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High-resolution SNP genotyping platform identified recurrent and novel CNVs in autism multiplex families
Accepted Manuscript High resolution SNP genotyping platform identified recurrent and novel CNVs in Autism Multiplex families Laila Y. AlAyadhi, Jamil A. Hashmi, Muhammad Iqbal, Alia M Albalawi, Mohammad I. Samman, Nadra E. Elamin, Shahid Bashir, Sulman Basit PII: DOI: Reference:
S0306-4522(16)30570-X http://dx.doi.org/10.1016/j.neuroscience.2016.10.030 NSC 17390
To appear in:
Neuroscience
Accepted Date:
11 October 2016
Please cite this article as: L.Y. AlAyadhi, J.A. Hashmi, M. Iqbal, A.M. Albalawi, M.I. Samman, N.E. Elamin, S. Bashir, S. Basit, High resolution SNP genotyping platform identified recurrent and novel CNVs in Autism Multiplex families, Neuroscience (2016), doi: http://dx.doi.org/10.1016/j.neuroscience.2016.10.030
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High resolution SNP genotyping platform identified recurrent and novel CNVs in Autism Multiplex families Laila Y. AlAyadhi1, Jamil A. Hashmi2, Muhammad Iqbal3, Alia M Albalawi2, Mohammad I. Samman2,4, Nadra E. Elamin1, Shahid Bashir1,5, Sulman Basit2 1
KSU-Autism Research & Treatment Center, AL-Amodi Autism Research Chair, Department of Physiology, Faculty of Medicine, King Saud University, Riyadh, Saudi Arabia 2
Center for Genetics and Inherited Diseases, Taibah University Almadinah Almunawwarah, Saudi Arabia 3
Aging Research Chair, Department of Physiology, Faculty of Medicine, King Saud University, Riyadh, Saudi Arabia 4
College of Applied Medical Sciences, Taibah University Almadinah Almunawwarah, Saudi Arabia 5
Berenson-Allen Center for Noninvasive Brain Stimulation, Division of Cognitive Neurology, Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. Laila Y. AlAyadhi; [email protected] Jamil A. Hashmi; [email protected] Muhammad Iqbal; [email protected] Alia Albalawi; [email protected] Mohammad I. Samman; [email protected] Nadra E. Elamin; Shahid Bashir; [email protected] Sulman Basit; [email protected]
Corresponding Author: Sulman Basit, PhD Center for Genetics and Inherited Diseases, Taibah University Almadinah Almunawwarah, Saudi Arabia Email: [email protected] Contact: 00966535370209
Competing Interests: The authors declare that they have no competing interests
ABSTRACT Single nucleotide polymorphisms (SNP) - based genotyping using microarray platform is now frequently used to detect copy number variants (CNVs) in the human genome. Here, we report CNVs identified using Illumina HumanOmni 2.5 M oligonucleotide microarrays in 11 multiplex families with autism spectrum disorder (ASD) referred to Autism Research and Treatment Center (ART) and Madinah Maternity and Children Hospital (MMCH). Of the 11 families, 22 patients with ASD (all males) and their parents, were recruited for the present study. In total, 43 individuals were genotyped with high resolution array. Abnormal microarray results were seen in all 22 patients with ASD. A total of 17 shared CNVs were selected for further analysis. Out of these 17 CNVs, we discovered one novel CNV, previously not described, and 16 recurrent CNVs that overlap with the genomic imbalances defined in the autism database (AutDB), autism chromosome rearrangement database (ACRD) and Database of Genomic Variants (DGV). Recurrent CNVs includes 11 common and 5 rare CNVs. All rare CNVs are duplications except a 16kb deletion on chr2q36.3. Rare gain of copy numbers includes a 2kb duplication on chr9q21.13, an overlapping duplications of 107kb and 181kb on chrXp22.33 in 2 different families and a 10kb duplication on chr18q21.13. A novel loss of copy number on chr3q23 was found in four ASD cases. This CNV results in deletion of intron 2 of calsyntenin 2 (CLSTN2) encoding synaptic protein calsyntenin 2. CLSTN2 is expressed exclusively in brain, with high levels occurring in cortical gamma-aminobutyric acid (GABA)ergic interneurons and in medial temporal lobe regions. These results verify the diagnostic relevance of genome-wide small common and rare CNVs and provides further evidence of the high diagnostic yield of microarray for genetic testing in children with ASD. Key Words: Autism, Copy Number Variations, SNP array, Multiplex families
INTRODUCTION Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder marked by deficit in the social behaviour and language development, as well as restricted or stereotyped interests. Multiple evidence exists for the association between copy number variants (CNVs) and ASD. CNVs are thought to provide explanation for almost 5-10% of the ASD cases depending on the cohort examined, though, no single candidate genetic locus has been shown as an underlying factor in more than 1% of ASD cases [Devlin and Scherer, 2012]. Most studies aiming to detect CNVs in ASD have been carried out in cohorts of European ancestry [Marshall et al., 2008; Pinto et al., 2010; Pinto et al., 2014]. Recently, genome wide CNV analysis in patients with ASD have been carried out in Chinese population [Yin et al., 2016; Gazzellone et al., 2014; Chong et al., 2014], however, studies describing the genetic architecture in other populations, particularly Middle Eastern populations, are limited specifically in the era where clinical microarray testing continues to be adopted as the standard of care across medical genetic laboratories worldwide [Miller, 2010]. Identifying CNVs using SNP and CGH arrays has proven to be a rapid method to detect small and large CNVs associated with ASD [Gazzellone et al., 2014; Chong et al., 2014; Marshall et al., 2008; Pinto et al., 2010; Christian et al., 2008; Shen et al., 2010; Sanders et al., 2011]. Some highly penetrant risk genes (e.g., the SHANK, Neurexin, and Neuroligin family members) and CNV loci (e.g., 1q21.1, 15q13.3, and 16p11.2) are now known [Sebat et al., 2007; Pinto et al., 2010; Glessner et al., 2009; Devlin and Scherer, 2012] as an underlying cause of ASD. Limited data is available on ASD prevalence in Arab countries. Recent study shows ASD prevalence of 1.4-4.3 per 10000 individuals in gulf cooperation countries [Salhia et al., 2014].
