Structural variation of chromosomes in autism spectrum disorder.

Structural variation (copy number variation [CNV] including deletion and duplication, translocation, inversion) of chromosomes has been identified in some individuals with autism spectrum disorder (ASD), but the full etiologic role is unknown. We performed genome-wide assessment for structural abnormalities in 427 unrelated ASD cases via single-nucleotide polymorphism microarrays and karyotyping. With microarrays, we discovered 277 unbalanced CNVs in 44% of ASD families not present in 500 controls (and re-examined in another 1152 controls). Karyotyping detected additional balanced changes. Although most variants were inherited, we found a total of 27 cases with de novo alterations, and in three (11%) of these individuals, two or more new variants were observed. De novo CNVs were found in approximately 7% and approximately 2% of idiopathic families having one child, or two or more ASD siblings, respectively. We also detected 13 loci with recurrent/overlapping CNV in unrelated cases, and at these sites, deletions and duplications affecting the same gene(s) in different individuals and sometimes in asymptomatic carriers were also found. Notwithstanding complexities, our results further implicate the SHANK3-NLGN4-NRXN1 postsynaptic density genes and also identify novel loci at DPP6-DPP10-PCDH9 (synapse complex), ANKRD11, DPYD, PTCHD1, 15q24, among others, for a role in ASD susceptibility. Our most compelling result discovered CNV at 16p11.2 (p = 0.002) (with characteristics of a genomic disorder) at approximately 1% frequency. Some of the ASD regions were also common to mental retardation loci. Structural variants were found in sufficiently high frequency influencing ASD to suggest that cytogenetic and microarray analyses be considered in routine clinical workup.

Contribution of SHANK3 mutations to autism spectrum disorder.

Mutations in SHANK3, which encodes a synaptic scaffolding protein, have been described in subjects with an autism spectrum disorder (ASD). To assess the quantitative contribution of SHANK3 to the pathogenesis of autism, we determined the frequency of DNA sequence and copy-number variants in this gene in 400 ASD-affected subjects ascertained in Canada. One de novo mutation and two gene deletions were discovered, indicating a contribution of 0.75% in this cohort. One additional SHANK3 deletion was characterized in two ASD-affected siblings from another collection, which brings the total number of published mutations in unrelated ASD-affected families to seven. The combined data provide support that haploinsufficiency of SHANK3 can cause a monogenic form of autism in sufficient frequency to warrant consideration in clinical diagnostic testing.

Mapping autism risk loci using genetic linkage and chromosomal rearrangements.

Autism spectrum disorders (ASDs) are common, heritable neurodevelopmental conditions. The genetic architecture of ASDs is complex, requiring large samples to overcome heterogeneity. Here we broaden coverage and sample size relative to other studies of ASDs by using Affymetrix 10K SNP arrays and 1,181 [corrected] families with at least two affected individuals, performing the largest linkage scan to date while also analyzing copy number variation in these families. Linkage and copy number variation analyses implicate chromosome 11p12-p13 and neurexins, respectively, among other candidate loci. Neurexins team with previously implicated neuroligins for glutamatergic synaptogenesis, highlighting glutamate-related genes as promising candidates for contributing to ASDs.

Absence of a paternally inherited FOXP2 gene in developmental verbal dyspraxia.

Mutations in FOXP2 cause developmental verbal dyspraxia (DVD), but only a few cases have been described. We characterize 13 patients with DVD--5 with hemizygous paternal deletions spanning the FOXP2 gene, 1 with a translocation interrupting FOXP2, and the remaining 7 with maternal uniparental disomy of chromosome 7 (UPD7), who were also given a diagnosis of Silver-Russell Syndrome (SRS). Of these individuals with DVD, all 12 for whom parental DNA was available showed absence of a paternal copy of FOXP2. Five other individuals with deletions of paternally inherited FOXP2 but with incomplete clinical information or phenotypes too complex to properly assess are also described. Four of the patients with DVD also meet criteria for autism spectrum disorder. Individuals with paternal UPD7 or with partial maternal UPD7 or deletion starting downstream of FOXP2 do not have DVD. Using quantitative real-time polymerase chain reaction, we show the maternally inherited FOXP2 to be comparatively underexpressed. Our results indicate that absence of paternal FOXP2 is the cause of DVD in patients with SRS with maternal UPD7. The data also point to a role for differential parent-of-origin expression of FOXP2 in human speech development.

