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PubMed2005

Localized breakdown in linkage disequilibrium does not always predict sperm crossover hot spots in the human MHC class II region.

To investigate the relationship between meiotic crossover hot spots and block-like linkage disequilibrium (LD), we have extended our high-resolution studies of the human MHC class II region to a 90-kb segment upstream of the HLA-DOA gene. LD blocks in this region are not as well defined as in the neighboring 210-kb DNA segment but do show two regions of LD breakdown in which coalescent analysis indicates substantial historical recombination. Sperm crossover analysis of one region revealed a novel localized hot spot similar in intensity and morphology to most other MHC hot spots. Crossovers at this hot spot are not obviously affected by a large insertion/deletion polymorphism near the hot spot. The second region of LD breakdown, within the DPB1 gene, shows an extremely low level of sperm crossover activity and does not contain a sperm crossover hot spot. These results highlight the complexity of LD patterns and the importance of experimentally verifying crossover hot spots.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

HAPLOT: a graphical comparison of haplotype blocks, tagSNP sets and SNP variation for multiple populations.

UNLABELLED: Understanding of human variation relevant to association studies can benefit from population comparison, especially comparing populations in the same geographical region. Variations in linkage disequilibrium patterns, in tagSNP sets, and in SNP heterozygosities among populations can be used to infer the evolutionary pattern. We present here a win32 system based Perl/Tk application for visual comparisons of these variations in different populations. AVAILABILITY: The application package is available at http://info.med.yale.edu/genetics/kkidd/programs.html CONTACT: sheng.gu@yale.edu.

Chromosome Mapping↗
PubMed1993

Genetics of salivary protein polymorphisms.

Human salivary PRPs are determined by six closely linked genes on chromosome 12p13.2. The many PRPs show complex electrophoretic patterns that differ between individuals and reflect numerous genetic polymorphisms. Frequent length and null polymorphisms are common among PRPs. Common themes emerge as a background for these PRP polymorphisms. First, posttranslational proteolysis occurs with double-banded patterns among acidic PRPs and the generation of numerous basic PRPs derived from precursor proteins. Specific mutations may interfere with proteolysis, preventing generation of double-banded acidic PRPs (as with the Pa protein) or of small basic PRPs from precursor proteins (as with Pm proteins). Second, single cysteine substitutions in PRPs (Pa from PRH1 and G1 8 from PRB3) may lead to disulfide bonded homodimers as well as heterodimers with salivary peroxidase. Third, frequent homologous and unequal crossing-over within the PRP gene cluster leads to frequent protein size-variants (intragenic events as with the G1 protein variants) and the generation of the PRB2/1 fusion gene (intergenic event) with deletion of the PRB1 coding region and absence of multiple PRB1 coded proteins (Ps, Pm, Pe) in PRB2/1 homozygotes. Fourth, null mutations may also be produced (as with PsO and G1 0) by single nucleotide changes.

Alleles↗
PubMed2006

SSR mining in coffee tree EST databases: potential use of EST-SSRs as markers for the Coffea genus.

Expressed sequence tags (ESTs) from Coffea canephora leaves and fruits were used to search for types and frequencies of simple sequence repeats (EST-SSRs) with a motif length of 1-6 bp. From a non-redundant (NR) EST set of 5,534 potential unigenes, 6.8% SSR-containing sequences were identified, with an average density of one SSR every 7.73 kb of EST sequences. Trinucleotide repeats were found to be the most abundant (34.34%), followed by di- (25.75%) and hexa-nucleotide (22.04%) motifs. The development of unique genic SSR markers was optimized by a computational approach which allowed us to eliminate redundancy in the original EST set and also to test the specificity of each pair of designed primers. Twenty-five EST-SSRs were developed and used to evaluate cross-species transferability in the Coffea genus. The orthology was supported by the amplicon sequence similarity and the amplification patterns. The >94% identity of flanking sequences revealed high sequence conservation across the Coffea genus. A high level of polymorphic loci was obtained regardless of the species considered (from 75% for C. liberica to 86% for C. canephora). Moreover, the polymorphism revealed by EST-SSR was similar to that exposed by genomic SSR. It is concluded that Coffea ESTs are a valuable resource for microsatellite mining. EST-SSR markers developed from C. canephora sequences can be easily transferred to other Coffea species for which very little molecular information is available. They constitute a set of conserved orthologous markers, which would be ideal for assessing genetic diversity in coffee trees as well as for cross-referencing transcribed sequences in comparative genomics studies.

