Abstract
Purpose To assess the contribution of rare variants in the genetic background towards variability of neurodevelopmental phenotypes in individuals with rare copy-number variants (CNVs) and gene-disruptive mutations.
Methods We analyzed quantitative clinical information, exome-sequencing, and microarray data from 757 probands and 233 parents and siblings who carry disease-associated mutations.
Results The number of rare secondary mutations in functionally intolerant genes (second-hits) correlated with the expressivity of neurodevelopmental phenotypes in probands with 16p12.1 deletion (n=23, p=0.004) and in probands with autism carrying gene-disruptive mutations (n=184, p=0.03) compared to their carrier family members. Probands with 16p12.1 deletion and a strong family history presented more severe clinical features (p=0.04) and higher burden of second-hits compared to those with mild/no family history (p=0.001). The number of secondary variants also correlated with the severity of cognitive impairment in probands carrying pathogenic rare CNVs (n=53) or de novo mutations in disease genes (n=290), and negatively correlated with head size among 80 probands with 16p11.2 deletion. These second-hits involved known disease-associated genes such as SETD5, AUTS2, and NRXN1, and were enriched for genes affecting cellular and developmental processes.
Conclusion Accurate genetic diagnosis of complex disorders will require complete evaluation of the genetic background even after a candidate gene mutation is identified.
Introduction
Significant advances in high-throughput genomic sequencing technologies have helped to identify hundreds of genes as risk factors for neurodevelopmental and neuropsychiatric disorders, including autism, intellectual disability, schizophrenia, and epilepsy. For example, in 2002, only 2-3% of autism cases were explained by genetic factors, whereas current studies suggest that rare disruptive mutations, including copy-number variants (CNVs) and single-nucleotide variants (SNVs), account for 10-30% of autism cases1. Despite initial claims of association with a specific disorder or syndrome, several of these pathogenic variants show incomplete penetrance and variable expressivity2–4. For example, the 16p11.2 BP4-BP5 deletion (OMIM #611913) was first described in children with autism, but further studies on other clinical and population cohorts demonstrated that this deletion is also associated with intellectual disability and developmental delay (ID/DD), obesity, epilepsy, cardiac disease, and scoliosis, and only about 24% of cases manifest an autism phenotype5–9. Phenotypic variability is not restricted to multi-genic CNVs but has also been reported for single genes with disruptive mutations, including DISC1, PTEN, SCN1A, CHD2, NRXN1, FOXP2, and GRIN2B3. While some of these effects could be due to allelic heterogeneity, phenotypic variability among carriers of the same molecular lesion suggests a strong role for variants in the genetic background10,11. For example, in a large family described by St. Clair and colleagues, carriers of a balanced translocation disrupting DISC1 manifested a wide range of neuropsychiatric features, including schizophrenia, bipolar disorder and depression12.
This phenomenon was exemplified by our delineation of a 520-kbp deletion on chromosome 16p12.1 (OMIM #136570) that is associated with developmental delay and extensive phenotypic variability13. Interestingly, in most cases, this deletion was inherited from a parent who also manifested mild neuropsychiatric features, and the severely affected children were more likely to carry another large (>500 kbp) rare CNV. We hypothesized that while each pathogenic primary mutation sensitizes the genome to varying extents, additional secondary variants in the genetic background modulate the ultimate clinical manifestation.
While previous studies have only assessed the role of large CNV second hits towards variability of disease-associated CNVs2, the combined effect of SNVs, insertion/deletions and CNVs in the genetic background towards variability of specific phenotypes among individuals carrying mutations (CNVs or SNVs) associated with neurodevelopmental disease has not been assessed. In this study, we evaluated 757 probands and 233 family members carrying disease-associated primary mutations (17 rare CNVs or disruptive mutations in 301 genes). A comparison of the genetic background between probands and parents or siblings showed that in the presence of the same primary mutation, variability and severity of neurodevelopmental disease correlates with the number of rare pathogenic variants, suggesting a global role of the genetic background towards phenotypic heterogeneity.
