Abdelmajid Belouchi

Genome-wide association study for Crohn's disease in the Quebec Founder Population identifies multiple validated disease loci

Genome-wide association (GWA) studies offer a powerful unbiased method for the identification of multiple susceptibility genes for complex diseases. Here we report the results of a GWA study for Crohns disease (CD) using family trios from the Quebec Founder Population (QFP). Haplotype-based association analyses identified multiple regions associated with the disease that met the criteria for genome-wide significance, with many containing a gene whose function appears relevant to CD. A proportion of these were replicated in two independent German Caucasian samples, including the established CD loci NOD2 and IBD5. The recently described IL23R locus was also identified and replicated. For this region, multiple individuals with all major haplotypes in the QFP were sequenced and extensive fine mapping performed to identify risk and protective alleles. Several additional loci, including a region on 3p21 containing several plausible candidate genes, a region near JAKMIP1 on 4p16.1, and two larger regions on chromosome 17 were replicated. Together with previously published loci, the spectrum of CD genes identified to date involves biochemical networks that affect epithelial defense mechanisms, innate and adaptive immune response, and the repair or remodeling of tissue.

Hotspots of Large Rare Deletions in the Human Genome

Background We have examined the genomic distribution of large rare autosomal deletions in a sample of 440 parent-parent-child trios from the Quebec founder population (QFP) which was recruited for a study of Attention Deficit Hyperactivity Disorder. Methodology/Principal Findings DNA isolated from blood was genotyped on Illumina Hap300 arrays. PennCNV combined with visual evaluation of images generated by the Beadstudio program was used to determine deletion boundary definition of sufficient precision to discern independent events, with near-perfect concordance between parent and child in about 98% of the 399 events detected in the offspring; the remaining 7 deletions were considered de novo. We defined several genomic regions of very high deletion frequency (‘hotspots’), usually of 0.4–0.6 Mb in length where independent rare deletions were found at frequencies of up to 100 fold higher than the average for the genome as a whole. Five of the 7 de novo deletions were in these hotspots. The same hotspots were also observed in three other studies on members of the QFP, those with schizophrenia, with endometriosis and those from a longevity cohort. Conclusions/Significance Nine of the 13 hotspots carry one gene (7 of which are very long), while the rest contain no known genes. All nine genes have been implicated in disease. The patterns of exon deletions support the proposed roles for some of these genes in human disease, such as NRXN1 and PARKIN, and suggest limited roles or no role at all, for others, including MACROD2 and CTNNA3. Our results also offer an alternative interpretation for the observations of deletions in tumors which have been proposed as reflecting tumor-suppressive activity of genes in these hotspots.

The aprt heterozygote/hemizygote system for screening mutagenic agents allows detection of large deletions

Frequencies of mutation at the hprt and aprt loci in various CHO cell lines were measured after exposure of the cells to ionizing radiation. In D423 and AA8-16, which are aprt+/- heterozygotes, the ratio of hprt- mutants to aprt- mutants ranged from 0.11 to 0.36. In D422 and AA8-5, which are aprt+/0 cell lines in which only one copy of the gene and its flanking sequences is present these ratios were greater than 5. In contrast, chemical mutagenesis generated mutations at both loci, in all cell lines, at equal frequencies. Southern blot analysis of DNA from hprt- and aprt- mutants of one of the aprt+/- heterozygous lines showed some apparently unaltered genes, some rearrangements and some complete deletions of hprt among hprt- mutants, but only complete deletions of aprt-linked sequences among aprt- mutants. These results strongly suggest that X-ray-induced mutational events are frequently larger than 40 kb (the length of the hprt gene) and that the difference among the frequencies observed at the two loci in the two types of cell lines were due to the presence of essential sequences close the respective target genes. The combined use of these cell lines in screening environmental mutagens should allow qualitative as well as quantitative analysis of the mutagenic potential of environmental agents.

A mutational hotspot in the aprt gene of Chinese hamster cells

Early work with adenine phosphoribosyltransferase-deficient mutants of CHO cells suggested that a site existed in the third exon of this gene which was preferentially susceptible to mutation by ethyl methanesulphonate. To determine whether this was real we analysed a large collection of induced mutants, and generated a high-density mutational spectrum for this exon. In addition, 4 sites outside exon 3 were analysed by blot. 37 mutations were found in 19 available sites, six of which were at nucleotide 1365, 1 of 2 sites in the putative hotspot (P less than 0.02). One other site, 1308, also was mutated in 6 cell lines and may also be preferentially mutable.

DNA Biomarkers for Pharmacogenomics and Personalized Medicine

Genome-wide association studies are expected to soon provide a significant increase in disease associated DNA sequences that can serve as biomarkers for diagnosis and treatment for psychiatric illnesses. These biomarkers have the potential to identify precisely defined disease phenotypes and to predict effective individually specific therapies. This chapter reviews the current knowledge of the association between DNA markers and disease phenotypes and discusses some of the anticipated problems that need to be overcome before DNA biomarkers can provide specific and sensitive diagnostic tests for both the presence of disease and the prediction of individual response to specific drugs.

Research paperThe aprt heterozygote/hemizygote system for screening mutagenic agents allows detection of large deletions

Frequencies of mutation at the hprt and aprt loci in various CHO cell lines were measured after exposure of the cells to ionizing radiation. In D423 and AA8-16, which are aprt+/− heterozygotes, the ratio of hprt− mutants to aprt− mutants ranged from 0.11 to 0.36. In D422 and AA8-5, which are aprt+/0 cell lines in which only one copy of the gene and its flanking sequences is present these ratios were > 5. In contrast, chemical mutagenesis generated mutations at both loci, in all cell lines, at equal frequencies. Southern blot analysis of DNA from hprt− and aprt− mutants of one of the aprt+/− heterozygous lines showed some apparently unaltered genes, some rearrangements and some complete deletions of hprt among hprt− mutants, but only complete deletions of aprt-linked sequences among aprt− mutants. These results strongly suggest that X-ray-induced mutational events are frequently larger than 40 kb (the length of the hprt gene) and that the difference among the frequencies observed at the two loci in the two types of cell lines were due to the presence of essential sequences close the the respective target genes. The combined use of these cell lines in screening environmental mutagens should allow qualitative as well as quantitative analysis of the mutagenic potential of environmental agents.