一个多发性内分泌腺瘤2A型家系和中国汉族人群慢性阻塞性肺疾病的遗传研究

发布时间：2018-05-17 11:05

本文选题：多发性内分泌腺瘤2A型 + RET　；参考：《中南大学》2012年博士论文

【摘要】：研究背景及目的：多发性内分泌腺瘤2型(multiple endocrine neoplasia type2, MEN2; MIM171400)是一种常染色体显性遗传的神经内分泌肿瘤易感性综合征,以可变外显率的甲状腺髓样癌(medullary thyroid carcinoma, MTC)、嗜铬细胞瘤(pheochromocytoma, PHEO)和甲状旁腺功能亢进(hyperparathyroidism, HPT)为特征。MEN2可分为三种亚型：MEN2A型(multiple endocrine neoplasia type2A; MIM171400)、MEN2B型(multiple endocrine neoplasia type2B; MIM162300)和家族性甲状腺髓样癌(familial medullary thyroid carcinoma, FMTC; MIM155240)。 MEN2A临床上主要表现为甲状腺髓样癌、嗜铬细胞瘤和原发性甲状旁腺功能亢进,约占MEN2的55%。FMTC一般不涉及其他内分泌病变,占MEN2的35%～40%,而MEN2B临床表现为甲状腺髓样癌、嗜铬细胞瘤和粘膜神经瘤,占MEN2的5%～10%。MEN2通常由RET原癌基因(the rearrangedduring transfection protooncogene gene, RET; MIM164761)或1型神经营养因子酪氨酸激酶受体基因(the neurotrophic tyrosine kinase receptor type1gene, NTRKI; MIM191315)突变引起。 MEN2是一种起病隐匿,易被延误诊治的疾病。对家系内未发病的携带者进行多发性内分泌瘤2型遗传检测,可为临床前诊断和早期治疗提供重要帮助。目前认为检测基因突变是诊断MEN2的金标准,并可用于指导临床选择治疗方案。对基因功能的更深入研究,将会使MEN2的基因治疗成为可能。为了调查中国汉族MEN2A家系中的遗传致病基因以及基因型和表型之间的关系,我们对一个3代MEN2A家系9名成员(5名患者)进行了遗传学分析。方法：收集1个3代中国汉族MEN2A家系9名家系成员的外周血,其中包括5名患者。抽取先证者和她妹妹的外周血做淋巴细胞培养,用植物凝集素刺激72小时后收集细胞中期分裂相,应用染色体核型分析吉姆萨胰蛋白酶显带(Giemsa-trypsin-Giemsa, GTG;G显带)技术对染色体分裂相进行分析。对MEN2A家系先证者进行RET基因和NTRK1基因全部编码区聚合酶链式反应(Polymerase Chain Reaction, PCR)扩增后直接测序。对家系成员进行突变共分离分析,对100名正常对照进行RET Cys634Arg位点突变检测。结果：对两名患者的G显带染色体核型分析发现均为正常女性染色体核型(46,XX)。对RET基因和NTRKI基因的整个编码区序列分析发现了9个核苷酸变异,包括5个RET变异：c.1296GA (Ala432Ala)、c.1900TC (Cys634Arg)、c.2071GA (Gly691Ser)、IVS12+47CT和c.2712CG(Ser904Ser);4个NTRK1变异：-5GA、c.1187CT (Ser396Leu)、IVS16-4delA和IVS18+6CT。其中RET c.1296GA(Ala432Ala)和c.2712CG (Ser904Ser), NTRK1C.1187CT (Ser396Leu)和IVS18+6CT为新的变异。对家系成员进一步分析只有RET突变Cys634Arg与疾病共分离,而在100个正常对照中未见该突变。5名已进行外科手术患者均为Cys634Arg杂合突变携带者,一名36岁未受累家系成员为RET Cys634Arg杂合突变携带者。结论：对一个中国汉族多发性内分泌腺瘤2A型家系进行G显带染色体核型分析和基因突变分析,发现了9个核苷酸变异,包括5个RET变异：c.1296GA (Ala432Ala)、c.1900TC (Cys634Arg)、 c.2071GA (Gly691Ser)、IVS12+47CT和c.2712CG (Ser904Ser);4个NTRK1变异：-5GA、c.1187CT (Ser396Leu)、IVS16-4delA和IVS18+6CT。其中RET c.1296GA (Ala432Ala)和C.2712CG (Ser904Ser), NTRK1c.1187CT (Ser396Leu)和IVS18+6CT为新的变异。RET c.1900TC (Cys634Arg)变异是致病突变。一名36岁Cys634Arg携带者无临床症状暗示该疾患可能存在外显不全或发病延迟。本研究有助于进一步了解多发性内分泌腺瘤2A型基因型和表型关系,同时对该家系遗传咨询和产前诊断有重要意义。研究背景及目的：慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD; MIM606963)是一种常见的、发病率和死亡率逐年上升的复杂性疾病,发病机制主要是多基因变异和环境因素相互作用。吸烟是COPD最主要的致病因素,不同个体之间存在COPD易感性差异。更好的理解COPD易感的遗传因素有助于发现新的药物靶点,从而促进COPD的早期诊断和治疗。