Cognitive and behavioural genetics J Med Genet: first published as 10.1136/jmedgenet-2019-106080 on 14 August 2019. Downloaded from http://jmg.bmj.com/ on April 26, 2021 at Univesity Autonomous of Barcelona. Original article Genetic factors contributing to autism spectrum disorder in Williams-B ­ euren syndrome Marta Codina-­Sola,1,2 Mar Costa-­Roger,1 Debora Pérez-­García,1 Raquel Flores,1 Maria Gabriela Palacios-­Verdú,1,3 Ivon Cusco ‍ ‍ ,1,2 Luis Alberto Pérez-­Jurado ‍ ‍ 1,4 ►► Additional material is ABSTRACT remaining 3% of patients with WBS show atypical published online only. To view, Background The hallmark of the neurobehavioural deletions mediated by other mechanisms.3 4 The please visit the journal online phenotype of Williams-­Beuren syndrome (WBS) is (http://d​ x.​doi.o​ rg/​10.​1136/​ WBS multisystemic phenotype is characterised by jmedgenet-​2019-​106080). increased sociability and relatively preserved language cardiovascular disease, distinctive facies, connec- skills, often described as opposite to autism spectrum tive tissue abnormalities and growth and endocrine For numbered affiliations see disorders (ASD). However, the prevalence of ASD in WBS alterations, among others.2 5 The main neurobe- end of article. is 6–10 times higher than in the general population. havioural hallmarks are mild to moderate intellec- We have investigated the genetic factors that could tual disability (ID), hypersociability and relative Correspondence to Dr Ivon Cusco, Clinical and contribute to the ASD phenotype in individuals with WBS. language preservation.6 Interestingly, the reciprocal Molecular Genetics Area, Vall Methods We studied four males and four females with duplication of 7q11.23 (OMIM#609757) results Hebrón Hospital Research WBS and a confirmed diagnosis of ASD by the Autism in a phenotype with speech delay, language impair- Institute (VHIR), Hospital de Diagnostic Interview-­Revised. We performed a detailed ment, milder learning problems and clear social la Vall d’Hebron, Hospital Vall molecular characterisation of the deletion and searched interaction deficits often associated with autism d’Hebron, Barcelona 08035, Spain; i​ cusco@​vhebron.​net for genomic variants using exome sequencing. spectrum disorder (ASD).6 7 Results A de novo deletion of 1.55 Mb (6 cases) or Due to their associated behavioural manifes- MC-­S and MC-­R are joint first 1.83 Mb (2 cases) at 7q11.23 was detected, being in tations, ASD and WBS have often been described authors. 7/8 patients of paternal origin. No common breakpoint, as diametric opposite disorders, although this Protected by copyright. Received 13 February 2019 deletion mechanism or size was found. Two cases consideration is an oversimplification of both Revised 11 July 2019 were hemizygous for the rare T allele at rs12539160 phenotypes.8 9 Moreover, several cases of WBS Accepted 16 July 2019 in MLXIPL, previously associated with ASD. Inherited with comorbid ASD have been reported and some Published Online First 14 August rare variants in ASD-­related or functionally constrained 2019 authors have suggested that ASD features should genes and a de novo nonsense mutation in the UBR5 be considered as part of the WBS phenotype.7–18 gene were identified in six cases, with higher burden in A recent meta-­analysis of the comorbidity of ASD females compared with males (p=0.016). features in several well-­defined genetic syndromes Conclusions The increased susceptibility to ASD in concluded that the prevalence of ASD features patients with WBS might be due to additive effects of among WBS individuals is as high as 12%.19 There- the common WBS deletion, inherited and de novo rare fore, the frequency of ASD is 6–10 times higher in sequence variants in ASD-­related genes elsewhere in the individuals with WBS than in the general popula- genome, with higher burden of deleterious mutations tion, a striking finding considering the typical WBS required for females, and possible hypomorphic variants neurocognitive profile. in the hemizygous allele or cis-­acting mechanisms on Potential modifiers of the common neurobe- imprinting. havioural phenotype of WBS include: (1) cis-­acting mechanisms due to variable breakpoints altering flanking genes, although most patients described Introduction present common deletions of identical size3 17; Williams-­Beuren syndrome (WBS, OMIM#194050) (2) trans-­acting factors present in the non-­deleted is a rare neurodevelopmental disorder resulting hemizygous allele; (3) genetic mutations and/or from an heterozygous deletion of 25–27 genes at structural variants elsewhere in the genome; (4) chromosome 7q11.23, estimated to affect approx- environmental events with or without epigenetic imately 1 in 7500 individuals.1 The WBS locus has effects, including medical complications during a complex genomic architecture with a single copy development and early life. All these factors can region flanked by three