Confirmation that the specific SSLP microsatellite allele 4qA161 segregates with fascioscapulohumeral muscular dystrophy (FSHD) in a cohort of multiplex and simplex FSHD families.

The molecular defect in fascioscapulohumeral muscular dystrophy (FSHD) has proved difficult to explain. Although contraction within a polymorphic tandem repeat located at 4q35.2 is unequivocally associated with disease expression, the specific biological mechanism involved in the phenotype has yet to be resolved. Several studies have demonstrated that a specific 4q35.2-located haplotype (4qA161) is also closely associated with FSHD expression. Therefore, in this study we analyzed the haplotype association in a large cohort of sporadic and familial FSHD families from the UK. In all cases the affected individuals displayed the 4qA161 haplotype.

High-resolution DNA copy number profiling of malignant peripheral nerve sheath tumors using targeted microarray-based comparative genomic hybridization.

PURPOSE: Neurofibromatosis type 1 (NF1) is an autosomal dominant condition that predisposes to benign and malignant tumors. The lifetime risk of a malignant peripheral nerve sheath tumor (MPNST) in NF1 is approximately 10%. These tumors have a poor survival rate and their molecular basis remains unclear. We report the first comprehensive investigation of DNA copy number across multitude of genes in NF1 tumors using high-resolution array comparative genomic hybridization (CGH), with the aim to identify molecular signatures that delineate malignant from benign NF1 tumors. EXPERIMENTAL DESIGN: We constructed an exon-level resolution microarray encompassing 57 selected genes and profiled DNA from 35 MPNSTs, 16 plexiform, and 8 dermal neurofibromas. Bioinformatic analysis was done on array CGH data to identify concurrent aberrations in malignant tumors. RESULTS: The array CGH profiles of MPNSTs and neurofibromas were markedly different. A number of MPNST-specific alterations were identified, including amplifications of ITGB4, PDGFRA, MET, TP73, and HGF plus deletions in NF1, HMMR/RHAMM, MMP13, L1CAM2, p16INK4A/CDKN2A, and TP53. Copy number changes of HMMR/RHAMM, MMP13, p16INK4A/CDKN2A, and ITGB4 were observed in 46%, 43%, 39%, and 32%, respectively of the malignant tumors, implicating these genes in MPNST pathogenesis. Concomitant amplifications of HGF, MET, and PDGFRA genes were also revealed in MPNSTs, suggesting the putative role of p70S6K pathway in NF1 tumor progression. CONCLUSIONS: This study highlights the potential of array CGH in identifying novel diagnostic markers for MPNSTs.

Further evidence for the association of MMP9 with nephropathy in type 2 diabetes and application of DNA pooling technology to candidate gene screening.

BACKGROUND: Diabetic nephropathy is characterised by extracellular matrix (ECM) expansion, a key modulator of which is TGF-b1. Glucose-stimulated transcriptional activation of the TGF-b1 gene is an important component of the pathogenesis of nephropathy, following which latent TGF-b1 protein is synthesised. Matrix metalloproteinase 9 (MMP9) remodels the ECM and has been implicated in TGF-b1 activation. The ECM glycosaminoglycan hyaluronan (HA) influences TGF-b1 generation and can modulate its signal transduction activity; renal HA is synthesised by HA synthases HAS2 and HAS3. METHODS: We report the first screening of the genes encoding HAS2 and HAS3 for sequence variants predisposing to nephropathy in UK type 2 diabetes patients, together with the MMP9 and TGF-b1 genes. Also for the first time, we used validated DNA pools to carry out association analyses of single nucleotide polymorphisms on nephropathic and non-nephropathic cohorts from a total of 199 type 2 diabetes patients, to increase the throughput and decrease the cost of genotype analysis. RESULTS: None of the 23 single nucleotide polymorphisms analysed in DNA pools were found to be associated with diabetic nephropathy. However, genotyping of alleles at the MMP9 promoter microsatellite locus D20S838 in individual genomic DNA samples supported previous evidence of association between this locus and diabetic nephropathy. CONCLUSIONS: The use of DNA pooling technology increased the throughput and decreased the cost of our association analysis of nephropathy in our type 2 diabetes sample, which demonstrated sufficient sensitivity to support previous positive findings of association with a microsatellite in the MMP9 promoter region.

Support for RGS4 as a susceptibility gene for schizophrenia.