Materials and methods
Cohorts analyzed
We analyzed clinical and genetic data in individuals carrying a disease-associated “primary variant” or “first-hit”, defined as follows: (a) rare CNVs previously associated with neurodevelopmental and neuropsychiatric disorders2, (b) previously reported de novo mutations in candidate genes14,15, and (c) likely damaging variants in recurrent genes associated with neurodevelopmental and neuropsychiatric disorders. Five subgroups of individuals carrying a disease-associated primary variant were analyzed (Figure 1): (a) 26 probands, 23 carrier parents and available family members carrying 16p12.1 deletion, (b) 53 autism probands from the Simons Simplex Collection (SSC) cohort who carry rare CNVs associated with syndromic and variably expressive genomic disorders2, (c) 84 probands and available family members with 16p11.2 BP4-BP5 deletion from the Simons Variation in Individuals Project (SVIP) cohort, (d) 295 autism probands from the SSC cohort reported to carry severe de novo loss-of-function mutations in neurodevelopmental genes14,15, (e) 184 probands and matched unaffected siblings from the SSC cohort who inherited the same rare (≤0.1% frequency) loss-of-function or likely pathogenic missense primary mutations (CADD ≥25) in genes recurrently disrupted in neurodevelopmental disorders16.
Patient recruitment and clinical data ascertainment
We obtained clinical and/or genomic data from 141 children carrying the 16p12.1 deletion, as well as 39 deletion carrier and 30 non-carrier parents. Probands and parents recruited through direct contact provided consent according to the protocol reviewed and approved by The Pennsylvania State University Institutional Review Board. When individuals were not contacted directly, de-identified phenotypic (case histories and clinical information) and genomic data were used; as such, these cases were exempt from IRB review and conformed to the Helsinki Declaration.
We extracted clinical information from medical records or clinical questionnaires completed by different physicians from 180 carrier individuals and available family members (Supplementary Method 1.1). We used a modified de Vries scoring system for quantifying the number and severity of phenotypic abnormalities in affected children, which allows for a uniform assessment of developmental phenotypes from clinical records (Table S1)17. Family history information was used to bin families with the deletion into strong and mild or negative family history categories based on the severity of neurodevelopmental or psychiatric features (Figure S1, Supplementary Method 1.1). Genomic and clinical data for SSC and SVIP cohort families were obtained from the Simons Foundation Autism Research Initiative (SFARI) following appropriate approvals (see Supplementary Method 1.1).
Secondary variant burden analysis
To identify all coding variants modulating the presentation of the 16p12.1 deletion, we performed exome sequencing and SNP arrays on 105 individuals from 26 families as previously reported (Supplementary Method 1.2). Variant calls (SNVs and CNVs) from 716 individuals in the SSC were obtained from exome and SNP microarray studies14,15,18, and variant call files (VCF) and SNP array data from 84 families with 16p11.2 BP4-BP5 deletion were obtained from the Simons Foundation. Single-nucleotide variants and CNVs were filtered following the same procedures as for the 16p12.1 deletion cohort (Supplementary Method 1.2). We defined “secondary variant” or “second-hit” as an additional rare likely pathogenic mutation (<0.1% frequency CNV or SNV with CADD≥25) affecting a functionally intolerant gene (RVIS≤20th percentile) in an individual who already carries a disease-associated primary mutation (Figure 1A, Supplementary Method 1.2). The function of second-hit genes was analyzed using Ingenuity Pathway Analysis (IPA, Qiagen Bioinformatics), expression data derived from the GTEx consortium19, and Gene ontology (GO) enrichment analysis20 (Supplementary Method 1.3).
