Identification of proteins interacting with the circadian clock protein PER1 in tumors using bacterial two-hybrid system technique / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify protein-protein interaction partners of PER1 (period circadian protein homolog 1), key component of the molecular oscillation system of the circadian rhythm in tumors using bacterial two-hybrid system technique.</p><p><b>METHODS</b>Human cervical carcinoma cell Hela library was adopted. Recombinant bait plasmid pBT-PER1 and pTRG cDNA plasmid library were cotransformed into the two-hybrid system reporter strain cultured in a special selective medium. Target clones were screened. After isolating the positive clones, the target clones were sequenced and analyzed.</p><p><b>RESULTS</b>Fourteen protein coding genes were identified, 4 of which were found to contain whole coding regions of genes, which included optic atrophy 3 protein (OPA3) associated with mitochondrial dynamics and homo sapiens cutA divalent cation tolerance homolog of E. coli (CUTA) associated with copper metabolism. There were also cellular events related proteins and proteins which are involved in biochemical reaction and signal transduction-related proteins.</p><p><b>CONCLUSION</b>Identification of potential interacting proteins with PER1 in tumors may provide us new insights into the functions of the circadian clock protein PER1 during tumorigenesis.</p>

Progress in epigenetic research on male infertility / 中华医学遗传学杂志

Male infertility is one of the major diseases that affect human health and social life, and is influenced by many genetic and environmental factors. Epigenetic modification on DNA strands in response to environmental factors plays an important role in the process of spermatogenesis. Abnormalities of epigenetic regulation may affect both the quantity and quality of sperm production and result in disorders of male reproduction. We hereby review recent progress made in research on epigenetic regulation including DNA methylation, histone modification and non-coding RNA related with male infertility.

Copy number variation of trinucleotide repeat in dynamic mutation sites of autosomal dominant cerebellar ataxias related genes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To standardize the experimental procedure of the gene test for autosomal dominant cerebellar ataxias (ADCA), and provide the basis for quantitative criteria of the dynamic mutation of spinocerebellar ataxia (SCA) genes in Chinese population.</p><p><b>METHODS</b>Genotyping of the dynamic mutation loci of the SCA1, SCA2, SCA3, SCA6 and SCA7 genes was performed, using florescence PCR-capillary electrophoresis followed by DNA sequencing, to investigate the variation range of copy number of CAG tandem repeat of the genes in 263 probands of ADCA pedigrees and 261 non-related normal controls. Based on the sequencing result, the bias of the CAG copy number estimation using capillary electrophoresis with different DNA controls was compared to analyze the technical detailes of the electrophresis method in testing the dynamic mutation sites.</p><p><b>RESULTS</b>PCR products containing dynamic mutation loci of the SCA genes showed significantly higher mobility than that of molecular weigh marker with relatively balanced GC content. This was particularly obvious in the SCA2, SCA 6 and SCA7 genes whereas the deviation of copy number could be corrected to +/-1 when known CAG copy number fragments were used as controls. The mobility of PCR products was primarily related to the copy number of CAG repeat when the fragments contained normal CAG repeat. In the 263 ADCA pedigrees, 6 (2.28%) carried SCA1 gene mutation, 8 (3.04%) had SCA2 mutation and 81 (30.80%) harbored SCA3 mutation. The gene mutation of SCA6 and SCA7 was not found. The normal variation range of the CAG repeat was 17-36 copies in SCA1 gene, 13-30 copies in SCA2, 14-39 copies in SCA3, 6-16 copies in SCA6 and 6-13 copies in SCA7. The heterozygosity was 76.1%, 17.7%, 74.4%, 72.1% and 41.3%, respectively. The mutation range of the CAG repeat was 49-56 copies in SCA1 gene, 36-41 copies in SCA2, 59-81 copies in SCA3. Neither homozygous mutation of an SCA gene nor double heterozygous mutation of the SCA genes was observed in the study.</p><p><b>CONCLUSION</b>The copy number of the CAG repeat in SCA genes could be calculated accurately based on the result of florescence PCR-capillary electrophoresis when limited amount of known repeat copy number controls were used. Our result supported that the notion that SCA3 gene mutation was the most common cause for ADCA, and the obtained data would be helpful for establishing quantitative criteria of the dynamic mutation of the SCA genes in Chinese.</p>