人类基因组计划和国际人类基因组单体型图计划(the International HapMap Project)的完成从全基因组水平提供了研究COPD遗传机制的平台。全基因组关联研究(genome-wide association study, GWAS)能对整个基因组成千上万个单核苷酸变异(single nucleotide polymorphism, SNP)研究,以确定那些与疾病症状同时出现的SNP。该技术作为一种前沿和有效的研究复杂疾病的方法,已经应用于COPD相关研究中,并已取得了一些阳性结果,但这些结果需要在大样本、多种族的人群中进行验证。最近GWAS发现在非亚洲人群中一些基因变异与COPD易感性相关,如铁效应元件结合蛋白基因(the iron-responsive element-binding protein2gene, IREB2; MIM147582)、家族序列相似性13A基因(the family with sequence similarity13member A gene,FAM13A; MIM613299)和X线修复交叉互补基因5(the X-ray repair cross-complementing protein5gene, XRCC5; MIM194364)。本研究对中国大陆汉族人群COPD患者和正常对照进行IREB2rs2568494变异、FAM13A rs2869967变异和XRCC5rs3821104变异DNA序列分析,以确定中国汉族人群中这些基因变异与COPD表型的相关性。方法：对275名COPD患者和434名性别、年龄和种族匹配的正常对照进行IREB2rs2568494变异、FAM13A rs2869967变异和XRCC rs3821104变异DNA序列PCR扩增后直接测序,应用Assign3.5分析软件、MT Navigator软件和DNAstar软件对测序结果进行分析。对正常人群进行Hardy-Weinberg平衡测试,应用SPSS16.0对病例-对照人群进行基因变异与COPD的相关性分析。结果：正常对照中所有测试的基因型频率符合Hardy-Weinberg平衡。FAM13A rs2869967变异基因型和等位基因频率分布在正常对照和COPD患者中存在统计学意义(基因型分布：X2=6.319,P=0.042；等位基因分布：X2=4.014,P=0.045)；XRCC5rs3821104变异变异基因型和等位基因频率分布在正常对照和COPD患者中也存在统计学意义(基因型分布：X2=6.062,P=0.048；等位基因分布：X2=5.607,P=0.018),与文献报道结果一致。IREB2rs2568494变异与COPD表型无统计学意义(基因型分布：X2=0.590,P=0.744；等位基因分布：X2=0.034,P=0.854)。结论：我们发现FAM13A rs2869967和XRCC5rs3821104这两个变异在中国汉族COPD患者和正常对照中的基因型和等位基因频率分布有统计学意义。IREB2rs2568494在中国汉族COPD患者和正常对照中的基因型和等位基因频率分布无统计学意义,中国人群在COPD患病遗传基础上可能和非亚洲人群存在差异。遗传风险变异的发现有助于建立新的COPD预警和治疗方法。
[Abstract]:Background and purpose: multiple endocrine adenoma type 2 (multiple endocrine neoplasia type2, MEN2; MIM171400) is an autosomal dominant neuroendocrine tumor susceptibility syndrome, with variable exostatic thyroid myeloid carcinoma (medullary thyroid carcinoma, MTC), pheochromocytoma (pheochromocytoma, PHEO) and Hyperparathyroidism (hyperparathyroidism, HPT) can be divided into three subtypes: MEN2A (multiple endocrine neoplasia type2A; MIM171400), MEN2B type (multiple endocrine) and familial thyroid medullary carcinoma. The main manifestations are medullary thyroid carcinoma, pheochromocytoma and primary hyperparathyroidism, about MEN2 of 55%.FMTC generally do not involve other endocrine diseases, accounting for 35% to 40% of MEN2, while MEN2B is a clinical manifestation of medullary thyroid carcinoma, pheochromocytoma and mucous neuroma, accounting for 5% to 10%.MEN2 of MEN2, usually by RET proto oncogene (the rear) Rangedduring transfection protooncogene gene, RET; MIM164761) or type 1 neurotrophic factor tyrosine kinase receptor gene (the neurotrophic tyrosine kinase receptor type1gene) mutation.