Statistical analyses
Proband-parent or proband-sibling secondary variant burdens and clinical severity scores were compared using Wilcoxon signed-rank test. Genetic burden and clinical severity scores between different categories of probands with related or shared primary variants were compared using non-parametric one-tailed Mann-Whitney tests, due to the hypothesis-driven nature of the comparison. The Kolmogorov-Smirnov test was used to assess normality distribution of the burden of secondary variants, FSIQ, SRS T-scores, BMI z-scores and head circumference (HC) z-scores. Correlation between number of secondary variants and quantitative phenotypes was assessed using Pearson’s correlation for normally distributed datasets, or Spearman’s correlation for datasets that were not normally distributed. Statistics were calculated using Minitab or R (v.3.4.2) software. Boxplots display the distribution of data from minimum to maximum.
Results
Secondary variants and disease expressivity in 16p12.1 deletion probands
We assessed how rare variants in the genetic background can modulate phenotypes in concert with a first-hit by evaluating 757 affected probands and 233 family members carrying disease-associated primary variants (rare CNVs or disruptive gene mutations) (Figure 1, see Methods). Using the 16p12.1 deletion as a paradigm for studying the genetic basis of variable expression of disease traits, we analyzed 180 individuals with the deletion and their non-carrier family members (Figure 1B). The 16p12.1 deletion was inherited in 92.4% of cases, with a significant maternal bias (57.6% maternal (n=53) vs 34.8% paternal (n=32), one-tailed binomial test p=0.01) (Table S2). In accordance with the female protective model described for neurodevelopmental disorders2,21,22, we observed a significant gender bias among probands with the 16p12.1 deletion (67.9% males versus 32.1% females, one-tailed binomial test p<0.0001). Detailed clinical analysis of 141 affected children with 16p12.1 deletion showed a wide heterogeneity of phenotypes, with a high prevalence of neurodevelopmental, craniofacial and musculoskeletal features (>50%), and variable involvement of other organs and systems (Figure 2A, Table S3). In contrast, 32 of 39 (82%) (61.5% females, 38.5% males) carrier parents showed mild cognitive, behavioral and/or psychiatric features (Table S4), consistent with previous reports of cognitive impairment and increased risk for schizophrenia in carriers of the 16p12.1 deletion23,24.
To identify variants within protein-coding regions that contribute to a more severe manifestation of the deletion in the affected children compared to their carrier parents, we performed exome sequencing and high-resolution SNP arrays in 26 families (n=105) with 16p12.1 deletion (23 inherited and 3 de novo cases, Table S5). We first evaluated whether the deletion could unmask recessive alleles, and found no rare pathogenic mutations within the seven 16p12.1 genes on the non-deleted chromosome (Table S6). We next performed a case-by-case analysis of families for second hits elsewhere in the genome by focusing on rare CNVs (<0.1%, ≥50 kbp), de novo or rare (ExAC frequency <0.1%) loss-of-function (LoF) mutations, and rare likely pathogenic missense variants (Phred-like CADD ≥25) in disease-associated genes (see Methods, Tables S7-S9). For example, in one proband we identified two disease-associated mutations, including a de novo LoF mutation in the intellectual disability-associated gene SETD5 (OMIM #615761, c.1623_1624insAC, p.Asp542Thrfs*3) and a LoF mutation in DMD (OMIM #310200, c.9G>A, p.Trp3X) transmitted from the 16p12.1-deletion carrier mother (Figure 2C). Similarly, a rare deletion at 2p16.3 encompassing NRXN1 (OMIM #614332), inherited from the non-carrier mother, was identified in another proband (Figure 2C).