In the present study, we used a high-resolution Illumina SNP array consisting 2.5 million (2.5M) probes to assay for SNPs and CNVs in a Saudi cohort of 11 unrelated multiplex families containing 2 ASD cases each. Our data, enabled detection of small common and rare CNVs in ASD sample examined, fine mapped already known CNVs breakpoints and identified novel potential CNV. MATERIALS AND METHODS Sample Collection and DNA extraction Twenty two subjects with ASD from 11 multiplex families and their parents were selected for genetic analysis from a cohort of Saudi patients visiting Autism Research and Treatment Center (ARTC) and Madinah Maternity and Children Hospital (MMCH). Pedigrees were drawn after interviewing parents (Figure 1). Families having 2 sibs sharing characteristics features of ASD were selected reasoning that some families would show shared genetic defects. All cases with ASD met the criteria for autism on one or both diagnostic measures - Autism Diagnostic Interview-Revised (ADI-R) [Lord et al., 1989] and Autism Diagnostic Observation Schedule (ADOS) [Lord et al., 1989]. The total cutoff scores used for case determination were between 78 for the communication and language domain, 10 for the social interaction domain and 3 for restricted and repetitive behaviors. Subjects who had associated neurological diseases such as cerebral palsy, schizophrenia and tuberous sclerosis, metabolic disorders (e.g., phenylketonuria), allergic manifestations or concomitant infection were excluded from the study. Autism diagnosis/study measurements Autism diagnosis was based on clinical examination, neuropsychiatric assessment and clinical history given by the caregivers. Autism severity was assessed by using Childhood Autism Rating
Scale (CARS-2) which rates the child on a scale from one to four of fifteen areas as described previously [Schopler and Van Bourgondien, 2010]. According to this scale, children acquiring the score of 30-36 were designated as having mild to moderate autism while those with scores ranging between 36.5 and above had severe autism (Table 4). A formal approval was obtained for this study from the Institutional Review Board of the king Khalid university hospital (KKUH). Phenotypic assessment information of patients included was noted down separately. Parents of all participating individuals gave informed written consent for genetic analysis. Genomic DNA was extracted using QIAamp DNA blood midi kit (Qiagen, UK) following manufacturer’s instruction. SNP Genotyping SNP genotyping was performed on genomic DNA of all 107 individuals including 22 cases, 21 parents and 64 unrelated controls. All samples have been analyzed by Illumina 2.5M array according to manufacturer’s protocol described elsewhere [Basit et al., 2016]. We used control data from 64 neurologically normal individuals who had been recruited from MMCH and genotyped previously on the same platform. Illumina 2.5 M array consists of 2,500,000 probes providing relatively uniform whole genome coverage with high resolution. The arrays were scanned using Illumina iScan and data were extracted using iScan control software. The array experiments for autism cases, parents and control individuals were performed at Center for Genetics and Inherited Diseases, Taibah University Almadinah Almunawwarah (Kingdom of Saudi Arabia). Detection of CNVs
All array SNP data (autism cases, their parents and controls) were analysed using two different plugins for Illumina’s GenomeStudio data analysis software to obtain high confidence calls. Plugins used were a likelihood-based method with cnvPartition v3.2.0 (Illumina) and a hidden Markov method with PennCNV to identify CNVs [Wang et al., 2007]. Criteria used to call a CNV includes a region involving at least five consecutive probe sets and a log2 ratio cut-off of 0.41 and +0.32 for loss and gain, respectively. Regions with absolute median log2 ratio to median absolute deviation value >2 was filtered out [Prasad et al., 2012]. The copy numbers detected by cnvPartition and PennCNV for each subject were combined using flanking SNP probes. A CNV was defined as being ‘stringent’ if it was detected by both cnvPartition and PennCNV at the sample level. The stringent dataset was utilized for common, rare and novel copy number variations discovery. Classification of CNVs Stringent CNV calls were classified as common, rare and novel based on comparisons with 64 in-house control individuals, database of genomic variants (DGV), Autism database (AutDB), Autism chromosome rearrangement database (ACRD) and DECIPHER. Moreover, CNVs were classified as inherited or de novo based on the presence and absence of CNV in parents. Furthermore, for each autism sample, we calculated total number of CNVs, exact size of the CNV and total number of genes harboring this CNV. Quantitative Real-time PCR All copy number changes were further evaluated by quantitative real-time PCR (qPCR). The qPCR was performed by designing primers evenly distributed over CNV and the flanking sequences which allowed detection of the breakpoint regions of the duplication/deletions.
Thermalcycler 7500 Fast real-time PCR system using SYBR-Green PCR master mix was used for the qPCR. Briefly, 25 µl of total reaction mixture was used including 12.5 µl of SYBR-Green PCR master mix, 50 ng of genomic DNA and 3 µM of primers. All reactions were carried out in triplicates in separate tubes to permit quantification of target regions. Using calibrator samples of normal control genomic DNA, the copy numbers were estimated based on the ∆Ct method. CFTR was used as a reference gene and copy numbers were measured relative to it. RESULTS Copy Number Variation Screening All 43 individuals including 22 autism patients were genotyped using the Illumina Human Omni 2.5-8 array which includes 2.5 million SNPs. A call rate of more than 99% was achieved throughout samples. SNP data analysis was conducted with Illumina GenomeStudio software (Genotyping module v1.9.4, Genome Viewer v1.9.0). CNVs were called using cnvPartition and PennCNV. The chromosome plots of all individuals were carefully examined to identify any copy number variations. Consistently high confidence CNVs were selected, meeting the following criteria; confidence score < 10, spanning at least five consecutive probes, predicted by both algorithms and shared by both individuals in a family. The consistent high confidence CNV calls were then compared with 64 in-house Saudi control individuals and those reported in DGV, AutDB, ACRD and DECIPHER and were allocated to the following categories: (i) common (overlapping 50% with 5 events in the database), (ii) rare (overlapping with < 5 known events) and (iii) novel (not overlapping with any known event). A total of 22 patients with autism (all males) were found to have 17 CNVs, selected on the basis of set criteria. Out of these 17 CNVs, 11 were common, 5 were rare and 1 was novel. Common and rare CNVs overlapped with the genomic imbalances defined in the AutDB, ACRD, DGV
and DECIPHER and a novel CNV not described previously. Validation as well as segregation analysis of all CNVs were performed using qPCR. Exact boundary of the each CNV was defined by Sanger sequencing. Common CNVs and fine mapping As all samples were genotyped using a high resolution array, therefore, we were able to define boundaries and fine map recurrent CNVs in our ASD population. Based on our fine mapping, we delineated potential genes which might have an involvement in generating autism phenotype. Previous studies failed to define exact boundaries of CNVs either because of low resolution arrays used (low SNP coverage) or high signal-to-noise ratio [Miller et al., 2010]. In two multiplex families (AUT2 and AUT9; 4 ASD individuals), an inherited recurrent CNV at chr4q35.2 was detected. This CNV is a duplication of 203 kb (chr4:186930190-187133901) (Figure 2). This region contains TLR3, FAM149A and CYP4V2. In two other families (AUT4 and AUT6; 4 ASD individuals) a recurrent de novo duplication of 114 kb on chr4p16.1 (chr4:9370980-9485300) was identified. This duplication encompass β-defensin 131 (DEFB131) gene. In AUT2, a 43kb deletion was detected on chr6p22.1 (chr6:29855945-29899389). This CNV causes deletion of HLA-H and 5th intron of HLA-G gene respectively. Another maternally inherited 73 kb deletion on chr6p21.32 (chr6:32458168-32520907) was found in AUT8 resulting in a deletion of HLA-DRB5 gene. In AUT10 family, 2 ASD patients share a common inherited deletion of 160 kb on chr8p11.22 (chr8:39225528-39285262) encompassing ADAM5 gene. In a family (AUT4), a de novo ~7 kb copy number loss on chr8q11.21 (51031221-51038149) was detected. This deletion affects intron 1 of syntrophin gamma 1 (SNTG1) gene. Moreover, an inherited deletion of 41430 bps was found on 1q31.2 (191826884-191868313) in four affected
