广西壮族自治区南宁市新生儿GJB2 致聋基因的携带研究

目的 探讨南宁市新生儿GJB2致聋基因携带状况和突变位点与耳聋相关性。方法 收集2016～2018年期间在南宁市新生儿疾病筛查中心进行听力筛查未通过的1 007例新生儿血液,制成干血斑纸片,提取DNA,采用测序法进行GJB2全基因测序。统计新生儿GJB2基因突变的携带率,并结合新生儿听力损失确诊情况分析致聋基因突变特点。结果 1 007例新生儿中GJB2致聋基因突变携带率为55.21%(556/1 007),检测出GJB2基因突变位点16个,其中GJB2 c.109 G>A 突变携带率较高,占20.36%,其次是c.79 G>A 占10.03%,c.608 T>C 占7.65%,c.217 C>A 占7.15%,c.341 A>G 占6.85%,其它位点(包括c.11 G>A,c.-23+1 G>A, c.235del C, c.299 del AT,c.-121 G>A,c.464 A>G,c.147 C>G, c.676 G>T,c.316 T>G,c.-23-35 G>T 和c.368 C>A)突变携带率<0.1%。556例GJB2致聋基因突变者中有31例确诊为不同程度耳聋,4个致聋突变位点包括GJB2 c.109 G>A(26例),c.11 G>A(2例),c.235del C(2例)和c.299 del AT(1例),分别占耳聋患者比率是83.87%,6.45%,6.45%和3.23%。81例检出6个新突变位点c.-23-35 G>T,c.-121 G>A,c.464 A>G, c.217 C>A,c.147 C>G和 c.676 G>T及4例检出1个未明确致病性位点c.-23+1 G>A,另外有250例检出5个常见多态性突变位点。GJB2 c.109 G>A携带率与北京、台湾和上海比较差异有统计学意义(χ²=133.364,11.724,32.449,均P<0.05)。结论 南宁市新生儿GJB2致聋基因携带率较高,筛查出4个致聋突变位点,6个新突变位点及1个未明确致病位点,明确了致聋突变位点以c.109 G>A纯合子突变为主,基因突变特点有明显的地域性差异。     

山东省30例苯丙酮尿症患者苯丙氨酸羟化酶基因的突变分析

目的 研究山东省30例苯丙酮尿症患儿苯丙氨酸羟化酶基因的突变特点。方法 使用二代测序技术结合生物信息学分析手段检测PAH缺乏型苯丙酮尿症患者PAH基因的变异位点。结果 30例患者的60个PAH等位基因中共检测出53个PAH基因位点突变,检出率为88.3%。检测出的27种PAH基因的突变类型包括17种错义突变、4种无义突变、4种剪接突变、1种同义突变和1种移码突变。突变分布在第1-3、5-7、10-12号外显子和第4号、5号、7号、12号内含子上。其中热点突变为c.728G>A(24.53%)、c.721C>T(7.55%)和c.1238G>C(5.66%),新突变位点为：c.155T>G和c.621T>A。结论 山东省30例PKU患儿的PAH基因突变以错义突变为主,主要发生在第7号外显子上。研究检测出PAH基因的27个突变位点,包括2个新的突变位点,为苯丙酮尿症的产前诊断和机制研究提供依据。     

石家庄市苯丙氨酸羟化酶缺乏症的基因突变研究

目的分析石家庄市新生儿疾病筛查诊治中心确诊的苯丙氨酸羟化酶缺乏症(PAHD)患儿中PAH基因的突变规律及特点,为石家庄市PAHD的产前诊断、治疗提供有力的科学依据。方法收集在石家庄市新生儿疾病筛查诊治中心确诊的PAHD患儿67例作为研究对象,提取患儿及其父母外周血DNA标本进行二代测序,测序范围包括13个外显子和外显子上、下游200个碱基对的内含子,对可疑突变位点进行一代测序验证,可疑大片段缺失重复进行多重连接探针扩增验证。结果67例PAHD患儿中61例患儿PAH基因检测到2个突变位点,3例患儿PAH基因检测到3个突变位点,3例患儿PAH基因只检测到1个突变位点。67例PAHD患儿中共检测到44种突变,134个PAH基因突变位点。高频突变位点主要是c.158G>A、c.728G>A、c.331C>T。16例经典型苯丙酮尿症患儿中c.1068C>A和c.331C>T突变频率最高(15.63%,5/32);11例轻度苯丙酮尿症患儿中c.721C>T突变频率最高(18.18%,4/22);40例轻度高苯丙氨酸血症患儿中c.158G>A突变频率最高(35.00%,28/80)。检测出的134个突变位点中有3个尚未见报道,分别为c.912+5G>T、c.61-1G>A和c.630T>G。结论石家庄市PAHD患儿PAH基因突变以复合杂合突变为主,该研究明确了石家庄市PAHD患儿基因的突变类型与特点,为深入开展PAHD的诊断及产前诊断、治疗和遗传咨询提供了有力的依据和帮助。     