While private disease-associated secondary mutations may explain the variable and severe features in the affected children on a case-by-case basis, we lacked the statistical power to implicate individual genes or variants that modulate specific 16p12.1 deletion phenotypes. Therefore, to globally assess the genome-wide contribution of rare pathogenic mutations affecting functionally relevant genes, we performed an integrative analysis and quantified rare (frequency ≤ 0.1%), likely pathogenic variants (CNVs or SNVs with CADD≥25) within genes intolerant to functional variation. The Residual Variation Intolerance Score (RVIS) has been shown to be a good predictor of intolerance of a gene to pathogenic mutations, and has been widely used by multiple studies for the recapitulation of known and the discovery of novel disease-associated genes15,25. For example, Krumm et al. showed that autism genes have an average RVIS of 26th percentile, and using classifications from the human disease network, Petrovski et al. showed that genes involved in developmental, skeletal, cardiac, neurological and muscular disorders show average scores in the 20th-30th percentile15,26. Therefore, to focus our assessment of secondary mutations to a subset of genes relevant to disease pathogenicity, we quantified rare likely pathogenic variants in genes with RVIS≤20th percentile, hereafter referred to as the “burden of secondary variants” or “second hits”. Intra-familial comparison of second-hit burden showed that probands have an excess of secondary variants compared to their carrier parents (Wilcoxon signed-rank test, p=0.004, Figures 2B and S2A-D), with no change in the number of synonymous mutations in all genes (p=0.29) or in RVIS≤20th genes (p=0.36, Figures S2E-F). Further, functional analysis of second-hit genes showed that probands presented an excess of second-hit genes that were preferentially expressed in the human brain (Wilcoxon signed-rank test, p=0.04, Figure S3) and enriched for developmental pathways (Table S10) compared to carrier parents.
The severity and variability of neurodevelopmental features is contingent upon family history of neuropsychiatric disease22. In fact, the cognitive and social outcomes in probands with de novo 16p11.2 BP4-BP5 deletion or 22q11.2 deletion have been reported to positively correlate with the cognitive and social skills of their parents27,28. However, the genetic basis of such background effects has not been specifically studied. We assessed the role of second-hit burden in the family-specific background effects and in the observed inter-familial variability of clinical features in probands with 16p12.1 deletion. We found that probands with a strong family history of neurodevelopmental and psychiatric disease presented a more severe and heterogeneous clinical presentation (Mann Whitney one-tailed, p=0.04) and a higher burden of secondary variants (p=0.001) than those with mild or negative family history (Figures 3A-C and S4A-C). Interestingly, probands with a strong family history also showed a higher difference in burden compared to their carrier parents than probands with a mild family history (p=0.003, Figure 3D). While we did not observe a difference in burden between carrier parents based on family history (p=0.68, Figure S4B), we found that non-carrier parents with a strong family history presented a significantly higher burden compared to those with a mild family history (p=0.01, Figure 3E). Therefore, in families with a strong history of neurodevelopmental and psychiatric disease, a higher number of rare and likely pathogenic secondary variants are more likely to be transmitted to the proband from the non-carrier parent, potentially contributing to a more severe manifestation of the disorder. These results suggest a potential role for rare pathogenic variants in the genetic background in modulating intra- and inter-familial clinical variability observed in families with the 16p12.1 deletion.
Secondary variant burden correlates with quantitative phenotypes among individuals with 16p11.2 deletion and other rare pathogenic CNVs
We next assessed whether the second-hit burden modulates quantitative phenotypes in carriers of other CNVs associated with neurodevelopmental phenotypes (Figure 1B). In probands with disease-associated rare CNVs (n=53, Table S11) from the Simons Simplex Cohort (SSC), we observed a modest but significant negative correlation (Pearson correlation, R=−0.36, p=0.004) between the number of secondary variants and full-scale IQ scores (FSIQ). This result held true when we separately analyzed individuals carrying 16p11.2 BP4-BP5 deletion (R=−0.68, p=0.04), but did not show statistical significance for 16p11.2 BP4-BP5 duplication (R =−0.34, p=0.17), 1q21.1 duplication (R =−0.36, p=0.32), or 7q11.23 duplication (R =−0.74, p=0.17), potentially due to a low sample size (Figures 4A and S5). Interestingly, probands with disease-associated CNVs and intellectual disability (FSIQ<70) showed a significant increase in the number of secondary variants compared to those without intellectual disability (FSIQ≥70, one-tailed Mann-Whitney, p=0.02, Figure S6).
