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Male infertility is a multifactorial syndrome encompassing a wide variety of disorders. In recent years, several genome-wide single-nucleotide polymorphism (SNP) association studies (GWAS) have been performed on azoospermia and/or oligozoospermia in different populations including two GWAS on nonobstructive azoospermia in China; however, the association of SNPs with idiopathic male infertility, especially asthenozoospermia and oligozoospermia, and their correlation with semen parameters are still not clear. To investigate genetic variants associated with idiopathic male infertility (asthenozoospermia, oligozoospermia, and oligoasthenozoospermia) in Chinese Han people, 20 candidate SNPs were selected from GWAS results and genotyped using the Sequenom MassARRAY assay. A total of 136 subfertile men and 456 healthy fertile men were recruited. rs6476866 in SLC1A1 (P = 1.919E-4, OR = 0.5905, 95% CI: 0.447–0.78) and rs10129954 in DPF3 (P = 0.0023, OR = 2.199, 95% CI: 1.311–3.689) were strongly associated with idiopathic male infertility. In addition, positive associations were observed between asthenozoospermia and rs215702 in LSM5 (P = 0.0016, OR = 1.479, 95% CI: 1.075–2.033) and between oligoasthenozoospermia and rs2477686 in PEX10 (P = 0.0011, OR = 2.935, 95% CI: 1.492–5.775). In addition, six SNPs (rs215702 in LSM5, rs6476866 in SLC1A1, rs10129954 in DPF3, rs1801133 in MTHFR, rs2477686 in PEX10, and rs10841496 in PED3A) were significantly correlated with semen quality alterations. Our results suggest that idiopathic male infertility in different ethnic groups may share the same mechanism or pathway. Cohort expansion and further mechanistic studies on the role of genetic factors that influence spermatogenesis and sperm progressive motility are suggested.

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.

To assess the association between genetic polymorphisms of 7 SNPs in PTPN22 and PADI4 genes and susceptibility to rheumatoid arthritis in Yunnan.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.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).Our results suggested that rs2240340 in PADI4 gene is associated with susceptibility to rheumatoid arthritis in Yunnan.

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.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.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.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.

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).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.No significant difference was found in the frequencies of R353Q, 5'F7, C46T, -148C/T, rs1333049 and rs4977574 loci between the two groups (P0.05). 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 (P0.05), whilst the frequencies of CT of rs1333040 and GT of rs10116277 were significantly lower in MI group compared with the controls (P0.05).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.

To investigate the frequencies of HLA-Alu repeat polymorphisms (AluMICB, AluTF, AluHJ, AluHG and AluHF) in Chinese Lisu and Nu ethnic populations.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.Frequencies of AluTF*2 and AluHF*2 were different between the two populations (P0.05), while those of other three insertions were similar. The strength of association between HLA-Alus and HLA alleles were different (P0.05) 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.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.

To investigate the distribution of human leukocyte antigen(HLA) class I genes and haplotypes in Jinuo, Maonan and Wa ethnic populations in southwest China.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.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.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.

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.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.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(P0.01).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.

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.

Immortalized cell lines of spinocerebellar ataxia type 2 (SCA2) with Parkinson disease symptoms were established in order to provide experimental material for future study.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.Twenty-five immortalized cell lines were established successfully from the family and the microsatellite markers were unchanged.The karyotypes of the immortal cell lines were normal and the cell lines were genetically stable.

The pathogenesis of schistosomiasis is mainly caused by egg-induced granuloma formation and subsequent fibrosis. If Schistosoma japonicum infections could be detected in the early stage, especially before the egg deposition in the host tissues, the development of severe pathologic lesions might be prevented efficiently. The present study identified and characterized S. japonicum Sjp40, a potential antigen candidate for the early diagnosis of schistosomiasis. From the S. japonicum cercariae cDNA library, a clone encoding Sjp40 was identified by screening with the pooled rabbit sera collected on day 21 postinfection. Then, the recombinant Sjp40 protein (rSjp40) and monoclonal antibodies (McAbs) anti-rSjp40 were developed. The expression profiles of Sjp40 at 3 stages of S. japonicum, including egg, cercariae, and adult, were also determined at both mRNA and protein level, which displayed that the expression pattern of Sjp40 varied at different stages. Quantification of circulatory anti-Sjp40 IgG in the infected mice sera by time-resolved fluoroimmunoassay showed a statistically significant increase on days 21, 28, 35, and 42 postinfection compared with the mice sera prior to infection and the control mice. It was further confirmed by Western blot that all 8 clones of anti-rSjp40 McAbs could react specifically with the native antigen in S. japonicum cercariae, and rSjp40 could be recognized by the pooled infected mouse sera on days 21, 28, 35, and 42 postinfection as well as the pooled patient sera with acute schistosomiasis japonica. These findings indicated that Sjp40 and its antibodies are detectable from the host at a relatively early phase (day 21 postinfection with S. japonicum) and suggested that Sjp40 is a potential antigen candidate for the early diagnosis of schistosomiasis.

