[Association study of telomere length with idiopathic male infertility].

Telomeres are evolutionary conserved, multifunctional DNA-protein complexes located at the ends of eukaryotic chromosomes. Telomeres maintain chromosome stability and genome integrity and also play an important role in meiosis which aid in synapsis, homologous recombination, and segregation. Sperm telomere has been reported to play an important role in fertilization and embryo development. Nowadays, the association between telomere and reproduction is one of the major areas of interest, however whether sperm telomere associated with male infertility is not clear. In this study, in order to find out the association between Chinese idiopathic infertility and sperm telomere length, we analyzed the difference of sperm telomere length between idiopathic infertile men and normal fertile men, as well as the correlations between sperm telomere length and human semen characteristics. We analyzed 126 Chinese idiopathic infertile men and 138 normal fertile men for sperm telomere length by using quantitative PCR. We found that the relative sperm mean telomere length of infertile men was significantly shorter than that of fertile men (2.894 ± 0.115 vs. 4.016 ± 0.603, P=5.097 x 10⁻5). Both sperm count and semen progressive motility are related with telomere length. Our results suggest that sperm telomere length is associated with idiopathic male infertility of China and we proposed the possibility that shorter telomeres in sperm chromosome will reduce spermatogenesis and sperm functions, which finally affected the fertility of male.

[Association of polymorphisms of PTPN22 and PADI4 genes with rheumatoid arthritis in Yunnan].

OBJECTIVE: To assess the association between genetic polymorphisms of 7 SNPs in PTPN22 and PADI4 genes and susceptibility to rheumatoid arthritis in Yunnan. METHODS: A case-control study was carried out on 192 patients of rheumatoid arthritis and 288 healthy controls. Genotypes of rs33996649 and 1858 loci within PTPN22 gene, and rs11203366 and rs874881 loci within PADI4 gene were determined with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Genotypes of rs1635579, rs2428736 and rs2240340 in PADI4 gene were determined with pyrosequencing. RESULTS: The frequencies of alleles and genotypes of rs2240340 locus in PADI4 gene showed a significant difference between rheumatoid arthritis and controls in Yunnan population (P U+003C 0.05). CONCLUSION: Our results suggested that rs2240340 in PADI4 gene is associated with susceptibility to rheumatoid arthritis in Yunnan.

[Association between polymorphisms of PSMB8, PSMB9 and TAP2 genes with rheumatoid arthritis in ethnic Han Chinese from Yunnan].

OBJECTIVE: To assess the association between single nucleotide polymorphisms (SNPs) of PSMB8, PSMB9 and TAP2 genes and rheumatoid arthritis (RA) in ethnic Han Chinese from Yunnan. METHODS: A case-control study was carried out using 177 RA patients and 288 healthy controls. Genotypes of rs2071543, rs55745125 and rs138635403 loci of PSMB8 gene, and rs17587 locus of PSMB9 gene were determined with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). And a polymerase chain reaction amplification refractory mutation system (ARMS-PCR) was used for typing rs2228396 locus of TAP2 gene. Genotypic and allelic frequencies were calculated. An Epi Info 7 software was used to calculate the Odds Ratio (OR) of above SNPs between the two groups. RESULTS: Allelic and genotypic frequencies of rs138635403 and rs17587 loci have differed significantly between the two groups (P<0.05). The frequency of GG genotype for rs17587 locus was also higher in the RA group (0.672) compared with control group (0.524) (OR=1.862, 95%CI: 1.261-2.749). CONCLUSION: Genetic polymorphisms of rs17587 appeared to be associated with RA in ethnic Han Chinese from Yunnan.

[Tumor genetic heterogeneity].