BACKGROUND: The gene encoding the regulator of G-protein signaling 4 has recently been associated with susceptibility to schizophrenia. This finding is particularly interesting, because it was replicated within the same study and also because there are functional, positional, and expression data to support the regulator of G-protein signaling 4 as a schizophrenia candidate gene. Although the original report was highly suggestive, a limitation was that the study was conducted on rather small samples. METHODS: We have examined a large case (n = 709) control (n = 710) sample for association between schizophrenia using four markers investigated in the earlier study, denoted single nucleotide polymorphisms 1, 4, 7, and 18. RESULTS: We were able to replicate the associations with single nucleotide polymorphisms 4 and 18 that had previously been reported individually and have also identified significant association with haplotypes constructed from single nucleotide polymorphisms 1 and 4. CONCLUSIONS: Our data give modest support for the hypothesis that the regulator of G-protein signaling 4 is a susceptibility gene for schizophrenia.

Genotype effects of CHRNA7, CNR1 and COMT in schizophrenia: interactions with tobacco and cannabis use.

BACKGROUND: Genetic variations might modify associations between schizophrenia and cannabis or tobacco use. AIMS: To examine whether variants within the cannabinoid receptor (CNR1) and alpha(7) nicotinic receptor (CHRNA7) genes are associated with schizophrenia, and whether these effects vary according to cannabis or tobacco use. We also examined a putative interaction between cannabis and Val(158)Met within the catechol-O-methyltransferase gene (COMT). METHOD: Genotype effects of CHRNA7 and CNR1were studied in a case-control sample of 750 individuals with schizophrenia and 688 controls, with interactions for these genes studied in small subsamples. A case-only design of 493 ofthe schizophrenia group was used to examine interactions between cannabis use and COMT. RESULTS: There was no evidence of association between schizophrenia and CNR1 (OR=0.97, 95% CI 0.82-1.13) or CHRNA7 (OR=1.07, 95% CI 0.77-1.49) genotypes, or of interactions between tobacco use and CHRNA7, or cannabis use and CNR1or COMT genotypes. CONCLUSIONS: Neither CNR1 nor CHRNA7 variation appears to alter the risk of schizophrenia. Furthermore, our results do not support the presence of different effects of cannabis use on schizophrenia according to variation within COMT.

Evidence that interaction between neuregulin 1 and its receptor erbB4 increases susceptibility to schizophrenia.

There is now strong evidence that Neuregulin 1 (NRG1) is a susceptibility gene for schizophrenia. NRG1 mediates some of its effects through the tyrosine kinase receptor erbB4, and analysis of gene knock-out animals suggests that the functional interaction of NRG1 and erbB4 mediates behaviors that may model some aspects of the schizophrenia phenotype in mice. Given these findings, we have sought evidence for association between schizophrenia and erbB4. Mutation screening of erbB4 in 14 DSMIV schizophrenics revealed 15 SNPs, none of which were nonsynonymous. Analysis of the allele frequencies of each SNP in pools of 368 DSMIV schizophrenics and 368 controls provided modest evidence for association with two of the SNPs, although individual genotyping in an extended sample of 680 cases did not confirm this. However, we did find evidence for a significant interaction between the NRG1 "Icelandic" schizophrenia risk haplotype and erbB4 (P = 0.019). The NRG1 and erbB4 interacting marker was further genotyped in an independent sample of 290 cases and 634 controls from Dublin. Interaction between NRG1 and erbB4 remained significant in the combined sample of 970 cases and 1,341 controls, OR = 2.98 (CI: 1.16-7.64), P = 0.01, although it only showed a trend in the Dublin sample alone (P = 0.11, two tailed). Our data require independent replication, but tentatively suggest that NRG1 may mediate its effects on schizophrenia susceptibility through functional interaction with erbB4, and that genetic interaction between variants at the two loci increases susceptibility to schizophrenia.

Universal, robust, highly quantitative SNP allele frequency measurement in DNA pools.

Detecting alleles that confer small increments in susceptibility to disease will require large-scale allelic association studies of single-nucleotide polymorphisms (SNPs) in candidate, or positional candidate, genes. However, current genotyping technologies are one to two orders of magnitude too expensive to permit the analysis of thousands of SNPs in large samples. We have developed and thoroughly validated a highly accurate protocol for SNP allele frequency estimation in DNA pools based upon the SNaPshot (Applied Biosystems) chemistry adaptation of primer extension. Using this assay, we were able to estimate the difference in allele frequencies between pooled cases and controls (Delta) with a mean error of 0.01. Moreover, when we genotyped seven different SNPs in a single multiplex reaction, the results were similar, with a mean error for Delta of 0.008. The assay performed well for alleles of low frequency alleles (f approximately 0.05) and was accurate even with relatively poor quality DNA template extracted from mouthwashes. Our assay conditions are generalisable, universal, robust and, therefore, for the first time, permit high-throughput association analysis at a realistic cost.