A population-based LD map of the human chromosome 6p

The recent publication of the complete sequence of human chromosome 6 provides a platform from which to investigate genomic sequence variation. We report here a detailed linkage disequilibrium (LD) pattern map across the entire human chromosome 6p by using a set of 1152 single nucleotide polymorphisms (SNPs) in a population of 198 Singaporean Chinese, with 326 SNPs focused in the major histocompatibility complex (MHC) region. Our analysis shows some unexpectedly high segments of strong LD in a 10-Mb region that includes the extremely polymorphic and gene-rich MHC loci and many non-MHC genes. These include the telomeric peri-MHC region that harbors olfactory receptors, histones and zinc finger clusters, and the centromeric peri-MHC region that contains several unknown open reading frames. The data also help refine a human–mouse synteny break in the region between 28.6 and 29.4 Mb. The population-based LD map presented here will provide an essential resource for understanding the genomic sequence variation of chromosome 6p and LD mapping of disease genes of complex genetic traits. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. H. Yu and J.-M. Chia should be regarded as joint first authors.     

Single nucleotide polymorphisms within the promoter region of the rhesus monkey tumor necrosis factor-alpha gene

The human TNF-alpha gene is characterized by several single nucleotide polymorphisms (SNPs) in its promoter region, in part having been shown to influence TNF-alpha expression and susceptibility to various diseases. The rhesus macaque is widely used as an animal model for a variety of TNF-alpha associated pathological conditions, but little is known about genetic variation within the TNF-alpha promoter region. In order to check for such polymorphisms, primers based on rhesus sequence within 5 UTR were designed and used to amplify a 1 kb product from genomic DNA of 29 animals. Sequencing and cloning revealed a total of 11 polymorphisms leading to five different haplotypes.     

Serum selenium and single-nucleotide polymorphisms in genes for selenoproteins: relationship to markers of oxidative stress in men from Auckland, New Zealand

There is controversy as to the recommended daily intake of selenium (Se), and whether current New Zealand diets are adequate in this nutrient. Various functional single-nucleotide polymorphisms (SNPs) polymorphisms may affect the efficacy of Se utilisation. These include the glutathione peroxidases GPx1 rs1050450, GPx4 rs713041, as well as selenoproteins SEPP1 rs3877899, SEL15 rs5845, SELS rs28665122 and SELS rs4965373. This cross-sectional study measured serum Se levels of 503 healthy Caucasian men in Auckland, New Zealand, between ages 20–81. The Se distribution was compared with activities of the antioxidant enzymes glutathione peroxidase and thioredoxin reductase, and DNA damage as measured by the single cell gel electrophoresis assay, both without and with a peroxide-induced oxidative challenge. Serum Se was measured using inductively coupled plasma-dynamic reaction cell-mass spectrometry, while selenoprotein SNPs were estimated using TaqMan^® SNP genotyping assays. While antioxidant enzyme activities and DNA damage recorded after a peroxide challenge increased with increasing serum selenium, the inherent DNA damage levels in leukocytes showed no statistically significant relationship with serum selenium. However, these relationships and dietary Se requirements at the individual level were modified by several different SNPs in genes for selenoproteins. The GPx1 rs1050450 C allele was significantly associated with GPx activity. Significant correlations between serum Se level and GPX activity were seen with all genotypes except for homozygous minor allele carriers, while the GPx1 rs1050450 CT genotype showed the highest correlation. Several genotypes showed significant correlations between serum Se and TR activity with SEPP1 rs3877899 GG genotype showing the highest correlation. A significant decreasing trend in DNA damage with increasing serum Se was seen among GPx1 rs1050450 CC and GPx4 rs713041 TT genotype carriers up to a serum Se level of 116 and 149 ng/ml, respectively. In the absence of this genetic information, we would recommend a serum Se concentration in the region of 100–150 ng/ml as providing a useful compromise.     

