REPORT

Biallelic Truncating Mutations in FMN2, Encodingthe Actin-Regulatory Protein Formin 2, CauseNonsyndromic Autosomal-Recessive Intellectual Disability

Rosalind Law,1,15 Tracy Dixon-Salazar,2,3,15 Julie Jerber,2,3 Na Cai,2,3 Ansar A. Abbasi,4 Maha S. Zaki,5

Kirti Mittal,1 Stacey B. Gabriel,6 Muhammad Arshad Rafiq,1 Valeed Khan,7 Maria Nguyen,2,3

Ghazanfar Ali,8 Brett Copeland,2,3 Eric Scott,2,3 Nasim Vasli,1 Anna Mikhailov,1

Muhammad Nasim Khan,8 Danielle M. Andrade,9,10 Muhammad Ayaz,11 Muhammad Ansar,7

Muhammad Ayub,11,12 John B. Vincent,1,13,14,* and Joseph G. Gleeson2,3,*

Dendritic spines represent the major site of neuronal activity in the brain; they serve as the receiving point for neurotransmitters and

undergo rapid activity-dependent morphological changes that correlate with learning and memory. Using a combination of homozy-

gosity mapping and next-generation sequencing in two consanguineous families affected by nonsyndromic autosomal-recessive intel-

lectual disability, we identified truncating mutations in formin 2 (FMN2), encoding a protein that belongs to the formin family of actin

cytoskeleton nucleation factors and is highly expressed in the maturing brain.We found that FMN2 localizes to punctae along dendrites

and that germline inactivation of mouse Fmn2 resulted in animals with decreased spine density; such mice were previously demon-

strated to have a conditioned fear-learning defect. Furthermore, patient neural cells derived from induced pluripotent stem cells showed

correlated decreased synaptic density. Thus, FMN2 mutations link intellectual disability either directly or indirectly to the regulation of

actin-mediated synaptic spine density.

Intellectual disability (ID), or early cognitive impairment,

is a common neurodevelopmental disorder that is esti-

mated to affect >1% of the population.1 Globally, ID re-

mains a healthcare challenge as well as a socioeconomic

burden in both developing and developed countries, and

it has immense effects on individuals and their families.

Although ID is defined by cognitive deficits (IQ < 70)

and limitations in adaptive behaviors (ICD-10), cases can

also be classified according to accompanying radiologic,

morphologic, and/or metabolic syndromes. When clas-

sification is made on the basis of such accompanying

syndromes, the disorder is known as syndromic ID (S-

ID); in the absence of other accompanying syndromes, it

is known as nonsyndromic ID (NS-ID [MIM 249500]).

Genetic heterogeneity, mode-of-inheritance heterogene-

ity, and gene-gene or gene-environment interactions in

NS-ID have made identification of causes difficult, but

more than 50 genes are now known to be mutated in a

biallelic fashion in autosomal-recessive ID (AR-ID).2,3

Accumulating evidence suggests that impaired synapse

formation and synaptic plasticity might underlie ID:

neuropathological examination of brains from affected

individuals consistently shows abnormalities in the den-

dritic and synaptic organization of the cortex.4 Further-

1The Campbell Family Mental Health Research Institute, The Centre for Addict

Neuroscience, University of California, San Diego, San Diego, CA 92093, USA;

Azad Jammu and Kashmir, 13100 Muzaffarabad, Pakistan; 5Clinical Genetics

Research Centre, Cairo 12311, Egypt; 6Broad Institute of Harvard and Massac

of Biochemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan; 8Depart

zaffarabad, Pakistan; 9Division of Neurology, Department of Medicine, Univers

Centre, Toronto Western Research Institute, Toronto, Ontario M5S 2J7, Canada12Department of Psychiatry, Queen’s University, Kingston, Ontario K7L 3N6, C

M5T 1R8, Canada; 14Institute of Medical Science, University of Toronto, Toro15These authors contributed equally to this work