large segmental duplica- have additive effects acting on a sensitised back- © Author(s) (or their tions, each composed of three major blocks (A, B ground caused by haploinsufficiency at the WBS employer(s)) 2019. Re-­use and C), located in the centromeric (c), medial (m) locus.10 17 Two individuals with WBS and co-­occur- permitted under CC BY-­NC. No and telomeric (t) sides.2 Two types of recurrent ring ASD previously reported presented hyperse- commercial re-­use. See rights and permissions. Published rearrangements promoted by non-­ allelic homol- rotonaemia and were homozygous for the short (s) by BMJ. ogous recombination (NAHR) can result in WBS allele in the promoter of the serotonin transporter syndrome. The most frequent is a 1.55 Mb deletion SLC6A4 (5-­HTTLPR), suggesting a possible modi- To cite: Codina-­Sola M, Costa-­Roger M, occurring between the medial and centromeric B fier role of this locus.14 17 Pérez-­García D, et al. blocks in 87% of cases. Around 10% of patients In the present work, we have investigated the J Med Genet present a larger 1.83 Mb deletion due to a crossing genetic factors that could contribute to the ASD 2019;56:801–808. over between centromeric and medial A blocks. The phenotype of eight individuals with WBS and Codina-­Sola M, et al. J Med Genet 2019;56:801–808. doi:10.1136/jmedgenet-2019-106080 801 Cognitive and behavioural genetics J Med Genet: first published as 10.1136/jmedgenet-2019-106080 on 14 August 2019. Downloaded from http://jmg.bmj.com/ on April 26, 2021 at Univesity Autonomous of Barcelona. controls. The nature of the changes was assessed by PolyPhen and Condel (http://​bg.​upf.​edu/​fannsdb/) protein effect predic- tion algorithms. For CNV detection, we applied ExomeDepth and compared our samples with a matched aggregate reference set of 248 house exomes captured and sequenced using the same in-­ protocol. CNVs were filtered based on their overlap with vari- ants previously described in the Database of Genomic Variants and DECIPHER.20 Rare variant analysis and validation We selected exonic variants with a minor allele frequency (MAF) lower than 0.002 according to several databases (previously Figure 1 Strategy followed for the identification of second-­hit genetic mentioned) for heterozygous variants following a dominant factors. DGV, Database of Genomic Variants; LoF, loss of function; MAF, inheritance model. For homozygous or compound heterozy- minor allele frequency; SNV, single nucleotide variant. gous variants, under a recessive inheritance model, we selected a MAF ≤0.01. Since second-­hit variants would act in the presence of a major hit and would not be expected to solely cause ASD, comorbid ASD. We completed a detailed molecular character- they might be present in the general population and inherited isation of the deletion, genotyped the reported polymorphism from unaffected progenitors. Moreover, given the high degree at SLC6A4 and performed a genome-­wide unbiased search of of genetic heterogeneity of ASD, they could affect hundreds of second-­hits by exome sequencing following the strategy depicted genes. Taking into account the prevalence of ASD in individuals in figure 1. with WBS and the large number of genes involved, we reasoned that the frequency of each individual variant should be relatively Methods rare and, consequently, set our MAF threshold for homozygous Patient selection or compound heterozygous variants at ≤0.01. From a cohort of 122 individuals with a diagnosis of WBS To validate variants and perform segregation studies in and confirmed 7q11.23 deletion by molecular techniques, we parental samples, we used Sanger sequencing by capillary elec- Protected by copyright. selected four males and four females aged 6–31 years with an trophoresis (ABI PRISM 7900HT, Applied Biosystems, Foster associated diagnosis of ASD. The diagnosis was based on the City, California, USA). Primers were designed with the PRIMER3 direct observation by a trained psychologist and clinicians and program (http://​​www.​​bioinformatics.​​nl/​​cgi-​bin/​​primer3plus/​​ confirmed in all of them using the Autism Diagnostic Interview-­ primer3plus.​​cgi/​ ) and PCR reactions were carried following Revised (ADI-­R). Written informed consent was obtained from standard conditions. all parents or legal caregivers. Epistatic effects of rare variants 7q11.23 deletion characterisation To study the putative contribution of epistatic effects of rare Blood samples from probands and parents were obtained and SNVs on gene expression deregulation in WBS, we compiled a genomic DNA was extracted using the Puregene DNA Purifica- list of genes previously altered in WBS by various mechanisms, tion Kit (Gentra Systems, Big Lake, Minnesota, USA). The size including transcriptional dysregulation, differential methylation and parental origin of the deletion was established by the analysis and the direct GTF2I targets. We performed a