a complex microcephaly syndrome in a pakistani family associated with a novel missense mutation in...

Gene xxx (2014) xxx–xxx

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A complex microcephaly syndrome in a Pakistani family associatedwith a novel missense mutation in RBBP8 and a heterozygousdeletion in NRXN1

Zehra Agha a,b,1, Zafar Iqbal b,1, Maleeha Azam a, Maimoona Siddique c, Marjolein H. Willemsen b,Tjitske Kleefstra b, Christiane Zweier d, Nicole de Leeuw b, Raheel Qamar a,e, Hans van Bokhoven b,f,⁎a Department of Biosciences, Faculty of Science, COMSATS Institute of Information Technology, Islamabad, Pakistanb Department of Human Genetics, Nijmegen Centre for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlandsc Shifa International Hospital, Islamabad, Pakistand Institute of Human Genetics, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germanye Al-Nafees Medical College & Hospital, Isra University, Islamabad, Pakistanf Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behavior, Nijmegen, The Netherlands

Abbreviations: ATR, ataxia–telangiectasia and RAD3-CBC, complete blood count; CNV, copy number variationDSB, Double-strand break; EGF, epidermal growth factormegabase; MIM, Mendelian Inheritance in Man; MRI,MRN, MRE11-RAD50-NBS1; NRXN1, neurexin 1; OFC, ocence; qPCR, quantitative polymerase chain reaction; Rprotein 8; SCKL, Seckel syndrome; SD, standarddeviation;phism; WMD, white matter disease.⁎ Corresponding author at: Department of Human Gen

Molecular Life Sciences, Radboud University Medical CNijmegen, The Netherlands. Tel.: +31 24 3616696; fax: +

E-mail address: [email protected] (1 The first two authors have contributed equally to this

0378-1119/$ – see front matter © 2014 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.gene.2014.01.027

Please cite this article as: Agha, Z., et al., A coRBBP8 and a heterozygous deletion in NRXN

a b s t r a c t
a r t i c l e i n f o
Article history:Accepted 8 January 2014Available online xxx

Keywords:Human congenital microcephaly syndromeSeckel syndromeJawad syndromeRBBP8Psychiatric disordersNRXN1Intellectual disability

We report on a consanguineous Pakistani family with a severe congenital microcephaly syndrome resemblingthe Seckel syndrome and Jawad syndrome. The affected individuals in this family were born to consanguineousparents of whom the mother presented with mild intellectual disability (ID), epilepsy and diabetes mellitus.The two living affected brothers presented with microcephaly, white matter disease of the brain, hyponychia,dysmorphic facial features with synophrys, epilepsy, diabetes mellitus and ID. Genotyping with a 250 K SNParray in both affected brothers revealed an 18 MB homozygous region on chromosome 18p11.21-q12.1encompassing the SCKL2 locus of the Seckel and Jawad syndromes. Sequencing of the RBBP8 gene, underlyingthe Seckel and Jawad syndromes, identified the novel mutation c.919A N G, p.Arg307Gly, segregating in a reces-sive manner in the family. In addition, in the two affected brothers and their mother we have also found a het-erozygous 607 kb deletion, encompassing exons 13–19 of NRXN1. Bidirectional sequencing of the coding exonsof NRXN1 did not reveal any other mutation on the other allele. It thus appears that the phenotype of the mildlyaffected mother can be explained by the NRXN1 deletion, whereas the more severe and complex microcephalicphenotype of the two affected brothers is due to the simultaneous deletion in NRXN1 and the homozygousmissense mutation affecting RBBP8.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

The etiology of intellectual disability (ID) is highly heterogeneous.Mutations in over 450 genes have been implicated in ID, which only rep-resents a minor part of all ID genes that have been predicted (Inlow andRestifo, 2004; Schuurs-Hoeijmakers et al., 2011; van Bokhoven, 2011).The sheer number of ID genes presents a challenge for identification of

related; BMI, Body mass index;; DDR, DNA-damage response;; ID, intellectual disability; Mb,magnetic resonance imaging;cipital–frontal head circumfer-BBP8, retinoblastoma-bindingSNP, single nucleotidepolymor-

etics 855, Nijmegen Centre forenter, P.O. Box 9101, 6500 HB31 24 3668752.