遗传性椭圆形红细胞增多症患者临床及基因突变特征:9例报告及文献复习北大核心CSCD

目的报告9例遗传性椭圆形红细胞增多症(hereditary elliptocytosis,HE)患者基因突变类型并分析HE致病性基因突变的特征。方法报告中国医学科学院血液病医院2018年6月至2022年2月临床诊断的9例HE患者临床及基因突变特征,应用Sanger测序方法进行验证,分析基因突变构成、突变类型、基因型及与临床表型之间的关系。结果9例HE患者中6例为SPTA1、1例为SPTB、1例为EPB41红细胞膜蛋白基因突变,另1例为20号染色体拷贝缺失。共涉及11个基因突变位点,包括6个已知突变和5个新发突变。5个新发突变分别为:SPTA1基因9号外显子c.1247A>C(p.K416T),15号外显子c.1891delG(p.A631fs*17),6~12号外显子Del;SPTB基因c.154C>T(p.R52W);EPB41基因c.1636A>G(p.I546V)。6例SPTA1突变患者中3例为SPTA19号外显子突变。结论SPTA1是HE患者最常见的突变基因。     

胃肠间质瘤患者肿瘤组织中c-Kit基因和PDGFRA基因突变的分子病理检测分析

目的:探讨胃肠间质瘤(gastrointestinal stomal tumors,GIST)患者肿瘤组织中c-Kit和PDGFRA基因各亚型突变情况的研究。方法:应用直接测序方法检测65例GIST石蜡组织中c-Kit基因9、11、1 3和1 7外显子和P D G F R A基因1 2和1 8外显子突变情况。结果:G I ST肿瘤组织中c-K i t基因总突变率为63.08%(41/65),外显子9、11、13和17的突变率分别为23.08%(15/65)、35.38%(23/65)、1.54%(1/65)和3.08%(2/65);c-Kit基因各外显子之间双重突变共2例(3.08%),其中9外显子与11外显子双重突变1例(1.54%),11外显子与17外显子双重突变1例(1.54%);c-Kit基因11号外显子内双重突变2例(3.08%),三重突变1例(1.54%)。PDGFRA基因总突变率为3.08%(2/65),均为外显子18突变。c-Kit基因和PDGFRA基因双突变共存型1例(1.54%),为c-Kit基因13外显子V654L点突变和PDGFR A基因18外显子D842V点突变。结论:GIST患者中c-Kit基因存在较高的突变率,尤其为9和11外显子突变,其基因突变亚型分类能指导精准医学下伊马替尼的肿瘤靶向治疗,PD GF R A基因突变率和存在两种及其以上突变发生的发生概率虽低但不容忽视。     

1例罕见Melnick-Needles综合征患者基因变异分析

摘要:目的研究 1例Melnick-Needles综合征家系的致病基因并为该家系提供遗传咨询依据。方法采集先证者即 1例不明原因骨骼发育异常、身材矮小的青年女性患者及其父母外周血标本,提取基因组DNA,采用家系全外显子组测序(Trio-WES)筛查先证者致病基因及突变位点，并通过Sanger测序进行验证。结果先证者位于 X染色体上的FLNA基因有一杂合变异位点:NM_ 001 110556.2: c.3562G>A(p.A1188T),父母均为野生型。根据ACMG指南,提示FLNA基因的c.3562G>A突变为1个致病突变位点,与X连锁显性遗传疾病Melnick-Needles综合征相关。国内未见该致病位点报道。结论确诊1 例罕见Melnick-Needles综合征,致病FLNA突变c.3562G>A在国内为首次报道,为患者及家属遺传咨询提供了依据。     

