[Study on the distribution of Y chromosome haplogroups in Sichuan Han population].

OBJECTIVE: To investigate the distribution characteristics of Y chromosome haplogroups in Sichuan Han population. METHODS: Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), denatured high performance liquid chromatography (DHPLC) and DNA sequencing methods, 18 Y-chromosome bialletic markers were detected to type Y chromosome haplogroups in 341 unselected men from Sichuan Han population. RESULTS: A total of 14 haplogroups were observed, in which haplogroups H2 and H4 were identified for the first time in Sichuan population, and haplogroups H14 and N* were found firstly in Chinese. There was a significant frequency difference of Y haplogroups between Sichuan Han population and Southern Han population. CONCLUSION: More comprehensive frequency distribution data of Y chromosome haplogroups are obtained in Sichuan Han population, which would be helpful for understanding the association of Y chromosome background and the susceptibility to male specific diseases such as spermatogenic failure, prostate cancer, testical cancer and so on in present population.

DAZ1/DAZ2 cluster deletion mediated by gr/gr recombination per se may not be sufficient for spermatogenesis impairment: a study of Chinese normozoospermic men.

AIM: To explore the possible effect of the deleted in azoospermia (DAZ) copy cluster deletion on spermatogenesis in the Chinese population, the deletion of the azoospermia factor c (AZFc) region was analyzed in 346 normozoospermic men. METHODS: Three DAZ single nucleotide variant loci and seven AZFc-specific sequence-tagged sites were examined with polymerase chain reaction (PCR)-restriction fragment length polymorphism and routine PCR. RESULTS: Five (1.4%) of the normozoospermic men were found to have deletion of gr/gr-DAZ1/DAZ2. None of the men were found to have b2/b4-entire DAZ deletion. CONCLUSION: The presence of gr/gr-DAZ1/DAZ2 deletion in five men with normozoospermia suggests that this deletion per se may not be sufficient for spermatogenic impairment in Chinese men.

[Important genetic etiology of infertile Chinese males: chromosome abnormality and deletion of DAZ gene copy in the AZFc region of Y chromosome].

OBJECTIVE: To investigate the correlation of male infertility with abnormality of chromosomal quantity and construction and with the deletion of DAZ gene copy in the AZFc region of Y chromosome. METHODS: Included in the study were 247 azoospermic and 206 severe oligozoospermic patients, as well as 210 fertile men as controls. Multi-PCR and PCR-RFLP were used to analyze the deletion of DAZ gene copies in the AZFc region of Y chromosome. Chromosomal quantity and construction were detected by G-band in the 453 patients. RESULTS: In the azoospermic and severe oligozoospermic patients, the incidences of chromosomal abnormality were 12.6% and 8.3%; the rates of complete DAZ deletion were 7.7% and 11.2%, and the rates of DAZ1/DAZ2 deletion were 7.3% and 4.9% respectively, but no deletion was detected in the controls. CONCLUSION: There is a high frequency of chromosomal abnormality and DAZ gene copy deletion in patients with azoospermia and oligospermia, which suggests that chromosomal abnormality and partial and complete deletion of DAZ gene copy might be important genetic causes of Chinese male infertility.

[A method of haplotype analysis for multiple single-nucleotide polymorphisms].

Haplotypes from multiple single nucleotide polymorphisms(SNPs) spaced in longer DNA were constructed by multi-step PCR and DNA sequencing methods. Two allele-specific primers were synthesized and used for long DNA fragments (approximately 10 kb) amplification from human genome DNA. Fragments within these long DNA fragments were amplified by using these PCR products as templates in the second round PCR. The second round PCR products were subsequently sequenced. Haplotype construction was performed based on the character of nucleotides at the 3'-end of the allele-specific primers and the sequencing results from the second round PCR products. The DNA fragment (approximately 16 kb) from promoter to exon 4 of lipoprotein lipase (LPL) gene was amplified by allele-specific primers,and the DNA fragments including exon 2 or exon 3 of LPL gene were amplified and sequenced. A SNP of +13,557G-->A within intron 2 was identified. Four haplotypes including -421 G/ +13557G/ + 15222A, - 421A/ +13557G/ +15222A, -421G/ +13557G/ + 15222G, - 421G/+ 13557A/ +15222A were detected. The method is effective and relatively simple for construction of haplotypes from multiple single nucleotide polymorphisms.