*Correspondence: john.vincent@camh.ca (J.B.V.), jogleeson@rockefeller.edu (J

http://dx.doi.org/10.1016/j.ajhg.2014.10.016. �2014 by The American Societ

The American

more, many of the NS-ID encoded proteins localize at or

near neuronal synapses, and most animal models show

defective synaptic structure or function. For instance, syn-

aptic spine morphological defects are linked with inactiva-

tion of murine Fmr1.5

Synaptogenesis initiates during human fetal life, peaks in

childhood, reaches a steady state in adolescence6 and for

some brain regions such as the hippocampus, continues

into adulthood. Modeling neurodevelopmental disease

with human induced pluripotent stem cells (hIPSCs) has

shown neuronal structural defects, including alterations in

synaptic density. For example, hIPSC ID models, including

those involving CDKL5 (MIM 300203) mutations, MECP2

(MIM 300005) mutations, and Down syndrome (MIM

190685), show reduced synaptic density,7–9 consistent

with defects in synapses as an underlying substrate.

In this study, we performed homozygosity mapping and

linkage analysis, followed by next-generation sequencing

in two independent, consanguineous ID-affected families

from Pakistan and Egypt. Participants were part of larger

cohorts originating from the Consortium for the Study

of Autosomal-Recessive Intellectual Disabilities (CARID).

These cohorts consisted of approximately 2,000 families,

most with documented parental consanguinity and two

ion andMental Health, Toronto, Ontario M5T 1R8, Canada; 2Department of3Howard Hughes Medical Institute; 4Department of Zoology, University of

Department, Human Genetics and Genome Research Division, National

husetts Institute of Technology, Cambridge, MA 02142, USA; 7Department

ment of Biotechnology, University of Azad Jammu and Kashmir, 13100 Mu-

ity of Toronto, Toronto, Ontario M5S 2J7, Canada; 10Krembil Neuroscience

; 11Lahore Institute of Research and Development, Lahore 51000, Pakistan;

anada; 13Department of Psychiatry, University of Toronto, Toronto, Ontario

nto, Ontario M5S 1A8, Canada

.G.G.)

y of Human Genetics. All rights reserved.

Journal of Human Genetics 95, 721–728, December 4, 2014 721

or more affected members with moderate to severe NS-ID,

presumed recessive in nature; exome sequencing was per-

formed or is being performed on most of these families.

Participants with major dysmorphism, structural brain

defects, syndromic features, metabolic derangements, or

demonstrable cytogenetic abnormalities were excluded.

The study was approved by the ethics committees of

the participating institutions, the procedures followed

were in accordance with the ethical standards of the

responsible committee on human experimentation (insti-

tutional and national), and proper informed consent was

obtained.

Extracted blood-derived DNA was subjected to a 5K SNP

array with the Illumina Linkage IVb mapping panel for all

informative members and analyzed with easyLinkage-

Plus software for calculation of multipoint LOD scores

(for ARID-628); the Affymetrix 500K Nsp GeneChip SNP

mapping panel was used for the three affected offspring

and one healthy offspring, and DNA was analyzed for

homozygosity with dCHIP and HomozygosityMapper

(for ARID-49, Figures 1A and 1D).10–12 ARID-628 demon-

strated the major linkage peak at chr1: q43–44, delin-

eated by rs1481141–rs6426327 (or chr1: 238,408,184–

246,089,811) (hg19), with a multipoint pLOD of 2.30,

which is similar to the predicted pLOD score13 (Figure S1

in the Supplemental Data available with this article

online). Several narrower peaks of similar significance

were located on other autosomes. ARID-49 demonstrated

two blocks of homozygosity, the major one delin-

eated by rs6702864–rs12137158 (or chr1: 230,161,767–

240,660,818) and another delineated by rs1019258–

rs7588049 (or chr2: 178,012,446–178,390,778) (Figure S2).

There was no shared haplotype between the two families,

and although the phenotypes were similarly severe with

respect to ID, there were some apparent differences.

