**2. Autism is a heritable disorder**

There is unequivocal evidence to support the role of genetic liability as the basis of familiality in the aetiology of ASD. In their seminal work, Folstein and Rutter [4] observed that 4 of 11 (36%) pairs of identical twins were concordant for strictly defined autism – whilst none of 10 pairs of fraternal twins were concordant. In a large follow-up study, which included these original twins, Bailey and colleagues observed a more striking concordance rate in identical twins (60%) with no concordance in the fraternal twins [5]. This highly cited study underlined ASD as the most heritable of the neurodevelopmental and psychiatric disorders with herita‐ bility estimates of 91 - 93%. A recent review of over 30 twin studies of ASD [6] noted that the early twin studies, which relied upon a strict diagnosis of autism, show a median identical and fraternal concordance of 76% and 0% respectively. The expansion of these and other studies to include a broader definition of ASD revealed a median concordance rate of 88% and 31%. In both cases, these data reveal that ASD is underpinned by a strong genetic component. More recently, Hallmayer and colleagues [7] using more stringent clinical diagnostic tools, such as the Autism Diagnostic Interview (ADI; [8]) and the Autism Diagnostic Observation Schedule (ADOS; [9]) in a group of 202 twin pairs from the California Twin Registry, identified similar concordance rate of ASD as previously attained; with identical twin boys showing a concord‐ ance rate of 77% and fraternal twin boys 31%.

#### **3. Genetic linkage and association**

The heritability estimates above calculated from twin studies imply that there is unknown genetic variation in children with ASD that confers risk. Several models may fit the observed family and twin data including, at the extremes, a 'common disorder-common-variant' model, with many DNA variants of small effect combining together to create risk; and a multiple rare variant model where ASD is in reality a large collection of different and individually rare disorders. The true picture is only slowly emerging, and as with many other common disor‐ ders, common low penetrant and rare higher penetrant DNA variants combine to jointly con‐ tribute to risk. Molecular genetic methods have developed exponentially over the last few decades with ever increasing knowledge of the structure and function of the human genome. For disorders with largely unknown aetiology, such as ASD, using human genetic studies to identify risk genes is a gateway to an understanding of underlying biology. Such studies may focus on individuals or families each using different but related genetic strategies to track down risk or causative genetic variation. These methods include genetic linkage, genetic as‐ sociation, and direct examination of structural and copy number variation.

The basis of genetic linkage is the physical co-location of genes or other DNA variation. Such DNA variation is more likely to be transmitted together (or linked) than those that are further apart. Linkage studies take advantage of this non-random assortment of genetic variation. A linkage study calculates whether a known genetic variant and a disease mutation (represented by the disease trait) are linked and if so, roughly localises the causative mutation. Following the successes of using these approaches in the discovery of multiple loci implicate in Mendelian disorders, researchers were encouraged to apply linkage methodology to more complex traits, such as ASD where Mendelian principles may apply, at least in a proportion of families. How‐ ever, in ASD, only a few scans have highlighted loci with *significant* linkage, highlighting loci including chromosome 7q, 2q and 3q.

knowledge at this time, anticipating future developments, and outlining the implications for

There is unequivocal evidence to support the role of genetic liability as the basis of familiality in the aetiology of ASD. In their seminal work, Folstein and Rutter [4] observed that 4 of 11 (36%) pairs of identical twins were concordant for strictly defined autism – whilst none of 10 pairs of fraternal twins were concordant. In a large follow-up study, which included these original twins, Bailey and colleagues observed a more striking concordance rate in identical twins (60%) with no concordance in the fraternal twins [5]. This highly cited study underlined ASD as the most heritable of the neurodevelopmental and psychiatric disorders with herita‐ bility estimates of 91 - 93%. A recent review of over 30 twin studies of ASD [6] noted that the early twin studies, which relied upon a strict diagnosis of autism, show a median identical and fraternal concordance of 76% and 0% respectively. The expansion of these and other studies to include a broader definition of ASD revealed a median concordance rate of 88% and 31%. In both cases, these data reveal that ASD is underpinned by a strong genetic component. More recently, Hallmayer and colleagues [7] using more stringent clinical diagnostic tools, such as the Autism Diagnostic Interview (ADI; [8]) and the Autism Diagnostic Observation Schedule (ADOS; [9]) in a group of 202 twin pairs from the California Twin Registry, identified similar concordance rate of ASD as previously attained; with identical twin boys showing a concord‐