This study was aimed to explore the effect of nerve growth factor (NGF) on erythropoiesis and to elucidate the underlying mechanism. Using flow cytometry, colony forming assay, blood cell counter, fluorescent real-time quantitation PCR, and enzyme-linked immunosorbent assay (ELISA), the changes in the bone marrow cells (BMCs) proliferation cycle, CFU-E and BFU-E counts, the peripheral blood erythroid related parameters, kidney EPO, BMC GM-CSF, spleen EPO receptor (EPOR) mRNA expression, and serum EPO, GM-CSF, and IL-1 concentrations were all determined after NGF was injected intramuscularly into the thigh of mice, meanwhile the change of BFU-E and CFU-E counts and its relationship with EPO, IL-3 were investigated. The results indicated that the cell proportion in S+G2/M phase, the CFU-E and BFU-E counts of BMCs and the spleen EPOR mRNA expression in injection of NGF (7.5 microg/kg) for 7 days were significantly higher than that in injection of physiological saline for 13-19 days; red blood cell, hemoglobin, and reticulocyte counts increased as well. In vitro, NGF stimulated a dose-dependent increase of CFU-E colonies formation in the semisolid culture system with or without exogenous EPO; the colony counts in the system with NGF alone were significantly higher than that in the system with exogenous EPO alone. The BFU-E counts in the system with exogenous NGF and IL-3 were significantly higher than that in the system with exogenous EPO and IL-3. It is concluded that the NGF promotes the responsibility of hematopoietic cells to EPO and activates the same signal transduction pathway as EPO in hematopoietic cells, and then accelerates the BMCs into mitosis, the HSCs differentiating into erythroid cells, and CFU-E and BFU-E formation.

To explore the effect of nerve growth factor (NGF) on hematopoiesis of granulocyte-macrophage and to elucidate the underlying mechanism.Using flow cytometry, colony forming assay, blood cell count, fluorescent real-time quantitation PCR, and enzyme-linked immunosorbent assay (ELISA), changes in the bone marrow cells (BMCs) proliferation cycle, CFU-GM counts, the peripheral white blood cell (WBC)counts, BMC GM-CSF mRNA quantities, and serum GM-CSF, IL-3 concentrations were observed after NGF was injected intramuscularly into the thigh of normal and combined 60Co gamma ray irradiated and cyclophosphamide peritoneal cavity injected(combined radiation and chemistry injury mice, 60Co-Cy injury) mice. The effect of NGF on CFU-GM formation in vitro cooperation with exogenous GM-CSF or alone was also observed.An injection of NGF [7.5 microg/(kg x d) or 10 microg/(kg x d)] lasted 7 days, the S+G2/M proportions and the CFU-GM counts of BMCs rose greater than injection of physiological saline (PS) in normal and 60Co-Cy injury mice; the serum GM-CSF,IL-3 concentration in 60Co-Cy injury mice rose obviously. In vitro, NGF stimulated a dose-dependent increase of CFU-GM colonies formation in the semisolid culture system with exogenous rmGM-CSF.NGF cooperates with hematopoietic stimulating factors in culture system to promote the CFU-GM colonies in normal and 60Co-Cy injury mice in vitro. NGF accelerates the BMCs into mitosis, the hematopoietic stem cells differentiation into granulocyte-macrophage hematopoietic cells, the CFU-GM formation and promotes the white blood cells count in normal and 60Co-Cy injury mice in vivo, also NGF stimulates the secreting of GM-CSF and IL-3 in 60Co-Cy injury mice,this may be one of the reasons why NGF stimulate the granulocyte-macrophage hematopoiesis repair after combined radiation and chemistry injury.

To investigate the polymorphism of DYS287 among 28 ethnic populations in 9 provinces of China.YAP element was detected by Touchdown PCR amplification and 2% agarose gel electrophoresis.YAP+ frequencies in these ethnic populations were as follows: Zang 36.7%, Tu 23.8%, Yi 18.4%, Pumi 11.3%, Tajik 7.4%, Bai 6.7%, Jino 5.1%, Shandong Han 4%, Mulao 2.7%, and Maonan 1.3%. The rest ethnic populations in our study, including Gansu Han, Yunnan Han, Zhuangzu, Daizu, Lizu, Nuzu, Lisu, Naxi, Lahu, Dulong, Hani, Shezu, Weiwuer, Sala, Kerkizi, Dongxiang, Vazu, and Korea didn't carry YAP + element.Zangzu, Tuzu, Yizu, Pumi, Jino, and Baizu, which belong to Sino-Tibetan language family, carry a high YAP + frequency. Sala, Tuzu, and Tajik, regarded as Central Asia by origin in history and linguistics, also have a high YAP + frequency. Mulao and Maonan, which origin from "Baiyue" ancient ethnic groups, also have a considerable YAP + frequency.

