Genetic ecotoxicology I: DNA integrity and reproduction in mosquitofish exposed in situ to radionuclides

Female mosquitofish (Gambusia affinis) were collected from two sites located on the US Department of Energy's Oak Ridge Reservation that are contaminated with 137Cs, 90Sr, other radionuclides and chemical genotoxicants. Fish from non- radionuclide contaminated environments located off the reservation were also collected. DNA, extracted from liver tissue and blood cells, was examined by gel electrophoresis for structural damage in the form of strand breakage. In general, the level of DNA strand breaks was elevated in fish from radionuclide-contaminated sites with observed differences in the number and type of strand breaks between liver tissue and blood cells. The number of malformed embryos was higher in fish at the contaminated sites, and varied with season. Fecundity was negatively correlated with the level of double strand breaks in the DNA of fish from one contaminated site. Females with broods that included malformed embryos had more DNA strand breakage than those that did not; and furthermore, a threshold effect was observed between the occurrence of malformed embryos and the presence of double strand breaks in the DNA of the mother. These findings have implications for both ecological risk assessment and evolutionary ecology     

Linkage exclusion and mutational analysis of the noggin gene in patients with fibrodysplasia ossificans progressiva (FOP)

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare and disabling genetic disorder characterized by congenital malformation of the great toes and by progressive heterotopic endochondral ossification in predictable anatomical patterns. Although elevated levels of bone morphogenetic protein 4 (BMP4) occur in lymphoblastoid cells and in lesional cells of patients with FOP, mutations have not been identified in the BMP4 gene, suggesting that the mutation in FOP may reside in a BMP4-interacting factor or in another component of the BMP4 pathway. A powerful antagonist of BMP4 is the secreted polypeptide noggin. A recent case report described a heterozygous 42-bp deletion in the protein-coding region of the noggin gene in a patient with FOP. In order to determine if noggin mutations are a widespread finding in FOP, we examined 31 families with 1 or more FOP patients. Linkage analysis with an array of highly polymorphic microsatellite markers closely linked to the noggin gene was performed in four classically-affected multigenerational FOP families and excluded linkage of the noggin locus to FOP (the multipoint lod score was -2 or less throughout the entire range of markers). We sequenced the noggin gene in affected members of all four families, as well as in 18 patients with sporadic FOP, and failed to detect any mutations. Single-strand conformation polymorphism (SSCP) analysis of 4 of these patients plus an additional 9 patients also failed to reveal any mutations. Among the samples analyzed by SSCP and DNA sequencing was an independently obtained DNA sample from the identical FOP patient previously described with the 42-bp noggin deletion; no mutation was detected. Examination of the DNA sequences of 20 cloned noggin PCR products, undertaken to evaluate the possibility of a somatic mutation in the noggin gene which could be carried by a small subset of white blood cells, also failed to detect the presence of the reported 42-bp deletion. We conclude that mutations in the coding region of noggin are not associated with FOP.     

Association of haplotypes in the beta-chemokine locus with multiple sclerosis

Linkage studies in multiple sclerosis (MS) identified several susceptibility loci. One of these regions includes chromosome 17q11 where a meta-analysis of data from three genome scans suggested linkage. This region encodes a cluster of genes for beta-chemokines or CC chemokine ligands (CCLs), which may be involved in the development of MS lesions. Here we aimed to test if CCL alleles and haplotypes are associated with MS. Using methods of linkage and association, we observed deviations from the expected 50% transmission of haplotypes from unaffected parents to their affected children at CCL2, CCL11-CCL8-CCL13 and CCL3 within the investigated 1.85 MB chromosomal segment. Analyses of the linkage disequilibrium map support that variants with possible relevance to MS can be located within these subregions. Identification of MS associated CCL variants may have direct clinical significance, as it can lead to the design of small competitive antagonists of these molecules with beneficial effects in the treatment of patients with early and active disease.     

Multipoint genomic scanning for quantitative loci: effects of map density, sibship size and computational approach

Multipoint interval mapping (MIM) and the MAPMAKER/SIBS program (M/S) are two methods of mapping quantitative loci by examining identity by descent (IBD) sharing in a region spanned by multiple microsatellite DNA markers. For the purpose of comparison, we simulated a quantitative trait controlled by a two-locus model, and evaluated the power and genome-wide false positive rate of both approaches. Based on our simulation, we examined the effects of marker density (5 cM, 10 cM and 20 cM) and sibship size (2, 3, 4 and 5) on the power to detect linkage. Our results indicate that a 10 cM map provides the optimal trade-off between power and type I error, and that the power of MIM increases with sibship size and, in general, performs better than MAPMAKER/SIBS. Furthermore, we conclude that using a reasonable sample of randomly ascertained sibships, it is possible to map a quantitative trait locus (QTL) which accounts for 25% of the phenotypic variance.     

