一例Crigler-Najjar综合征Ⅱ型患儿的基因突变分析北大核心CSCD

目的对1例Crigler-Najjar综合征Ⅱ型患儿的尿苷二磷酸葡萄糖醛酸转移酶1A1（UGT1A1）基因进行突变分析,探讨其分子发病机制,为家系的遗传咨询及产前基因诊断提供依据。方法抽取患儿及其父母的外周静脉血提取基因组DNA,应用聚合酶链反应扩增UGT1A1基因的相应片段,经DNA测序法对UGT1A1基因进行突变检测,并随访患儿至3岁6个月。结果患儿为持续高未结合胆红素血症,DNA测序结果显示患儿UGT1A1基因的第1外显子存在c．610A〉G（p．Met 204 Val）杂合突变,第4外显子存在c．1091C〉T（p．Pro 364 Leu）杂合错义突变,患儿为UGT1A1基因复合突变杂合子。患儿父亲UGT1A1基因的第4外显子存在C．1091C〉T（p．Pro 364 Leu）的杂合错义突变,患儿母亲UGT1A1基因的第1外显子存在c．610A〉G（p．Met204 Val）杂合错义突变。结论UGT1A1基因第1外显子c．610A〉G、第4外显子c．1091C〉A复合杂合突变为该患儿的发病原因,为其家系的遗传咨询和产前基因诊断提供了依据。     

一例火棉胶样儿TGM1基因的突变分析CSCD

目的对1例维吾尔族火棉胶样患儿的TGM1基因进行突变分析,寻找其病因。方法对患儿及其父母进行常规染色体G显带核型分析,应用芯片高通量测序方法对患儿进行鱼鳞病及鱼鳞病样皮肤病25个相关基因的检测。 结果染色体G显带核型分析结果显示患儿及父母核型均正常;芯片捕获高通量测序检测出患儿TGM1基因的c.919C〉T（p.Arg307Trp）和c.856C〉T（p.Arg286Trp）复合杂合突变;前者为已知致病突变,后者为临床意义未明突变。Sanger测序验证,患儿父亲携带TGM1基因c.856C〉T（p.Arg286Trp）杂合突变,未检出患儿母亲TGM1基因c.919C〉T、c.856C〉T的位点突变。结论TGM1基因c.919C〉T和c.856C〉T复合杂合突变可能是患儿的致病原因。     

一个琥珀酸半醛脱氢酶缺陷症家系ALDH5A1基因的突变检测北大核心CSCD

目的对1个琥珀酸半醛脱氢酶缺陷症家系进行ALDH5A1基因突变分析,为疾病的诊断及遗传咨询提供依据。方法收集患者及其父母外周血,提取基因组DNA,应用PCR扩增产物直接测序法对致病基因ALDH5A1的11个外显子序列及其两侧内含子区域进行分析,以100名健康人为正常对照。结果在患者的ALDH5A1基因上发现第2外显子C．398_399delAA以及第4外显子c．638G〉T（p．R213L）复合杂合突变,家系成员检测证实其中c.398—399delAA突变来自母亲,而c．638G〉T（p．R213L）突变来自父亲。100名健康对照未见上述突变。结论c．398—399delAA和c．638G〉T复合杂合突变是该家系患儿的致病原因,上述突变尚未见报道,丰富了琥珀酸半醛脱氢酶缺陷症致病基因ALDH5A1的突变谱。     

单纯型甲基丙二酸血症家系MUT基因变异的分析及产前诊断CSCD

目的对20个单纯型甲基丙二酸血症家系MUT基因的变异进行测序分析,为家系产前诊断提供依据。方法应用PCR产物直接测序法对20例单纯型甲基丙二酸血症患儿及其父母的MUT基因进行变异检测和分析,明确基因变异情况,并对9名孕妇进行产前诊断。结果20例患儿的家系共检测出19种MUT基因变异,最常见的变异为C.323G〉A（P．Arg108His）、c．1106G〉A（P．Arg369His）、C．729_730insTT（P．D244Lfs＊39）和c．1107dupT（P．T370Yfs＊22）。C．920_923delTCTT（P．F307SIs＊6）、C．419T〉C（P．Leu140Pro）和C．613G〉A（P．Glu205Lys）为未报道过的新变异。Polyphen2和Mutationtaster软件预测这3个变异均可能致病。产前诊断结果显示1例胎儿未检测到MUT基因变异,3例胎儿为MUT基因杂合变异携带者,5例胎儿为MUT基因复合杂合变异或纯合变异患儿。MUT基因正常或杂合变异携带者胎儿的家系选择继续妊娠,而MUT基因纯合变异或复合杂合变异胎儿的家系均选择终止妊娠,胎儿娩出后随访结果与产前诊断结果一致。结论MUT基因突变分析结果为家系的产前诊断提供了依据,新变异的检出丰富了MUT基因突变谱。     