H. van Bokhoven).work.

ights reserved.

mplex microcephaly syndrom1, Gene (2014), http://dx.doi

the primary genetic defect in individual families and isolated cases. Alsoin their clinical presentation, ID disorders show extensive phenotypicvariability. A plethora of associated features can be seen in most individ-uals presenting with ID. Facial dysmorphism and neurological features,such as autism, epilepsy, attention deficit hyperactivity disorder and be-havioral anomalies are particularly common. These associated featurescan be highly characteristic for a specific syndrome caused by mutationsin a particular gene. However, a large degree of clinical variability is oftenseen formutations in a specific gene,which can hamper clinical diagnosisin isolated patients and small families.

Microcephaly is seen in a variety of syndromes comprising ID. Thehead circumference in microcephaly is reduced three standard devia-tions below the age- and sex-related means and reflects the reductionin underlying brain volume (Woods et al., 2005).Microcephaly associat-ed syndromes differ from each other on the basis of other clinicalmanifestations (Woods et al., 1992, 2005). The Seckel syndrome (MIM210600), Filippi syndrome (MIM 272440) and Jawad syndrome (MIM251255) are three separate syndromes in which microcephaly, ID,short stature and digital malformations are common hallmarks.

e in a Pakistani family associated with a novel missense mutation in.org/10.1016/j.gene.2014.01.027

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2 Z. Agha et al. / Gene xxx (2014) xxx–xxx

Qvist et al. recently reported RBBP8 (MIM 604124), as the causativegene for both the Jawad syndrome and Seckel syndrome (Qvist et al.,2011). This gene is located on chromosome 18q11.2 and encodes theubiquitously expressed retinoblastoma-binding protein 8, a nuclearprotein that is involved in the regulation of cell proliferation. It is a con-served DNA repair protein that facilitates DNA end re-sectioning in thedouble-strand break (DSB) repair pathway (D'Andrea, 2010).

Another important ID associated gene is NRXN1 (neurexin 1, MIM600565), which is one of the largest human genes (1.1 Mb). It codesfor a neuronal cell adhesion molecule, and is located on chromosome2p16.3 (Boucard et al., 2005). Recent data suggest that heterozygousmutations in NRXN1 represent a susceptibility factor for a broad spec-trum of neurological and neuropsychological disorders for example,schizophrenia (Ching et al., 2010; Kirov et al., 2008; Rujescu et al.,2009), ID disorders (McIntosh et al., 2006; Zahir et al., 2008; Zweieret al., 2009), autism, (Gregor et al., 2011) and various other neuropsy-chiatric disorders (Ching et al., 2010). A number of studies reveal reces-sive genetic defects inNRXN1 associatedwith severe ID and dysmorphicfeatures resembling the Pitt–Hopkins like syndrome in some cases(Duong et al., 2012; Harrison et al., 2011; Zweier et al., 2009).

In the current study, we have identified a homozygous mutation inRBBP8, which co-segregateswithmicrocephaly-associated ID syndromein the Pakistani family (MRQ12). In addition, we have also identified aheterozygous deletion encompassing the NRXN1 in this family, whichis present in two affected sibs with a complex phenotype, as well asthe mother with a mild phenotype.

2. Methods

2.1. Clinical analysis

The proband of MRQ12 was ascertained at the National TrainingCentre for Special Persons, Islamabad, Pakistan, where he wasreferred for evaluation of ID and microcephaly. Informed writtenconsent was obtained from participants to this study or their

Fig. 1. Pedigree of MRQ12. The filled squares are affected males and the unfilled circles and sqdifferent and mild phenotype. The small sized square with diagonal line represents prenatal dindicates the proband. The symbols R+/M, RM/M represent the RBBP8 unaffected carriers and hoand normal copy number of NRXN1, respectively.