The A-204C Polymorphism in CYP7A1 Gene Affects Its Promoter Activity / 中国生物化学与分子生物学报

cholesterol 7α-hydroxylase gene ( CYP7A 1 ) plays a key role in the catabolism of cholesterol into bile acids. To investigate whether the A-204C polymorphism in CYP7A1 gene affects the gene expression,using luciferase as the reporter gene, four recombinants were constructed by inserting forward or reverse sequence with A or C allele at the polymorphism site into the promoter-less vector pGL3-basic. The constructs were then transfected into four cell lines and the luciferase activity of each expression vector was examined by dual luciferase reporter gene assay system. The results showed that activities of the forward sequence of both genotypes were higher than that of reverse sequence. Promoter activity of the recombinants with A allele was about one third lower than that with C allele. According to the analysis with TRANSFAC database, there may exist a Zic3 binding site when there is the C allele at -204. Our study indicates that the A-204 C polymorphism in CYP7A1 promoter region decreases its promoter activity and thus represses the gene expression, possibly due to the lack of a potential Zic3 binding site.

No evidence for genetic association between alpha-2 macroglobulin I1000V polymorphism and sporadic Alzheimer's disease in two independent Chinese populations / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Alpha-2 macroglobulin (alpha2M) is a proteinase inhibitor found in association with senile plaques in Alzheimer's disease (AD). Also alpha2M has been implicated in several pathophysiological processes in AD. In view of the recent contradictory reports on the relationship between AD and a common polymorphism I1000V in A2M gene, the present authors studied a relatively large sample, determined the genotype of the I1000V polymorphism in A2M gene in sporadic AD patients and age-matched controls with normal cognition, and examined the possible association of the polymorphism with AD.</p><p><b>METHODS</b>Genotypes of A2M and apolipoprotein E (apoE) were detected by polymerase chain reaction combined with restriction fragment length polymorphism in 257 patients and 242 controls in Guangzhou, and 112 patients and 113 controls in Chengdu.</p><p><b>RESULTS</b>The 1000Val allele frequencies in the merged AD and control groups were 7.7% and 8.7%, respectively. The differences of allelic and genotypic frequencies between the patients and control subjects were not statistically significant, even after stratification by apoE epsilon4 status or by age-of-onset of the disease.</p><p><b>CONCLUSION</b>The results of this study revealed no association between the I1000V polymorphism of A2M and Chinese sporadic AD in Guangzhou and Chengdu.</p>

Idiopathic male infertility and partial copy deletion of DAZ gene family / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the pattern and prevalence of partial copy deletion of deleted-in-azoospermia (DAZ) gene in the azoospermia factor C(AZFc) region of patients with idiopathic azoospermia or severe oligozoospermia.</p><p><b>METHODS</b>sY581 and sY587 in DAZ gene region were analyzed by polymerase chain reaction-restriction length polymorphism(PCR-RFLP) for its deletion in 197 patients with azoospermia, 166 patients with severe oligozoospermia, and 210 fertile men as controls.</p><p><b>RESULTS</b>Deletion of both DAZ1 and DAZ2 was detected in 18 patients with azoospermia and 10 with severe oligozoospermia, and the prevalence was 9.1% and 6.0% respectively. There was significant difference in deletion rate between the cases and controls.</p><p><b>CONCLUSION</b>The frequency of partial copy deletion of DAZ gene in Chinese idiopathic azoospermia or severe oligozoospermia patients is much higher than that of fertile controls, suggesting that the deletion of DAZ1/DAZ2 may be one of the important genetic etiological factors of spermatogenesis damage. The pattern and prevalence of DAZ partial copy deletion are similar to those of Caucasians populations, and detection of DAZ gene partial copy deletion by PCR-RFLP may be adopted as an additional clinical gene diagnostic measure after AZF microdeletion detection.</p>

