CN CN CN

Control Unaff. Aff.

48kDa

fraction:

-ACTIN45kDa62kDa

37kDa GAPDH

figure 2. CLP1 p.r140H is stable but functionally compromised. (a) Homology modeling of human CLP1 with the crystal structure of yeast Clp1 (left). Lower panels: Magnified images of the p.140r and p.140H residues in human and p.149K residue in yeast. substitution predicted to disrupt conserved hydrogen bond (red arrowhead) formed between p.r140 and gLU (red arrow, residue 48 in human and 59 in yeast). (B) Unaltered levels of CLP1 protein from affected cells. (C) defective kinase activity of recombi-nant human CLP1 p.r140H mutation (purification shown in figure s2), against rna poly(a)20, rna syBr staining (bottom), quantified at right. (d) nuclear localization of CLP1 p.r140H is reduced in affected cells (schematic and validation shown in figure s2). (e) Western blot of cellular fractions showing CLP1 mislocalization in patient fibroblasts. TfiiH, β-aCTin, and gaPdH: loading controls, quantified below. n = nuclear, C= cytoplasmic, Unaff. = Unaffected, aff. = affected. error bar: seM. * p < 0.05 student’s t-test. scale bar = 10 µm.

74

biopsy (figure 2B), suggesting protein stability was unaltered in the presence of the mutation.

The p.r140H mutation occurred right after the aTP-binding P-loop (i.e. Walker a motif), conserved in all kinases. To determine if the mutation alters CLP1 kinase activ-ity we tested recombinant wild type (wt) and mutant gsT-tagged human CLP1 protein against a poly(a) rna oligonucleotide natural kinase substrate in an established assay (ramirez et al. , 2008). recombinant mutant p.r140H CLP1 was stable to purification (figure s2a) but displayed defective kinase activity, reduced by more than half of wt levels (figure 2C). We conclude that CLP1 kinase activity was functionally impaired as a result of the p.r140H mutation.

We found that CLP1, each of the known TSENs, and HSPC117 were expressed ubiq-uitously in 14 human tissues and ages tested (figure s2B), suggesting a conserved function. Therefore, to understand the selective cellular vulnerability, we generated induced neurons (ineurons) from affected and unaffected fibroblasts, using recently published methods (Xue et al. , 2013), yielding approximately 80% neural cells in culture (figure s2C-e). Both patient fibroblasts and ineurons showed reduced nuclear localized CLP1, supported by nuclear/cytoplasmic fibroblast cell fractionation (fig-ure  2d-e). We conclude that the p.r140H mutation results in both failed nuclear localization and impaired kinase activity, as a mechanism of impaired function.

mutant zebrafish phenocopy CLP1R140H mutant patients

recent work in mice suggests an essential function for Clp1 even before zygotic im-plantation; however, mice with a homozygous kinase-dead mutant allele (p.K127a) survived embryogenesis but died perinatally with spinal motor neuron degeneration (Hanada et al. , 2013). To test for central nervous system requirements in a model vertebrate, we generated a germline clp1 p.r44X mutation by enU mutagenesis in zebrafish and bred to homozygosity. Wild type fish showed strong neural clp1 expres-sion by in situ hybridization (isH), which was severely reduced in mutants (figure s3a). Heterozygous and wt clutchmates were indistinguishable, but homozygous mutants did not survive past 5 days post fertilization (dpf), displayed abnormal swimming behavior, abnormal head shape and curved tail consistent with a neuromotor defect (figure 3a-B). a second allele, representing a missense mutation near the kinase do-main (p.L35r) showed a similar uniform lethality by 5 dpf (figure s3B). We conclude that clp1 is essential in zebrafish.

from timed larvae, whole mount isH for the midbrain marker, otx2, demonstrated unremarkable expression level and distribution at 24 hours post-fertilization (hpf) in mutants (figure 3C). However, by 48 hpf mutants displayed weak, spatially restricted expression of otx2, and by 72 hpf, otx2 was not evident in the mutant zebrafish. since

hier hoofdstuk 4 figuur 2

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initial otx2 expression was initially unremarkable in mutant animals, our data sug-gests the progressive loss of expression results from cell loss as opposed to defec-tive patterning. To further differentiate between these possibilities, we performed terminal deoxynucleotidyl transferase dUTP nick end labeling (TUneL), to detect dna fragmentation (Chen et al. , 2010). We noted a dramatic increase in staining specific to the forebrain and hindbrain (figure 3d). Therefore, clp1 mutant zebrafish showed evi-dence of hindbrain neurodegeneration, similar to tsen54 zebrafish morphant (Kasher et al. , 2011). The similarity in brain phenotype between the human p.r140H and the zebrafish mutations suggests loss-of-function as the disease mechanism in humans.

We next tested for a spinal motor neuron phenotype, since both mouse and human show loss of this class of neurons with CLP1 mutations. Zebrafish larvae were fixed at 72 hpf and stained with the motor neuron marker sv2 (Boon et al. , 2009). specifi-cally in the clp1 p.r44X mutants, we found altered motor neuron morphology with full penetrance (figure s3C), similar to established zebrafish motor neuron mutants (fassier et al. , 2010).

in mouse, multiple phenotypes observed in Clp1 mutants are rescued by allelic removal of p53, suggesting that the cellular apoptosis observed in clp1 mutants is p53-dependent (Hanada et al. , 2013). in order to test this in zebrafish, we performed p53 morpholino knockdown using a published reagent (Langheinrich et al. , 2002) in clp1 wt and mutants, then performed otx2 isH as a marker for cell loss. We observed rescued otx2 expression in p53-knockdown clp1 mutant zebrafish, suggesting the neu-ral apoptosis is p53-dependent (figure s3d). We also performed zygotic knockdown of the single hspc117 zebrafish orthologue, the gene proposed to mediate the redundant splicing pathway, with either aTg- or splice-blocking morpholinos, achieving near complete loss of spliced transcript with the latter. However, we found no phenotype in any of the morphants (not shown). We additionally performed hspc117 knockdown in clp1 p.r44X mutants, but found no exacerbation of the clp1 mutant phenotype (not shown), suggesting that, at least in zebrafish, hspc117 does not play an essential role, or genetically interact with clp1.

To determine if human CLP1 is a functional orthologue of zebrafish clp1 we injected wt and mutant human CLP1 mrna into mutant clp1 p.r44X mutant fish zygotes. The curved tail phenotype was apparent by 48 hpf so we utilized this as a readout as measured by the depth of the vertebral curve from highest to lowest point at 3 dpf. average height was less than 50 µm in wt fish but over 300 µm in mutant fish (figure 3e). injection of wt human CLP1 mrna partially rescued the average curve height to less than 200 µm, although variable from fish-to-fish. injection of the hu-man p.r140H mutant CLP1 mrna did not mediate such rescue, with peak average height not less than 300 µm. in addition, we performed isH for otx2 on wt and clp1 p.r44X mutants injected with human CLP1 mrna. as expected, human wt CLP1, but

76

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figure 3. Zebrafish clp1R44X homozygous mutant show gross brain defects, reduced survival and neuro-degeneration. (a) gross morphology of wt and clp1R44X/R44X zebrafish mutant showed misshapen head, small eye and curved tail (arrow), suggesting neuromotor defects. scale bar = 500 µm (B) Kaplan–Meier curve showed reduced survival of clp1R44X/R44X fish (additional allele shown in figure s3). (C) Progressively reduced otx2 expression in developing clp1R44X/R44X zebrafish brains. Broad otx2 expression domain at 24 hpf was unremarkable in mutant (bracket), suggesting initial patterning was not disrupted. from 48-72 hpf, wt fish showed expression restricted to midbrain-hindbrain organizer (bracket), whereas mutant showed weak expression, completely absent by 72 hpf (#). (d) TUneL positive cells were dramatically increased in mutant at 48 hpf in both the hindbrain (arrow) and the midbrain/diencephalon (double

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not CLP1R140H was sufficient to prevent most loss of otx2 expression in clp1 p.r44X mutants (figure 3f). We conclude that human CLP1 can at least partially replace the zebrafish clp1, suggesting the human and zebrafish CLP1 genes are orthologues, and that the human mutation lacks activity in vivo.

Patient CLP1R140H ineurons show intron-containing pre-trnas accumulation and mature trna depletion

due to the shared hindbrain phenotype observed in zebrafish and the established role of the Tsen/CLP1 complex in trna splicing, we pursued defective trna splicing as a potential disease mechanism. in order to study the relative expression of the known 32 intron-containing trna isoforms (i.e. genes), following reverse transcription (rT) of total rna, we generated isoform-specific pre-trna primers to each, utilizing the unique intronic sequence for the reverse primer (figure  4a). excluding those that failed PCr or where expression was undetectable, using rT-PCr we profiled the ex-pression of the remaining 15 in human cortex, ineurons, and fibroblasts (figure s4a). expression in brain more closely mirrored expression in ineurons than fibroblasts, supporting the use of ineurons for these experiments.

To test for defective trna splicing, we generated cdna from fibroblasts and ineurons, then analyzed expression of these pre-trnas using qrT-PCr to test for differences between affected and unaffected. normalizing to the unaffected in fibroblasts, in the half of pre-trnas where a difference was noted, the differences were roughly equally distributed into those where the pre-trna was higher in the affected and those where it was lower in the affected (figure 4B, s4B). in ineurons, however, where a difference in pre-trnas level was noted, the majority (6 of 8) showed an accumulation of pre-trna in affected cells compared with unaffected (figure 4C, s4C). We conclude that there is an accumulation of some pre-trnas in CLP1 patient cells.

To exclude that the qPCr data was biased due to impaired rT of fully modified trnas, we utilized northern blotting, the most established method for trna assessment, gen-erating probes to interrogate representative unique isoforms for pre-trna for Tyrosine Chr14:trna19 and isoleucine Chr19:trna10, as well as to detect all isoacceptor (pan: all isoacceptor, i.e. same anticodon) for pre-trna and mature trna for Tyrosine, isoleucine and Leucine. Most probes showed some evidence for expression in both

hier hoofdstuk 4 figuur 3

arrow), further investigated in figure s3. (e) Partial rescue of the clp1R44X/R44X phenotype with human wt but not p.r140H CLP1 mrna, by measuring curved tail height. no significance difference was detected between uninjected and injected with p.r140H mrna, whereas wt mrna partially recovered curved tail phenotype. p <0.01 student t-test. (f) In situ for otx2 in wt, clp1R44X/R44X, and clp1R44X/R44X injected with human CLP1 mrna. Human CLP1, but not CLP1R140H, prevented the loss of otx2 expression at 48 hpf in clp1 mutants, quantified at right. n>25 embryos each condition, p < 0.05 student’s t-test. error bar: seM. scale bar = 100 µm.

78

fibroblasts and ineurons with the exception of pre-trna for Chr14:trna19-TyrgTa, which was detectable only in ineurons, run in duplicate to avoid sample loading vari-ability. We identified both accumulation of pre-trnas as well as depletion of mature trnas specifically in patient ineurons. Pre-trna Chr14:trna19-TyrgTa, pre-trna Chr19:trna10-ileTaT, as well as pre-trna ileTaT (pan) and pre-trna LeuCaa (pan) showed stronger bands in affected vs. unaffected ineurons (figure 4d). We also noted an accumulation of Chr19.trna10-ileTaT intron (data not shown). Correspondingly, there was a reduction in the amounts of mature trnas for isoleucine and Leucine for mature trna ileTaT (pan) and mature trna LeuCaa (pan). These results were quanti-fied as the percent mature trna or pre-trna of total trna for each isoacceptor, and demonstrate neural specific changes in pre- and mature trna transcript levels.

next we analyzed whether the alterations in pre-trna levels were due to impaired Tsen-CLP1 complex formation resulting from the CLP1 p.r140H mutation. Using an established assay (figure  s4d) (Paushkin et al. , 2004), we found reduced amounts of the active site containing Tsen complex proteins, Tsen2 and Tsen34 co-purified with double-affinity tagged CLP1r140H, compared to complexes purified with wt CLP1 or Tsen54 (figure 4e, s4e). additionally, we generated two control mutants, CLP1r140a and CLP1K127a, and observed a less severe reduction of Tsen2 and Tsen34 bound com-pared to CLP1r140H, indicating the patient substitution p.140H is particularly damaging to Tsen complex formation (figure 4e, s4e) (Hanada et al. , 2013). We conclude that mutant CLP1 has altered affinity for the Tsen complex.

To determine if CLP1r140H-, CLP1r140a- and CLP1K127a-bound protein complexes display altered endonuclease activity we performed an in vitro trna cleavage assay using the co-purified protein complexes isolated from the two step elution (figure s4d) and radiolabeled Tyrosine pre-trna with yeast trna endonuclease as a positive enzymatic control (Trotta et al. , 2006). Consistent with a severe defect in Tsen complex affin-ity, CLP1r140H-associated protein complexes were unable to cleave pre-trna Tyr as compared to wt CLP1-purified complexes at all time points analyzed (figure 4f). as expected, CLP1r140a and CLP1K127a co-purified complexes displayed more endonuclease activity than CLP1r140H-bound proteins, but less activity than wt (figure  s4f). The data suggest that loss of the Tsen complex from mutant CLP1-associated protein complexes impairs pre-trna cleavage activity.

the tyr trna 5’-Phospho-3’-exon fragment protects CLP1R104H mutant cells from stress-induced cell death

as there are at least two proposed mechanisms of re-ligating “half” trnas in mam-mals we set out to uncover the requirement for CLP1 in human cells. To test for altered cell viability of CLP1 mutant cells, we performed growth analysis of fibroblasts and ineurons under basal conditions, but detected no differences (figure s5a-B). We next

hier hoofdstuk 4 figuur 4

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Figure 4

5’ 3’

Exon Exon

pre-tRNA

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3’ primer

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- pre-tRNA IleTAT (pan)

- pre-tRNA TyrGTA (pan)

- mature tRNA TyrGTA (pan)

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- mature tRNA LeuCAA (pan)

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F

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Time:(min)

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Yeast CLP1 CLP1R140H

YeastCLP1CLP1R140H

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CLP1R

140H

CLP1R

140A

CLP1K

127A

TSEN

2/Fl

agH

is P

rote

in

FlagHis Pulldown

**** *******

figure 4. increased intron-containing pre-trna in CLP1 mutant patient cells and loss of Tsen complex affinity resulting in reduced pre-trna cleavage with CLP1-purified complexes. (a) schematic pre-trna with location of the intron, occurring one base 3’ to the anticodon. Primers designed to the individual trna homologue exon sequence and the unique consensus intron (red). amplifications with the 5’-primer and any of the 3’-primers. (B, C) qrT-PCr results showed variable changes in pre-trna expression in affected fibroblasts (i.e. about half were increased and half were decreased in affected), while most pre-trna transcripts from affected ineurons were increased (i.e. six of eight were increased). Validation and unchanged pre-trnas are shown in figure s4. (d) northern blot analysis of Chr14:trna19-TyrgTa and

80

tested whether overexpression of pre-trnas compared with mature trnas were toxic in patient ineurons in culture, since ineurons displayed an accumulation of these pre-trnas. Using a lentiviral delivery system expressing Chr14:trna19-TyrgTa or Chr19:trna10-ileTaT pre- or processed trna (both pre isoforms accumulated by northern analysis in patient ineurons), we found no difference in viability between patient and control ineurons 24h after transduction (figure s5C). We conclude that the patient ineurons do not show a dose-dependent toxicity to pre-trnas under these conditions.

oxidative stress can up-regulate trna cleavage (Thompson et al. , 2008). We detected no differences in basal protein or dna oxidation in patient fibroblasts or ineurons compared with controls, but noted that ineurons showed higher basal oxidation than fibroblasts, irrespective of genotype (figure s5d-e). Therefore to determine whether CLP1R140H patient cells display heightened sensitivity to oxidative stress-induced cell death, we performed a dose-dependent cell viability assay using hydrogen peroxide. (HP). as previously reported in murine fibroblasts (Hanada et al. , 2013), we found that human CLP1R140H mutant fibroblasts and ineurons showed compromised cell survival to HP at the highest 50 uM concentration, indicating sensitivity to oxidative stress (figure s5f-g).

it was recently proposed that excessive 5’-half-trnas were responsible for p53-dependent activation of cellular stress in mice (Hanada et al. , 2013). Therefore, we hypothesized that inadequate CLP1 kinase activity might result in 3’-half-trnas that lacked a phospho group at the 5’-end (figure 5a). We used a stress-induced viability assay to determine whether the patient mutation affected cell survival in the pres-ence of various trna fragments, testing whether the addition of 3’- or 5’-half-trna fragments might exacerbate the HP-induced phenotype in human cells. We predicted that the substrate but not the product of CLP1 modification (i.e. the unphosphorylated but not the phosphorylated 3’-exon) would be toxic.