systematic litera- of multiple ligation-­dependent probe amplification and several ture review and selected only high-­quality studies done in human single and multiple-­ copy microsatellites. Refined mapping of subjects, obtaining 2251 candidate genes (online supplementary deletion breakpoints was also performed by quantitative analysis table 1).21–24 of paralogous sequence variants as previously described.3 Analysis of ASD susceptibility loci SLC6A4 genotyping To study the potential contribution of common variants, we The polymorphism in the serotonin transporter promoter selected loci previously associated with ASD by Genome-­Wide SLC6A4 (5-­HTTLPR) was genotyped by PCR and agarose gel Association Studies (GWAS). Variants containing the term electrophoresis, using primers previously described.17 ‘Asperger’ or ‘Autism’ were extracted from the Genome-­Wide Repository of Associations between SNPs and phenotypes Exome sequencing and analysis (GRASP) database V.2.0 (online supplementary table 2).25 Allele Exomes were captured using the SureSelect Human All Exon V5 frequency in the WBS cohort was then compared with non-­ capture kit (Agilent, Santa Clara, California, USA) and libraries Finnish European (NFE) population data from ExAC by Fisher’s were sequenced on an Illumina MiSeq platform. Paired-­ end exact test and q-­value and false discovery rate (FDR) values were sequences were obtained with a read length of 250 bp. calculated using the R package qvalue.26 To avoid population Mapping, variant calling and filtering were performed using stratification, ExAC allele frequencies were compared with that BWA and GATK’s standard parameters. The hg19 human reported in Spanish Variant Server, including data from 578 genome reference version was used. Variant annotation was Spanish individuals (http://​csvs.​babelomics.​org/). performed using ANNOVAR (http://www.​ openbioinformatics.​ org/​annovar/), considering the variant frequency in control data- Results bases: dbSNP137 (http://www.​ncbi.​nlm.​nih.​gov/​SNP/), ExAC Clinical characteristic of selected patients (http://​exac.​broadinstitute.​org/), Kaviar (http://​db.​systemsbi- The study was performed in eight Spanish patients with WBS ology.​net/​ kaviar/) and an in-­ house database of 248 Spanish (four males and four females), which had periodic follow-­up in 802 Codina-­Sola M, et al. J Med Genet 2019;56:801–808. doi:10.1136/jmedgenet-2019-106080 Cognitive and behavioural genetics J Med Genet: first published as 10.1136/jmedgenet-2019-106080 on 14 August 2019. Downloaded from http://jmg.bmj.com/ on April 26, 2021 at Univesity Autonomous of Barcelona. Table 1 Clinical characteristics of the WBS individuals with associated ASD WBS1 WBS2 WBS3 WBS4 WBS5 WBS6 WBS7 WBS8 Birth year 1987 1985 2000 2006 2002 2008 2001 2010 Gender F M M M F M F F Relevant family history No No Yes* No No Yes† No No Age at WBS diagnosis 7y 3y 5m 3m 3m 2y 5y 1y Age at ASD diagnosis 4y 10 y 6y 5y 12 y 5y 3y 5y ADI-­R    A 22 11 11 22 14 10 19 18  B (V) – 7 4 – – – – –  B (NV) 10 – – 12 7 4 14 9  C 8 4 6 8 8 6 9 5  D 5 4 1 5 3 4 4 5 IQ (WISC-­R)     40   <40 41 <40   Other neurological symptoms   ADHD ADHD   Behavioural Epilepsy, ADHD   ADHD problems Cardiovascular disease ND SVAS Coarctation of SVAS SVAS SVAS SVAS ND aorta Endocrine abnormalities ND ND ND ND Early puberty ND Early puberty Subclinical hypothyroidism ADI-­R scores: A. Social interaction (cut-­off: 10); B. Communication and language, (V): verbal (cut-­off: 8), (NV): non-­verbal (cut-­off: 7); C. Restricted and repetitive behaviours (cut-­ off: 3); D. Developmental alterations earlier than 36 months (cut-­off: 1). *Paternal aunt with ASD and severe ID. †Father committed suicide. ADHD, attention deficit hyperactivity disorder; ADI-­R, Autism Diagnostic Interview-­Revised; ASD, autism spectrum disorder; F, female; IQ, intellectual quotient; M, male; m, month; ND, not detected by standard testing; SVAS, supravalvular aortic stenosis; WBS, Williams-­Beuren syndrome; WISC-­R, Wechsler Intelligence Scale for Children-­Revised; y, year. Protected by copyright. a multidisciplinary clinic and complete neurobehavioural evalu- and all but one (7/8) were of paternal origin (table 2). The most ation due to a comorbid diagnosis of ASD. Their main clinical common 1.55 Mb deletion was found in 6/8 patients, whereas characteristics are summarised in table 1. All patients fulfilled 2/8 carried the larger recurrent 1.83 Mb deletion mediated by criteria in the social interaction and restrictive behaviour NAHR between A blocks (figure 2). In two patients (WBS1 and domains while two of them (WBS2 and WBS3) did not reach the WBS3), the common 1.55 Mb deletion had been mediated by an verbal communication domain threshold. Family history of ASD inversion in the transmitting progenitor. or psychiatric disorders was positive on the paternal side in two Since GTF2IRD2 has been postulated as a possible modulator