members of two autism families (AUT1 and AUT7). This region has no UCSC gene. Table 1 contains list of all common CNVs found in this study. Rare CNVs In two families (AUT8 and AUT10, 4 ASD individuals) a previously reported duplication on Xp22.33 was identified. The largest duplication of 181 kb was detected in a family AUT10 (chrX:417861-599626). This duplication encompass SHOX gene. A ~10kb de novo copy number gain was identified in a family (AUT11) on chr18q21.31 (57652638-57663179). This duplication affects exon 21-24 of ATP8B1 (ATPase phospholipid transporting 8B1) gene. Table 2 contains list of all rare CNVs detected in Saudi families. Novel CNV The novel copy number loss (deletion) identified in this study have not been recorded in the AutDB, ACRD, DECIPHER and DGV before in autism cases. In AUT5 and AUT9, a small (~7kb) inherited loss of copy number (chr3q23) was detected in 4 individuals. This CNV results in deletion of intron 2 of calsyntenin 2 (CLSTN2) gene (Figure 3). The novel deletion identified in this study is presented in Table 3. DISCUSSION Our high-resolution SNP array data have identified shared recurrent, novel and multiple rare CNVs in families having 2 individuals each with autism spectrum disorder. Novel CNV (chr3q23) identified in this study was not reported before, hence providing an additional valuable resource for autism risk gene discovery. Moreover, identification of common and rare CNVs validates previously reported CNVs. Though hundreds of autism susceptibility genes, loci and CNVs [Betancur, 2011] are currently known, no single gene/locus or CNV is considered as an underlying factor for more than 0.8% of cases in a particular cohort. Majority of the genetic
defects, identified so far, contribute to less than 0.1% of ASD cases [Devlin and Scherer, 2012]. Furthermore, large scale CNV studies [Pinto et al., 2010; Sanders et al., 2011; Neale et al., 2012; O'Roak et al., 2012] and recently performed whole exome sequencing studies [Sanders et al., 2011; Neale et al., 2012; O'Roak et al., 2012] suggests that perhaps hundreds of autism associated genes are yet to be discover. These data show that diverse experimental approaches will probably be required to define all autism susceptibility genes and CNVs. We found that the Illumina HumanOmni 2.5M bead array is sensitive for discovery of many smaller copy number variations, which are frequently missed by low-resolution SNP and CGH microarrays. Smaller CNVs went undetected in previous studies either due to use of low SNP coverage or high signal-to-noise ratio [Miller et al., 2010]. The Illumina HumanOmni 2.5M array is able to detect small CNVs because the median probe spacing is < 2 kb. Four recurrent CNVs have been identified in more than one family. This includes a duplications on 4p16.1, 4q35.2 and Xp22.33 and a deletion on chr6p22.1. A common duplication of 114kb on chr4p16.1 encompass β-defensin 131 (DEFB131) gene. β-defensin are cysteine-rich cationic polypeptides that are important in the immunologic response. Recently, Kanduri et al [2016] and Engchuan et al [2015] have reported duplications in ASD patients in 4p16.1 region. A common recurrent duplication of 203 kb at chr4q35.2 was found in 2 autism families (4 Autism individuals). This region contains TLR3, FAM149A and CYP4V2 genes. Duplication of this region has previously been reported in patients with developmental delay and intellectual disability [Cuturilo et al., 2011; Youngs et al., 2012; Chong et al., 2014]. A rare duplication of Xp22.33 has been detected in 2 autism families (4 Autism individuals). Duplications of this region in patients with severe ID, ADHD and ASD has been reported previously [Boycott et al., 2003; Kaminsky et al., 2011]. This duplication affects first three exons of the SHOX gene. In
families AUT2 and AUT11, a copy number loss of 43kb was detected on chr6p22.1. This CNV causes deletion of HLA-H and 5th intron of HLA-G gene. Another maternally inherited 73 kb deletion on chr6p21.32 was found in AUT8 resulting in a deletion of HLA-DRB5 gene. Multiple evidence exists that inflammatory responses are involved in the development and progression of autism [Michel et al., 2012; Bashir and Al-Ayadhi, 2015; Mostafa et al., 2016]. Maternal immune system stimulation throughout gestation is thought to trigger fetal inflammatory responses, in some cases with harmful effects on brain development in the fetus, leading to ASD [Matsunami et al., 2013]. This environmental insult could be mediated or enhanced by genomic changes that predispose the fetus to high inflammatory responses. It is noteworthy that a number of genes, affected by copy number gains and losses, identified in this study, are involved in producing inflammatory responses. Examples of these include DEFB131, HLA-G, and HLADRB5. Some common shared CNVs have also been detected in our cohort. For instance, in 2 individuals of a family AUT1, a common inherited CNV on chromosome 15q11.2 was identified. CNVs in this region has previously been associated with several neurodevelopmental diseases including ASD, bipolar 1 and bipolar 2 disorders [Burnside et al., 2011; Leblond et al., 2012; De Wolf et al., 2013; Rosenfeld et al., 2013]. It has been postulated that 15q11.2 deletion have moderate or mild effect and it probably require other genetic (or non-genetic) factors to take the phenotype across the ASD threshold [Devlin and Scherer, 2012]. In the same family (AUT1), another paternally inherited CNV, a deletion on chromosome 6p22.1 might explain the ASD phenotype. A common copy number loss of ~7 kb on chr8q11.21 affecting intron 1 of syntrophin gamma 1 (SNTG1) gene was identified in AUT4. It has been shown that SNTG1 localizes in the brain [Piluso et al., 2000] suggesting that the SNTG1 protein may have a neurological function.
Rare CNVs identified in this study includes chr2q36.3 deletion and duplications of chr9q21.13, 18q21.31 and Xp22.33. Rare copy number loss of ~ 16 kb at chr2q36.3 (chr2: 228241621228258288), identified in AUT5, overlaps with CNVs in AutDB, DECIPHER and DGV (Figure 4). This deletion affects intron 3 and exon 4 of the TM4SF20 (transmembrane 4 L six family member 20) gene. TM4SF20 expression is known in the brain, with high levels in the parietal lobe, hippocampus, pons, white matter and cerebellum. Deletion of exon 3 of TM4SF20 is known to cause susceptibility to early language delay in children (Wiszniewski et al., 2013). A small paternally inherited duplication of ~2 kb has been detected in 2 members of an autism family on chromosome 9q21.13 (chr9:73907625-73909871) encompassing TRPM3 gene. Deletion of exon 1-9 of TRPM3 has previously been reported in a family with autism [Pagnamenta et al., 2011]. A rare ~10kb de novo copy number gain was identified in a family (AUT11) on chr18q21.31. This duplication affects exon 21-24 of ATP8B1 (ATPase phospholipid transporting 8B1) gene. A novel loss of copy number (~7kb) was detected in four ASD cases. This CNV results in deletion of intron 2 of calsyntenin 2 (CLSTN2) gene. A CLSTN2 encodes the synaptic protein calsyntenin 2. CLSTN2 is expressed exclusively in brain, with high levels occurring in cortical gamma-aminobutyric acid (GABA)ergic interneurons and in medial temporal lobe regions [Hintsch et al., 2002]. Polymorphisms at a locus within CLSTN2 has been shown to modulate early and delayed recall of words in a large young samples of Swiss origin [Papassotiropoulos et al., 2006]. Studies have shown that metabolism of calsyntenins and amyloid precursor protein (APP) is coordinated in neurons and nAChRs might play role in regulating this coordination [Ikeda et al., 2008; Araki et al., 2003, 2007; Snyder et al., 2005; Nie et al., 2008] suggesting that calsyntenins are essential for learning.