Cloning, Molecular↗
PubMed2003

Overexpression of S100A4 is closely related to the aggressiveness of gastric cancer.

Elevated levels of the calcium-binding protein S100A4 cause metastasis of benign rat mammary tumor cells. To investigate whether S100A4 plays an important role in the invasion and metastasis of gastric cancers, we examined the gene mutations in the coding regions and expression patterns of the S100A4 in gastric adenocarcinoma in Korea. Moderate to strong expression of S100A4 was found in 53 (68.8%) of the 77 gastric adenocarcinomas, whilst normal gastric epithelium either failed to stain or showed weak staining. Interestingly, S100A4 expression was more frequently observed in gastric cancer patients with advanced gastric cancer (p=0.039), positive lymph node metastasis (p=0.001), and peritoneal dissemination (p=0.022). No gene mutations were found in the analyzed genomic area in 77 gastric adenocarcinomas and 15 gastric cancer cell lines. We found one single nucleotide polymorphism without an amino acid change, A99G, in two cases. These data suggest that the overexpression of S100A4 may be closely related to the aggressiveness of gastric cancer in Korea.

Adenocarcinoma↗
PubMed2002

Can long-range microsatellite data be used to predict short-range linkage disequilibrium?

The distribution of linkage disequilibrium (LD) across the genome is highly complex. Little is known about the relationship between long-range and short-range LD in a genomic region. We assessed whether a dense set of microsatellite data could be used to predict short-range LD in family samples. We analyzed intermarker LD in data derived from chromosomal regions 18q22 and 10q25-26, densely genotyped with microsatellite markers. The pattern of LD was highly heterogeneous within and between both chromosomal regions. On 10q25-26, very little LD was detected. On 18q22, where marker density was higher, many marker pairs were in LD. We modeled the decay of LD over distance in this region. A classical model accounted for most of the relationship between LD and distance (R (2)=63%). We used this model to predict the proportion of markers expected to show useful levels of LD at short distances. This prediction agreed with estimates based on single-nucleotide polymorphism (SNP) marker genotypes in the region. Both microsatellite and SNP data predict that about 80% of marker pairs would display levels of LD that are useful for association studies at distances of up to 15 kb in this region. These projections also agree with levels of LD directly measured in a 10 kb set of SNP genotypes generated in a nearby region of finished sequence. Our results suggest that existing sets of microsatellite data, if sufficiently dense, may be used to develop good initial estimates of the density of additional markers needed to screen a region for disease alleles by association analysis.

Chromosome Mapping↗
PubMed2006

Identification of a genomic subgroup of BK polyomavirus spread in European populations.

BK polyomavirus (BKV) is highly prevalent in the human population, infecting children without obvious symptoms and persisting in the kidney in a latent state. In immunosuppressed patients, BKV is reactivated and excreted in urine. BKV isolates worldwide are classified into four serologically distinct subtypes, I-IV, with subtype I being the most frequently detected. Furthermore, subtype I is subdivided into subgroups based on genomic variations. In this study, the distribution patterns of the subtypes and subgroups of BKV were compared among four patient populations with various immunosuppressive states and of various ethnic backgrounds: (A) Finnish renal-transplant recipients; (B) Irish/English haematopoietic stem-cell transplant recipients with and without haemorrhagic cystitis; (C) Japanese renal-transplant recipients; and (D) Japanese bone-marrow transplant recipients. The typing sequences (287 bp) of BKV in population A were determined in this study; those in populations B-D have been reported previously. These sequences were subjected to phylogenetic and single nucleotide polymorphism analyses. Based on the results of these analyses, the BKV isolates in the four patient populations were classified into subtypes and subgroups. The incidence of subtype IV varied significantly among patient populations. Furthermore, the incidence of subgroup Ib-2 within subtype I was high in populations A and B, whereas that of Ic was high in populations C and D (P<0.01). These results suggest that subgroup Ib-2 is widespread among Europeans, whereas Ic is unique to north-east Asians. Furthermore, a phylogenetic analysis based on complete BKV DNA sequences supported the hypothesis that there is geographical separation of European and Asian BKV strains.