Genetic variations of TLRs and their association with HPV/EBV,co-infection along with nicotine exposure in the development of premalignant/malignant lesions of the oral cavity in Indian population

Background: Despite being most preventable malignancies associated with smoked and smokeless tobacco products, squamous cell carcinoma of oral cavity is one of the most common malignancy in India. The aim of the present study was to evaluate the role of TLRs in oral pre-cancerous, cancerous cases and their genotypic correlation with HPV/EBV, co-infection & lifestyle habits in Indian population.Methods: The present study was conducted on 300 subjects (100 OSCC, 50 pre-cancer & 150 controls). The amplification of TLRs gene and HPV/EBV co-infection was assessed by Nested PCR, PCR–RFLP and further confirmation by direct sequencing.Results: The TLR 9(−1486 T/C), revealed that the TT vs. CT + CC genotype had a ˜5-fold increased risk for the development of pre-cancerous lesions as compared to controls (p = 0.0001). Further analysis showed that the risk of cancer was extremely pronounced in HPV/EBV, co-infection (p = 0.0141), implicating the possible interaction between TLR 9(−1486T/C) genotype and HPV infection in increasing cancer/pre-cancer risk. The ‘G’ allele of TLR 4(+896A/G) was also a higher risk of developing pre-cancerous lesions with 4.5 fold and statistically significant (p = 0.0001). The genotypic association of TLR 9(-1486T/C) in OSMF cases showed ˜8 fold increased risk and TLR 4(+896A/G) showed fourteen fold higher risk for leukoplakia (p < 0.0001, OR = 14.000).Conclusion: Genetic polymorphism of TLR 9(−1486 T/C) and TLR 4(+896A/G) may influence the effects of HPV/EBV, co-infection and play the significant role in development of the disease. The significance of these TLRs seemed to be enhanced by tobacco chewing and smoking habits also, which act as an important etiological risk factor for OSCC.     

Genetic mapping of expressed sequences in onion and in silico comparisons with rice show scant colinearity

The Poales (which include the grasses) and Asparagales [which include onion (Allium cepa L.) and other Allium species] are the two most economically important monocot orders. Enormous genomic resources have been developed for the grasses; however, their applicability to other major monocot groups, such as the Asparagales, is unclear. Expressed sequence tags (ESTs) from onion that showed significant similarities (80% similarity over at least 70% of the sequence) to single positions in the rice genome were selected. One hundred new genetic markers developed from these ESTs were added to the intraspecific map derived from the BYG15-23×AC43 segregating family, producing 14 linkage groups encompassing 1,907 cM at LOD 4. Onion linkage groups were assigned to chromosomes using alien addition lines of Allium fistulosum L. carrying single onion chromosomes. Visual comparisons of genetic linkage in onion with physical linkage in rice revealed scant colinearity; however, short regions of colinearity could be identified. Our results demonstrate that the grasses may not be appropriate genomic models for other major monocot groups such as the Asparagales; this will make it necessary to develop genomic resources for these important plants. Electronic Supplementary Material Supplementary material is available for this article at     

Tumour necrosis factor-alpha receptor 1 polymorphisms and serum soluble TNFR1 in early spontaneous miscarriage

OBJECTIVES: The study investigated the association of TNFR1 gene polymorphism with early recurrent spontaneous miscarriage (ERSM) in Chinese women, and soluble TNFR1 (sTNFR1) expression in ERSM women. STUDY DESIGN: Two single nucleotide polymorphisms (SNPs) located at -383 (AGA to AGC) in the promoter region and +36 (CCA to CCG) in exon 1 of TNFR1 were investigated in 188 non-pregnant ERSM Chinese women. The serum sTNFR1 was measured by the ELISA method. RESULTS: Both SNPs were not associated with ERSM. The non-pregnant ERSM women had significantly higher levels of serum sTNFR1, compared with the non-pregnant, normal women (1.84+/-0.54 ng/ml versus 1.62+/-0.38 ng/ml; t=-2.053; p<0.05). CONCLUSIONS: The data do not provide evidence that TNFR1 gene polymorphism is etiologically important for ERSM in Chinese women. But, a significantly raised sTNFR1 level in non-pregnant ERSM women was recorded compared to women with normal pregnancies. The result suggests that pregnancy failure is associated with an increase of sTNFR1.     