一个先天性肌营养不良症家系的POMT1基因突变鉴定与产前诊断CSCD

目的确定1个先天性肌营养不良症（congenital muscular dystrophy, CMD）家系的POMT1致病基因突变,并对该家系中孕11周的胎儿进行产前诊断。方法收集1例CMD患者及其表型正常父母的外周血标本,抽取母亲孕11周胎儿的绒毛标本。采用PCR扩增POMT1基因的第19和第20外显子,对PCR产物进行双向测序检测基因突变,明确致病突变来源后,进一步对胎儿进行产前诊断。结果先证者POMT1基因第19外显子检测到C．1939G〉A（P．Ala647Thr）杂合错义突变,来自其母亲;第20外显子检测到C．2141delG（P．Trp714Ter）杂合框移突变,导致蛋白质翻译的提前终止,该突变来自其父亲。产前诊断结果显示胎儿携带POMT1基因第19外显子c．1939G〉A杂合错义突变,推测其为与母亲相同POMn基因致病突变携带者的可能性大。结论POMT1基因第19外显子C．1939G〉A错义突变和第20外显子C．2141delG移码突变的复合杂合突变可能是该CMD家系的致病原因,符合常染色体隐性遗传的规律,通过基因产前诊断可以有效阻止致病突变的传递。     

SACS基因复合杂合突变导致Charlevoix-Saguenay型痉挛性共济失调一家系两例CSCD

目的对1个常染色体隐性遗传痉挛性共济失调Charlevoix-Saguenay型（autosomal recessive spastic ataxia of Charlevoix-Saguenay, ARSACS）家系进行SACS基因突变分析,探讨其遗传学病因。方法应用目标区捕获高通量靶向测序对SACS基因进行突变筛查,用Sanger测序对突变位点进行验证。结果测序结果显示先证者和弟弟存在SACS基因C．13085T〉G（P．14362R）和C．5236dupA（P．T1746fs）复合杂合突变,父亲携带SACS基因c．5236dupA（P．T1746fs）杂合突变,母亲携带SACS基因C．13085T〉G（p．14362R）杂合突变,因此患者的C．5236dupA（P．T1746fs）和C．13085T〉G（P．14362R）突变分别来自父母。经检索人类基因突变数据库这两个变异均为未报道过的新突变,根据美国医学遗传学及基因组学会遗传变异解读指南,提示均为可能的致病性突变。结论C．5236dupA（P．T1746fs）和C．13085T〉G（P．14362R）突变为该ARSACS家系患者的遗传学病因,新突变的检出丰富了SACS基因突变谱。     

一例皮肤松弛症患儿的临床与遗传学分析CSCD

目的采用遗传病相关基因外显子测序的方法确定1例皮肤松弛症患儿的致病基因,并对其相关的临床表型及基因型进行总结。方法收集先证者及其父母的临床资料,首先对先证者进行遗传病相关基因的外显子测序,确定了可能的致病突变,对先证者及其父母进行Sanger测序验证,确定基因突变位点。结果先证者存在ATP6VOA2基因C．187C〉T（P．R63X）杂合突变和C．1189G〉C（P．A397P）杂合突变,先证者父母分别携带ATP6VOA2基因c．1189G〉c（P．A397P）杂合突变和C．187C〉T（P．R63X）杂合突变。结合患儿临床表型,诊断为常染色体隐性遗传皮肤松弛症2A型（autosomal recessive cutis laxa type2A,ARCL2A）。结论通过遗传学方法确诊了ARCL2A型患儿,总结了ARCL2A患儿的典型临床特征,新突变的发现扩大了ATP6VOA2基因的突变谱。外显子测序可作为诊断复杂遗传病致病基因的重要工具。     

一个琥珀酸半醛脱氢酶缺陷病家系ALDH5A1基因突变分析

目的 对1例临床怀疑且经尿筛查诊断为琥珀酸半醛脱氢酶缺陷病的患儿及家系进行ALDH5A1基因突变分析,以进一步明确诊断和辅助遗传咨询.方法 应用聚合酶链反应及DNA直接测序技术对1例琥珀酸半醛脱氢酶缺陷病患儿及其父母的ALDH5A1基因进行突变位点检测,同时检测100名健康对照者的ALDH5A1基因以排除多态性变异.结果 患儿携带有ALDH5A1基因编码区序列第3外显子c.527G＞A(p.Gly176Glu)和第4外显子c.691G＞A(p.Glu231Lys)两种杂合突变,其母亲为c.527G＞A杂合突变携带者,父亲为c.691G＞A杂合突变携带者.另外患儿还存在两种已报道的核酸多态性改变:c.545C＞T杂合突变和c.538C＞T纯合突变.患儿的c.545C＞T突变来源于父亲,c.538C＞T突变分别来源于父母.100名健康对照者中未检测到c.527G＞A和c.691G＞A突变.结论 c.527G＞A(p.Gly176Glu)和c.691G＞A(p.Glu231 Lys)错义突变可能是该患儿的致病突变.     