Asian People↗
PubMed2003

Silencing-specific methylation and single nucleotide polymorphism of hMLH1 promoter in gastric carcinomas.

AIM: To investigate CpG methylation and single nucleotide polymorphism (SNP) of a specific promoter region of hMLH1 in primary gastric carcinoma. METHODS: Primary gastric carcinomas (n=80), their corresponding normal mucosal samples, and gastric mucosal biopsies from normal/gastritis control patients (n=54) were used. Hypermethylation at -253 nt and -251 nt in relation with the translational start site and SNP of a silencing specific region (-339 nt-46 nt) in the hMLH1 promoter were analyzed by Bst UI-combined bisulfite assay (COBRA), denaturing high performance liquid chromatogram (DHPLC), and sequencing. RESULTS: (A) The specific methylation at -253 nt and -251 nt was observed in 2 of 60 primary gastric carcinomas, but neither in all of the corresponding mucosa nor in normal/gastritis samples, by Bst UI-COBRA and DHPLC. (B) The hMLH1 promoter was methylated homogeneously in the xenograft of the primary gastric carcinoma with the methylated and unmethylated hMLH1. (C) The pattern of SNP at -93 nt of the hMLH1 promoter in 54 Chinese patients with gastric carcinoma was the same as that in the control patients: 51 % was A/G heteroalleles, 34 % and 15 % were A/A and G/G homoalleles, respectively. CONCLUSION: Biallelic inactivation of hMLH1 by epigenetic silencing existed in human primary gastric carcinoma homogeneously. Hypermethylation of hMLH1 may play a role in the early stage of development of a few gastric carcinomas. The SNP at -93 nt is not related to the susceptibility of gastric carcinomas.

Adaptor Proteins, Signal Transducing↗
PubMed2004

TTF-1 and RET promoter SNPs: regulation of RET transcription in Hirschsprung's disease.

Single nucleotide polymorphisms (SNPs) of the coding regions of receptor tyrosine kinase gene (RET) are associated with Hirschsprung's disease (HSCR, aganglionic megacolon). These SNPs, individually or combined, may act as a low penetrance susceptibility locus and/or be in linkage disequilibrium (LD) with another susceptibility locus located in RET regulatory regions. Because two RET promoter SNPs have been found associated with HSCR, in LD with HSCR-associated RET coding region haplotypes, their implication in the transcriptional regulation of RET is of major interest. Analysis of 172 sporadic HSCR patients also revealed the presence of HSCR-associated RET promoter SNPs in LD with the main coding region RET haplotype observed in Chinese patients. By using a weighted logistic regression approach, we determined that of all SNPs tested in our study, the promoter SNPs are the most correlated to the disease. Functional analysis of the RET promoter SNPs in the context of additional 5' regulatory regions demonstrated that the HSCR-associated alleles decrease RET transcription. These SNPs overlap a TTF-1 binding site and TTF-1-activated RET transcription is also decreased by the HSCR-associated SNPs. Moreover, we identified an HSCR patient with a Gly322Ser TTF-1 mutation that compromises activation of transcription from HSCR-associated RET promoter haplotypes. Interestingly, we show that the pattern of RET and TTF-1 expression is coincident in developing human gut. We also present a detailed profile of the RET gene in our population, which provides an insight into the higher incidence of the disease in China.

Alleles↗
PubMed2000

Analysis of the NF1 gene by temperature gradient gel electrophoresis reveals a high incidence of mutations in exon 4b.

A total of 196 unrelated patients with neurofibromatosis type 1 (NF1) was screened for mutations in exons 4a-c of the NF1 gene by temperature gradient gel electrophoresis (TGGE) of polymerase chain reaction (PCR)-amplified genomic DNA fragments using intron-based primers. DNA samples with abnormal TGGE band patterns were subjected to sequence analysis. Sequence alterations were identified in ten patients (5.1%): 496delGT (1), 499delTGTT (4), T528A = D176E (2), T539A = L180X (1), 540insA (1), C574T = R192X (1). Thus, a total of six different mutations was identified in exon 4b but none in exons 4a and 4c. Only the missense mutation D176E, which we assume to be a nonpathogenic polymorphism, and the 4-base pair (bp) deletion 499delTGTT have been described before. The reason for the high incidence of mutations in exon 4b is obviously a tetranucleotide tandem repeat comprising nucleotides 495-502 (TGTTTGTT) that may give rise to slipped mispairing and subsequent deletion of one repeat unit during replication. Additionally, the recurrent 4 bp deletion was found as a second hit in a malignant schwannoma of a further NF1 patient, suggesting that microlesions may be as frequent among somatic as among germline mutations. This is the first report of a systematic study of NF1 exons 4a-c in a large group of NF1 patients.

