ORIGINAL ARTICLE

Novel variants identified in methyl-CpG-binding domaingenes in autistic individuals

Holly N. Cukier & Raquel Rabionet & Ioanna Konidari & Melissa Y. Rayner-Evans &

Mary L. Baltos & Harry H. Wright & Ruth K. Abramson & Eden R. Martin &

Michael L. Cuccaro & Margaret A. Pericak-Vance & John R. Gilbert

Received: 1 May 2009 /Accepted: 26 October 2009# Springer-Verlag 2009

Abstract Misregulation of the methyl-CpG-binding protein2 (MECP2) gene has been found to cause a myriad ofneurological disorders including autism, mental retardation,seizures, learning disabilities, and Rett syndrome. Wehypothesized that mutations in other members of themethyl-CpG-binding domain (MBD) family may also causeautistic features in individuals. We evaluated 226 autisticindividuals for alterations in the four genes most homolo-gous to MECP2: MBD1, MBD2, MBD3, and MBD4. Atotal of 46 alterations were identified in the four genes,including ten missense changes and two deletions that altercoding sequence. Several are either unique to our autisticpopulation or cosegregate with affected individuals within afamily, suggesting a possible relation of these variations todisease etiology. Variants include a R23M alteration in twoaffected half brothers which falls within the MBD domainof the MBD3 protein, as well as a frameshift in MBD4 that

is predicted to truncate almost half of the protein. Theseresults suggest that rare cases of autism may be influencedby mutations in members of the dynamic MBD proteinfamily.

Keywords Autism . Rett syndrome .MeCP2

Introduction

Autism is a neurodevelopmental disorder with a triad oftypical features: (1) impairments in social relatedness, (2)problems in communication, and (3) the presence ofrepetitive stereotyped behaviors or restricted interests [1].Autism has an incidence of approximately one in every1,000 individuals in the general population and has askewed male to female ratio of about 4:1 [2]. Autism is partof a spectrum of disorders which includes Asperger’ssyndrome, pervasive developmental disorder not otherwisespecified, childhood disintegrative disorder, and Rettsyndrome. Combined, autism spectrum disorders occur ata rate of approximately one in 150 individuals [3]. A highconcordance of autism in monozygotic twins signifies astrong genetic component and heritability of the disorder[4–6]; however, linkage and association studies have beenlargely unable to uncover a single causative gene thataccounts for an appreciable percentage of autism cases [7].This is thought to result from one of two scenarios: (1)distinct, rare variants in an unknown number of geneswhose strong effect results in a clinical phenotype or (2)common variants that have little effect by themselves, butacting in concert with modifiers or additional loci, mayconverge to promote the manifestation of autism. The firsthypothesis is demonstrated by distinct cases of autism thathave been shown to stem from single gene mutations, such

Electronic supplementary material The online version of this article(doi:10.1007/s10048-009-0228-7) contains supplementary material,which is available to authorized users.

H. N. Cukier : I. Konidari :M. Y. Rayner-Evans :M. L. Baltos :E. R. Martin :M. L. Cuccaro :M. A. Pericak-Vance :J. R. Gilbert (*)John P. Hussman Institute for Human Genomics,University of Miami,1501 NW 10th Avenue,Miami, FL 33136, USAe-mail: jgilbert@med.miami.edu

R. RabionetGenes and Disease Program, Centre de Regulació Genòmicaand CIBER en Epidemiología y Salud Pública,Barcelona, Spain

H. H. Wright : R. K. AbramsonUniversity of South Carolina School of Medicine,Columbia, SC, USA

NeurogeneticsDOI 10.1007/s10048-009-0228-7

as those in NLGN3, NLGN4, and SHANK3 [8, 9]. Evidencesupporting the latter idea comes from studies that haveidentified a common CNTNAP2 variant in families withmale affected individuals and from genome-wide associationstudies that recognized a novel region of interest onchromosome 5 [10–13].

While there are many autism candidate genes, the autismspectrum disorder Rett syndrome results almost exclusivelyfrom alterations in a single gene, methyl-CpG-bindingprotein 2 (MECP2) [14]. Rett syndrome differs from classicautism in several ways, the most obvious being that amajority of patients are female. Due to MEPC2’s locationon the X chromosome, many mutations are heterozygousviable in females but hemizygous lethal in males. Rettpatients typically acquire the hallmark feature of repetitivehand wringing or clasping [15, 16]. Rett syndrome is alsocharacterized by developmental regression, a feature onlyseen in a subset of autistic patients [1, 17]. On the otherhand, Rett patients are similar to autistic individuals in thatthey demonstrate increased anxiety in novel situations andapathy toward people and objects around them [18].

Point mutations, deletions, and duplications in MECP2cause more than 85% of Rett syndrome cases [14, 19]. Whilethese mutations have been found throughout the gene in Rettpatients, they tend to occur in domains of functionalimportance: the methyl-CpG-binding domain (MBD) or thetranscriptional repression domain (TRD) [20]. Currentevidence demonstrates that the MeCP2 protein modulatesgene expression through chromatin remodeling, acting bothas an enhancer and a repressor of gene activity [21].