Nevertheless, the two families shared a minimal candi-

date interval defined as chr1: 238,408,184–240,660,818,

or 2.25 Mb, containing three genes, CHRM2, FMN2,

and GREM2 (MIM 118493, 606373, and 608832, respec-

tively), and the long intergenic non-protein-coding RNA

LINC01139.

We generatedwhole-exome sequence for two affected in-

dividuals fromARID-628 and for one individual fromARID-

49 by using the Agilent SureSelect HumanAll Exome 50Mb

kit. We performed sequencing with an Illumina HiSeq2000

or ABI SOLiD 5500 instrument, resulting in ~94% recovery

at >103 coverage. GATK was used for SNP and INDEL

variant identification, and SeattleSeq was used for annota-

tion; variants were then filtered with >1% allele frequency

according to presence in dbSNP, the Exome Variant Server,

or in our in-house exome data set of more than 2,000

individuals. In addition, they were filtered if annotated

as benign according to functional prediction scores

(PolyPhen, Grantham, Phastcon, GERP).14,15 We analyzed

variants from both families with HomozygosityMapper to

confirm linkage intervals (Figures 1B and 1E).16 We tested

segregation of all exonic homozygous rare variants pre-

722 The American Journal of Human Genetics 95, 721–728, Decemb

dicted to be deleterious, and we only report the variant as

causative if it is the single segregating variant. Affected

members of both families harbored a homozygous pre-

dicted frameshift variant in FMN2 (MIM606373), encoding

formin2 (FMN2) (Figures 1C and1F).None of the other var-

iants considered as candidates showed patterns consistent

with a recessive mode of inheritance for affectation status

(Tables S1 and S2).

ARID-628 was recruited from Lower Egypt as a consan-

guineous pedigree with two affected members, aged 16

and 14 years (Table 1). The children were delivered at

term but experienced delayed milestones, primarily in

cognition and speech, whereas gross-motor and fine-motor

development were not as dramatically affected. IQ was

50–60, tested in the teenage years, and both children

remain illiterate past adolescence and speak in only 1–2

word phrases. Independent gait was obtained at 2 years.

Rare partial complex seizures developed at age 10 years,

controlled with carbamazepine. The older individual has

a history of asymptomatic mitral valve prolapse. ARID-49

was recruited from a remote village in Azad Jammu and

Kashmir, Pakistan as a consanguineous pedigree with three

affected members, aged 30, 25, and 17 years, and three

healthy siblings. All were born after normal pregnancies.

Individual IV-1 was independent in self-care, and speech

was well developed, but he was unable to read or write

and has never been gainfully employed. He frequently dis-

appeared from home for days at a time, wandered in the

local area, and functioned in the mildly intellectually

disabled range. The other two affected members never

developed speech beyond 2 or 3 word phrases, nor reading

or writing, and were unable to care for themselves. There

were no hearing or visual impairments evident and no his-

tory of seizures. For both families, no dysmorphic features

were noted, and occipital frontal head circumference was

within the normal range. MRI and EEG analyses were

normal in one affected member.

The putative causative variant in ARID-628 was in

the first exon and was annotated in hg19 as chr1:

240,256,799insG, a single G nucleotide insertion, predict-

ing a frameshift at amino acid 466 of 1,726 residues

(c.1394_1395insC [p.Ala466Glyfs*483], RefSeq transcript

NM_020066.4). The variant in ARID-49 was in the fifth

constitutive exon, chr1:240,370,627delA, a single A nucle-

otide deletion, predicting a frameshift at amino acid 839

(c.2515_2515delA [p.Thr839Argfs*848], RefSeq transcript

NM_020066.4). We confirmed both variants with Sanger

sequencing, and additionally confirmed that the variants

segregated according to a strict recessive model with full

penetrance. The ARID-49 variant occurred on the minor

allele of a common synonymous SNP, rs10926166, and

thus sequences were compared with two different healthy

individuals, one displaying the major and one displaying

the minor allele (Figures 1C and 1F). Neither frameshifting

variant was predicted to affect several shorter FMN2 tran-

script variants (from mRNAs AK297755 and AK298141,

derived from coronary artery smooth muscle cells and

er 4, 2014

ARID-628

1 2 3 4 5 6

1 2 3 4 5 6

ARID-49

Co

ntr

ol

Aff

ecte

dC

on

tro

lA

ffec

ted

Co

ntr

ol (

MA

)