Base Sequence↗
PubMed2004

Allelic genes of blood group antigens: a source of human mutations and cSNPs documented in the Blood Group Antigen Gene Mutation Database.

In this report, we analyze data assembled in the Blood Group Antigen Gene Mutation Database (www.bioc.aecom.yu.edu/bgmut/index.htm), which describes sequence information on human genes associated with expression of the various serologically-determined blood group phenotypes. The database documents 38 genetic loci and a total of 624 alleles that together encode a large repertoire of proteins and constitute 27 serologically-defined blood group systems. Analysis of sequence variation patterns across alleles of a number of genes is focused on their molecular profiles, including mutational sites and recurrence, patterns of gene rearrangements in duplicated gene families, correlation of predicted location of epitopes in extracellular loops with sites of alterations, and effects of mutations on protein expression. That information, and the relative ease of identifying individuals bearing variant alleles, has led to the proposal that genes encoding blood group antigens are an important and unique resource for studies of human DNA variation. Another focus is on mutations in regions that encode the antigenic epitopes and on their occurrence in world populations. These mutations may be viewed as coding single nucleotide polymorphisms (cSNPs). We propose that one group of these cSNPs, which are known to occur with significant frequency in all world populations, could serve as well-validated genetic markers. In addition, specific mutations in a number of "low incidence" and rare alleles could serve as cSNPs specific for a given population. The allelic frequencies of these mutations and knowledge of their world-wide occurrence add a valuable dataset to the existing cSNP pools documented in SNP databases.

Alleles↗
PubMed1998

Characterisation of anisakid nematodes with zoonotic potential by nuclear ribosomal DNA sequences.

Larvae of three species of anisakid nematode from fish, Anisakis simplex, Hysterothylacium aduncum and Contracaecum osculatum, were characterised genetically using a molecular approach. The nuclear ribosomal DNA region spanning the first internal transcribed spacer, the 5.8S gene and the second internal transcribed spacer was amplified and sequenced. The lengths of the first and second internal transcribed spacer sequences of the three species ranged from 392 to 449 bp and 262 to 347 bp, respectively, whereas the 5.8S sequence was 157 bp. For the three species, the G+C contents for the three regions of ribosomal DNA ranged from 42.4 to 52.2%. While no intraspecific variation was detected in the second internal transcribed spacer or 5.8S sequence of any species examined, one polymorphic nucleotide position was detected in the first internal transcribed spacer sequence for A. simplex and H. aduncum. The extent of sequence differences in the first (approximately 34-45%) and second (approximately 50-53%) internal transcribed spacers among the species was greater than in the 5.8S gene (approximately 3-5%). Based on the sequence differences, PCR-based restriction fragment length polymorphism and single-strand conformation polymorphism methods were established for the unequivocal delineation of the three species. These methods should provide valuable tools for studying the life-cycle, transmission pattern(s) and population structure of each of the three anisakid nematodes examined herein, and for the diagnosis of anisakiasis in humans and animals.

Animals↗
PubMed1990

Identification of human acrosomal antigen SP-10 in primates and pigs.