Chr19:trna10-ileTaT pre-trna (intron probes) and trna-TyrgTa, trna-ileTaT, trna-LeuCaa pan mature trna (5’-exon probes) transcripts relative to U6 loading control, in duplicate showed similar amounts of pre-trna and mature trna for all transcripts tested, while affected ineurons show a increase in Tyr, ile, and Leu pre-trna transcripts, with a corresponding reduction in processed, mature trna. Quantification below, displayed as percent mature or pre-trna of total trna. (e) Western blot of CLP1-purified com-plexes showing CLP1 p.r140H, p.r140a, and p.K127a with reduced bound Tsen2 and Tsen34 compared to wt CLP1 (Tsen54 served as a positive control). Quantification of the amount of Tsen2 normalized to flagHis-tagged protein from four independent replicates (below). (f) Time-course of trna endonuclease reactions performed on exogenous radiolabeled Tyrosine (Tyr) pre-trna with double affinity purified CLP1-bound complexes. Buffer serves as a negative control and yeast trna endonuclease as a positive control. reduced endonuclease activity observed with CLP1r140H bound complexes compared with wt, quantified at left. *= p < 0.05, **= p < 0.01, ***= p < 0.001, ****= p < 0.0001 students t-test (qPCrs) or one-way anoVa (pulldown). error bar: seM.

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We transfected control and CLP1R140H mutant fibroblasts with rna oligonucleotides corresponding to the most abundantly expressed Tyr trna intermediates, either in the absence or presence of HP and evaluated cell viability after 24h (figure 5B-C). Control fibroblasts and neural progenitor cells showed little toxicity in response to the trna intermediates, either in the absence or presence of HP (figure s5H-i, K). Co-treatment with the 5’-exon containing the terminal 2’-3’-cyclic phosphodiester did not have a notable effects on survival (figure s5H). Most strikingly, the unphosphorylated 3’-exon was the most lethal of the trna halves, particularly in the presence of HP (figure 5B, s5i). remarkably, the 5’-phosphorylated 3’-exon did not cause toxicity and in fact significantly improved viability in the presence of HP (figure 5C, s5J). These results are consistent with the model whereby the loss of the phosphorylated-3’-exon required for HsPC117-independent trna ligation (and therefore production of mature trnas) or the toxic hydroxyl-3’-exon (incapable of further processing in mutant cells), may contribute significantly to the disease phenotype in patients.

hier hoofdstuk 4 figuur 5

Control

5’ 3’

5’-Exon 3’-Exon

Anticodon

pre-tRNA

Intron

5’ 3’

5’-Exon 3’-Exon

Anticodon

5’ 3’

5’-Exon 3’-Exon

Anticodon

5’ 3’

5’-Exon 3’-Exon

Anticodon

mature tRNA

TSENComplex

CLP1

P-OH

2’,3’-P 2’,3’-P

tRNA ligase

Affected

Viab

ility

0 0.2 0.4 0.6 0.8

1 1.2 1.4

Mock 3'-Exon H2O2+3'-Exon

0 0.2 0.4 0.6 0.8

1 1.2

0 0.2 0.4 0.6 0.8

1 1.2

Mock H2O2 P-3'-Exon H2O2+P-3'-Exon

0 0.2 0.4 0.6 0.8

1 1.2

3’-Exon

-OH

P-3’-Exon

P

H2O2 Mock 3'-Exon H2O2+3'-Exon

H2O2

Mock H2O2 P-3'-Exon H2O2+P-3'-Exon

Treatment

A

B

C

Figure 5

*

* *

figure 5. Toxic effects of unprocessed 3’-“half” trna exon in CLP1 patient cells. (a) Model of trna splicing. The pre-trna contains an intron (red) 1 base 3’ of the anticodon. The Tsen complex excises the intron, leaving two “half” trnas, the 5’-exon containing a 2’-3’-phosphodiester, and the 3’-exon containing an hydroxyl group. CLP1 is capable of phosphorylating the 5’-end of the 3’-exon, then a still unknown ligase repairs the break. (B, C) Control (green) and CLP1 patient cells (red) transfected with either the unphos-phorylated 3’-exon (3’-exon), or the phosphorylated 3’-exon (P-3’-exon), in the presence or absence of hydrogen peroxide. none of the conditions were adverse to control cells (shown in figure s5), whereas patient cells showed reduced viability to hydrogen peroxide and 3’-exon transfection, and improved vi-ability upon P-3’-exon transfection. * p < 0.05 2-way anoVa. error bar: seM.

82

disCUssion

our results report the effect of a CLP1 mutation in humans. Based upon this, and previously published data, we propose that patients with a CLP1R140H founder muta-tion display neurodegeneration due to defects in trna splicing. Because CLP1, in at least one capacity, associates with the Tsen complex and because patients display features similar to PCH, we propose that the condition we describe here should be considered an alternative form of PCH, designated as PCH type 10. The appearance of the brain scan is very distinct from the previously published PCH forms, does not show the “dragonfly” sign (namavar et al. , 2011a), and shows equal involvement of the hindbrain and forebrain. further characterization of this clinical phenotype could help better define similarities/differences from other forms of PCH.

While CLP1 kinase-dead mice develop progressive loss of spinal motor neurons mimicking the pathology of amyotrophic lateral sclerosis, our patients showed instead a neurodegenerative disease with loss of cerebellar, brainstem and cortical volume, thinning of the corpus callosum, and loss of acquired motor and cognitive skills, with evidence for later loss of motor neurons. Certain forms of PCH show co-existent spinal motor neuron degeneration, notably PCH type i (also known as norman’s disease), and PCH due to EXOCS3 mutations (goutieres et al. , 1977; Wan et al. , 2012), suggesting that both hindbrain and motor neurons may share susceptibility across the mutation spectrum. Cerebellar Purkinje cells and spinal motor neurons are among the largest neurons and thus likely most metabolically challenged of the cells in the nervous system, but why these specific neuronal cell types are vulnerable to reduced CLP1 activity is unclear.

The CLP1 p.r140H mutation does not destabilize the protein, but substantially impairs kinase activity, alters the nuclear localization, reduces the affinity for Tsen proteins affecting trna endonuclease activity, and when expressed in Clp1 mutant zebrafish, fails to rescue in vivo phenotypes. in an accompanying manuscript in this issue of Cell (Karaca et al. , 2014), show similar biochemical findings in addition to evidence of microcephaly in Clp1-kinase dead mice. Together, our findings support a mechanism by which the patient mutation impacts several of the known functions of CLP1.

in this study, we focus on the role of CLP1 in trna processing and show elevated levels of some unspliced pre-trnas and depletion of some mature trnas for several different isoacceptors including Tyrosine, isoleucine and Leucine in ineurons. This re-sult implies reduced processivity of pre-trnas to mature trnas when CLP1 is mutated, which we predict occurs at both the pre-trna cleavage step and the ligation step directly downstream of CLP1 in the processing cascade. The presence of the HsPC117 redundant splicing pathway may account for the rescue of processed trna levels in

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the presence of the CLP1 mutation in fibroblasts, although we found no phenotype upon hspc117 knockdown in fish, and no genetic interaction with clp1. Thus the rela-tive contributions of the two trna splicing pathways, or others yet undefined, will require further investigation.

a reduction in trna ligation results in the accumulation of trna half fragments, which inhibit protein translation and cause cell death in yeast and mammalian cells (sobala and Hutvagner, 2013; Thompson et al. , 2008; Thompson and Parker, 2009; yamasaki et al. , 2009). The CLP1 kinase-dead mice accumulated a 5’-trna exon deriva-tive fragment, but we found no evidence of accumulation or toxicity of this fragment in human cells. instead, we found that unphosphorylated 3’-exon (i.e. a substrate for CLP1) but not the phosphorylated 3’-exon exacerbated toxicity. our findings support a required role for CLP1 in the 5’-end phosphorylation-dependent ligation of “half”-trnas in humans. Cumulatively, we uncover a cell-type specific requirement for the HsPC117-independent trna maturation pathway in human development, which when perturbed causes a new PCH-like neurological disease.

in addition to the trna processing defects described here, the combined effect of the CLP1 loss-of-function phenotypes may be due to impaired phosphorylation of tar-get rna. in particular, CLP1 functions as an sirna kinase required for loading sirna onto the risC complex (Weitzer and Martinez, 2007), and thus there may be other cellular effects on these pathways as a result of the mutation. CLP1 also functions as part of a multi-protein complex required for 3’-end cleavage in pre-mrna processing and maturation (de Vries et al. , 2000; Wickens and gonzalez, 2004), and not surpris-ingly we observed CLP1r140H-bound protein complexes were depleted of mrna 3’-end processing proteins (data not shown). further experimentation will be required to test whether these other cellular mechanisms contribute to the disease pathogenesis in these patients.

eXPeriMenTaL ProCedUres

Methods and any associated references are available in the online version of the paper at http://www.cell.com

Patient recruitment.

Patients were enrolled and sampled by standard practice in approved human subjects protocols at the University of California, san diego.

84

exome sequencing.

genomic dna was extracted and subjected to exon capture, sequencing, variant iden-tification, and computational filtering as previously described (akizu et al. , 2013). see extended experimental Procedures for more detail.

Zebrafish.

Zebrafish work was performed in accordance with aMC iaCUC regulations. enU muta-genesis was performed to create two independent clp1 mutations. gene knockdown was performed with morpholino oligonucleotides (gene-Tools). Lifespan analysis, in situ hybridization, TUneL, rna rescue, and immunostaining were performed with standard protocols. additional detail can be found in the extended experimental Procedures.

trna analysis.

rna from patient fibroblasts or ineurons was used for cdna generation and quantita-tive rT-PCr of pre-trnas using standard methods. northern blot for intron contain-ing pre- and mature trnas were assessed by standard protocols. Probe and primer sequences, and detailed protocols are listed in the extended experimental Procedures.

cell culture.

fibroblasts were generated from Unaffected and affected skin punch biopsies. in-duced neurons (ineurons) were generated as previously described (Xue et al. , 2013). HeK293T, HeK293H, primary fibroblasts, neural progenitor cells, and ineurons were cultured using well-established techniques. Basal protein and dna oxidation and rna transfection were performed as suggested by the manufacturer. Cell viability assays were performed as previously described (Carmichael et al. , 1987). details can be found in the extended experimental Procedures.

clP1 Protein assays.

recombinant wt and mutant gsT-CLP1 was purified from E. coli and tested for kinase activity as previously described (ramirez et al. , 2008). double affinity purification of flagHis-CLP1 was carried out as previously described (Trotta et al. , 2006; Volta et al. , 2005). associated protein complexes were used in trna endonuclease assays and analyzed by Western blot. detailed experimental techniques can be found in the extended experimental Procedures.

computation.

CLP1 mutations were modeled onto the crystalized yeast structure with Phyre2 (Kel-ley and sternberg, 2009). PLinK was used to create autozygosity maps from exome

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sequence (Purcell et al. , 2007). The age of the CLP1 p.r140H mutation was calculated according to published methods (fu et al. , 2013). additional details can be found in the extended experimental Procedures.

statistical analysis.

anoVa (1- or 2-way) or student’s two-tailed non-paired t tests were carried out to determine the statistical significance of differences between samples. P < 0.05 was considered nominally statistically significant for all tests.

accession numbers.

exome sequencing data have been deposited to dbgaP phs000288.v1.p1. accession numbers for sequences used in the study are as follows: refseq id human CLP1: gene: nM_006831.2; Protein: nP_006822.1, yeast clp1 Q08685, Zebrafish clp1 (LOC565621): XM_688892.4, Zebrafish hspc117 (rtcb): nM_213103.1.

Supplemental Information is linked to the manuscript.

Web resources

The UrLs for data presented herein are as follows:online Mendelian inheritance in Man (oMiM), http://www.omim.orgseattleseq annotation, http://gvs.gs.washington.eduUCsC genome browser, http://www.genome.ucsc.eduUniversal Protein resource, http://uniprot.orgHuman Brain Transcriptome database, http://www.humanbraintranscriptome.orgexome data deposited at dbgaP : http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000288.v1.p1

acknowledgements

supported by the niH (P01Hd070494, r01ns048453, and P30ns047101 for imag-ing support to J.g.g., Broad institute grant U54Hg003067 to eric Lander, gM049369 to X.-d.f., the yale Center for Mendelian disorders U54Hg006504 to r. Lifton and M.g., rC2ns070477), the gregory M. Kiez and Mehmet Kutman foundation to M.g. We acknowledge M. gerstein, s. Mane, a. B. ekici, ad s. Uebe for sequencing support and analysis, the yale Biomedical High Performance Computing Center for data analysis and storage, the yale Program on neurogenetics, and the yale Center for Human genetics and genomics, the Center for inherited disease research for genotyping; the simons foundation autism research initiative. Consortium for autosomal reces-sive intellectual disability (Carid) supported patient ascertainment. e.W.e. Verweij for zebrafish analysis, e. Cuppen and H. van roekel for support with enU Tilling, and

86

Zf Health funding for tilling. a.e.s. is the recipient of an a.P. gianinni fellowship. V.C. was supported by the french national research agency (anr-rVP13016KKa). J.g.g. is an investigator of the Howard Hughes Medical institute. We thank patients and parents for participation, and J. Lupski, J. Martinez and s. Weitzer for communicating unpublished results and sharing reagents.

author contributions

J.L.s, a.o.C. and M.s.r. independently identified CLP1 mutation in patient cohorts. a.e.s performed all experiments except those involving zebrafish and tandem affinity purification, performed by V.r.C.e and C.T, respectively. a.e.s, n.g.C. and y.X performed ineuron experiments. f.B. and J.g.g. coordinated the fish study. a.e.s and J.g.g. wrote the manuscript, edited by all co-authors.

competing financial interests

The authors declare no competing financial interests.