Although the majority of spontaneous tumors derive from a single cell, people have come to realize intra-tumor heterogeneity of individual tumors. Human cancers frequently display substantial difference in phenotypic features, such as the degree of differentiation, cell proliferation rate, invasion and metastatic potential, response to therapy and many other aspects. Molecular biology studies have confirmed the occurrence of new mutations during the process of tumor progression, which provide more powerful evidences to show the existence of intra-tumor genetic heterogeneity. This re-view will focus on recent major advances in the study of tumor genetic heterogeneity. Considering that genetic heterogene-ity analysis can provide important information to indicate how long normal cells transform into tumor cells and how to spread and migrate, we firstly describe experimental evidences of intra-tumor genetic heterogeneity. Then we discuss the research value of genetic diversity in the evolutionary history of human individual tumor, introduce the two modes of the genetic heterogeneity - cancer stem cell model and the clonal evolution model, and summarize the implications of in-tra-tumor heterogeneity studies in metastasis and therapy. In addition, the article presents the research methods of genetic heterogeneity, including specific gene and genome-wide level, pointing out their strengths and limitations.

[Distribution of HLA-G 14 bp insertion/deletion polymorphism and their associations with HLA-A alleles in four Chinese ethnic groups].

Recently, a 14 bp insertion/deletion polymorphism (+14 bp/-14 bp) in exon 8 of the human leucocyte antigen-G (HLA-G) gene has been widely recognized to associate with recurrent miscarriage, autoimmune diseases, hepatocellular carcinoma and other diseases. Our previous studies have shown the distribution characteristics of linguistic family for HLA-G 14 bp insertion/deletion in different ethnic groups. In the present study, we investigated the distribution of HLA-G 14 bp insertion/deletion polymorphism and their subsequent associations with HLA-A alleles in Tu, Yugu, Lisu and Nu ethnic populations based upon the HLA-A genotyping data. Our results showed that the frequencies of the 14 bp insertion/deletion polymorphism were diverse in these four populations while that in the same linguistic subfamily was similar. The significant difference in different linguistic subfamily except for Han and Mongolian language subfamily was identified. In addition, the 14 bp insertion was found to associate with HLA-A alleles in different ethnic populations.

[Association between six single nucleotide polymorphisms of EGLN1 gene and adaptation to high-altitude hypoxia].

To investigate the association between SNPs located in 5'UTR and intron of prolyl hydroxylase 2 (EGLN1 or PHD2) and adaptation to high-altitude hypoxia, the SNPs (rs2066140, rs2808584, rs2491405, rs2486741, rs2486734 and rs21533646) of EGLN1 gene were genotyped using Sequenom MassArray genotyping system in 152 unrelated healthy Tibetan individuals (3 650 m altitude) and 192 Han (5 00 m altitude), and the haplotypes of these SNPs were constructed and analyzed. Our results showed all the homozygous genotypes of six SNPs loci were significantly different between the two groups (P<0.05). The frequencies of haplotypes G-G (rs2066140 and rs2808584) and G-C (rs2486741 and rs2486734) of high-altitude group were significantly different from low-altitude group (P<0.05). In addition, the frequencies of haplotypes C-A (rs2066140 and rs2808584) and C-T (rs2486741 and rs2486734) of high-altitude group were significantly lower than those in low-altitude group (P<0.05). Our results indicate that the polymorphism of homozygous genotype in six SNPs and their haplotypes were associated with adaptation to high-altitude hypoxia.

[Association between gene polymorphisms and myocardial infarction in Han Chinese of Yunnan province].

OBJECTIVE: To determine frequencies of genetic polymorphisms of coagulation factor VII (FVII), coagulation factor FXII (FXII), fibrinogen (FBG) and 9p21 in ethnic Han Chinese from Yunnan province, and to assess the association between such polymorphisms and onset of myocardial infarction (MI). METHODS: One hundred and forty-two patients with MI and 192 healthy controls were analyzed. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and pyrosequencing were used to determine the genotypes of FVII, FXII, FBG and 9p21. RESULTS: No significant difference was found in the frequencies of R353Q, 5'F7, C46T, -148C/T, rs1333049 and rs4977574 loci between the two groups (P> 0.05). However, the frequencies of AA of -455G/A, T and TT of rs1333040, T and TT of rs10116277 and G and GG of rs2383207 were significantly higher in MI group compared with the controls (P< 0.05), whilst the frequencies of CT of rs1333040 and GT of rs10116277 were significantly lower in MI group compared with the controls (P<0.05). CONCLUSION: Polymorphisms of FVII, FXII, -148C/T of FBG and rs1333049 of 9p21 were not associated with myocardial infarction. Polymorphisms of -455G/A of FBG and rs1333040, rs10116277 and rs2383207 of 9p21 may be associated with MI in ethnic Han Chinese from Yunnan province.