一个白化病家系的TYR基因突变分析北大核心CSCD

目的对1个白化病家系的TYR基因进行突变检测,为遗传咨询和产前诊断提供参考。方法应用PCR技术扩增TYR基因的全部外显子区和外显子-内含子交界区序列,进行DNA测序。结果测序结果显示家系2例患者的TYR基因第2外显子存在c．896G〉A（p．Arg299His）纯合突变,7名表型正常的家系成员的TYR基因第2外显子存在c．896G〉A（Arg299His）杂合突变,7名家系成员和4名正常对照者则均未检测到该突变。结论TYR基因第2外显子c．896G〉A（p．R299H）突变应为该白化病家系的致病原因。     

眼皮肤白化病Ⅰ型产前基因诊断二例

目的对生育过眼皮肤白化病（oculocutaneous albinism,OCA）患儿的2个家系进行基因诊断分型,并在此基础上提供产前基因诊断。方法采用PCR扩增先证者OCA 1型疾病相关基因TYR的所有5个编码外显子,PCR产物直接测序,在确定致病突变的基础上对及家系成员进行综合分析。结果 2个OCA先证者均携带TYR基因复合杂合突变,确定2例先证者均为OCA1型患者。TYR基因共检测到3种突变：c.71G〉A,c.896G〉A和c.929ins C。产前诊断：第1个家系提示胎儿基因型与先证者一致,家属选择终止妊娠;第2个家系中胎儿为TYR基因野生型携带者,继续妊娠至足月分娩,新生儿随访正常。结论利用基因检测可为眼皮肤白化病患者提供确切的临床分型,并在此基础上提供有效的产前基因诊断。     

Maternal isodisomy of the telomeric end of chromosome 2 is responsible for a case of primary hyperoxaluria type 1

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder of glyoxylate metabolism, in which excessive oxalates are formed by the liver and excreted by the kidneys, causing a wide spectrum of disease, ranging from renal failure in infancy to mere renal stones in late adulthood. This disease is caused by a deficiency of alanine:glyoxylate aminotransferase (AGT), which is encoded by a single copy gene, AGXT, located in 2q37.3. We identified an apparently homozygous, loss-of-function, mutation in a patient; the gene defect was present in the heterozygous mother but not in the patient's father. We performed a microsatellite repeat analysis using 13 specific chromosome 2 markers and non-chromosome 2 minisatellites. Six specific chromosome 2 markers showed an apparently homozygous maternal inheritance while four showed a biparental transmission consistent with paternity (confirmed by minisatellite analysis). Quantitative PCR of AGXT exons 1 and 3 on the patient's and parents genomic DNA revealed the presence of two copies of the gene. This is the first case of PH1 caused by segmental maternal isodisomy of 2q37.3.     

Linkage of microsatellites to the AGXT gene on chromosome 2q37.3 and their role in prenatal diagnosis of primary hyperoxaluria type 1

Defects in the AGXT gene mapped to chromosome 2q37.3 cause primary hyperoxaluria type 1 (PH1), one of the inherited disorders of endogenous oxalate overproduction. In order to identify diagnostically useful linkage markers in this region of chromosome 2 we have typed three microsatellite loci mapping to the q37 region of chromosome 2 in 192 individuals from 30 families. They were additionally studied for mutations and polymorphisms in the AGXT gene. Maximum lod scores of 29·1, 22·8 and 15·8 were obtained for D2S140, D2S125 and D2S395 respectively at recombination fractions (theta) of 0·001, 0·015 and 0·02. Confidence intervals for recombination as determined by the 'lod-1 rule' were 0·015, 0·05 and 0·06. Three recombinants were identified between AGXT and D2S125/D2S395, whereas no recombination between AGXT and D2S140 was observed. These data allow the calculation of the risk of incorrect prenatal diagnosis of PH1 based solely on linkage analysis with these extragenic markers.     