MeCP2 is a member of the MBD family of proteins.Along with MeCP2, there are four other closely relatedMBD proteins designated in humans: MBD1, MBD2,MBD3, and MBD4 (Fig. 1) [22]. MBD1, MBD2, MBD4,and MeCP2 each have the ability to bind DNA at methylatedCpG sites while MBD3 requires additional factors in order tobe recruited to DNA [23]. All of the MBD proteins havedemonstrated transcriptional repression activity. The MBD4protein is unique in that it has preferential binding for G–Tmismatches and acts to repair DNA damage [24]. TheMBD proteins are also capable of simultaneously bindingthe same promoter regions, implying either a functionalinterdependence or redundancy between these proteins [25,

26]. The latter can be seen where MBD2 and MBD3 act ina mutually exclusive manner to form distinct Mi-2/NuRDchromatin-remodeling complexes [27]. Therefore, it ispossible that dysfunction in one of these MBD proteinscould titrate away core components of the Mi-2/NuRDcomplex, leading to an imbalance of the protein componentsavailable to bind to the second MBD protein.

Significantly, mutations in the MECP2 gene have alsobeen identified in autistic patients without classic Rettsyndrome [28–30]. Specifically, our laboratory identifiedtwo autistic females carrying de novo MECP2 alterationsthat result in coding changes [28]. Further studies haveshown that MECP2 mutations can cause a spectrum ofclinical presentations including mild learning disabilities,mental retardation, schizophrenia, Angelman-like symptoms,and mental retardation presenting with infantile seizures [29–37]. These findings demonstrate that there is a broad range ofphenotypes caused by misregulation of the MeCP2 protein.Moreover, a recent study demonstrated an associationbetween markers within MECP2 and autism [38].

In addition to clinical data, there is accumulatingevidence from animal models demonstrating the severeeffects of MBD misregulation [39–44]. The Mecp2308/y

knock-in model generated by Shahbazian and colleaguescreates a truncated protein similar to that found in Rettpatients, and the mice exhibit anxiety, seizures, andabnormal social and nesting behavior [40, 45, 46].Furthermore, Mecp2308/y mice are defective in social,spatial, and contextual fear memory [47]. Another MBDmodel, the Mbd1−/− mouse, is also deficient in a wide rangeof social behaviors, showing increased anxiety, greatersusceptibility to depression, and a reduced prepulseinhibition of the startle response, a feature found in autisticpatients [41, 48]. Interestingly, these mice were also found tohave an increase in the RNA and protein levels of serotoninreceptor 2c [48]. Both of these models demonstrate thatmutating or completely eliminating a single MBD proteincan result in autistic features in mice.

Based on the findings of autistic patients with mutationsin MeCP2 and results from animal models, we decided toinvestigate additional members of the MBD family todetermine whether rare variations within these genes mightbe associated with autism. It is possible that the MBD

Fig. 1 The methyl-CpG-binding domain (MBD) protein family. The five MBD proteins, each containing an MBD domain, are illustrated. Alongwith this domain, each protein contains at least one other functional domain that contributes to the protein's function

Neurogenetics

domain may perform a similar function within each MBDprotein, and when the gene is misregulated, could lead tosymptoms that fall along the autism spectrum. To date, onlya single study limited to a Japanese population of 65autistic cases has evaluated the possible role of mutations inthe MBD genes in autistic patients [49]. One variation ofinterest was identified, R269C in MBD1 [49]. In this study,we evaluated MBD1, MBD2, MBD3, and MBD4 and foundmultiple alterations that cause amino acid changes andpotentially lead to autistic features.

Materials and methods

Patient ascertainment We randomly selected DNA from226 unrelated autistic individuals (195 Caucasian (CA) and31 African-American (AA) probands) and their familymembers, when available, from our dataset of autismspectrum disorder families. This cohort was comprised of180 males and 46 females.

Participants were recruited, enrolled, and sampledaccording to the Institutional Review Board protocols forthe University of Miami and the University of SouthCarolina. Individuals entered the study through well-developed referral sources (e.g., clinics) as well as throughfamily support groups, schools, and practitioners. Partic-ipants with autism were ascertained on the basis of apreviously existing diagnosis of autism and met thefollowing minimal inclusion criteria: (1) age between 3and 21 years, (2) absence of severe sensory or significantmotor impairments, and (3) absence of identified metabolic,genetic, or progressive neurological disorders. The researchdiagnosis for participants with autism was assigned usingDiagnostic and Statistical Manual of Mental Disorders IVcriteria [50], supported in most cases by the AutismDiagnostic Interview-Revised, and when available, theAutism Diagnostic Observation Schedule [51, 52]. In aminority of cases, neither measure was available, and theresearch diagnosis was made by our expert clinical panel.We also administered the Vineland Adaptive BehaviorScales (VABS) [53], a measure of developmental function-ing to assure that the participants with autism met a basicdevelopmental age of 18 months. This developmentalthreshold ensures that the diagnostic instruments are valid.Detailed family and medical histories were obtained,including information about psychiatric, developmental,and behavioral problems in family members. A completedescription of the study was provided to the subjects, andwritten informed consent was obtained. Whole blood wascollected from all participants by venipuncture.