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92

sUPPLeMenTaL eXPeriMenTaL ProCedUres

Patient ascertainment. Patients were identified from genetics and/or neurology clin-ics from several world-wide recruitment efforts targeting patients with intellectual disability, structural brain diseases or neurodevelopmental disorders. all patients/families enrolled in irB approved protocols based at referral institutions, and each family provided consent for study. sequencing was performed on at least one but sometimes two affecteds or the father-mother-affected trio per family.

dna extraction and Whole exome sequencing. genomic dna was extracted (Qiagen or oragene), subject agilent Human all exon 50 Mb kit library preparation, then paired-end sequencing (2x100bp) on an illumina Hiseq 2000 or soLid5500 xl instrument. for deceased individual 1810-iV-2, dna was extracted from an archived umbilical cord sample (sidransky and stubblefield, 1993). for each patient sample,  >90% of the exome was covered at  >30x. gaTK (dePristo et al. , 2011) was used for variant identification, and then filtered for homozygous variants using custom Python scripts (available upon request), to remove alleles with over 1% frequency in the population, not occurring in homozygous intervals, or without high scores for likely damage to protein function.

founder haplotype analysis. The region surrounding CLP1 was extracted from exome sequencing VCf files to include both common and rare polymorphisms.

Zebrafish stocks and maintenance. Zebrafish work was in accordance with aMC iaCUC regulations. Two mutations in clp1 were identified from an enU mutagenized stock: c.130C>T (p.r44X) and c.104T>g (p.L35r) and genotyped using standard methods (de Bruijn et al. , 2009), and outcrossed for 4 generations. Zygotes (1-cell stage) were injected with morpholino oligonucleotides (gene-Tools) targeting hspc117 (aTg Mo: 5’-gCT CaT CgT TgT aag agC gaC TCa T-3’; exon 6 splice Mo: 5’-TaT TTT CCC aTC aTg aCa CTg Tgg C-3’), p53 (5 ng; 5’- gCg CCa TTg TTT gCa aga aTT g-3’) or a control morpholino (5’-CCT CTT aCC TCa gTT aCa aTT TaT a-3’). Lifespan was assessed in larvae from a clp1R44X/+ or clp1L35R/+ intercross. dead larvae were removed from the tank and genotyped daily, until all clp1-mutant fish were represented. Kaplan-Meier curves were generated with graphPad Prism, ver5.

Zebrafish in situ hybridization, tunel, and immunostaining. In situ was performed with a standard protocol (Thisse and Thisse, 2008). TUneL assay was performed on fixed, bleached and permeabilized larvae (roche). for immunostaining, larvae were incubated overnight in anti-sv2 (dsHB) following permeabilization. fluorophore-

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conjugated secondary antibodies were used, and images were captured on a confocal microscope (Leica Microsystems).

cloning. Human CLP1 was amplified from human fetal brain into the gateway system for mammalian expression, site-directed mutagenized (stratagene) to introduce the patient mutation, then subcloned into pgeX-6P-1 (addgene) for gsT-tagged expres-sion and pCs2+ for mrna production. sequence-verified flagHis-tagged constructs were generated using whole-vector PCr techniques (geneWiZ, inc.) oligos with overhangs complementary to pLKo.1 for pre-trna and mature trna to Chr19.trna10-ileTaT and Chr14.trna19-TyrgTa were annealed then ligated to BamHi and agei sites using gibson assembly methods (neB). expression of the pre-trnas was validated in HeK293T cells by northern analysis before lentivirus production. pLKo.1-scramble (sigma) was used as a control.

Zebrafish clp1 rescue. Human CLP1 in pCs2+ plasmid was linearized using aflii re-striction enzyme and in vitro transcription was performed using the sP6 mMessage mMaCHine kit (ambion). Zebrafish zygotes from a clp1R44X/+ intercross were injected with 400 pg wt or mutant human CLP1 mrna and scored at 48 hpf. for otx2 rescue, ‘normal’ was defined as intense purple staining with distinct boundaries, ‘reduced’ as pinkish staining with vague boundaries, intensity reduced by at least half of normal levels, ‘absent’ as little evidence of signal above background.

rna extraction. rna was extracted from cells using Trizol (Life Technologies), treated with dnase (neB) for 30 min at 37oC, phenol/chloroform extracted, precipitated, and then rehydrated for analysis.

cdna synthesis and Quantitative Pcr. cdna synthesis of 3 µg patient rna was per-formed with the superscript iii first-strand cdna synthesis system for rT-PCr (Life Technologies). qrT-PCr was performed in triplicate on 10 ng human cdna (primer sequences available upon request). trna dCT values were normalized to gaPdH as a cdna loading control and fold change calculated in reference to the unaffected individual.

northern blot. rna (3µg) was separated in a 12% 8 M urea polyacrylamide gel, transferred to Hybond-n+ membrane (ge Healthcare), fixed by UV-crosslinking, pre-hybridized with 0.1 mg/ml salmon sperm dna in express-Hyb (Clontech) for 2h at 50oC, hybridized in the same solution at 50oC with 100 pmol 5’-32P labeled Chr14.trna19-TyrgTa intron probe 5’-gaT gTC CaC aaa TgT TTC TaC agg CTa C-3’, Chr19.trna10-ileTaT intron probe 5’-TgC TCC gCT CgC aCT gTC a-3’, Tyr-gTa 5’exon probe

94

5’-CTa Cag TCC TCC gCT CTa CC-3’, ile-TaT 5’-exon probe 5’-TaT aag TaC CgC gCg CTa aC-3’, Leu-Caa 5’-exon probe 5’-CTT gag TCT ggC gCC TTa gaC-3’, or U6 probe 5’-gCa ggg gCC aTg CTa aTC TTC TCT gTa TCg-3’ as a loading control, washed with 1x ssC, 1% sds for 10 min and developed. Band densitometry was calculated by imageJ (niH).

lentivirus and transduction. Lentivirus was created as previously described (Xue et al. , 2013), using co-transfection of the pLKo.1-PTB shrna vector with pCMVr8.74 and pCMV-VsVg in HeK293T cells. Virus containing media was collected at 48 h and 72 h, filtered and stored at 4oC or -80oC, mixed 1:1 with fresh growth media plus 8 µg/ml polybrene, and added to cells for 24 h, then selected for 72 h with hygromycin.

cell culture and neural induction: Primary fibroblast cell lines were established from skin biopsies according to standard methods. neural induction was performed as pre-viously described (Xue et al. , 2013). ineurons were maintained in n3 media (dMeM/f12 with 25 mg/ml insulin, 50 mg/ml transferrin, 30 nM sodium selenite, 20 nM pro-gesterone, and 100 nM putrescrine) supplemented with Bdnf, gdnf, nT3 and CnTf (r&d). neural cells (ineurons) induced for 10 days or more were used for analysis.

cell fractionation and Western blot. Primary fibroblasts were washed with PBs and lysed in riPa buffer (50 mM Tris-HCl pH 7.4, 1% nP-40, 0.25% na-deoxycholate, 150 mM naCl, 1 mM edTa) with protease inhibitors or fractionated with ne-Per nuclear and cytoplasmic extraction kit (Pierce). Protein concentration was calculated by Bradford assay, separated by 10% Page and electroblotted to a PVdf membrane (immobilon-P, Millipore), probed with rabbit anti-flag (sigma), rabbit anti-HeaB/CLP1 (abcam), mouse anti-β actin (sigma), rabbit anti-TfiiH/gTf2H1 (Proteintech), rabbit anti-Tsen2 (Proteintech), mouse anti-Tsen34 (abnova), mouse anti-α TUBULin (sigma), or mouse anti-gaPdH (Millipore), irdye secondary antibodies (Li-Cor) and quantified with odyssey (Li-Cor).

immunocytochemistry. Cells were fixed 10 min with 4% Pfa, washed with PBs, permeabilized with 0.15% Triton-X-100, and blocked in the same solution with 5% normal donkey serum. Primary antibodies, rabbit anti-HeaB/CLP1 (abcam), mouse anti-Tuj1 (Covance), or rabbit anti-MaP2 (Cell signaling Technologies), were incubated overnight at 4oC. donkey anti-rabbit alexa 555 (invitrogen) or donkey anti-mouse alexa 488 (invitrogen) were used for detection. images were captured on an olympus iX51 or eVos-ci (aMg).

transfection, electroporation and cell Viability. H2o2 was added to the media for 1h and viability assessed at 24h by MTT method (Carmichael et al. , 1987). for the rna

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transfection and electroporation experiments, ten wells per genotype were plated at a density of 2000 cells per well (fibroblasts) and 15,000 cells per well (neural progeni-tor cells), into a 96-well plate the day before, transfected or electroporated with 300 ng of rna (Valuegene, Chemgenes), 5’-CCg CUC CCU UCg aUa gCU Cag CUg gUa gag Cgg agg aCU gUa g-2’,3’Po4 3’ (5’-exon), 5’-Po4-aUC CUU agg UCg CUg gUU Cga UUC Cgg CUC gaa gga-CCa-3’ (P-3’ exon), 5’-aUC CUU agg UCg CUg gUU Cga UUC Cgg CUC gaa gga-CCa-3’ (3’-exon) using Lipofectamine rnaiMaX in opti-MeM media (Life Technologies), or the nucleofector kit for neural Progenitor Cells (amaxa). The media was changed to growth media or 50 µM H2o2-containing media for 1 h then replaced with growth media, and MTT was used to assess viability.

affinity purification and trna endonuclease assay. double affinity purification (Hisflag-tagged CLP1) complexes isolation and trna endonuclease reactions were carried out as previously described (Trotta et al. , 2006; Volta et al. , 2005). Briefly, pre-trnaTyr was generated by in vitro transcription using T7 rna polymerase and labeled with α32P UTP as previously described (rauhut et al. , 1990). yeast trna endonuclease was used as a positive control (Trotta et al. , 2006). reactions were stopped with 10µl of 20mg/ml proteinase K (invitrogen), phenol/chloroform extracted, precipitated and rna products were separated on 16% 8 M urea polyacrylamide gels and exposed to phosphorimager (Typhoon 8600).

recombinant Protein Kinase assay. gsT-CLP1 or mutant gsT-CLP1r140H protein was isolated and tested for kinase activity against 80 pmol 5’-oH oligonucleotide ra20 (Val-uegene) as described (ramirez et al. , 2008), then quantified by densitometry (imageJ), calculated as the ratio of radiolabelled to total rna.

Protein and dna Oxidation analysis. one 10 cm plate of cells was used per replicate. eLisa assays were performed to detect the levels of Protein Carbonyl or 8-oHdg (CellBio Labs, inc).

computation. CLP1 mutations were modeled onto the structure of the partially crys-talized yeast Clp1 using the Phyre2 software engine (Kelley and sternberg, 2009). PLinK was used to construct autozygosity maps from exome sequence (Purcell et al. , 2007). The age of mutation (t_n,b denoting the age of a mutant having b copies in a sample of n genes) was calculated according to published methods (fu et al. , 2013). gene trees were produced using n individuals across a 5 kb region, and growth parameters from published demographic models (Plagnol and Wall, 2006; Tennessen et al. , 2012). n_0 was selected based on the number of haploblocks identified by phas-ing the Middle eastern cohorts around the target mutation. one hundred replicates

96

were run for each demographic, and used to estimate sTar-species trees using the nJ algorithm and calculate expected coalescent times. finally, these expected times were used as input into the formula published by (griffiths and Tavaré, 1998). selective pressure was not included in the calculations as fu et al. 2013 found no significant difference in estimates when included, largely due to the overriding impact of ex-ponential population growth. sample size (n) was selected based on the number of unique individuals from the three cohorts, while b represents the number of carriers of the mutant allele in this population (not including the affected). We calculated the expected mutation age for the mutant allele e(t_n,b) and the standard error Var(t_n,b).

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B

A

C 1810-V-8-Father

1810-V-9-Mother

1810-VI-1-Affected

1810-VI-2-Affected

Figure S1

Genotype Chrom Pos dbSNP Ref Alt Genotype Chrom Pos dbSNP Ref Alt Genotype Chrom Pos dbSNP Ref Alt Genotype Chrom Pos dbSNP Ref AltHet 11 57004386 G A 11 57004386 G NC 11 57004386 G NC Hom 11 57004386 G G

11 57077351 G C Het 11 57077351 G C 11 57077351 Hom 11 57077351 G CHom 11 57081197 4939134 G C Hom 11 57081197 4939134 G C Hom 11 57081197 4939134 G C Hom? 11 57081197 4939134 G CHom 11 57117385 2276042 C T Hom 11 57117385 2276042 C T Hom 11 57117385 2276042 C T Hom 11 57117385 2276042 C C

11 57135663 78580111 A NC Hom 11 57135663 78580111 A AG Hom 11 57135663 78580111 A AG 11 57135663 78580111 A NCHom 11 57137371 2276039 G A Hom 11 57137371 2276039 G A Hom 11 57137371 2276039 G A Hom 11 57137371 2276039 G GHom 11 57137424 2276038 C T Hom 11 57137424 2276038 C T Hom 11 57137424 2276038 C T Hom 11 57137424 2276038 C C

11 57137538 74770235 A NC Hom 11 57137538 74770235 A AG Hom 11 57137538 74770235 A AG 11 57137538 74770235 A NCHom 11 57147016 540687 A G Hom 11 57147016 540687 A G Hom 11 57147016 540687 A G Hom 11 57147016 540687 A G

11 57148053 571651 G T Hom 11 57148053 571651 G T Hom 11 57148053 571651 G T Hom 11 57148053 571651 G THom 11 57148175 669661 A G Hom 11 57148175 669661 A G Hom 11 57148175 669661 A G Hom 11 57148175 669661 A GHom 11 57155001 536455 G A Hom 11 57155001 536455 G A Hom 11 57155001 536455 G A Hom 11 57155001 536455 G AHom 11 57155288 630396 T C 11 57155288 630396 T NC Hom 11 57155288 630396 T C Hom 11 57155288 630396 T CHet 11 57156438 3741089 A G Hom 11 57156438 3741089 A G Hom 11 57156438 3741089 A G Hom 11 57156438 3741089 A GHom 11 57157405 490358 A G Hom 11 57157405 490358 A G Hom 11 57157405 490358 A G Hom 11 57157405 490358 A A