广州市地区7234例新生儿耳聋基因突变分析

目的分析广州地区7234例新生儿4个常见遗传性耳聋基因热点突变情况,为广州地区遗传性耳聋的防治提供依据。方法收集广州市天河区中山大学附属第三医院2015年6月至2019年1月分娩的7234例新生儿脐带血液标本进行耳聋基因热点检测,基因突变新生儿行Sanger测序验证及基因外显子测序。结果检测到耳聋基因突变携带者266例(3.68%),其中GJB2基因突变159例(22.0%),SLC26A4基因突变85例(1.18%)。在266例的基因携带者中,GJB2 c.235delC杂合突变占49.6%(132/266),SLC26A4 IVS7-2A>G突变占28.2%(75/266),其余位点占22.2%。携带者一代测序验证结果与耳聋基因筛查结果一致,基因外显子测序中发现有3名双重突变患儿,两例为GJB2 c.235 del C与GJB2 c.109G>A双重杂合突变患儿,另一例为SLC26A4 IVS 7-2 A>G与SLC26A4 c.1983C>A双重杂合突变患儿,3例患儿经父母血液标本验证,双重杂合位点分别遗传自父母。结论本研究7234例新生儿聋基因筛查中,GJB2基因突变频率最高,其次是SLC26A4基因,为耳聋儿童的早期发现与早期干预提供参考与指导。现行热点突变检测位点可能漏检其他突变位点,可根据本地检测情况调整或者制定适合本地的筛查方案。     

原发、复发及转移胃肠道间质瘤KIT及PDGFRA突变与临床病理特征间的关系

目的:探讨原发、复发及转移性胃肠道间质瘤(gastrointestinal stromal tumor,GIST)中的KIT及PDGFRA基因突变与免疫表型、临床病理学特征之间的相关性。方法:收集2004年到2020年本院167例资料完整的原发GIST患者,其中复发及转移患者22例。运用免疫组织化学(免疫组化)染色及第一代测序技术对167例原发GIST病灶及22例复发或转移转移灶进行CD117、DOG1、CD34和Ki-67增殖指数检测,以及KIT基因第9、11、13、17号外显子和PDGFRA基因第12、18号外显子测序。结果:167例原发GIST中KIT和PDGFRA基因的突变率分别为83.8%(140/167)和3.0%(5/167)。KIT外显子11突变率为74.9%(125/167),外显子9突变率为8.4%(14/167),外显子17突变率为0.6%(1/167)。5例GIST原发灶中存在PDGFRA基因外显子18突变,其中4例为D842V突变。KIT外显子突变类型包括点突变(34.3%,48/140)、缺失突变(40.7%,57/140)、重复突变(3.6%,5/140)、混合突变(插入合并缺失,12.1%,17/140),其中携带KIT外显子11非点突变的GIST(n=76)Ki-67增殖指数(10.7%)高于携带外显子11点突变(n=45)的GIST(8.1%)(P=0.005 2)。22例复发及转移GIST病灶中,KIT双外显子突变共有9例,其中6例为外显子11和17双突变,2例为外显子11和13双突变,1例为外显子9和13双突变。在22例复发、转移GIST患者中,与KIT单突变(中位时间60个月)及野生型(中位时间30个月)相比,KIT双外显子突变患者的无疾病进展时间显著延长(中位时间为108个月,P=0.029 9和P=0.011 1),而Ki-67表达值、CD117、DOG1、CD34、肿瘤直径及核分裂象与前二者间差异无统计学意义。结论:原发GIST灶中,以KIT基因外显子11突变为主,缺失突变最多,占40.7%;原发灶KIT基因外显子11非点突变的肿瘤患者,其Ki-67表达值高于点突变类型患者,可能提示该类患者的肿瘤生物学行为更差。与原发灶KIT基因突变模式均为单外显子突变不同,复发及转移灶存在KIT基因双外显子突变,且占比较高(9/22)。在肿瘤转移、复发患者中,与野生型及单突变者相比,KIT基因双外显子突变者无疾病进展时间较长。     