Primary hyperoxaluria type 1: a cluster of new mutations in exon 7 of the AGXT gene.

Primary hyperoxaluria type 1 (PH1) is a severe autosomal recessive inborn error of glyoxylate metabolism caused by deficiency of the hepatic peroxisomal enzyme alanine:glyoxylate aminotransferase. This enzyme is encoded by the AGXT gene on chromosome 2q37.3. DNA samples from 79 PH1 patients were studied using single strand conformation polymorphism analysis to detect sequence variants, which were then characterised by direct sequencing and confirmed by restriction enzyme digestion. Four novel mutations were identified in exon 7 of AGXT: a point mutation T853C, which leads to a predicted Ile244Thr amino acid substitution, occurred in nine patients. Two other mutations in adjacent nucleotides, C819T and G820A, mutated the same codon at residue 233 from arginine to cysteine and histidine, respectively. The fourth mutation, G860A, introduced a stop codon at amino acid residue 246. Enzyme studies in these patients showed that AGT catalytic activity was either very low or absent and that little or no immunoreactive protein was present. Together with a new polymorphism in exon 11 (C1342A) these findings underline the genetic heterogeneity of the AGXT gene. The novel mutation T853C is the second most common mutation found to date with an allelic frequency of 9% and will therefore be of clinical importance for the diagnosis of PH1.     

一个遗传性凝血因子Ⅴ缺乏症家系的临床表型及基因分析

目的探讨一个遗传性凝血因子V缺乏症家系的表型特征及其分子致病机制。方法综合分析患儿及其家系成员的临床表现及辅助检查结果,并应用靶向捕获高通量测序及Sanger测序进行变异位点分析和家系验证。结果患儿凝血酶原时间、活化部分凝血活酶时间延长,Ⅴ因子活性仅为0.1%,但无任何出血征象;基因检测结果显示患儿F5基因上携带父源性c.653T>C(p.F218S)杂合变异和母源性c.3642_3643del(p.P1215Rfs*175)杂合变异,患儿哥哥携带父源性的c.653T>C(p.F218S)杂合变异,符合常染色体隐性遗传规律。其中c.653T>C(p.F218S)为已报道的致病性变异,c.3642_3643del(p.P1215Rfs*175)为国际上未见报道的可疑致病性变异。结论明确了F5基因为该患儿的致病基因,靶向捕获高通量测序结合Sanger测序可以快速准确的对该病进行基因变异检测。     

Clinical characteristics and molecular analysis of 21 Chinese children with congenital agammaglobulinemia

Congenital agammaglobulinemia is a humoral primary immunodeficiency and affected patients have extremely low levels of peripheral B cells and profound deficiency of all immunoglobulin isotypes. Mutations of the Bruton's tyrosine kinase (BTK) gene are responsible for most of the congenital agammaglobulinemia. In this study, the phenotypes of congenital agammaglobulinemia were investigated in 21 male children from 21 unrelated Chinese families. Sixteen different mutations of BTK gene were identified in 18 patients, and three patients did not have BTK gene mutations. Nine mutations had been reported previously including one gross deletion (c.722_2041del), one missense mutation (c.1764G>T), three non-sense mutations (c.194C>A, c.895C>T and c.1821G>A) and four invariant splice-site mutations (c.971+2T>C, c.1481+2T>A, c.1482-2A>G, c.1699-2A>G). Seven novel mutations were identified (c.373_441del, c. 504delG, c.537delC, c.851delA, c.1637G>A, c.1879T>C and c. 1482_1882 del). Ten of the eighteen mutations of BTK gene were located in the TK domain, four in the PH domain, three in the SH3 domain and one spanned the TH, SH3, SH2 and TK domain. Candidate genes of autosomal-recessive agammaglobulinemia, including IGHM, CD79a, CD79b and IGLL1, were screened in three patients without mutations in the BTK gene. A compound heterozygosity mutation in the IGHM gene (c.1956G>A, c.175_176insC) was identified in one patient. The results of our study further support that molecular genetic testing represents an important tool for early confirmed diagnosis of congenital agammaglobulinemia and may allow accurate carrier detection and prenatal diagnosis.     