MEN2 is a disease of insidious onset and easily delayed diagnosis and treatment. Genetic detection of multiple endocrine neoplasia type 2 can provide important help for preclinical diagnosis and early treatment. It is considered that detection of gene mutation is the gold standard for diagnosis of MEN2 and can be used to guide clinical selection treatment. More in-depth study will make the gene therapy for MEN2 possible. In order to investigate the genetic pathogenicity of the MEN2A family in the Chinese Han family and the relationship between genotype and phenotype, we have carried out a genetic analysis of 9 members of the 3 generation MEN2A family (5 patients).
Methods: the peripheral blood of 1 family members of the MEN2A family of 3 generations of Han Chinese was collected, including 5 patients, including 5 patients. The lymphocyte culture of the precursor and her sister's peripheral blood was collected. The metaphase phase of the cell was collected after 72 hours of plant agglutinin stimulation, and the Giemsa-trypsin-Giemsa was analyzed by karyotype. GTG; G ribbon) technique was used to analyze the chromosomal division phase. The RET gene and the NTRK1 gene were amplified by polymerase chain reaction (Polymerase Chain Reaction, PCR) for direct sequencing of the MEN2A family precursor. The mutation co separation of the family members was analyzed and the RET Cys634Arg site mutation was detected in 100 normal controls.
Results: the karyotype analysis of the G banding chromosomes of two patients was found to be normal female chromosome karyotype (46, XX). 9 nucleotide variations, including 5 RET variants, c.1296GA (Ala432Ala), c.1900TC (Cys634Arg), c.2071GA (Gly691Ser), IVS12+47CT, and NTRKI, were found in the sequence analysis of the whole coding region of the RET and NTRKI genes. 4Ser); 4 NTRK1 variants: -5GA, c.1187CT (Ser396Leu), IVS16-4delA, and IVS18+6CT. in which RET c.1296GA (Ala432Ala) and c.2712CG (Ser904Ser) are new variations. All the patients who had undergone surgery were carriers of Cys634Arg heterozygous mutation. A 36 year old unaffected family member was a RET Cys634Arg heterozygous mutation carrier.