[Possible association between 278C/A single nucleotide polymorphism of FKBP6 and idiopathic azoospermia].

OBJECTIVE: To investigate 2 polymorphism sites in exon 3 and intron 2 of FKBP6 in Chinese population, while screening the gene mutations and polymorphisms in exons 3, 4 of FKBP6, and the association of these polymorphisms with azoospermia. METHODS: Possible variations of exons 3, 4 and genotypes and frequencies of 2 polymorphic loci were examined by denaturing high-performance liquid chromatography(DHPLC) and PCR-restriction fragment length polymorphism(PCR-RFLP) technique in 177 azoospermia patients and 231 control individuals. RESULTS: The observed allele frequencies conformed well to Hardy-Weinberg equilibrium. The frequency of 278A allele was significantly higher in controls than that in patients (P<0.05). C/T(s7797242) polymorphism was not found in either group and variations in exons 3, 4 were not detected. CONCLUSION: 278A polymorphism of FKBP6 gene was associated with idiopathic azoospermia, while C/T, 370G/A, 430G/C, 467T/C, 468G/A polymorphisms might be very rare in Chinese population.

[Association of APOA5 gene single nucleotide polymorphism with levels of lipids and coronary heart disease in Chinese].

OBJECTIVE: To investigate the single nucleotide polymorphism 4 (SNP4) of the apolipoprotein A5 (APOA5) gene possible association with coronary heart disease(CHD) and its distribution of in Chinese Han population. METHODS: APOA5 SNP4 genotyping was performed using polymerase chain reaction and Hae III restriction fragment length polymorphism analysis. RESULTS: APOA5 allelic frequencies of T, C were 0.435, 0.565 and 0.374, 0.626 in CHD group and control group, respectively. There is significant difference in allele and genotype frequencies between CHD group and control group (P<0.05). The levels of plasma high density lipoprotein in CHD patients with CC genotype were higher than those in CHD patients with other genotypes (P<0.01). The frequencies of T allele and C allele in Chinese was significantly different from those in Caucasians (0.374 vs 0.663, 0.626 vs 0.337, P<0.01). The C allele was much more common in Chinese population. CONCLUSION: The association is found between the Hae III polymorphism and CHD, There is a significant correlation between the CC genotype of the APOA5 and the levels of plasma high density lipoprotein-cholosteal in the CHD group.

[Mouse whole mount RNA in situ hybridization: an effective technique for analyzing gene expression].

OBJECTIVE: To set up a method of analyzing gene expression profile from mouse whole embryos. METHODS: Mouse whole mount RNA in situ hybridization(WM-ISH) of E10.5-E14 embryos was carried out by using digoxigenin-labeled Runx1 and Runx3 RNA probes and their expression profile was observed by detecting the existence and status of corresponding mRNAs in the embryonic tissues. RESULTS: Clear hybridization signals were observed in different tissues and organs hybridized by Runx1 or Runx3 RNA probe. Different probes and ages of embryos had need of their own optimal proteinase K treatment conditions. CONCLUSION: Mouse whole mount RNA in situ hybridization is an effective method of analyzing gene expression. It is useful for revealing whole gene expression profile and has a great potentiality in the era of functional genomics. It provides an alternative method of studies on gene expression which is at least as good as LacZ staining and immunohistochemistry. The key factor of the success to mouse whole mount RNA in situ hybridization is whether the proteinase K treatment conditions are optimal or not.

[Association of 7alpha-hydroxylase gene polymorphism with levels of plasma lipids].

