本文選題：不育癥 + 非梗阻性無精子癥（NOA）��； 參考：《安徽醫(yī)科大學》2014年碩士論文

【摘要】：第一部分中國漢族男性非梗阻性無精子癥ETV5，HORMAD1和HORMAD2基因相關性研究 目的：探討ETV5，HORMAD1和HORMAD2基因多態(tài)性與非梗阻性無精子癥的易感性。 方法：應用Sequenom MassArray質(zhì)譜陣列技術對368例已生育的漢族男性人群(對照組)和361例漢族男性非梗阻性無精子癥（實驗組）ETV5基因的5個標簽單核苷酸多態(tài)(single nucleotide polymorphism,SNP)位點(rs12631658, rs6444106, rs7430047,rs7433760, rs9824882)，HORMAD1基因3個SNP位點（rs1336900，rs16840074，rs6694531），HORMAD2基因8個SNP位點（rs8135823，rs11090601，rs4823073，rs5763779， rs718772，rs9620953，rs9625930， rs975704）進行基因型檢測。應用Plink1.07軟件對數(shù)據(jù)資料進行統(tǒng)計分析，比較對照組與病例組最小等位基因頻率（MAF）及基因型差異，運用Haploview軟件對ETV5、HORMAD1和HORMAD2基因進行單體型分析。 結果：ETV5基因5個標簽SNP，HORMAD13個SNP和HORMAD28個標簽SNP的等位基因頻率、基因型分布在實驗組和對照組均無統(tǒng)計學差異（P＞0.05），進一步的單體型分析亦未顯示統(tǒng)計學差異（P＞0.05）。當分析各SNP位點基因型在NOA亞組分布情況，我們發(fā)現(xiàn)HORMAD2基因rs718772位點最小等位基因頻率（MAF）分布在雙側(cè)平均睪丸體積大于10ml組與小于10ml組中有顯著性差異（P=0.035）。 結論：檢測的ETV5基因，HORMAD1和HORMAD2基因SNP位點多態(tài)性與漢族男性非梗阻性無精子癥的發(fā)生可能不相關, HORMAD2基因rs718772位點SNP可能與NOA患者的睪丸發(fā)育有關。 第二部分CREM信號通路基因單核甘酸多態(tài)性與NOA發(fā)生的關聯(lián)性研究 目的比較CREM信號通路相關基因CREM、ACT、Kif17b和SPAG8基因17個SNPs（single nucleotide polymorphisms, SNPs）位點的基因型頻率、等位基因頻率和單體型頻率等在特發(fā)性非梗阻性無精子癥（non-obstructive azoospermia，NOA）患者及正常生育男性間的分布，探討這4種基因突變是否為NOA的致病因素。同時運用q-RT-PCR方法，研究CREM在NOA（生精阻滯）患者睪丸組織及正常睪丸組織的表達差異，探討CREM基因在精子發(fā)生過程的作用及意義。 方法我們選擇361例特發(fā)性非梗阻性無精子癥（NOA）作為實驗組，368例精液質(zhì)量正常的男性作為對照組。采用Sequenome Massarray質(zhì)譜陣列技術對CREM、ACT、Kif17b和SPAG8基因的17個SNPs位點進行了基因型分型。選取30例生精阻滯患者及30例正常精子發(fā)生的睪丸組織，運用q-RT-PCR的方法檢測CREM基因的在兩組的mRNA表達水平，T檢驗進行數(shù)據(jù)統(tǒng)計，P0.05為具有統(tǒng)計學意義。 結果CREM基因共檢測4個SNP位點（rs4934540，rs2295415，rs11592356，rs1148247），其中rs4934540位點C等位基因頻率（24.7%VS34.4%，P=4.9*10-5，OR=0.624,95%CI=(0.497-0.784)，rs2295415位點的G等位基因頻率（18.0%VS24.2%，P=0.003，OR=0.686,95%CI=(0.532-0.885)，rs11592356位點G等位基因頻率（6.9%VS10%，P=0.036，OR=0.672,95%CI=(0.461-0.978)均在NOA組顯著低于正常生育組，提示該位點的變異可能對正常精子的發(fā)生具有保護作用，進一步的單體型分析提示TATG（rs493454, rs2295415, rs11592356and rs1148247）單體型的比例在NOA組顯著高于正常生育組（P=0.011，OR=1.317，95%CI=1.064-1.631）。而ACT基因9個標簽SNP（srs2273621，rs9373985，，rs2252816，rs9398152，rs9398148，rs4839688，rs17056756，rs3798292，rs9486705）、KIF17b3個SNP（rs631357，rs522496，rs2296225）、SPAG8基因1個SNP（rs3763631）基因型分布在實驗組及對照組均未見差異。CREM基因在NOA（生精阻滯）組及正常精子發(fā)生中睪丸組織的表達用明顯差異，CREM基因在NOA（生精阻滯）患者睪丸組織中低表達（P=0.00001）。 結論CREM基因多態(tài)性可能是中國精子發(fā)生障礙患者的遺傳易感因素，rs4934540，rs2295415，rs11592356三個位點對精子發(fā)生可能起保護作用，而ACT、KIF17b和SPAG8基因單核苷酸多態(tài)性與NOA的發(fā)生無關。CREM的正常表達對精子的正常發(fā)生具有重要的作用，CREM的低表達可能是精子發(fā)生障礙的一個重要原因。
[Abstract]:Part one: correlation between ETV5, HORMAD1 and HORMAD2 genes in Chinese Han men with non obstructive azoospermia.