*

A B C

D E

F

G

chr1:240256799insGc.1394_1395insC

chr1:240370627delAc.2515_2515delA

FMN2

GAPDH

ARID-628C IV-4 III-2 C IV-4 III-2

Formin 2

GAPDH

ARID-628I J

P P P

p.Ala466Glyfs*483 p.Thr839Argfs*48

H

FH2DEP

1 2

1 2

398 bp

37 kD

195 kD

383 bp

1 250 500 750 1000 1250 1500 1726aa

FH1

FSI

NM_020066.1NM_020066.2NM_020066.3NM_020066.4

200kb

5’ 3’

Figure 1. ARID-628 and ARID-49 with Homozygous Inactivating Mutations in FMN2(A and D) Consanguineous parents and multiple affected individuals suggest recessive inheritance.(B and E) Homozygous exome haplotypes containing FMN2 in telomeric chromosome 1 (arrows).(C and F) ARID-628 homozygous chr1:240,256,799insG (arrow) and ARID-49 showing major-allele control TTCAAACGA, minor-allelecontrol (MA) TTCAAACCA, and diseased TTCAACGA (arrow) chr1:240,370,627delA.(G) Genomic organization and location of mutations for chr1q43–44, including FMN2, from the UCSC Genome Browser; mutations arenot predicted to interfere with shorter transcripts.(H) FMN2 encodes a 1,726 residue protein with DEP, FH1, and FH2 domains, several putative sites of phosphorylation (small verticalticks), and an FMN2-SPIRE1-interacting domain (FSI).(I) RT-PCR of FMN2 primary fibroblasts shows that products are absent in IV-4 but present in the control (C), III-2, and GAPDHfibroblasts, suggesting nonsense-mediated decay. Primer sequencing: FMN2-F, 50-CCGTCTCAGTCCCCTAATCA-30; FMN2-R, 50-ATCCGGGAGCAAAACTTCTC-30; GAPDH-F, 50-ATCCCATCACCATCTTCCAG-30; and GAPDH-R, 50-CCATCACGCCACAGTTTCC-30.(J) An immunoblot of FMN2 in primary fibroblasts shows an absence of protein in IV-4 but presence in the control, III-2. GAPDH wasused as a loading control. Antibodies: FMN2, Sigma HPA004937, 1:1,000; and GAPDH, Millipore mab374, 1:5,000.

The American Journal of Human Genetics 95, 721–728, December 4, 2014 723

Table 1. Clinical Description of Affected Individuals from Families ARID-628 and ARID-49

ARID-628-IV-4 ARID-628-IV-5 ARID-49-IV-1 ARID-49-IV-2 ARID-49-IV-3

Family Information

Consanguineous þ þ þ þ þ

Multiplex þ þ þ þ þ

Number of affectedindividuals

2 2 3 3 3

Extended family historyof ID and/or epilepsy

þ þ - - -

Clinical Information

Age at examination 16 years 14 years 30 years 25 years 17 years

Sex female male male female female

Intellectual disability þ þ þ þ þ

Speech 1–2 words 1–2 words full sentences 2–3 words single words

Hearing normal normal normal normal normal

Daily activities (eating,toilet)