To study the distribution of Eco31I restriction polymorphism in nucleotide -204 of 7alpha-hydroxylase gene(CYP7A1)in Sichuan Han population of China and association of the polymorphism with coronary heart disease(CHD),CYP7A1 genotyping was performed by using PCR-RFLP approach in 183 CHD patients and 101 control subjects. 7alpha-hydroxylase gene allele frequencies of C,A were 0.840 and 0.160 in CHD group and 0.822 and 0.178 in control group,respectively. There was no significant difference in frequencies of allele and genotypes in A-204C polymorphism between CHD group and control group (P>0.05). However, in CHD patients there was significant difference in total cholesterol (TC) levels among CC,CA and AA genotypes (P<0.05) ,and the levels of high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) in CHD patients with AA genotype were lower than those in CHD patients with CC and CA genotypes(P<0.05). In control group there was significant difference in TC levels between CC and CA genotypes (P<0.05) . The frequencies of C,A alleles at A-204C polymorphic site were significantly different from those reported in white people(P<0.05). The results indicating that no direct association was found between the A-204C polymorphism and CHD,but there was significant correlation between this polymorphism and the levels of TC ,and there was significant correlation in CHD patient group between this polymorphism and levels of HDL-C and LDL-C.

A novel polymorphism A(+884)-->G in the hepatic lipase gene and its association with coronary artery disease.

Hepatic lipase (HL) activity may influence susceptibility to coronary artery disease (CAD). Association between the single nucleotide polymorphisms (SNPs) in the HL gene with the occurrence of CAD has been investigated thoroughly, but to date most studies focused on the base variation in the promoter of HL gene, little is known about the variation in the coding region. In present study, the SNP in all exons of the HL gene were analyzed. All 9 exons with their flanking sequences of the HL gene were amplified from the Chinese patients with CAD and normal controls by PCR technique, and the PCR products were detected by denaturing high performance liquid chromatography (DHPLC) and sequenced with a dideoxy terminal termination method. As the result, a novel SNP A(+884)-->G within the sixth exon of HL gene was found, the 276 codon AAA was changed into AGA and resulted in the substitution of arginine for lysine. Compared with the control group, more CAD patients carried the G+884 allele (AG+GG) (54.9% vs. 41.5%, chi(2)=6.164, df=2, P=0.046). The prevalence of the G+884 allele was significantly higher in the CAD patients than that in control subjects (31.4% vs. 21.3%, chi(2) =4.652, df=1, P=0.031). Data from the linkage disequilibrium analysis showed that the A(+884)-->G polymorphism was strong in linkage disequilibrium with the T(-2)-->C variation we identified previously(D'=0.699, 0.742 in CAD patients and controls, respectively), and the frequency of the C(-2)/G(+884) haplotype (mutation) is significantly higher in CAD patients than that in controls (0.253 vs. 0.172, P<0.05).

Relationship between a novel polymorphism of hepatic lipase gene and coronary artery disease.

Hepatic lipase (HL) is a lipolytic enzyme involved in the catabolism of plasma lipoproteins, and is an important determinant of high density lipoproteins(HDL) concentration and low density lipoproteins(LDL) subclass distribution. Accordingly, HL activity may influence body's susceptibility to coronary artery disease (CAD). Association on the single nucleotide polymorphisms (SNPs) in the HL gene to post-heparin plasma HL activity and the plasma HDL-cholesterol concentration have been investigated thoroughly, but to date, little is known about th is in Chinese. In present study, the SNPs of the HL gene were analyzed. The promoter region and all the 9 exons with their flanking sequences of the HL gene were amplified from the Chinese patients with CAD and normal controls by PCR technique, and the PCR products were detected by denaturing high performance liquid chromatography (DHPLC) and sequenced with a dideoxy terminal termination method. As the result, a novel SNP-2T right curved arrow C in the promoter of HL gene was found. Compared with the control group, more CAD patients carried the -2C allele(TC+CC) (57.9% versus 42.7%, chi(2) =4.181, df=2, =0.041). The prevalence of the -2C allele was significantly higher in the CAD patients than in control subjects (chi(2)=3.988, df=1, P=0.046) and the odds ratio(OR) of -2C allele associated with the risk of CAD is 1.58 [95% confidence interval(CI): 1.01-2.47]. The -2C allele homozygous carriers in the CAD patients had a significantly higher HDL-cholesterol level than the noncarriers [(1.13-/+0.24) mmol/L versus (0.91-/+0.14) mmol/L, P<0.05]. These suggest that a T right curved arrow C substitution at -2 of the HL promoter may be associated with th e variation of HDL-cholesterol concentration and therefore affect the risk of CAD in Chinese.