Using Whole-Exome Sequencing to Identify Inherited Causes of Autism

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5' and 3' RACE confirmed at least one polyadenylated transcript from the P3 promoter, corresponding to GenBank clone BC039121 and encompassing exons 57-63 of the predicted full-length SYNE1 mRNA.

“5′ and 3′ RACE (data not shown) confirmed the existence of at least one polyadenylated transcript emanating from this promoter, corresponding to the GenBank deposited mRNA clone BC039121, encompassing exons 57–63 of the predicted full-length SYNE1 mRNA.”

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AMT p.D198G is poorly soluble and has a temperature-sensitive stability defect, while enzyme specific activity is preserved, suggesting pathogenicity through misfolding or instability rather than catalytic dysfunction.

“Functional assays of AMT p.D198G demonstrated that, like p.I308F and other NKH-associated mutations, p.D198G is poorly soluble (Figure S5). AMT p.D198G also exhibited a temperature-sensitive protein stability defect (Figure S5). Enzyme specific activity was preserved (Figure S5), suggesting that pathogenicity may be due to protein misfolding/stability and not catalytic dysfunction, similar to what”

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Two consanguineous families had homozygous nonsense or frameshift mutations in PAH and were subsequently confirmed to have phenylketonuria by clinical laboratory testing.

“Two consanguineous families had homozygous nonsense or frameshift mutations in PAH (Table 1A; Figure S4), the cause of phenylketonuria and one of the earliest neurometabolic syndromes described as a cause of ASD (Zecavati and Spence, 2009). These families were subsequently confirmed to have phenylketonuria by clinical laboratory testing.”

• cites Zecavati and Spence, 2009 sourceNot checked yet

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Whole-exome sequencing of ASD families selected for inherited causes through consanguinity identified familial ASD associated with biallelic mutations in AMT, PEX7, SYNE1, VPS13B, PAH, and POMGNT1, with some of these genes not previously implicated in ASD.

“We apply whole exome sequencing (WES) to ASD families enriched for inherited causes due to consanguinity and find familial ASD associated with biallelic mutations in disease genes (AMT, PEX7, SYNE1, VPS13B, PAH, POMGNT1), some implicated for the first time in ASD.”

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SYNE1 p.L3206M alters a highly conserved residue within a spectrin repeat and has a SIFT score of 0.01.

“SYNE1 p.L3206M alters a highly conserved residue that lies within a spectrin repeat (Figure 3D; SIFT score 0.01).”

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One consanguineous family had a homozygous AMT p.D198G missense change in a child with ASD and intellectual disability; the variant was heterozygous in both parents and an unaffected sibling and disrupts a conserved residue with SIFT score 0.01.

“In one consanguineous ASD family, we identified a linked homozygous missense change in AMT (p.D198G) in a single affected child with ASD and intellectual disability (Table 1B). This variant was heterozygous in both parents and an unaffected sibling, and disrupts a highly conserved residue of AMT (SIFT score 0.01).”

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AMT p.I308F alters an isoleucine residue that is highly conserved across AMT orthologs and is packed into a hydrophobic pocket.

“The mutation resulted in p.I308F, altering an Ile residue that is highly conserved in all AMT orthologs (Figure 1C), and is packed tightly into a hydrophobic pocket (Figure 1D)(Okamura-Ikeda et al., 2005).”

• cites Okamura-Ikeda et al., 2005 sourceNot checked yet

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ChIPseq in human neurons identified at least four major transcriptional start sites within the SYNE1 locus, one of which, P3, lies immediately upstream of the p.L3206M mutation.

“ChIPseq using antibodies to H3K4Me3, a mark associated with active promoter sites (Ernst et al., 2011), and to H3K27Ac, a mark associated with enhancer elements (Heintzman et al., 2009), demonstrated mapped read peaks corresponding to at least four major transcriptional start sites within the SYNE1 locus (P1–P4), one of which (P3) lies immediately upstream of the p.L3206M mutation (Figure 3E).”

• cites Ernst et al., 2011 sourceNot checked yet

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• cites Heintzman et al., 2009 sourceNot checked yet

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In the second family, parametric mapping excluded 97.2% of the genome and established linkage to a homozygous interval on 6p23 with a maximum LOD score of 2.78, indicating a greater than 600:1 likelihood that the interval contained the disease-causing mutation.

“Parametric mapping excluded 97.2% of the genome and established linkage to a homozygous interval on 6p23 (Figure 2B) (LOD 2.78, the maximum obtainable in the pedigree, under a recessive model, indicating a >600:1 likelihood that this interval contained the disease-causing mutation).”

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In a semi-quantitative peroxisomal import assay, wildtype PEX7 fully restored processing of thiolase, PhyH, and AGPS, whereas PEX7 p.W75C produced only partial processing.