dependent dependent Independent dependent dependent

Dysmorphic facies - - - - -

Hypotonia þ þ - - -

Microcephaly - - - - -

Seizures (partial complex) controlled controlled - - -

Electroencephalogram normal N/A N/A N/A N/A

Medication Carbamazepine Carbamazepine

Spasticity - - - - -

Visual abnormalities - - - - -

Head circumference 52.4 cm 50.3 cm 51.7 cm 51.6 cm 50.4 cm

Other systemic features mitral valve prolapse - - - -

Brain MRI normal N/A N/A N/A normal

Homozygous mutation(hg19 coordinates)

chr1:240,256,799insG chr1:240,256,799insG chr1:240,370,627delA chr1:240,370,627delA chr1:240,370,627delA

cDNA mutation(NM_020066.4)

c.1394_1395insC c.1394_1395insC c.2515_2515delA c.2515_2515delA c.2515_2515delA

Protein alteration(NP_064450.3)

p.Ala466Glyfs*483 p.Ala466Glyfs*483 p.Thr839Argfs*848 p.Thr839Argfs*848 p.Thr839Argfs*848

N/A indicates ‘‘not assessed.’’

synoviocytes, respectively (Figure 1G), when alternative

start sites were used. The variants were not identified in

any other individuals from more than 3,000 whole-exome

sequences, in 200 healthy control geographically matched

individuals, or in any public database. There were no other

potentially deleterious FMN2 variants in the CARID data-

base or in private databases consisting of more than

4,000 intellectually disabled individuals who underwent

exome sequencing.

The NHLBI study identified several different potentially

deleterious variants in the database of >4,000 individuals,

but none argued strongly against FMN2 as a potential cause

of ID in these families. The rs375865107 variant predicts

724 The American Journal of Human Genetics 95, 721–728, Decemb

p.Trp601* and was found to be heterozygous in one of

6,502 individuals, and the chr1:240286646_240286647insT

(RefSeq accession number NM_020066.4; c.1782þ1_

1782þ2insT) variant was found to be heterozygous

in one of 6,295 individuals; probably both variants

are valid. However, the chr1:240371554_240371558del5

(RefSeq NM_020066.4; c.3442_3446del5) variant, pre-

dicting p.Arg1148Glyfs*103, and the chr1:240371609_

240371610insT (RefSeq NM_020066.4; c.3497_3498insT)

variant, predicting p.Leu1168Serfs*85, have average read

depth below 10 and were far outside of Hardy-Weinberg

equilibrium (p value < 1 3 10�311), and the latter occurred

ina stretchofhomocytosines.Nonewere found inanyother

er 4, 2014

public databases (or in our internal database), suggesting

sequencing errors. Finally, several variants, including

rs141879002, rs370558399, rs142397272, rs147210965,

rs199721402, rs146873580, rs374743076, rs150801382,

and rs183078344, modify well-conserved residues, but

none are homozygous in any retrievable database.

To investigate the effect of the mutation on mRNA and

protein, we tested expression of FMN2 in control lympho-

cytes but were unable to detect any expression, precluding

analysis from family ARID-49, where only lymphocytes

were available. Thus, skin biopsy was obtained from family

ARID-628, RNA was reverse transcribed, and primers were

designed to span coding exons. FMN2 encodes a 1,726

amino acid polypeptide as the major isoform (Figure 1H).

We amplified a 398 bp product in control and III-2 individ-

uals but no product in affected member IV-4, consistent

with mRNA nonsense mediated decay, whereas GAPDH

amplified a 383 bp product similarly in the three individ-

uals (Figure 1I). Total cellular protein from confluent fibro-

blasts was analyzed by SDS-PAGE and blotted with a

commercial FMN2 antibody, yielding a 195 Kd protein in

control and III-2 individuals but no product in affected

member IV-4, whereas GAPDH yielded similar band inten-

sities in all (Figure 1J). We conclude that the ARID-628

mutation results in the absence of detectable full-length

protein in cells from the affected individual.

FMN2 is one of 15 formin proteins identified in humans.