11 57184218 4938865 C NC Hom 11 57184218 4938865 C T Hom 11 57184218 4938865 C T 11 57184218 4938865 C NCHom 11 57243588 2258835 A G Hom 11 57243588 2258835 A G 11 57243588 2258835 A NC Hom 11 57243588 2258835 A G

11 57268170 2250673 T NC Hom 11 57268170 2250673 T C Hom 11 57268170 2250673 T C Hom 11 57268170 2250673 T CHom 11 57310307 80123614 A G Hom 11 57310307 80123614 A G Hom 11 57310307 80123614 A G Hom 11 57310307 80123614 A A

11 57317640 8890 C NC Hom 11 57317640 8890 C T Hom 11 57317640 8890 C T Hom 11 57317640 8890 C C11 57320044 2234412 T C Hom 11 57320044 2234412 T C Hom 11 57320044 2234412 T C Hom 11 57320044 2234412 T C

Hom 11 57427367 G A Hom 11 57427367 G A Hom 11 57427367 G A Hom 11 57427367 G A11 57461472 TG NC Het 11 57461472 TG T 11 57461472 TG NC Hom 11 57461472 TG T11 57472141 G NC Het 11 57472141 G GTTTTTTTT 11 57472141 G NC Hom 11 57472141 G G11 57472167 G NC Het 11 57472167 G T 11 57472167 G NC Hom 11 57472167 G G11 57472168 C NC Het 11 57472168 C T 11 57472168 C NC Hom 11 57472168 C C

Hom 11 57509429 635663 A G Hom 11 57509429 635663 A G Hom 11 57509429 635663 A G Hom 11 57509429 635663 A GHom 11 57563991 10896644 C T Hom 11 57563991 10896644 C T Hom 11 57563991 10896644 C T Hom 11 57563991 10896644 C THom 11 57571232 11229137 C T Hom 11 57571232 11229137 C T Hom 11 57571232 11229137 C T Hom 11 57571232 11229137 C T

11 57799108 34846253 AC NC Hom 11 57799108 34846253 AC A 11 57799108 34846253 AC NC Hom 11 57799108 34846253 AC AHom 11 57799371 1374570 G C Hom 11 57799371 1374570 G C Hom 11 57799371 1374570 G C Hom 11 57799371 1374570 G CHom 11 57947264 11229273 C G Hom 11 57947264 11229273 C G Hom 11 57947264 11229273 C G Hom 11 57947264 11229273 C GHom 11 57958775 7120468 C T Hom 11 57958775 7120468 C T Hom 11 57958775 7120468 C T Hom 11 57958775 7120468 C T

NC Het 11 57971171 C T 11 57971171 C NC Hom 11 57971171 C CHom 11 57971195 11229279 G A Hom 11 57971195 11229279 G A 11 57971195 11229279 G NC Hom 11 57971195 11229279 G AHom 11 57971201 11229280 G A Hom 11 57971201 11229280 G A 11 57971201 11229280 G NC Hom 11 57971201 11229280 G AHom 11 57982229 1966836 A G Hom 11 57982229 1966836 A G Hom 11 57982229 1966836 A G Hom 11 57982229 1966836 A GHom 11 57982584 1966835 T C Hom 11 57982584 1966835 T C 11 57982584 1966835 T NC Hom 11 57982584 1966835 T CHom 11 57982620 1966834 A G Hom 11 57982620 1966834 A G Hom 11 57982620 1966834 A G Hom 11 57982620 1966834 A GHet 11 57982726 1993089 G T Hom 11 57982726 1993089 G T 11 57982726 1993089 G NC Hom 11 57982726 1993089 G T

Family-1810-6-1 Family-1327-2 Family-57718Family-1337-2

chr pos1 pos2 kb nSNP density chr pos1 pos2 kb nSNP density

1 156131095 158736215 2605.12 120 21.71 2 1.86E+08 1.88E+08 1278.95 201 6.362 230663576 233243793 2580.22 41 62.93 2 2.36E+08 2.41E+08 4937.64 1394 3.543 153839960 158538056 4698.10 53 88.64 4 24451810 31785258 7333.45 1647 4.45

11 43776481 48346932 4570.45 96 47.61 4 58036217 79877936 21841.72 4830 4.5211 56510672 59224720 2714.05 55 49.35 4 1.2E+08 1.27E+08 6749.94 1423 4.7413 31495733 33704065 2208.33 46 48.01 7 96434961 1.15E+08 18318.38 1330 13.7716 55866894 58566304 2699.41 95 28.41 11 45006741 51563636 6556.90 788 8.3221 37425955 41450573 4024.62 70 57.49 11 54828062 74913756 20085.69 3341 6.01

22 18541497 26759278 8217.78 1577 5.21

chr pos1 pos2 kb nSNP density chr pos1 pos2 kb nSNP density

1 155629483 156130654 501.17 518 0.97 1 9416346 15723843 6307.50 1111 5.6811 55110770 58345418 3234.65 2079 1.56 1 1.96E+08 2.05E+08 8842.50 1454 6.0811 59426278 60617812 1191.53 626 1.90 2 2.12E+08 2.24E+08 11547.29 1349 8.5614 21559714 22102315 542.60 632 0.86 3 1.19E+08 1.22E+08 3717.27 571 6.5119 11708361 12502315 793.95 585 1.36 4 85704 4322981 4237.28 1009 4.2022 31673111 32340837 667.73 631 1.06 7 538191 2998173 2459.98 667 3.6922 41925207 42524310 599.10 577 1.04 11 1246941 7335198 6088.26 1744 3.49X 69504091 70586311 1082.22 577 1.88 11 45671280 61300476 15629.20 1964 7.96

12 11992168 55421250 43429.08 4227 10.2716 64981764 78466521 13484758 2267 5948.2822 17956759 32815398 14858640 2922 5085.09

Family 1810

Family 1327

Family 72597

Family 1337

supplemental figure 1. Confirmation of the CLP1 founder mutation, related to figure 1. (a) Whole exome analysis from family 1810, 1327, 1337, and 72597 using PLinK, demonstrating that all contain a homozy-gous block (red) around the CLP1 locus. The pos1 and pos2: top and bottom limits of the interval (hg19); kb: size of interval in kilobases; nsnP: number of single nucleotide polymorphisms (snP) in run; density: average snP density (1 snP per kb). for family 1327 and 1337, only intervals with nsnP>500 are shown. (B) shared haplotype block (red) between the four Turkish families containing the CLP1 mutation (bold). (C) sequence chromatogram from family 1810 showing site of heterozygous and homozygous mutation (arrow).

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A

Fibroblast

day 1: PTB shRNA

day 3: Doxycyline CHIR99021 A8301 Noggin DbcAMP LDN-193189

day 10: Doxycyline BDNF GDNF NT3 CNTF

NEURAL INDUCTION NEURAL MATURATION

Immature iNeuron Mature iNeuron

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supplemental figure 2. generation of induced neurons (ineurons), related to figure 2. (a) Coomassie stained sds-Page gel showing recombinant CLP1 wt and mutant protein. (B) rT-PCr of transcripts en-coding proteins involved in trna processing showing ubiquitous expression across human tissues. (C) schematic of neural induction from primary fibroblasts to produce patient-derived neural cells (ineu-rons). (d) expression of neural markers TUJ1 (green) and MaP2 (red) counterstained with dapi (blue) in ineurons at day (d) 10. (e) Brightfield images of Control, Unaffected, and affected fibroblast-to-ineuron conversion at d0, d5, and d10. Cells show similar morphology during reprogramming regardless of geno-type. gdna=genomic dna; scale bars = 100 µm.

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AFigure S3

clp1+/+

clp172

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0 1 2 3 4 50

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supplemental figure 3. clp1 mutant zebrafish showed reduced lifespan, motor neuron fragmentation, and p53-mediated neural loss; related to figure 3. (a) In situ hybridization showed clp1 expressed in developing brain and motor neurons of clp1+/+ zebrafish at 72 hours post fertilization (hpf) but not cl-p1R44X/R44X. (B) Lifespan of clp1L35R/L35R mutant zebrafish is reduced to 4.5 days compared to heterozygous or wt clutchmates. (C) sv2 immunostaining showed motor neuron fragmentation in clp1R44X/R44X zebrafish compared to control clutchmates at 72 hpf, concordant with neural degeneration. (d) In situ hybridiza-tion at 72 hpf for otx2 in clp1+/+ and clp1R44R/R44X zebrafish injected with p53 antisense morpholino, or uninjected, showed p53 knockdown restores otx2 expression in mutant zebrafish to near wt levels. Black scale bar = 100 µm, white scale bar = 50 µm.

100

Figure S4

0 0.2 0.4 0.6 0.8

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Collect Eluate for Western Blot and Cleavage Assay

ImidazoleLysis

supplemental figure 4. trna expression in human cells, related to figure 4. (a) Tyrosine (Tyr), isoleu-cine (ile) and Leucine (Leu) pre-trna expression in primary adult human cortex, induced neural cells (ineurons), and fibroblasts. (B, C) expression of certain isoforms of Tyr, ile, and Leu pre-trnas were not significantly changed in CLP1r140H patient cells compared to unaffected control cells. (d) schematic of double affinity purification of flagHis-tagged protein complexes. (e) replicate of Western blot analysis of CLP1-purified complexes showing p.r140H, p.r140a and p.K127a CLP1 mutants had reduced affinity for Tsen2 and Tsen34 compared to wt CLP1 and Tsen54. (f) trna endonuclease time-course performed on exogenous radiolabeled Tyrosine (Tyr) pre-trna with double affinity purified CLP1-bound complexes. CLP1r140H, CLP1r140a, and CLP1K127a bound complexes showed reduced endonuclease activity compared to wt CLP1 bound complexes. error bar: seM.

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supplemental figure 5. Viability output from hydrogen peroxide and transfection or transduction of trna species, related to figure 5. (a) Viability curve over 4 days showed equivalent growth of affected fibroblasts compared to Unaffected and unrelated Control fibroblasts. (B) Viability curve during neu-ral induction showed no loss of induced neural cells (ineurons). (C) Transduction of pre- and mature trnas for Chr14:trna19-TyrgTa and Chr19:trna10-ileTaT in Unaffected and affected ineurons, where no significant difference in viability was observed. (d, e) Basal protein and dna oxidation analysis show-ing ineurons had higher levels of protein carbonyl and 8-oHdg (8-hydroxydeoxyguanosine) compared with fibroblasts. no genotype-specific differences were detected. (f, g) dose dependent decline of fibro-blast and ineuron viability after 24h from hydrogen peroxide treatment to culture media. affected cells showed reduced cell survival compared with cells from Control and Unaffected. (H) Hydrogen peroxide reduced viability of affected cells, but not transfection with the Tyr trna 5’-exon containing 2’-3’-phos-phodiester bond. neither hydrogen peroxide nor 5’-exon rna altered Control or Unaffected cell viability (i, J) Transfection of the 3’-exon reduced cell viability in the presence of hydrogen peroxide in Unaffected cells, while all other treatments had no effect. (K) electroporation of Tyr trna exons did not affect human neural progenitor cell viability. *p<0.05, **p<0.01 student’s t-test (2-tailed). C = Control, U = Unaffected, a = affected. error bar: seM.

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fu, W., o’Connor, T.d., Jun, g., Kang, H.M., abecasis, g., Leal, s.M., gabriel, s., rieder, M.J. , altshuler, d., shendure, J., et al. (2013). analysis of 6,515 exomes reveals the recent origin of most human protein-coding variants. nature 493, 216-220.

griffiths, r.C., and Tavaré, s. (1998). The age of a mutation in a general coalescent tree. stochastic Models 14, 273-295.

Kelley, L.a., and sternberg, M.J. (2009). Protein structure prediction on the Web: a case study using the Phyre server. nat Protoc 4, 363-371.

Plagnol, V., and Wall, J.d. (2006). Possible ancestral structure in human populations. PLos genet 2, e105.Purcell, s., neale, B., Todd-Brown, K., Thomas, L., ferreira, M.a., Bender, d., Maller, J. , sklar, P., de Bakker,

P.i. , daly, M.J., et al. (2007). PLinK: a tool set for whole-genome association and population-based linkage analyses. am J Hum genet 81, 559-575.

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rauhut, r., green, P.r., and abelson, J. (1990). yeast trna-splicing endonuclease is a heterotrimeric enzyme. J Biol Chem 265, 18180-18184.

sidransky, e., and stubblefield, B. (1993). dried umbilical cords as a source of dna for genetic studies. BioTechniques 15, 30.

Tennessen, J.a., Bigham, a.W., o’Connor, T.d., fu, W., Kenny, e.e., gravel, s., Mcgee, s., do, r., Liu, X., Jun, g., et al. (2012). evolution and functional impact of rare coding variation from deep sequencing of human exomes. science 337, 64-69.

Thisse, C., and Thisse, B. (2008). High-resolution in situ hybridization to whole-mount zebrafish em-bryos. nat Protoc 3, 59-69.

Trotta, C.r., Paushkin, s.V., Patel, M., Li, H., and Peltz, s.W. (2006). Cleavage of pre-trnas by the splicing endonuclease requires a composite active site. nature 441, 375-377.

Volta, V., Ceci, M., emery, B., Bachi, a., Petfalski, e., Tollervey, d., Linder, P., Marchisio, P.C., Piatti, s. , and Biffo, s. (2005). sen34p depletion blocks trna splicing in vivo and delays rrna processing. Biochem Biophys res Commun 337, 89-94.

Xue, y., ouyang, K., Huang, J. , Zhou, y., ouyang, H., Li, H., Wang, g., Wu, Q., Wei, C., Bi, y., et al. (2013). di-rect conversion of fibroblasts to neurons by reprogramming PTB-regulated microrna circuits. Cell 152, 82-96.