The heritability estimates above calculated from twin studies imply that there is unknown genetic variation in children with ASD that confers risk. Several models may fit the observed family and twin data including, at the extremes, a 'common disorder-common-variant' model, with many DNA variants of small effect combining together to create risk; and a multiple rare variant model where ASD is in reality a large collection of different and individually rare disorders. The true picture is only slowly emerging, and as with many other common disor‐ ders, common low penetrant and rare higher penetrant DNA variants combine to jointly con‐ tribute to risk. Molecular genetic methods have developed exponentially over the last few decades with ever increasing knowledge of the structure and function of the human genome. For disorders with largely unknown aetiology, such as ASD, using human genetic studies to identify risk genes is a gateway to an understanding of underlying biology. Such studies may focus on individuals or families each using different but related genetic strategies to track down risk or causative genetic variation. These methods include genetic linkage, genetic as‐

The basis of genetic linkage is the physical co-location of genes or other DNA variation. Such DNA variation is more likely to be transmitted together (or linked) than those that are further

sociation, and direct examination of structural and copy number variation.

clinical management.

**2. Autism is a heritable disorder**

300 Recent Advances in Autism Spectrum Disorders - Volume I

ance rate of 77% and fraternal twin boys 31%.

**3. Genetic linkage and association**

In 1998, the International Molecular Genetic Study of Autism Consortium (IMGSAC) [10] re‐ ported modest evidence for linkage. This included *significant* linkage arising on chromosome 7q32-q34. Supplemented analyses of IMGSAC families provided additional support for link‐ age at 7q22, 16p13, and 2q31 [11, 12]. The long arm of chromosome 7 has received particular attention with additional support reported for 7q21 [13], 7q22 [14], 7q31 [15, 16], 7q32 [17] and 7q36 [16].

Few individual families exist where ASD segregates in an obvious Mendelian fashion that is large enough to provide significant evidence for linkage by themselves. Most linkage studies required the assumption that a significant proportion of the families in the sample might be linked to a given locus and few were sufficiently large to accommodate even modest locus heterogeneity. Under a 'common disorder - common variant' model, multiply affected families will occur but linkage methods would be considerably underpowered. The effects of DNA variation with low penetrance are more easily identified using a genetic association study design in a sample of cases drawn from a population.

Fine-mapping and candidate gene association studies at implicated regions on 7q have impli‐ cated a number of potential susceptibility genes including *RELN*, *MET*, *CNTNAP2* and *EN2*. Persico and colleagues [18] studied five DNA variants or polymorphisms across the *RELN* gene locus, including a GGC repeat variant located close to the *RELN* gene translation initiator codon. Located at 7q22, *RELN* encodes an extracellular matrix protein Reelin, which plays a pivotal role in the development of laminar structures including cerebral cortex, cerebellum and hippocampus. Using a genetic association approach, Persico and colleagues identified a nominally significant association with this 5'-UTR GCC-triplet-repeat polymorphism [18]. This finding was further supported by some studies [19-25] but not others [26-31]. *MET*, located at 7q31 received considerable attention following a high-profile association reported by Camp‐ bell and colleagues. The *MET* gene encodes a protein involved in MET (Mesenchymal epithelial transition factor) receptor tyrosine kinase signalling which has been implicated in brain growth and maturation – offering biological plausibility to its candidature. As with other candidate genes in ASD, the original findings have been supported in some [32], but not other studies [33] [34]. A similar scenario played out for the *EN2* homeobox gene located at 7q36 [35, 36]. Arking and colleagues [37] observed an association at *CNTNAP2* in the NIHM/AGRE collec‐ tion. The main association observed was for rs7794745 located in intron 2 of the gene (*Discovery P* = 0.00002, *Validation P* = 0.005). The *CNTNAP2* (7q35) gene encodes the contactin-associated protein-like 2 protein, which is a member of the neurexin family and thought to play a role in axonal differentiation and guidance. Li and colleagues also found mild support for *CNTNAP2* where they observed a weak association with a haplotype containing the SNP rs7794745 [38]. More recently, a large GWAS of 2705 families identified a strong association for the SNP rs1718101 in the *CNTNAP2* gene with a subset of individuals without intellectual disability and of European ancestry (*P* = 7.78 x 10-9) [39].