We further expanded our analysis by evaluating a larger set of 84 families with 16p11.2 BP4-BP5 deletion from the Simons Variation in Individuals Project (SVIP). We observed a higher median number of secondary variants in probands carrying the 16p11.2 deletion that had intellectual disability (FSIQ<70, median=8) compared to those with no intellectual disability (FSIQ≥70, median=7 one-tailed Mann-Whitney, p=0.08, Figure 4B), without a difference in the number of synonymous mutations between the two subgroups (median of 9,957 synonymous changes for FSIQ<70 group versus 10,052 for the FSIQ≥70 group, two-tailed Mann-Whitney, p=0.51, Figure S7A). Notably, we observed only a mild negative correlation between second-hit burden and FSIQ, which did not attain statistical significance (Pearson correlation, R=−0.16, p=0.08, Figure S7B). We hypothesized that this marginal significance compared to 16p11.2 deletion probands from the SSC cohort (Figures 4A and S5B) could be due to differences in clinical ascertainment, as the SVIP cohort was selected for individuals carrying a 16p11.2 deletion who manifested a more heterogeneous set of phenotypes, while individuals from the SSC cohort were specifically ascertained for idiopathic autism29. These differences in ascertainment were evident by different distributions of quantitative phenotypes, including BMI, FSIQ, and SRS T-scores, in both populations (see Figure S8).
After adjusting for age to allow for full manifestation of the head phenotype, we identified a negative correlation between the number of secondary variants in SVIP probands with 16p11.2 deletion and their head circumference (HC) z-scores (age≥12 months, n=80, Pearson’s R=−0.26, p=0.009, Figure 4C)8,30. The observation that HC z-scores decline steadily (from >2 to <-2 scores) as second hits accumulate confirms that the deletion primarily leads to macrocephaly phenotypes, and suggests that the second-hits could explain the incomplete penetrance of this phenotype among carriers of the deletion30. We note that the second-hit burden did not correlate with Social Responsiveness Scale (SRS) T-scores or Body-Mass Index (BMI) z-scores, measures for autism and obesity, among probands with rare CNVs from the SSC cohort (Figures S9A-B) or those with 16p11.2 deletion from the SVIP cohort (Figures S9C-D), suggesting other mechanisms for the variability of these phenotypes.
Secondary variants modulate disease manifestation among individuals with disruptive mutations in disease-associated genes
We next analyzed 295 simplex cases from the SSC cohort with previously identified de novo gene-disruptive mutations within 271 genes14,15, and observed a moderately negative correlation between secondary variant burden and FSIQ scores (Spearman’s correlation, R=−0.25, p<0.0001, Figure 4D). Within this cohort, individuals with intellectual disability (FSIQ<70, n=93) presented an enrichment of second hits compared to those without intellectual disability (FSIQ≥70, n=197) (one-tailed Mann-Whitney, p=0.001, Figure S10A). We did not observe a role for secondary variant burden in modulating BMI z-scores (Spearman’s R=−0.038, p=0.27, Figure S10B), although we did find a mild but significant positive correlation with SRS T-scores (Spearman’s R=0.12, p=0.02, Figure S10C). Moreover, when probands were separated by gender, we observed a higher burden of secondary variants in females compared to males (one-tailed Mann-Whitney, p=0.02, Figure S11). This supports the hypothesis that females require a higher contribution from the genetic background to reach the genetic threshold for pathogenesis of neurodevelopmental disease than males22.