cases (WBS3 and WBS6) and negative in the others (table 1). of WBS cognitive phenotype,27 we analysed the deletion break- points to assess the number of functional copies of this gene Detailed characterisation of the 7q11.23 deletion in the patients (figure 2). As expected, the deletions mediated To define potential cis-­acting factors in the deleted allele, we by A blocks included the entire medial B block with a copy of characterised all deletions at the molecular level including deter- GTF2IRD2, resulting in a loss of one functional copy (1M/2T: mination of the parental origin. All rearrangements were de novo 1 medial (GTF2IRD2)/2 telomeric (GTF2IRD2B)). The two Table 2 Summary of the main genetic findings per patient Hemizygous 7q11.23 deletion allele Genome-­wide Genome-­wide rare LoF SNVs Individual Origin GTF2IRD2 Size (Mb) SNVs CNVs ASD candidate genes LoF intolerant genes WBS1 Pat 1M/3T 1.55 WBS2 Pat 2M/2T 1.55 T at rs12539160 WBS3 Pat 1M/3T 1.55 SIK1 partial dupPI WBS4 Pat 1M/2T 1.83 CC2D1A c.1357-­2A>CPI WBS5 Pat 2M/2T 1.55 PYHIN1 p.(Arg373*)MI SEC24C c.2682+2A>GMI UBR5 p.(Arg633*)DN WBS6 Pat 1Ch/1M/2T 1.55 T at rs12539160 AGAP1 c.1051-­2A>TPI WBS7 Mat 2M/2T 1.55 DUSP22 delNM USP45 p.(Glu220*)MI CXXC1 p.(Ser222Leufs*7)NM PI WBS8 Pat 1M/2T 1.83 PIK3CG p.(Glu14Glyfs*147) MED26 c.147+2T>CPI EPHB1 p.(Ser435ProfsTer13)MI *Stop codon. Ch, chimeric-­type copies; DN, de novo; del, deletion; dup, duplication; LoF, loss of function; M, medial-­type copies; MI, maternally inherited; Mat, de novo, maternal chromosome; NM, not maternal (father not available); PI, paternally inherited; Pat, de novo, paternal chromosome; T, telomeric-­type copies; WBS, Williams-­Beuren syndrome. Codina-­Sola M, et al. J Med Genet 2019;56:801–808. doi:10.1136/jmedgenet-2019-106080 803 Cognitive and behavioural genetics J Med Genet: first published as 10.1136/jmedgenet-2019-106080 on 14 August 2019. Downloaded from http://jmg.bmj.com/ on April 26, 2021 at Univesity Autonomous of Barcelona. synonymous SNVs with no functional effect, we remained with two non-­ synonymous variants in genes MLXIPL and TBL2 predicted as tolerant by various protein effect prediction algo- rithms ((SIFT (sorting intolerant from Tolerant), PolyPhen (Polymorphism Phenotyping) and Condel)). No rare variants were identified at GTF2I, NCF1 and GTF2IRD2, the genes in the flanking segmental duplications. Genome-wide analysis of rare variants Copy number variants We also studied the presence of additional rearrangements that could explain the autistic symptoms in our cohort. In addition Figure 2 Schematic representation of the Williams-­Beuren syndrome to the WBS 7q11.23 deletion, we observed an average of 25 (WBS) locus, showing the two most common deletions and the gene CNVs per patient ranging from 170 bp to 334 kb. None of the content of the B block. The B block contains three genes: GTF2I, NCF1 additional rearrangements overlapped with known genomic and GTF2IRD2. Whereas GTF2I and NCF1 have a single functional copy disorders or was previously associated with neurodevelopmental located at the medial B block, GTF2IRD2 has two functional copies, disorders and all overlapped with previous CNVs described in located at the medial (GTF2IRD2) and telomeric (GTF2IRD2B) B blocks. The the general population.29 Only two CNVs comprised ASD candi- 1.55 Mb deletion is mediated by B blocks and results in a chimeric medial-­ date genes (SIK1 and DUSP22) (online supplementary table 3).30 centromeric block, with the number of functional copies of GTF2IRD2 and The CNV involving SIK1 was a partial duplication affecting the NCF1 depending on the deletion breakpoint. In contrast, the 1.83 Mb last two exons of the gene, paternally inherited in patient WBS3. deletion is mediated by A blocks, resulting in the loss of the medial and The heterozygous deletion completely containing DUSP22 was centromeric B blocks with functional copies of GTF2IRD2 and NCF1. found in patient WBS7 and not in her mother, but paternal sample was unavailable. patients with inversion-­mediated deletions had a 1M/3T geno- type, originated from the loss of a medial copy and a gain of a telomeric copy of GTF2IRD2. Among the patients with 1.55 Mb Rare single nucleotide variants Protected by copyright. deletions, three had breakpoints before GTF2IRD2, with no We prioritised deleterious variants present in a list of candi- change in the number of functional copies (2M/2T), whereas in date genes from -­Simons Foundation Autism Research Initiative one patient the breakpoint occurred within GTF2IRD2, creating (SFARI) (n=791) (online supplementary table 3) and/or highly a chimeric copy (Ch) between the medial gene and the centro- constrained genes, as previous studies have shown that genes meric pseudogene (1Ch/1M/2T) (table 2). The breakpoints also predisposing to ASD carry a low burden of disrupting mutations affect the number of NCF1 copies, by either deleting or not