proach than linkage to identify genetic risk. However, the transition from candidate gene to genome-wide association studies was not realised until technological advances firstly enriched the maps of common variation across the genome and secondly enabled the interrogation of

The Genetic Architecture of Autism and Related Conditions

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

303

Wang and colleagues [56] performed one of the first GWAS on individuals in European an‐ cestry individuals from the AGRE collection, the Autism Case Control Collection and unaf‐ fected controls from the Children's Hospital of Philadelphia control collection. Neither familybased nor case-control analysis alone yielded genome-wide significant findings. However, in a combined analyses the authors identified genome-wide significant association on chromo‐ some 5p14.1 (rs4307059; *P* =3.4 x 10-8) and a number of additional association signals on chro‐ mosome 13q33.3, 14q21.1 and Xp22.32. The 5p14.1 association was validated in the Collaborative Autism Project (CAP) and Centre for Autism Research and Treatment (CART) study. The authors found a modest to strong replication of the association signal on chromo‐ some 5p14.1. In a reciprocal study with the CAP and CART study as the discovery sample followed by validation using the AGRE dataset was published in parallel by Ma and colleagues [57]. The authors examined approximately 500k SNPs, more than in the Wang report and albeit not genome-wide significant, they retained the association signal on chromosome 5p14.1.

A second independent GWAS was reported by Weiss and colleagues [54]. In the initial scan the authors did not find any GW-significant associations. However, as with the previous GWAS, additional supplementation of their family-based studies with a case-control set de‐ rived from 90 probands without parental data garnered some additional signal for the top hits. A replication consortium of greater than 2000 trios was genotyped for 45 SNPs across all of the top associated regions. The only marker that showed evidence of replication resides on the short arm of chromosome 5 at 5p15. Although, like Ma and colleagues, this report has consid‐ erable overlap with the AGRE families reported by Wang and colleagues, the authors did not observe strong association at 5p14.1. The chromosome 5p association lies in close proximity to *TAS2R1*. The *TAS2R1* gene encodes a G-protein coupled receptor that is involved in bitter taste recognition. The authors highlight a more biologically plausible ASD candidate gene approximately 80 Kb telomeric, *SEMA5A. SEMA5A* encodes a gene important in axonal guid‐ ance and has been shown to be down regulated in the occipital lobe cortex, lymphoblast cell

Two additional GWAS followed by the Autism Genome Project (AGP) [39, 58]. In the first report of 1369 families, Anney and colleagues identified a single GW-significant finding on chromosome 20 at position 20p12 within the *MACROD2* (MACRO-domain containing 2) gene locus (rs4141463; *P* = 2.1 x 10-8; OR = 0.56). Weak statistical support was observed for *MAC‐ ROD2* in an AGRE validation sample, albeit showing the same direction of effect for the risk allele. In a follow-up study using an additional 1301 families the authors showed little if any signal of association (rs4141463; *P* = 0.206, OR=0.91). In a combined analyses the association at *MACROD2* was less compelling (rs4141463; *P* = 1.2 x 10-6; OR=0.77). The role of *MACROD2* is largely unknown although structural variation in this gene and specifically the region har‐ bouring the ASD association signal have been implicated in schizophrenia [59, 60] and epilepsy [61]. However, this same region has been identified as a hotspot for deletions in the genome

these variants *en masse* as part of genome-wide SNP arrays.

lines and lymphocytes from individuals with autism.