[Association between Alu insertion polymorphisms and HLA class I alleles in Chinese Lisu and Nu ethnic populations].

OBJECTIVE: To investigate the frequencies of HLA-Alu repeat polymorphisms (AluMICB, AluTF, AluHJ, AluHG and AluHF) in Chinese Lisu and Nu ethnic populations. METHODS: The frequencies of HLA-Alu repeat polymorphisms in above populations were determined with polymerase chain reaction (PCR). The associations between HLA-Alu repeat polymorphisms and HLA-A, HLA-B and HLA-C alleles were also analyzed. Phylogenetic trees were constructed with genetic distance calculated from the frequencies of HLA-Alu repeat polymorphisms. RESULTS: Frequencies of AluTF*2 and AluHF*2 were different between the two populations (P< 0.05), while those of other three insertions were similar. The strength of association between HLA-Alus and HLA alleles were different (P< 0.05) in the two populations. Although AluMICB*2 were associated with HLA-B*56:01 in both populations, the association was stronger in Lisu population (74.0%) but moderate in Nu population (30.7%). HLA-Alus were associated with particular HLA subtypes, e.g., AluHG*2 with certain HLA-A*02 subtypes. By phylogenetic analysis, Lisu and Nu were clustered together with southern Chinese and Thai populations. CONCLUSION: The distribution of HLA-Alus and the strength of associations between HLA-Alus and HLA class I alleles have varied between the two populations. Study of this association may facilitate identification of origins, evolution, progenitor haplotypes and recombination within the HLA class I region.

[Detection and preliminary study of a family carrying a CCR5Δ32 deletional mutation].

OBJECTIVE: To investigate the frequencies of chemokine (C-C motif) receptor 5 gene (CCR5)Δ32 deletional mutation of in Han and Dai populations from Yunnan province. Immortalized cell lines were derived from a family carrying the CCR5Δ32 mutation. METHODS: Blood samples of 346 Han and 355 Dai individuals were collected for genotyping. The coding regions of CCR5 gene were amplified with PCR followed by agarose gel electrophoresis. Suspected mutations were verified with DNA sequencing. Immortalized cell lines were constructed by using Epstain Barr virus and cyclosporine A. The difference between the cell lines and original blood samples was verified with PCR. RESULTS: One ethnic Han individual was confirmed to be heterozygous for a deletional mutation by sequencing, which has led to discovery of a family with CCR5Δ32. Nine immortalized cell lines were established from this family, and no difference between the cell lines and original blood samples was detected by PCR. CONCLUSION: Together with previous reports, this study has indicated a significant difference in CCR5Δ32 among different ethnic groups in China. Established immortalized cell lines can also provide material for future research.

[The research progress of human genetic diversity in China].

Human genetic diversity refers to genomic variation among races, ethnic groups, isolated populations and individuals worldwide, and is one major resource and tool on discovering human evolution and migration, interaction between genetic background and environment, and factors associated with human diseases and health. China has abundant and valuable resource of human genetic diversity due to 56 ethnic groups and a large population accounting for one fifth of the total population in the world. After decades of efforts, a large number of research data on human genetic diversity have been accumulated in China, and some of outcomes reach advanced international level. This review mainly focuses on the recent progress and outcomes achieved in applying genetic markers including morphological markers, biochemical and immunological markers and DNA markers in research of genetic diversity, and the application of mitochondrial DNA, Y chromosomal DNA, HLA and others in research of the origin and relationship of Chinese ethic groups, and the origin and mi-of modern East Asian populations. This review also summarizes the advances in the research fields of preservation and utilization of Chinese genetic resource, identification of genes associated with disease selective and adaptive for natural pressure, application of whole genome association study and next generation sequencing, and Chinese human genome as well.

[Association between single nucleotide polymorphisms of 5'-untranslated region of GPx4 gene and male infertility].

