X-连锁迟发性脊髓骨骺发育不良家系的基因诊断

目的 探讨一个X-连锁迟发性脊髓骨骺发育不良(X-linked spondyloepiphyseal dysplasia tarda,SEDL)家系发病的分子机制,建立基因诊断方法 .方法 采用身高测量、影像学检查殚及家系分析进行临床诊断.收集相关家系成员的外周血标本.提取基因组DNA后,采用PCR-SSCP和DNA测序分析家系中先证者、携带者和健康人SEDL基因的第3～6外显子.利用微卫星位点DXS16进行连锁分析.结果 PCR-SSCP检测到先证者第4外显子有异常泳动带;第4外显子的序列分析表明3例先证者均存在C.218C＞T突变,导致氨基酸序列S73L改变,3例携带者均存在该核苷酸位点的杂合突变,健康人未见突变;先证者的第3、5和6外显子核酸序列未见突变.序列分析证实家系中3名未婚女性Ⅲ10、Ⅳ6和Ⅳ7为致病基因携带者.结论 C.218C＞T突变是导致该家系发病的分子机制,可采用第4外显子序列分析对该家系进行基因诊断和产前基因诊断.     

X-连锁迟发性脊髓骨骺发育不良家系的基因诊断

ObjectiveTo investigate the molecular pathogenesis of a pedigree of X-linked spondyloepiphyseal dysplasia atarda (SEDL) and to establish methods of gene diagnosis. Methods Clinical diagnosis was made based on height measurement, radiological examination and pedigree analysis. Peripheral blood samples of relevant family members were collected. After genomic DNA extraction, single strand conformation polymorphism (SSCP) followed with DNA sequencing was used to detect SEDL gene exons 36. Microsatellite marker DXS16 was selected for linkage analysis. Results The abnormal electrophoretic bands were detected in exon 4 of probands by PCR-SSCP. A c. 218C ＞ T mutation in exon 4 of SEDL gene was found in three probands, which resulted in a change in amino acid sequence S37L. The heterozygous exon 4 mutation was identified in three carriers, but not in healthy individuals, and no mutations were detect in exon 3, 5 and 6 of probands. Three unmarried young females (Ⅲ10, Ⅳ6 and Ⅳ7) were found to harbor the mutation by DNA sequencing analysis. ConclusionsA c. 218C ＞ T missense mutation in exon 4 of SEDL gene is the cause of molecular pathogenesis of the pedigree. SSCP and DNA sequencing can be used for prenatal gene diagnosis.     

连锁分析在X-连锁迟发性脊椎骨骺发育不良诊断中价值

目的探讨微卫星连锁分析方法对X-N锁迟发性脊椎骨骺发育不良（X—linked spondyloepiphyseal dysplasia tarda,SEDL）家系症状前患者实施间接基因诊断的可行性。方法应用聚合酶链反应、变性聚丙烯酰胺凝胶电泳及银染色技术检测微卫星DXS16的片段长度多态性,通过连锁分析对4个SEDL家系中11例年幼待诊对象进行SEDL症状前患者或携带者的诊断。结果11例中2例为症状前患者,3例女性为致病基因携带者,其余6例基本排除SEDL症状前患者或致病基因携带者。结论微卫星连锁分析方法对SEDL症状前患者有较高诊断价值。     

PCR-SSCP对子宫内膜癌nm23-H1基因突变的检测

子宫内膜癌是女性生殖道常见的恶性肿瘤 ,其发生多脏器转移是死亡的主要原因。 nm2 3- H1基因作为近年来所发现的众多转移抑制基因之一 ,受到广泛的关注。 nm2 3- H1基因在信号传递、转录调控、细胞粘附等多个环节中影响染色体合成 [1 ] ,进而影响细胞增殖与分化。在对结肠癌、卵巢癌等 [2 ] 的研究中发现随癌灶进展 ,nm 2 3- H1突变率增加。对 1998- 0 9～ 2 0 0 2 - 0 5我们应用 PCR- SSCP技术检测人子宫内膜癌组织中 nm2 3- H1基因突变检测 ,分析如下。1　对象和方法1.1　对象　正常子宫内膜组织 2 0例 ;增生过长内膜组织 35例 ,其…     