Please cite this article as: Agha, Z., et al., A complex microcephaly syndromRBBP8 and a heterozygous deletion in NRXN1, Gene (2014), http://dx.doi

guardians prior to blood sampling. The study has been approved bythe Ethics Review Committee of COMSATS Institute of InformationTechnology, Islamabad, Pakistan and Radboud University, Nijmegen,The Netherlands.

FamilyMRQ12 is a consanguineous Pakistani family (Fig. 1) from thenortheastern region of Pakistan. At the age of 72 years, the mother pre-sented with mild learning disabilities and epilepsy. She also sufferedfrom insulin dependent diabetes mellitus since the age of 22 years.The father had normal intellectual functioning throughout his life. Atthe age of 76 years he had symptoms of Parkinson's disease but withno family history of the disease. The mother gave birth to six children,and one pregnancy was spontaneously terminated at four months dueto growth arrest of themale fetus. Four of the six childrenwere intellec-tually normal without any congenital abnormality. One of her sons(IV:2: Fig. 1) died at the age of 18 years from leukemia but he did nothave ID nor any psychiatric problems. The two youngest sons (IV:5and IV:6; Fig. 1) suffered from microcephaly-associated ID syndrome.

The patient IV:5 (proband: Fig. 2a) was born after 40 weeks of ges-tation of an uneventful pregnancy. Microcephaly was noted at birthbut detailed clinical data of birth weight and height of the probandand the other affected brother were not available. The proband startedwalking at the age of 11 months and speaking at the age of four years.At three months of age his intellectual weakness was noted due to de-layed milestones, at age 10 years he had symptoms of progressivehyponychia (Fig. 2b). Upon clinical evaluation at the age of 40 yearsthe proband (Fig. 2a) had relatively short stature and obesity (height162 cm, weight 76 kg) while his BMI was 27.8 (average BMI forPakistani adults is 18.8 ± 3.6). He had symmetrical microcephaly witha head circumference of 46 cm b −2 SD for occipital–frontal headcircumference (OFC). He also had dysmorphic facial features withsynophrys, a prominent irregular shaped long nose and a short philtrum.He also had deep set eyes and bilateral convergent strabismus. His earswere rotated posteriorly and he had a highly pitched and shrill voice.On his fingers and toes complete hyponychia was observed, and hesuffered from hyperhidrosis and could not count or write. His gait was

uares are unaffected females and males, respectively. The circle filled in gray indicates theeath of the fetus. Symbols with a diagonal line represent deceased individuals. The arrowmozygousmutants, respectively. Ndel/+ and N+/+reflect the NRXN1 heterozygous deletion



Fig. 2. Photographs showing the clinical features of the patients. (a) Frontal picture of 40-year old proband. The figure shows the synophrys and clearly depicts the deeply set eyes of theproband. The proband showsmicrocephaly, squinting of the eyes and a prominent nose on an elongated face. The ears of the patient are projected backward. (b) Picture of the hand of the40-year old proband showing complete hyponychia of the finger nails. (c) Frontal picture of the 39-year old brother of the proband.

3Z. Agha et al. / Gene xxx (2014) xxx–xxx

normal and he could visualize things normally. Speech was normal, buthis comprehension was poor. Cardiologic evaluation, metabolic screens,complete blood count (CBC) and liver function tests were all normal,while his brain MRI revealed accentuated white matter of the cerebrumand the cerebellum (Fig. 3), suggestive of an underlying white matterdisease (WMD) of the brain. Otherwise the ventricle and cortical sulciwere normal with no focal mass lesion or midline shift and no abnormalcollection or hemorrhage.

Patient IV:6, the proband's 39 years old affected brother (Fig. 2c),had a similar phenotype as his brother, including obesity and shortstature (height 156 cm, and weight 69 kg) while his BMI was 25.1 (aver-age BMI at adult age for Pakistani population is 18.8 ± 3.6). He had mi-crocephaly with a head circumference of 47 cm b −2 SD OFC. He hadsynophrys and his eyes were deeply set but he did not have a squint.His nose was large, prominent and irregularly shaped, while his earswere rotated posteriorly. His voice was high pitched and shrill, he hadcomplete hyponychia of fingers and toes, had hyperhidrosis since anearly age and also had seizures. His EEG was not recorded hence the ep-ileptic seizures cannot be diagnosed. Intellectually he was weaker thanhis brother, he could speak only a fewwords and hewas very aggressive.