OBJECTIVE: To study the association between the single nucleotide polymorphisms (SNPs) of the 5'-untranslated region (5'-UTR) of phospholipid hydroperoxide glutathione peroxidase (GPx4 or PHGPx) gene and oligo- or asthenozoospermic male infertility. METHODS: The 5'-UTR region of the GPx4 gene was amplified from infertile men and controls using the polymerase chain reaction and was analyzed for polymorphisms by direct sequencing. RESULTS: A total of 9 SNPs were present in the cohort, however there were no significant differences in these 9 SNPs between the case and control groups. According to the results of linkage disequilibrium analysis and haplotype construction, one haplotype (rs757229-rs757230-rs4588110-rs3746165-rs3746166: C-G-G-T-A) was present only in the control men, and significant difference was detected(P< 0.01). CONCLUSION: The SNPs of 5'-UTR region of the GPx4 gene might not be associated with oligo- or asthenozoospermic male infertility. However, the haplotype (rs757229-rs757230-rs4588110- rs3746165-rs3746166: C-G-G-T-A) might be a protective haplotype.

[Distribution of HLA-C genes and HLA C-B, A-C-B haplotypes in Jinuo, Maonan and Wa ethnic populations in southwest China].

OBJECTIVE: To investigate the distribution of human leukocyte antigen(HLA) class I genes and haplotypes in Jinuo, Maonan and Wa ethnic populations in southwest China. METHODS: Polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) typing by Luminex was performed to genotype the HLA-C alleles in unrelated healthy individuals in the three populations. HLA C-B, A-C-B haplotypes were computed by combining the previous HLA-A and -B genotyping data using Pypop7.0 software. RESULTS: Eighteen HLA-C genes were identified in the three populations, with 17, 13 and 15 HLA-C genes in Jinuo, Maonan and Wa populations respectively. The alleles with frequency of more than 10% from high to low were C*08:01, C*01:02, C*03:04 and C*07:02 in the Jinuo, C*03:04, C*01:02, C*07:02 and C*08:01 in the Maonan, and C*12:03, C*08:01, C*07:02 and C*04:01 in the Wa. The predominant HLA A-C-B haplotypes were A*02:07-C*01:02-B*46:01, A*11:01-C*08:01-B*15:02 and A*11:01-C*03:04-B*13:01 in the Jinuo, A*11:01-C*03:04-B*13:01, A*02:07-C*01:02-B*46:01, A*11:01-C*08:01-B*15:02 and A*02:03-C*07:02-B*38:02 in the Maonan, and A*11:01-C*08:01-B*15:02, A*11:01-C*12:03-B*15:32 and A*11:01-C*04:01-B*35:01 in the Wa, respectively. CONCLUSION: There were different characteristics in the distributions of HLA-C genes and HLA C-B, A-C-B haplotypes in the Jinuo, Maonan and Wa populations. However, haplotypes C*08:01-B*15:02 and A*11:01-C*08:01-B*15:02 with high frequencies were common in the three populations, which might be the common ancient haplotypes of southern Chinese population. The study of HLA genes and haplotypes in these populations may be of significance in the study of population genetics, transplantation and disease association.

[Association between diversity of hypoxia at different altitude and the polymorphism of EPAS1 gene].

OBJECTIVE: To study the selection effect of endothelial PAS domain protein 1 (EPAS1) gene induced by high altitude hypoxia environment. METHODS: Fourteen single nucleotide polymorphism sites (SNPs) of the EPAS1 gene were genotyped using PCR-restriction fragment length polymorphism (PCR-RFLP) in three Tibetan groups (58 samples from Tibetan living in an altitude of about 3700 meters above sea level, 47 from Qinghai province, about 3100 meters above sea level, 43 from Yunnan province, about 2500 meters above sea level), and Han of Shandong (47 samples, about 50 meters above sea level). RESULTS: There were significant differences of most SNP allelic, genotypic and haplotypic frequencies when comparing Han of Shandong, Tibetan of Yunnan with Tibetan of Tibetan and Qinghai. But no difference between Han of Shandong and Tibetan of Yunnan was found. CONCLUSION: The EPAS1 gene might be under hypoxic selection induced by high altitude.