武汉地区葡萄糖-6-磷酸脱氢酶缺乏症患者基因突变分析

目的:探讨武汉地区葡萄糖-6-磷酸脱氢酶(G6PD)缺乏症患者的基因突变特征。方法:对湖北省妇幼保健院1 321例筛查阳性的新生儿及门诊患者进行G6PD基因检测,首选多色探针熔解曲线法(MMCA)检测中国人群常见的12种G6PD基因突变,对于MMCA法未检出者,分析其酶活性和临床信息,必要时进行测序检测。结果:在1 321例受检者中共检出768例突变,检出率58.1%。共检出18种G6PD基因变异类型,包括c.1388G>A、c.1376G>T、c.95G>A、c.1024C>T、c.871G>A、c.392G>T、c.487 G>A、c.1360C>T、c.1004C>A、c.517T>C、c.592C>T、c.94C>G、c.152C>T、c.320A>G、c.1028A>G、c.1316G>A、c.1327G>C和c.1376G>C,其中男性半合子683例,女性纯合子3例,女性杂合子80例,女性复杂杂合2例。结论:本研究共检出18种G6PD基因变异类型,并首次在中国人群中报道了c.94C>G、c.1028A>G和c.1327G>C突变类型。武汉地区人群常见G6PD基因突变类型为c.1388G>A、c.1376G>T和c.95G>A。     

番禺地区无偿献血人群中Jk(a-b-)表型的筛选与研究

目的研究番禺地区献血人群中Jk(a-b-)表型的分布特点。方法用2mol/L尿素攻膜试验(96孔微量板法)筛选出阴性(不溶血)样本,然后作血清学表型鉴定,对Jk(a-b-)表型进行基因分型及基因组DNA编码区外显子4-11及其侧翼区的分段扩增和测序。结果从本站2004年6月-2006年8月献血的50034名汉族人中筛选出10例Jk(a-b-)表型,频率为0.02%;发现3种突变序列:1)Intron5:3'剪切位点AG>AA,Exon7:nt588A>G、AA196CCA(Pro)>CCG(Pro),推测基因型为JKb(△6)/JKb(△6);2)Exon5:nt222C>A、AA74AAC(Asn)>AAA(Lys),Exon7:nt536C>G、AA179CCT(Pro)>CGT(Arg),Exon7:nt588A>G、AA196CCA(Pro)>CCG(Pro),推测基因型为JKb(222A)/JKb(536G);3)Intron5:3'剪切位点AG>AA,Exon7:nt499A>G,AA167ATG(Met)>GTG(Val),Exon7:nt588A>G、AA196CCA(Pro)>CCG(Pro),推测基因型为JKb(△6)/JKb(499G)。结论在番禺地区献血人群Jk(a-b-)表型查中发现的Exon7:nt499A>G和Exon7:nt536C>G2种突变为首次报道。     

Two novel Jk(null) alleles derived from 222C>A in Exon 5 and 896G>A in Exon 9 of the JK gene

BACKGROUND: Polynesian Jk^null is well known for its mutation as Intron 5 g>a at the 3' splice acceptor site. After sequencing analysis, however, it was noticed that only three of eight samples with the Jknull phenotype carried typical homozygous Polynesian Jk^null mutation. Five others were noted to be unreported heterozygous Polynesian Jk^null mutation. An investigation was then conducted to characterize the underlying mechanism leading to this particular Jk^null genotype.
STUDY DESIGN AND METHODS: Genomic DNA covering 5'-untranslated region exons and intervening introns of the JK gene was amplified by polymerase chain reaction, and the fragments were directly sequenced. The sequencing results were compared with those published in literature and related biologic Web sites.
RESULTS: In all five samples with a heterozygous Polynesian Jk^null mutation, additional mutations were identified. Two samples carried missense mutations: 222C>A (Asn74Lys) in Exon 5 and 499A>G (Met167Val) in Exon 7. Three others had missense mutation 896G>A (Gly299Glu) in Exon 9. These substituted amino acids were located either near or at transmembrane domains, respectively. In addition, two polymorphic nucleotides at positions −103 (a>g) and −119(c>a) from the 3' end of Intron 1 were also Polynesian mutation–related.
CONCLUSIONS: In contrast to the typical homozygous Polynesian Jk^null mutation, two novel heterozygous Jk^null alleles were noted to be associated with the Jknull phenotype. One carried missense mutation 222C>A in Exon 5, and the other had 896G>A missense mutation in Exon 9. These findings may have implications in designing a molecular screening assay for people with the Jknull phenotype.     

