Abdul Noor

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.

Peptidyl argininedeiminase 2 CpG island in multiple sclerosis white matter is hypomethylated

In previous studies, we documented increased citrullinated myelin basic protein (MBP) was present in MBP isolated from multiple sclerosis (MS) normal appearing white matter (NAWM). This increase was due to the myelin enzyme peptidyl argininedeiminase 2 (PAD2). In this study, we show that methylation of cytosine of the PAD2 promoter in DNA from MS NAWM was decreased to one‐third of the level of that in DNA from normal white matter. The PAD2 promoter in DNA from thymus obtained from the same MS patients and white matter DNA from Alzheimers, Huntingtons, and Parkinsons was not hypomethylated. DNA demethylase activity in supernatants prepared from NAWM of MS patients was 2‐fold higher than the DNA demethylase from normal, Alzheimers, Huntingtons and Parkinsons disease white matter. The amount of PAD2 enzyme and citrullinated MBP was increased in MS NAWM. The decreased methylation of cytosines in the PAD2 promoter may explain the increased synthesis of PAD2 protein that is responsible for the increased amount of citrullinated MBP, which in turn results in loss of myelin stability in MS brain.

Disruption at the PTCHD1 locus on Xp22.11 in autism spectrum disorder and intellectual disability

Mutations of the X-linked gene PTCHD1 are associated with autism spectrum disorders and intellectual disability. A Patch in the Fabric of Autism What causes autism? This disabling disorder is characterized by severe language and social impairment and is now included under the umbrella term “autism spectrum disorder” (ASD), which also includes milder deficits in communication and social development. Numerous theories have been advanced as to its causes. These have ranged from discredited concepts—“refrigerator” mothers and vaccines—to the modern idea of gene-environment interactions. Although no one gene simply explains the predisposition of patients for ASD, these disorders are wellknown to have a strong genetic component. Here, Noor et al. report the results of genetic analysis in thousands of patients and control subjects: Mutations at the PTCHD1 (patched-related gene) locus are associated with the inheritance of ASD and with intellectual disability in a small fraction of cases. In this study, the authors analyzed the PTCHD1 gene from 1896 patients with ASD and 246 with intellectual disability, and compared these to more than 10,000 control individuals, and found mutations in various parts of this gene in 25 affected individuals in 20 different families, but not in any of the controls. Some patients had large deletions, in one case spanning the entire gene, and in others the culprit was a missense mutation. A result of this gene’s location on the X chromosome, the affected patients were almost all male, and most had unaffected mothers and other female relatives. The authors also present evidence that the PTCHD1 gene may be part of the Hedgehog signaling pathway, which is important in embryonic development. Autism and intellectual disability are not straightforward disorders that can be attributed to mutations in a single gene. Even when candidate genes such as PTCHD1 are known, differences in the gene sequence do not perfectly correlate with phenotype, because there are many as yet undefined additional genes and environmental influences that dictate the ultimate characteristics of the person. Identifying some of these genes, as Noor et al. have done in this study, allows a better understanding of the disorder and the development of ways to compensate for its disabilities. Autism is a common neurodevelopmental disorder with a complex mode of inheritance. It is one of the most highly heritable of the complex disorders, although the underlying genetic factors remain largely unknown. Here, we report mutations in the X-chromosome PTCHD1 (patched-related) gene in seven families with autism spectrum disorder (ASD) and in three families with intellectual disability. A 167-kilobase microdeletion spanning exon 1 was found in two brothers, one with ASD and the other with a learning disability and ASD features; a 90-kilobase microdeletion spanning the entire gene was found in three males with intellectual disability in a second family. In 900 probands with ASD and 208 male probands with intellectual disability, we identified seven different missense changes (in eight male probands) that were inherited from unaffected mothers and not found in controls. Two of the ASD individuals with missense changes also carried a de novo deletion at another ASD susceptibility locus (DPYD and DPP6), suggesting complex genetic contributions. In additional males with ASD, we identified deletions in the 5′ flanking region of PTCHD1 that disrupted a complex noncoding RNA and potential regulatory elements; equivalent changes were not found in male control individuals. Thus, our systematic screen of PTCHD1 and its 5′ flanking regions suggests that this locus is involved in ~1% of individuals with ASD and intellectual disability.

