**5. Copy number variation**

identified as a region of large rare deletions [62]. MACROD-like proteins are highly conserved across evolutionary time, which may indicate an essential role. The MACRO-domain is an ADP-ribose binding module [63] and has been implicated in the ADP-ribosylation of proteins, an important post-translational modification that occurs in a variety of biological processes such as DNA repair [64], heterochromatin formation, histone modification and sirtuin biology [65-67] as well as long-term memory formation [68]. The association signal observed at rs4141463, albeit tagged to the *MACROD2* gene, resides in an intronic region near an intragenic non-protein-coding-RNA, *NCRNA00186* (*MACROD2-AS1*). Two *MACROD2-AS1* transcripts have been reported of 673bp and 1230bp in length, located on the reverse strand between exon 5 and 6 of *MACROD2*. Anti-sense RNAs typically interact with mRNA, resulting in transcrip‐ tional or post-transcriptional effects and have been linked to brain development and plasticity [69]. However, unlike *MSNP1AS* described for the 5p14.1 association observed by Wang and

The strongest association signal observed by the AGP combined analyses was rs1718101 (*P* = 7.8 x 10-9; OR=2.13 (1.63-2.80)), a SNP within the *CNTNAP2* gene which was previously impli‐ cated in ASD through linkage analyses. This association was observed in a secondary analysis which was restricted to ASD individuals of European ancestry with a higher IQ. Anney and colleagues suggest that with the current data "few if any common variants have an impact on risk exceeding (an effect size of) 1.2 (or below its inverse)." In an attempt to seek evidence for or against common variants having an impact on risk, the authors constructed an allele-score. The allele-score method, as previously described by Purcell and colleagues [70], calculates a score for each individual based on number of risk associated alleles that the individual pos‐ sesses. This score is then used to either calculate the predictive value of the score between cases and controls or estimate the amount of variance that this score predicts for the disease. Allelescores derived from the transmission of common alleles from the families described in the AGP stage 1 GWAS [58] could significantly predict case-status in the independent Stage 2 sample. The authors concluded however that despite the limited findings for individual loci from GWAS studies to date, *en masse*the top results exert a detectable impact suggesting that as the sample sizes increase, additional significant loci will emerge. Putting together samples of the size seen in successful GWAS studies in other disorders (n>20,000) is challenging for a disorder

colleagues, a function for this non-coding RNA has not yet been reported.

**4. Structural variation: Chromosomal abnormalities in autism**

Autism has been frequently associated with chromosome abnormalities, such as deletions, duplications, inversions, or translocations; with abnormalities on the long arm of Chromosome 15 and with numerical and structural abnormalities of the sex chromosomes. Reddy in a survey of chromosomal abnormality in autism found one in 14 of 421 individuals (3.33%) [71]. These fourteen cases broke down into 4 supernumerary chromosome markers, 4 deletions, 3 inver‐ sions and 3 duplications. Several reviews have since confirmed that such abnormalities can be identified in ~3-5% of these patients [72, 73]. The regions commonly reported include 2q37,

with a complex phenotype such as ASD.

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The human genome has both sequence and structural variation, the majority of which has no functional consequences and is unrelated to any disorder. Structural variation may be bal‐ anced, such as is the case for inversions and balanced translocations or may alter DNA copy number. The later, referred to as copy number variants (CNVs) extend from the duplication or deletion of a single base pair to whole chromosome abnormalities. The term CNV is gen‐ erally used to indicate the larger changes. Initially, large CNVs were identified with classical chromosomal staining and light microscopy. As many of these abnormalities were initially identified in sub-telomeric regions, there was interest in knowing if such deletions, duplica‐ tions and other re-arrangements might occur throughout the genome. This hypothesis was confirmed with the development of high resolution array based comparative genomic hy‐ bridization (aCGH) [75].

In the arrays, comparative hybridization is performed with DNA immobilized on a platform such as a glass slide. Initially the DNA arrays consisted of human DNA cloned into bacterial artificial chromosomes (BAC arrays) representing the human genome at approximately 1Mb intervals. The present arrays consist of 25-base pair oligonucleotides (probes). In the last dec‐ ade the resolution of the arrays have improved to the extent that the number of smaller CNVs identified in the genome that were previously invisible to microscopy has increased enor‐ mously. Deletions and duplications at least as small as 10kb are now known to occur through‐ out the genome.

Interestingly, some CNVs appear to differ from single nucleotide polymorphisms (SNPs) in terms of locus specific mutation rates. Rates for genomic re-arrangements range from 10-4 to 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 and recent mutation in neurodevelopmental disorders.

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

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previously associated with schizophrenia.