Alles is een raadsel en ik weet nog nietIn welke vorm of in welke taal- Spinvis

5lOst in translatiOn:

POtential PathOmechanisms underlying POntOcerebellar

hyPOPlasia

Veerle rC eggens

107

Chapter

5

discussion

LosT in TransLaTion: PoTenTiaL PaTHoMeCHanisMs UnderLying PonToCereBeLLar HyPoPLasia

The last decade, a number of genes has been identified as PCH-causing. The majority of these genes functions in rna processing and/or protein synthesis (Table 5.1), sug-gesting these processes play part in the disease mechanism of PCH. Protein synthesis is crucial in all types of cells, so why would the brain –  and particularly the pons and cerebellum - be so heavily affected? The whole brain increases exponentially in volume during the first eight weeks of development [1]. The cerebellum undergoes an additional period of extensive growth in the third trimester; a fivefold growth in vol-ume and up to a 30-fold growth in surface from 27 to 40 weeks gestational age [2,3] in which it requires a tremendous amount of proteins. Possibly, the developing brain of a PCH patient cannot cope with this high demand for de novo protein synthesis.

in this chapter, i will review the present knowledge about reduced protein synthesis and brain atrophy. i will explore several hypotheses how mutations in PCH-associated genes could cause reduced protein synthesis and how this could eventually lead to neurodegeneration.

reduced protein synthesis

figure  5.1 shows an overview of potential direct and indirect pathways between mutated genes found in PCH patients and neurodegeneration. given the functions of

hier tabel 5.1

table 5.1: PCH-associated genes and their function.

gene functionKey references on gene function

TSEN54, TSEN2, TSEN34 trna splicing endonuclease [4]

CLP1 phosphorylates trna exon after splicing, maintains Tsen complex integrity

[5,6]

RARS2 mitochondrial arginyl-trna synthetase [7,8]

EXOSC3, EXOSC8 components 3 and 8 of the exosome complex, which degrades and processes small rna molecules

[9,10]

TOE1 has deadenylating activity and maintains Cajal body integrity, therefore porentially involved in mrna splicing

[11,12]

AMPD2 Maintains cellular guanine nucleotide levels, involved in gTP dependent protein translation initiation

[13,14]

CHMP1A cell proliferation, chromatin modelling [15,16]

PCLO regulates presynaptic proteins and vesicles [17]

VRK1 kinase involved in cell cycle (of neuronal progenitors) and dna damage, also involved in Cajal body dynamics

[18,19]

108

PCH-associated genes, suboptimal protein synthesis seems a plausible mechanism in view of genetic mutations and excessive cerebellar growth. a first question that has to be answered is whether protein synthesis is indeed reduced in PCH patients. interest-ingly, already in 1990 Barth described a PCH2 patient which showed accumulation of ribosomes at the endoplasmic reticulum (er) on electron microscopy images of corti-cal neurons [20], indicative for a problem at the level of protein translation. indeed, yeast strains of tsen2 and ampd2 mutants show a decrease in actively translating ribosomes and a pronounced decrease in [35]s-methionine incorporation was found in tsen2 mutant yeast [21]. another clue for deficient protein translation machinery underlying neurodegeneration comes from gTP-binding protein 2 (GTPBP2) knockout mice, that have variations in the gene encoding trna-arg-UCU [22]. These mice pres-ent ataxia, and neurodegeneration in the cerebellum and cortex. The variation in the trna-arg-UCU gene induced ribosome stalling, which normally would be resolved by gTPBP2 and a protein named Pelota. since the gTPBP2 protein is absent in these mice, ribosomal stalling could not be resolved. although ribosome stalling and re-duced protein synthesis has not yet been extensively demonstrated in patients, these first results in yeast and mice suggest it is a plausible mechanism in PCH and other forms of neurodegeneration.

hier figuur 5.1

REDUCED PROTEIN SYNTHESIS

MUTATIONS IN tRNA PROCESSING GENES

tRNA MOLECULES tRNA FRAGMENTS

SHORTAGE OF FUNCTIONAL tRNAS

AAR

NEURODEGENERATION

figure 5.1: Potential pathways leading to neurodegeneration in pontocerebellar hypoplasia. defects in trna processing and protein synthesis seem recurring mechanisms in PCH. The two mechanisms can be linked directly (shortage of functional trna molecules, inhibiting effects of trna fragments) or indirectly (via the amino acid response (aar)).

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lack of functional trnas

in case reduced protein synthesis indeed occurs in patients with PCH, this reduction could have various origins. The TSEN genes, CLP1 and RARS2 are directly involved in trna processing. When trna processing occurs incorrect or inefficient – which could be the case in patients with a TSEN or CLP1 mutation - or when trna molecules are not attached to their corresponding amino acid – in case of RARS2 mutations, a shortage of functional trna could arise. This could subsequently lead to a reduction of the capacity for protein synthesis. a straightforward question is whether functional trna levels are indeed decreased in PCH patients. it seems that the overall abundance of mature trnas is not altered in TSEN of CLP1 fibroblasts. However, the efficiency of trna splicing is reduced in these cells [23,24]. interestingly, in induced neurons (ineurons) with a CLP1 mutation, there is a difference in mature trna levels between affected and unaffected cells [25]. Pre-mature trna levels are higher and mature trna levels are lower in affected ineurons compared to unaffected ineurons. The mutation in CLP1 has an apparently more severe effect on neurons compared to fibroblasts, which is in line with the neurospecificity of PCH. Concerning rars2, reduced levels of mitochondrial trna-arg transcript are seen in fibroblasts of a small number of patients [7,26]. However, nearly all residual trna-arg was acetylated. This suggests that unacetylated trnas are unstable.

supporting evidence that a sufficient number of trnas is important in nervous system development comes from trnaser/sec knockout mice, which show cerebellar hypoplasia and Purkinje cell loss and early death [27]. additionally, mutations in the rna Polymerase iii transcription initiation factor BRF1 lead to reduced pol iii related transcription, intellectual disability and cerebellar hypoplasia [28]. one of the main targets of rna Pol iii are trna genes.

The regional specificity of the trna processing defects remains enigmatic. not all mutations that affect protein synthesis give rise to cerebellar atrophy. Mutations in aminoacyl-trna synthetases (arss) are reported in a number of diseases. some of these diseases have a neurological component, as glycyl-trna synthestase (GARS) and alanyl-trna synthetase (AARS) have been associated with Charcot-Marie-Tooth disease [29,30]. Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBsL), caused by mutations in mitochondrial aspartyl-trna synthetase (DARS2) is nonetheless a brain disease, but a disorder of the white mat-ter rather than grey matter [31]. Mutations in mitochondrial seryl-trna synthetase (SARS2) results in the multisystem disorder Hyperuricemia, Pulmonary Hypertension, renal failure and alkalosis (HUPra syndrome) [32]. To summarize, mutations in aminoacyl-trna synthetases have been associated with a number of disease. Many of these - but certainly not all - include a neurological disease component. This wide

110

range of phenotypes could have various explanations, e.g. tissue specific variations in aminoacyl-trna synthetase expression or genetic redundancy.

it is important to keep in mind that only a subset of trnas should be affected by mutations in the TSEN complex or CLP1. since only 6% of all human trna genes contain an intron (http://gtrnadb.ucsc.edu/) only these would encounter problems in case of a TSEN or CLP1 mutation. an explanation for the neuronspecificity would be that intron-containing trna genes are primarily expressed in the brain, but measure-ment of trna abundance in different human tissues did not reveal such elevated expression [33]. nevertheless, TSEN54 is highly expressed in human cerebellum at 8 weeks gestational age (fig. 5.2). This could be interpreted as if there is a need for efficient trna processing in this region. on the other hand, it cannot be excluded that Tsen54 has another function in this brain area and developmental stage besides trna splicing.

integrated stress response

The most important function of trnas is to transport amino acids to the ribosomes to facilitate protein synthesis. However, alternative biological functions for trna molecules and trna fragments have been described [35]. one of these functions is induction of the amino acid response (aar) (fig. 5.3). The aar is a signaling path-way that senses a shortage of amino acids by measuring the amount of uncharged trnas. general control nonderepressible 2 (gCn2) functions as a sensor of amino acid deprivation, as it is activated by these uncharged trna molecules. This leads to phosphorylation of translation initiation factor eif2alpha. Consequently, eif2alpha represses general protein synthesis, but increases translation of a subset of mrnas

hier figuur 5.2

figure 5.2: TSEN54 mrna expression in a human embryo, 8 weeks gestational age. In situ hybridisation with a human Tsen54 specific Lna/2oMe probe was performed (5’-TcuTucTcuTgcCauCucC-3’ (ribotask aps, denmark) with Lna residues in capital letters and 2oMe in lower case) [34] . a series of sec-tions were stained and 3d images were constructed using amira® software. yellow=Tsen54 expression; green=brain tissue.

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among which activating transcription factor 4 (ATF4) and 5 (ATF5) and growth ar-rest and dna-damage inducible 34 (GADD34). This selective upregulation brings the cell in survival state when deprived from sufficient amino acids. However, when cell homeostasis cannot be restored, aTf4-induced apoptosis can be initiated [36]. This makes the cell go in survival state. This invoked response is called the integrated stress response (isr).

as the isr can be triggered by uncharged trna molecules [36] (fig. 5.3) it would be interesting to investigate whether the increased levels of pre-trnas seen in ineurons of PCH10 patients could elicit the aar as well [25]. activation by uncharged trnas is not the only trigger for the isr. a second factor that initiates the isr is er stress, which is on its turn induced by unfolded or misfolded proteins [37]. as deficiencies in splicing could lead to misfolded proteins, mutations in genes linked to splicing processes as TOE1, EXOSC3 and EXOSC8 could induce er stress. Thirdly, double strand (ds)rna molecules can trigger the isr pathway. one of the functions of the rna exo-some is to degrade small rna molecules. Possibly, when these molecules accumulate in patients with mutations in EXOSC3 or EXOSC8, the isr can be initiated.

UNCHARGED tRNAS

GCN2

P-EIF2-ALPHA

ADAPTATION TO STRESS

ER STRESS DSRNA

PERK PKR

REDUCTION OF GLOBAL TRANSLATION

ENHANCEMENT OF ATF4 INDUCED TRANSLATION

APOPTOSIS

AAR UPR

figure 5.3: The integrated stress response (isr). The isr can be induced via the amino acid response (aar, orange), the unfolded protein response (UPr, blue) or by dsrna molecules. all pathways stimulate phosphorylation of eif2-alpha. Consequently, global protein synthesis reduces and aTf4 induced trans-lation increases.

112

a thorough examination of the role of isr in neurodegenerative disease as PCH still has to be set up. nevertheless, it is a promising mechanism, linking trna processing and rna degradation defects to neuronal cell death.

trna fragments and modifications

above we discussed an alternative function of trna molecules, namely induction of the aar upon amino acid limitation. Previous research has shown that not only intact trna molecules have a biological function, trna fragments can also play a role in cell homeostasis (reviewed in [38]). These trna fragments can derive from various events. for example, several sources of cellular stress (arsenite, heat shock and UV irradiation) promote trna cleavage by the ribonuclease angiogenin, resulting in accumulation of stress-induced small trnas (tirnas) and ultimately global translational arrest by the arisen 5’ tirna [39]. an example an association between angiogenin-derived 5’ trna exons and neurodegeneration is shown in mice with a mutation in the gene NSun2, which methylates trna molecules [40]. it was shown that decreased methylation of trna molecules due this mutation leads to increased trna cleavage by angiogenin. The accumulation of 5’ exon of the trna leads to a decrease in protein synthesis and induction of stress pathways resulting in reduction of cell size and apoptosis. as a result, these mice present microcephaly and other neurological abnormalities.

Could trna fragments also play part in the disease mechanism of PCH? trna intron accumulation was obviously seen in CLP1 mutants, and moderately in TSEN54 mutants [24]. These trna fragments could, alike angiogenin-induced trna fragments, induce stress pathways. as shown in Chapter 4 of this thesis, phosphorylation of the 3’ trna exon seems to have a protective effect [25]. future research could elucidate the cel-lular effects of trna intron and exon fragments in PCH and other neurodegenerative diseases.

This chapter, as most of the research on the pathomechanism of PCH, has focussed on how trna processing deficiencies could lead to reduced protein synthesis and/or neurodegeneration. However, it has to be kept in mind that defects in other rna spe-cies could underlie the origin of PCH. for example, the exosome complex is involved in processing of various kinds of small rnas [10], as is TOE1 possibly has a general rna deadenylation activity [41]. Both TOE1 and VRK1 are involved in maintaining Cajal bodies [11,19], which makes mrna splicing an interesting process to explore in the disease mechanism of PCH.

To conclude, impaired protein synthesis is an interesting and promising hypothesis underlying PCH and other neurodegenerative diseases. a global decrease in protein synthesis could have various origins, e.g. scarcity of mature trnas, induction of the amino acid response, inhibition of translation by trna fragments, or it could arise from processing deficiencies of other rna species. supporting evidence can be found

hier figuur 5.3

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for each of these potential mechanisms, and it will be an interesting and dynamic field of research in the following years. not only will it elucidate more about the disease mechanism of PCH, it will also teach us fundamental knowledge of the process of neurodegeneration, applicable in many other diseases.

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32. Belostotsky r, Ben-shalom e, rinat C, Becker-Cohen r, feinstein s, Zeligson s et al.: Mutations in the mitochondrial seryl-trna synthetase cause hyperuricemia, pulmonary hypertension, renal failure in infancy and alkalosis, HUPra syndrome. Am J Hum Genet 2011, 88: 193-200.

33. dittmar Ka, goodenbour JM, Pan T: Tissue-specific differences in human transfer rna expres-sion. PLoS Genet 2006, 2: e221.

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34. Kasher Pr, namavar y, van TP, fluiter K, sizarov a, Kamermans M et al.: impairment of the trna-splicing endonuclease subunit 54 (tsen54) gene causes neurological abnormalities and larval death in zebrafish models of pontocerebellar hypoplasia. Hum Mol Genet 2011, 20: 1574-1584.