[Variation of CAG repeats in coding region of ATXN2 gene in different ethnic groups].

Toinvestigate CAG repeats variation of ATXN2 gene coding region in six ethnic groups that live in comparatively different environments, to evaluate whether these variations are under positive selection, and to find factors driving selection effects, 291 unrelated healthy individuals were collected from six ethnic groups and their STR geneotyping was performed. The frequencies of alleles and genotypes were counted and thereby Slatkin's linearized Fst values were calculated. The UPGMA tree against this gene was constructed. The MDS analysis among these groups was carried out as well. The results from the linearized Fst values indicated that there were significant evolutionary differences of the STR in ATXN2 gene between Hui and Yi groups, but not among the other 4 groups. Further analysis was performed by combining our data with published data obtained from other groups. These results indicated that there were significant differences between Japanese and other groups including Hui, Hani, Yunnan Mongolian, and Inner Mongolian. Both Hui and Mongolian from Inner Mongolia were significantly different from Han. In conclusion, the six ethnic groups had their own distribution characterizations of allelic frequencies of ATXN2 STR, and the potential cause of frequency changes in rare alleles could be the consequence of positive selection.

[Polymorphic Alu insertions and their associations with HLA I alleles in Yugu and Zhuang ethnic populations].

Many studies have show that the structurally polymorphic Alu insertion within HLA class I region are useful tools for investigating the origin, evolution and recombination of HLA class I progenitor haplotypes and gene diversity in different ethnic populations. In the present study, we determined the frequencies of HLA-Alus (i.e., AluMICB, AluTF, AluHJ, AluHG, and AluHF) in Zhuang and Yugu ethnic populations at first. Then, combined with HLA genotyping data, we studied associations between HLA-Alus and HLA-A alleles in Zhuang, Yugu, Bulang, Dai, and Hani ethnic populations. Our results showed that (1) the frequencies of five HLA-Alus were 1.5%~35.8% and 9.2%~34.8% in Zhuang and Yugu, respectively; and (2) the results of association between HLA-A alleles and HLA-Alu showed strong association between AluHG insertion and HLA-A 02 subtypes in all populations, association between AluHJ insertion and HLA-A 2402 in all populations, and association between AluHJ insertion and HLA-A 1101, -A 2407 in Bulang. The present study suggested that the distribution of HLA-Alus as well as the associations between HLA-Alus and HLA class I alleles are variable in different ethnic populations. HLA Alus alone or together with the HLA class I alleles are informative genetic markers for the identification of HLA class I allele and variation of haplotype lineages in different populations.

[Study of nine single nucleotide polymorphism loci of human HIF1A gene in three Tibetan groups].

OBJECTIVE: To investigate the effect of hypoxia environment induced by altitude on hypoxia inducible factor 1α (HIF1A) gene. METHODS: Nine single nucleotide polymorphism (SNP) loci of the HIF1A gene from three Tibetan groups (Tibet, Qinghai Province and Yunnan Province) were genotyped using PCR-restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: For non-synonymous mutation SNP site, there was no significant difference among the three Tibetan groups, except for SNP rs11549465 between Tibet Tibetan and Yunnan Tibetan, as well as between Qinghai Tibetan and Yunnan Tibetan. Frequencies of genotypes and alleles in rs4899056, rs1957757, rs10873142 and rs3783752 had significant differences between Tibet Tibetan and Yunnan Tibetan, and between Qinghai Tibetan and Yunnan Tibetan (all P<0.05). We also observed that the difference was negatively correlated with the altitude. CONCLUSION: The results suggested that the HIF1A gene might be under hypoxic selection induced by high altitude in the three groups.

[Analysis of the 14 bp insertion/deletion polymorphism in human leukocyte antigen-G gene in Chinese Dai and Han ethnic groups in Yunnan Province].