An X-Linked Myopathy with Postural Muscle Atrophy and Generalized Hypertrophy, Termed XMPMA, Is Caused by Mutations in FHL1

We have identified a large multigenerational Austrian family displaying a novel form of X-linked recessive myopathy. Affected individuals develop an adult-onset scapulo-axio-peroneal myopathy with bent-spine syndrome characterized by specific atrophy of postural muscles along with pseudoathleticism or hypertrophy and cardiac involvement. Known X-linked myopathies were excluded by simple-tandem-repeat polymorphism (STRP) and single-nucleotide polymorphism (SNP) analysis, direct gene sequencing, and immunohistochemical analysis. STRP analysis revealed significant linkage at Xq25-q27.1. Haplotype analysis based on SNP microarray data from selected family members confirmed this linkage region on the distal arm of the X chromosome, thereby narrowing down the critical interval to 12 Mb. Sequencing of functional candidate genes led to the identification of a missense mutation within the four and a half LIM domain 1 gene (FHL1), which putatively disrupts the fourth LIM domain of the protein. Mutation screening of FHL1 in a myopathy family from the UK exhibiting an almost identical phenotype revealed a 3 bp insertion mutation within the second LIM domain. FHL1 on Xq26.3 is highly expressed in skeletal and cardiac muscles. Western-blot analysis of muscle biopsies showed a marked decrease in protein expression of FHL1 in patients, in concordance with the genetic data. In summary, we have to our knowledge characterized a new disorder, X-linked myopathy with postural muscle atrophy (XMPMA), and identified FHL1 as the causative gene. This is the first FHL protein to be identified in conjunction with a human genetic disorder and further supports the role of FHL proteins in the development and maintenance of muscle tissue. Mutation screening of FHL1 should be considered for patients with uncharacterized myopathies and cardiomyopathies.

Identification of Mutations in TRAPPC9, which Encodes the NIK- and IKK-β-Binding Protein, in Nonsyndromic Autosomal-Recessive Mental Retardation

Mental retardation/intellectual disability is a devastating neurodevelopmental disorder with serious impact on affected individuals and their families, as well as on health and social services. It occurs with a prevalence of approximately 2%, is an etiologically heterogeneous condition, and is frequently the result of genetic aberrations. Autosomal-recessive forms of nonsyndromic MR (NS-ARMR) are believed to be common, yet only five genes have been identified. We have used homozygosity mapping to search for the gene responsible for NS-ARMR in a large Pakistani pedigree. Using Affymetrix 5.0 single nucleotide polymorphism (SNP) microarrays, we identified a 3.2 Mb region on 8q24 with a continuous run of 606 homozygous SNPs shared among all affected members of the family. Additional genotype data from microsatellite markers verified this, allowing us to calculate a two-point LOD score of 5.18. Within this region, we identified a truncating homozygous mutation, R475X, in exon 7 of the gene TRAPPC9. In a second large NS-ARMR/ID family, previously linked to 8q24 in a study of Iranian families, we identified a 4 bp deletion within exon 14 of TRAPPC9, also segregating with the phenotype and truncating the protein. This gene encodes NIK- and IKK-beta-binding protein (NIBP), which is involved in the NF-kappaB signaling pathway and directly interacts with IKK-beta and MAP3K14. Brain magnetic resonance imaging of affected individuals indicates the presence of mild cerebral white matter hypoplasia. Microcephaly is present in some but not all affected individuals. Thus, to our knowledge, this is the sixth gene for NS-ARMR to be discovered.

Mutation in NSUN2, which Encodes an RNA Methyltransferase, Causes Autosomal-Recessive Intellectual Disability

Causes of autosomal-recessive intellectual disability (ID) have, until very recently, been under researched because of the high degree of genetic heterogeneity. However, now that genome-wide approaches can be applied to single multiplex consanguineous families, the identification of genes harboring disease-causing mutations by autozygosity mapping is expanding rapidly. Here, we have mapped a disease locus in a consanguineous Pakistani family affected by ID and distal myopathy. We genotyped family members on genome-wide SNP microarrays and used the data to determine a single 2.5 Mb homozygosity-by-descent (HBD) locus in region 5p15.32-p15.31; we identified the missense change c.2035G>A (p.Gly679Arg) at a conserved residue within NSUN2. This gene encodes a methyltransferase that catalyzes formation of 5-methylcytosine at C34 of tRNA-leu(CAA) and plays a role in spindle assembly during mitosis as well as chromosome segregation. In mouse brains, we show that NSUN2 localizes to the nucleolus of Purkinje cells in the cerebellum. The effects of the mutation were confirmed by the transfection of wild-type and mutant constructs into cells and subsequent immunohistochemistry. We show that mutation to arginine at this residue causes NSUN2 to fail to localize within the nucleolus. The ID combined with a unique profile of comorbid features presented here makes this an important genetic discovery, and the involvement of NSUN2 highlights the role of RNA methyltransferase in human neurocognitive development.