“Transfection of wildtype PEX7 fully restores processing, whereas transfection of PEX7 p.W75C produced only partial processing (Figure 2F).”

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In the first family, linkage mapping under a single-locus biallelic model excluded 99.3% of the genome and found a single peak at 3p21.31 within a large homozygous interval, with a maximum LOD score of 2.96, indicating a greater than 900:1 likelihood that the responsible mutation lay in that interval.

“Mapping under a single locus, biallelic model (i.e., allowing for both homozygous and compound heterozygous mutations) excluded 99.3% of the genome and revealed a single linkage peak centered at 3p21.31, in a large homozygous interval, reaching the maximum LOD score obtainable in the pedigree, 2.96 (Figure 1B), suggesting a >900:1 likelihood that the responsible mutation was contained within this”

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One family had a nonsense NLGN4X p.Q329X mutation in a single affected male child, inherited from an unaffected mother.

“One of these was a nonsense mutation in NLGN4X (p.Q329X), found in a single affected male child. NLGN4X is an X-linked gene encoding a neuronal synaptic adhesion molecule, and mutations in NLGN4X have been described in individuals with autism, Asperger syndrome, and intellectual disability (Jamain et al., 2003). This mutation was inherited from an unaffected mother, consistent with prior”

• cites Jamain et al., 2003 sourceNot checked yet

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In a screen of 163 consanguineous and/or multiplex families, five families had novel molecular genetic diagnoses due to severe loss-of-function hemizygous or homozygous mutations in known genes.

“In five families (Tables 1A and S4), our screen revealed novel molecular genetic diagnoses due to severe loss of function (nonsense or frameshift) hemizygous or homozygous mutations in known genes.”

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In RCDP fibroblasts, PTS2-mCherry fails to be imported into peroxisomes and remains cytosolic; wildtype PEX7 fully restores import, whereas PEX7 p.W75C fails to rescue in most cells and partially rescues a fraction.

“In RCDP fibroblasts, fluorescent mCherry fused to the PTS2 peroxisomal targeting sequence fails to be imported into peroxisomes and remains cytosolic (Figure 2E). Co-transfection of wildtype PEX7 fully restores peroxisomal import (Figure 2E). In contrast, transfection with PEX7 p.W75C failed to rescue (Figure 2E): the majority of cells showed cytosolic PTS2-mCherry, although a fraction showed”

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Whole-exome sequencing of the entire third family found no rare protein-altering variants in the 7q33 interval, and only one protein-altering variant in 6q25 that segregated with disease: a homozygous SYNE1 p.L3206M missense change.

“No rare, protein-altering variants were found in the 7q33 linkage interval, whereas 6q25 harbored only one protein-altering variant, absent from known databases and population-matched controls, that segregated with disease: a homozygous missense change in SYNE1 (p.L3206M) (Table 1).”

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RNAseq in cultured human primary neurons showed that depolarization induced full-length SYNE1 transcription by 1.27-fold (n=5, SE 0.06, p=0.0203) and BC039121 transcription by 1.50-fold (n=5, SE 0.11, p=0.0225).

“Transcription of full-length SYNE1 was induced 1.27-fold (n=5, S.E. 0.06, p=0.0203, t test, one-tailed) by neuronal activity, and transcription of BC039121 was induced by 1.50-fold (n=5, S.E. 0.11, p=0.0225, t test, one-tailed) across five biological replicates (Figures 3E and S2).”

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One consanguineous family had a homozygous frameshift VPS13B/COH1 p.A3943fs mutation in a proband with ASD and mild dysmorphic features; the mutation truncates the C-terminal 54 amino acids.

“We found a homozygous frameshift alteration in VPS13B/COH1 in the proband in a consanguineous family who had ASD and mild dysmorphic features (Figure 5A; Table 1A). The mutation (p.A3943fs) causes truncation of the C-terminal 54 amino acids of VPS13B/COH1.”

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Eleven families had rare segregating homozygous or hemizygous missense changes in known genes, and clinical and/or biochemical evidence supported pathogenicity in at least four instances.

“Eleven families were found to have rare, segregating, homozygous or hemizygous missense changes in known genes (Tables 1B, S3, and S4). While some of these may be expected to be functionally silent, we found clinical and/or biochemical evidence supporting their pathogenicity in at least four instances (Table 1B).”

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Direct biochemical analysis showed that AMT p.I308F has reduced activity: it is poorly soluble at 30°C, can be rescued by coexpressing GroES and GroEL at 22°C, and retains only 45% of wildtype glycine cleavage activity and 1.8% of wildtype glycine synthesis activity.

