**6. CNVs in autism**

As discussed above, autism is a phenotypic feature of many genomic disorders. Betancur [77], in her review, lists 103 disease genes and 44 genomic loci where autism or autistic like behaviors have been described. ASD is diagnosed in ~30% of males with Fragile X syndrome and in reverse, Fragile X mutations are found in as many as 7–8% of individuals with ASD [78]. Sim‐ ilarly, mutations in MECP2, the Rett Syndrome gene, have been found among cases of autism that do not have the classical Rett phenotype and autism patients have an increased risk for neurofibromatosis and other rare monogenic diseases like tuberous sclerosis and Joubert's Syndrome, again inversely patients with these disorders have an increased risk for having autism [79, 80].

The genes and loci listed in the Betancur study are all causally implicated in learning disability (LD), indicating that these two neurodevelopmental disorders share some ge‐ netic risk factors. Early use of aCGH in non-syndromic autism suggested the method had promise in detecting hitherto unrecognized CNVs. For example Jacquemont et al [81] identified 6 deletions and 2 duplications in 29 patients presenting with syndromic ASD where previous high resolution karyotyping was reported as normal. Another study showing the potential for CNV analysis was a large linkage study [14] using a 10k SNP array where intensity data was used to determine copy number. The authors highlight some individual findings including a family with two sisters with ASD, both of whom had a ~300kb deletion on Ch. 2p16 that included the coding exons of the neu‐ rexin 1 gene (*NRXN1*). A second finding was a recurrent 1.1Mb duplication at Ch. 1q2.1 in four affected individuals from three families, a third was a ~900kb de novo duplica‐ tion at 17p12 in an affected sib-pair, and with the same region appearing as a maternal‐ ly inherited deletion in two male siblings, with a paternally inherited deletion in a further female. Duplications in this region cause Charcot-Marie-Tooth 1A (*CMT1A*) and hereditary neuropathy with pressure palsies when deleted, and overlapping deletions are seen in Smith-Magenis syndrome that includes autism symptoms in many cases [82].

A key development was the report of de novo copy number variants in autism [83] using aCGH. These authors showed that individually rare CNVs, and in particular ones that affect neurodevelopmental genes, were enriched in cases. They further suggested that the rate of de novo CNVs differed between simplex cases, where they occurred in 10% of families in the sample, and familial cases where they occurred in 3% of families, suggesting that sporadic and familial cases of ASD might have different underlying genetic mechanisms, although not all studies since then have found this distinction. Several studies of CNVs in large autism case and family series have followed. Marshall et al. [84] examined 427 ASD families using a 500k SNP array and karyotyping by standard clinical diagnostic method. A de novo rate of 7.1% and 2.0% in simplex and multiplex families respectively was observed, supporting the previ‐ ous findings [83]. Families occasionally showed more than a single de-novo event where both may combine to produce risk. A further set of loci were identified in two or more unrelated families, increasing the evidence supporting a pathogenic role. As with the LD literature, at some loci, both deletions and duplication were found suggesting a more complex mechanism than simple over or under-expression of gene products. Of the 196 inherited CNVs confirmed experimentally, 90 were of maternal and 106 of paternal origin. The authors list numerous potential ASD candidate genes where a structural change was either de-novo, found in two or more unrelated ASD cases, or, in the case of the X-chromosome, transmitted from an unaffected mother. Given their rarity, very few individual CNVs in this study provided statistical evi‐ dence to support their role in autism. For example, 4 CNVs from 427 cases were found at the *DPP6* – a subunit that affects the function of Kv4.2 channels at the same site of expression as *SHANK3* and *NLGN* gene products. Only one similar CNV was found in 1652 controls (Fisch‐ er's exact test p = 0.016). In keeping with previous cytogenetic findings and the emerging overlap in disorders involved, CNVs were found in ASD cases that involved known loci or genes in disorders such as Waardenburg Type IIa, Speech and language disorder, learning disability and VCFS. A further study [85] found a total of 51 CNVs in 46 cases and not in the controls. 42 of these were familial and 9 de novo with recurrence in two or more cases at three loci. In total, case specific CNVs were found in 11.6% of cases, although, in keeping with the Marshall et al [84] study, none were individually associated with case status with the majority being observed in only a single case. Pinto et al [86] compared CNVs in 996 ASD cases of European ancestry to 1,287 matched controls, using the Illumina 1M SNP array. Cases were found to carry a higher global burden of rare, genic CNVs, especially so for loci previously implicated in either ASD and/or intellectual disability. Nearly 6% of the cases had de-novo mutations with some having two or more events. Novel candidate genes were identified that were de-novo in cases and not controls, including *SHANK2*, *SYNGAP1* and *DLGAP2*. In keep‐ ing with previous studies only one novel CNV (maternally inherited X-linked deletions at *PTCHD1*) occurred statistically more frequently in cases compared to controls (7 vs 0). *PTCHD1* involvement in autism and LD was further extended and confirmed in a focused examination of the *PTCHD1* locus in cohorts of autism and LD cases and extending the study of CNVs to sequence data, identifying additional maternally inherited missense mutations in 8 probands not seen in controls [87]. In the Pinto et al study[86], certain gene sets were found to be enriched for case deletions but not duplications. These included sets involved in cell and neuronal de‐ velopment, projection, motility and proliferation; GTPase/Ras signaling known to be involved in regulating dendrite and spine plasticity; and kinase activity/regulation. There was addi‐ tional overlap with gene sets thought to be involved in LD including microtubule cytoskeleton, glycosylation and CNS development/adhesion. More recently, Salyakina and colleagues [88] have shown the value of extended multiply affected families in a CNV study of 42 families. They found 5 deletions and 7 duplications that co-segregated with ASD, two overlapping with known autism CNVs on 7p21.3 and 15q24.1 and two near regions on 3p26.3 and 12q24.32 previously associated with schizophrenia.