The Caucasian control group consisted of 245 individualsthat had no known indicators of behavioral, developmental, orcognitive abnormalities (100 males and 145 females). The

human random control panel (Sigma-Aldrich) encompassedDNA from 96 Caucasian individuals collected in the UnitedKingdom (66 males and 30 females). The African-Americancontrols were a combination of the Coriell African-Americancontrol panel (NDPT031), which was comprised of 94individuals (25 males and 69 females) and 86 samplescollected at the University of Miami (49 males and 37females). Both of these datasets only contained individualsthat were negative for evidence of neurological disease.

Statistical analysis In order to determine our ability todetect variants within our autistic patient dataset, we usedthe binomial distribution to compute the probability that atleast one variant allele (with frequency p) was observed in asample of N chromosomes. We assumed sample sizes ofN=390 and N=62, which represent the number of chromo-somes in our affected CA and AA samples, respectively, andconsidered allele frequencies of 0.01 and 0.05. Theprobabilities were computed using the binomial cumulativedensity function.

Denaturing high-performance liquid chromatography GenomicDNA was isolated from whole frozen blood according tostandard protocol [54]. DNA from 226 probands wasassembled into 47 distinct pools of three to five sampleseach. Thirty-two Caucasian control samples were assembledinto five pools of three to five samples. Mutation screeningwas performed by denaturing high-performance liquidchromatography (DHPLC) using the WAVE™ DNA frag-ment analysis system (Transgenomic, Omaha). Primers weredesigned to amplify all exons for the MBD1, MBD2, MBD3,and MBD4 genes in each proband and control pool(Supplementary Table 1). The polymerase chain reaction(PCR) products were evaluated via agarose gel electropho-resis. For heteroduplex molecule formation, all PCR prod-ucts were subjected to denaturation for 3 min followed bygradual reannealing in the thermocycler for 45 min. Thetemperature for successful resolution of the heteroduplexmolecules was selected using Transgenomic software beforeproceeding to DHPLC analysis. Homozygous and heterozy-gous peak profiles were identified using Transgenomicsoftware for all proband and control pools. Pools of interestfor both homozygous and heterozygous profiles were brokendown, and each sample within the pool was sequenced.

Sequencing Individuals with possible variants identified byDHPLC were sequenced using Applied Biosystems BigDye Terminator version 3.1 on the ABI 3730 sequencer,and the primers listed in Supplementary Table 1. Wheneverpossible, family members of the proband sample were alsoexamined by sequencing. Additionally, a custom TaqManallelic discrimination assay (Applied Biosystems) wasdesigned for each novel variant. These newly identified

Neurogenetics

variants were evaluated in 150 Caucasian control individuals,and when appropriate, 180 African-American individuals.Three variants were tested in the Sigma-Aldrich Caucasianhuman random control panel instead of the 150 CA controls.For novel variants that altered the amino acid sequence andwere absent in the initial controls tested, an additional 95Caucasian controls were sequenced across the region.

Cloning mutations All insertion/deletion variants wereverified by cloning. Exons for MBD genes were amplifiedby PCR; products were cloned into the Invitrogen pCR2.1-TOPO high copy plasmid vector and transformed in Top10Escherichia coli chemically competent cells. The standardTOPO TA cloning protocol was followed with a 30-minligation using 4µl of PCR product, 30-minute incubation onice, and 75µl of transformation plated on Luria–Bertanimedium (LB)/ampicillin/X-gal. Individual colonies wereselected and grown in LB ampicillin medium, and plasmidDNAwas extracted using the Qiagen Miniprep Spin kit. Theplasmid DNA samples were sequenced on ABI 3100 usingBigDye 3.1 Sequencing kit (Applied Biosystems) and M13forward and reverse primers (Integrated DNATechnologies).

Prediction of splice factor binding sites Sequences con-taining variations within intronic and exonic regions in thisstudy were entered into the Human Splicing Analyzerversion 2.3 to reveal potential binding sites for splicingfactors (http://www.umd.be:2300/) [55].

Results

We identified 198 autistic individuals carrying geneticalterations at 46 different locations in the four MBD genesthat we studied (Table 1, Fig. 2). Twenty-five of thesechanges represent novel variations, while twenty-one aresingle nucleotide polymorphisms (SNPs) that have beenpreviously identified and incorporated into the NationalCenter for Biotechnology Information SNP database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=snp). The frequen-cies of previously recognized SNPs in both AA and CApopulations can be found in Supplementary Table 2. Of thenovel changes that were identified, 20 were singlenucleotide alterations, and five were insertion/deletions.For these 25 new variants, we tested 150 control Caucasianindividuals (300 chromosomes), and when appropriate, anadditional 180 African-American control individuals (360chromosomes) to evaluate the relative frequency of eachvariation in the general population. Fourteen of the novelsingle nucleotide changes and three of the deletions werenot found in 150 Caucasian or 180 African-Americancontrols. When a novel variant was identified that alteredthe amino acid sequence, it was tested in an additional 95

Caucasian controls (190 chromosomes). We did not detectthe MBD1 R269C variation that was previously reported ina study of Japanese autistic patients, suggesting differencesin variations due to ethnicity [49].