Analysis of genotype polymorphism of tumor-related genes harbored in chromosome arm 1p and 8p in hepatocellular carcinoma patients by cSNP chip

The majority of single nucleotide polymorphisms (SNPs) found in the coding region (cSNPs) are single base substitutions that may or may not lead to amino acid substitutions, most of which are related to diseases. Some cSNPs may prove useful for their potential links to functional cSNPs via linkage disequilibrium mapping. We have selected 48 cSNPs located in the coding regions of 25 genes to construct the cSNP chip. These genes are harbored in the high frequency loss regions of the chromosome 1p and 8p and related with apoptosis, cell cycles, signal transduction, oncogene, tumor suppressor genes and so on. All of the cSNPs can lead to amino acid substitutions except TP73 (rs1801174). The PCR products amplified from 31 hepatocellular carcinoma (HCC) specimens were labeled with Dig-dUTP and then hybridized with the cSNP chips. The results showed that there was no hybridization signal when there was more than one site of mutation in the amplification sequence, indicating that the cSNP chip had a high sensitivity. The statistic data of the SNP (MT, homozygous and HT, heterozygous) in the HCC patients with different phenotypes (HBV+/−, differentiation stage, family history positive or negative, tumor size) indicated that the number of MT was distinctly different between patients with positive HBV and negative HBV. The MT and HT numbers of all the 48 cSNPs were significantly different between low differentiation and high differentiation HCC patients. The numbers of MT and HT were not different between positived and negative family history groups and between tumor size >3 cm and ⩽ 3 cm groups. The study results provided useful information for understanding the molecular mechanisms of HCC development.     

延边朝鲜族和汉族脂联素启动子SNPs与原发性高血压的相关性

为了探讨延边朝鲜族和汉族脂联素基因启动子单核苷酸多态性(SNPs)与原发性高血压(EH)的关系, 文章采用PCR产物直接测序方法检测了220例EH患者和268例对照个体的脂联素启动子5个SNPs位点: -11426A>G(rs16861194)、-11391G>A(rs17300539)、-11377C>G(rs62620185)、-11156insCA(rs60806105)、-11043C>T(rs76786086), 氧化酶法测定空腹血糖、甘油三酯、总胆固醇、低密度脂蛋白、高密度脂蛋白, 酶联免疫吸附法(ELISA)测定血浆脂联素和胰岛素。结果显示: (1) -11426A>G、-11377C>G 和-11156insCA 3个位点具有多态性, 且它们的基因型频率分布符合Hardy-Weinberg平衡定律(P>0.05), -11391G>A和-11043C>T位点无多态性; (2) -11426A>G和-11156insCA呈完全连锁不平衡(D’=1; r2=1); (3) -11426G基因频率比较, 朝鲜族(21.10%)高于汉族(12.05%), 汉族EH组高于对照组; -11377C>G的基因型和基因频率在朝鲜族和汉族间及同一民族内EH组和对照组间比较均无统计学意义(P>0.05); (4)单倍型?11426G -11377C的频率, 汉族EH组高于对照组(P<0.05), 朝鲜族EH组和对照组比较无统计学意义(P>0.05); (5)EH组的血浆脂联素水平明显低于对照组(P<0.001)。据此得出结论: (1)首次发现?11426A>G和?11156insCA呈完全连锁不平衡, -11426 A>G的多态性在朝鲜族和汉族中存在民族差异; (2) -11426 G和-11426G -11377C是延边汉族EH的危险因子和危险单倍型, 但不是朝鲜族的; (3)低血浆脂联素是延边朝鲜族和汉族EH的重要危险因素; (4)血浆脂联素水平与-11426A>G基因型无关。     

A polymorphism of matrix Gla protein gene is associated with kidney stone in the Chinese Han population

Purpose

Matrix Gla protein (MGP) is a molecular determinant regulating the extracellular matrix calcification. To further confirm whether the MGP genetic polymorphism was universally associated with the risk of kidney stone, we investigated the association of genetic polymorphisms of MGP with kidney stone in the Chinese Han population.