A 14 bp insertion/deletion polymorphism (+14 bp/-14 bp) in exon 8 of HLA-G gene has been investigated extensively because this polymorphism was associated with HLA-G mRNA stability and could influence HLA-G mRNA expression. In the present study, we genotyped 14 bp insertion/deletion polymorphisms in the HLA-G gene in Dai and Han populations in Yunnan Province. The frequencies of +14 bp in Dai and Han were 31.97% and 40.87%, respectively. The frequency of +14 bp/+14 bp in Dai was lower (8.20%) than that of Han (17.31%). Nevertheless, the frequency of +14 bp/-14 bp was similar in both populations (47.54% in Dai and 47.11% in Han). When compared with other populations, the frequency of 14 bp insertion/deletion of Han was similar to others, whereas the Dai showed significant difference from others and presented it's specific characteristic. We assumed that the distribution characteristic of 14 bp insertion/deletion in Dai might be the results of random genetic drift, but the role of natural selection cannot be excluded.

Amplification of PRKCI, located in 3q26, is associated with lymph node metastasis in esophageal squamous cell carcinoma.

DNA amplification is one of the mechanisms to activate genes that are implicated in neoplastic transformation and gain of chromosome band 3q26 is a common event in squamous cell carcinomas. The aim of the present work was to identify the specific target gene from four candidates (MDS1, PRKCI, ECT2, and PIK3CA) located on 3q26 amplification in esophageal squamous cell carcinomas (ESCCs). To assess the prevalence of copy number gains of putative genes, fluorescence in situ hybridization (FISH) was applied on 108 ESCCs and 9 ESCC cell lines. Our data showed that MDS1 and PRKCI were more frequently gained. Positive correlation was found only for PRKCI between amplification and tumor size (P = 0.043), lymph node metastasis (P = 0.015) and clinical stage (P = 0.002). PRKCI gene amplification was highly correlated with protein overexpression (P = 0.009), suggesting that gene amplification is one important mechanism involved in PRKCI overexpression. To investigate further the role of PRKCI alteration in esophageal tumors, a tissue microarray containing samples from 180 ESCCs was used for immunohistochemistry analysis. Statistical analysis revealed that PRKCI overexpression was correlated with lymph node metastasis (P = 0.002) and higher stage (P = 0.004). Performing multivariate logistic regression analysis, a significant association between PRKCI overexpression and presence of lymph node metastasis was found, which was independent of T-stage of the primary tumors (P = 0.004). Our results indicate that PRKCI is an attractive target in the 3q26 amplicon and that it may serve as a molecular marker for metastasis and occult advanced tumor stages in ESCC.

[Establishment of immortalized cell lines and genetic stability evaluation for a family with spinocerebellar ataxia type 2].

OBJECTIVE: Immortalized cell lines of spinocerebellar ataxia type 2 (SCA2) with Parkinson disease symptoms were established in order to provide experimental material for future study. METHODS: The immortalized cell lines were constructed by using Epstein Barr virus and cyclosporine A. Microsatellite markers were detected to see whether there is any change between the cell lines and the original blood samples, and the genetic stability of the cell lines were evaluated. RESULTS: Twenty-five immortalized cell lines were established successfully from the family and the microsatellite markers were unchanged. CONCLUSION: The karyotypes of the immortal cell lines were normal and the cell lines were genetically stable.

[Comparison on mitochondrial ATP6, ATP8 and Cyt b genes between Chinese Tibetans in three different zones: detecting the signature of natural selection on mitochondrial genome].

Mitochondrial DNA (mtDNA) differs from nuclear genome in many aspects such as lack of recombination, thus the investigation of mtDNA plays an essential role in human evolutionary history. We compared different sequences (approximately 2 kb) of ATP6, ATP8 and Cyt b genes in mtDNA among Tibetans in three different zones and found that the whole mtDNA sequences of the three genes, ATP6 and ATP 8 genes deviate gradually from neutral model with the increase of altitudes, yet no differences were observed. Also we found that the effect of purifying selection on Cyt b gene was elevated with the decrease of altitudes. Meanwhile, there was a possibility for the adaptive selection in ATP6 gene, which had an enhanced trend with the increase of altitudes. Thus, the geographic environment is the main determinant for selection, namely, different geographic environment has direct effect on selection.