Jennifer Skaug

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.

A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2

Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.

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.

Molecular basis for expression of common and rare fragile sites.

ABSTRACT 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.

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.

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 Crohns 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 Crohns disease.

Murine phosphatidylserine-specific phospholipase A1 (Ps-pla1) maps to chromosome 16 but is distinct from the lpd (lipid defect) locus.

Abstract. We have previously generated a mouse transgenic line with an insertional mutation designated lpd that demonstrates a phenotype of hypertriglyceridemia and fatty liver. Since the recently identified phosphatidylserine-specific phospholipase A1 (PS-PLA1) demonstrates significant homology to triglyceride lipases, we reasoned that the mouse Ps-pla1 gene may be the disrupted gene within the lpd locus. Using a rat PS-PLA1 cDNA sequence to search the EST database, we identified a mouse EST homolog AA839424. Sequencing analysis of AA839424 revealed a putative Ps-pla1 protein of 456 amino acids with extensive overall structural conservation with human and rat PS-PLA1 and with triglyceride lipases. Conserved sequences in Ps-pla1 include a lipase consensus sequences G×S×G, a catalytic triad, and eight of the ten conserved cysteine residues that are required for tertiary structure. Mouse Ps-pla1 carries a phosphatidylserine-binding motif that is absent in all triglyceride lipases. Using a mouse whole-genome radiation hybrid (WG-RH) mapping panel (T31), we mapped mouse Ps-pla1 to Chromosome (Chr) 16 between genetic markers D16Mit194 and D16Mit38, which is 17.1 cM centromeric to the lpd locus. On the basis of chromosome location, we conclude that Ps-pla1 and lpd are distinct genes in lipid metabolism.