“Direct biochemical analysis of the p.I308F mutation confirms that it has reduced activity. While wildtype AMT is fully soluble at 30°C when expressed in bacteria, mutant AMT p.I308F was poorly soluble (Figure 1E), indicating a protein folding defect, similar to that observed with NKH-associated AMT mutations (Figure S1; Table S1). This defect could be rescued by coexpressing GroES and GroEL heat”

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One child with ASD and moderate intellectual disability had a homozygous POMGNT1 p.R367H missense change disrupting a conserved residue; this exact allele was previously reported as causative in a relatively mild case as a compound heterozygous mutation with a splice site mutation.

“A child affected with ASD and moderate intellectual disability was found to have a homozygous missense change (p.R367H) in POMGNT1 (Table 1B; Figure S4). POMGNT1 is responsible for an inherited dystroglycanopathy characterized by brain malformation, intellectual disability, developmental delay, hypotonia, and myopia; interestingly, rare patients have been reported with severe autistic features (”

• cites Godfrey et al., 2007 sourceNot checked yet

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The genes implicated in this study include MECP2 and SYNE1, which are known to regulate or be regulated by synaptic activity, and AMT, PEX7, and VPS13B/COH1, which have not traditionally been considered synaptic.

“Genes implicated in this study include ones known to regulate or be regulated by synaptic activity (MECP2, SYNE1) but also genes not traditionally thought of as having synaptic roles (AMT, PEX7, VPS13B/COH1).”

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One consanguineous family had a single affected child homozygous for a rare VPS13B p.S824A missense variant; the proband had some but not all features of Cohen syndrome, including autism, mild facial dysmorphism, and joint laxity, consistent with mild previously reported versions.

“Finally, in another consanguineous family, the single affected child was homozygous for a rare missense variant (p.S824A) in VPS13B (Table 1B; Figure S4). The proband had in retrospect some but not all features of Cohen syndrome (autism with mild facial dysmorphism and joint laxity), consistent with mild versions reported previously (Hennies et al., 2004).”

• cites Hennies et al., 2004 sourceNot checked yet

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Transmitted rare copy number variants such as 16p11.2 microdeletion/duplication and 15q11.2-q13 duplication contribute to ASD, but account for only a small number of cases.

“Transmitted, rare copy number variants (CNVs), such as 16p11.2 microdeletion/duplication and 15q11.2-q13 duplication have been found to contribute, although the total number of cases accounted for by these conditions is small (Levy et al., 2011; Pinto et al., 2010; Weiss et al., 2008).”

• cites Levy et al., 2011 sourceNot checked yet

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• cites Pinto et al., 2010 sourceNot checked yet

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• cites Weiss et al., 2008 sourceNot checked yet

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Studies have implicated homozygous copy number variants and long homozygous intervals as significantly associated with ASDs.

“Additional evidence supporting a role of biallelic mutations comes from studies that have implicated homozygous CNVs (Levy et al., 2011; Morrow et al., 2008) and long homozygous intervals as significantly associated with ASDs (Casey et al., 2012).”

• cites Levy et al., 2011 sourceNot checked yet

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• cites Morrow et al., 2008 sourceNot checked yet

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• cites Casey et al., 2012 sourceNot checked yet

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ASDs are significantly heritable, but the basis of this heritability remains largely unexplained.

“Despite studies suggesting that autism spectrum disorders (ASDs) are significantly heritable, the basis of this heritability remains largely unexplained (Devlin and Scherer, 2012).”

• cites Devlin and Scherer, 2012 sourceNot checked yet

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The identified mutations are often only partially disabling or present atypically, and affected individuals lack diagnostic features of the Mendelian disorders classically associated with the mutated genes.

“These mutations are often only partially disabling or present atypically, with patients lacking diagnostic features of the Mendelian disorders with which these genes are classically associated.”

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De novo copy number variants and de novo protein-altering point mutations have significant roles in ASD.

“Significant roles have also been demonstrated for diverse, de novo CNVs (Levy et al., 2011; Sanders et al., 2011; Sebat et al., 2007) and more recently, de novo, protein-altering point mutations (Iossifov et al., 2012; Neale et al., 2012; O’Roak et al., 2011; O’Roak et al., 2012; Sanders et al., 2012).”

• cites Levy et al., 2011 sourceNot checked yet

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• cites Sanders et al., 2011 sourceNot checked yet

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• cites Sebat et al., 2007 sourceNot checked yet

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Sanger validation confirmed that AMT p.I308F was heterozygous in both parents, homozygous in all affected children, and absent or heterozygous in the two unaffected children.

“Sanger validation confirmed that the mutation was heterozygous in both parents, homozygous in all affected children, and absent or heterozygous in the two unaffected children.”

Classified as probably not a substantial claim.

A recent whole-exome sequencing study suggested a role for biallelic point mutations in a subset of ASD patients with long runs of homozygosity.

“Finally, a recent whole exome sequencing (WES) study has suggested a role for biallelic point mutations in a subset of patients with ASDs that show long runs of homozygosity (Chahrour et al., 2012).”