While there is a consensus on the pathogenic role of de novo gene-disruptive mutations in simplex families, the interpretation of inherited disruptive variants within the same genes is challenging. To understand the role of the genetic background in the penetrance of inherited disruptive mutations in disease-associated genes, we analyzed 184 pairs of probands and unaffected siblings who inherited the same pathogenic mutations in genes recurrently disrupted in neurodevelopmental disorders (Table S12). We found a greater enrichment of second hits in probands compared to their unaffected siblings (Wilcoxon signed-rank test p=0.03, Figure 4E), suggesting that second hits likely contribute to increased penetrance of neurodevelopmental phenotypes in children with inherited pathogenic single-gene mutations. When we analyzed probands carrying disruptive mutations in specific neurodevelopmental genes, we found that the severity of cognitive deficits in individuals with damaging mutations in SCN1A was concordant with an excess of secondary variants (probands with FSIQ<70, n=8, median=16.5, versus those with FSIQ≥70, n=8, median=8.5, one-tailed Mann-Whitney, p=0.02) (Figure 4F). This observation could also explain the diversity of other phenotypes co-occurring with the disruption of the epilepsy-associated SCN1A gene, such as motor delay and autism31.
Secondary variants involve disease associated genes and affect core cellular and developmental processes
To understand how secondary variants could modulate phenotypes among probands with pathogenic first-hit variants, we explored the functionality of second-hit genes identified in all probands analyzed in our study. Overall, we identified 3,197 rare likely-pathogenic mutations encompassing a diverse set of 1,615 functionally intolerant genes. Of these, 40.9% (660/1,615) were found to be extremely intolerant to loss-of-function mutations (pLI metric ≥0.9). These genes were also enriched for postsynaptic density genes, genes encoding FMRP protein targets, chromatin-associated genes, genes embryonically expressed, and essential genes, compared to the whole genome (52% vs. 26%, Chi-squared test, p<0.0001)32,33. Interestingly, 44 of these second-hit genes (such as CNTNAP2, MBD5, SCN1A, CHD8 and AUTS2) have been recurrently associated with neurodevelopmental disorders16 (Figure S12), 58 genes have been previously identified as a causative gene in simplex autism cases14,15 (Table S13), and 50 genes have been associated with skeletal, muscular, cardiovascular or renal disorders, as classified in the human disease network from the OMIM database (Table S14)34. We further assessed the location of second-hit mutations within a subset of genes recurrently associated with disease, including RIMS1, DIP2A, KDM5B and ACOX2, and found no specificity for the location of the secondary variants within the protein sequences compared to previously reported de novo disruptive mutations within these genes (Figure 5A). In fact, in some cases, we observed stopgain secondary variants that were more premature in the protein sequence than previously reported disruptive mutations in these genes, suggesting that the second-hit can potentially exert an effect as severe as the first-hit, if these are true loss-of-function variants. The allelic diversity of second hits within these genes suggests that further functional analysis should be performed in order to understand their specific effects on modulating developmental phenotypes.
To further characterize the function of second-hit genes, we performed Gene Ontology enrichment analysis of second-hit genes in probands with de novo mutations from the SSC and 16p11.2 deletion probands from the SVIP cohort. We found that genes carrying secondary variants in probands from both cohorts were enriched for core processes, including cell signaling and adhesion and developmental processes (Figures 5B-C, Table S15-S16). Although some of these genes have been individually associated with a disease phenotype, further functional analyses are required to understand potential interactions between genes affected by primary and secondary variants, their cumulative burden, and ultimately their potential contribution towards phenotypic variability.
Discussion
Our results support an oligogenic model for neurodevelopmental disorders, where the primary variant provides a sensitized genomic background for disease and secondary variants within functionally intolerant genes modify the phenotypic trajectory of the disorder. We propose that a higher burden of secondary variants increases the likelihood of involving a disease-associated modifier gene within disease-related pathways, as well as allows for a higher number of oligogenic combinations potentially modulating the phenotype associated with the first-hit. Some primary variants that are more tolerant to changes in the genetic background, such as the 16p12.1 deletion, are transmitted through generations and only surpass the threshold for severe disease with the accumulation of several rare pathogenic mutations. Other primary variants which are often de novo, such as the 16p11.2 deletion, push the genetic background closer to the threshold for severe manifestation and therefore require a lesser contribution from secondary variants. Similarly, highly penetrant syndromic CNVs such as Smith-Magenis syndrome and Sotos syndrome, which are mostly de novo and encompass genes more intolerant to functional variation compared to variably expressive CNVs (p=0.03, one-tailed Mann-Whitney, Table S17, Figure S13), would push the genetic liability beyond the threshold for severe disease2,13. While additional mutations may not be necessary for complete penetrance of these disorders, these variants can modify specific phenotype traits when present. For example, deleterious mutations in histone modifier genes have been reported to contribute to the conotruncal heart manifestation of 22q11.2 deletion syndrome35. This model would also apply for single-gene disorders, where second hits potentially explain discordant clinical features reported among affected carriers of the same molecular alteration, as described for Rett syndrome and individuals with disruptive mutations in the intellectual disability gene PACS136,37.