Materials and methods

728 subjects were recruited for the study. We firstly re-sequenced the human genomic MGP gene including the 1500 bp promoter, 5′-UTR, 4 exons and 3′-untranslated regions, identified single nucleotide polymorphisms (SNPs) in MGP, and performed an association analysis with kidney stones in 54 subjects of the Chinese Han population. A candidate tag SNP was genotyped in total subjects using an allele specific PCR, and further analyzed the association with kidney stone.

Results

We identified 18 polymorphisms including four tag SNPs. A tag SNPrs4236 was associated with kidney stones. The G allele carrier had a 1.373-fold reduced kidney stone risk compared with A allele carriers in SNPrs4236 (odds ratios (OR) = 1.373; 95%CI, 1.051–1.793; p = 0.019). However, we did not find an association between the polymorphism and clinical characteristics of kidney stones.

Conclusions

Our findings showed that SNPrs4236 of the MGP gene is associated with kidney stones in the Chinese Han population, and influences the genetic susceptibility to kidney stones. In the future, functional assays of the polymorphism should permit a better understanding of the role of MGP genetic variants and kidney stones.     

Interleukin-10 and interleukin-18 promoter polymorphisms in an Italian cohort of patients with undifferentiated carcinoma of nasopharyngeal type

Purpose: Cytokines such as IL-10 and IL-18 seem to be involved in the inflammatory response of undifferentiated carcinoma of nasopharyngeal type (UCNT). The aim of this study was to evaluate the correlation between functional single nucleotide polymorphisms (SNPs) in the promoter region of IL-10 and IL-18 genes and the virological and clinical characteristics in a large case series of Caucasian patients suffering from UCNT, a tumor regularly associated with the Epstein Barr Virus (EBV). Methods: Eighty-nine patients with histologically confirmed UCNT and 130 healthy donors were included in our study. DNA was examined for the polymorphisms of IL-10 gene at positions –1082, −819, −592 by direct sequencing and IL-18 gene at position −607 and −137 by allele –specific PCR. EBV DNA serum viremia was evaluated by QC-PCR. Results: The distributions of the IL-10 and IL-18 genetic variants were not different between UCNT patients and healthy controls. The frequency of IL-10 –1082G allele, which is associated with high IL-10 expression, showed a nearly statistically significant increase in UCNT patients EBV DNA-negative as compared to healthy controls (OR=3.3 95% CI: 1.2–9.8). Subjects with C/C or C/G combined IL-18 genotypes showed an increased risk of being with Stages III-IV (OR=2.1 95% CI: 1.2–6.6). Conclusion: This study was performed to improve the definition of the pathogenetic factors implicated in UCNT by addressing the correlation between cytokine polymorphisms and clinical parameters. This is the first study investigating the possible role of the IL-18 and IL-10 polymorphisms in the development and outcome of UCNT. In our genetic analysis there is no evidence for involvement of IL-10 promoter polymorphisms alone in the genetic predisposition to this tumor. On the other hand, IL18 genetic variants may represent a genetic risk factor for tumor aggressiveness.     

FOXO3 gene polymorphisms influence the risk of acute lymphoblastic leukemia in Chinese children