Molecular basis for expression of common and rare fragile sites.

Fragile sites are specific loci that form gaps, constrictions, and breaks on chromosomes exposed to partial replication stress and are rearranged in tumors. Fragile sites are classified as rare or common, depending on their induction and frequency within the population. The molecular basis of rare fragile sites is associated with expanded repeats capable of adopting unusual non-B DNA structures that can perturb DNA replication. The molecular basis of common fragile sites was unknown. Fragile sites from R-bands are enriched in flexible sequences relative to nonfragile regions from the same chromosomal bands. Here we cloned FRA7E, a common fragile site mapped to a G-band, and revealed a significant difference between its flexibility and that of nonfragile regions mapped to G-bands, similar to the pattern found in R-bands. Thus, in the entire genome, flexible sequences might play a role in the mechanism of fragility. The flexible sequences are composed of interrupted runs of AT-dinucleotides, which have the potential to form secondary structures and hence can affect replication. These sequences show similarity to the AT-rich minisatellite repeats that underlie the fragility of the rare fragile sites FRA16B and FRA10B. We further demonstrate that the normal alleles of FRA16B and FRA10B span the same genomic regions as the common fragile sites FRA16C and FRA10E. Our results suggest that a shared molecular basis, conferred by sequences with a potential to form secondary structures that can perturb replication, may underlie the fragility of rare fragile sites harboring AT-rich minisatellite repeats and aphidicolin-induced common fragile sites.

Human chromosome 7: DNA sequence and biology.

DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate genes for developmental diseases including autism.

Identification of a novel lipase gene mutated in lpd mice with hypertriglyceridemia and associated with dyslipidemia in humans.

Triglyceride (TG) metabolism is crucial for whole body and local energy homeostasis and accumulating evidence suggests an independent association between plasma TG concentration and increased atherosclerosis risk. We previously generated a mouse insertional mutation lpd (lipid defect) whose phenotype included elevated plasma TG and hepatic steatosis. Using shotgun sequencing (approximately 500 kb) and bioinformatics, we have now identified a novel lipase gene lpdl (lpd lipase) within the lpd locus, and demonstrate the genetic disruption of exon 10 of lpdl in the lpd mutant locus. lpdl is highly expressed in the testis and weakly expressed in the liver of 2-week old mice. Human LPDL cDNA was subsequently cloned, and was found to encode a 460AA protein with 71% protein sequence identity to mouse lpdl and approximately 35% identity to other known lipases. We next sequenced the human LPDL gene exons in hypertriglyceridemic subjects and normal controls, and identified seven SNPs within the gene exons and six SNPs in the adjacent introns. Two hypertriglyceridemic subjects were heterozygous for a rare DNA variant, namely 164G>A (C55Y), which was absent from 600 normal chromosomes. Two other coding SNPs were associated with variation in plasma HDL cholesterol in independent normolipidemic populations. Using bioinformatics, we identified another novel lipase designated LPDLR (for 'LPDL related lipase'), which had 44% protein sequence identity with LPDL. Together with the phospholipase gene PSPLA1, LPDL and LPDLR form a new lipase gene subfamily, which is characterized by shortened lid motif. Study of this lipase subfamily may identify novel molecular mechanisms for plasma and/or tissue TG metabolism.

A third human carnitine/organic cation transporter (OCTN3) as a candidate for the 5q31 Crohn's disease locus (IBD5).

Organic cation transporters function primarily in the elimination of cationic drugs in kidney, intestine, and liver. The murine organic cation/carnitine (Octn) transporter family, Octn1, Octn2, and Octn3 is clustered on mouse chromosome 11 (NCBI Accession No. NW_000039). The human OCTN1 and OCTN2 orthologs map to the syntenic IBD5 locus at 5q31, which has been shown to confer susceptibility to Crohn's disease. We show that the human OCTN3 protein, whose corresponding gene is not yet cloned or annotated in the human reference DNA sequence, does indeed exist and is uniquely involved in carnitine-dependent transport in peroxisomes. Its functional properties and inferred chromosomal location implicate it for involvement in Crohn's disease.