Genetic Polymorphism of CYP2C19 gene in the Stanislas cohort. A link with inflammation.

CYP2C19, a member of the cytochrome P450 family, metabolises arachidonic acid to produce epoxyeicosanoid acids, which are involved in vascular tone and inflammation. Thus, this study describes the possible relationship between a CYP2C19 polymorphism (681G>A) and three inflammatory markers: interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and high sensitivity C-reactive protein (hs-CRP) in healthy individuals. In a sub-sample of 178 men and 181 women from the Stanislas study, we quantified plasma IL-6 and TNF-alpha concentrations by using an enzyme-linked immunosorbent assay, and serum hs-CRP concentration by immunonephelometry. The CYP2C19 681G>A polymorphism was genotyped using the kinetic thermocycling allele specific PCR method. In the Stanislas cohort, the frequency of the allele CYP2C19*2 (681A) was 17.8%. Circulating levels of inflammatory factors were increased in individuals homozygous for the defective allele CYP2C19*2 (A) notably IL-6 in the whole sample (P= 0.0008) and hs-CRP only in women (P= 0.008), with a significant interaction with sex (P= 0.005), in comparison to carriers of one copy or more of the wild type allele CYP2C19*1 (G). Only a trend of association (P= 0.089) was found between this polymorphism and TNF-alpha concentration in the whole sample. The association between CYP2C19*2 polymorphism and inflammatory markers' concentrations could suggest that CYP2C19 may be considered as a new candidate gene for cardiovascular risks via inflammation.

Lack of association between the SOST gene and bone mineral density in perimenopausal women: analysis of five polymorphisms.

Osteoporosis is a common disease characterized by a decrease in bone mass, architectural deterioration of the bone tissue, and an increased risk of fracture. The condition is under strong genetic control, involving a large variety of gene products, but to date the genes responsible remain poorly defined. Although population-based studies have identified polymorphisms in several candidate genes that are associated with bone mineral density (BMD), these account for only a small proportion of the population variance in bone mass. In this study, we looked for evidence of an allelic association between polymorphisms in the SOST gene and BMD. This gene was analyzed because loss-of-function mutations in SOST cause sclerosteosis, a sclerosing bone dysplasia associated with increased bone mass due to increased bone formation. We identified 26 different polymorphisms in the SOST gene and selected 5 of these for association analysis in a case-control study of 619 women with either high or low BMD, drawn from a random population-based survey of 5119 perimenopausal white women. The high BMD group comprised 326 women in whom lumbar spine BMD values adjusted for age, height, and weight were in the highest 16% of the population distribution, and the low BMD group comprised 293 women in whom BMD values were in the lowest 16% of the population distribution. The distribution of genotypes and alleles for each Single Nucleotide Polymorphism (SNP) examined did not differ in the low and high BMD groups. We conclude that, in this population, common allelic variations in the SOST gene do not contribute significantly to the regulation of high or low BMD.

Identification of a 52 kb deletion downstream of the SOST gene in patients with van Buchem disease.

Van Buchem disease is an autosomal recessive skeletal dysplasia characterised by generalised bone overgrowth, predominantly in the skull and mandible. Clinical complications including facial nerve palsy, optic atrophy, and impaired hearing occur in most patients. These features are very similar to those of sclerosteosis and the two conditions are only differentiated by the hand malformations and the tall stature appearing in sclerosteosis. Using an extended Dutch inbred van Buchem family and two inbred sclerosteosis families, we mapped both disease genes to the same region on chromosome 17q12-q21, supporting the hypothesis that van Buchem disease and sclerosteosis are caused by mutations in the same gene. In a previous study, we positionally cloned a novel gene, called SOST, from the linkage interval and identified three different, homozygous mutations in the SOST gene in sclerosteosis patients leading to loss of function of the underlying protein. The present study focuses on the identification of a 52 kb deletion in all patients from the van Buchem family. The deletion, which results from a homologous recombination between Alu sequences, starts approximately 35 kb downstream of the SOST gene. Since no evidence was found for the presence of a gene within the deleted region, we hypothesise that the presence of the deletion leads to a down regulation of the transcription of the SOST gene by a cis regulatory action or a position effect.

Nontruncating APC germ-line mutations and mismatch repair deficiency play a minor role in APC mutation-negative polyposis.

Familial adenomatous polyposis, an autosomal-dominantly inherited colorectal cancer predisposition syndrome, is caused by germ-line mutations in the adenomatous polyposis coli (APC) gene. Despite the use of different screening methods, studies worldwide fail to identify APC mutations in 20-50% of all familial adenomatous polyposis patients (APC mutation-negatives). In this study, missense mutations in the coding region of the APC gene, which would have been missed by the protein truncation test, as well as mutations in the APC promoter and the 3' untranslated region, were determined by the single nucleotide polymorphism discovery assay and direct DNA sequencing in 31 mutation-negative polyposis patients. Seventeen gene alterations were identified, whereof four (12.9%) represent possibly pathogenic germ-line mutations: silent A290T (promoter) and A8822G (3' untranslated region) as well as missense R99W and E1317Q (coding region). The 27 remaining, truly APC mutation-negative polyposis patients displayed a significantly later age at diagnosis compared with APC mutation carriers (46.1 versus 35.2 years; P < 0.01). APC mutation-negative individuals with >100 colonic polyps were more likely to present with extracolonic disease (P < 0.05) than those with <100. Assessment of microsatellite instability (MSI), a hallmark of mismatch repair deficiency, in 68 tumors from 21 truly APC mutation-negative patients, identified 4 (5.9%) unstable tubulo-villous adenomas (3 MSI-High and 1 MSI-Low), stemming from 4 (19%) unrelated individuals and likely to be caused by hMLH1 promoter hypermethylation. In conclusion, only a small proportion of APC germ-line mutation carriers is missed by the protein truncation test, and mismatch repair deficiency does not seem to substantially contribute to tumor development in APC mutation-negative polyposis patients.