Its identification is based upon the presence of a catalytic

actin-nucleating and polymerizing formin homology

2 (FH2) domain.17 FMN2 additionally encodes a DEP

(Dishevelled, Egl-10, and Pleckstrin) domain, suggesting

a role in G-protein-coupled receptor signaling; a proline-

rich FH1 domain; a formin-Spire1 interaction (FSI)

domain18 involved in actin nucleation; and several phos-

phorylation sites, which probably regulate its activity.

FMN2 has 18 exons, encodes three major isoforms, and is

predicted to have important roles in actin cytoskeletal or-

ganization and the establishment of cellular polarity.

Although FMN2 is expressed predominantly in the devel-

oping and mature brain,19 Fmn2 knockout mice (Neo

cassette replacing 433 amino acids at the FH1 domain,

Jackson labs, stock #016264, maintained on a pure

129S6/SvEvTac background) survive without major defects

and have neither gross nor histological brain differences

when they are compared with littermates.19 Fmn2�/�

germline female mutant mice show decreased fertility

and abnormal positioning of the metaphase spindle and

formation of the first polar body during oogenesis, but

males show no corresponding defect.20 Alhough FMN2

overexpression in NIH 3T3 cells showed dramatic disrup-

tion of the actin and microtubule network,21 we noted

no major cellular morphology defect in FMN2 mutant

fibroblasts under basal growth conditions.

Fmn2 was identified a ‘‘learning-regulated gene’’ as part

of a screen for genes that were upregulated and for which

H4K12 acetylation of the coding region increased upon

fear conditioning in 3-month-old but not 16-month-old

The American

mice. Furthermore, Fmn2�/� 8-month-old but not 3-

month-old mice showed impaired associative learning,

whereas pain sensation, explorative behavior, and basal

anxiety were similar among groups.22 Because fear condi-

tioning is hippocampal dependent, we studied FMN2 in

the hippocampus at P14 in home-cage-reared mice and

found that the protein within the granule neuron den-

dritic tree colocalized with the actin cytoskeleton, on the

basis of phalloidin colocalization; as a control, we found

no staining in Fmn2�/� mice (Figure 2A). Hippocampal

neuroanatomy was grossly intact, arguing against a role

for Fmn2 in neuronal migration or differentiation.

Most CNS excitatory synapses are located on small

membrane protrusions called dendritic spines, in which a

dynamic F-actin matrix is linked to postsynaptic-density

scaffold proteins, signaling proteins, and NMDA and

AMPA receptors. Actin dynamics are of great importance

for synaptic plasticity andmemory formation, and formins

have been proposed as candidates for regulating the nucle-

ation and polymerization of actin filaments in dendritic

filopodia. In fact, the formin member mDia2 localizes to

hippocampal synaptic spines, and knockdown results in

altered spine morphology.23 In cultured murine primary

hippocampal neurons, we found partially adjacent and

overlapping protein localization between FMN2 and

the postsynaptic-density marker PSD95 and the presynap-

tic markers VGLUT1 and SYNAPSIN, suggesting that

FMN2 also localizes to intracellular sites nearby spines

(Figure 2B), although determining precise subspine

compartmental localization will require additional work.

To determine whether Fmn2 is required for the

morphology of hippocampal dendritic spines, we per-

formed Golgi staining in P47 wild-type mice and their

Fmn2�/� littermates, then measured spine density. We

found that overall neuronal architecture was intact and

that there were no notable differences in dendritic branch-

ing or length. However, dendritic spines were reduced in

density by 32% on mutant hippocampal dentate granule

neurons (Figures 2C–2E). We conclude that hippocampal

dendritic-spine density is reduced in Fmn2�/� mice.