35. raina M, ibba M: trnas as regulators of biological processes. Front Genet 2014, 5: 171. 36. Kilberg Ms, shan J, su n: aTf4-dependent transcription mediates signaling of amino acid

limitation. Trends Endocrinol Metab 2009, 20: 436-443. 37. shah sZ, Zhao d, Khan sH, yang L: Unfolded Protein response Pathways in neurodegenerative

diseases. J Mol Neurosci 2015. 38. gebetsberger J, Polacek n: slicing trnas to boost functional ncrna diversity. RNA Biol 2013, 10:

1798-1806. 39. yamasaki s, ivanov P, Hu gf, anderson P: angiogenin cleaves trna and promotes stress-induced

translational repression. J Cell Biol 2009, 185: 35-42. 40. Blanco s, dietmann s, flores JV, Hussain s, Kutter C, Humphreys P et al.: aberrant methylation

of trnas links cellular stress to neuro-developmental disorders. EMBO J 2014, 33: 2020-2039. 41. Wagner e, Clement sL, Lykke-andersen J: an unconventional human Ccr4-Caf1 deadenylase

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Ieder verhaal eindigt gelukkig, als je maar vroeg genoeg ophoudt- Annie M.G. Schmidt

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on THe origin of PonToCereBeLLar HyPoPLasia:

finding genes for a rare disease

This thesis describes novel genes involved in pontocerebellar hypoplasia (PCH). The identification of TOE1 (Chapter 2) and CLP1 (Chapter 4) as PCH causing genes is an important finding for PCH research and is directly implementable in PCH diagnostics. furthermore, we elucidated genotype-phenotype correlations in these patients and in patients with mutations in EXOSC3 (Chapter 3). With this knowledge, relatives of pa-tients suffering from PCH can be better informed about the expected disease progress. Here, i will summarise and discuss our findings, elaborate on the underlying disease mechanism of PCH and propose ideas for further research.

chapter 1 provides an introduction of the clinical and genetic aspects of pontocer-ebellar hypoplasia. Ten subtypes of the disease have been described so far (PCH1-10). Common features in all subtypes include hypoplasia of the pons and cerebellum and severe mental and motor disabilities. onset of the disease is premature, and life expectancy ranges from few days to late twenties. The spectrum of PCH is expanding, which is partly a matter of nomenclature. in some subtypes hypoplasia of the pons and cerebellum is the main hallmark, whereas in other subtypes it solely one aspect of the disease manifestation. in around half of PCH and PCH-like patients, a genetic mutation is identified. in 2008, the first gene causing PCH was discovered by linkage analysis [1]. nowadays, novel techniques as next generation sequencing allow us to identify more genes for rare diseases as PCH. The majority of genes involved in PCH play a role in rna processing, which seems to be a common pathomechanism in the disease.

in chapter 2 we present the TOE1 gene as the locus for PCH7. We started our search for a gene by performing exome sequencing in a Turkish family with two children with PCH plus genital abnormalities. selecting for rare non-synonymous variants under an autosomal recessive model revealed three candidate genes: SLC39A1, CLK2 and TOE1. The latter two genes have a potential role in rna processing, an apparently common disease pathway in PCH, which made us decide to follow up both genes. injecting an antisense morpholino (Mo) against the aTg start site region of clk2 gave a brain phenotype in zebrafish embryos. injected zebrafish embryos showed a small head, an abnormally shaped midhindbrain boundary and increased cell death. However, we were unable to prove the specificity of the aTg Mo induced phenotype, thus we cannot conclude that the abnormal brain in these fish is due to CLK2 knockdown. on a biochemical level, we showed that the p.a390s variation in CLK2 does not abolish

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kinase activity of the CLK2 protein in vitro despite it being a highly conserved residue. CLK2 knockout mice did not show brain or genital abnormalities. Last but not least, we identified CLK2 variations in only a single family with PCH7. Based on these results we concluded that CLK2 is not the gene causing PCH7.

in the meantime, we collected a cohort of ten patients (eight families) with PCH and genital abnormalities. We identified rare homozygous and compound heterozygous missense and nonsense mutations in the TOE1 gene in all affected individuals. Muta-tions in this gene seem to be restricted to PCH7, since no potentially pathogenic TOE1 variations were identified in PCH patients with other subtypes. analysis of the protein structure of Toe1 revealed that the identified mutations possibly interrupt enzymatic activity and/or protein-protein interactions of Toe1.

all 46,Xy patients in our PCH7 cohort have some degree of undervirilisation of the internal and external genitals. The two patients with karyotype 46,XX had normal female external genitalia and in one 46,XX patient no ovaries could be identified by medical imaging. endocrinological investigations in six patients revealed hyper-gonadotrophic hypogonadism. neurological manifestations included axial hypotonia with increased tone in the limbs and seizures. development was severely delayed with no or very limited ability to sit, walk, talk and interact. Three patients died at the age of 24 weeks, 2 years and 3 years. The patients alive vary in age from one year to late twenties. Brain Mri of the patients with TOE1 mutations showed pontocerebellar hypoplasia, ventriculomegaly and decreased white matter including a thin corpus callosum.

With the identification of TOE1 as PCH7-causing gene, we confirm the presence of PCH plus genital abnormalities as a monogenetic syndrome.

The brain anomalies seen in PCH7 patients are not restricted to the pons and the cerebellum. one could argue that this is not within the spectrum of PCH, and also the prominent disorders of sex development make PCH7 an outsider in the spectrum. it is interesting to investigate whether overlap exists in disease mechanism between PCH7 and the other PCH subtypes. alike the other genes causing PCH, TOE1 is involved in several rna processing events: it has deadenylating activity [2] and is involved in Cajal body maintenance, which might implicate a role in rna splicing [3]. it was recently shown that Toe1 can penetrate cells and impair transcription and replication of viruses [4]. Possibly, TOE1 has an effect on transcription of other genes as well.

Many questions remain to be answered about the disease mechanism of PCH7. TOE1s function in mrna splicing and deadenylation are interesting to follow up, as well as its role in transcription regulation. another clue that might lead to elucidating the mechanism is the finding that both 46,Xy and 46,XX seem to have aberrations in genital development. This suggests that the onset of deviations in gonadal develop-ment is already before the formation of the bipotential gonad. This bipotential gonad

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is formed in the fifth week of development and gives rise to either testes or ovaries [5]. Comparing TOE1 with genes as the homeobox gene EMX2, which is involved in forma-tion of the bipotential gonad, mrna transport and is associated with schizencephaly [6] could be very worthwhile.

in chapter 3 describes a cohort of patients with EXOSC3-related PCH. it has been previously shown that EXOSC3 mutations account for around half of all PCH1 patients [7-9]. We show that mutations in this gene are restricted to this subtype and we pres-ent the disease course of twelve additional families with a genetic mutation in this gene. With this we show that the specific genetic mutation in EXOSC3 can largely predict the disease course. Patients with a homozygous p.d132a mutation in this gene display a prolonged disease course compared to other PCH patients, with possible survival into puberty. These patients can achieve some milestones as crawling and sitting, which are often lost when disease progresses. The pons can be unaffected in patients with this mutation. The p.g31a mutation is prevalent amongst the roma population and lead to death in infancy. Contractures are reported in these patients and pontine hypoplasia is more pronounced than in the p.d132a group. Patients with a p.d132a and a nonsense or p.y109n mutation on the other allele, or with a homozygous p.g135e mutation, present a severe phenotype as in the homozygous p.g31a group. our study helps in predicting the disease course of PCH1 patients, and broadens the phenotype of PCH1, since EXOSC3 mutations can be considered in case of an unaffected pons.

Mutations in either EXOSC3 or EXOSC8 lead to PCH1 [10]. Both genes are com-ponents of the exosome complex, which main function is to process and degrade various species of small rnas [11]. a signal for decreased exosome function is the accumulation of unprocessed ribosomal rna [12]. This was not seen in fibroblasts of patients with an EXOSC3 mutation [13]. Perhaps, we should look for another, or a more subtle cellular effect in patients, or shift to studies in neurons rather than fibroblasts. in view the other genes involved in PCH, it is interesting to further investigate the role of the exosome complex in trna processing and the amino acid response.

chapter 4 describes PCH10 caused by mutations in the CLP1 gene. exome sequenc-ing revealed nine families with a homozygous p.r140H mutation, all from the same region in Turkey [14,15]. Patients present pontocerebellar hypoplasia, a thin corpus callosum and both central and peripheral nervous system abnormalities. We mimicked the disease in zebrafish with a homozygous nonsense allele in Clp1. The fish die in early larval stage, and show brain atrophy and disorganised motor neurons. inject-ing wild-type CLP1 mrna rescues this phenotype, while injecting mrna with the p.r140H mutation does not, demonstrating the deleterious effect of the mutation.

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CLP1 is physically and functionally connected to the Tsen complex, fulfilling a role in trna splicing. after trna intron cleavage by the Tsen complex, CLP1 phosphorylates the 5’ end of the 3’ trna exon. Hereafter, the two exon halves can be ligated to form a functional trna molecule. We show that the p.r140H mutation affects the function of CLP1 on various levels. due to a disturbed interaction with the Tsen complex, trna cleavage is decreased in mutant cells. furthermore, the kinase activity of CLP1 is reduced and the nuclear localisation of the CLP1 protein is impaired. our results sug-gest that the mutation in CLP1 has a cell specific effect. in fibroblasts, no difference in levels of intron-containing pre-trna or mature trna was seen in patient versus control samples. in contrast, ineurons show higher levels of pre-trna and lower levels of mature trna in patients cells versus control cells. This specific effect in neuronal cells is in line with the neurospecificity seen in PCH. furthermore, trna fragments could play a role in CLP1-associated PCH. firstly, accumulation of trna introns has been found [14], which possibly have a deleterious effect on the cell. secondly, our experiments show that phosphorylation of the 3’ trna exon by CLP1 has a protective effect on stress-induced cell death. it remains elusive to which extent each of the aberrant functions –  trna cleavage, nuclear localisation, kinase activity and trna fragments – account for the development of PCH10.

an interesting finding is that in PCH1 and PCH10, in contrast to other PCH subtypes, motor neurons are affected. a possible explanation is that the mechanism in PCH1 and PCH10 works two-fold: on one hand there is disturbed rna processing potientially leading to reduced protein synthesis. on the other hand there are rna fragments that become toxic for motor neurons in particular. in case of EXOSC3 and EXOSC8 these fragments could arise from defective clean-up by the exosome, in case of CLP1 they could arise from accumulating trna introns or trna exons. rna foci composed of rna fragments are often seen in motor neurons diseases as amyotrophic lateral sclerosis [16] and spinal cerebellar ataxia [17,18].

chapter 5 discusses potential mechanisms underlying PCH. in the last few years, a start has been made in elucidating the pathomechanism of PCH. Most of the genes involved in PCH point towards a defect in (t)rna processing and protein synthesis. re-duced protein synthesis can have various origins, for example from a lack of functional (t)rnas. However, a global inhibition of translation can also arise via the intergrated stress response or due to effects of trna fragments. future research might bring more clarity in the disease mechanism behind PCH and similar diseases.

future directions

as a results of the developments in next generation sequencing, enormous progress is made in finding new genes for rare diseases as PCH. This is very useful for diagnostics

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and prenatal counselling, but does not provide a treatment for a disease as PCH. The next challenging step is to elucidate the pathomechanism. as described in this thesis, the first steps are made in understanding the disease on cellular and molecular level, but many questions remain. investigating whether the hypotheses about reduced protein synthesis and rna metabolism are indeed the main bottleneck in PCH is the most important step in future research. When we discover more about what processes are affected in PCH, we can proceed with finding ways to intervene these processes. nevertheless, finding a treatment for PCH will be challenging, considering the early - in most cases prenatal - onset of the disease.

conclusions

This thesis describes several new genetic mutations causative for pontocerebellar hypoplasia and associated genotype-phenotype relations. it thereby provides directly implacable tools for improving diagnostics and genetic counselling of PCH. further-more, the studies in this thesis provide new clues for the disease mechanism of PCH and other neurodegenerative diseases.

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referenCes

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2. Wagner e, Clement sL, Lykke-andersen J: an unconventional human Ccr4-Caf1 deadenylase complex in nuclear cajal bodies. Mol Cell Biol 2007, 27: 1686-1695.

3. fong KW, Li y, Wang W, Ma W, Li K, Qi rZ et al.: Whole-genome screening identifies proteins localized to distinct nuclear bodies. J Cell Biol 2013, 203: 149-164.

4. sperandio s, Barat C, Cabrita Ma, gargaun a, Berezovski MV, Tremblay MJ et al.: Toe1 is an inhibitor of HiV-1 replication with cell-penetrating capability. Proc Natl Acad Sci U S A 2015, 112: e3392-e3401.

5. eggers s, ohnesorg T, sinclair a: genetic regulation of mammalian gonad development. Nat Rev Endocrinol 2014, 10: 673-683.

6. Brunelli s, faiella a, Capra V, nigro V, simeone a, Cama a et al.: germline mutations in the homeobox gene eMX2 in patients with severe schizencephaly. Nat Genet 1996, 12: 94-96.

7. rudnik-schoneborn s, senderek J, Jen JC, Houge g, seeman P, Puchmajerova a et al.: Pontocer-ebellar hypoplasia type 1: clinical spectrum and relevance of eXosC3 mutations. Neurology 2013, 80: 438-446.

8. Biancheri r, Cassandrini d, Pinto f, Trovato r, di rM, Mirabelli-Badenier M et al.: eXosC3 muta-tions in isolated cerebellar hypoplasia and spinal anterior horn involvement. J Neurol 2013, 260: 1866-1870.

9. schwabova J, Brozkova ds, Petrak B, Mojzisova M, Pavlickova K, Haberlova J et al.: Homozygous eXosC3 mutation c.92g—>C, p.g31a is a founder mutation causing severe pontocerebellar hypoplasia type 1 among the Czech roma. J Neurogenet 2013, 27: 163-169.

10. Boczonadi V, Muller Js, Pyle a, Munkley J, dor T, Quartararo J et al.: eXosC8 mutations alter mrna metabolism and cause hypomyelination with spinal muscular atrophy and cerebellar hypoplasia. Nat Commun 2014, 5: 4287.

11. Liu Q, greimann JC, Lima Cd: reconstitution, activities, and structure of the eukaryotic rna exosome. Cell 2006, 127: 1223-1237.

12. Mitchell P, Petfalski e, shevchenko a, Mann M, Tollervey d: The exosome: a conserved eukaryotic rna processing complex containing multiple 3’—>5’ exoribonucleases. Cell 1997, 91: 457-466.

13. Wan J, yourshaw M, Mamsa H, rudnik-schoneborn s, Menezes MP, Hong Je et al.: Mutations in the rna exosome component gene eXosC3 cause pontocerebellar hypoplasia and spinal motor neuron degeneration. Nat Genet 2012, 44: 704-708.

14. Karaca e, Weitzer s, Pehlivan d, shiraishi H, gogakos T, Hanada T et al.: Human CLP1 mutations alter trna biogenesis, affecting both peripheral and central nervous system function. Cell 2014, 157: 636-650.

15. schaffer ae, eggens Vr, Caglayan ao, reuter Ms, scott e, Coufal ng et al.: CLP1 founder muta-tion links trna splicing and maturation to cerebellar development and neurodegeneration. Cell 2014, 157: 651-663.

16. dejesus-Hernandez M, Mackenzie ir, Boeve Bf, Boxer aL, Baker M, rutherford nJ et al.: ex-panded ggggCC hexanucleotide repeat in noncoding region of C9orf72 causes chromosome 9p-linked fTd and aLs. Neuron 2011, 72: 245-256.

17. Chen iC, Lin Hy, Lee gC, Kao sH, Chen CM, Wu yr et al.: spinocerebellar ataxia type 8 larger triplet expansion alters histone modification and induces rna foci. BMC Mol Biol 2009, 10: 9.

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18. White MC, gao r, Xu W, Mandal sM, Lim Jg, Hazra TK et al.: inactivation of hnrnP K by expanded intronic aUUCU repeat induces apoptosis via translocation of PKCdelta to mitochondria in spinocerebellar ataxia 10. PLoS Genet 2010, 6: e1000984.

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nederlandse samenVatting

oVer HeT onTsTaan Van PonToCereBeLLaire HyPoPLasie:

HeT Vinden Van genen Voor een ZeLdZaMe ZieKTe

dit proefschrift gaat over de zoektocht naar genen die de ziekte pontocerebellaire hypoplasie (PCH) veroorzaken. Zoals beschreven in hoofdstuk 1 hebben patiënten met PCH ernstige mentale en lichamelijke handicaps. Ze kunnen vaak niet zelfstandig zit-ten of lopen en kunnen slechts zeer beperkt communiceren. de ziekte begint al voor de geboorte en heeft een progressief verloop. Patiënten met PCH overlijden erg jong, variërend van vlak na de geboorte tot op ongeveer 30 jarige leeftijd, maar de meeste patiënten overlijden in hun kinderjaren. PCH is een ziekte waarbij bepaalde delen van de hersenen, met name de pons en het cerebellum, te klein zijn. Tot nu toe zijn er tien subtypes beschreven, die van elkaar zijn te onderscheiden op grond van klinische symptomen en/of genetische achtergrond. in ongeveer de helft van de gevallen is de genetische achtergrond bekend. Van de andere helft van de patiënten is onbekend welke genetische mutatie er aan hun ziekte ten grondslag ligt. in deze laatste groep patiënten hebben we gezocht naar nieuwe genen die PCH veroorzaken.