To investigate the polymorphism of nine Y-short tandem repeat loci in 117 male individuals from four minority populations of China, and to obtain the polymorphism information in these populations.Nine loci in all samples were amplified by PCR. Products were electrophoresed on ABI PRISM 377 DNA sequencer. The electrophoresis result was analyzed by Genotyper 2.0 software. Allele frequencies, diversities and genetic distance were calculated.Forty-three alleles and 32 haplotypes were found in Manchu population, 43 alleles and 33 haplotypes in Uygur population, 31 alleles and 15 haplotypes in Kirgiz population and 34 alleles and 23 haplotypes in Zhuang population. The closest genetic distance is 0.02530 between Manchu and Uygur, the highest genetic distance is 0.34590 between Kirgiz and Zhuang.High haplotype diversities were found in four populations. The study of genetic diversity among different populations is useful in research of their origins, migrations and their relationships.

To study the genetic polymorphism of Y chromosome in different Chinese ethnic group populations.Genotypes of 17 biallelic markers located in the nonrecombining portion of the Y chromosome in 76 men from 3 Chinese ethnic group populations (Han in Shandong, Bai in Yunnan, and Tu in Qinghai) were examined with polymerase chain reaction (PCR) and allelic-specific PCR (ASPCR). Their haplotypes made of these 17 binary markers were constructed. The principle component (PC) analysis was conducted based on the haplotype frequency distribution among these 3 and other 15 published Chinese ethnic group populations.The diversities of M50, M110, M103, M88, M3, and M7 were not found in these 3 populations. The frequencies of YAP+ were 23.8%, 6.7%, and 4% respectively in Tu, Bai, and Shandong Han. Eleven haplotypes were found in 3 populations--7 haplotypes (H1, H3, H5, H6, H8, H9, and H11) in Shandong Han (Han.SD), 8 haplotypes (H1, H2, H3, H5, H6, H8, H11, and H16) in Tu, and 9 haplotypes (H1, H3, H4, H5, H6, H8, H9, H11, and H13) in Bai. The predominant haplotypes were H1, H3, H5, H6, H8, and H11. According to PC analysis, Bai was close to Northern Han; Shandong Han, Southern Han (Han.S), Bai and Yunnan Tibetan clustered together; and Tu was close to Yi, Hui and Manchurian.Shandong Han may have had genetic exchanges with southern populations in China. It has been confirmed that some gene components of Han had flowed into Bai's gene pool. Gene flowed from Central Asia had impacted Chinese western populations.

PURPOSE. Telomerase is a specialized polymerase that catalyzes synthesis of telomeres in most eukaryotes. When introduced into somatic cells, it extends the proliferative lifespan and prevents replicative senescence. Whether it has similar functions in lens epithelial cells, especially in human lens epithelial cells (HLECs) remains to be determined. In this study, the human telomerase reverse transcriptase (hTERT) catalytic subunit was introduced into HLECs. A stable cell line expressing hTERT was established and the functions of hTERT were studied. METHODS. The telomeric repeat amplification protocol (TRAP) assay was used to analyze the telomerase activity. Western blot analysis was used to examine hTERT expression. Southern blot analysis was used to detect telomere length. HLECs isolated from intact lenses were cultured in DMEM and transfected with hTERT cDNA. The expression of the exogenous hTERT was examined with RT-PCR, Western blot analysis, and TRAP assay. The functions of hTERT were examined with various techniques. RESULTS. Among the human, bovine, and rabbit lenses examined, only the central epithelium from the 6-month rabbit lens displayed telomerase activity. In both transparent and cataractous human lenses, hTERT activity and expression were not detected. However, the template RNA was present in both types of human lenses. The telomeres in transparent lenses were approximately 1 kb longer than those in cataractous lenses. The primary cultures and later passages of HLECs also displayed no detectable telomerase activity. Introduction of hTERT cDNA into HLECs followed by G418 selection yielded a stable line of HLECs expressing hTERT. In this line, hTERT has supported normal growth after 48 population doublings (PDs) to date and also enhanced antiapoptotic activity against oxidative stress. CONCLUSIONS. Telomere lengths may be associated with cataractogenesis. hTERT introduced into HLECs prevents replicative senescence through telomere synthesis. Furthermore, hTERT displays functions beyond telomere synthesis in normal HLECs.