先天性甲状腺功能减退症伴发育不全患儿FOXE1基因突变的初步研究

目的：研究先天性甲状腺功能减退症(CH)伴甲状腺发育不全患儿转录因子2( FOXE )的基因突变。方法：选取90 例CH伴甲 状腺发育不全患儿及90 例正常儿童作为对照,提取外周静脉血基因组DNA,采用PCR扩增与直接测序技术,对基因外 显子进行突变筛查。结果：分别在1 例先天性甲状腺功能减退症伴甲状腺发育不全患者外显子测序中发现一杂合错义变体c. A3401G (p.K1134R),在1 例患者中发现1 个已知的单核苷酸多态性(single nucleotide polymorphisms,SNP)位点(rs755282859, c. 483G>C),在正常对照组中未发现以上变化。结论：在先天性甲状腺功能减退症(CH) 伴甲状腺发育不全患儿中发现新的关于FOXE1 杂合错义变体。     

先天性甲状腺功能减退症伴发育不全患儿FOXE1基因突变的初步研究（英文）

目的:研究先天性甲状腺功能减退症(CH)伴甲状腺发育不全患儿转录因子2(FOXE1)的基因突变。方法:选取90例CH伴甲状腺发育不全患儿及90例正常儿童作为对照,提取外周静脉血基因组DNA,采用PCR扩增与直接测序技术,对FOXE1基因外显子进行突变筛查。结果:分别在1例先天性甲状腺功能减退症伴甲状腺发育不全患者外显子测序中发现一杂合错义变体c.A3401G(p.K1134R),在1例患者中发现1个已知的单核苷酸多态性(single nucleotide polymorphisms,SNP)位点(rs755282859,c.483GC),在正常对照组中未发现以上变化。结论:在先天性甲状腺功能减退症(CH)伴甲状腺发育不全患儿中发现新的关于FOXE1杂合错义变体。     

男性性腺功能减退症的致病基因分析

[目的]通过全外显子组测序(WES)技术筛选男性性腺功能减退症的致病基因,并对基因突变位点进行生物信息学分析。[方法]收集5例男性性腺功能减退症患者临床及遗传学检测资料。采用WES技术筛选相关致病基因,并通过PCR扩增、Sanger测序以及生物信息学分析等验证突变位点。[结果]先证者1为PROKR2基因c.533G>C(p.W178S)纯合突变,家系验证结果发现其父母均为PROKR2基因c.533G>C(p.W178S)杂合突变携带者,符合常染色体隐性遗传。先证者2为ZFPM2基因c.1498C>G(p.Q500E)杂合突变,生物信息学分析发现,该突变位点编码的氨基酸在不同物种中高度保守,并在人类外显子数据库、参考人群千人基因组1000G、SNP数据库及人群基因组突变频率数据库中未发现该突变位点,该突变经SIFT、Polyphen2和Mutation Taster软件预测结果均为有害。[结论]PROKR2基因c.533G>C(p.W178S)和ZFPM2基因c.1498C>G(p.Q500E)突变可能是男性性腺功能减退症的致病原因。     

一个中国Gilbert综合征家系的遗传学分析

对一个中国汉族Gilbert综合征遗传家系致病基因突变位点进行鉴定,以期了解该病的分子遗传学基础。首先提取先证者基因组DNA,PCR扩增尿苷二磷酸葡萄糖醛酸转移酶UGT1A1基因的5个外显子,以琼脂糖电泳鉴定PCR产物,纯化后直接测序鉴定。基因扫描显示,与血清胆红素水平密切相关的UGT1A1基因在第1和第5外显子存在纯合突变,而 UGT1A1基因启动子区域和内含子/外显子剪接边界位点序列未检测到突变。进一步对其他家系成员该基因的相应位点进行突变检测,结果显示他们在第1和第5外显子也存在杂合突变,其中还有两个成员在启动子区域检测到(TA)插入突变。对家系成员未抗凝新鲜血液进行生化检测证实了基因突变分析的结果。综合以上结果发现该家系三种突变并存,致病因素为第1和/或第5外显子突变,为显性遗传,两种突变位点纯合导致先证者出现严重胆红素代谢功能障碍。该家系因此成为Gilbert综合征突变位点及其致病机理研究的一个典型临床病例。     