CC2D2A, Encoding A Coiled-Coil and C2 Domain Protein, Causes Autosomal-Recessive Mental Retardation with Retinitis Pigmentosa

Autosomal-recessive inheritance is believed to be relatively common in mental retardation (MR), although only four genes for nonsyndromic autosomal-recessive mental retardation (ARMR) have been reported. In this study, we ascertained a consanguineous Pakistani family with ARMR in four living individuals from three branches of the family, plus an additional affected individual later identified as a phenocopy. Retinitis pigmentosa was present in affected individuals, but no other features suggestive of a syndromic form of MR were found. We used Affymetrix 500K microarrays to perform homozygosity mapping and identified a homozygous and haploidentical region of 11.2 Mb on chromosome 4p15.33-p15.2. Linkage analysis across this region produced a maximum two-point LOD score of 3.59. We sequenced genes within the critical region and identified a homozygous splice-site mutation segregating in the family, within a coiled-coil and C2 domain-containing gene, CC2D2A. This mutation leads to the skipping of exon 19, resulting in a frameshift and a truncated protein lacking the C2 domain. Conservation analysis for CC2D2A suggests a functional domain near the C terminus as well as the C2 domain. Preliminary functional studies of CC2D2A suggest a possible role in Ca(2+)-dependent signal transduction. Identifying the function of CC2D2A, and a possible common pathway with CC2D1A, in correct neuronal development and functioning may help identify possible therapeutic targets for MR.

Oligodontia Is Caused by Mutation in LTBP3, the Gene Encoding Latent TGF-β Binding Protein 3

We have identified a consanguineous Pakistani family where oligodontia is inherited along with short stature in an autosomal-recessive fashion. Increased bone density was present in the spine and at the base of the skull. Using high-density single-nucleotide polymorphism microarrays for homozygosity mapping, we identified a 28 Mb homozygous stretch shared between affected individuals on chromosome 11q13. Screening selected candidate genes within this region, we identified a homozygous nonsense mutation, Y774X, within LTBP3, the gene for the latent TGF-beta binding protein 3, an extracellular matrix protein believed to be required for osteoclast function.

Genome-wide association study of bipolar disorder in Canadian and UK populations corroborates disease loci including SYNE1 and CSMD1

BackgroundRecently, genome-wide association studies (GWAS) for cases versus controls using single nucleotide polymorphism microarray data have shown promising findings for complex neuropsychiatric disorders, including bipolar disorder (BD).MethodsHere we describe a comprehensive genome-wide study of bipolar disorder (BD), cross-referencing analysis from a family-based study of 229 small families with association analysis from over 950 cases and 950 ethnicity-matched controls from the UK and Canada. Further, loci identified in these analyses were supported by pathways identified through pathway analysis on the samples.ResultsAlthough no genome-wide significant markers were identified, the combined GWAS findings have pointed to several genes of interest that support GWAS findings for BD from other groups or consortia, such as at SYNE1 on 6q25, PPP2R2C on 4p16.1, ZNF659 on 3p24.3, CNTNAP5 (2q14.3), and CDH13 (16q23.3). This apparent corroboration across multiple sites gives much confidence to the likelihood of genetic involvement in BD at these loci. In particular, our two-stage strategy found association in both our combined case/control analysis and the family-based analysis on 1q21.2 (closest gene: sphingosine-1-phosphate receptor 1 gene, S1PR1) and on 1q24.1 near the gene TMCO1, and at CSMD1 on 8p23.2, supporting several previous GWAS reports for BD and for schizophrenia. Pathway analysis suggests association of pathways involved in calcium signalling, neuropathic pain signalling, CREB signalling in neurons, glutamate receptor signalling and axonal guidance signalling.ConclusionsThe findings presented here show support for a number of genes previously implicated genes in the etiology of BD, including CSMD1 and SYNE1, as well as evidence for previously unreported genes such as the brain-expressed genes ADCY2, NCALD, WDR60, SCN7A and SPAG16.

Mutations in the Alpha 1,2-Mannosidase Gene, MAN1B1, Cause Autosomal-Recessive Intellectual Disability

We have used genome-wide genotyping to identify an overlapping homozygosity-by-descent locus on chromosome 9q34.3 (MRT15) in four consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability (NS-ARID) and one in which the patients show additional clinical features. Four of the families are from Pakistan, and one is from Iran. Using a combination of next-generation sequencing and Sanger sequencing, we have identified mutations in the gene MAN1B1, encoding a mannosyl oligosaccharide, alpha 1,2-mannosidase. In one Pakistani family, MR43, a homozygous nonsense mutation (RefSeq number NM_016219.3: c.1418G>A [p.Trp473*]), segregated with intellectual disability and additional dysmorphic features. We also identified the missense mutation c. 1189G>A (p.Glu397Lys; RefSeq number NM_016219.3), which segregates with NS-ARID in three families who come from the same village and probably have shared inheritance. In the Iranian family, the missense mutation c.1000C>T (p.Arg334Cys; RefSeq number NM_016219.3) also segregates with NS-ARID. Both missense mutations are at amino acid residues that are conserved across the animal kingdom, and they either reduce k(cat) by ∼1300-fold or disrupt stable protein expression in mammalian cells. MAN1B1 is one of the few NS-ARID genes with an elevated mutation frequency in patients with NS-ARID from different populations.