2.2. Genetic analysis

Genomic DNAwas extracted from the peripheral blood of the partic-ipating familymembers (III:1, III:2, IV:1, IV:5 and IV:6), using a standardphenol–chloroform extraction method (Sambrook and Russell, 2006).Genotyping of the affected members (IV:5 and IV:6), their parents(III:1 and III:2) and a healthy sib (IV:1) of the family MRQ12 was per-formed using Affymetrix 250 K single nucleotide polymorphisms(SNP) microarray (Affymetrix Inc. Santa Clara, CA) in order to obtainthe copy number variation (CNV) data while the HomozygosityMapper

Fig. 3.Magnetic resonance imaging (MRI) of the family members. (a) Normal brain, (b, c) Turbrows in the figures are pointing toward the area of the brain where abnormal white matter depreader is referred to the web version of this article.)


(http://www.homozygositymapper.org/) was used to find commonhomozygous regions among affected family members. The 250 K dataalso revealed heterozygous deletion in the affectedmembers of MRQ12.

Genomic real-time qPCR analysis was performed for family mem-bers III:1, III:2, IV:1, IV:5 and IV:6 on a 7500 Fast Real-Time PCR System(Applied Biosystems, Foster City, CA, US) by using Power SYBR GreenPCR Master Mix (Applied Biosystems, Foster City, CA, US) according tothe manufacturer's instructions. The melting curves of all PCR productsshowed a single PCR product, while the controls were negative. Copynumbers were measured relative to the reference gene cystic fibrosistransmembrane conductance regulator (CFTR).

The coding exons (2–19) of RBBP8, and (2–24) of NRXN1 of theproband and familymemberswere screened by bidirectional sequencingusing ABI-PRISM 3730 (Life Technologies, Foster City, CA) as describedpreviously (Gregor et al., 2011).

3. Results

Genotyping of III:1, III:2, IV:1, IV:5 and IV:6 family members ofMRQ12 by 250 K SNP array revealed four homozygous regions i.e. Chr.6(3.15 Mb); Chr.7 (5.51 Mb); Chr.10 (3.63 Mb) and Chr.18 (17.71 Mb) intwo affected IV:5 and IV:6 while these regions were not homozygous intheir healthy sister IV:1 nor in the parents III:1 and III:2. The other livinghealthy siblings (IV:4 and IV:7)were unavailable for testing. The region atchromosome 18p21.2–q12.2 with flanking SNPs rs8060649 andrs16954273 contained the RBBP8, which has been previously reportedto be mutated in both the Seckel and Jawad syndromes (Qvist et al.,2011). In order to screen for a mutation in the patients of familyMRQ12, we sequenced all the coding exons of RBBP8 and found a homo-zygous missense change (c.919A N G, p.Arg307Gly) in exon 11 (Fig. 4).The substitution co-segregatedwith the phenotype in a recessivemanner

o FLAIR region of the brain of MRQ12's proband showing diffuse white matter. The red ar-osition can be seen. (For interpretation of the references to color in this figure legend, the



Fig. 4. Sequencing chromatograms. Theupper chromatogram represents theheterozygouschange c.919A N G in the unaffected father while the lower chromatogram shows the ho-mozygous change c.919A N G in the affected proband.


(Fig. 1) in the two sons (IV:5 and IV:6) with the severe phenotype, whilethemother (III:2), father (III:1) and healthy daughter (IV:1)were hetero-zygous for the substitution. This RBBP8 variant was not seen in N13,000alleles in the Exome Variant Server database (http://evs.gs.washington.edu/EVS/) [August 2013].