one in the general population.30 31 Constrained genes were defined of the functional copies. as those with a probability of being loss of function (LoF) intol- erant (pLI >0.9) according to ExAC.32 LoF and missense vari- Hemizygous variants in the 7q11.23 region ants predicted as damaging by both SIFT and PolyPhen were To look for trans-­acting factors in the 7q11.23 allele present in considered deleterious variants. hemizygosity, we analysed the entire captured region within the We detected a total of 38 rare deleterious SNVs in 34 candi- WBS common deletion locus (chr7:72 700 000–74 250 000) date genes (online supplementary table 4). All LoF variants looking for over-­represented rare and common variants, shared (n=6) were validated and segregation studies were performed haplotypes and rare deleterious SNVs. For over-­represented on all samples, showing a de novo variant in the UBR5 gene variants, we compared allele frequencies of all described in case WBS5 (table 2). De novo variants in the UBR5 gene, hemizygous variants (n=32) in our cohort to those reported two missense and one LoF, have been previously described in in ExAC for European population. Variants with significantly ASD individuals.33–35 WBS5 is a severely affected female who different frequencies in ExAC and Spanish Variant Server were presented an obsessive ritualistic behaviour, hypersensitivity to excluded to avoid population stratification. Due to the small noise and physical stimuli, had not developed language and did sample, none of the variants reached statistical significance, but not show eye contact. we identified a nearly significant association (p=0.076) with As for highly constrained genes, excluding candidate genes, rs12539160, which was present in two individuals (WBS2 and we detected 68 SNVs in 65 genes, including four LoF mutations WBS6). This synonymous variant located near an exon-­intron (table 2). All genes selected on the basis of being LoF intolerant boundary of MLXIPL had been previously associated with but harbouring a LoF in our cohort were brain-­expressed and ASD in a GWAS.28 Taking advantage of the hemizygosity of some had been previously associated with psychiatric and neuro- SNPs in the deleted single-­copy region, we extracted phased developmental disorders, such as SEC24C, CXXC1 and EPHB1, haplotypes in this interval to study if a common haplotype was which was mutated in two patients with WBS.36–39 shared between individuals. Two linkage disequilibrium blocks were identified from rs1128349 to rs13227841 (DNAJC30 to WBSCR28) and from rs17851629 to rs2074667 (located Epistatic effects of rare variants in GTF2IRD1). Allele frequencies of the tag markers did not To study if second-­hit variants could be disrupting genes already differ significantly from those in the general population and altered in WBS and act by an epistatic effect, we intersected a no common shared haplotype was identified in our cohort. In manually curated list of genes with altered expression in WBS addition, we looked for rare SNVs in the single-­copy region of with the list of mutated genes (figure 3). The results showed the WBS locus. Only hemizygous variants with a MAF <0.01 that 20 of mutated genes were also altered in WBS. Two of them were selected. After filtering for exonic variants and excluding harboured a LoF in our cohort: AGAP1 and EPHB1 in two cases. 804 Codina-­Sola M, et al. J Med Genet 2019;56:801–808. doi:10.1136/jmedgenet-2019-106080 Cognitive and behavioural genetics J Med Genet: first published as 10.1136/jmedgenet-2019-106080 on 14 August 2019. Downloaded from http://jmg.bmj.com/ on April 26, 2021 at Univesity Autonomous of Barcelona. over-­represented allele corresponded to the risk allele, whereas it was the protector allele for the 3 remaining. Three of the markers had been previously described as Expression Quantita- tive Trait Loci (eQTL) in brain, with rs2275477 associated with increased expression of OSCP1 and rs4823086 and rs5749088 with transcript RP1.130H16.16/CCDC157.41 We also identified a nominally significant increase in the frequency of rs2135720, a non-­synonymous SNP within PCDH15 associated with lipid traits.42 Finally, we also genotyped two promoter variants at SLC6A4 not captured in exome data that had been proposed as modifiers for the phenotypic outcome in WBS in two patients with autistic symptoms and hyperserotonaemia. In our cohort, the genotype frequencies were similar to those found in population of Euro- pean origin, with 3/8 individuals homozygous for the major long variant (l), 4/8 presenting an heterozygous genotype (l/s) and 1/8 Figure 3 Overlap between candidate and functionally constrained genes individual being homozygous for the s allele. mutated in our cohort and genes altered in Williams-­Beuren syndrome (WBS). Discussion Burden of rare deleterious mutations, increased in females We have performed a genome-­wide comprehensive analysis