Our observations that probands with a strong family history exhibit severe clinical manifestation and a higher second-hit burden also provide insights into the role of rare variants in the genetic background towards the reported correlation between parental profiles and clinical outcome in probands carrying rare CNVs27,28. Moreover, the observed higher burden of secondary variants in the non-carrier parents in families with strong family history suggests assortative mating, and transmission of these hits to the proband potentially explains the increased clinical severity. Similarly, in 16p11.2 deletion, we observed that children who inherited the CNV presented lower FSIQ scores (n=8, median FSIQ=75) than probands with a de novo deletion (n=57, median FSIQ=85, one-tailed Mann-Whitney, p=0.006, Figure S14A-B), in agreement with previous reports8. This was also consistent with a non-significant excess of the second-hit burden among probands with an inherited 16p11.2 deletion (one-tailed Mann-Whitney, p=0.06, Figure S14B) compared to de novo probands. These results highlight the importance of eliciting family history of psychiatric and neurodevelopmental disease for more accurate diagnostic assessment of the affected children.
Overall, these results suggest a multi-dimensional effect of secondary variants towards clinical features, and that their contribution to specific phenotypic domains depends on the extent to which the primary variant sensitizes an individual towards a specific phenotypic trajectory. An important observation from our study is that a large number of disease-associated variants that were deemed to be solely causative for the disorder are in fact accompanied by substantial amount of rare genetic variation. Longitudinal and quantitative phenotyping across multiple developmental domains in all family members, along with whole genome sequencing studies in affected and seemingly asymptomatic individuals with a primary variant, are necessary for a more accurate understanding of these complex disorders. Therefore, it is critical that even after identifying a likely diagnostic disruptive mutation, further analysis of the genetic background must be performed in order to provide appropriate counseling and management.
Acknowledgements
We are grateful to all of the families in each cohort (16p12.1 deletion, SVIP and SSC) who participated in the study. We thank the SSC principal investigators (A. Beaudet, R. Bernier, J. Constantino, E. Cook, E. Fombonne, D. Geschwind, R. Goin-Kochel, E. Hanson, D. Grice, A. Klin, D. Ledbetter, C. Lord, C. Martin, D. Martin, R. Maxim, J. Miles, O. Ousley, K. Pelphrey, B. Peterson, J. Piggot, C. Saulnier, M. State, W. Stone, J. Sutcliffe, C. Walsh, Z. Warren, E. Wijsman) as well as the Simons VIP Consortium. We appreciate obtaining access to genomic and phenotypic data on SFARI Base. Approved researchers can obtain the SSC and SVIP population datasets described in this study by applying at https://base.sfari.org.
This work was supported by NIH R01-GM121907, Brain and Behavior Foundation (NARSAD#22535), SFARI Pilot Grant (SFARI #399894, SG) and resources from the Huck Institutes of the Life Sciences to S.G. L.P. was supported by Fulbright Commission Uruguay – ANII and the Huck Institutes of the Life Sciences. M.J. was supported by NIH T32-GM102057. C.R., L.C., O.G. and E.A. were supported by the Italian Ministry of Health and ‘5 per mille’ funding. K.M. is a Jacobs Foundation Research Fellow. A.R. is supported by the Swiss National Science Foundation (31003A_160203). Dedicated to the memory of Ethan Francis Schwartz, 1996-1998.