Study on the association of lecithin cholesterol acyltransferase gene polymorphisms with the lipid metabolism in coronary atherosclerotic heart disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To examine the distribution of 3 polymorphisms of lecithin cholesterol acyltransferase gene in Chinese population and the association of these polymorphisms with lipid metabolism in patients with atherosclerotic heart disease (CHD).</p><p><b>METHODS</b>Genotypes and frequencies of 3 sites were examined by PCR-restriction fragment length polymorphism technique in 209 unrelated normal control individuals and 203 CHD patients.</p><p><b>RESULTS</b>The observed allele frequencies conform well to Hardy-Weinberg equilibrium. The frequency of 608T allele was significantly higher in controls than that in patients (P=0.034). Compared with the CHD patients without 608T, the CHD patients with 608T exhibited a significant increase in plasma HDL-C concentration (P=0.015). 911T/C and 1188C/T polymorphisms were not found in either group.</p><p><b>CONCLUSION</b>608T polymorphism of LCAT gene was associated with higher plasma HDL-C level in CHD patients, while 911T/C and 1188C/T polymorphisms maybe very rare in Chinese population.</p>

Advances in modulation of mammalian sperm function by fertilization promoting peptide / 中华医学遗传学杂志

Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss by modulation of the adenylyl cyclase (AC)/cAMP signal transduction pathway. This is a review aimed at analyzing the function of fertilization promoting peptide during this process. The possible molecular basis is also discussed.

Polymorphisms of four microsatellite markers tightly linked with polycystic kidney disease 2 gene in Chinese / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the polymorphisms of four microsatellites, D4S1534, D4S1563, D4S423 and D4S414, which are tightly linked to polycystic kidney disease 2 (PKD2) gene, and hence to provide a basis for studying the heterogeneity of adult polycystic kidney disease (APKD).</p><p><b>METHODS</b>An analysis on the DNA of some unrelated Chinese people was performed using polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and silver staining.</p><p><b>RESULTS</b>In Chinese Hans, there are 11 alleles of D4S1534, and their sizes are 142-162 bp 14 alleles of D4S1563, 205-235 bp 17 alleles of D4S423, 103-135 bp; and 15 alleles of D4S414, 236-264 bp. The polymorphism information contents of the four microsatellites are 0.872, 0.844, 0.921 and 0.871 respectively.</p><p><b>CONCLUSION</b>In the Chinese Han people studied above, the four microsatellite markers that have many alleles are highly polymorphic genetic markers and may serve as the data of population genetics, suggesting that all four microsatellites could be used in studies on heterogeneity of APKD, linkage analysis of APKD and forensic personal identification.</p>

Fluorescence probe two-hybridization technique and its application in medical genechips / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a new nucleic acid hybridization detection technique which may be used in medical genechips.</p><p><b>METHODS</b>The specific DNA fragment was detected by sequential two hybridization of fluorescence probe with template DNA and fixed DNA probe.</p><p><b>RESULTS</b>Fluorescence probe two-hybridization (FPTH) was applied to genechips for the detection of sex-transmitted pathogens from culture strains, and the results showed that the values of fluorescence density of the positive groups decreased remarkably when compared with those of the negative group. Both the sensitivity and specificity for detecting clinical samples are higher than 90%. There is no need of any additional reagent in hybridization procedure, and the hybridization detection can be accomplished in 40 minutes.</p><p><b>CONCLUSION</b>The FPTH technique is rapid, simple and reliable, it can also make the clinical detection process completely automatic and integrative.</p>