Conclusion: the karyotype analysis and gene mutation analysis of the G banding chromosome of a Chinese Han 2A family with multiple endocrine adenomas found 9 nucleotide variations, including 5 RET variations: c.1296GA (Ala432Ala), c.1900TC (Cys634Arg), c.2071GA (Gly691Ser), IVS12+47CT and c.2712CG (Ser904Ser); 4 variations. CT (Ser396Leu), IVS16-4delA and IVS18+6CT. in which RET c.1296GA (Ala432Ala) and C.2712CG (Ser904Ser), NTRK1c.1187CT (Ser396Leu) and IVS18+6CT are new variants are pathogenic mutations. A 36 year old carrier without clinical symptoms suggests that the disease may have extic or delay in the disease. It is helpful for us to further understand the genotype and phenotype relationship of multiple endocrine neoplasia type 2A, and is of great significance for genetic counseling and prenatal diagnosis of this family.
Background and purpose: chronic obstructive pulmonary disease (COPD; MIM606963) is a common complex disease with increasing incidence and mortality year by year. The main pathogenesis is the interaction of multi gene mutation and environmental factors. Smoking smoking is the most important pathogenic factor of COPD, and the survival of different individuals is stored among different individuals. A better understanding of the genetic factors of COPD susceptibility helps to identify new drug targets, thereby promoting early diagnosis and treatment of COPD. COPD is more susceptible to the disease.
The completion of the human genome project and the the International HapMap Project provides a platform for studying the genetic mechanism of COPD from the whole genome level. The whole genome association study (genome-wide association study, GWAS) can make up thousands of single nucleotide variations (single nucleoti) for the whole gene. De polymorphism, SNP) studies have been applied to the research of COPD related studies and have obtained some positive results in the study of the SNP., which is associated with the symptoms of disease, as a frontier and effective method for the study of complex diseases, but these results need to be verified in large sample and multiracial groups.
GWAS recently found that some of the genetic variations in non Asian populations are associated with COPD susceptibility, such as the iron effect element binding protein gene (the iron-responsive element-binding protein2gene, IREB2; MIM147582), and the family sequence similarity 13A gene (the family with) and X-ray repair. The cross complementation gene 5 (the X-ray repair cross-complementing protein5gene, XRCC5; MIM194364). This study conducted IREB2rs2568494 variation, FAM13A rs2869967 variation and XRCC5rs3821104 variant sequence analysis for COPD patients and normal controls in Chinese mainland Han population to determine the genetic variation and phenotype in Chinese Han population. Relevance.
Methods: 275 COPD patients and 434 normal controls of sex, age and race matched with IREB2rs2568494 mutation, FAM13A rs2869967 variation and XRCC rs3821104 variant DNA sequence PCR were amplified and sequenced directly. Assign3.5 analysis software, MT Navigator software and DNAstar soft parts were used to analyze the sequencing results. Rdy-Weinberg balance test and SPSS16.0 were used to analyze the correlation between gene mutation and COPD in case-control population.
Results: the genotype frequencies of all tests in the normal control were consistent with the Hardy-Weinberg balanced.FAM13A rs2869967 variant genotype and the allele frequency distribution in normal controls and COPD patients (genotype distribution: X2=6.319, P=0.042; allele distribution: X2=4.014, P=0.045); and XRCC5rs3821104 variant variant The frequency distribution of genotype and allele was also statistically significant in normal controls and COPD patients (genotype distribution: X2=6.062, P=0.048; allele distribution: X2=5.607, P=0.018). There was no statistical significance between.IREB2rs2568494 variation and COPD phenotypes (genotype distribution: X2=0.590, P=0.744; allele). B: X2=0.034, P=0.854).
Conclusion: we found that the frequency distribution of genotype and allele of the two mutations of FAM13A rs2869967 and XRCC5rs3821104 in Chinese Han COPD patients and normal controls had statistical significance.IREB2rs2568494 in the genotype and allele frequency distribution of COPD patients and normal controls in Chinese Han people, and there was no statistical significance in the genotype and allele frequency distribution in Chinese Han people and normal controls. There may be differences in the genetic basis of COPD and non Asian populations. The discovery of genetic risk variants helps to establish new COPD early warning and treatment methods.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R563.9

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