Taking into account all de novo and inherited, common and rare genomic variations together, no single variant accounts for more than 1% of the etiology in autism. This emphasize the complex genetic heterogeneity of autism. Therefore, it is important to unravel the entire spectrum of genomic variations contributing to autism susceptibility to explain the missing heritability. Although, only 11 families (22 ASD patients) have been analyzed in this study and the rarity of the novel CNV identified here, it is likely that high resolution genome-wide analysis of thousands of autism patients are required to confirm this finding. The recurrent CNVs identified in this study validates autism genetic risk factors detected earlier using whole genome SNP genotyping, WES and WGS approach. This data allowed us to significantly expand the profile of genetic variants that are possibly causal of ASD in this cohort. In eleven such families with severe ASD, we show the phenotype is associated with de novo and inherited CNVs. Acknowledgments: The volunteer participation of all families in the study is highly appreciated. Competing Interests: The authors declare that they have no competing interests Authors’ Contributions: SB and MI conceived the idea and designed the study. LA and NEE recruited patients and performed phenotyping. MI extracted DNA and performed DNA quantification. JAH and AMA analyzed microarray data and compiled results. AMA and MIS performed experiments. AMA performed DNA extraction, SNP genotyping and exome sequencing. SB performed whole genome SNP genotyping data analysis and wrote manuscript. All authors read and approved the final manuscript.
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Legends to Figures
Figure 1: Pedigrees of 11 families studied. Open and filled symbols represent unaffected and affected subjects respectively.
Figure 2: B allele frequency and Log R ratio for three recurrent copy number variations located on chromosome 4 and chromosome 6 are shown. The upper panel (A) shows the region between 180 Mb to 191 Mb on chromosome 4 in affected individuals from family AUT2 and AUT9. A 203 kb maternally inherited heterozygous duplications identified in both families is on chr4q35.2 (chr4:186930190-187133901). The lower panel (B) shows the region between 28 Mb to 33 Mb on chromosome 6 in affected individuals from family AUT1, AUT2 and AUT8. In family AUT1 and AUT2 a 10 kb inherited deletion identified in both families is on chr6p22.1 (chr6:2985594529899389) while in family AUT8 a 62 kb maternally inherited deletion is on chr6p21.32 (chr6:32458168-32520907).
Figure 3: B allele frequency and Log R ratio for some of the novel copy number variations located on chr1q31.2, chr3q23 and chr18q21.31 are shown. The upper panel (A) shows the region between 190 Mb to 196 Mb on chromosome 1 in affected individuals from family AUT1. A 41 kb maternally inherited heterozygous deletion identified in this family is on chr1q31.2 (chr1:191826884-191868313). The middle panel (B) shows the region between 136 Mb to 141 Mb on chromosome 3 in affected individuals from family AUT5 and AUT7. A 7 kb maternally inherited deletion identified in these families is on chr3q23 (chr3:140290973-140323704). The lower panel (C) shows the region between 54 Mb to 60 Mb on chromosome 18 in affected
individuals from family AUT11. A 10 kb de novo duplication identified in this family is on chr18q21.31 (chr18:57652638-57663179).
Figure 4: Schematic representation of the chromosomal region around 2q36.3 between 228.10 Mb to 229.10 Mb region. Deletion identified in this study and overlapping deletions mapped to 2q36.3 region reported in AutDB, DECIPHER, DGV database.
Table 1: Common CNVs in autism multiplex families Fam ID AUT1 AUT2 AUT3 AUT4 AUT5 AUT6 AUT7 AUT8 AUT9 AUT10
Cytoband chr6p22.1 chr15q11.2 chr4q35.2 chr6p22.1 chr14q21.1 chr15q14 chr4p16.1 chr8q11.21 chr14q11.2 chr4p16.1 chr1q31.2 chr6p21.32 chr4q35.2 chr8p11.22
Chromosomal Location chr6:29855945-29899389 chr15:24381205-24438156 chr4:186930190-187133901 chr6:29855945-29899389 chr14:41615839-41661685 chr15:34736064-34810762 chr4:9370980-9485300 chr8:51031221-51038149 chr14:20197311-20422799 chr4:9370980-9485300 chr1:191826884-191868313 chr6:32458168-32520907 chr4:186930190-187133901 chr8:39225528-39385262
Size (bps) 43445 bps 56952 bps 203712 bps 43445 bps 45847 bps 74699 bps 114321 bps 6929 bps 225489 bps 114321 bps 41430 bps 62740 bps 203712 bps 159735 bps
CNV type Deletion Deletion Duplication Deletion Deletion Deletion Duplication Deletion Duplication Duplication Deletion Deletion Duplication Deletion
Genes in the region HLA-H PWRN2 TLR3, FAM149A, CYP4V2 HLA-H, HLA-G LRFN5 GJD2, ACTC1 DEFB131 SNTG1 TTC5, CCNB1IP1, PARP2, TEP1 DEFB131 No known gene HLA-DRB5 TLR3, FAM149A, CYP4V2 ADAM5
Inheritance PI PI MI PI PI de novo de novo de novo PI de novo MI de novo I I
PI; paternally inherited, MI; maternally inherited, I; Inherited from both, de novo; not found in both parents
Table 2: Rare CNVs in autism multiplex families
Fam ID AUT4 AUT5 AUT8 AUT10 AUT11
Cytoband chr9q21.13 chr2q36.3 chrXp22.33 chrXp22.33 chr18q21.31
Chromosomal Location chr9:73907625-73909871 chr2:228241621-228258288 chrX:477422-585125 chrX:417861-599626 chr18:57652638-57663179
Size (bps) 2247 bps 16668 bps 107704 bps 181765 bps 10542 bps
CNV type Duplication Deletion Duplication Duplication Duplication
Genes in the region TRPM3 TM4SF20 No known gene SHOX ATP8B1
Inheritance de novo de novo MI MI de novo
CNV type Deletion Deletion
Genes in the region CLSTN2 CLSTN2
Inheritance de novo de novo
MI; materna lly inherite d, de novo;
not found in both parents
Table 3: Novel CNV identified in autism multiplex families Family ID AUT5 AUT9
Cytoband chr3q23 chr3q23
not found in both parents
Chromosomal Location chr3:140290973-140323704 chr3:140290973-140323704
CNV size 7276 bps 7276 bps
de novo;
Table 4: CARS, SRS and sensory profile scores of children with autism spectrum disorder
S.No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Patient ID AUT1-1 AUT1-2 AUT2-1 AUT2-2 AUT3-1 AUT3-2 AUT4-1 AUT4-2 AUT5-1 AUT5-2 AUT6-1 AUT6-2 AUT7-1 AUT7-2 AUT8-1 AUT8-2 AUT9-1 AUT9-2 AUT10-1 AUT10-2 AUT11-1 AUT11-2
Age
CARS* 30.5 37.0 35.5 36.0 33.0 35.0 36.5 33.5 36.0 33.0 34.5 34.5 37.0 40.0 43.0 40.0 28.5 35.5 36.0 33.0 35.0 35.5
SRSα Sensory profile 138 76 137 74 139 76 140 80 133 78 134 79 123 80 154 79 137 80 133 157 125 81 75 77 70 72 73 185 76 141 77 136 75 180 -
*CARS; Childhood Autism Rating Scale, αSRS; Social Responsiveness Scale
•
Novel and recurrent CNVs in 11 multiplex families with Autism were identified.
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A novel loss of copy number CNV affecting intron 2 of CLSTN2 gene was detected in 2 autistic families.