to In total, six patients with WBS with ASD carried, in addition investigate possible genetic factors in eight individuals presenting to the specific de novo 7q11.23 deletion, one to three strong with both diagnoses of WBS and ASD. To date, the analysis of candidate rare genetic variants (CNV and/or SNV) that could exome data failed to reveal genetic variants that could explain contribute to the ASD phenotype, either inherited or de novo the variance of the social behaviour phenotype in a cohort of 85 (table 2). patients with WBS without comorbid ASD,43 and the only locus Since ASD is four times more prevalent in males than females, suggested to act as a modifying factor was the serotonin trans- it has been suggested that females have a higher risk threshold porter SLC6A4 (5-­ HTTLPR), based on two individuals with Protected by copyright. and require a higher genetic load to develop the disorder.40 WBS and ASD who had hyperserotonaemia and were homozy- To examine if females in our cohort carried a higher genetic gous for the ss polymorphism at the gene promoter.17 However, burden, we compared the frequency of rare deleterious vari- the results in our cohort show genotype frequencies similar to ants in ASD candidate genes between males (n=4) and females those of the general population and do not support a role of (n=4) (figure 4). The results showed a statistically significant SLC6A4. increase (p=0.016) in the number of total mutations per patient The characterisation of the deletion showed no atypical rear- − − in females (‍x = 6‍, SD=1.7) compared with males (‍x = 3‍, rangements and different breakpoints in the patients, excluding SD=1.2). This effect was not seen when comparing the burden the alteration of flanking genes as a major cause. In the studied of all rare variants between genders (p=0.51). patients the deletion had originated in the paternal allele in seven of eight, suggesting that epigenetic control mechanisms Association study with candidate ASD genes could influence ASD risk in WBS. This parental bias was not To study the potential role of common variants, we analysed the present in our larger cohort of WBS without comorbid ASD loci previously associated with ASD and compared their varia- (n=374 trios) as the origin of the deletion was 50% maternal tion frequency in our cohort with that of NFE ExAC popula- and 50% paternal. There are some evidences supporting the tion. A total of 645 single-­nucleotide markers located in coding imprinting of the 7q11.23 region based on expression of FKBP6 regions were analysed, of which only 13 obtained a significant and GTF2I.44–46 However, in a recent study of six patients with p value (p<0.05) and none passed FDR correction. For 10 of WBS and ASD, the deletion was of maternal origin in four out of the significantly over-­represented SNVs in our cohort, the six patients.47 Collectively, there is suggestive evidence regarding a possible role of genetic imprinting, but not enough data to confirm it, which demands characterisation of the deletion and parental origin in additional individuals with WBS and ASD to confirm whether imprinting can influence phenotypic variability. Besides deletion origin and breakpoints, hemizygous variants unmasked in the deleted region could also act as second-­hit modifying factors. The detailed analysis of SNVs did not reveal any common haplotype or any rare variant in the coding regions of GTF2I or GTF2IRD2. However, the analysis of common vari- ants revealed an over-­representation of a relatively rare hemizy- gous variant previously associated with ASD (rs12539160).28 It has a MAF of 0.06 in European population and of 0.03 in the Figure 4 Gender (male vs female) comparison of the average number Spanish population but it was found in two patients (0.25) of our of rare variants in Williams-­Beuren syndrome-­autism spectrum disorders cohort, representing an eightfold increase in frequency.32 While cases. Statistical significance in all tests was calculated using a two-­sided not meeting strict criteria for statistical significance (p=0.076), Student’s t-­test, excluding SNVs of the X and Y chromosomes from the this finding deserves additional investigation in future studies. analysis. (A) Deleterious variants in candidate genes. (B) All rare variants. In a recent study, exome data of 85 patients with WBS was anal- SNV, single nucleotide variant. ysed in order to search for common and rare variants that could Codina-­Sola M, et al. J Med Genet 2019;56:801–808. doi:10.1136/jmedgenet-2019-106080 805 Cognitive and behavioural genetics J Med Genet: first published as 10.1136/jmedgenet-2019-106080 on 14 August 2019. Downloaded from http://jmg.bmj.com/ on April 26, 2021 at Univesity Autonomous of Barcelona. account for the variance in the social behaviour of these patients.43 a more severe presentation than males. Further studies assessing Together with the genotyping of our cohort of