Selected exonic sequencing of the AGXT gene provides a genetic diagnosis in 50% of patients with primary hyperoxaluria type 1

BACKGROUND: Definitive diagnosis of primary hyperoxaluria type 1 (PH1) requires analysis of alanine:glyoxylate aminotransferase (AGT) activity in the liver. We have previously shown that targeted screening for the 3 most common mutations in the AGXT gene (c.33_34insC, c.508G>A, and c.731T>C) can provide a molecular diagnosis in 34.5% of PH1 patients, eliminating the need for a liver biopsy. Having reviewed the distribution of all AGXT mutations, we have evaluated a diagnostic strategy that uses selected exon sequencing for the molecular diagnosis of PH1. METHODS: We sequenced exons 1, 4, and 7 for 300 biopsy-confirmed PH1 patients and expressed the identified missense mutations in vitro. RESULTS: Our identification of at least 1 mutation in 224 patients (75%) and 2 mutations in 149 patients increased the diagnostic sensitivity to 50%. We detected 29 kinds of sequence changes, 15 of which were novel. Four of these mutations were in exon 1 (c.2_3delinsAT, c.30_32delCC, c.122G>A, c.126delG), 7 were in exon 4 (c.447_454delGCTGCTGT, c.449T>C, c.473C>T, c.481G>A, c.481G>T, c.497T>C, c.424-2A>G), and 4 were in exon 7 (c.725insT, c.737G>A, c.757T>C, c.776 + 1G>A). The missense changes were associated with severely decreased AGT catalytic activity and negative immunoreactivity when expressed in vitro. Missense mutation c.26C>A, previously described as a pathological mutation, had activity similar to that of the wild-type enzyme. CONCLUSIONS: Selective exon sequencing can allow a definitive diagnosis in 50% of PH1 patients. The test offers a rapid turnaround time (15 days) with minimal risk to the patient. Demonstration of the expression of missense changes is essential to demonstrate pathogenicity.     

二叶式主动脉瓣主动脉病变相关基因突变者的影像学特征与遗传学分析

目的探讨主动脉病变相关基因与二叶式主动脉瓣(BAV)相关性主动脉病变的关系。方法选取行Bentall手术并检测到主动脉病变相关基因突变的8例BAV患者,应用超声心动图评估其BAV分型、瓣膜功能及升主动脉内径等指标;收集其外周血并对主动脉疾病相关基因进行筛查,对变异进行致病性预测;记录患者一般临床参数及实验室检查结果。结果 8例患者中5例主动脉最宽处位于升主动脉管部,3例位于主动脉窦部。2例以主动脉瓣狭窄表现为主,5例以主动脉瓣关闭不全表现为主,1例主动脉瓣狭窄与关闭不全程度相当。4例患者FBN1基因突变(分别为c.2926C>Tp.Arg976Cys、c.2374T>Cp.Cys792Arg、c.2639G>Ap.Gly880Asp、c.6700G>Ap.Val2234Met),2例COL3A1基因突变(分别为c.2190A>Tp.GLU730Asp、c.2181G>Ap.Met727Ile),1例MYLK基因突变(c.1414C>Tp.Leu472Phe),1例TGFBR1基因突变(c.134A>Gp.Asn45Ser)。8例患者中1例FBN1基因突变患者为Sievers0型BAV,余7例均为SieversⅠ型BAV,其中RL亚型6例,RN亚型1例。3例FBN1基因突变患者(c.2926C>Tp.Arg976Cys,c.2374T>Cp.Cys792Arg,c.2639G>Ap.Gly880Asp)经3个突变评价软件预测均为可能致病性突变,该3例患者胸主动脉最大径显著高于其他患者。结论基因变异相关的解剖结构异常及主动脉瓣功能改变所引起的血流动力学变化可能共同导致了BAV患者不同类型的主动脉病变。     

RAG1基因新发变异致重症联合免疫缺陷1例报告及文献复习

目的 提高重症联合免疫缺陷临床表型及基因型的认识.方法 回顾性分析1例重症联合免疫缺陷患儿的临床资料及基因检测结果.结果 患儿,女性,1月6天,发现体质量不增半月余.住院后并发感染且不易控制,血常规复查提示中性粒细胞绝对值波动于(0.34～5.51)×109/L,骨髓穿刺未见明显异常,粒细胞集落刺激因子治疗效果欠佳,淋...     