10-5, a rate that is considerably more frequent than point mutations [76]. This high mutation rate, coupled with reduced fecundity in some carriers, and the fact that comparatively, they affect a larger proportion of the genome, make CNVs a potentially important source of new

As discussed above, autism is a phenotypic feature of many genomic disorders. Betancur [77], in her review, lists 103 disease genes and 44 genomic loci where autism or autistic like behaviors have been described. ASD is diagnosed in ~30% of males with Fragile X syndrome and in reverse, Fragile X mutations are found in as many as 7–8% of individuals with ASD [78]. Sim‐ ilarly, mutations in MECP2, the Rett Syndrome gene, have been found among cases of autism that do not have the classical Rett phenotype and autism patients have an increased risk for neurofibromatosis and other rare monogenic diseases like tuberous sclerosis and Joubert's Syndrome, again inversely patients with these disorders have an increased risk for having

The genes and loci listed in the Betancur study are all causally implicated in learning disability (LD), indicating that these two neurodevelopmental disorders share some ge‐ netic risk factors. Early use of aCGH in non-syndromic autism suggested the method had promise in detecting hitherto unrecognized CNVs. For example Jacquemont et al [81] identified 6 deletions and 2 duplications in 29 patients presenting with syndromic ASD where previous high resolution karyotyping was reported as normal. Another study showing the potential for CNV analysis was a large linkage study [14] using a 10k SNP array where intensity data was used to determine copy number. The authors highlight some individual findings including a family with two sisters with ASD, both of whom had a ~300kb deletion on Ch. 2p16 that included the coding exons of the neu‐ rexin 1 gene (*NRXN1*). A second finding was a recurrent 1.1Mb duplication at Ch. 1q2.1 in four affected individuals from three families, a third was a ~900kb de novo duplica‐ tion at 17p12 in an affected sib-pair, and with the same region appearing as a maternal‐ ly inherited deletion in two male siblings, with a paternally inherited deletion in a further female. Duplications in this region cause Charcot-Marie-Tooth 1A (*CMT1A*) and hereditary neuropathy with pressure palsies when deleted, and overlapping deletions are seen in Smith-Magenis syndrome that includes autism symptoms in many cases [82].

A key development was the report of de novo copy number variants in autism [83] using aCGH. These authors showed that individually rare CNVs, and in particular ones that affect neurodevelopmental genes, were enriched in cases. They further suggested that the rate of de novo CNVs differed between simplex cases, where they occurred in 10% of families in the sample, and familial cases where they occurred in 3% of families, suggesting that sporadic and familial cases of ASD might have different underlying genetic mechanisms, although not all studies since then have found this distinction. Several studies of CNVs in large autism case and family series have followed. Marshall et al. [84] examined 427 ASD families using a 500k

and recent mutation in neurodevelopmental disorders.