1个X-连锁迟发性脊椎骨骺发育不良家系的TRAPPC2基因突变分析

目的确定1个迟发性脊椎骨骺发育不良(spondyloepiphyseal dysplasia tarda,SEDT)家系的致病基因。方法收集先证者及家系的临床资料,提取先证者及亲属外周血DNA,用高通量测序技术对先证者的COL2A1、COL1A1、MATN3、TRAPPC2、FGFR3等189个骨骼相关基因的外显子编码区测序,对发现的致病突变进行Sanger测序验证,并对家系其他成员进行该突变的检测。结果在先证者TRAPPC2基因第5外显子上发现了1个移码突变c.271_275del CAAGA半合子缺失,为X-SEDT的致病性突变,同时发现患者母亲为该突变的携带者,而患者父亲和妹妹未检测到该突变。结论用靶向二代测序和Sanger测序结合的方法确定了1个X-SEDT家系的移码突变c.271_275del CAAGA半合子缺失,为临床遗传咨询提供了分子依据。     

fas基因和p53基因在瘢痕疙瘩成纤维细胞中的结构异常与其功能的关系

目的：人体内瘢痕疙瘩成纤维细胞对生长因子的需要量，以及对生长抑制因子的反应都与正常成纤维细胞不同。探讨，fas基因和P53基因在瘢痕疙瘩成纤维细胞中的结构变化与其功能的关系。方法：实验于2001-03／09在解放军第一军医大学热带病研究所完成。采用聚合酶链反应-单链构象多态性及基因测序技术，以增生性瘢痕及正常皮肤为对照，检测瘢痕疙瘩组织标本中成纤维细胞fas全基因编码序列和p53基因突变高发区外显子4～6的基因结构，以其变化判断在瘢痕形成中抑制细胞进程及介导细胞凋亡的功能。结果：瘢痕疙瘩fas基因外显子6，8，9及p53基因外显子4，5，6均存在突变，对照组未发现突变。结论：正常皮肤fas和P53基因结构无变异，瘢痕疙瘩成纤维细胞，fas基因和p53基因外显子的突变，致介导细胞凋亡及抑制细胞进程等功能丧失．与瘢痕疹瘩的形成有关。     

载脂蛋白E基因多态性检测技术的对比性研究

目的建立并比较载脂蛋白Ｅ（ＡｐｏＥ）基因多态性检测技术,为ＡｐｏＥ基因分型提供良好的方法。方法应用聚合酶链反应（ＰＣＲ）限制性片段长度多态性（ＲＦＬＰ）和ＰＣＲ单链构象多态性（ＳＳＣＰ）检测技术对１１０例患者及相同人数的正常人进行ＡｐｏＥ基因型检测和分析。结果应用ＲＦＬＰ、ＳＳＣＰ技术测得正常对照组：ε２／２（０,０．０２０）,ε３／３（０．７００,０．７６０）,ε４／４（０,０．０４０）,ε２／３（０．１００,０．０６０）,ε２／４（０,０．０２０）,ε３／４（０．２００,０．１００）,ε２（０．０５０,０．０６０）,ε３（０．８５０,０．８４０）,ε４（０．１００,０．１００）。经χ２检验,ＡｐｏＥ基因型频率和等位基因频率的差异无显著意义（Ｐ＞０．０５）。上两种检测技术与既往两种检测技术（ＩＥＦ,ＷｅｓｔｅｒｐＢｌｏｔｉｎｇ）的结果经χ２检验,其ＡｐｏＥ等位基因频率的差异也无显著意义（Ｐ＞０．０５）,均符合中国汉族人ＡｐｏＥ基因频率分布。结论ＰＣＲＲＦＬＰ方法是一种稳定可靠的ＡｐｏＥ基因多态性分析方法,多重ＰＣＲＳＳＣＰ技术更加简便有效,使ＡｐｏＥ基因多态性的研究更加完善。     