先天性缝性白内障一家系中晶体蛋白基因的突变筛查

目的：对一中国常染色体显性先天性缝性白内障家系进行晶体蛋白基因（CRYAA、CRYAB、CRYBB2、CRYGC、CRYGD）的突变筛查。方法：对一中国先天性白内障家系进行研究,通过直接测序,筛查此家系中全部患者的CRYAA、CRYAB、CRYBB2、CRYGC和CRYGD基因外显子以及临近的内含子的剪接位点。结果：直接测序后发现该缝性白内障家系基因的外显子及其临近的内含子中．均未发现任何突变。结论：CRYAA、CRYAB、CRYBB2、CRYGC和CRYGD为该先天性白内障家系的非致病基因。     

先天性缝性白内障一家系中晶体蛋白基因的突变筛查

目的:对一中国常染色体显性先天性缝性白内障家系进行晶体蛋白基因(CRYAA、CRYAB、CRYBB2、CRYGC、CRYGD)的突变筛查。方法:对一中国先天性白内障家系进行研究,通过直接测序,筛查此家系中全部患者的CRYAA、CRYAB、CRYBB2、CRYGC和CRYGD基因外显子以及临近的内含子的剪接位点。结果:直接测序后发现该缝性白内障家系基因的外显子及其临近的内含子中,均未发现任何突变。结论:CRYAA、CRYAB、CRYBB2、CRYGC和CRYGD为该先天性白内障家系的非致病基因。     

家族性高胆固醇血症患者低密度脂蛋白受体基因复合杂合突变分析

目的:对1例临床确诊为纯合型家族性高胆固醇血症(FH)先证者及其核心家系成员进行基因检测分析,探讨患儿发病的分子病理基础.方法:收集先证者及父母血标本及临床资料,酚氯仿法提取基因组DNA,DNA直接测序方法检测低密度脂蛋白受体(LDL-R)基因18个外显子和启动子及载脂蛋白B(ApoB100)R3500Q位点,核苷酸序列分析结果与Gen Bank比对寻找突变.结果:(1)先证者三尖瓣轻度关闭不全,先证者父母双侧颈总动脉内-中膜增厚,先证者母亲左侧颈内动脉起始处后壁多发混合回声斑块(2)该家系排除ApoB100基因R3500Q突变;(3)先证者LDL-R基因第13外显子发生A606T和D601Y复合杂合突变,前者第1879位G→A碱基置换,导致丙氨酸改变为苏氨酸,后者为1864位G>T碱基置换,导致天冬氨酸改变为酪氨酸,其父为携带A606T突变的杂合子,其母为携带D601Y突变的杂合子.结论:先证者LDL-R基因存在A606T和D601Y复合杂合突变,它们分别来源于父系及母系遗传.     

一性连锁Alport综合征中国家系COL4A5基因新突变位点的检测

通过PCR和直接测序的方法,对一性连锁Alport综合征家系17个受检个体的COL4A5基因所有51个外显子及其相邻内含子的DNA序列进行检测。结果发现,在第26外显子2240位点,男患者存在C碱基缺失(2240delc),女患者存在杂合缺失,同时对女患者相应的PCR产物进行克隆和测序以验证PCR测序结果的可靠性,而在正常家系成员和80例对照中均未发现此位点异常,说明2240delc为引起该家系临床病变的突变位点,不是多态性位点。在性连锁Alport综合征中,COL4A5基因的这个单碱基缺失突变位点为首次报道。     

心脏肌球蛋白重链基因c.1273G>A突变与肥厚型心肌病的关联分析

为研究中国人家族性肥厚型心肌病(HCM)的致病基因突变位点, 分析基因型与临床表型的相互关系, 文章在1个中国汉族HCM家系中进行心脏肌钙蛋白T (TNNT2) 基因、心脏肌球蛋白结合蛋白C (MYBPC3) 基因和心脏β-肌球蛋白重链 (MYH7) 基因的突变筛查, 聚合酶链式反应(PCR)扩增基因功能区外显子片段并对PCR产物进行测序分析。结果表明: 在该家系接受调查的7名成员中有4名成员携带MYH7基因c.1273G>A杂合突变, 该突变位点位于MYH7基因的14号外显子并使425位的甘氨酸(Gly)转换为精氨酸(Arg)。该突变首次在国内HCM家系中发现, 突变携带者的临床表型在家系内部呈现明显的异质性。该家系成员TNNT2及MYBPC3基因未发现突变且正常对照组相同位置未发现异常。MYH7基因是我国家族性 HCM的致病基因之一, 携带c.1273G>A突变的肥厚型心肌病患者临床表型差异明显, 提示可能有其它因素参与了肥厚型心肌病的发展过程。     