306 Recent Advances in Autism Spectrum Disorders - Volume I

**6. CNVs in autism**

autism [79, 80].

As the resolution of the probe arrays improves, smaller CNVs will be detected, and the boun‐ daries of previously identified CNVs will become more refined [89]. Nord et al. [90], examined genomic DNA of 41 children with autism and 367 healthy controls for rare CNVs using a very high-resolution aCGH platform. They found that cases were more likely than controls to have CNVs as small as ~10 kb, likely to affect genes involved in transcription, nervous system de‐ velopment, and receptor activity. They found that expression of *CNTNAP2*, *ZNF214*, *PRODH* and *ARID1B* genes affected by CNVs were decreased in probands compared with controls suggesting reduced expression as a potentially aetiological factor during development.

lopmental disorder. In this study, individuals with childhood developmental delay are en‐ riched approximately fourfold for a rare 520-kb 16p12 deletion. In nearly all cases examined (22/23), the deletion was inherited. Thus, 16p12 deletions appear to be an example of inherited predisposition to neurodevelopmental disorder with dominant transmission. However, these individuals were more likely to carry a second large (>500 kb) CNV compared to matched controls, and clinical features of those with a second large CNV were typically more severe than those with the 16p12 deletion alone. Itsara et al. [95] suggest that multiply affected autism pedigrees segregate an existing inherited mutation of low penetrance which by itself is rarely sufficient to cause disease. Secondary mutations, such as de-novo mutations are required to manifest as disorder. Whether or not these second hits are disease specific remains to be ex‐ amined. The authors propose that the excess of *de novo* CNVs among cases may be due to a depletion of second-hits in the unaffected sibling due to the initial low penetrant mutation segregating in the family. The abundance of inherited low penetrance mutations and the high rate of de novo CNVs in the population enable multiple cases to appear within families with

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http://dx.doi.org/10.5772/54854

309

There is an increasing literature describing the occurrence of single nucleotide mutation in ASD. Unlike the common polymorphisms described earlier, these variants are often rare or private to an individual and considered to have a large impact on the gene's function. Early studies have focused on candidate genes with strong *a priori* evidence to suggest causation, such as the Neuroligin and Shank encoding genes. Sanger sequencing of the exons of families with ASD have identified many putatively causative mutation in these synaptic proteins [96, 97]. As with the transition from candidate gene association studies to GWAS, the advent of more sophisticated high-throughput next-generation sequencing approaches have enabled many genes to be screened in a single experiment. This has extended to the systematic scanning

In April 2012, three key manuscripts were published in Nature describing a considerable exome sequencing effort of 622 family trios and 250 unaffected siblings [98-100]. In these stud‐ ies the focus was on de novo mutation in the exome of individuals with ASD. *De novo* mutations are those changes that occurred in the child which are not inherited from the parent. Given the natural mutation rate, the number of families studied and the number of genes under investigation it was estimated that three independent mutations would provide strong evi‐ dence implicating a gene in ASD, whilst two independent mutations would be suggestive but not definitive. Unfortunately in these studies no gene was burdened with three independent mutations, whilst several revealed two independent de novo mutations. Follow-up analyses across the studies of the two-hit genes in a cohort of over 2600 individuals with ASD and over 1600 typically developing controls identified additional non-synonymous mutations in *KAT‐ NAL2* (katanin p60 subunit A-like 2), *CHD8* (Chromodomain-helicase-DNA-binding protein 8), *GRIN2B* (Glutamate [NMDA] receptor subunit epsilon-2), *LAMC3* (Laminin subunit gam‐ ma-3), *SCN1A* (sodium channel, voltage-gated, type I, alpha subunit) and *SCN2A* (sodium

of the whole exome of individuals with ASD to identify putative risk mutations.

apparently unusual patterns of inheritance.