凝血因子Ⅴ及凝血因子Ⅷ联合缺陷患者四例的基因诊断

目的 对4例FⅤ及FⅧ联合缺陷患者及其家系成员进行基因诊断及分子发病机制研究.方法 测定先证者及家系成员APTT、PT、FⅧ:C及FⅤ:C等进行表型诊断;采用凝血酶生成试验检测先证者及健康对照者的凝血酶生成情况;Tiangen试剂盒法抽提先证者及家系成员全血基因组DNA;平衡酚-乙醚法抽提羊水DNA;PCR扩增4例先证者及家系成员的LMAN1基因及MCFD2基因,用末端标记双脱氧法检测核酸序列查找基因突变.结果 先证者1的APTT显著延长,为88.2 s,PT 延长至19.6 s,FⅧ:C降至24.2%,FⅤ:C为9.1%.先证者2与先证者3为亲姐妹,两人的APTF均明显延长,分别为71.6 s及74.6 s,PT分别延长至22.1 s及18.3 s,FⅧ:C均降低,分别为25%及19.6%,FⅤ:C分别降至7.6%及14.5%.先证者4的AFTT及PT均延长,分别为70.3 s及18.2 s,其FⅦ:C降至15.7%,FV:C降至9.4%,4例先证者的其余实验室表型检测指标均正常,临床诊断为FⅤ及FⅧ联合缺陷性疾病.对先证者1进行LMAN1及MCFD2基因直接测序,显示其LMAN1基因存在双杂合突变:突变1位于第8号外显子,为插入突变:nt912insA(X71661.1),导致第305位氨基酸发生移码突变,并在编码20个氨基酸后终止,其母亲该位点亦为杂合突变;先证者1的另一个杂合突变位于第11号外显子:nt1366C>CT(X71661.1),导致第456位精氨酸发生无义突变(p.Arg456X),其父亲及胎儿该位点均为杂合突变.先证者1及其父母的MCFD2基因测序均未发现突变.先证者2及3的LMAN1基因测序均未发现突变,MCFD2基因直接测序检测发现两者均在该基因的第4号外显子处存在1个纯合突变:nt411T>C(NM_139279),导致第136位异亮氨酸突变成苏氨酸(p.Ile136Thr).先证者2的女儿该位点为杂合突变.先证者4的LMAN1基因测序显示其在该基因的第5号外显子存在1个纯合突变:nt615C>T,导致第202位的精氨酸无义突变;其MCFD2基因测序未发现突变.凝血酶生成试验检测显示4例FⅤ及FⅧ联合缺陷患者的凝血酶生成潜力较健康对照者均有不同程度的下降.结论 先证者1是由LMAN1基因的双杂合突变nt1366C>CT及nt912insA引起,突变分别遗传自其父亲及母亲,对其母亲进行产前基因诊断发现胎儿为1名女性携带者,该胎儿遗传了其父亲的1个杂合突变nt1366C>CT.先证者2及3是由MCFD2基因上的纯合突变(nt411T>C,p.ne136Thr)所导致的.先证者2的女儿遗传了其母亲的一个杂合突变,为携带者.先证者4是由LMAN1基因的纯合无义突变(nt615C>T,p.Arg202X)所导致的.     