The intra-acrosomal human sperm protein SP-10 was previously designated a "primary vaccine candidate" by a World Health Organization Taskforce on Contraceptive Vaccines. In the present study, a monoclonal antibody to SP-10 (MHS-10) was employed on Western blots to identify immunoreactive SP-10 in sperm extracts from baboon (Papio cyanocephalus anubis) and two macaques (Macaca mulatta and Macaca fascicularis). In each of these primates, the MHS-10 monoclonal antibody recognized a polymorphic pattern of immunoreactive peptides similar to that in humans. Immunoreactive SP-10 was also demonstrated in pig sperm. Using purified preparations of the previously described intra-acrosomal molecules acrosin and sperminogen in the pig, we observed that the MHS-10 monoclonal antibody did not react with these proteins, indicating SP-10 is distinct from these known acrosomal components. Sperm from several common species including the rabbit, bull, rat, guinea pig and cat did not immunoreact with the MHS-10 monoclonal antibody. By use of a radioactive probe spanning 628 nucleotides of the open reading frame for SP-10 on Northern blots of poly A + RNA obtained from testes of Macaca fascicularis, Papio papio, and Papio cyanocephalus anubis, a 1.35-kb mRNA of identical size to the mRNA from human testes was identified. These results indicate that baboons, macaques, and pigs may be appropriate models for testing an SP-10-based contraceptive vaccine.

Acrosome↗
PubMed1997

Secondary structure of the 3'-untranslated region of yellow fever virus: implications for virulence, attenuation and vaccine development.

A genetic algorithm-based RNA secondary structure prediction was combined with comparative sequence analysis to construct models of folding for the distal 380 nucleotides of the 3'-untranslated region (3'-UTR) of yellow fever virus (YFV). A number of structural elements that are thermodynamically stable, conserved in shape, and confirmed by compensatory mutations were revealed. At the same time structural polymorphisms were observed among strains of YFV. These polymorphisms showed an association with virulence: all wild and pathogenic strains were likely to be folded in a significantly different way from vaccine strains with reduced virulence. Structural divergence was also found among vaccine strains, with 17DD, the most virulent in the mouse model, exhibiting an intermediate pattern of folding, combining structural features of both wild and vaccine strains. The observation of a strong association between secondary structure of the 3'-UTR and virulence of YFV may help elucidate the molecular mechanisms of virus attenuation and lead to new strategies of vaccine development directed towards rational modification of secondary structure of the 3'-UTR.

Base Sequence↗
PubMed2006

Mutational spectrum and linkage disequilibrium patterns at the ornithine transcarbamylase gene (OTC).

Ornithine transcarbamylase (OTC; EC 2.1.3.3) is a hepatic enzyme involved in ammonia elimination via the urea cycle. Since the sequence of the OTC gene was reported many types of mutations continue to be found in OTC deficiency patients, continuing to increase the already wide mutational spectrum known for this gene. In this study we present the clinical, biochemical and molecular features of thirteen late-onset OTC deficiency patients. Mutations were identified in all these patients, among which six were novel point substitutions (L59R, A137P, L148S, Y176L, L186P, and K210N) and one was a 2-bp deletion at exon 4 (341-342delAA). In addition, a de novo genomic deletion of maternal origin encompassing exons 1 to 5 was also identified by the analysis of LD patterns using intragenic polymorphic markers. This work exemplifies the potential value of population genetic studies for the detection of large deletions.

Exons↗
PubMed2005

Balinese Y-chromosome perspective on the peopling of Indonesia: genetic contributions from pre-neolithic hunter-gatherers, Austronesian farmers, and Indian traders.

The island of Bali lies near the center of the southern chain of islands in the Indonesian archipelago, which served as a stepping-stone for early migrations of hunter-gatherers to Melanesia and Australia and for more recent migrations of Austronesian farmers from mainland Southeast Asia to the Pacific. Bali is the only Indonesian island with a population that currently practices the Hindu religion and preserves various other Indian cultural, linguistic, and artistic traditions (Lansing 1983). Here, we examine genetic variation on the Y chromosomes of 551 Balinese men to investigate the relative contributions of Austronesian farmers and pre-Neolithic hunter-gatherers to the contemporary Balinese paternal gene pool and to test the hypothesis of recent paternal gene flow from the Indian subcontinent. Seventy-one Y-chromosome binary polymorphisms (single nucleotide polymorphisms, SNPs) and 10 Y-chromosome-linked short tandem repeats (STRs) were genotyped on a sample of 1,989 Y chromosomes from 20 populations representing Indonesia (including Bali), southern China, Southeast Asia, South Asia, the Near East, and Oceania. SNP genotyping revealed 22 Balinese lineages, 3 of which (O-M95, O-M119, and O-M122) account for nearly 83.7% of Balinese Y chromosomes. Phylogeographic analyses suggest that all three major Y-chromosome haplogroups migrated to Bali with the arrival of Austronesian speakers; however, STR diversity patterns associated with these haplogroups are complex and may be explained by multiple waves of Austronesian expansion to Indonesia by different routes. Approximately 2.2% of contemporary Balinese Y chromosomes (i.e., K-M9*, K-M230, and M lineages) may represent the pre-Neolithic component of the Indonesian paternal gene pool. In contrast, eight other haplogroups (e.g., within H, J, L, and R), making up approximately 12% of the Balinese paternal gene pool, appear to have migrated to Bali from India. These results indicate that the Austronesian expansion had a profound effect on the composition of the Balinese paternal gene pool and that cultural transmission from India to Bali was accompanied by substantial levels of gene flow.