Objective: To investigate the association of ETV5, HORMAD1 and HORMAD2 gene polymorphisms with non obstructive azoospermia.
Methods: the 5 labelling single nucleotide polymorphisms (single nucleotide polymorphism, SNP) loci (rs12631658, rs6444106, rs7430047, rs7433760, rs9824882) of 368 Han male and 361 Han male non obstructive azoospermia (experimental group) ETV5 genes were used by Sequenom MassArray mass spectrometry array technique. The 3 SNP loci of the MAD1 gene (rs1336900, rs16840074, rs6694531), and the 8 SNP loci of the HORMAD2 gene (rs8135823, rs11090601, rs4823073, rs5763779, rs718772, etc.) were used for genotyping. And genotype differences, the haplotypes of ETV5, HORMAD1 and HORMAD2 genes were analyzed by Haploview software.
Results: the allele frequency of ETV5 gene 5 label SNP, HORMAD13 SNP and HORMAD28 label SNP was not statistically different between the experimental group and the control group (P > 0.05), and the further monosomatograph analysis did not show statistical difference (P > 0.05). When the distribution of the SNP loci genotype in the NOA subgroup was analyzed, we found HORM The minimum allele frequency (MAF) of rs718772 locus in AD2 gene was significantly higher in the bilateral mean testicular volume than in the 10ml group and smaller than that in the 10ml group (P=0.035).
Conclusion: the detection of ETV5 gene, HORMAD1 and HORMAD2 gene polymorphisms may not be related to the occurrence of non obstructive azoospermia in Han men. The rs718772 locus SNP of the HORMAD2 gene may be related to the testicular development of NOA patients.
The second part is the association between CREM signal pathway gene mononuclear nucleotide polymorphism and the occurrence of NOA.
Objective to compare the genotype frequencies of the 17 SNPs (single nucleotide polymorphisms, SNPs) loci of CREM, ACT, Kif17b and SPAG8 genes, and the distribution of allelic frequencies and haplotype frequencies between the allele frequencies and the haplotype frequencies of the CREM signaling pathway related genes in idiopathic non obstructive azoospermia (non-obstructive azoospermia) and normal fertile men. Whether the 4 kinds of gene mutations are the pathogenic factors of NOA, and the difference in the expression of CREM in the testis and normal testicular tissues of the patients with NOA (spermatogenesis block) by using the q-RT-PCR method, and to explore the role and significance of the CREM gene in the process of spermatogenesis.
Methods we selected 361 cases of idiopathic non obstructive azoospermia (NOA) as the experimental group and 368 men with normal semen quality as the control group. The genotyping of 17 SNPs loci of CREM, ACT, Kif17b and SPAG8 genes was performed by Sequenome Massarray mass spectrometry. 30 cases of spermatogenesis block and 30 normal spermatozoa were selected. The testis tissue of the newborn was detected by q-RT-PCR. The expression level of CREM gene in the two groups was detected by T, and the data were statistically analyzed by T test.
Results 4 SNP loci (rs4934540, rs2295415, rs11592356, rs1148247) were detected in the CREM gene, and the frequency of rs4934540 loci C allele frequencies (24.7%VS34.4%, P=4.9*10-5, OR=0.624,95%CI= (0.497-0.784), and allele frequencies of the loci. 6.9%VS10%, P=0.036, and OR=0.672,95%CI= (0.461-0.978) were significantly lower in the NOA group than in the normal growth group, suggesting that the mutation of the loci may have protective effects on normal sperm. Further haplotype analysis suggests that the proportion of TATG (rs493454, rs2295415, rs11592356and rs1148247) in the NOA group is significantly higher than that in the normal growth group (P=). 0.011, OR=1.317,95%CI=1.064-1.631) and ACT gene 9 label SNP (srs2273621, rs9373985, rs2252816, rs9398152, rs9398148, rs4839688, rs17056756, rs3798292, rs9486705). There was a significant difference in the expression of testicular tissue between normal group and normal spermatogenesis group. The expression of CREM gene was low in testicular tissue of NOA (spermatogenesis arrest) (P=0.00001).
Conclusion CREM gene polymorphism may be a genetic predisposing factor in Chinese spermatogenesis disorders. The three loci of rs4934540, rs2295415, and rs11592356 may play a protective role in spermatogenesis, while the positive expression of ACT, KIF17b and SPAG8 single nucleotide polymorphisms is not related to the occurrence of NOA, and it has an important role in the normal occurrence of sperm. The low expression of CREM may be an important cause of spermatogenesis disorders.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R698

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