The Paris Autism Research International Sibpair (PARIS) study [15], that supported the find‐ ings on 7q, noted additional loci at 2q31-q32, 16p13 and 19p13, that overlapped to some extent with regions identified in the IMGSAC analyses. Fine mapping of the 2q31-q32 led to addi‐ tional evidence implicating the genes SLC25A12, STK39 and ITGA4 as putative risk genes for ASD. As with most candidate genes examined in small samples, these associations were vali‐ dated by some [40-42] but not all studies [43-46]. The positional candidature of the 2q31-q32 was further supported by a case study of a young Irish male with high-functioning autism with a complex translocation traversing chromosome 2q32 (46,XY,t(9;2)(q31.1;q32.2q31.3)) [47]. Fine mapping and mutation analysis identified an association with a polymorphism within the splice donor sequence of exon 16 of the ITGA4 gene and ASD (rs12690517; P = 0.008) [48].

In a study of Finnish families, Auranen and colleagues [49, 50], who included individuals with autism, infantile autism, Asperger syndrome (AS) and developmental dysphasia reported a *significant* linkage to 3q25-q27. This was supported by *suggestive* evidence at 3q25-q27 in a study of AS also in the Finnish population [51] as well as linkage at 3q25-q27 in a single large extended Utah pedigree of Northern European ancestry [52].

Interestingly, the advent of large collaborative studies by the Autism Genome Research Exchange (AGRE), the Autism Genome Project (AGP) and the National Institute of Men‐ tal Health (NIMH) has not yielded stronger evidence in favour of specific loci. Liu and colleagues using data from 110 multiplex families from the AGRE collection [16] ob‐ served only *suggestive* linkage on chromosomes 5p13, Xq26-qtel, and 19q12 alongside modest support for previously reported linkage on 7q (7q31 and 7q36) and 16p13. A fol‐ low-up analysis including 235 additional AGRE multiplex families was again limited to only *suggestive* loci at chromosomes 17q11, 5p13, 11p11-p13, 4q21-q22 and 8q24 [53]. In a much larger collection of 1168 multiply-affected families from the AGP (including fami‐ lies previously included in the AGRE, CPEA, IMGSAC, PARIS and Seaver linkage stud‐ ies), Szatmari and colleagues identified *suggestive* linkage to chromosome 11p12-p13 and a large region on chromosome 15q23-q25 [14]. Of the regions that were featured promi‐ nently in previous linkage analyses, there was only modest support for previously high‐ lighted linkage regions on chromosome 2q31 (female autism-probands) and 7q22 (male ASD-probands) from families of European ancestry [14]. A similarly large linkage study of 1031 families, including 1553 affected offspring, Weiss and colleagues identified *sug‐ gestive* linkage at 6p27 and *significant* linkage at 20p13 [54].

Early association studies examined whether specific variation in genes was associated with the disease and focused on candidate loci identified from linkage and cytogenetic studies (po‐ sitional candidate genes) as well as in genes within biological processes that we perceived as having a role in ASD. In the mid-1990's Risch and Merikangas [55] demonstrated that where genetic variants have only small effect on risk the association study is a more powerful ap‐ proach than linkage to identify genetic risk. However, the transition from candidate gene to genome-wide association studies was not realised until technological advances firstly enriched the maps of common variation across the genome and secondly enabled the interrogation of these variants *en masse* as part of genome-wide SNP arrays.

where they observed a weak association with a haplotype containing the SNP rs7794745 [38]. More recently, a large GWAS of 2705 families identified a strong association for the SNP rs1718101 in the *CNTNAP2* gene with a subset of individuals without intellectual disability

The Paris Autism Research International Sibpair (PARIS) study [15], that supported the find‐ ings on 7q, noted additional loci at 2q31-q32, 16p13 and 19p13, that overlapped to some extent with regions identified in the IMGSAC analyses. Fine mapping of the 2q31-q32 led to addi‐ tional evidence implicating the genes SLC25A12, STK39 and ITGA4 as putative risk genes for ASD. As with most candidate genes examined in small samples, these associations were vali‐ dated by some [40-42] but not all studies [43-46]. The positional candidature of the 2q31-q32 was further supported by a case study of a young Irish male with high-functioning autism with a complex translocation traversing chromosome 2q32 (46,XY,t(9;2)(q31.1;q32.2q31.3)) [47]. Fine mapping and mutation analysis identified an association with a polymorphism within the splice donor sequence of exon 16 of the ITGA4 gene and ASD (rs12690517; P = 0.008)