27例雄激素不敏感综合征患儿AR基因突变

雄激素受体(AR)基因突变是雄激素不敏感综合征(AIS)的主要直接原因,本研究是为了了解中国病人AR基因突变谱.本研究根据病史、查体、激素检测、染色体核型分析及影像学检查结果,确诊AIS患儿27例.收集患儿及其部分家属静脉抗凝血,提取DNA,合成外显子1~8及外显子和内含子剪切处引物, PCR扩增,扩增产物送公司测序,分析AR基因突变情况.结果提示, 27例AIS患儿共发现突变23个, 10例携带5个相同突变(p.R841H, p.P914S,p.S176R, p.Y572S和p.Y782N),未报道突变11个. 8/27例家族史阳性. 12个已知突变中,仅1个为内含子4的剪切突变:c.2173+1GT;余均为点突变,含1个已报道的第6外显子的新生突变:p.R775C. 2例病人具有相同第8外显子突变:p.P914S,患者均表现为CAIS,与报道的PAIS不符;其余临床分型与已经报道的分型一致.未报道突变中,点突变为8/11个,另有2个碱基缺失导致的移码突变:p.345fs和p.828_829del, 1个碱基插入导致的移码突变:p.885fs, 1例病人携带两个点突变:p.Y365C和p.E898D.突变由多至少在外显子的分布:外显子7有6例患儿5个不同突变;外显子8有5例患儿4个突变,外显子1和5各有3个突变;外显子2, 3, 4和6各有2个不同突变. 23个突变在功能区的分布情况:16个突变位于LBD, 4个位于DBD, 3个位于NTD.本研究报道了不同AIS表型AR的突变谱, 50%为未报道突变,进一步丰富了AR数据库.错义突变是主要形式,且大部分位于LBD区.携带相同突变的病人可呈现不同表型.     

Two novel ANK1 loss‐of‐function mutations in Chinese families with hereditary spherocytosis

Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia disorder. ANK1 mutations account for most HS cases, but pathogenicity analysis and functional research have not been widely performed for these mutations. In this study, in order to confirm diagnosis, gene mutation was screened in two unrelated Chinese families with HS by a next‐generation sequencing (NGS) panel and then confirmed by Sanger sequencing. Two novel heterozygous mutations (c.C841T, p.R281X and c.T290G, p.L97R) of the ANK1 gene were identified in the two families respectively. Then, the pathogenicity of the two new mutations and two previously reported ANK1 mutations (c.C648G, p.Y216X and c.G424T, p.E142X) were studied by in vitro experiments. The four mutations increased the osmotic fragility of cells, reduced the stabilities of ANK1 proteins and prevented the protein from localizing to the plasma membrane and interacting with SPTB and SLC4A1. We classified these four mutations into disease‐causing mutations for HS. Thus, conducting the same mutation test and providing genetic counselling for the two families were meaningful and significant. Moreover, the identification of two novel mutations enriches the ANK1 mutation database, especially in China.     

Whole-Exome Sequencing Efficiently Detects Rare Mutations in Autosomal Recessive Nonsyndromic Hearing Loss

Identification of the pathogenic mutations underlying autosomal recessive nonsyndromic hearing loss (ARNSHL) is difficult, since causative mutations in 39 different genes have so far been reported. After excluding mutations in the most common ARNSHL gene, GJB2, via Sanger sequencing, we performed whole-exome sequencing (WES) in 30 individuals from 20 unrelated multiplex consanguineous families with ARNSHL. Agilent SureSelect Human All Exon 50 Mb kits and an Illumina Hiseq2000 instrument were used. An average of 93%, 84% and 73% of bases were covered to 1X, 10X and 20X within the ARNSHL-related coding RefSeq exons, respectively. Uncovered regions with WES included those that are not targeted by the exome capture kit and regions with high GC content. Twelve homozygous mutations in known deafness genes, of which eight are novel, were identified in 12 families: MYO15A-p.Q1425X, -p.S1481P, -p.A1551D; LOXHD1-p.R1494X, -p.E955X; GIPC3-p.H170N; ILDR1-p.Q274X; MYO7A-p.G2163S; TECTA-p.Y1737C; TMC1-p.S530X; TMPRSS3-p.F13Lfs*10; TRIOBP-p.R785Sfs*50. Each mutation was within a homozygous run documented via WES. Sanger sequencing confirmed co-segregation of the mutation with deafness in each family. Four rare heterozygous variants, predicted to be pathogenic, in known deafness genes were detected in 12 families where homozygous causative variants were already identified. Six heterozygous variants that had similar characteristics to those abovementioned variants were present in 15 ethnically-matched individuals with normal hearing. Our results show that rare causative mutations in known ARNSHL genes can be reliably identified via WES. The excess of heterozygous variants should be considered during search for causative mutations in ARNSHL genes, especially in small-sized families.     