Genetic polymorphisms of <Emphasis Type="Italic">TLR3</Emphasis> are associated with Nasopharyngeal carcinoma risk in Cantonese population

Background  

Nasopharyngeal carcinoma is endemic in Southern China, displays a strong relationship with genetic susceptibility and associates with Epstein-Barr virus infection. Toll-like receptor 3 (TLR3) plays an important role in the antivirus response. Therefore, we examined the association between TLR3 gene polymorphisms and NPC susceptibility.     

A new locus for dominant drusen and macular degeneration maps to chromosome 6q14

PURPOSE: To report the localization of a gene causing drusen and macular degeneration in a previously undescribed North American family. METHODS: Genetic mapping studies were performed using linkage analysis in a single family with drusen and atrophic macular degeneration. RESULTS: The clinical manifestations in this family ranged from fine macular drusen in asymptomatic middle-aged individuals to atrophic macular lesions in two children and two elderly patients. We mapped the gene to chromosome 6q14 between markers D6S2258 and D6S1644. CONCLUSIONS: In a family with autosomal dominant drusen and atrophic macular degeneration, the gene maps to a 3.2-cM region on chromosome 6q14. This locus appears to be distinct from, but adjacent to, the loci for cone-rod dystrophy 7 (CORD7) and North Carolina macular dystrophy (MCDR1). Future identification of the gene responsible for the disease in this family will provide a better understanding of macular degeneration.     

Genetic Ecotoxicology III: the Relationship Between DNA Strand Breaks and Genotype in Mosquito Fish Exposed to Radiation

DNA polymorphism in mosquito fish (Gambusia affinis), as revealed by RAPD (randomly amplified polymorphic DNA) and allozyme analysis, was compared to relative amounts of DNA strand breakage in blood and liver tissues. Mosquito fish were exposed to radionuclide contamination in situ and to X-rays in the laboratory. The types of RAPD metrics used were the number of RAPD bands per individual and the frequency of certain RAPD bands. In a previous study, it was noted that in some instances the number of RAPD bands and the frequency of certain RAPD bands were elevated in radionuclide-contaminated sites relative to reference sites. In the present study, it was found that the median molecular length (MML) of the DNA (which is inversely proportional to the amount of DNA strand breakage) was correlated in several cases to the number of RAPD bands per individual. In addition, for those RAPD bands that occurred at a higher frequency in mosquito fish from radionuclide-contaminated sites, DNA strand breakage was often lower for those fish with than without these RAPD bands. RAPD data obtained on mosquito fish exposed to X-rays in the laboratory paralleled those from the field. Furthermore, analysis showed that heterozygotes for the allozyme locus nucleoside phosphorylase were more prevalent in radionuclide-contaminated sites and had fewer DNA strand breaks than did homozygotes. These results provide additional evidence that changes in population genetic structure of mosquito fish exposed to a genotoxicant (radiation) can be detected at the DNA level.     

A gender-specific association of CNV at 6p21.3 with NPC susceptibility

Copy number variations (CNVs), a major source of human genetic polymorphism, have been suggested to have an important role in genetic susceptibility to common diseases such as cancer, immune diseases and neurological disorders. Nasopharyngeal carcinoma (NPC) is a multifactorial tumor closely associated with genetic background and with a male preponderance over female (3:1). Previous genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) that are associated with NPC susceptibility. Here, we sought to explore the possible association of CNVs with NPC predisposition. Utilizing genome-wide SNP-based arrays and five CNV-prediction algorithms, we identified eight regions with CNV that were significantly overrepresented in NPC patients compared with healthy controls. These CNVs included six deletions (on chromosomes 3, 6, 7, 8 and 19), and two duplications (on chromosomes 7 and 12). Among them, the CNV located at chromosome 6p21.3, with single-copy deletion of the MICA and HCP5 genes, showed the highest association with NPC. Interestingly, it was more specifically associated with an increased NPC risk among males. This gender-specific association was replicated in an independent case-control sample using a self-established deletion-specific polymerase chain reaction strategy. To the best of our knowledge, this is the first study to explore the role of constitutional CNVs in NPC, using a genome-wide platform. Moreover, we identified eight novel candidate regions with CNV that merit future investigation, and our results suggest that similar to neuroblastoma and prostate cancer, genetic structural variations might contribute to NPC predisposition.