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CLSTN2 gene encodes synaptic protein calsyntenin 2.
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CLSTN2 is expressed exclusively in brain.
•
Multiple evidence shows that calsyntenins are essential for learning.
S0306-4522(16)30570-X http://dx.doi.org/10.1016/j.neuroscience.2016.10.030 NSC 17390
To appear in:
Neuroscience
Accepted Date:
11 October 2016
Please cite this article as: L.Y. AlAyadhi, J.A. Hashmi, M. Iqbal, A.M. Albalawi, M.I. Samman, N.E. Elamin, S. Bashir, S. Basit, High resolution SNP genotyping platform identified recurrent and novel CNVs in Autism Multiplex families, Neuroscience (2016), doi: http://dx.doi.org/10.1016/j.neuroscience.2016.10.030
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High resolution SNP genotyping platform identified recurrent and novel CNVs in Autism Multiplex families Laila Y. AlAyadhi1, Jamil A. Hashmi2, Muhammad Iqbal3, Alia M Albalawi2, Mohammad I. Samman2,4, Nadra E. Elamin1, Shahid Bashir1,5, Sulman Basit2 1
KSU-Autism Research & Treatment Center, AL-Amodi Autism Research Chair, Department of Physiology, Faculty of Medicine, King Saud University, Riyadh, Saudi Arabia 2
Center for Genetics and Inherited Diseases, Taibah University Almadinah Almunawwarah, Saudi Arabia 3
Aging Research Chair, Department of Physiology, Faculty of Medicine, King Saud University, Riyadh, Saudi Arabia 4
College of Applied Medical Sciences, Taibah University Almadinah Almunawwarah, Saudi Arabia 5
Berenson-Allen Center for Noninvasive Brain Stimulation, Division of Cognitive Neurology, Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. Laila Y. AlAyadhi; [email protected] Jamil A. Hashmi; [email protected] Muhammad Iqbal; [email protected] Alia Albalawi; [email protected] Mohammad I. Samman; [email protected] Nadra E. Elamin; Shahid Bashir; [email protected] Sulman Basit; [email protected]
Corresponding Author: Sulman Basit, PhD Center for Genetics and Inherited Diseases, Taibah University Almadinah Almunawwarah, Saudi Arabia Email: [email protected] Contact: 00966535370209
Competing Interests: The authors declare that they have no competing interests
ABSTRACT Single nucleotide polymorphisms (SNP) - based genotyping using microarray platform is now frequently used to detect copy number variants (CNVs) in the human genome. Here, we report CNVs identified using Illumina HumanOmni 2.5 M oligonucleotide microarrays in 11 multiplex families with autism spectrum disorder (ASD) referred to Autism Research and Treatment Center (ART) and Madinah Maternity and Children Hospital (MMCH). Of the 11 families, 22 patients with ASD (all males) and their parents, were recruited for the present study. In total, 43 individuals were genotyped with high resolution array. Abnormal microarray results were seen in all 22 patients with ASD. A total of 17 shared CNVs were selected for further analysis. Out of these 17 CNVs, we discovered one novel CNV, previously not described, and 16 recurrent CNVs that overlap with the genomic imbalances defined in the autism database (AutDB), autism chromosome rearrangement database (ACRD) and Database of Genomic Variants (DGV). Recurrent CNVs includes 11 common and 5 rare CNVs. All rare CNVs are duplications except a 16kb deletion on chr2q36.3. Rare gain of copy numbers includes a 2kb duplication on chr9q21.13, an overlapping duplications of 107kb and 181kb on chrXp22.33 in 2 different families and a 10kb duplication on chr18q21.13. A novel loss of copy number on chr3q23 was found in four ASD cases. This CNV results in deletion of intron 2 of calsyntenin 2 (CLSTN2) encoding synaptic protein calsyntenin 2. CLSTN2 is expressed exclusively in brain, with high levels occurring in cortical gamma-aminobutyric acid (GABA)ergic interneurons and in medial temporal lobe regions. These results verify the diagnostic relevance of genome-wide small common and rare CNVs and provides further evidence of the high diagnostic yield of microarray for genetic testing in children with ASD. Key Words: Autism, Copy Number Variations, SNP array, Multiplex families
INTRODUCTION Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder marked by deficit in the social behaviour and language development, as well as restricted or stereotyped interests. Multiple evidence exists for the association between copy number variants (CNVs) and ASD. CNVs are thought to provide explanation for almost 5-10% of the ASD cases depending on the cohort examined, though, no single candidate genetic locus has been shown as an underlying factor in more than 1% of ASD cases [Devlin and Scherer, 2012]. Most studies aiming to detect CNVs in ASD have been carried out in cohorts of European ancestry [Marshall et al., 2008; Pinto et al., 2010; Pinto et al., 2014]. Recently, genome wide CNV analysis in patients with ASD have been carried out in Chinese population [Yin et al., 2016; Gazzellone et al., 2014; Chong et al., 2014], however, studies describing the genetic architecture in other populations, particularly Middle Eastern populations, are limited specifically in the era where clinical microarray testing continues to be adopted as the standard of care across medical genetic laboratories worldwide [Miller, 2010]. Identifying CNVs using SNP and CGH arrays has proven to be a rapid method to detect small and large CNVs associated with ASD [Gazzellone et al., 2014; Chong et al., 2014; Marshall et al., 2008; Pinto et al., 2010; Christian et al., 2008; Shen et al., 2010; Sanders et al., 2011]. Some highly penetrant risk genes (e.g., the SHANK, Neurexin, and Neuroligin family members) and CNV loci (e.g., 1q21.1, 15q13.3, and 16p11.2) are now known [Sebat et al., 2007; Pinto et al., 2010; Glessner et al., 2009; Devlin and Scherer, 2012] as an underlying cause of ASD. Limited data is available on ASD prevalence in Arab countries. Recent study shows ASD prevalence of 1.4-4.3 per 10000 individuals in gulf cooperation countries [Salhia et al., 2014].