The 250 K array CNV data also revealed a 610 kb deletion in2p16.3 (Chr2:50.21–50.82 Mb UCSC Human Genome Browser build19; Fig. 5), resulting in a heterozygous deletion of exon 13 to exon 19of NRXN1, which was confirmed by qPCR. The deletion was inheritedby the severely affected sons (IV:5 and IV:6) in a dominant mannerfrom the mother (III:2), who was suffering from mild ID and epilepsy,while both the father (III:1) and daughter (IV:1) had normal copy num-ber of NRXN1. No mutation on the other allele of NRXN1 was detectedfrom the paternal chromosome.


4. Discussion

RBBP8 point mutations have previously been reported in the Seckelsyndrome and Jawad syndrome (Qvist et al., 2011). The Seckel syn-drome belongs to the group of genome instability disorders, collectivelyreferred to as DNA-damage response (DDR) and repair defectivesyndromes (O'Driscoll et al., 2003). While, cancer predisposition isoften associated with such syndromes, only a few cancers have been re-ported for the Seckel syndrome patients. Instead, the Seckel syndromepathogenesis is primarily based on marked growth and neurologicalimpairments (O'Driscoll et al., 2003). The Jawad syndrome is anotherautosomal recessive congenital microcephaly syndrome that sharesthe SCKL2 locus with the Seckel syndrome (Hassan et al., 2008).

The Seckel syndrome also known as dwarfism syndrome has variousdysmorphic facial features such as bird shaped appearance that is adiagnostic feature of this syndrome. The syndrome is usually character-ized by intrauterine growth retardation and postnatal microcephalyand dwarfism associated with ID (Borglum et al., 2001; Filippi, 1985;Goodship et al., 2000; Kilinc et al., 2003; Sharif and Donnai, 2004),while the Jawad syndrome is characterized by microcephaly, growthretardation, café au lait white pigmentation on the skin, congenitalanonychia and digital malformation (Hassan et al., 2008).

For the Seckel syndrome five different forms have been identified,i.e. SCKL1, characterized by amutation that creates an alternative splicesite in the ATR (ataxia telangiectasia and Rad3 related; MIM 601215)(Borglum et al., 2001), SCKL2 (MIM 606744) mapping to 18p11.31–q11.2 (O'Driscoll et al., 2003), and SCKL3 (MIM 608664), mappedto 14q23–q24 (Goodship et al., 2000). SCKL4 (MIM 613676) maps to13q12.2 and is caused by recessivemutations in the CENPJ (centromericprotein J, MIM 609279) (Griffith et al., 2008), and SCKL5 (MIM 613823)is caused by mutations in the CEP152 (centrosomal protein 152-KD,MIM 613529), which maps to 15q21 (Kalay et al., 2011).

The affected males in family MRQ12 shared the bird shaped appear-ance of the Seckel syndrome andmicrocephalywith both the Jawad andSeckel syndromes, while congenital anonychia is shared by the Jawadsyndrome and the MRQ12 family. ID and microcephaly are commonfeatures shared between the Seckel syndrome, Jawad syndrome andthe present family (Table 1). Microcephaly is thought to result fromreduced proliferative potential in the developing nervous system,most likely due to increased cell death of the neuronal stem cells or pro-genitor cells in the rapidly expanding fetal brain (O'Driscoll and Jeggo,2008).

RBBP8 has been observed towork in DSB repair processing as licens-ing of DNA-end resection that requires cell-cycle dependent phosphor-ylation of RBBP8, and in the absence of this protein, DSB processing isimpaired and the activation of ATR, which is a DNA damage responsiveprotein, is blocked (Qvist et al., 2011). Based on these observations,RBBP8was screened by Qvist et al. in two families, one with the Seckelsyndrome and the other with the Jawad syndrome. Bothwere previous-ly linked to the SCKL2 locus at 18p11.31–q11.2 (Qvist et al., 2011). Theseanalyses revealed two independent homozygous mutations in RBBP8, anintronic mutation in the Seckel syndrome leading to an alternativelyspliced transcript and the appearance of a premature stop codonpredicting a loss-of-function allele due to nonsense-mediated RNAdecay and/or the generation of a C-terminally truncated form of RBBP8.While in the Jawad syndrome there was a two base pair deletion inexon 11, causing a frame-shift and the appearance of a premature stopcodon, either way, a complete loss of RBBP8 activity would be predictedaffecting DSB resection and ATR activation (Qvist et al., 2011). The ATRprotein orchestrates cellular responses to DNA damage and replicationstress. Complete loss of ATR function thus leads to chromosomal instabil-ity and cell death (Fang et al., 2004).