In a study of Finnish families, Auranen and colleagues [49, 50], who included individuals with autism, infantile autism, Asperger syndrome (AS) and developmental dysphasia reported a *significant* linkage to 3q25-q27. This was supported by *suggestive* evidence at 3q25-q27 in a study of AS also in the Finnish population [51] as well as linkage at 3q25-q27 in a single large extended

Interestingly, the advent of large collaborative studies by the Autism Genome Research Exchange (AGRE), the Autism Genome Project (AGP) and the National Institute of Men‐ tal Health (NIMH) has not yielded stronger evidence in favour of specific loci. Liu and colleagues using data from 110 multiplex families from the AGRE collection [16] ob‐ served only *suggestive* linkage on chromosomes 5p13, Xq26-qtel, and 19q12 alongside modest support for previously reported linkage on 7q (7q31 and 7q36) and 16p13. A fol‐ low-up analysis including 235 additional AGRE multiplex families was again limited to only *suggestive* loci at chromosomes 17q11, 5p13, 11p11-p13, 4q21-q22 and 8q24 [53]. In a much larger collection of 1168 multiply-affected families from the AGP (including fami‐ lies previously included in the AGRE, CPEA, IMGSAC, PARIS and Seaver linkage stud‐ ies), Szatmari and colleagues identified *suggestive* linkage to chromosome 11p12-p13 and a large region on chromosome 15q23-q25 [14]. Of the regions that were featured promi‐ nently in previous linkage analyses, there was only modest support for previously high‐ lighted linkage regions on chromosome 2q31 (female autism-probands) and 7q22 (male ASD-probands) from families of European ancestry [14]. A similarly large linkage study of 1031 families, including 1553 affected offspring, Weiss and colleagues identified *sug‐*

Early association studies examined whether specific variation in genes was associated with the disease and focused on candidate loci identified from linkage and cytogenetic studies (po‐ sitional candidate genes) as well as in genes within biological processes that we perceived as having a role in ASD. In the mid-1990's Risch and Merikangas [55] demonstrated that where genetic variants have only small effect on risk the association study is a more powerful ap‐

and of European ancestry (*P* = 7.78 x 10-9) [39].

302 Recent Advances in Autism Spectrum Disorders - Volume I

Utah pedigree of Northern European ancestry [52].

*gestive* linkage at 6p27 and *significant* linkage at 20p13 [54].

[48].

Wang and colleagues [56] performed one of the first GWAS on individuals in European an‐ cestry individuals from the AGRE collection, the Autism Case Control Collection and unaf‐ fected controls from the Children's Hospital of Philadelphia control collection. Neither familybased nor case-control analysis alone yielded genome-wide significant findings. However, in a combined analyses the authors identified genome-wide significant association on chromo‐ some 5p14.1 (rs4307059; *P* =3.4 x 10-8) and a number of additional association signals on chro‐ mosome 13q33.3, 14q21.1 and Xp22.32. The 5p14.1 association was validated in the Collaborative Autism Project (CAP) and Centre for Autism Research and Treatment (CART) study. The authors found a modest to strong replication of the association signal on chromo‐ some 5p14.1. In a reciprocal study with the CAP and CART study as the discovery sample followed by validation using the AGRE dataset was published in parallel by Ma and colleagues [57]. The authors examined approximately 500k SNPs, more than in the Wang report and albeit not genome-wide significant, they retained the association signal on chromosome 5p14.1.

A second independent GWAS was reported by Weiss and colleagues [54]. In the initial scan the authors did not find any GW-significant associations. However, as with the previous GWAS, additional supplementation of their family-based studies with a case-control set de‐ rived from 90 probands without parental data garnered some additional signal for the top hits. A replication consortium of greater than 2000 trios was genotyped for 45 SNPs across all of the top associated regions. The only marker that showed evidence of replication resides on the short arm of chromosome 5 at 5p15. Although, like Ma and colleagues, this report has consid‐ erable overlap with the AGRE families reported by Wang and colleagues, the authors did not observe strong association at 5p14.1. The chromosome 5p association lies in close proximity to *TAS2R1*. The *TAS2R1* gene encodes a G-protein coupled receptor that is involved in bitter taste recognition. The authors highlight a more biologically plausible ASD candidate gene approximately 80 Kb telomeric, *SEMA5A. SEMA5A* encodes a gene important in axonal guid‐ ance and has been shown to be down regulated in the occipital lobe cortex, lymphoblast cell lines and lymphocytes from individuals with autism.