To determine whether this defect in synaptic density

occurred in human cells, we reprogrammed fibroblasts

from one diseased and one healthy member of ARID-628

and a healthy age- and sex-matched control by using inte-

gration-free episomal methods,24 excluded gross chromo-

somal postreprogramming rearrangements, and generated

neural cells by using a dual-SMAD inhibition protocol.25

There was no defect noted in the efficiency of reprogram-

ming or in the formation of neural rosettes, neural precur-

sor cells (NPCs), or mature neuronal cells (Figure S3A), nor

was any defect noted in the differentiation of hIPSCs into

mesodermal or endodermal lineages (Figure S3B), suggest-

ing that the hIPSCs reprogrammed fully and generated

healthy neural cells. In neurons cultured for 4 weeks, there

were no defects in survival, differentiation, dendritic

complexity, or nuclear morphology among the three lines.

We subsequently stained neural cells for F-actin and

Journal of Human Genetics 95, 721–728, December 4, 2014 725

Fmn2

+/+

Fmn2

-/-

anti-FMN2 PhalloidinP14 Hippocampus

DAPI

0

0.2

0.4

0.6

0.8

1.0

Fmn2

+/+

Fmn2

-/-

Fmn2-/-Fmn2+/+

# Sp

ines

/um

*

FMN2

FMN2

FMN2

PSD-95

VGLUT1

SYNAPSIN

FMN2

FMN2

FMN2

PSD-95

VGLUT1

SYNAPSIN

/

/

/

A

B

C C’

D D’

E

Figure 2. FMN2 Is Required for Dendritic-Spine Morphogenesis in Mice(A) P14 hippocampus from Fmn2þ/þ andFmn2�/�. Immunofluorescence showedFMN2 staining in the granule neuron den-dritic region (the highlighted section isthe CA2 region) of Fmn2þ/þ mice but nostaining in knockout mice. Phalloidinstaining was not overtly different betweengenotypes. The scale bar represents150 mm.(B) FMN2 punctae localize alongdendrites of cultured murine primary hip-pocampal neurons at 21 days in vitro.Staining was adjacent to both post-(PSD-95) and pre-synaptic (VGLUT1 andSYNAPSIN) markers but did not fullycolocalize. The scale bar represents5 mm. FMN2: Sigma HPA004937, 1:200.PSD95 Thermo MA1-045, VGLUT1 Syn-aptic Systems, Inc. #135302, SYNAPSINCell Signaling #5297.(C and D) Golgi-stained images of Fmn2þ/þ

and Fmn2�/� are shown at low and highmagnification (FDneurotech Rapid GolgiStain method). Neuronal morphology wasindistinguishable, but spine density wasreduced in knockout neurons when Sholl’smethod for dendritic arborization wasused. The scale bars in (D) and (E) represent50 mm; in (D0) and (E0) it represents 20 mm.(E) Quantification of spine density permicron of dendrite of hippocampal granule

neuron from the CA2 region from three mice of each genotype, two sections per animal, >1,000 spines per animal. *p < 0.001 by Stu-dent’s t test. The error bar shows SEM (0.80 5 0.05 versus 0.54 5 0.04 spines/mm, n ¼ 3 mice of each genotype).

VGlut1 in order to identify excitatory synapses (Figure S4),

then segmented punctae to determine synaptic density

(Figure 3A). We found that synaptic density in diseased

cells was reduced by 43% in comparison with healthy con-

trols (Figure 3B), suggesting reduced synaptic density in

human IPSC-derived neural cells in the absence of FMN2.

In summary, we report homozygous truncating muta-

tions in FMN2 as a cause of autosomal-recessive nonsyn-

dromic ID. The work is corroborated by an absence of

protein in cells from affected members, by study of expres-

sion and localization of the endogenous gene and protein

in mice, by synaptic spine defects in knockout mice, and

by synaptic density defects in mutant human neural cells.

We suggest that FMN2 might be required for regulation of

the actin cytoskeleton during spine development, matura-

tion, or remodeling. Previously, two individuals with de

novo interstitial deletions involving FMN2, among other

genes, demonstrated intellectual disability,26,27 suggesting

that haploinsufficiency, perhaps in combination with

other gene deficiencies, might similarly correlate with ID.

