Methylation of the <Emphasis Type="Italic">RASSF1A</Emphasis> promoter region and the allelic imbalance frequencies in chromosome 3 critical regions correlate with progression of clear cell renal carcinoma

The short arm of chromosome 3 (3p) contains several critical regions that have increased frequencies of allelic deletions and harbor a set of tumor suppressor genes. In particular, the range of functions performed by RASSF1A (LUCA region, 3p21.31) includes those potentially associated with carcinogenesis. Among 3p genes, RASSF1A has the highest methylation frequency in epithelial tumors of various locations. For the first time, two different methods (methylation-specific PCR and methylation-sensitive restriction analysis) independently showed that the methylation level of the CpG island in the RASSF1A promoter region significantly correlated with grade and clinical stage of clear cell renal cell carcinoma (RCC). An analysis of 23 3p polymorphic markers in a representative set of 80 RCC cases characterized clinically and histologically revealed that RCC progression significantly correlated with the frequency of allelic imbalances in some critical regions of 3p (LUCA and AP20), but not in 3p as a whole. These data suggest that RCC progression is associated with the methylation of the RASSF1A promoter and, possibly, with structural and functional alterations in other 3p genes. In addition, significant correlation between RASSF1A methylation and allelic losses at the nearby polymorphic marker locus suggests the “two hit” model for the inactivation of this tumor suppressor gene in RCC.     

Identification of Stmm3 locus Conferring Resistance to Late-stage Chemically Induced Skin Papillomas on Mouse Chromosome 4 by Congenic Mappingand Allele-specific Alteration Analysis

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to chemically induced skin papillomas on chromosome 4 and 7 with a large number of [(FVB/N × MSM/Ms) F₁ × FVB/N] backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 4. We used linkage analysis and a congenic mouse strain, FVB.MSM-Stmm3 to refine the location of Stmm3 (Skin tumor modifier of MSM 3) locus within a physical interval of about 34 Mb on distal chromosome 4. In addition, we used patterns of allele-specific imbalances in tumors from N₂ and N₁₀ congenic mice to narrow down further the region of Stmm3 locus to a physical distance of about 25 Mb. Furthermore, immunohistochemical analysis showed papillomas from congenic mice had less proliferative activity. These results suggest that Stmm3 responsible genes may have an influence on papilloma formation in the two-stage skin carcinogenesis by regulating papilloma growth rather than development.     

Analysis of NAT2 point mutations with biological microchips

The NAT2 product, N-acetyltransferase 2, is involved in biotransformation and detoxification of several aromatic amines (in particular, 2-aminofluorene, 4-aminobiphenyl, and 4-naphthylamine), which are strongly mutagenic and carcinogenic, and acetylates some drugs, affecting their metabolism. A biological microchip was developed to detect 16 point mutations, which determine 36 alleles and 660 genotypes of NAT2. The genotypes can be divided into four groups according to the acetylator phenotype: groups with rapid (R/R), intermediate (R/S), or slow (S/S) acetylation and a group combining intermediate and slow alleles (“R/S or S/S”). The last group includes the alleles determined by combinations of seven mutations (191G/A, 282C/T, 341T/C, 481C/T, 590G/A, 803A/G, and 857G/A), whose cis or trans position is detectable by restriction enzyme analysis. The NAT2 genotype was unequivocally established for 37 out of 71 DNA specimens, while the other 34 specimens were characterized by more than two genotypes. By the acetylator phenotype, 16 out of the 34 genotypes were assigned to the group “R/S or S/S,” combining mutations 282C/T, 341T/C, 481C/T, 590G/A, and 803A/G. Thus, the biochip allows primary analysis of most NAT2 polymorphic substitutions, the acetylator genotype being important to know in predictive medicine and individualized therapy.     

等位基因特异性引物PCR技术及其应用研究

目的：研究建立等位基因特异性引物PCR技术体系,并将其应用于基因单核苷酸多态性研究工作。方法：通过美国国家生物信息中心(NCBI)的genBank获取基因序列及其相应位点的SNP信息。利用Primer5．0软件设计引物,并经NCBI的Blast2．0软件检验其特异性。结果：建立了单一等位基因特异性引物PCR(SASP—PCR)与嵌套式等位基因特异性引物PCR(NASP-PCR)两种技术,并应用于β2肾上腺素受体及内皮源性一氧化氮合酶基因单核苷酸多态性的研究,证实该技术的稳定性和优越性。结论：等位基因特异性引物PCR技术是一种更为简便、特异性较高、费用少的、便于推广的SNP检测方法,特别是在群体基因单核苷酸多态性研究中更有优势。     

Transformation of QTL genotypic effects to allelic effects

The genotypic and allelic effect models are equivalent in terms of QTL detection in a simple additive model, but the QTL allelic model has the advantage of providing direct information for marker-assisted selection. However, the allelic matrix is four times as large as the genotypic IBD matrix, causing computational problems, especially in genome scans examining multiple positions. Transformation from genotypic to allelic effects, after estimating the genotypic effects with a smaller IBD matrix, can solve this problem. Although the validity of transformation from genotypic to allelic effects has been disputed, this work proves that transformation can successfully yield unique allelic effects when genotypic and allelic IBD matrixes exist.     