Two additional GWAS followed by the Autism Genome Project (AGP) [39, 58]. In the first report of 1369 families, Anney and colleagues identified a single GW-significant finding on chromosome 20 at position 20p12 within the *MACROD2* (MACRO-domain containing 2) gene locus (rs4141463; *P* = 2.1 x 10-8; OR = 0.56). Weak statistical support was observed for *MAC‐ ROD2* in an AGRE validation sample, albeit showing the same direction of effect for the risk allele. In a follow-up study using an additional 1301 families the authors showed little if any signal of association (rs4141463; *P* = 0.206, OR=0.91). In a combined analyses the association at *MACROD2* was less compelling (rs4141463; *P* = 1.2 x 10-6; OR=0.77). The role of *MACROD2* is largely unknown although structural variation in this gene and specifically the region har‐ bouring the ASD association signal have been implicated in schizophrenia [59, 60] and epilepsy [61]. However, this same region has been identified as a hotspot for deletions in the genome 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 colleagues, a function for this non-coding RNA has not yet been reported.

5p14, 5p15, multiple locations on chromosome 7, 11q25, 15q11-q13, 16q22.3, 17p11.2, 18q21.1,

The Genetic Architecture of Autism and Related Conditions

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

305

Many studies have converged on particular chromosomal abnormalities in autism, the most common of which are maternally inherited duplications at 15q11-13. These duplications are

Another relatively common chromosomal variant is the 22q11.2 microdeletion syndrome, or velocardiofacial syndrome (VCFS) that occurs in ~1/4000 live births. This syndrome is also identified in the context of learning disability or in schizophrenia and has a complex pheno‐ typic expression affecting multiple organs. The physical features include a typical facial ap‐ pearance, (long face, narrow palpebral fissures, flattened malar eminences, prominent nose and small mouth); anatomical and/or functional abnormalities of the palatal shelves such as cleft palate and velopharyngeal insufficiency; lymphoid tissue hypoplasia and heart defects. A wide range of childhood onset developmental symptoms and disorders are described in association with the VCFS mutation [74] including attention deficit hyperactivity disorder (ADHD), oppositional defiant disorder, phobias, anxiety, obsessive compulsive disorder and

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‐

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‐

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

18q23, 22q11.2, 22q13.3 and Xp22.2–p22.3.

autism spectrum disorders.

bridization (aCGH) [75].

out the genome.

**5. Copy number variation**

found in as many as 1–3% of patients diagnosed with autism.

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 with a complex phenotype such as ASD.

### **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, 5p14, 5p15, multiple locations on chromosome 7, 11q25, 15q11-q13, 16q22.3, 17p11.2, 18q21.1, 18q23, 22q11.2, 22q13.3 and Xp22.2–p22.3.

Many studies have converged on particular chromosomal abnormalities in autism, the most common of which are maternally inherited duplications at 15q11-13. These duplications are found in as many as 1–3% of patients diagnosed with autism.

Another relatively common chromosomal variant is the 22q11.2 microdeletion syndrome, or velocardiofacial syndrome (VCFS) that occurs in ~1/4000 live births. This syndrome is also identified in the context of learning disability or in schizophrenia and has a complex pheno‐ typic expression affecting multiple organs. The physical features include a typical facial ap‐ pearance, (long face, narrow palpebral fissures, flattened malar eminences, prominent nose and small mouth); anatomical and/or functional abnormalities of the palatal shelves such as cleft palate and velopharyngeal insufficiency; lymphoid tissue hypoplasia and heart defects. A wide range of childhood onset developmental symptoms and disorders are described in association with the VCFS mutation [74] including attention deficit hyperactivity disorder (ADHD), oppositional defiant disorder, phobias, anxiety, obsessive compulsive disorder and autism spectrum disorders.