non-­autistic WBS, the difference in prevalence and severity of ASD features this variant (rs12539160) was found with a frequency of 0.0573 between genders in individuals with WBS will help clarify if the among 157 WBS individuals which is similar to general popula- female protective effect has a role in the expressivity of disorders tion. Although this variant lies in the intronic region of MLXIPL, of full penetrance. mainly involved in lipid abnormalities, deregulation of the same Finally, we assessed the contribution of common variation by or other nearby gene either by this SNP or other risk variants in looking for over-­represented variants previously associated with linkage disequilibrium not detected by exome sequencing might ASD. Although none of the variants passed FDR correction, three contribute to ASD risk in WBS. markers with nominal p values had been previously described as We also studied genetic variation at a genome-­ wide level. eQTLs in brain and may have a direct functional effect. However, Regarding CNVs, no additional large (>500 kb) or pathogenic our study is limited by the fact that most common variation is rearrangement was identified. Likewise, previous studies eval- not covered by exome sequencing, as it resides in non-­coding uating second-­ hit CNVs in genomic disorders showed that regions. Currently, common variation seems to explain at least additional rearrangements were more frequent in disorders of 20% of ASD liability.52 Therefore, further studies regarding the variable expressivity than in syndromic entities. In fact, only 5% role of common variants in autism will provide a necessary basis of individuals with WBS carried a second event, similar frequency for future studies. than in the control population, suggesting that additional large In summary, our work represents a thorough assessment of CNVs may cause a more severe phenotype and/or be incompat- second-­ hit modifier genetic factors in individuals with WBS ible with life.48 However, relatively small CNVs altering ASD and associated ASD. Similar to previous reports, patients did candidate genes and therefore potential contributors to the ASD not differ in deletion breakpoints, discarding the role of atyp- phenotype were found in two patients, a partial duplication of ical rearrangements. However, in seven of eight the deletion the last two exons of SIK1, and a complete heterozygous dele- had originated in the paternal allele, a factor that had not been tion of DUSP22. addressed in previous studies. In addition, two individuals The analysis of rare deleterious SNVs on ASD candidate carried a relatively rare hemizygous variant previously associ- and functionally constrained genes uncovered several brain-­ ated with ASD, representing an eightfold increase with respect expressed genes that harbour LoF variants in our cohort. Among to the general population. Inherited rare variants and a de novo those, we detected a de novo stop mutation affecting UBR5, an nonsense mutation in ASD-­related or functionally constrained E3 ubiquitin-­protein ligase in a severely affected female. There- genes were identified in six cases, with higher burden in females Protected by copyright. fore, the presence of WBS and ASD in this individual is probably compared with males. Taken together, these results suggest that due to the co-­occurrence of two mechanistically unrelated de imprinting mechanisms, trans-­ acting factors in the remaining novo events, the WBS deletion and the UBR5 mutation. allele and inherited or de novo rare sequence variants elsewhere However, the increased prevalence of ASD features in WBS in the genome may play a role in the susceptibility to ASD in and the low frequency of de novo events suggest that other WBS. Therefore, similar factors influencing ASD risk in the genetic factors of smaller effect may be influencing the ASD risk general population also shape phenotypic variability in disorders in most cases. In fact, we have identified several rare variants in with full penetrance. ASD candidate genes. Two of the genes harbouring LoF muta- tions, EPHB1 and AGAP1, have been found deregulated in WBS, Author affiliations 1 suggesting a possible epistatic effect. AGAP1 was hypermethyl- Genetics Unit, Universitat Pompeu Fabra, Hospital del Mar Research Institute (IMIM), and Centro de Investigación Biomédica en Red de Enfermedades Raras ated in patients with WBS compared with individuals with the (CIBERER), Universitat Pompeu Fabra Departament de Ciences Experimentals i de la reciprocal duplication, which is associated with a phenotype of Salut, Barcelona, Spain language impairment, anxiety and increased risk of ASD and 2 Clinical and Molecular Genetics Area, Vall Hebrón Hospital Research Institute schizophrenia.24 49–51 EPHB1 was underexpressed in patients (VHIR), Hospital Vall d’Hebron, Barcelona, Spain 3 with WBS and individuals with atypical deletions and low IQ. Fundacio