With our cohort of 226 autistic individuals, we calculatedthat we would have a 98.0% probability of detecting rarevariants that occurred at a 1% frequency in our 390 CAchromosomes. This probability increases to 99.9% for alleleswith a 5% frequency. We would also have a 95.8% likelihoodof detecting rare alleles occurring at a 5% frequency in our 62AA chromosomes.

MBD1

Eleven SNPs were detected in MBD1, four known andseven novel. Three of the novel SNPs as well as rs12555resulted in nonsynonymous amino acid changes (R147K,R428H, K558N, and P401A, respectively). The novelalteration c.440G>A (R147K) was identified in the Cauca-sian family no. 7801 (Fig. 2a). This alteration was found intwo of three affected brothers and an unaffected sister.Interestingly, the two affected individuals carrying thevariant appeared to be more severely affected clinicallythan the brother without the variant; while all three brotherswere found to have speech difficulties, the probanddeveloped speech later than the brother without the variant(III:5), and the third brother (III:4) remained nonverbal at45 months and had irregular breathing and gait (Table 2).The proband also demonstrated regression in language aswell as social interest and play skills, all before 5 years ofage. This alteration was inherited from the paternalgrandfather; while the father was not reported to haveautism, he did have a history of seizures. The variant wasnot identified in 245 Caucasian controls.

The second novel missense mutation, c.1283G>A(R428H), was identified in a single affected individual inthe African-American family no. 7992 and was nottransmitted maternally (Fig. 2b). This male proband wasnonverbal at the time of examination (40 months, Table 2).The third novel nonsynonymous change occurred atc.1674A>T (K558N) and was passed maternally in familyno. 7947 to a single affected son as well as an unaffectedson and daughter (Fig. 2c, Table 2). Neither of these novelSNPs were identified in control individuals. The finalnonsynonymous variant, rs125555, causes a c.1201C>Galteration and results in a P401A change. This variant wasfound in a singleton Caucasian family (no. 7978) and wascarried by two daughters—one diagnosed with autism andthe other with anxiety and speech delay (Fig. 2d). Theautistic proband presented with regression in language andsocial interest prior to 5 years of age as well as seizures andan unusual gait (Table 2). The allele is also present in boththe mother, who presented with anxiety and depression, and

Neurogenetics

Tab

le1

MBD

variations

identifiedin

autistic

prob

ands

andcontrolindividu

als

Gene

Autistic

individuals

Controls(frequency)

Chrom

osom

ePosition

Location

Nucleotide

Aminoacid

SNP/novel

Probands

Male/female

Ethnicity

CA

AA

MBD118q21.1

Exon4

46,057,352

G>A

–rs140686

54/1

1AA,4CA

Exon5

46,057,065

G>A

R147K

Novel

11/0

1CA

0/245

Intron

1046,055,301

C>T

–Novel

2a2/0

2CA

6/149(0.04)

Exon12

46,054,177

C>G

P401A

rs125555

10/1

1CA

Exon12

46,054,095

G>A

R428H

Novel

11/0

1AA

0/242

0/180

Exon12

46,053,968

G>A

–Novel

11/0

1CA

0/245

Intron

1346,053,335

T>C

–Novel

10/1

1AA

0/150

0/179

Exon14

46,053,234

A>T

K558N

Novel

11/0

1CA

0/239

Intron

1546,051,935

C>T

–Novel

11/0

1AA

0/146

0/178

Exon17

46,050,015

C>T

–rs72923678

44/0

4CA

Exon17

46,049,627

G>T

–rs11663629

3929/10

13AA,26

CA

MBD218q21.2

Exon1

50,005,135

C>T

–Novel

44/0

4CA

3/150(0.02)

Exon1

50,005,114

T>C

–Novel

44/0

4CA

1/142(0.01)

Intron

249,985,346

G>A

–rs16957946

5a5/0

1AA,4CA

Exon7

49,935,242

A>G

–rs7614

32/1

3CA

Exon7

49,934,805

G>A

–rs62099763

11/0

1CA

Exon7

49,934,755

G>A

–rs1259938

85a

63/22

4AA,81

CA

MBD319p13.3

Exon1

1,543,563

G>T

R23M

Novel

11/0

1AA

0/243

0/172

Intron

11,543,502

C>T

–Novel

11/0

1AA

0/234

3/172(0.017)

Intron

41,532,272

G>A

–Novel

32/1

3CA

0/146

Exon5

1,532,144

C>T

–Novel

11/0

1CA

0/150

Exon6

1,529,372-1,529,374

delGAG

E281del

Novel

11/0

1AA

0/187b

3/167(0.018)

Exon6

1,529,321

C>T

–Novel

22/0

2AA

1/190b

(0.005)

6/162(0.037)

Exon7

1,529,191

A>G

–Novel

11/0

1CA

0/148

Exon7

1,529,075-1,529,077

delGCG

–Novel

3a2/1

1AA,2CA

1/142(0.007)

10/178

(0.056)