Mutations in other FH2-domain-containing proteins

have been implicated in human disease: for example, mu-

tations in INF2 (MIM 610982) cause autosomal-dominant

intermediate Charcot-Marie-Tooth disease type E (MIM

614455) and dominant focal segmental Glomerulosclero-

sis type 5 (MIM 613237), and mutations in DIAPH2

(MIM 300108) cause X-linked premature ovarian failure

(MIM 200511). Recently, a biallelic null mutation in

726 The American Journal of Human Genetics 95, 721–728, Decemb

DIAPH1 (MIM 602121), encoding a diaphanous-related

formin-family-of-Rho effector protein, was identified in a

family affected by microcephaly.28 FMN1 (MIM 136535)

shows embryonic expression,29 targeted deletion in mouse

shows oligodactylism and alters BMP signaling,30 and a

genomic rearrangement including FMN1 shows non-

syndromic oligosyndactyly.31 FMN2 now joins a group of

genes implicated in neuronal functions mediating higher

cognition in humans.

Supplemental Data

Supplemental Data include four figures and two tables and can be

found with this article online at http://dx.doi.org/10.1016/j.ajhg.

2014.10.016.

Acknowledgments

We thank the study participants and their families for their invalu-

able contributions to this study. This work was supported by Na-

tional Institutes of Health grants R01NS041537, R01NS048453,

R01NS052455, P01HD070494, and P30NS047101, the Simons

Foundation Autism Research Initiative, the Howard Hughes

Medical Institute (J.G.G.), the California Institute of Regenerative

Medicine, the Canadian Institutes of Health Research (grant MOP-

102758 to J.B.V.), and the Pakistan Higher Education Commission

(HEC, A.A.A.). We thank the Broad Institute (grant U54HG003067

to E. Lander), the Yale Center for Mendelian Disorders grant

(U54HG006504 to R. Lifton and M. Gunel) for sequencing

er 4, 2014

Control Unaffected Affected

0.00

0.25

0.50

0.75

1.00

AffectedUnaffectedControl

VGlu

t1+

punc

tae/

um2

A

B

F-ac

tin/V

Glu

t1

*

Figure 3. Reduced Synaptic Density in Human FMN2 MutantIPSC-Derived Neural Cells(A) FMN2 mutant cells exhibit reduced density of excitatory syn-apses as assessed by VGlut1 (green) staining of neural cells (seealso Figure S4). F-actin was visualized via phalloidin staining (red).(B) Quantification of excitatory synapses per square micronshowed reduced density in cells from affected members specif-ically under the Imaris MeasurementPro module. *p < 0.01 byStudent’s t test. The error bar represents SEM (0.83 5 0.085 versus0.81 5 0.065 versus 0.47 5 0.110 in the control individual, unaf-fected individual, and affected individual, respectively; n ¼ 10dendrites segmented from each of four neurons from two inde-pendent cultures per individual).

support, the Consortium for Autosomal Recessive Intellectual

Disability (CARID) for supporting ascertainment, and Katie Hsiao

(Nathanial Heintz lab) for sharing hippocampal cultures.

Received: June 6, 2014

Accepted: October 29, 2014

Published: December 4, 2014

The American

Web Resources

The URLs for data presented herein are as follows:

NHLBI Exome Sequencing Project (ESP) Exome Variant Server,

http://evs.gs.washington.edu/EVS/

Online Mendelian Inheritance in Man (OMIM), http://www.

omim.org/

PolyPhen-2, www.genetics.bwh.harvard.edu/pph2/

Quantsmooth,http://www.bioconductor.org/packages/release/bioc/

html/quantsmooth.html

RefSeq, http://www.ncbi.nlm.nih.gov/RefSeq

SeattleSeq,http://snp.gs.washington.edu/SeattleSeqAnnotation137/

Accession Numbers

RefSeq NCBI transcript FMN2: NM_020066.4

Exome data for ARID-628 have been deposited into dbGaP

(phs000288).

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