In our study,we have identified a novel homozygousmutation in theRBBP8 gene(c.919ANG) that predicts an amino acid substitutionp.Arg307Gly. The arginine at this position is highly conserved. Thechange of a large positively charged arginine by a small and highly



Fig. 5. NRXN1 deletion region of family MRQ12. (a) Original data of CNV analysis derived from 250 k Affymetrix SNP array of proband, showing heterozygous deletion in the indicatedregion (chr2:502, 10–508, 17 Mb UCSC Human Genome Browser version 19). (b) Figure showing the NRXN1 exons from 1–24 with its two isoforms, alpha-isoform and beta-isoform,it represents the 607 kb microdeletion in family MRQ12 covering exons 13 to 19 coding for both isoforms i.e. part of alpha and promoter of beta.

5Z. Agha et al. / Gene xxx (2014) xxx–xxx

flexible glycine residue is significant and generally predicts the creationof instability due to the increased flexibility. Arg307 is not located in aknown functional domain and there is no crystal structure availablefor RBBP protein family members to make further predictions aboutthe functional consequences of this mutation.

Inactivating mutations of RBBP8 are expected to impair the capacityof cells to respond optimally to endogenously arising DNA damage. Thiswould lower the apoptotic threshold of the cells and cause reduction intheir proliferative potential, which can cause retardation in the growthof the patient (Qvist et al., 2011). Our results are in agreement withthe findings of Qvist et al. (2011) as our mutation is also located in theC-terminal part of the RBBP8 protein, which could result in the produc-tion of an abnormal protein. Since RBBP8 acts together with the MRN(MRE11-RAD50-NBS1) complex to promote DNA end resection andthe generation of single-stranded DNA, this function could be impaired,and this is critically important for the homologous recombination repair(Yuan and Chen, 2009). Based on these factors we also propose that themutation we have identified in MRQ12 is responsible for the severephenotype of the two sibs as this region bears an MRN interactiondomain (Sartori et al., 2007) that is crucial for DNA-end resection.Although a second MRN interaction point has been found in theN-terminal part of the protein (Yuan and Chen, 2009), the C-terminalregion is essential for RBBP8-mediated activation of MRN-associatednuclease activity. Therefore it is probable that this mutation can render

Table 1Comparison of clinical and morphometric findings in the Seckel (SCKL2), Jawad and MRQ12 pa

Seckel (SCKL2) Jawad

Appearance of symptoms Infancy InfancBirth weight (kg) Low (1 to 1.5) Data nHeight (SD) Reduced (−3.5 to−5.5) NormaHead circumference (SD) Reduced (−4.7 to−5.0) ReducFacial characteristics Narrow, not receding, forehead and

prominent noses, small craniumsSharplnoses,

Global developmental delay Mild ModerSkin abnormalities Café au lait spots Café auDigital malformation Phalangeal joint swellings, clinodactyly Phalan

polydaVoice Normal Norma


theMRN-RBBP8 complex ineffective and cause the impaired productionof single stranded DNA. This results in the inactivation of ATR that ulti-mately cause defective apoptosis activity in patients due to hypersensi-tivity to DNA damage.

In addition to the RBBP8mutationwe identified an intragenic 610 kbdeletion comprising exons 13–19 of NRXN1, including a promoterwhich is used to generate β-neurexin of NRXN1. A number of studieshave shown that neurexins have an essential role in the developmentof synapse, not in its initial adhesion but in the recruitment ofmolecularcomponents and its maturation (Zhang et al., 2005). There are threeneurexin genes in mammals, which have the capacity to generate astunning variety of distinct transcripts by using alternate promoters,splice sites and exons (Rowen et al., 2002; Tabuchi and Sudhof, 2002).For NRXN1, most transcripts use the upstream promoter and encodealpha-neurexin isoforms; fewer transcripts are produced from thedownstream promoter yielding the beta-neurexin isoforms. Alpha-neurexins contain epidermal growth factor-like (EGF-like) sequencesand laminin G domains, and they interact with neurexophilins. Beta-neurexins lack EGF-like sequences and contain fewer laminin G do-mains than alpha-neurexins (Kirov et al., 2008). Interestingly, for eachof the three Neurexin genes, knockout of the α-neurexins, leaving theβ-neurexins intact, leads to perinatal lethality due to the loss of presyn-aptic Ca2+ channel function (Missler et al., 2003). Different studies haveshown that α-neurexins have a unique function that is not provided