Molecular characterization of phenylketonuria in Japanese patients

We characterized phenylalanine hydroxylase (PAH) genotypes of Japanese patients with phenylketonuria (PKU) and hyperphenylalaninemia (HPA). PKU and HPA mutations in 41 Japanese patients were identified by denaturing gradient gel electrophoresis and direct sequencing, followed by restriction fragment length polymorphism analysis to find a large deletion involving exons 5 and 6. Of 82 mutant alleles, 76 (92%) were genotyped showing 21 mutations. The major mutations were R413P (30.5%), R243Q (7.3%), R241 C (7.3%), IVS4nt-1 (7.3%), T278I (7.3%), E6nt-96A→g (6.1%), Y356X (4.9%), R111X (3.7%), and 442–706delE5/6 (2.4%). Eight new mutations (L52 S, delS70, S70P, Y77X, IVS3nt-1, A132 V, W187 C, and C265Y) and a polymorphism of IVS10nt-14 were detected. In vitro PAH activities of mutant PAH cDNA constructs were determined by a COS cell expression system. Six mutations, viz., R408Q, L52 S, R241 C, S70P, V388 M, and R243Q, had 55%, 27%, 25%, 20%, 16% and 10% of the in vitro PAH activity of normal constructs, respectively. The mean pretreatment phenylalanine concentration (0.83±0.21 mmol/l) of patients carrying the R408Q, R241 C, or L52 S mutation and a null mutation was significantly lower (P<0.0005) than that (1.99±0.65 mmol/l) of patients with both alleles carrying mutations associated with a severe genotype. Simple linear regression analysis showed a correlation between pretreatment phenylalanine concentrations and predicted PAH activity in 29 Japanese PKU patients (y=31.9–1.03x, r=0.59, P<0.0001). Genotype determination is useful in the prediction of biochemical and clinical phenotypes in PKU and can be of particular help in managing patients with this disorder. Received: 24 July 1998 / Accepted: 12 September 1998     

Molecular analysis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency in Hong Kong Chinese patients

BackgroundCongenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency (21OHD) is an autosomal recessive disorder due to mutation in the CYP21A2 gene.ObjectiveTo elucidate the genetic basis of 21-hydroxylase-deficient CAH in Hong Kong Chinese patients.Patients and methodsMutational analysis of the CYP21A2 gene was performed on 35 Hong Kong Chinese patients with 21OHD using direct DNA sequencing and multiplex ligation-dependent probe amplification (MLPA).ResultsThe genetic findings of 21 male and 14 female patients are the following: c.293-13A/C>G (intron 2 splice site; 20 alleles), p.I172N (13), p.R356W (7), p.Q318X (4). A total of 20 mutant alleles contained gross deletion/conversion of all or part of the CYP21A2 gene. A novel mutation, c.1367delA (p.D456fs), was detected in one patient. One patient had only a heterozygous mutation detected. Out of 35 patients, 16 would have been incorrectly genotyped if either DNA sequencing or MLPA alone was used for molecular analysis.ConclusionsThe frequency of various mutations in the studied patients differs from those reported in other Asian populations. Gross deletion/conversion accounts for nearly one-third of the genetic defects. Therefore, laboratories must include methods for detecting point mutations as well as gross deletions/conversions to avoid misinterpretation of genotype. Genotyping has increasingly been proven to be a useful tool for supplementing, if not replacing, hormonal profiling for the diagnosis of 21OHD.     