**8. Next-generation sequencing studies**

Larger samples, particularly those based on families, will also enable the improved estimation of the overall effects of de-novo mutations and the assessment of rare recurrent events as disease associated mutations [91]. These authors studied 1124 autism families containing pro‐ bands, unaffected parents and an unaffected sibling using the Illumina 1M SNP array. In a related paper [92] they were able to confirm de novo CNVs identified using the SNP array with those detected using a Nimblegen 2.1M aCGH platform. A combined total of 58 rare de novo CNVs were identified across the two studies with each array type identifying 95% of the total. However, the sensitivity for smaller CNVs was low for both arrays. Overall, the burden of rare de novo CNVs in the Sanders et al [91] study was greater in probands than in siblings for total number, size and gene content. Using the rate in siblings as a control to evaluate findings in the cases, there was strong individual statistical support for recurrent de novo duplications at 7q11.23, the locus at which deletions cause Williams-Beuren syndrome; dele‐ tions at 16p11.2 and duplications at 16p11.2. In addition the authors observed 8 loci at which rare transmitted CNVs, present only in probands, overlapped with one of the 51 regions in probands containing one or more rare *de novo* CNVs. However, the rare transmitted CNVs were not more likely to be in cases than in unaffected siblings, even when subdivided into genic, exonic, brain-expressed or previously identified as ASD related. This suggested that the excess burden in their sample was due to rare de novo events, although when the gene sets were applied to gene pathway analysis, more pathways showed enrichment in the case set compared to the sibling set. To date, the number of definitive replicated findings for ASD from all studies has been small, with the data suggesting an extreme heterogeneity model with no single risk variant occurring in more than 1% of cases.

### **7. Mutation rates and models of risk in autism**

Given the replicated finding that de-novo mutations are more frequent in simplex cases com‐ pared to familial cases, Zhao et al [93] have suggested a model of autism risk in which families fall into two groups; those in which the overall risk for autism is low, representing the majority of families, and those in which the risk is higher due to a disease mutation with a dominant mode of transmission with greater penetrance in males compared to females. Under this mod‐ el, sporadic cases of autism occur in low-risk families due to a de novo mutation of relatively high penetrance, whereas familial autism occurs due to the inheritance of an existing mutation from a clinically unaffected or asymptomatic parent. In another model, Girirajan et al. [94] proposed the necessity in some families for a second mutation to lead to severe neurodeve‐ lopmental disorder. In this study, individuals with childhood developmental delay are en‐ riched approximately fourfold for a rare 520-kb 16p12 deletion. In nearly all cases examined (22/23), the deletion was inherited. Thus, 16p12 deletions appear to be an example of inherited predisposition to neurodevelopmental disorder with dominant transmission. However, these individuals were more likely to carry a second large (>500 kb) CNV compared to matched controls, and clinical features of those with a second large CNV were typically more severe than those with the 16p12 deletion alone. Itsara et al. [95] suggest that multiply affected autism pedigrees segregate an existing inherited mutation of low penetrance which by itself is rarely sufficient to cause disease. Secondary mutations, such as de-novo mutations are required to manifest as disorder. Whether or not these second hits are disease specific remains to be ex‐ amined. The authors propose that the excess of *de novo* CNVs among cases may be due to a depletion of second-hits in the unaffected sibling due to the initial low penetrant mutation segregating in the family. The abundance of inherited low penetrance mutations and the high rate of de novo CNVs in the population enable multiple cases to appear within families with apparently unusual patterns of inheritance.

### **8. Next-generation sequencing studies**

As the resolution of the probe arrays improves, smaller CNVs will be detected, and the boun‐ daries of previously identified CNVs will become more refined [89]. Nord et al. [90], examined genomic DNA of 41 children with autism and 367 healthy controls for rare CNVs using a very high-resolution aCGH platform. They found that cases were more likely than controls to have CNVs as small as ~10 kb, likely to affect genes involved in transcription, nervous system de‐ velopment, and receptor activity. They found that expression of *CNTNAP2*, *ZNF214*, *PRODH* and *ARID1B* genes affected by CNVs were decreased in probands compared with controls suggesting reduced expression as a potentially aetiological factor during development.