In the present study, we used a high-resolution Illumina SNP array consisting 2.5 million (2.5M) probes to assay for SNPs and CNVs in a Saudi cohort of 11 unrelated multiplex families containing 2 ASD cases each. Our data, enabled detection of small common and rare CNVs in ASD sample examined, fine mapped already known CNVs breakpoints and identified novel potential CNV. MATERIALS AND METHODS Sample Collection and DNA extraction Twenty two subjects with ASD from 11 multiplex families and their parents were selected for genetic analysis from a cohort of Saudi patients visiting Autism Research and Treatment Center (ARTC) and Madinah Maternity and Children Hospital (MMCH). Pedigrees were drawn after interviewing parents (Figure 1). Families having 2 sibs sharing characteristics features of ASD were selected reasoning that some families would show shared genetic defects. All cases with ASD met the criteria for autism on one or both diagnostic measures - Autism Diagnostic Interview-Revised (ADI-R) [Lord et al., 1989] and Autism Diagnostic Observation Schedule (ADOS) [Lord et al., 1989]. The total cutoff scores used for case determination were between 78 for the communication and language domain, 10 for the social interaction domain and 3 for restricted and repetitive behaviors. Subjects who had associated neurological diseases such as cerebral palsy, schizophrenia and tuberous sclerosis, metabolic disorders (e.g., phenylketonuria), allergic manifestations or concomitant infection were excluded from the study. Autism diagnosis/study measurements Autism diagnosis was based on clinical examination, neuropsychiatric assessment and clinical history given by the caregivers. Autism severity was assessed by using Childhood Autism Rating
Scale (CARS-2) which rates the child on a scale from one to four of fifteen areas as described previously [Schopler and Van Bourgondien, 2010]. According to this scale, children acquiring the score of 30-36 were designated as having mild to moderate autism while those with scores ranging between 36.5 and above had severe autism (Table 4). A formal approval was obtained for this study from the Institutional Review Board of the king Khalid university hospital (KKUH). Phenotypic assessment information of patients included was noted down separately. Parents of all participating individuals gave informed written consent for genetic analysis. Genomic DNA was extracted using QIAamp DNA blood midi kit (Qiagen, UK) following manufacturer’s instruction. SNP Genotyping SNP genotyping was performed on genomic DNA of all 107 individuals including 22 cases, 21 parents and 64 unrelated controls. All samples have been analyzed by Illumina 2.5M array according to manufacturer’s protocol described elsewhere [Basit et al., 2016]. We used control data from 64 neurologically normal individuals who had been recruited from MMCH and genotyped previously on the same platform. Illumina 2.5 M array consists of 2,500,000 probes providing relatively uniform whole genome coverage with high resolution. The arrays were scanned using Illumina iScan and data were extracted using iScan control software. The array experiments for autism cases, parents and control individuals were performed at Center for Genetics and Inherited Diseases, Taibah University Almadinah Almunawwarah (Kingdom of Saudi Arabia). Detection of CNVs
All array SNP data (autism cases, their parents and controls) were analysed using two different plugins for Illumina’s GenomeStudio data analysis software to obtain high confidence calls. Plugins used were a likelihood-based method with cnvPartition v3.2.0 (Illumina) and a hidden Markov method with PennCNV to identify CNVs [Wang et al., 2007]. Criteria used to call a CNV includes a region involving at least five consecutive probe sets and a log2 ratio cut-off of 0.41 and +0.32 for loss and gain, respectively. Regions with absolute median log2 ratio to median absolute deviation value >2 was filtered out [Prasad et al., 2012]. The copy numbers detected by cnvPartition and PennCNV for each subject were combined using flanking SNP probes. A CNV was defined as being ‘stringent’ if it was detected by both cnvPartition and PennCNV at the sample level. The stringent dataset was utilized for common, rare and novel copy number variations discovery. Classification of CNVs Stringent CNV calls were classified as common, rare and novel based on comparisons with 64 in-house control individuals, database of genomic variants (DGV), Autism database (AutDB), Autism chromosome rearrangement database (ACRD) and DECIPHER. Moreover, CNVs were classified as inherited or de novo based on the presence and absence of CNV in parents. Furthermore, for each autism sample, we calculated total number of CNVs, exact size of the CNV and total number of genes harboring this CNV. Quantitative Real-time PCR All copy number changes were further evaluated by quantitative real-time PCR (qPCR). The qPCR was performed by designing primers evenly distributed over CNV and the flanking sequences which allowed detection of the breakpoint regions of the duplication/deletions.
Thermalcycler 7500 Fast real-time PCR system using SYBR-Green PCR master mix was used for the qPCR. Briefly, 25 µl of total reaction mixture was used including 12.5 µl of SYBR-Green PCR master mix, 50 ng of genomic DNA and 3 µM of primers. All reactions were carried out in triplicates in separate tubes to permit quantification of target regions. Using calibrator samples of normal control genomic DNA, the copy numbers were estimated based on the ∆Ct method. CFTR was used as a reference gene and copy numbers were measured relative to it. RESULTS Copy Number Variation Screening All 43 individuals including 22 autism patients were genotyped using the Illumina Human Omni 2.5-8 array which includes 2.5 million SNPs. A call rate of more than 99% was achieved throughout samples. SNP data analysis was conducted with Illumina GenomeStudio software (Genotyping module v1.9.4, Genome Viewer v1.9.0). CNVs were called using cnvPartition and PennCNV. The chromosome plots of all individuals were carefully examined to identify any copy number variations. Consistently high confidence CNVs were selected, meeting the following criteria; confidence score < 10, spanning at least five consecutive probes, predicted by both algorithms and shared by both individuals in a family. The consistent high confidence CNV calls were then compared with 64 in-house Saudi control individuals and those reported in DGV, AutDB, ACRD and DECIPHER and were allocated to the following categories: (i) common (overlapping 50% with 5 events in the database), (ii) rare (overlapping with < 5 known events) and (iii) novel (not overlapping with any known event). A total of 22 patients with autism (all males) were found to have 17 CNVs, selected on the basis of set criteria. Out of these 17 CNVs, 11 were common, 5 were rare and 1 was novel. Common and rare CNVs overlapped with the genomic imbalances defined in the AutDB, ACRD, DGV
and DECIPHER and a novel CNV not described previously. Validation as well as segregation analysis of all CNVs were performed using qPCR. Exact boundary of the each CNV was defined by Sanger sequencing. Common CNVs and fine mapping As all samples were genotyped using a high resolution array, therefore, we were able to define boundaries and fine map recurrent CNVs in our ASD population. Based on our fine mapping, we delineated potential genes which might have an involvement in generating autism phenotype. Previous studies failed to define exact boundaries of CNVs either because of low resolution arrays used (low SNP coverage) or high signal-to-noise ratio [Miller et al., 2010]. In two multiplex families (AUT2 and AUT9; 4 ASD individuals), an inherited recurrent CNV at chr4q35.2 was detected. This CNV is a duplication of 203 kb (chr4:186930190-187133901) (Figure 2). This region contains TLR3, FAM149A and CYP4V2. In two other families (AUT4 and AUT6; 4 ASD individuals) a recurrent de novo duplication of 114 kb on chr4p16.1 (chr4:9370980-9485300) was identified. This duplication encompass β-defensin 131 (DEFB131) gene. In AUT2, a 43kb deletion was detected on chr6p22.1 (chr6:29855945-29899389). This CNV causes deletion of HLA-H and 5th intron of HLA-G gene respectively. Another maternally inherited 73 kb deletion on chr6p21.32 (chr6:32458168-32520907) was found in AUT8 resulting in a deletion of HLA-DRB5 gene. In AUT10 family, 2 ASD patients share a common inherited deletion of 160 kb on chr8p11.22 (chr8:39225528-39285262) encompassing ADAM5 gene. In a family (AUT4), a de novo ~7 kb copy number loss on chr8q11.21 (51031221-51038149) was detected. This deletion affects intron 1 of syntrophin gamma 1 (SNTG1) gene. Moreover, an inherited deletion of 41430 bps was found on 1q31.2 (191826884-191868313) in four affected