Exon7

1,529,140

C>T

–Novel

11/0

1CA

0/144

Exon7

1,528,326

C>T

–Novel

11/0

1AA

0/140

0/178

Exon7

1,528,325

G>A

–Novel

11/0

1CA

0/145

Exon7

1,528,208

C>T

–rs1053151

151a

118/33

28AA,123CA

Exon7

1,528,117

G>A

–rs9585

154a

120/34

31AA,123CA

Exon7

1,528,025-1,528,045

del20insCTG

–Novel

10/1

1AA

0/137

0/176

Exon7

1,527,932

A>G

–rs16994495

51a

40/11

6AA,45

CA

Exon7

1,527,928

A>G

–rs2238588

51a

40/11

6AA,45

CA

Exon7

1,527,924

G>C

–rs12884

51a

40/11

6AA,45

CA

Exon7

1,527,834

G>A

–Novel

11/0

1CA

6/94

b(0.064)

Neurogenetics

the father, who presented with anxiety/panic and bipolardisorders.

A possible case of germline mosaicism was identified inMBD1. A novel synonymous change at c.1410G>A wasfound within exon 12 in the Caucasian family no. 7660 inaffected identical twin brothers and one unaffected sisterwhich was not identified in either of the parents (Fig. 2e,Table 2). Previous genetic testing with this family did notdemonstrate Mendelian inconsistencies, ruling out non-paternity as the cause for unusual results and suggestinginstead germline mosaicism at this locus. While thisvariation does not produce a change in amino acidsequence, the alteration is predicted to affect binding sites ofsplicing factors (Supplementary Table 2). A second novelnoncoding variant of interest was identified within intron 15in the African-American family no. 7948 (Fig. 2f). Thec.1779-27C>T change (chr18:46,051,935) is concordant withdisease in three affected brothers, two of whom are identicaltwins. While the mother does not carry the change, the fatherwas unavailable for testing. Neither variant was present inCaucasian controls nor was the c.1779-27C>T alterationpresent in African-American controls.

MBD2

We identified two novel and four previously reported SNPswithin MBD2. Five of the six changes occurred inuntranslated regions of the gene, and the remainingalteration did not result in a change of amino acid sequenceor generate a readily identified cryptic splice site.

MBD3

Variations discovered in the MBD3 gene included fiveknown SNPs, ten novel SNPs, and four insertions/deletions.Only three variants occurred in the coding region, two ofwhich altered the amino acid sequence of MBD3. Ofparticular interest is a c.68G>T (R23M) change identifiedin two affected half brothers in an African-American family(no. 7974, Fig. 2g). This variation falls within the methyl-CpG-binding domain and changes the charged arginine to anonpolar methionine. The alteration was inherited from thematernal grandmother, suggesting a possible sex-specificeffect. Both brothers were late to acquire speech and onlyone developed functional language (Table 2). The motherwas diagnosed with anxiety/panic disorder. This variationwas not identified in either the African-American orCaucasian controls.

MBD4

Alterations in our autistic population were discoveredwithin the MBD4 gene at eight previously identified SNPsT

able

1(con

tinued)

Gene

Autistic

individuals

Controls(frequency)