A study of the relationship between MICA gene and systemic lupus erythematosus / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between major histocompatibility complex class I chain-related A(MICA) gene and systemic lupus erythematosus (SLE).</p><p><b>METHODS</b>The alleles and frequencies of exons 4 and 5 of MICA gene were determined in 70 cases of SLE and 152 controls of Yunnan Hans by STR genotyping, polymerase chain reaction, single strand conformation polymorphism and bidirection DNA sequencing.</p><p><b>RESULTS</b>Five alleles of exon 5 and 10 alleles of exon 4 were found in this study. The frequency of each allele was determined in patients and controls. There was no significant difference between the two groups in exons 4 and 5 of MICA gene.</p><p><b>CONCLUSION</b>Exons 4 and 5 of MICA were not related to SLE in Yunnan Hans.</p>

Nested coamplification polymerase chain reaction / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a simple and reliable method for intensifying the hybridization signals of gene chips.</p><p><b>METHODS</b>The authors added EDTA and another FAM-labeled probe to the normal PCR products, denatured the mixture by heat, and then let the mixture hybridize with the fastened probes on the chip.</p><p><b>RESULTS</b>With the use of EDTA and another FAM-labeled probe, the hybridization signals increased by 6 times or greater.</p><p><b>CONCLUSION</b>Adding EDTA and another probe to the normal PCR products is a simple and efficient method to intensify the hybridization signal of chips.</p>

Relationship between a novel polymorphism of lipoprotein lipase gene and coronary heart disease / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate polymorphisms in the gene for lipoprotein lipase (LPL) in Chinese populations with coronary heart disease (CHD) and to inquire into the relationship between these polymorphisms in LPL gene and CHD.</p><p><b>METHODS</b>Genomic DNA was extracted from patients with CHD and normal control subjects using a salting out method. The entire coding region and flanking sequences of all coding exons of the LPL gene were amplified by PCR technique and PCR products were detected by denaturing high-performance liquid chromatography (DHPLC) and sequenced with a dideoxy terminal termination method.</p><p><b>RESULTS</b>A novel polymorphic site, G830A, that is within the fifth exon of the LPL gene was found. The 192 codon CGA was changed into CAA and resulted in the substitution of glutamine for arginine. Between the control and CHD groups, chi-square test showed no significant difference in the frequencies of the A/A genotype and A allele (P > 0.05). However, the frequencies of A/A genotype and A allele (0.653 and 0.786) in CHD patients with high plasma triglyceride/lowed plasma high density lipoprotein cholesterol were higher than those (0.415 and 0.642) in CHD patients without hyperlipidemia (P < 0.05).</p><p><b>CONCLUSION</b>No direct association was found between the LPL Arg192-->Gln substitution polymorphism and CHD, but there is a significant positive correlation between the A/A genotype of the LPL gene and CHD associated with high triglyceride/lowed high density lipoprotein cholesterol. This study may provide new data for exploring the molecular mechanism of CHD.</p>

The relationship between haplotypes of angiotensinogen gene and essential hypertension / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the polymorphism of angiotensinogen gene (AGT) and the risk for hypertension in a Chinese population.</p><p><b>METHODS</b>Three polymorphisms of AGT gene were analyzed in 335 patients with documented essential hypertension and 196 control subjects by using PCR-restriction fragment length polymorphism. Expectation maximization(EM) algorithm was then used for pairwise linkage disequilibrium test and haplotype analysis of AGT polymorphisms.</p><p><b>RESULTS</b>Linkage disequilibrium between M235T and A-20C, between M235T and A-6G, between A-20C and A-6G was observed (P<10(-4)). The case-control analysis revealed that the frequency of T235 is significantly higher in essential hypertension patients than in control subjects. But all haplotype frequencies showed no significant difference between the patient and control groups.</p><p><b>CONCLUSION</b>No association was noted between the haplotypes of AGT gene and hypertension in tested people, but T235 allele might play an important role in increased risk for essential hypertension.</p>

COLONY HYBRIDIZATION WITH BIOTIN LABELED DNA PROBE / 微生物学通报

A simple, rapid method for colony hybridization has been developed. The DNA probes were labeled by digo xigenin. The signal of hybridization was detected by streptavidin and poly (AP) system. The results showed that this method is sensitive, specific and repoducible, it can be used for colony hybridization instead of isotopic.