Acute lymphoblastic leukemia (ALL) is the most frequently diagnosed cancer in children and single-nucleotide polymorphisms (SNPs) in certain genes influence risk of ALL. Although FOXO3 had been demonstrated to be involved leukemia, the role of FOXO3 polymorphisms was still not clear. In the present study, we explored the association of FOXO3 SNPs with ALL risk in Chinese children. We genotyped four polymorphisms (rs17069665 A>G, rs4945816 T>C, rs4946936 C>T, and rs9400241 A>C) of FOXO3 in 425 ALL cases and 1339 health controls. The associations were estimated by odds ratios (ORs) with their 95% confidence intervals (CIs). Further analyses were performed to explore associations of rs17069665 and rs9400241 with ALL susceptibility in terms of age, gender, immunophenotype, minimal residual disease (MRD), and other clinical characteristics. We found rs17069665 related to the increased ALL risk (OR = 1.76; 95% CI = 1.02-3.04), rs9400241 related to decreased ALL risk (OR = 0.80; 95% CI = 0.64-0.99). The effects of rs17069665 on ALL risk were more predominant in males and children < 10 years, and patients with lower rates of platelet or neutrophil. As for rs9400241, the effects were more predominant in children < 10 years, and in patients with pre B ALL, positive MRD, anemia, or hepatomegaly. In conclusion, FOXO3 gene polymorphisms influence the risk of ALL in children and might be a potential biomarker for ALL susceptibility.     

Analysis of genotype polymorphism of tumor-related genes harbored in chromosome arm lp and 8p in hepatocellular carcinoma patients by cSNP chip

The majority of single nucleotide polymorphisms (SNPs) found in the coding region (cSNPs) are single base substitutions that may or may not lead to amino acid substitutions,most of which are related to diseases.Some cSNPs may prove useful for their potential links to functional cSNPs via linkage disequilibrium mapping.We have selected 48 cSNPs located in the coding regions of 25 genes to construct the cSNP chip.These genes are harbored in the high frequency loss regions of the chromosome 1p and 8p and related with apoptosis,cell cycles,signal transduction,oncogene,tumor suppressor genes and so on.All of the cSNPs can lead to amino acid substitutions except TP73 (rs1801174).The PCR products amplified from 31 hepatocellular carcinoma (HCC) specimens were labeled with Dig-dUTP and then hybridized with the cSNP chips.The results showed that there was no hybridization signal when there was more than one site of mutation in the amplification sequence,indicating that the cSNP chip had a high sensitivity.The statistic data of the SNP (MT,homozygous and HT,heterozygous) in the HCC patients with different phenotypes (HBV +/-,differentiation stage,family history positive or negative,tumor size) indicated that the number of MT was distinctly different between patients with positive HBV and negative HBV.The MT and HT numbers of all the 48 cSNPs were significantly different between low differentiation and high differentiation HCC patients.The numbers of MT and HT were not different between positived and negative family history groups and between tumor size＞3 cm and≤3 cm groups.The study results provided useful information for understanding the molecular mechanisms of HCC development.     

Allelic variants of ABC drug transporter Cdr1p in clinical isolates of Candida albicans

Candida drug resistance protein (Cdr1p) is a major drug efflux protein, which plays a key role in commonly encountered clinical azole resistance in Candida albicans. We have analyzed its sequence in several azole resistant clinical isolates to evaluate the allelic variation within CDR1 gene and to relate it to its functional activity. The sequence analysis revealed 53 single nucleotide polymorphisms (SNPs), out of which six were non-synonymous single nucleotide polymorphisms (NS-SNPs) implying a change in amino acid and were found in two or more than two allelic combinations in different sensitive or resistant isolates. We have identified three new NS-SNPs namely, E948P, T950S, and F1399Y, in isolates wherein F1399Y appeared to be unique and was present in one of the naturally occurring azole resistant isolates obtained from Indian diabetic patients. However, site-directed mutagenesis showed that the residue F1399 in between TMS 11 and TMS 12 does not affect the functionality of Cdr1p. Taken together, our SNPs analyses reveal that unlike human P-gp, the naturally acquired allelic variations are mostly present in non-conserved regions of the protein which do not allow Cdr1p to genetically evolve in a manner, that would allow a change in its functionality to affect substrate recognition, specificity, and drug efflux activity of C. albicans cells.     

INDEL variation in the regulatory region of the major flowering time gene LanFTc1 is associated with vernalization response and flowering time in narrow‐leafed lupin (Lupinus angustifolius L.)