Characterization of CADASIL among the Han Chinese in Taiwan: Distinct Genotypic and Phenotypic Profiles

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is originally featured with a strong clustering of mutations in NOTCH3 exons 3–6 and leukoencephalopathy with frequent anterior temporal pole involvement. The present study aims at characterizing the genotypic and phenotypic profiles of CADASIL in Taiwan. One hundred and twelve patients with CADASIL from 95 families of Chinese descents in Taiwan were identified by Sanger sequencing of exons 2 to 24 of NOTCH3. Twenty different mutations in NOTCH3 were uncovered, including 3 novel ones, and R544C in exon 11 was the most common mutation, accounting for 70.5% of the pedigrees. Haplotype analyses were conducted in 14 families harboring NOTCH3 R544C mutation and demonstrated a common haplotype linked to NOTCH3 R544C at loci D19S929 and D19S411. Comparing with CADASIL in most Caucasian populations, CADASIL in Taiwan has several distinct features, including less frequent anterior temporal involvement, older age at symptom onset, higher incidence of intracerebral hemorrhage, and rarer occurrence of migraine. Subgroup analyses revealed that the R544C mutation is associated with lower frequency of anterior temporal involvement, later age at onset and higher frequency of cognitive dysfunction. In conclusion, the present study broadens the spectrum of NOTCH3 mutations and provides additional insights for the clinical and molecular characteristics of CADASIL patients of Han-Chinese descents.     

IDH1 and IDH2 mutations are frequent in Chinese patients with acute myeloid leukemia but rare in other types of hematological disorders

Frequent mutations in the isocitrate dehydrogenase 1 and 2 genes (IDH1 and IDH2) have been identified in gliomas and acute myeloid leukemia (AML). Our aim is to assess whether IDH mutations were presented in Chinese patients with various hematological disorders. In this study, we screened the IDH1 and IDH2 mutations in a cohort of 456 Chinese patients with various hematological malignancies and disorders. We found three missense (p.R132C, p.R132G, and p.I99M; occurred in five patients) and one silent mutation (c.315C>T; occurred in two patients) in the IDH1 gene and two missense mutations (p.R140Q and p.R172K; occurred in four AML patients) and one silent mutation (c.435G>A) in the IDH2 gene. Except for one non-Hodgkin lymphoma (NHL) patient harboring IDH1 mutation p.R132C, all IDH1 and IDH2 missense mutations were observed in patients with AML. Intriguingly, the IDH2 mutation p.R140Q and novel IDH1 mutation p.I99M co-occurred in a 75-year-old patient with AML developed from myelodysplastic syndromes (MDS). The frequency of IDH1 and IDH2 missense mutations in Chinese AML patients reached 5.9% and 8.3%, respectively. Our results supported the recent findings that IDH gene mutations were common in AML. Conversely, IDH mutations were rather rare in Chinese patients with other types of hematological disorders.     

Mutation spectrum of the PAH gene in the PKU patients from Khorasan Razavi province of Iran

Background

Characterization of the molecular basis of phenylketonuria (PKU) in North-east of Iran has been accomplished through the analysis of 62 unrelated chromosomes from 31 Iranian PKU patients.

Methods

Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by direct DNA sequencing exons 6, 7, 10 and 11.

Results

A mutation detection rate of 74% was achieved. Eleven different mutations were found, with the most frequent mutation, IVS10-11G > A, accounting for 19% of Khorasan-Razavi PKU alleles. Ten mutations (R176X, E280K, IVS11 + 1G > C, S231P, Q383X, R243X, I224T, E390G, R252W and P281L) represent the rest PKU chromosomes. One novel mutation, Q383X in the homozygote form was identified which is located in the catalytic domain (residues143–410).

Conclusion

With this high detection rate of mutations in North-east of Iran, new strategy for carrier testing could be DNA sequencing of these four exons. The other exons and boundaries will be studied only when either one or no mutations are detected in the initial screen.     

Identification of two novel mutations in the first Sicilian APECED patient with no R203X mutation in AIRE gene and review of Italian APECED genotypes

Aims of the present study are: 1) to report an additional Sicilian patient with autoimmune polyendocrinopathy-candidiasis-ectodermal-dystrophy (APECED), whose genotypic features are original, in that she is the first APECED patient with three coexisting mutations of Autoimmune Regulator (AIRE) gene, two of which had never been reported to date; 2) to review the genotypic epidemiology of APECED in Italy, in order to underline the great genetic heterogeneity of this disorder in Italian patients. Both the novel mutations of our new patient, named S107C and Q108fs respectively, were detected on exon 3 and were inherited by the mother, whereas the third one (T16M) was inherited by the father. Phenotypically, the present case is quite characteristic, in that she exhibited a classical clinical picture, with no original features. Conclusions: a) this is the seventh Sicilian APECED patient identified to date, and the first one with no R203X mutation; b) our data confirm that distribution of mutations may considerably vary according to different geographical distribution, within the same country, thus suggesting a potential founder effect.