members of two autism families (AUT1 and AUT7). This region has no UCSC gene. Table 1 contains list of all common CNVs found in this study. Rare CNVs In two families (AUT8 and AUT10, 4 ASD individuals) a previously reported duplication on Xp22.33 was identified. The largest duplication of 181 kb was detected in a family AUT10 (chrX:417861-599626). This duplication encompass SHOX gene. A ~10kb de novo copy number gain was identified in a family (AUT11) on chr18q21.31 (57652638-57663179). This duplication affects exon 21-24 of ATP8B1 (ATPase phospholipid transporting 8B1) gene. Table 2 contains list of all rare CNVs detected in Saudi families. Novel CNV The novel copy number loss (deletion) identified in this study have not been recorded in the AutDB, ACRD, DECIPHER and DGV before in autism cases. In AUT5 and AUT9, a small (~7kb) inherited loss of copy number (chr3q23) was detected in 4 individuals. This CNV results in deletion of intron 2 of calsyntenin 2 (CLSTN2) gene (Figure 3). The novel deletion identified in this study is presented in Table 3. DISCUSSION Our high-resolution SNP array data have identified shared recurrent, novel and multiple rare CNVs in families having 2 individuals each with autism spectrum disorder. Novel CNV (chr3q23) identified in this study was not reported before, hence providing an additional valuable resource for autism risk gene discovery. Moreover, identification of common and rare CNVs validates previously reported CNVs. Though hundreds of autism susceptibility genes, loci and CNVs [Betancur, 2011] are currently known, no single gene/locus or CNV is considered as an underlying factor for more than 0.8% of cases in a particular cohort. Majority of the genetic
defects, identified so far, contribute to less than 0.1% of ASD cases [Devlin and Scherer, 2012]. Furthermore, large scale CNV studies [Pinto et al., 2010; Sanders et al., 2011; Neale et al., 2012; O'Roak et al., 2012] and recently performed whole exome sequencing studies [Sanders et al., 2011; Neale et al., 2012; O'Roak et al., 2012] suggests that perhaps hundreds of autism associated genes are yet to be discover. These data show that diverse experimental approaches will probably be required to define all autism susceptibility genes and CNVs. We found that the Illumina HumanOmni 2.5M bead array is sensitive for discovery of many smaller copy number variations, which are frequently missed by low-resolution SNP and CGH microarrays. Smaller CNVs went undetected in previous studies either due to use of low SNP coverage or high signal-to-noise ratio [Miller et al., 2010]. The Illumina HumanOmni 2.5M array is able to detect small CNVs because the median probe spacing is < 2 kb. Four recurrent CNVs have been identified in more than one family. This includes a duplications on 4p16.1, 4q35.2 and Xp22.33 and a deletion on chr6p22.1. A common duplication of 114kb on chr4p16.1 encompass β-defensin 131 (DEFB131) gene. β-defensin are cysteine-rich cationic polypeptides that are important in the immunologic response. Recently, Kanduri et al [2016] and Engchuan et al [2015] have reported duplications in ASD patients in 4p16.1 region. A common recurrent duplication of 203 kb at chr4q35.2 was found in 2 autism families (4 Autism individuals). This region contains TLR3, FAM149A and CYP4V2 genes. Duplication of this region has previously been reported in patients with developmental delay and intellectual disability [Cuturilo et al., 2011; Youngs et al., 2012; Chong et al., 2014]. A rare duplication of Xp22.33 has been detected in 2 autism families (4 Autism individuals). Duplications of this region in patients with severe ID, ADHD and ASD has been reported previously [Boycott et al., 2003; Kaminsky et al., 2011]. This duplication affects first three exons of the SHOX gene. In
families AUT2 and AUT11, a copy number loss of 43kb was detected on chr6p22.1. This CNV causes deletion of HLA-H and 5th intron of HLA-G gene. Another maternally inherited 73 kb deletion on chr6p21.32 was found in AUT8 resulting in a deletion of HLA-DRB5 gene. Multiple evidence exists that inflammatory responses are involved in the development and progression of autism [Michel et al., 2012; Bashir and Al-Ayadhi, 2015; Mostafa et al., 2016]. Maternal immune system stimulation throughout gestation is thought to trigger fetal inflammatory responses, in some cases with harmful effects on brain development in the fetus, leading to ASD [Matsunami et al., 2013]. This environmental insult could be mediated or enhanced by genomic changes that predispose the fetus to high inflammatory responses. It is noteworthy that a number of genes, affected by copy number gains and losses, identified in this study, are involved in producing inflammatory responses. Examples of these include DEFB131, HLA-G, and HLADRB5. Some common shared CNVs have also been detected in our cohort. For instance, in 2 individuals of a family AUT1, a common inherited CNV on chromosome 15q11.2 was identified. CNVs in this region has previously been associated with several neurodevelopmental diseases including ASD, bipolar 1 and bipolar 2 disorders [Burnside et al., 2011; Leblond et al., 2012; De Wolf et al., 2013; Rosenfeld et al., 2013]. It has been postulated that 15q11.2 deletion have moderate or mild effect and it probably require other genetic (or non-genetic) factors to take the phenotype across the ASD threshold [Devlin and Scherer, 2012]. In the same family (AUT1), another paternally inherited CNV, a deletion on chromosome 6p22.1 might explain the ASD phenotype. A common copy number loss of ~7 kb on chr8q11.21 affecting intron 1 of syntrophin gamma 1 (SNTG1) gene was identified in AUT4. It has been shown that SNTG1 localizes in the brain [Piluso et al., 2000] suggesting that the SNTG1 protein may have a neurological function.
Rare CNVs identified in this study includes chr2q36.3 deletion and duplications of chr9q21.13, 18q21.31 and Xp22.33. Rare copy number loss of ~ 16 kb at chr2q36.3 (chr2: 228241621228258288), identified in AUT5, overlaps with CNVs in AutDB, DECIPHER and DGV (Figure 4). This deletion affects intron 3 and exon 4 of the TM4SF20 (transmembrane 4 L six family member 20) gene. TM4SF20 expression is known in the brain, with high levels in the parietal lobe, hippocampus, pons, white matter and cerebellum. Deletion of exon 3 of TM4SF20 is known to cause susceptibility to early language delay in children (Wiszniewski et al., 2013). A small paternally inherited duplication of ~2 kb has been detected in 2 members of an autism family on chromosome 9q21.13 (chr9:73907625-73909871) encompassing TRPM3 gene. Deletion of exon 1-9 of TRPM3 has previously been reported in a family with autism [Pagnamenta et al., 2011]. A rare ~10kb de novo copy number gain was identified in a family (AUT11) on chr18q21.31. This duplication affects exon 21-24 of ATP8B1 (ATPase phospholipid transporting 8B1) gene. A novel loss of copy number (~7kb) was detected in four ASD cases. This CNV results in deletion of intron 2 of calsyntenin 2 (CLSTN2) gene. A CLSTN2 encodes the synaptic protein calsyntenin 2. CLSTN2 is expressed exclusively in brain, with high levels occurring in cortical gamma-aminobutyric acid (GABA)ergic interneurons and in medial temporal lobe regions [Hintsch et al., 2002]. Polymorphisms at a locus within CLSTN2 has been shown to modulate early and delayed recall of words in a large young samples of Swiss origin [Papassotiropoulos et al., 2006]. Studies have shown that metabolism of calsyntenins and amyloid precursor protein (APP) is coordinated in neurons and nAChRs might play role in regulating this coordination [Ikeda et al., 2008; Araki et al., 2003, 2007; Snyder et al., 2005; Nie et al., 2008] suggesting that calsyntenins are essential for learning.