Chrom

osom

ePosition

Location

Nucleotide

Aminoacid

SNP/novel

Probands

Male/female

Ethnicity

CA

AA

Exon7

1,527,785-1,527,805

Deletion

–Novel

11/0

1AA

0/145

0/163

MBD43q21.3

Intron

2130,639,226

T>C

–rs140692

3829/9

11AA,27

CA

Intron

2130,638,887

C>G

–rs3138340

55/0

4AA,1CA

Exon3

130,638,360

G>A

A273T

rs10342

2719/8

2AA,25

CA

Exon3

130,638,232-130,638,235

delAAGA

E314fsX

316

Novel

10/1

1AA

0/242

0/163

Exon3

130,638,153

T>C

S342P

rs2307289

9a9/0

8AA,1CA

Exon3

130,638,141

G>A

E346K

rs140693

33/0

3CA

Exon3

130,638,104

T>C

I358T

rs2307298

5a3/2

5CA

Intron

3130,637,926

T>G

–rs3138341

55/0

4AA,1CA

Exon5

130,635,394

A>G

N467S

Novel

21/1

2CA

0/244

Exon6

130,634,779

C>T

–rs140696

3829/9

11AA,27

CA

aThese

variations

werealso

identifiedin

theDHPLCCaucasian

controlsamples

bThe

Caucasian

Sigmacontrolsamples

wereused

forthesevariations

Neurogenetics

E. Family #7660 (MBD1 c.1410G>A)

G/A G/A G/A

G/G G/G

A. Family #7801 (MBD1 R147L) B. Family #7992 (MBD1 R428H)

G/A

G/G

C. Family #7947 (MBD1 K558N)

A/T

A/T

A/T A/A A/T

A/A

D. Family #7978 (MBD1 P401A)

C/GC/G bipolar

C/G C/G

I. Family #7605 (MBD4 E314fsX316)

+/del

+/del

+/+

A/G

A/G simple phobias

A/A

J. Family #7941 (MBD4 N467S) K. Family #7921 (MBD4 N467S)

A/G

A/AA/G

A/A

H. Family #7730 (MBD3 E281del)

+/+

+/+

+/+

+/del +/del

+/+

anxiety/panic disorder

depression

speech delay

autism

anxiety & depression

anxiety & speech delay

G. Family #7974 (MBD3 R23M)

G/G G/T G/T

G/T

G/Tschizophrenia

F. Family #7948 (MBD1 c.1779-27C>T)

C/C fear of heights claustrophobia

C/T C/T C/T

L. Family #7710 (MBD4 E346K) N. Family #7970 (MBD4 E346K)

G/A

G/A learning

disabilities

G/Glearning

disabilities ADHD

G/A

G/GG/A

G/A

M. Family #7785 (MBD4 E346K)

G/A

G/GG/A

G/G G/AG/G

depression & speech delay

G/G G/A

G/G

G/G G/GG/A

G/A G/GG/A G/G

G/G

G/A seizures

Fig. 2 Pedigrees of autistic families identified with mutations inMBD genes. Alterations identified in MBD1 (a–f), MBD3 (g–h), andMBD4 (i–n) are shown. Each nucleotide change is marked under the

individual in whom it occurs. In families with multiple affectedindividuals, the initially identified autistic individual (proband) issignified with an arrow

Neurogenetics

Tab

le2

Clin

ical

features

ofaffected

individu

alsidentifiedwith

variations

inMBD

genes

Variant

Fam

ilyIndividu

alAllele

Age

atexam

aAge

towalka

1st

words

aPhrase

speech

aVerbal

VABS

Regression

Midlin

emov

ements

Seizures

Irregu

lar

breathing

Unu

sual

gait

Head

circum

ference

incm

(age

a )

MBD1R14

7L78

01Maleprob

and

G/A

126

1160

96Y

–Y

NN

NY

57.0

(114

)

Brother

(III:4)

G/A

4514

––

N68

NN

NY

Y52

.5(36)

Brother

(III:5)

G/G

6212

3648

Y–

NN

NN

N50

.5(21)

MBD1P40

1A79

78Fem

aleprob

and

C/G

9213

2272

Y63

YN

YN

Y–

MBD1R

428H

7992

Maleprob

and

G/A

4013

––

N–

NN

NN

Y52

.5(46)

MBD1K

558N

7947

Maleprob

and

A/T

140

1536

40Y

54N

NN

NN

53.2

(148

)

MBD1c.14

10G>A

7660

Maleprob

and

G/A

123

–36

54Y

35–

––

––

–

Identical

twin

brother

G/A

178

1148

57Y

31N

NY

NY

–

MBD1c.17

79-27C

>T

7948

Maleprob

and

C/T

192

1836

60Y

–N

NN

NY

–

MBD3R23

M79

74Maleprob

and

G/T

9812

78-

N43

NY

NN

N54

.0(91)

Halfbrother

G/T

4111

3636

Y93

NN

NN

N48

.26(18)

MBD3E28

1del

7730

Maleprob

and

+/del

209

--

--

--

--

--

-

Brother

(III:3)

+/del

176

–84

–N

–Y

NN

NN

–

MBD4E31

4fsX

316

7605

Fem

aleprob

and

+/del

142

1348

60N

–Y

NN

NY

53.0

(132

)

MBD4E34

6K77

10Maleprob

and

G/A

5617

3942

N72

NN

NN

Y55

.9(140

)

Brother

(II:2)

–60

––

––

––

––

––

56.1

(109

)

MBD4E34

6K77

85Maleprob

and

G/A

818

3636

Y–

NN

NN

N–

Brother

(II:2)

G/G

5310

36–

N–

YN

NN

Y–

Brother

(II:3)

G/G

4910

––

N–

YY

NN

Y–

MBD4E34

6K79

70Maleprob

and

G/A

3213

––

N73

YN

NN

N49

.5(31)

MBD4N46

7S79

41Fem

aleprob

and

A/G

109

1857

57N

34Y

NN

NN

56.0

(184

)

MBD4N46

7S79

21Maleprob

and

A/G

63–

––

Y48

––

––

––

End

ashthetraitwas

notevaluated

aAge

measuredin

mon

ths

Neurogenetics

and two novel variants—a deletion and a single nucleotidechange. Six of these variants result in a coding change. Ofspecial interest is the deletion, c.942_945delAAGA(E314fsX316), transmitted maternally in a single femaleproband in the African-American family no. 7605 (Fig. 2i).The affected daughter demonstrated regression in play,social skills, and communication to the extent of becomingnonverbal (Table 2). This alteration is predicted to cause aframeshift within exon 3 at amino acid 314, encoding twoaberrant amino acids and then a premature stop codon.While retaining the methyl-CpG-binding domain, thistruncation is expected to remove almost half of the protein’s580 amino acids including the DNA glycosylase domain.No Caucasian or African-American controls were identifiedwith the alteration.