tients.

MRQ12

y Infancyot available Data not availablel Reduceded (−5.0 to −7.0) Reducedy receding foreheads, prominentsmall craniums

Narrow, bird shaped, deeply set eyes,squint, prominent nose, hypertelorism,low ears

ate to severe Moderate–severelait-like spots of white appearance Hyperhydrosisgeal joint swellings, clinodactyly,ctyly, syndactyly, total absence of nails

Anonychia congenita

l Shrilled, high pitched




by β-neurexins. Moreover, a patient with mild ID and autistic featuresreported by Zahir et al. (2008) had a heterozygous deletion that specif-ically affected NRXN1-α, leaving NRXN1-β intact. The heterozygous lossof exon 13 to exon 19 ofα-neurexin, aswell as deletion of the promoterfor β-neurexin is likely to be causative for themild ID of themother. TheNRXN1 deletion may also explain her epilepsy, since heterozygousexon-disrupting deletions ofNRXN1 have been shown to represent a ge-netic risk factor common in idiopathic generalized epilepsy (Molleret al., 2013). Moreover, epilepsy is seen in 43% of NRXN1 deletioncarriers (Bena et al., 2013). From the current work the etiology of diabe-tes mellitus in the mother is not clear. Interestingly in a recent study afamily was reported in which a heterozygous deletion comprising theupstream promoter and first intron of NRXN1 was seen in a patientwith diabetes mellitus (Duong et al., 2012). In addition, it is of notethat the neurexins are expressed in theβ-cells of the pancreas, thereforeany aberrations in the protein could potentially lead to a decrease infunction of the pancreas (Suckow et al., 2008). Together with our obser-vations, these data warrant further investigation of the involvement ofNRXN1 in diabetes. Finally, it has been shown that a NRXN1 polymor-phism was associated with white matter abnormalities in autismand schizophrenia (Voineskos et al., 2011), suggesting that the NRXN1deletion might also contribute to the white matter anomalies thatwere observed in the two affected males.

5. Conclusion

In conclusion, we propose that the severe Seckel/Jawad syndrome-like phenotype of the two brothers in MRQ12 is attributed to the homo-zygous RBBP8 missense mutation combined with a heterozygous exonicdeletion of NRXN1. Their mother is comparatively less affected and hasmild learning disability and epilepsy that can be explained by a NRXN1deletion. This deletion could be a contributing factor to the mild ID anddiabetesmellitus of themother. Clinical variability is typical for heterozy-gous NRXN1 deletions, suggesting modulation by other genetic factors. Itis also conceivable that modifier loci are linked to genes such as RBBP8that have a role in neurodevelopment.

Conflict of interest

The authors state that they have no conflict of interest.

Acknowledgments

We are grateful to all the family members for their participation inthis study. We thank Dr. Hanka Venselaar for helpful comments aboutthe predicted effect of the amino acid change. This study was funded bythe European Union's Seventh Framework Program under grant agree-ment number 241995, project GENCODYS and grant agreement number223143, project TECHGENE. Zehra Agha was supported by the IRSIP pro-gramof theHigher Education Commission (HEC), Islamabad, Pakistan. CZwas funded by a grant from the Deutsche Forschungsgemeinschaft(Zw184/1-1). This study was also supported by funds to RQ from theHEC National Research Program for Universities under grant no 2155. ZIwas supported by HEC.

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a complex microcephaly syndrome in a pakistani family associated with a novel missense mutation in...

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