Larger samples, particularly those based on families, will also enable the improved estimation of the overall effects of de-novo mutations and the assessment of rare recurrent events as disease associated mutations [91]. These authors studied 1124 autism families containing pro‐ bands, unaffected parents and an unaffected sibling using the Illumina 1M SNP array. In a related paper [92] they were able to confirm de novo CNVs identified using the SNP array with those detected using a Nimblegen 2.1M aCGH platform. A combined total of 58 rare de novo CNVs were identified across the two studies with each array type identifying 95% of the total. However, the sensitivity for smaller CNVs was low for both arrays. Overall, the burden of rare de novo CNVs in the Sanders et al [91] study was greater in probands than in siblings for total number, size and gene content. Using the rate in siblings as a control to evaluate findings in the cases, there was strong individual statistical support for recurrent de novo duplications at 7q11.23, the locus at which deletions cause Williams-Beuren syndrome; dele‐ tions at 16p11.2 and duplications at 16p11.2. In addition the authors observed 8 loci at which rare transmitted CNVs, present only in probands, overlapped with one of the 51 regions in probands containing one or more rare *de novo* CNVs. However, the rare transmitted CNVs were not more likely to be in cases than in unaffected siblings, even when subdivided into genic, exonic, brain-expressed or previously identified as ASD related. This suggested that the excess burden in their sample was due to rare de novo events, although when the gene sets were applied to gene pathway analysis, more pathways showed enrichment in the case set compared to the sibling set. To date, the number of definitive replicated findings for ASD from all studies has been small, with the data suggesting an extreme heterogeneity model with no

Given the replicated finding that de-novo mutations are more frequent in simplex cases com‐ pared to familial cases, Zhao et al [93] have suggested a model of autism risk in which families fall into two groups; those in which the overall risk for autism is low, representing the majority of families, and those in which the risk is higher due to a disease mutation with a dominant mode of transmission with greater penetrance in males compared to females. Under this mod‐ el, sporadic cases of autism occur in low-risk families due to a de novo mutation of relatively high penetrance, whereas familial autism occurs due to the inheritance of an existing mutation from a clinically unaffected or asymptomatic parent. In another model, Girirajan et al. [94] proposed the necessity in some families for a second mutation to lead to severe neurodeve‐

single risk variant occurring in more than 1% of cases.

308 Recent Advances in Autism Spectrum Disorders - Volume I

**7. Mutation rates and models of risk in autism**

There is an increasing literature describing the occurrence of single nucleotide mutation in ASD. Unlike the common polymorphisms described earlier, these variants are often rare or private to an individual and considered to have a large impact on the gene's function. Early studies have focused on candidate genes with strong *a priori* evidence to suggest causation, such as the Neuroligin and Shank encoding genes. Sanger sequencing of the exons of families with ASD have identified many putatively causative mutation in these synaptic proteins [96, 97]. As with the transition from candidate gene association studies to GWAS, the advent of more sophisticated high-throughput next-generation sequencing approaches have enabled many genes to be screened in a single experiment. This has extended to the systematic scanning of the whole exome of individuals with ASD to identify putative risk mutations.

In April 2012, three key manuscripts were published in Nature describing a considerable exome sequencing effort of 622 family trios and 250 unaffected siblings [98-100]. In these stud‐ ies the focus was on de novo mutation in the exome of individuals with ASD. *De novo* mutations are those changes that occurred in the child which are not inherited from the parent. Given the natural mutation rate, the number of families studied and the number of genes under investigation it was estimated that three independent mutations would provide strong evi‐ dence implicating a gene in ASD, whilst two independent mutations would be suggestive but not definitive. Unfortunately in these studies no gene was burdened with three independent mutations, whilst several revealed two independent de novo mutations. Follow-up analyses across the studies of the two-hit genes in a cohort of over 2600 individuals with ASD and over 1600 typically developing controls identified additional non-synonymous mutations in *KAT‐ NAL2* (katanin p60 subunit A-like 2), *CHD8* (Chromodomain-helicase-DNA-binding protein 8), *GRIN2B* (Glutamate [NMDA] receptor subunit epsilon-2), *LAMC3* (Laminin subunit gam‐ ma-3), *SCN1A* (sodium channel, voltage-gated, type I, alpha subunit) and *SCN2A* (sodium channel, voltage-gated, type II, alpha subunit). Despite the early caution regarding the number of de novo events observed per gene, the burgeoning evidence from focused re-sequencing of these suggests that other families with ASD have damaging loss-of-function mutation in these genes.

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