Dent’s disease and prevalence of renal stones in dialysis patients in Northeastern Italy

Dents disease (DD) involves nephrocalcinosis, urolithiasis, hypercalciuria, LMW proteinuria, and renal failure in various combinations. Males are affected. It is caused by mutations in the chloride channel CLCN5 gene. It has been suggested that DD is underdiagnosed, occurring in less overt forms, apparently without family history. A possible approach to this problem is to search for CLCN5 mutations in patients who may have a high prevalence of mutations: end-stage renal disease (ESRD) patients with previous calcium, struvite, or radio-opaque (CSR) stones. We looked for CLCN5 mutations in 25 males with ESRD–CSR stones selected from all of the patients (1,901 individuals, of which 1,179 were males) of 15 dialysis units in the Veneto region. One DD patient had a new DD mutation (1070 G>T) in exon 7. The new polymorphism IVS11–67 C>T was detected in intron 11 in one patient and one control. We also found 28 females with ESRD and stone history, and seven more males with ESRD and non-CSR stones. The prevalence of stone formers among dialysis patients in our region was 3.2%, much lower than the prevalence observed in older studies. Struvite stones continue to play a major role in causing stone-associated ESRD .Preliminary data from this paper were presented at the 10th International Symposium on Urolithiasis, Hong Kong, May 25–28, 2004, and published in abstract form in Urol Res 32:152–153, 2004     

一例罕见的新的TCIRG1基因杂合性突变引起的婴儿恶性石骨症

目的:研究1例婴儿恶性石骨症患者的致病基因及其突变。TCIRG1和CLCN7是婴儿恶性石骨症最常见的致病基因。前者被认为是纯合性致病基因,国外只有6例其杂合性改变也致本病的报导,而我国无杂合性突变导致本病的报道。方法:通过骨组织X线检查结合临床症状及体征确诊1例散发性婴儿恶性石骨症患者。提取患者及其父母的外周血基因组DNA,PCR扩增TCIRG1和CLCN7基因外显子及其剪切位点序列,对PCR产物直接测序。用TCRIG1基因附近的微卫星标记和SNP构建患者及其父母的单倍型。用染色体微阵列分析技术对患者及其母亲进行TCIRG1基因拷贝数目变异的检测。结果:患者TCIRG1基因第5号外显子内发现一个4个碱基的缺失突变c.449_452del AGAG(p.Gln149Glnfs16),该突变使得基因3’端编码的666个氨基酸被截断,失去了整个ATP酶V0复合结构域。患儿双亲TCIRG1和CLCN7基因的突变检测及单倍体构建证实该突变来源于患者父亲。染色体微阵列分析未发现患儿及其母亲携带有任何累及TCIRG1及CLCN7基因的拷贝数目变异。结论:本研究发现了1例TCIRG1基因新的杂合性突变所致的婴儿恶性石骨症。这是我国TCIRG1基因杂合性突变引起婴儿恶性石骨症的首例报道。这个发现可用于婴儿恶性石骨症的分子诊断。     

X-连锁无汗性外胚层发育不良一家系EDA新发突变

目的检测一X连锁无汗性外胚层发育不良家系EDA基因突变。方法提取先证者、其姐和父母外周血基因组DNA,PCR扩增EDA基因编码区的8个外显子,PCR产物测序,明确突变位点。结果先证者EDA基因6号外显子第781位胞嘧啶C突变成胸腺嘌呤T,使其编码的蛋白第261位谷氨酰胺密码子CAA变成终止密码子UAA,蛋白表达提前终止;患者的姐姐和母亲在该位点均为C/T杂合子,患者父亲在该位点为C纯合子。结论本家系中c.781C>T突变为国内外首报,是导致X连锁无汗性外胚层发育不良临床表型的原因。     

一例视网膜色素变性患者 C2ORF71基因的变异分析

目的:分析1例视网膜色素变性(retinitis pigmentosa, RP)患者的基因变异,明确其可能的遗传学病因。方法:应用全外显子测序技术对先证者进行致病基因筛查,结合临床表型确定可疑变异,应用Sanger测序法验证检出的变异,分析双亲携带变异位点的情况。采用多种软件对所检出的变异进行致病性分析。结果:全外显子...     

X-linked Opitz G/BBB syndrome: identification of a novel mutation and prenatal diagnosis in a Korean family

X-linked Opitz G/BBB syndrome (XLOS; MIM 300000) is a rare multiple congenital anomaly disorder that is characterized by facial anomalies, laryngeal/tracheal/esophageal defects and genitourinary abnormalities. XLOS is caused by mutations in the MID1 gene which encodes a microtubule-associated RING-Bbox-Coiled-coil (RBCC) protein. We recently found a four-year Korean male patient who was suspected of having XLOS. Mutation analysis of the MID1 gene in the patient and his mother demonstrated that the patient had a novel insertion mutation (c.1798_1799-insC), and his mother was a heterozygous carrier of the mutation. After identification of the causative mutation in this family, prenatal diagnosis of two consecutive fetuses were successfully undertaken. This is the first report on a genetically confirmed case of XLOS in Korea.