fas基因和p53基因在瘢痕疙瘩成纤维细胞中的结构异常与其功能的关系

目的:人体内瘢痕疙瘩成纤维细胞对生长因子的需要量,以及对生长抑制因子的反应都与正常成纤维细胞不同。探讨fas基因和p53基因在瘢痕疙瘩成纤维细胞中的结构变化与其功能的关系。方法:实验于2001-03/09在解放军第一军医大学热带病研究所完成。采用聚合酶链反应-单链构象多态性及基因测序技术,以增生性瘢痕及正常皮肤为对照,检测瘢痕疙瘩组织标本中成纤维细胞fas全基因编码序列和p53基因突变高发区外显子4～6的基因结构,以其变化判断在瘢痕形成中抑制细胞进程及介导细胞凋亡的功能。结果:瘢痕疙瘩fas基因外显子6,8,9及p53基因外显子4,5,6均存在突变,对照组未发现突变。结论:正常皮肤fas和p53基因结构无变异,瘢痕疙瘩成纤维细胞fas基因和p53基因外显子的突变,致介导细胞凋亡及抑制细胞进程等功能丧失,与瘢痕疙瘩的形成有关。     

HLA-A、B、C基因第二外显子的单链构象多态性分析及骨髓移植受供者基因配型

用与HLA-A、B、C座位第二外显子两末端稳定部位互补的,对引物扩增基因组DNA得到HLA-A、B、C三座位的第二外显子包含高变区在内的196bp片断,进行单链构象多态性分析。以此检测HLA-Ⅰ类基因多态性的一致性,为20例骨髓移植供受者对配型,其中11对相合,9对不合。与其它配型结果比较表明,HLA-Ⅰ类基因的聚合酶链反应一单链构象多态性(PCR-SSCP)配型在区分同胞供受者Ⅰ类抗原多态性上有较高的灵敏度。     

X-linked spondyloepiphyseal dysplasia tarda: Identification of a TRAPPC2 mutation in a Korean pedigree

Spondyloepiphyseal dysplasia (SED) comprises a heterogeneous group of skeletal dysplasias that primarily affect the epiphyses and vertebral bodies. Patients affected by SED usually exhibit short stature and experience early development of degenerative osteoarthritis. SED is subdivided into congenita and tarda forms according to the age at onset and clinical severity, and further subdivided into genetically different forms according to the mode of inheritance and the gene involved. We report a 14-yr-old Korean male who presented with a disproportionately short stature and a short trunk. A pedigree analysis of 3 generations with 6 affected persons revealed an X-linked recessive mode of inheritance. Mutation analysis of the TRAPPC2 (previously called SEDL) gene, the only gene associated with X-linked spondyloepiphyseal dysplasia tarda (X-linked SEDT; MIM 313400), was performed, and a splice-donor site mutation in intron 3 of the TRAPPC2 gene (c.93+5G>A) was identified in the proband and in his unaffected mother (a heterozygote). This mutation is one of the 2 most frequent mutations reported in the medical literature, and is known to result in exon 3 skipping. This is the first report of a genetically confirmed X-linked SEDT case in Korea and highlights the importance of recognizing the mode of inheritance in the diagnosis of X-linked SEDT.     

Molecular prenatal diagnosis in 2 pregnancies at risk for spondyloepiphyseal dysplasia congenita

BACKGROUND: Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant skeletal dysplasia characterized by short stature, abnormal epiphyses, and flattened vertebral bodies. Secondary prevention of SEDC can be achieved by prenatal diagnosis. Reports of antenatally-diagnosed SEDC fetuses have been very rare and molecular prenatal diagnosis even rarer. We previously reported a familial G504S mutation in the type II collagen (COL2A1) gene resulting in SEDC. In this study, molecular prenatal diagnosis was performed to 2 couples in this family with pregnancies at risk for SEDC. METHODS: Amniotic fluid was sampled by amniocentesis under ultrasound guidance at 19+3 and 18+6 weeks' gestation, respectively. Karyotype and molecular genetic analysis were performed on cultured amniotic fluid cells. Maternal cell contamination was excluded by short tandem repeat (STR) analysis. Direct DNA sequencing and DHPLC were conducted to detect the potential mutation in exon 23 of COL2A1 gene. Both women underwent serial sonograms because they insisted that the molecular diagnosis should be confirmed by another method, although they had been informed that mutation analysis is predictive of the disease. RESULTS: Karyotype of both fetuses was normal and molecular genetic analysis revealed that fetus 1 carried a G504S mutation in exon 23, while fetus 2 was normal. In case 1, femur length of the fetus was markedly below the 5th centile at 23 weeks' gestation, which confirms the accuracy of molecular diagnosis. A medical termination was carried out at 27+5 weeks' gestation and a male fetus with a relatively large head and short limbs was delivered. The fetal radiograph demonstrated a number of features, including generalised platyspondyly, absent ossification of the vertebral bodies in the cervical region and significant shortening of the long bones. The diagnosis of SEDC was thus confirmed clinically. Ultrasound monitoring of fetus 2 showed that its femur length was normal for gestational age at repeated scans, which was consistent with the molecular diagnosis. CONCLUSIONS: Molecular analysis allows early and accurate prenatal diagnosis for SEDC once mutation is known in a family. However, considering the poor genotype/phenotype correlation in many cases of SEDC, the combination of ultrasound as well as molecular genetic approach might be needed.     