Dexeus Salut de la Dona, Barcelona, Spain 4 SA Clinical Genetics, Women’s and Children’s Hospital, South Australian Health Inherited mutations in those genes would result in further dereg- and Medical Research Institute (SAHMRI) and University of Adelaide, South ulation of the expression already altered by the WBS deletion21 Australian Health and Medical Research Institute, Adelaide, South Australia, and higher risk for a more severe phenotype. Australia Globally, six of the eight patients with WBS with ASD carried, in addition to the specific de novo deletion at the 7q11.23 locus, Acknowledgements The authors would like to thank the patients and their one to three rare genetic variants (CNV and/or SNV) altering families for their support. The authors would also like to thank Marcos López-­ Sánchez for his help with the data acquisition and handling. candidate genes that could contribute to the ASD phenotype. Consequently, a proportion of cases of WBS with associated Contributors MC-­S and MC-­R analysed the exome data, performed the experiments and drafted the manuscript. DP-­G and MGP-­V contributed with ASD could be explained by the same factors influencing ASD the clinical and phenotypic information. RF contributed with detailed molecular risk in the general population with a threshold model. Given the characterisation of deletions. IC and LAP-­J conceived the study and participated increased risk for ASD in WBS, the WBS deletion would act as a in the design and data interpretation, and helped in drafting the manuscript. All predisposing factor facilitating the role of other genetic (inher- authors read and approved the manuscript. ited or de novo) and/or environmental factors by acting on an Funding This work was funded by grants from the Spanish Ministry of Economy already sensitised background. and Competiveness (FIS PI16/00369 and PI1302481 cofunded by FEDER, and ’Programa de Excelencia María de Maeztu’ MDM-2014-0370), and the Generalitat Interestingly, the average number of rare deleterious vari- de Catalunya (2017SRG01974 and ICREA-­Acadèmia program). MC-­R had a ants in candidate genes was significantly higher in females than predoctoral fellowship of Ministry of Education, Culture and Sport (FPU16/06907) males, an effect not observed when considering all rare variants. and DP-­G had a predoctoral fellowship from the Instituto de Salud Carlos III Although our small sample size requires caution interpreting the (FI11/00656). results, this difference could support the higher risk threshold Competing interests LAP-­J is scientific advisor of qGenomics SL. The remaining requiring higher genetic load in females to develop ASD, or authors declare no conflict of interest. could just be explained by the fact that females in our cohort had Patient consent for publication Not required. 806 Codina-­Sola M, et al. J Med Genet 2019;56:801–808. doi:10.1136/jmedgenet-2019-106080 Cognitive and behavioural genetics J Med Genet: first published as 10.1136/jmedgenet-2019-106080 on 14 August 2019. Downloaded from http://jmg.bmj.com/ on April 26, 2021 at Univesity Autonomous of Barcelona. Ethics approval The study was approved by the Clinical Research Ethics 23 Adamo A, Atashpaz S, Germain P-­L, Zanella M, D’Agostino G, Albertin V, Chenoweth Committee of the Parc Salut Mar. J, Micale L, Fusco C, Unger C, Augello B, Palumbo O, Hamilton B, Carella M, Donti E, Pruneri G, Selicorni A, Biamino E, Prontera P, McKay R, Merla G, Testa G. Provenance and peer review Not commissioned; externally peer reviewed. 7Q11.23 dosage-­dependent dysregulation in human pluripotent stem cells affects Data availability statement All data relevant to the study are included in the transcriptional programs in disease-­relevant lineages. Nat Genet 2015;47:132–41. article or uploaded as supplementary information. 24 Strong E, Butcher DT, Singhania R, Mervis CB, Morris CA, De Carvalho D, Weksberg R, Open access This is an open access article distributed in accordance with the Osborne LR. Symmetrical dose-­dependent DNA-­methylation profiles in children with Creative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which deletion or duplication of 7q11.23. Am J Hum Genet 2015;97:216–27. permits others to distribute, remix, adapt, build upon this work non-­commercially, 25 Eicher JD, Landowski C, Stackhouse B, Sloan A, Chen W, Jensen N, Lien J-­P, Leslie R, and license their derivative works on different terms, provided the original work is Johnson AD. Grasp v2.0: an update on the genome-­wide Repository of associations properly cited, appropriate credit is given, any changes made indicated, and the use between SNPs and phenotypes. Nucleic Acids Res 2015;43:D799–D804. is non-­commercial. See: http://​creativecommons.​org/​licenses/​by-​nc/​4.​0/. 26 Bass J, Dabney A, Robinson D. qvalue: Q-­value estimation for false discovery rate control, 2015. 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