Narrow‐leafed lupin (Lupinus angustifolius L.) cultivation was transformed by 2 dominant vernalization‐insensitive, early flowering time loci known as Ku and Julius (Jul), which allowed expansion into shorter season environments. However, reliance on these loci has limited genetic and phenotypic diversity for environmental adaptation in cultivated lupin. We recently predicted that a 1,423‐bp deletion in the cis‐regulatory region of LanFTc1, a FLOWERING LOCUS T (FT) homologue, derepressed expression of LanFTc1 and was the underlying cause of the Ku phenotype. Here, we surveyed diverse germplasm for LanFTc1 cis‐regulatory variation and identified 2 further deletions of 1,208 and 5,162 bp in the 5' regulatory region, which overlap the 1,423‐bp deletion. Additionally, we confirmed that no other polymorphisms were perfectly associated with vernalization responsiveness. Phenotyping and gene expression analyses revealed that Jul accessions possessed the 5,162‐bp deletion and that the Jul and Ku deletions were equally capable of removing vernalization requirement and up‐regulating gene expression. The 1,208‐bp deletion was associated with intermediate phenology, vernalization responsiveness, and gene expression and therefore may be useful for expanding agronomic adaptation of lupin. This insertion/deletion series may also help resolve how the vernalization response is mediated at the molecular level in legumes.     

Linkage disequilibrium and founder effect analysis of the NF1 gene in French Canadians from the Quebec population

We genotyped 19 neurofibromatosis type 1 (NF1) families from French Canadians of the Quebec population with four intragenic microsatellites (IVS26-2.3, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0). Linkage analysis of the four microsatellite markers among the 19 NF1 families indicates that the four microsatellites are strongly linked with NF1 disease (LOD = 2.76-3.64). The four markers are associated (P = 0-0.077) except marker pair IVS26-2.3/IVS27AC33.1 (P = 0.18 or 0.17). However, perhaps due to the high mutation rate of the NF1 gene, no founder effect for NF1 was detected in the Quebec French Canadians.     

Cleaved amplified polymorphic sequence markers in sugi, Cryptomeria japonica D. Don, and their locations on a linkage map

Sugi, Cryptomeria japonica D. Don, is one of the most important forestry species in Japan. We here report the development of 217 CAPS markers derived from sugi cDNA libraries. More than half of a set of STS markers produced could be converted into CAPS markers using restriction endonuclease analysis. Of the 217 markers, 71 showed different patterns of polymorphism when they were digested with a range of endonucleases and, in total, 347 polymorphisms were found in the various combinations of STSs and endonucleases. When the polymorphisms gave co-dominant patterns in a screening program, the polymorphic information content (PIC) used to evaluate the value of the polymorphisms was relatively high (0.33, on average) compared to the information yielded by commonly used markers, like isozymes. The results of a segregation analysis suggest that approximately 80% of the CAPS markers developed here will show co-dominant inheritance. From logistic regression analysis, the polymorphisms were found to be associated more strongly with intron than with exon regions. Sixty two markers were subsequently mapped on the previously reported linkage map, 15 of which showed abnormal segregation, presumably caused by linkage with lethal factors. Received: 7 December 2000 / Accepted: 23 January 2001     

Stability of the T-DNA flanking regions in transgenic Arabidopsis thaliana plants under influence of abiotic stress and cultivation practices

Genetic transformation is often associated with different rearrangements of the plant genome at the site of insertion. Therefore the question remains weather these T-DNA insertion sites are more prone to genotoxic stresses. Here, we studied the impact of propagation through generations, the influence of gene stacking and of photo oxidative stress caused by high light intensity on the stability of the transgene flanking regions in the model plant Arabidopsis thaliana. Conformational Sensitive Capillary Electrophoresis (CSCE), RFLP and sequencing were deployed in this analysis in order to study the proximal 100 bp and the long-range T-DNA flanking sequences. By screening seven transgenic lines no evidence for occurrence of mutation events were found, implying that the nucleotide sequence of the T-DNA flanking regions of the studied events is unlikely to be unstable. N. Papazova and R. Ghedira have equally contributed to the paper.