A semi-automated system for analysis and storage of SNPs.

The discovery of single nucleotide polymorphisms ( SNPs) is currently pursued with a tremendous effort. SNPs represent a rich source for molecular markers, since estimations predict six to seven million of these DNA variations in the human genome. A subset of these genetic variants is thought to have a pervasive impact on modern medicine, be it for the elucidation of differential pharmacological response or for the facilitated identification of genes involved in monogenetic and complex human diseases. Here we describe the overall process that leads to the set up of a SNP database. We describe a high-throughput sequencing assay for SNP discovery, automation of the dataflow from the DNA sequencer to the SNP analysis, and the tools to facilitate it. At the end of the process, a web-accessible interface collects the SNP information, which is processed in order to be written into the SNP database and to be available for end users who would like to select appropriate SNPs for their special screening needs.

Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST).

Sclerosteosis is a progressive sclerosing bone dysplasia with an autosomal recessive mode of inheritance. Radiologically, it is characterized by a generalized hyperostosis and sclerosis leading to a markedly thickened and sclerotic skull, with mandible, ribs, clavicles and all long bones also being affected. Due to narrowing of the foramina of the cranial nerves, facial nerve palsy, hearing loss and atrophy of the optic nerves can occur. Sclerosteosis is clinically and radiologically very similar to van Buchem disease, mainly differentiated by hand malformations and a large stature in sclerosteosis patients. By linkage analysis in one extended van Buchem family and two consanguineous sclerosteosis families we previously mapped both disease genes to the same chromosomal 17q12-q21 region, supporting the hypothesis that both conditions are caused by mutations in the same gene. After reducing the disease critical region to approximately 1 Mb, we used the positional cloning strategy to identify the SOST gene, which is mutated in sclerosteosis patients. This new gene encodes a protein with a signal peptide for secretion and a cysteine-knot motif. Two nonsense mutations and one splice site mutation were identified in sclerosteosis patients, but no mutations were found in a fourth sclerosteosis patient nor in the patients from the van Buchem family. As the three disease-causing mutations lead to loss of function of the SOST protein resulting in the formation of massive amounts of normal bone throughout life, the physiological role of SOST is most likely the suppression of bone formation. Therefore, this gene might become an important tool in the development of therapeutic strategies for osteoporosis.

Isolation of zebrafish gdf7 and comparative genetic mapping of genes belonging to the growth/differentiation factor 5, 6, 7 subgroup of the TGF-beta superfamily.

The Growth/differentiation factor (Gdf) 5, 6, 7 genes form a closely related subgroup belonging to the TGF-beta superfamily. In zebrafish, there are three genes that belong to the Gdf5, 6, 7 subgroup that have been named radar, dynamo, and contact. The genes radar and dynamo both encode proteins most similar to mouse GDF6. The orthologous identity of these genes on the basis of amino acid similarities has not been clear. We have identified gdf7, a fourth zebrafish gene belonging to the Gdf5, 6, 7 subgroup. To assign correct orthologies and to investigate the evolutionary relationships of the human, mouse, and zebrafish Gdf5, 6, 7 subgroup, we have compared genetic map positions of the zebrafish and mammalian genes. We have mapped zebrafish gdf7 to linkage group (LG) 17, contact to LG9, GDF6 to human chromosome (Hsa) 8 and GDF7 to Hsa2p. The radar and dynamo genes have been localized previously to LG16 and LG19, respectively. A comparison of syntenies shared among human, mouse, and zebrafish genomes indicates that gdf7 is the ortholog of mammalian GDF7/Gdf7. LG16 shares syntenic relationships with mouse chromosome (Mmu) 4, including Gdf6. Portions of LG16 and LG19 appear to be duplicate chromosomes, thus suggesting that radar and dynamo are both orthologs of Gdf6. Finally, the mapping data is consistent with contact being the zebrafish ortholog of mammalian GDF5/Gdf5.

Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene.

Although disorders of iron metabolism are prevalent, iron transport remains poorly understood. To address this problem, we undertook a positional cloning strategy to identify the causative mutation in mice with microcytic anaemia (mk). Homozygous mk/mk mice have microcytic, hypochromic anaemia due to severe defects in intestinal iron absorption and erythroid iron utilization. We report the identification of a strong candidate gene for mk, and suggest that the phenotype is a consequence of a missense mutation in Nramp2 (ref. 5), a previously identified gene of unknown function. Nramp2 is homologous to Nramp1, a gene activa in host defense. If Nramp2 is mk, as the cumulative evidence suggests, our findings have broad implications for the understanding of iron transport and resistance to intracellular pathogens.