Wat wordt er precies bedoeld met de genetische achtergrond van een ziekte? Het erfelijk materiaal van ieder mens bestaat uit ongeveer 30.000 genen. door middel van specifieke enzymen kan een gen worden ‘afgelezen’. er wordt dan mrna gevormd, wat op zijn beurt codeert voor een eiwit. er zijn dus heel veel verschillende eiwitten die er samen voor zorgen dat een cel, en daarmee het lichaam, goed kan functioneren. Het eiwit dat wordt gevormd vanuit het gen is een reeks van aminozuren. genen bestaan uit dna, en dna is weer opgebouwd uit vier verschillende bouwstenen (nucleotiden) die we a, T, g en C noemen. een gen bestaat uit duizenden van die nucleotiden achter elkaar. Mensen verschillen van elkaar doordat ze kleine variaties hebben in hun dna, op de plek waar bij de één een T staat, staat bij de ander bijvoorbeeld een g. deze variaties zorgen ervoor dat de één blond haar heeft en de ander bruin, dat de één een grotere neus heeft dan de ander, etc. af en toe zit er echter ook een variatie tussen die een ziekte veroorzaakt, zo’n variatie met een negatief effect heet een mutatie. als je op zoek bent naar een mutatie die PCH veroorzaakt, moet je dus zoeken naar een verschil in al die miljoenen dna-nucleotiden tussen patiënten en gezonde mensen. dat lijkt misschien onbegonnen werk, maar gelukkig zijn er methoden ontwikkeld om dit redelijk snel te doen. Zie hier het resultaat

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in hoofdstuk 2 gaan we op zoek naar zo’n nieuw gen, in dit geval een die PCH subtype 7 veroorzaakt. Patiënten met PCH7 hebben zowel hersenafwijkingen als afwijkingen in hun genitale ontwikkeling (disorders of sex development, dsd). in een broer en zus met PCH7 vonden we variaties in twee kandidaatgenen die de ziekte zouden kunnen veroorzaken: CLK2 en TOE1. Welke van de twee genen zou de boosdoener zijn? Uit ons onderzoek bleek dat CLK2 met of zonder de mutatie even goed z’n werk kan doen. Bovendien hebben we in slechts één familie met PCH7 variaties in CLK2 gevonden; variaties in TOE1 vonden we in alle tien families met PCH7 waarvan we dna tot onze beschikking hadden. dit maakt het heel waarschijnlijk dat mutaties in TOE1 de ziekte PCH7 veroorzaken. alle TOE1-patiënten met een X en een y chromosoom, wat dus gewoonlijk jongens zouden zijn, hebben een zekere vorm van dsd, wat zich uit in een geslachtsontwikkeling die tussen mannelijk en vrouwelijk in zit. Bij deze kinderen is het dus moeilijk om het geslacht vast te stellen op basis van hun externe genitalia. daarnaast vertonen alle TOE1-patiënten gelijkende hersenafwijkingen: naast een kleine pons en een klein cerebellum hebben ze een dunne verbinding tussen de twee hersenhelften (een dun corpus callosum) en grote ventrikels (met vocht gevulde holtes) in de hersenen.

in hoofdstuk 3 zoomen we verder in op een ander gemuteerd gen, namelijk EXOSC3. Mutaties in dit gen veroorzaken PCH subtype 1. We zien dat er verschillende mutaties kunnen optreden in het EXOSC3 gen. Zoals eerder gezegd codeert elk gen voor een eiwit. een genetische mutatie kan dus leiden tot een eiwit waarin een aminozuur is veranderd. de mutaties in EXOSC3 leiden ook tot aminozuurveranderingen, meer specifiek wordt de glycine op positie 31 van het eXosC3 eiwit een alanine (p.g31a) of de aspartaat op positie 132 wordt een alanine (p.d132a). Het blijkt dat patiënten met een p.g31a mutatie vaak contracturen hebben (afwijkende positie van gewrichten) en in hun eerste levensjaar al overlijden. Patiënten met een p.d132a mutatie hebben een iets langere levensduur (enkele patiënten tot in de puberteit) en kunnen soms zelfstandig kruipen of lopen. Bovendien is in deze groep de pons van normale grootte. dit is belangrijk om te weten, omdat clinici nu weten dat ook bij patiënten zonder verkleinde pons PCH een mogelijke diagnose kan zijn. door ons onderzoek naar de correlatie tussen genetische mutatie en klinische uitkomst weten families van PCH1 patiënten nu beter wat ze kunnen verwachten van het ziekteverloop.

in hoofdstuk 4 gaan we nog wat dieper in op een gemuteerd gen, in dit geval CLP1. We hebben een diermodel gemaakt voor PCH door zebravissen te kweken die het CLP1 gen missen. Het blijkt dat deze vissen een verstoorde ontwikkeling hebben van hun hersenen en motorneuronen en vroegtijdig doodgaan, vergelijkbaar met de PCH patiënten. Wanneer je bij deze vissen gezond mrna (het tussenproduct van gen naar

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eiwit) van CLP1 injecteert, vertonen de vissen deze symptomen in mindere mate, je kunt ze er als het ware een beetje beter mee maken. door injectie van mrna met de mutatie erin lukt dit niet, wat erop duidt dat de mutatie in CLP1 inderdaad PCH vero-orzaakt. Verder hebben we gekeken naar de gevolgen van een gemuteerd CLP1 gen op celniveau. Hierbij ontdekten we dat de mutatie ervoor zorgt dat er minder functionele trnas zijn, dat zijn moleculen die essentieel zijn bij eiwitsynthese. ook kan gemuteerd CLP1 minder goed naar de celkern getransporteerd worden dan gezond CLP1. een interessante bevinding is dat we deze effecten in neuronen (hersencellen) sterker zien dan in fibroblasten (huidcellen). dit klopt met wat we zien in PCH: hoewel de mutatie in elke cel van het lichaam zit, hebben de patiënten alleen een hersenziekte, de andere organen functioneren goed.

hoofdstuk 5 gaat over PCH op cel- en molecuulniveau. Zoals beschreven hebben we ontdekt dat mutaties in bepaalde genen leiden tot de ziekte PCH, maar hoe kan dat eigenlijk? Wat vindt er in hersenen van deze patiënten plaats wat maakt dat er hersencellen afsterven? Helaas kunnen we deze vraag nog niet beantwoorden, maar we hebben wel aanwijzingen in welke richting we moeten zoeken. Veel genen die PCH veroorzaken zijn betrokken bij de vorming van allerlei eiwitten. de hersenen hebben ontzettend veel eiwitten nodig in vergelijking met andere organen. als de productie hiervan is verstoord, zullen de hersenen daar dus als eerste last van hebben. in dit hoofdstuk bespreken we de hypothese dat een algemeen eiwittekort inderdaad de oorzaak is van PCH en zetten we uiteen welke cellulaire processen daaraan ten grondslag kunnen liggen.

Wat is eigenlijk het nut van dit onderzoek? Het is niet zo, dat als de genetische achter-grond van een ziekte bekend is, patiënten direct genezen kunnen worden. Toch is het kennen van de genetica achter een ziekte erg belangrijk. Ten eerste helpt het bij het diagnosticeren van een kind met PCH, wat erg belangrijk is voor de familie van PCH-patiënten. Zo heeft het onderzoek naar TOE1 mutaties veel duidelijkheid gebracht. Het was voor de ontdekking van TOE1 als PCH-veroorzakend gen nog niet duidelijk of PCH7 een aparte ziekte was, of dat de patiënten toevallig zowel brein- als genitale afwijkingen hadden. nu is aangetoond dat het één ziekte is, kan het ziekteverloop van deze patiënten in kaart worden gebracht.

een tweede gebruik van genetische kennis is counseling en prenatale diagnostiek. Wanneer de genetische mutatie in een familie met PCH bekend is, kan er bij een volgende (aankomende) zwangerschap gecounseld worden. de ouders kunnen dan een overwogen besluit maken om al dan niet in verwachting te raken. Wanneer de vrouw al zwanger is kan de foetus op PCH worden getest en behoort beëindiging van de zwangerschap tot de mogelijkheden.

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Ten derde is de identificatie van de genetische achtergrond van groot belang voor verder onderzoek naar PCH. Het vinden van een gen kan als een startpunt worden gezien: er zijn nu veel genen bekend die PCH veroorzaken, maar hoe de ziekte precies ontstaat, op cellulair en moleculair niveau, is nog onduidelijk. Voor sommige PCH subtypes is met dat onderzoek al een begin gemaakt, zo denken we dat een tekort aan algehele eiwitproductie een van de problemen is in PCH. als je weet wat er precies misgaat in de cel, weet je ook op welke punten in dat proces je eventueel zou kunnen interfereren. dit zijn punten die wellicht interessant zijn voor toekomstige medicijn-ontwikkeling. omdat PCH al vroeg in de foetale ontwikkeling begint, is het echter de vraag of volledige genezing ooit mogelijk zal zijn.

in dit proefschrift zijn meerdere nieuwe genen beschreven die PCH kunnen veroorza-ken. Hiermee levert het een bruikbare bijdrage aan snellere diagnose van PCH en beter inzicht in het verloop van deze ziekte.

Want in Mokum ben ik rijkEn gelukkig tegelijk- Johnny Jordaan

list Of authOrs

POrtfOliO

danKWOOrd

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list Of authOrs

mostafa abdellateefneurogenetics Laboratory, Howard Hughes Medical institute, department of neurosciences, University of California, san diego, Ca 92093, Usa

fesih aktar department of Pediatrics, diyarbakir state Hospital, 21100 diyarbakir, Turkey

Kimberly a aldinger Center for integrative Brain research, seattle Children’s research institute, seattle, Washington, Usa

umut altunoglu Medical genetics department, istanbul Medical faculty, is-tanbul University, Millet Caddesi, 34093 fatih/istanbul, Turkey

bart appelhof department of genome analysis, academic Medical Center, University of amsterdam, amsterdam, the netherlands

eleonora aronica department of (neuro)Pathology, academic Center, University of the netherlands, amsterdam, the netherlands

frank baas department of genome analysis, academic Medical Center, University of amsterdam, amsterdam, the netherlands

Peter g barth division of Pediatric neurology, emma’s Children’s Hospital, academic Medical Centre, amsterdam, the netherlands

Jonathan n berg division of Pathology and neuroscience, University of dundee, dundee, United Kingdom

Kaya bilguvar yale Program on neurogenetics, departments of neurosur-gery, neurobiology and genetics, yale University, school of Medicine, new Haven, Connecticut 06510, Usa.

caner caglar yale Program on neurogenetics, departments of neurosur-gery, neurobiology and genetics, yale University, school of Medicine, new Haven, Connecticut 06510, Usa.

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ahmet Okay caglayan yale Program on neurogenetics, departments of neurosur-gery, neurobiology and genetics, yale University, school of Medicine, new Haven, Connecticut 06510, Usa.

na cai neurogenetics Laboratory, Howard Hughes Medical institute, department of neurosciences, University of California, san diego, Ca 92093, Usa

huseyin caksen department of Pediatrics, Meram Medical school, necmettin erbakan University, 42080 Konya, Turkey

Vincent cantagrel (1)neurogenetics Laboratory, Howard Hughes Medical insti-tute, department of neurosciences, University of California, san diego, Ca 92093, Usa; (2) institut iMagine,  inserM U1163, faculté Paris-descartes, Paris, france

J leonie cazemier department of genome analysis, academic Medical Center, University of amsterdam, amsterdam, the netherlands

david chitayat department of Pediatrics, (1)division of Clinical and Meta-bolic genetics and (2)neuroimiging, sickkids Hospital and (3)The Prenatal diagnosis and Medical genetics Program, de-partment of obstetrics and gynecology, Mount sinai Hospital, University of Toronto, Toronto, ontario, Canada

nicole g coufal neurogenetics Laboratory, Howard Hughes Medical institute, department of neurosciences, University of California, san diego, Ca 92093, Usa

niklas darin department of Paediatrics, University of gothenburg, The Queen silvia’s Children Hospital, gothenburg, sweden

Justin h davies department of Paediatric Medicine, University Hospital southampton nHs foundation Trust, southampton

tessa van dijk department of genome analysis, academic Medical Center, University of amsterdam, amsterdam, the netherlands

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marit b dijns-de Wissel department of genome analysis, academic Medical Centre, amsterdam, the netherlands

abhijit dixit Clinical genetics, nottingham City Hospital, nottingham, United Kingdom

William b dobyns departments of Pediatrics and neurology, University of Wash-ington and Center for integrative Brain research, seattle Children’s research institute, seattle, Washington, Usa

Veerle rc eggens department of genome analysis, academic Medical Center, University of amsterdam, amsterdam, the netherlands

chistina fagerberg department of Clinical genetics, odense University Hospital, odense, denmark

Joel fluss Pediatric neurology, Children’s Hospital, geneva, switzerland

nicola foulds Wessex Clinical genetics services, University Hospital south-ampton nHs foundation Trust, southampton UK.

darren fowler Paediatric Pathology, University Hospital southampton nHs Trust, southampton, UK

mary louise freckmann department of Clinical genetics, The Canberra Hospital, Woden aCT 2606 australia

xiang-dong fu Cellular Molecular Medicine, University of California, san diego, Ca 92093, Usa.

stacey gabriel Broad institute of Harvard and Massachusetts institute of Technology, Cambridge, Ma 02142, Usa

Joseph g. gleeson neurogenetics Laboratory, Howard Hughes Medical institute, department of neurosciences, University of California, san diego, Ca 92093, Usa

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murat gunel yale Program on neurogenetics, departments of neurosur-gery, neurobiology and genetics, yale University, school of Medicine, new Haven, Connecticut 06510, Usa

tibor hortobágyi department of neuropathology, institute of Pathology, Uni-versity of debrecen, debrecen, Hungary

thomas Jacques neural development Unit, UCL institute of Child Health and the department of Histopathology, great ormond street Hospital for Children nHs foundation Trust, London, United Kingdom

Paul r Kasher department of genome analysis, academic Medical Center, University of amsterdam, amsterdam, the netherlands

hülya Kayserili (1)Medical genetics department, Koc University school of Medicine, sarıyer 34450 İstanbul Turkey (2) Medical genetics department, İstanbul Medical faculty, İstanbul University, fatih 34093 İstanbul Turkey

maria Kibaek department of Pediatric, odense University Hospital, odense, denmark

mary d King Paediatric neurology, Childrens University Hospital, Temple st. , dublin, ireland