Denaturing high-performance liquid chromatography mutation analysis in patients with reduced Protein S levels

BACKGROUND: Patients with congenital Protein S deficiency have increased risk of venous thromboembolism. However, Protein S levels show large intra-individual variation and the biochemical assays have low accuracy and a high interlaboratory variability. Genetic analysis might aid in a more precise diagnosis and risk estimation. The aim was to design a high-throughput genetic analysis based on denaturing high-performance liquid chromatography to identify sequence variations in the gene coding for Protein S. PATIENTS: In total, 55 patients referred to the Section of Thrombosis and Haemostasis, Odense University Hospital, in the period 1998-2004 were included in the study. RESULTS: Mutations were found in ten of the 55 patients: Six different variants were identified, of which four were not previously reported: One were a nonsense mutation substituting a glutamine with a stopcodon (c.790C>T) and the rest were missense mutations (c.932T>G; c.1367A>G; c.1378T>C). Furthermore, four patients carried the same mutation (c.1045G>A), while two carried the Heerlen mutation (c.1378T>C). CONCLUSIONS: The reported method will be useful for rapidly detecting sequence variations in the gene coding for Protein S, giving a precise diagnosis and subsequently a better risk estimation.     

自贡地区HBV基因BCP区1762/1764及前C区1896位点突变与HBV相关性肝癌的相关性研究

目的通过分析自贡地区乙肝患者乙型肝炎病毒HBV基因BCP区1762/1764及前C区1896位点的突变情况,探讨其与HBV相关性肝癌的关系。方法收集2015年9月至2018年6月141例经本院确诊的乙肝性相关疾病患者血清标本(HBV DNA≥103IU/mL):慢性乙肝(CHB组)50例、肝硬化(LC组)45例、肝癌(HCC组)46例,并收集临床相关资料。采用荧光定量PCR法检测患者的HBV DNA水平,多通道荧光PCR法检测HBV基因型,然后采用ARMS-PCR法检测HBV基因BCP区1762/1764及前C区1896突变情况。采用SPSS17.0软件对数据进行统计学分析。结果HCC组和CHB组在e抗原阳性率、HBV DNA水平及HBV DNA>105IU/mL方面对比发现差异均具有统计学意义(P<0.05);HCC组和LC组在e抗原阳性率方面比较发现差异无统计学意义(P>0.05),而HBV DNA水平方面差异具有统计学意义(P<0.05)。男性、女性乙肝患者HBV基因BCP区1762/1764位点突变率分别为67.0%、57.1%(P>0.05),前C区1896位点突变率分别为61.6%、66.7%(P>0.05)。HCC组患者、LC组患者、CHB组患者的HBV基因BCP区1762/1764突变率分别为91.3%、84.4%、22%,HCC组与CHB组比较差异具有统计学意义(P<0.05),而HCC组与LC组比较差异无统计学意义(P>0.05);HBV基因前C区1896突变率分别为84.8%、62.2%、42.0%,HCC组与CHB组、LC组比较差异均具有统计学意义(P<0.05)。HCC组患者的HBV基因前C区1896和BCP区1762/1764位点同时突变率为78.3%,要高于LC组和CHB组,差异均具有统计学意义(P<0.05)。HBV基因型方面比较,无论是基因B型还是C型的乙肝相关患者,疾病进程较重(HCC组、LC组)的受试者携带BCP区1762/1764突变型或前C区1896突变型的比例都要高于慢性乙肝患者(CHB组)。结论自贡地区HBV基因BCP区1762/1764和前C区1896突变率高,无性别和基因型差异,其中HBV基因BCP区1762/1764位点和前C区1896位点联合突变与HBV相关性HCC发生可能存在一定关系。