Five out of the nine single nucleotide variants result inmissense changes within MBD4. The novel c.1400A>G(N467S) variant is interesting due to its location within thefunctional DNA glycosylase domain [24]. This alterationoccurred in two Caucasian singleton families, one beinginherited from mother to daughter (family no. 7941, Fig. 2j)and the other passing from father to son (family no. 7921,Fig. 2k). The novel variation changes an asparagine to aserine and was not found in 244 Caucasian controlindividuals. The remaining four missense changes occur atc.817G>A (A273T), c.1024T>C (S342P), c.1036G>A(E346K), and c.1073T>C (I358T). Each change alterseither the polarity or charge of the original residue. TheE346K alteration (rs140693) was found in three familiesand was absent in the DHPLC control DNA (Fig. 2l–n).While this variant is not concordant with disease in thefamilies, it is interesting that in family no. 7710, theunaffected sister who also inherited the E346K change wasfound to have a delayed speech acquisition. The S342P andI358T variants were each identified in controls, suggestingthat these alterations are likely not related to autism.

Discussion

While it is unlikely that every alteration identified in thisstudy is causative of autism, several variants appearespecially interesting in their relationship to this disease.Specifically, there are two alterations which may alter theamino acid sequence sufficiently to interfere with proteinfunction. The first is the R23M alteration in MBD3. Thisalteration falls within the methyl-CpG-binding domain ofthe protein and replaces the basic arginine with a nonpolarmethionine. While this amino acid is not completelyconserved in the other members of the MBD family,another basic amino acid, lysine, is found in the sameposition for the MeCP2, MBD1 and MBD2 proteins. Analteration of the charge at this position could interfere with

the function of the MBD domain. It is also interesting thatthe variation is inherited from the maternal grandmotherthrough a mother presenting with an anxiety disorder to twoaffected half brothers. This leaves open the possibility thatthere is a sex-specific effect for this variation.

The second variation of interest is E314fsX316 withinMBD4. This maternally inherited allele is predicted tocause a frameshift at amino acid 314, encode two aberrantamino acids, and then a stop codon, resulting in aprematurely truncated protein. The mutant protein wouldcontain the MBD domain, but the DNA glycosylasedomain would be absent. While it is likely that much ofthe abnormal RNA would be removed by nonsense-mediated decay [56], it is conceivable that any truncatedprotein generated with an intact MBD domain may be ableto interfere in the normal function of all of the MBDproteins. Indeed, Bader and colleagues recently demon-strated in vitro that a MBD4 protein truncated at amino acid313 could inhibit the glycosylase activity of the wild-typeMBD4 protein as well as uracil DNA glycosylase [57]. Analternative scenario is that an irregular phenotype couldsimply result from haploinsufficiency of the wild-typeprotein. This change warrants further investigation.

There are additional examples of amino acid changesdetected in the MBD proteins that could result in functionalchanges. There are three nonsynonymous changes, MBD1R147K, MBD1 K558N, and MBD4 E346K, which eitherremove or create lysine residues. Lysine is an amino acidsubject to dramatic posttranslational modifications includingmethylation, acetylation, sumoylation, ubiquitination, andneddylation [58]. The MBD proteins have demonstrated thatthey are susceptible to such modifications [59–62]. Alteringthis key amino acid could dramatically influence the functionof a protein, affect protein regulation, or interfere withprotein–protein interactions. Another example of a variationthat may affect posttranslational modification is S342P inMBD4. The disruption of a serine could remove a potentialsite of phosphorylation or glycosylation. Likewise for thecreation of a serine site, as occurs in the MBD4 N467Svariation within the glycosylase domain. Other variantswhich alter the polarity and charge of the residues, as seenin missense mutations in MBD1 and MBD4, could interferewith a binding site on the protein or cause misfolding.

It is also possible that some of the detected MBDvariations could result in RNA processing errors bydestroying splice sites, creating cryptic splice sites, or evenchanging the ratios of alternative isoforms by eliminating orgenerating binding sites for splicing factors (SupplementaryTable 3). One example of this is the MBD1 intronic variantc.1585-12T>C, just 12 bp upstream of exon 14. Thealteration causes a change in a highly conserved nucleotide,potentially breaking a consensus site for the SRp55 splicingfactor and creating new binding sites.

Neurogenetics

Surprisingly, there appears to be a higher occurrence ofvariations with potential effects on amino acid functionwithin the African-American autistic patients as comparedto Caucasian patients. African-Americans comprise only 31of the 226 (13.7%) families participating in this study, yetthese families represent a disproportionate number of thosewith nonsynonymous alterations (24%). This could be due,in part, to the higher genetic diversity found in Africanpopulations [63, 64]. Furthermore, missense changes thatwere most abundant varied between the ethnicities, with theMBD4 A273T (rs10342) change being the most commonfor Caucasian families and the MBD4 S342P (rs2307289)alteration being predominantly found in African-Americanfamilies. These results are consistent with the HapMap datafor these known SNPs in the AA and CA populations. Inaddition, four of the five deletions identified in this studyoccurred only in African-American families, three of whichwere not identified in any control individuals. The remainingMBD3 GCG deletion was identified in both AA and CAaffected and controls while the MBD3 GAG deletion wasfound exclusively in AA affected and controls, suggestingthat both deletions are most likely not associated withautism. Nonetheless, these racial differences in variantfrequencies add to previous findings in which African-Americans with autism showed subtle differences in pheno-type and association to SNPs in GABAergic genes [65, 66].