Asia, Southeastern↗
PubMed2004

Single-nucleotide polymorphism mutation spectra and resistance to quinolones in Salmonella enterica serovar Enteritidis with a mutator phenotype.

Resistance to quinolone antibiotics has been associated with single-nucleotide polymorphisms (SNPs) in the quinolone resistance-determining region (QRDR) of gyrA. Mutations in the gyrA gene were compared by using mutant populations derived from wild-type Salmonella enterica serovar Enteritidis and its isogenic mutS::Tn10 mutator counterpart. Spontaneous mutants arising during nonselective growth were isolated by selection with either nalidixic acid, enrofloxacin, or ciprofloxacin. QRDR SNPs were identified in approximately 70% (512 of 695) of the isolates via colony hybridization with radiolabeled oligonucleotide probes. Notably, transition base substitution SNPs in the QRDR were dramatically increased in mutants derived from the mutS strain. Some, but not all, antibiotic-resistant mutants lacking QRDR SNPs were resistant to tetracycline and chloramphenicol, consistent with alterations in nonspecific efflux pumps or other membrane transport mechanisms. Changing the selection conditions shifted the mutation spectrum. Selection with ciprofloxacin was least likely to yield a mutant harboring either a QRDR SNP or chloramphenicol resistance. Selection with enrofloxacin was more likely to yield mutants containing Ser83-->Phe mutations, whereas selection with ciprofloxacin or nalidixic acid favored recovery of Asp87-->Gly mutants. Fluoroquinolone-resistant Salmonella strains isolated from veterinary or clinical settings frequently display a mutational spectrum with a preponderance of transition SNPs in the QRDR, the pattern found in vitro among mutS mutator mutants reported here. Both the preponderance of transition mutations and the varied mutation spectra reported for veterinary and clinical isolates suggest that bacterial mutators defective in methyl-directed mismatch repair may play a role in the emergence of quinolone and fluoroquinolone resistance in feral settings.

Anti-Bacterial Agents↗
PubMed2001

Application of single-strand conformation polymorphism to the study of bovine viral diarrhea virus isolates.

Single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products is a genetic screening technique for rapid detection of nucleotide substitutions in PCR-amplified genomic DNA or cDNA. It is based on the observation that partially formamide-denatured double-stranded DNA migrates as 2 single-stranded DNA molecules when electrophoresed in nondenaturing polyacrylamide gels. The mobility depends on the 3-dimensional conformation of the strand under the conditions used. It is possible to discriminate between DNA strands differing in only 1 nucleotide. The method was applied to the analysis of Bovine Viral Diarrhea Virus (BVDV) isolates. Reference and Argentinian strains were assessed for variations in their 5' untranslated region (5'-UTR). The PCR products of the 5'-UTR ends were formamide denatured and compared by SSCP analysis in nondenaturing 15% polyacrylamide and 15% polyacrilamide-5% glycerol gels. The reference strains SD-1, Singer, and Oregon C24V had differences in electrophoretic patterns. Despite the high conservation among the 5'-UTR of pestiviruses, the method allowed discrimination among all 9 Argentinian isolates. The 5'-UTR of a fetal kidney-derived isolate (1R93) was PCR amplified and cloned in a plasmid vector; the SSCP analysis of 30 PCR products obtained by direct amplification over randomly selected clones produced 5 different banding patterns, indicating the existence of viral quasispecies. The results show that SSCP may be used to identify and differentiate among BVDV isolates.

5' Untranslated Regions↗