Taking into account all de novo and inherited, common and rare genomic variations together, no single variant accounts for more than 1% of the etiology in autism. This emphasize the complex genetic heterogeneity of autism. Therefore, it is important to unravel the entire spectrum of genomic variations contributing to autism susceptibility to explain the missing heritability. Although, only 11 families (22 ASD patients) have been analyzed in this study and the rarity of the novel CNV identified here, it is likely that high resolution genome-wide analysis of thousands of autism patients are required to confirm this finding. The recurrent CNVs identified in this study validates autism genetic risk factors detected earlier using whole genome SNP genotyping, WES and WGS approach. This data allowed us to significantly expand the profile of genetic variants that are possibly causal of ASD in this cohort. In eleven such families with severe ASD, we show the phenotype is associated with de novo and inherited CNVs. Acknowledgments: The volunteer participation of all families in the study is highly appreciated. Competing Interests: The authors declare that they have no competing interests Authors’ Contributions: SB and MI conceived the idea and designed the study. LA and NEE recruited patients and performed phenotyping. MI extracted DNA and performed DNA quantification. JAH and AMA analyzed microarray data and compiled results. AMA and MIS performed experiments. AMA performed DNA extraction, SNP genotyping and exome sequencing. SB performed whole genome SNP genotyping data analysis and wrote manuscript. All authors read and approved the final manuscript.
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Legends to Figures
Figure 1: Pedigrees of 11 families studied. Open and filled symbols represent unaffected and affected subjects respectively.
Figure 2: B allele frequency and Log R ratio for three recurrent copy number variations located on chromosome 4 and chromosome 6 are shown. The upper panel (A) shows the region between 180 Mb to 191 Mb on chromosome 4 in affected individuals from family AUT2 and AUT9. A 203 kb maternally inherited heterozygous duplications identified in both families is on chr4q35.2 (chr4:186930190-187133901). The lower panel (B) shows the region between 28 Mb to 33 Mb on chromosome 6 in affected individuals from family AUT1, AUT2 and AUT8. In family AUT1 and AUT2 a 10 kb inherited deletion identified in both families is on chr6p22.1 (chr6:2985594529899389) while in family AUT8 a 62 kb maternally inherited deletion is on chr6p21.32 (chr6:32458168-32520907).
Figure 3: B allele frequency and Log R ratio for some of the novel copy number variations located on chr1q31.2, chr3q23 and chr18q21.31 are shown. The upper panel (A) shows the region between 190 Mb to 196 Mb on chromosome 1 in affected individuals from family AUT1. A 41 kb maternally inherited heterozygous deletion identified in this family is on chr1q31.2 (chr1:191826884-191868313). The middle panel (B) shows the region between 136 Mb to 141 Mb on chromosome 3 in affected individuals from family AUT5 and AUT7. A 7 kb maternally inherited deletion identified in these families is on chr3q23 (chr3:140290973-140323704). The lower panel (C) shows the region between 54 Mb to 60 Mb on chromosome 18 in affected
individuals from family AUT11. A 10 kb de novo duplication identified in this family is on chr18q21.31 (chr18:57652638-57663179).
Figure 4: Schematic representation of the chromosomal region around 2q36.3 between 228.10 Mb to 229.10 Mb region. Deletion identified in this study and overlapping deletions mapped to 2q36.3 region reported in AutDB, DECIPHER, DGV database.
Table 1: Common CNVs in autism multiplex families Fam ID AUT1 AUT2 AUT3 AUT4 AUT5 AUT6 AUT7 AUT8 AUT9 AUT10
Cytoband chr6p22.1 chr15q11.2 chr4q35.2 chr6p22.1 chr14q21.1 chr15q14 chr4p16.1 chr8q11.21 chr14q11.2 chr4p16.1 chr1q31.2 chr6p21.32 chr4q35.2 chr8p11.22
Chromosomal Location chr6:29855945-29899389 chr15:24381205-24438156 chr4:186930190-187133901 chr6:29855945-29899389 chr14:41615839-41661685 chr15:34736064-34810762 chr4:9370980-9485300 chr8:51031221-51038149 chr14:20197311-20422799 chr4:9370980-9485300 chr1:191826884-191868313 chr6:32458168-32520907 chr4:186930190-187133901 chr8:39225528-39385262
Size (bps) 43445 bps 56952 bps 203712 bps 43445 bps 45847 bps 74699 bps 114321 bps 6929 bps 225489 bps 114321 bps 41430 bps 62740 bps 203712 bps 159735 bps
CNV type Deletion Deletion Duplication Deletion Deletion Deletion Duplication Deletion Duplication Duplication Deletion Deletion Duplication Deletion
Genes in the region HLA-H PWRN2 TLR3, FAM149A, CYP4V2 HLA-H, HLA-G LRFN5 GJD2, ACTC1 DEFB131 SNTG1 TTC5, CCNB1IP1, PARP2, TEP1 DEFB131 No known gene HLA-DRB5 TLR3, FAM149A, CYP4V2 ADAM5
Inheritance PI PI MI PI PI de novo de novo de novo PI de novo MI de novo I I
PI; paternally inherited, MI; maternally inherited, I; Inherited from both, de novo; not found in both parents
Table 2: Rare CNVs in autism multiplex families
Fam ID AUT4 AUT5 AUT8 AUT10 AUT11
Cytoband chr9q21.13 chr2q36.3 chrXp22.33 chrXp22.33 chr18q21.31
Chromosomal Location chr9:73907625-73909871 chr2:228241621-228258288 chrX:477422-585125 chrX:417861-599626 chr18:57652638-57663179
Size (bps) 2247 bps 16668 bps 107704 bps 181765 bps 10542 bps
CNV type Duplication Deletion Duplication Duplication Duplication
Genes in the region TRPM3 TM4SF20 No known gene SHOX ATP8B1
Inheritance de novo de novo MI MI de novo
CNV type Deletion Deletion
Genes in the region CLSTN2 CLSTN2
Inheritance de novo de novo
MI; materna lly inherite d, de novo;
not found in both parents
Table 3: Novel CNV identified in autism multiplex families Family ID AUT5 AUT9
Cytoband chr3q23 chr3q23
not found in both parents
Chromosomal Location chr3:140290973-140323704 chr3:140290973-140323704
CNV size 7276 bps 7276 bps
de novo;
Table 4: CARS, SRS and sensory profile scores of children with autism spectrum disorder
S.No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Patient ID AUT1-1 AUT1-2 AUT2-1 AUT2-2 AUT3-1 AUT3-2 AUT4-1 AUT4-2 AUT5-1 AUT5-2 AUT6-1 AUT6-2 AUT7-1 AUT7-2 AUT8-1 AUT8-2 AUT9-1 AUT9-2 AUT10-1 AUT10-2 AUT11-1 AUT11-2
Age
CARS* 30.5 37.0 35.5 36.0 33.0 35.0 36.5 33.5 36.0 33.0 34.5 34.5 37.0 40.0 43.0 40.0 28.5 35.5 36.0 33.0 35.0 35.5
SRSα Sensory profile 138 76 137 74 139 76 140 80 133 78 134 79 123 80 154 79 137 80 133 157 125 81 75 77 70 72 73 185 76 141 77 136 75 180 -
*CARS; Childhood Autism Rating Scale, αSRS; Social Responsiveness Scale
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Novel and recurrent CNVs in 11 multiplex families with Autism were identified.
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A novel loss of copy number CNV affecting intron 2 of CLSTN2 gene was detected in 2 autistic families.
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CLSTN2 gene encodes synaptic protein calsyntenin 2.
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CLSTN2 is expressed exclusively in brain.
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Multiple evidence shows that calsyntenins are essential for learning.