DNA-Based Individual and Sex Identification from Wolverine (Gulo Gulo) Faeces and Urine

Non-invasive genetic analyses are important for studies of species that are rare, sensitive or at risk of extinction. This study investigates the possibility of using faeces and urine to obtain microsatellite genotypes for individual identification of wolverines (Gulo gulo). The reliability of the employed method was assessed by analysing independent amplifications of non-invasive samples (a multiple-tube approach) and by comparing genotypes obtained from faeces to genotypes obtained from blood or tissue of the same individual. Ten microsatellite markers were successfully amplified in 65% of the faecal samples (n = 32) and 40% of the urine samples (n = 22). Allelic dropout was found in 12 and 14% of the amplifications from extracts of faeces and urine, respectively. Nevertheless, all multi-locus genotypes were correct, as judged from comparison to data from tissue or blood samples, after three replicates. These results suggest that a non-invasive approach based on DNA-analysis of faeces can be a powerful tool in population monitoring of wolverines, potentially providing reliable estimates of population size and immigration rate. A second objective of the study was to develop markers for DNA-based sex identification in wolverines using non-invasive samples. We developed two Y-linked markers, one that was specific to wolverine and one that also successfully identified sex in another mustelid. Importantly, none of the markers amplified potential prey species such as reindeer or rodents.     

Prediction of early termination from the self-regulatory treatment of chronic headache

The ability of demographic, psychological testing and history information to predict which patients will terminate early from nonpharmacological treatment of headache (relaxation and biofeedback) was tested. Information from each of these areas was initially examined for differences between dropouts and treatment completers using univariate analyses. These analyses were followed by a canonical discriminate function analysis that predicted whether patients would complete treatment or drop out. Information from the three predictor sets combined resulted in 77.4% of the patients being correctly classified.This research was supported by a grant from the NINCDS, NS15235.     

Gene regulation,quantitative genetics and the evolution of reaction norms

Summary The ideas of phenotypic plasticity and of reaction norm are gaining prominence as important components of theories of phenotypic evolution. Our understanding of the role of phenotypic plasticity as an adaptation of organisms to variable environments will depend on (1) the form(s) of genetic and developmental control exerted on the shape of the reaction norm and (2) the nature of the constraints on the possible evolutionary trajectories in multiple environments. In this paper we identify two categories of genetic control of plasticity: allelic sensitivity and gene regulation. These correspond generally to two classes of response by the developmental system to environmental change: phenotypic modulation, in which plastic responses are a continuous and proportional function of environmental stimuli and developmental conversion, where responses tend to be not simply proportional to the stimuli. We propose that control of plasticity by regulatory actions has distinct advantages over simple allelic sensitivity: stability of phenotypic expression, capacity for anticipatory response and relaxation of constraints due to genetic correlations. We cite examples of the extensive molecular evidence for the existence of environmentally-cued gene regulation leading to developmental conversion. The results of quantitative genetic investigations on the genetics and evolution of plasticity, as well as the limits of current approaches are discussed. We suggest that evolution of reaction norms would be affected by the ecological context (i.e. spatial versus temporal variation, hard versus soft selection, and fine versus coarse environmental grain). We conclude by discussing some empirical approaches to address fundamental questions about plasticity evolution.     

SINGLE LOCUS MICROSATELLITES IN GRACILARIALES (RHODOPHYTA): HIGH LEVEL OF GENETIC VARIABILITY WITHIN GRACILARIA GRACILIS AND CONSERVATION IN RELATED SPECIES1

Four single locus microsatellites identified in the red alga Gracilaria gracilis (Stackhouse) Steentoft, Irvine, et Farnham (Rhodophyta) were examined for allelic diversity at different spatial and taxonomic levels. First, because simple morphological diagnostic characters are often missing within the Gracilariaceae, we developed a simple and rapid method based on rDNA ITS size variation in order to verify the taxonomic status of the samples used in this study. All European (including Mediterranean samples), Argentinian, and Namibian samples used in our study were confirmed to be a homogenous G. gracilis group. By contrast, our results on rDNA ITS sizes showed that Gracilaria from Japan, initially identified as G. gracilis, was different from the rest of the G. gracilis group. Secondly, microsatellite polymorphism and conservation at the species level was tested on the worldwide collection of G. gracilis and within a single population. The loci Gv1AAG and Gv1AAC showed no allelic variation, whereas two others, Gv1CT and Gv2CT, were highly polymorphic. All microsatellite loci were conserved within G. gracilis, except in the sample from Japan. The taxonomic status of G. gracilis from Japan is thus questionable. This study revealed a high level of within-population polymorphism (52 alleles for Gv1CT and 12 for Gv2CT). Moreover, the combination of these two loci was shown to be very powerful for identifying individuals within a population, that is, 93% of the individuals were characterized by a unique genotype. Finally, conservation of the four loci was tested in taxonomically related species of Gracilaria (G. chilensis, G. pacifica, and G. tikvahiae) and two Gracilariopsis species (Gs. sp. and Gs. longissima). The results suggest that the polymorphic locus Gv2CT may provide a valuable genetic marker within the different species of the Gracilariaceae.