中国人2型糖尿病患者胰岛素受体底物-1基因变异的筛选研究

目的研究胰岛素受体底物 -1(IRS -1)基因变异与 2型糖尿病 (NIDDM )发生的关系。方法 :采用聚合酶链反应 -单链构象多态性 (PCR -SSCP)分析方法筛选了 68例中国人 (来自长沙的病例 ) 2型糖尿病患者和 68例正常对照的胰岛素受体底物-1基因的 + 170 0～ + 44 3 7bp片段。结果中国人2型糖尿病患者胰岛素受体底物 -1基因变异的发生频率明显高于正常对照 (3 8.2 %vs . 7.4% ,χ2 =18.42 ,P <0 .0 1)。结论胰岛素受体底物 -1基因的核苷酸变异可能与中国人 (来自长沙的病例 ) 2型糖尿病的发生相关     

迟发性骨骺发育不良1例

患者男,29岁。三年来无明显诱因出现腰背部及髋、膝关节疼痛,最近一年腰背部伸展活动受限,久坐后需缓慢活动后方可行动。体检：患者身高约145cm,躯干短小,四肢相对较长,立位时手指尖几达膝部,肘关节略有膨大,行走时步态未见明显异常,智力发育正常。     

应用单链构象多态性和异源双链分析研究拉米夫定耐药变异

目的 应用单链构象多态性/异源双链分析技术研究慢性乙型肝炎患者拉米夫定治疗前后乙肝病毒准种组成的改变。方法 聚合酶链反应扩增6例患者治疗前后共12份血清标本中目的片段并逐一分析。结果 拉米夫定治疗前患者体内乙肝病毒聚合酶基因序列准种组成较复杂,准种数在7-14(平均9.8),均高于治疗后准种数4-8(平均5.7),t=3.98,P<0.05。6例患者治疗前的准种分布中有一种或两种优势准种,但比例较低(33.3％-81.80％);治疗后显著升高(78.80％-90.9％),t=3.24,P<0.05。选择优势克隆测序,6例患者经拉米夫定治疗后2例在酪氨酸-蛋氨酸-天冬氨酸-天冬氨酸(YMDD)序列出现M550V/L528M,3例为M5501变异,1例无变异。结论 采用单链构象多态性/异源双链分析技术对人体内病毒作整体性的研究,可避免其他研究方法的片面性,为发现新的变异点提供机会,进一步解释拉米夫定的耐药机制。     

脊柱骨骺发育不良的影像学表现

目的：分析脊柱骨骺发育不良的影像学特征。方法：12例脊柱骨骺发育不良患儿，其主要临床症状为躯干与四肢不成比例的矮小身材、背部和肢体疼痛、脊柱侧弯及畸形足等。所有患儿进行一系列的X线检查。结果：检查发现头颅直径增大3例，齿状突发育不良2例，扁平椎12例，椎间隙狭窄12例，髋内翻11例，股骨近端骨骺骨化延迟12例．青少年髋关节退行性改变6例。结论：脊柱骨骺发育不良的影像学表现有其特征性，结合其临床表现能够做出明确诊断。