• cites Chahrour et al., 2012 sourceNot checked yet

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MECP2 p.E483X is likely hypomorphic because MECP2 nonsense mutations are typically lethal in males and p.E483X is a late truncation predicted to remove only the last four amino acids.

“The finding of MECP2 nonsense mutations in this family was unusual since these are typically lethal in males (Chahrour and Zoghbi, 2007), suggesting that this allele is likely hypomorphic. Consistent with this idea, p.E483X is a late truncation predicted to remove only the last four amino acids of the full-length protein.”

• cites Chahrour and Zoghbi, 2007 sourceNot checked yet

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PEX7 p.W75C is pathogenic but causes partial loss of function, consistent with affected individuals not exhibiting the full RCDP syndrome.

“These results demonstrate that this allele is pathogenic, but partial loss-of-function, consistent with these individuals not exhibiting full features of the RCDP syndrome.”

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Several neurometabolic disorders have been linked to autistic symptoms.

“Although several neurometabolic disorders have been associated with autistic symptoms (Zecavati and Spence, 2009)”

• cites Zecavati and Spence, 2009 sourceNot checked yet

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PEX7 regulates peroxisomal protein import, and peroxisomes are abundant in dendrites.

“PEX7 regulates peroxisomal protein import, and peroxisomes are abundant in dendrites (Kou et al., 2011).”

• cites Kou et al., 2011 sourceNot checked yet

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Biallelic mutations are also present in nonconsanguineous autism cohorts, but much larger analyses of genes and patients are needed to quantify their prevalence.

“These data confirm that biallelic mutations are also found in nonconsanguineous autism cohorts, but analysis of much larger numbers of genes and patients will be needed to quantify their prevalence.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Very mild mutations of this kind may be rare, particularly in populations without consanguinity.

“although such very mild mutations may be quite rare, especially in nonconsanguineous populations.”

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A common theme for ASD mutations in general may be partial loss of gene function and/or dosage sensitivity.

“Hypomorphic, biallelic mutations, combined with CNVs and heterozygous stop mutations (Neale et al., 2012; O’Roak et al., 2012; Sanders et al., 2012) which completely inactivate one of two alleles, suggest that a common theme for ASD mutations in general might be partial loss of gene function and/or dosage sensitivity.”

• cites Neale et al., 2012 sourceNot checked yet

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• cites O’Roak et al., 2012 sourceNot checked yet

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• cites Sanders et al., 2012 sourceNot checked yet

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Null mutations in these genes affect many tissues, whereas hypomorphic mutations may cause subtler defects largely confined to neurons.

“while null mutations in these genes have effects in many tissues, hypomorphic mutations may cause subtler defects primarily limited to neurons.”

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A common theme among many identified mutations is hypomorphic mutations that partially impair gene function, although one or two null mutations were also identified.

“A common theme of many of these mutations is hypomorphic mutations that partially impair gene function, though one or two null mutations are also identified.”

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Peroxisomal import was verified by co-localization with the peroxisome membrane protein PEX14.

“Peroxisomal import was confirmed by co-localization of the peroxisome membrane protein PEX14.”

Classified as probably not a substantial claim.

Sanger sequencing was used to confirm every reported variant.

“All reported variants were confirmed by Sanger sequencing.”

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A common deletion in TMLHE has been implicated as a risk factor for autism.

“a common deletion in TMLHE, encoding the first enzyme in carnitine biosynthesis, has been recently implicated as a risk factor for autism (Celestino-Soper et al., 2012).”

• cites Celestino-Soper et al., 2012 sourceNot checked yet

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The exome sequencing had a mean target coverage of 85.6% at 20X or more and a mean read depth of 158X.

“with a mean target coverage of 85.6% at >=20X and a mean read depth of 158X.”

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ASD associations with Mendelian genes such as FMR1 or TSC2 show near-complete penetrance for syndromic features but only partial penetrance for ASD.

“This variable expressivity has parallels in known associations of ASD with Mendelian genes like FMR1 or TSC2, which are near fully penetrant for syndromic features of Fragile X or Tuberous Sclerosis, respectively, but only partially penetrant for ASD (Hagerman et al., 2010; Wiznitzer, 2004).”

• cites Hagerman et al., 2010 sourceNot checked yet

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• cites Wiznitzer, 2004 sourceNot checked yet

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Recovery of peroxisomal import was scored blindly in 100 cells from each of 3 separate transfections.

“Recovery of peroxisomal import was assessed by blinded visual scoring of 100 cells each from 3 separate transfections.”

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VPS13/COH1 plays essential roles in vesicle trafficking through the Golgi apparatus.

“VPS13/COH1 has essential roles in vesicle trafficking through the Golgi apparatus (Seifert et al., 2011).”

• cites Seifert et al., 2011 sourceNot checked yet

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