Kanako Kurata department of Pediatrics, gunma University graduate school of Medicine, 3-39-22, showa-machi, Maebashi, gunma 371-8511, Japan

charles blm majoie department of radiology, academic Medical Center, amster-dam, the netherlands

Periklis makrythanasis department of genetic Medicine and development, Univer-sity of geneva, geneva, switzerland

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david manchester department of Pediatrics, Clinical genetics and Metabolism, University of Colorado school of Medicine, Children’s Hospital Colorado, aurora, Colorado, Usa

henk a marquering department of Biomedical engineering and Physics, aMC, amsterdam, the netherlands; department of radiology, aca-demic Medical Center, amsterdam, the netherlands

adrienn máté department of neurosurgery, University of szeged, szeged, Hungary

naomichi matsumoto department of Human genetics, yokohama City University graduate school of Medicine, 3-9 fukuura, Kanazawa-ku, yo-kohama 236-0004, Japan

mia t van meegen department of genome analysis, academic Medical Center, University of amsterdam, amsterdam, the netherlands

linda de meirleir Pediatric neurology and Metabolic diseases, Universi-tair Ziekenhuis Brussel (UZ Brussel), Vrije Universiteit Brussel (VUB), Brussels, Belgium

Kazuhiro muramatsu department of Pediatrics, gunma University graduate school of Medicine, 3-39-22, showa-machi, Maebashi, gunma 371-8511, Japan

yasmin namavar department of genome analysis, academic Medical Center, University of amsterdam, amsterdam, the netherlands

marie-cecile nassogne Pediatric neurology Unit, Cliniques Universitaires saint-Luc, Brussels, Belgium

James ar nicoll Clinical and experimental sciences, University of southamp-ton, southampton, UK

Jikke-mien f niermeijer division of Pediatric neurology, emma’s Children’s Hospital, academic Medical Centre, amsterdam, the netherlands

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Kazuhiro Ogata department of Biochemistry, yokohama City University gradu-ate school of Medicine, yokohama, 236-0004, Japan

declan O’rourke Paediatric neurology, Childrens University Hospital, Temple st. , dublin, ireland

bwee-tien Poll-the division of Pediatric neurology, emma’s Children’s Hospital, academic Medical Centre, amsterdam, the netherlands

sue Price Virtual academic Unit, Child development Centre, northamp-ton, northants, UK

andré reis institute of Human genetics, Universität erlangen-nürnberg, schwabachanlage 10, erlangen 91054, germany

miriam s reuter institute of Human genetics, Universität erlangen-nürnberg, schwabachanlage 10, erlangen 91054, germany

rasim Ozgur rosti neurogenetics Laboratory, Howard Hughes Medical institute, department of neurosciences, University of California, san diego, Ca 92093, Usa

fred van ruissen department of genome analysis, academic Medical Centre, amsterdam, the netherlands

hirotomo saitsu department of Human genetics, yokohama City University graduate school of Medicine, 3-9 fukuura, Kanazawa-ku, yo-kohama 236-0004, Japan

n bilge satkin Medical genetics department, istanbul Medical faculty, is-tanbul University, Millet Caddesi, 34093 fatih/istanbul, Turkey

ashleigh e schaffer neurogenetics Laboratory, Howard Hughes Medical institute, department of neurosciences, University of California, san diego, Ca 92093, Usa

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eric scott neurogenetics Laboratory, Howard Hughes Medical institute, department of neurosciences, University of California, san diego, Ca 92093, Usa

masaaki shiina department of Biochemistry, yokohama City University gradu-ate school of Medicine, yokohama, 236-0004, Japan

Jennifer l silhavy neurogenetics Laboratory, Howard Hughes Medical institute, department of neurosciences, University of California, san diego, Ca 92093, Usa

mohnish suri Clinical genetics, nottingham City Hospital, nottingham, United Kingdom

laszlo sztriha department of Paediatrics, University of szeged, szeged, Hungary

dirk troost department of (neuro)Pathology, academic Center, University of the netherlands, amsterdam, the netherlands

christopher trotta PTC Therapeutics, south Plainfield, nJ 07080, Usa

beyhan tuysuz department of Pediatric genetics, Cerrahpaşa Medical school, istanbul University, 34098 istanbul, Turkey

linda Warwick aCT genetic service, The Canberra Hospital, australia.

marian aJ Weterman department of genome analysis, academic Medical Center, University of amsterdam, amsterdam, the netherlands

andrew n Williams Virtual academic Unit, Child development Centre, northamp-ton, northants, UK

louise Wilson Clinical genetics, great ormond street Hospital, London, United Kingdom

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yuanchao xue Cellular Molecular Medicine, University of California, san diego, Ca 92093, Usa

cengiz yalcinkaya department of neurology, division of Child neurology, Cerrahpaşa Medical school, istanbul University, 34098 istan-bul, Turkey

Katsuhito yasuno yale Program on neurogenetics, departments of neurosur-gery, neurobiology and genetics, yale University, school of Medicine, new Haven, Connecticut 06510, Usa

christiane Zweier institute of Human genetics, Universität erlangen-nürnberg, schwabachanlage 10, erlangen 91054, germany

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Phd POrtfOliO

name: Veerle rosa Catelijne eggensPhd period: 2011-2016Promotor: prof. dr. f. BaasCo-promotor: prof. dr. B.T. Poll-The

courses

year Workload

Laboratory animal course (article 9) 2011 100 hours

Biostatistics 2012 40 hours

radiation protection level 5B 2012 48 hours

functional neuroanatomy (onWar) 2012 40 hours

degenerative diseases of the nervous system (onWar) 2012 40 hours

developmental neurobiology (onWar) 2012 50 hours

teaching

year Workload

lecturing

- Lectures for master students about laboratory animals 2014 2 hours

supervising

- Master student 2012 6 months

- Bachelor student 2012 4 months

- Master student 2013 6 months

- Master student 2014 8 months

Other- Labtours and mini-internships for high school students 2011-2015 several days

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conferences and seminarsyear Presentation

onWar Phd retreat, Zeist 2011 poster

genetics retreat rolduc, Kerkrade 2012 oral

american society of Human genetics, san francisco, Usa 2012 oral

onWar Phd retreat, Zeist 2012 poster

genetics retreat rolduc, Kerkrade 2013 oral

european society of Human genetics, Paris, france 2013 poster

onWar Phd retreat, Zeist 2013 poster

Pediatric neurological disorder with cerebellar involvement, rome, italy

2013 oral

gene translation: fidelity and quality control, Barcelona, spain 2013 poster

dutch society for Human genetics, arnhem 2013 poster

onWar Phd retreat, Zeist 2014 oral

genetics retreat rolduc, Kerkrade 2014 oral

trna Conference, Kyllini, greece 2014 oral

dutch society for Human genetics, arnhem 2014 oral

Joshua deeth foundation, London, england 2014 oral

Weekly seminars dept. of genome analysis 2011-2015 oral (6 times)

grants and Prizes

year

grants

eufishBioMed grant to do research on the institute for Translational neuroscience, University of sheffield, england

2011

awards and Prizes

Best oral presentation – 1st prize, dutch genetics retreat, 2012 2012

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list of publications

eggens VrC, Chitayat d, Kayserili H, foulds n, van dijk T, Muramatsu K, et al. Toe1 mutations cause pontocerebellar hypoplasia and disorders of sex development. Manuscript in preparation.

eggens Vr, Barth Pg, niermeijer JM, Berg Jn, darin n, dixit a, et al. eXosC3 mutations in pontocerebellar hypoplasia type 1: novel mutations and genotype-phenotype correlations. Orphanet Journal of Rare Disease. 9:23 (2014)

schaffer ae, eggens Vr, Caglayan ao, reuter Ms, scott e, Coufal ng, et al. CLP1 founder mutation links trna splicing and maturation to cerebellar development and neurodegeneration. Cell. 157:651-63 (2014)

eggens VrC, Barth Pg, Baas f. EXOSC3-related Pontocerebellar Hypoplasia. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong CT, Smith RJH, Stephens K, editors.  GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2014. (2014)

eggens VrC, Barth Pg, Baas f. Update on pontocerebellar hypoplasia: novel subtypes, genes and insights. In: Peadiatric neurological disorders with cerebellar involvement. Diagnosis and management. Mariani Foundation Peadiatric Neurology: 27 (2014)

namavar y, eggens VrC, Barth Pg, Baas f. TSEN54-related Pontocerebellar Hypoplasia .In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong CT, Smith RJH, Stephens K, editors. GeneReviews®[Internet]. Seattle (WA): University of Washington, Seattle; 1993-2014.2009 Sep 08 (updated 2013)

namavar y, Barth Pg, Kasher Pr, van ruissen f, Brockmann K, Bernert g, Writzl K, Ventura K, Cheng ey, ferriero dM, Basel-Vanagaite L, eggens Vr, et al. Clinical, neuroradiological and genetic findings in pontocerebellar hypoplasia. Brain. 134:143-56 (2011)

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danKWOOrd

Voor je ligt een boekje met daarin de studies en experimenten die tijdens mijn pro-motietraject geslaagd zijn. Zo lijkt het voor een buitenstaander misschien alsof je als onderzoeker aan de lopende band nieuwe genen voor zeldzame ziektes ontdekt. Van dag tot dag bestaat het leven van een promovendus echter uit zwemmen tussen lek-kende sds-Page gels, proeven doen met imaginaire rna-pellets (gewoon doorgaan!), vissen redden die uit hun aquarium zijn gesprongen, aio-nootjes halen bij de buren, en in vier jaar tijd ruim 16.000 km fietsen tussen het lab en thuis. Kortom, het was fantastisch!

in het bijzonder wil ik mijn promotor bedanken, prof. dr. frank Baas. frank, bedankt dat ik dit onderzoek in jouw lab mocht uitvoeren. dank je wel voor het creëren van een informele sfeer en je eeuwige vertrouwen in een goede afloop.

Mijn co-promotor, prof. dr. Bwee-Tien Poll-The, bedankt voor uw hulp en uw begelei-ding vanaf de klinische kant.

Prof. dr. Peter Barth, dank u voor uw enorme geduld, uw hulp en uw bevlogenheid.

een groot cohort aan collega’s heeft ervoor gezorgd dat ik een ontzettend leuke tijd heb gehad in het lab:

Bart en Tessa! Wat waren we een goed team! superfijn om alle mutatiemisère en vissenvreugde met elkaar te kunnen delen. Bart, gezellig dat jij de wetenschap ook zo emotioneel beleeft. dank je wel dat je mijn paranimf wil zijn! Tessa, bedankt voor je vrolijke lach, ik ben blij dat ik nog een congres met je heb meegemaakt!

Martin, wat tof om iemand in het lab te hebben die als het nodig is (en vooral als het niet nodig is) een gezichtje op een mandarijn tekent :). superbedankt voor alle pret! Jelly, dank je voor alle belachelijke anekdotes die je hebt laten ontstaan, en ook bedankt voor je serieuzere kant. dr. Mook! ik hoop dat als ik later zo oud ben als jij, ik ook nog steeds zo’n goed gevoel voor humor heb.

Judith, dank je voor het delen van beginnende-aio-onzekerheden, en voor het evalu-eren van de Boer-zoekt-vrouw-kalender. anna, many thanks for apple times and your company when working after six. nawal, iliana and Valeria, thank you for bringing Mediterranean vibes into the room! anna, wat leuk dat we nog wat labtijd hebben gedeeld! Bedankt voor je gehoepel en andere vrolijkheid.

150

Hyung, iets met zingen op vrijdag, printeronderdelen, maar ook een luisterend oor. Wat een figuur ben jij, bedankt daarvoor! Katja, dank je voor je rust, geduld en gezelligheid bij het op weg helpen van jonge aio’s en ook zeker daarna! Joeri, dank je voor de vele afleveringen van Kloneren met Joeri Both. yasmin, dank je wel voor je begeleiding bij de eerste stapjes in de wondere wereld van PCH, je hulp en je humor. nerd.

rob en anneloor, ik denk dat er zonder jullie geen enkele proef gelukt zou zijn, super-bedankt voor ontzettend veel hulp!

Leonie, dankzij je pijlsnelle vissenfotografie shinen onze beestjes nu in Cell, yeah! noortje, dank je voor de gezellige en nuttige TLC-uurtjes. Mirte, met jouw enthousi-asme en slimme kop red je het wel! dank je voor de goede samenwerking.

Marit, dank je voor je goede geheugen en gezang in het lab. susan, met jou wordt een Western Blotten een feest! Wiep, bedankt voor het theater in en buiten het lab. Jeroen, dank je voor je gevatte grappen. Maaike en Carin, dank jullie wel voor de goede zorgen voor de mens. Wim, dank je wel voor de goede zorgen voor de vis.

alle andere collega’s uit het lab, heel erg bedankt voor jullie hulp en gezelligheid: diana, Line, Céline, silvana, Marleen, Paul Kasher, Marian, fred, ruud, Kees, Lou, Patrick, Marja, Hamid, Jenny, rebecca, francesco, Mia, Linda, ferry, Coby, Lydia, Ted, et al.

ook buiten het lab zijn er veel mensen die de afgelopen jaren een belangrijke rol hebben gespeeld:

Brenda! Vriendinnetje, reismattie, buuf, aMC-collega, paranimf, dank je wel voor alle belevenissen de afgelopen jaren! de andere meisjes - Marja, shannon, stijntje, esther, Janneke, eva – ook al zijn we allemaal heel verschillend, het werkt goed samen in ons mini-ecosysteem. Laten we dat nog heel lang zo doen!

de neuro’s, wat een gezelligheid dat veel van ons gelijk opgaan met promoveren en dat we lekker kunnen klagen en jubelen onder het genot van wijn en kaas.

Céline, wat een briljant idee was het om ons in hetzelfde lab stage te laten lopen. Wij laten zien dat je met minimale input, maximale pret kunt hebben. dank je wel voor alle ontspanning! Merel, dank je voor je openheid en geen-gezeik-mentaliteit. dat is precies wat je af en toe nodig hebt. dorota, Marlies en renate, superbedankt voor alle België-tripjes met bonzende temporaalkwabben tot gevolg. Lau, wat hyggelig

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dat we elkaar steeds ergens in europa tegenkomen! Marcel, je weet het, een dag niet gespeedvact is een dag niet geleefd.

Papa en mama, bedankt dat ik van kleine Veer helemaal tot dr. eggens kon worden! finbar, Patricia en Julian, dank voor alle viscadeautjes! sietses familie, bedankt dat jul-lie zo meeleefden met de totstandkoming van mijn proefschrift. Hij is nu echt af hoor!

en natuurlijk sietse! Lieve sietse! onze homologie in nerdschap tussen theatermensen bleek een transcriptiefactor voor heel veel leuks. en het allerbeste is: dat gaat gewoon door nu we allebei het lab uit zijn. dank je voor het meemokken en meejuichen in dalen en pieken met wat ik deed, doe en zal doen. Cheers love, absolute cheers!