In the families with nonsynonymous mutations, psychi-atric disorders were reported (on family history interview)in first degree relatives who carry a variation. This isconsistent with previous reports of an increased prevalenceof neuropsychiatric disorders including schizophrenia,depression, and anxiety in the parents of autistic children[67–70]. Inheritance of MBD variants from parents diag-nosed with psychiatric disorders was found in familiesnumbers 7978, 7974, 7941, and 7785 (Fig. 2d, g, j, m).Moreover, two families have additional offspring who carrythe same variant found in their affected sibling and haspsychiatric problems without a diagnosis of autism. Thesister in family no. 7978 was diagnosed with anxiety andspeech delay, and a brother in family no. 7710 alsopresented with delayed speech (Fig. 2d, l). This lendssupport to the idea that the variants identified here couldcontribute to autism and other psychological defects.Indeed, some variants were identified in both affected andunaffected individuals within a family and multiplexfamilies were not always concordant for the variant beingidentified. In some instances, as outlined above, the variantsappear in people negative for autism, but demonstratingclinical characteristic found in autism. Such is the case forfamily no. 7974 where a mother with anxiety/panic disorderpasses along the MBD3 R23M variant to two autistic halfbrothers. The variation in phenotypic presentation may alsobe related to incomplete penetrance of genetic factors. This

can be demonstrated by the fact that there is an increase inthe concordance of autism in monozygotic twins ascompared to dizygotic twins, but there is not 100%concordance in MZ twins [4–6]. Therefore, althoughgenetics plays a strong role in autism etiology, it is notthe sole component influencing presentation of disease.Lack of perfect concordance should not be used as ameasure to rule out causal mutations within complexdiseases. Other factors such as environmental or epigeneticevents may also contribute to the intricacy of autism.

It is intriguing that we observed clinical similarities inselect patients to the Rett syndrome phenotype. While, bydefinition, Rett syndrome and autism overlap clinically,there are features common to Rett syndrome which are lessfrequent in the general autistic population. For example,regression is found in most classical manifestations of Rett,but only in a minority of autistic individuals [1, 17]. Yet,we identified eight cases with a regression in language, insome cases accompanied by losses in social responsivenessor play skills. Furthermore, select individuals were reportedto have irregular breathing, unusual gait, and seizures, alltraits are commonly found in Rett patients. Also of notewere two individuals identified with unusual midlinemovements, reminiscent of the classic Rett feature wheregirls clasp their hands together at the center of their body[15, 16]. Perhaps, these patients are presenting clinically ina manner more similar to Rett individuals due to thealterations identified in this study within the MBD genes. Itis also interesting that the MBD1 R147K alterationidentified in family no. 7801 was found in the two moreseverely affected of three autistic brothers as well as anunaffected sister. This could suggest that this alterationresults in a sex-specific effect that may contribute to theautism phenotype without being the sole causative factor.

While there is mounting evidence from human andmouse studies implicating MBD genes in autism, fewpatients have been identified with alterations in MBD1,MBD2, MBD3, and MBD4 [28, 29, 39, 45, 48, 71]. Thisseeming inconsistency may be clarified by evaluating themurine phenotypes. While the Mbd1−/− mouse exhibitsdeficiencies in a wide range of social behaviors, it is able tolive an almost normal life span [48]. In contrast, Mecp2−/−

mice have a severe phenotype that is usually lethal by10 weeks of age [39, 71]. Observing the relatively mildphenotype of the Mbd1 null mice in relation to the Mecp2null mice suggests that there may be an ascertainment biasin identifying patients with recognized MECP2 mutations.MBD1 mutations may lead to such a mild phenotype thatthey rarely present as clinical abnormalities [48].

In summary, we identified genetic alterations at 46unique loci in the MBD genes. Twenty-one were previouslyrecognized SNPs, while 25 represent novel variations (fiveinsertion/deletions and 20 SNPs). Although the majority of

Neurogenetics

alterations were synonymous or noncoding variants, weidentified ten nonsynonymous changes in MBD1, MBD3,and MBD4, the deletion of a single amino acid in MBD3,and a frameshift mutation in MBD4 that is predicted totruncate almost half of the protein. These results suggestthat rare variants in these genes may contribute to autismsusceptibility. Further studies utilizing larger cohorts will berequired to determine the potential contribution of thesegenes to the autism clinical spectrum, but evidence to dateindicates that the entire MBD family may play a role inautism etiology.

Acknowledgements We are grateful to the patients with autism andtheir families for their participation, without whom, this work wouldnot be possible. A subset of the participants was ascertainedwhile several of the authors (RR, IK, MYR, ERM, MLC, MAPV,and JRG) were at Duke University. We also acknowledge fellow labmembers for their critical review of this manuscript. This research wassupported by grants from the National Institutes of Health (NS26630,NS36768, and MH080647), Autism Speaks, and by a gift from theHussman Foundation. RR is supported by the Ramon y Cajalprogram.

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