20例先天性甲状腺功能减退症患者甲状腺过氧化物酶基因突变分析

检测20例先天性甲状腺功能减退症患者甲状腺过氧化物酶(TPO)基因突变.发现1例患者在TPO基因第13外显子处存在c.2268insT纯合性突变,另1例为TPO基因c.2268insT突变和第9外显子c.1477G＞A突变复合杂合子.TPO基因可能是中国人群先天性甲状腺功能减退症发生的原因之一.

Abstract：

Thyroid peroxidase(TPO) gene was detected in 20 patients with congenital hypothyroidism. An insertion c. 2268insT of TPO gene was found in one of them, and c. 2268insT combined with c. 1477G＞C mutation in another. TPO gene mutation may be related to pathogenesis of congenital hypothyroidism in Chinese.     

TSH受体基因突变致先天性甲状腺功能减退症一例及其家系分析

目的 研究先天性甲状腺功能减退症（甲减）患者的TSH受体（TSHR）基因突变与家系遗传规律。方法 用TKM法提取18例先天性甲减患者、35名正常对照者外周血白细胞DNA，PCR-SSCP分析TSHR基因第1、4、6、10号外显子，突变经正反向测序证实。分析先证者家系成员TSHR基因与甲状腺功能情况。结果 发现1例先天性甲减患儿在TSHR基因第10号外显子有2个位点纯合子突变：450位密码子CGC置换为CAC，Arg450→His（R450H）；727位密码子GAC置换为GAG，Asp727→Glu（D727E）。调查家系成员10人，6人为R450H／D727E复合杂合子，以女性携带为主，先证者的父母均为复合杂合子，杂合子中5人血清sTSH轻度升高，为亚临床甲状腺功能减退症（亚临床甲减）。结论 R450H／D727E纯合子突变导致先天性甲减，R450H／D727E杂合子突变可发生亚临床甲减。     

两个MODY2中国家系葡萄糖激酶基因杂合突变的研究

目的 寻找两个典型的MODY2家系的责任基因. 方法 抽提两个MODY2家系成员基因组DNA,PCR扩增、直接测序候选基因葡萄糖激酶(GCK)基因5′端、3′端非翻译区及1～10号外显子,确认突变. 结果 家系1中4人携带GCK基因杂合突变c.661G>A(E221K),先证者为MODY2,另2名突变携带者表现为糖调节受损(IGR),1名突变携带者血糖正常.家系2中2人携带GCK基因杂合突变c.771G>A(W257ter),先证者为MODY2,另1名突变携带者表现为IGR.在这些患者中,饮食控制和增强运动能收到良好效果. 结论 GCK基因突变c.661G>A(E221K)和c.771G>A(W257ter)可能是两个MODY2家系的主要致病基因,其中c.771G>A(W257ter)是一个新发现的突变位点.     

汉族肥厚型心肌病患者中Fabry病的基因突变鉴定和家系研究

目的探讨基因检测在汉族肥厚型心肌病(hypertrophic cardiomyopathy,HCM)患者中Fabry病的基因突变情况及家系筛查中的应用,并分析基因型与表型的关系。方法应用半导体靶向二代测序平台筛查在阜外医院诊断为HCM的217例患者,应用Sanger测序验证先证者和家系内成员的GLA基因突变位点,收集GLA突变携带者的临床资料并进行基因型与表型关联分析。结果发现2例男性Fabry病先证者(在HCM中占比0.93%)。1例携带GLA基因错义突变c.887TC(p.M296T),表现为迟发心脏型Fabry病;对其一家四代中的25个家庭成员进行家系突变筛查,结果发现有4个女性杂合突变携带者,其中1个确诊为HCM。另1例携带GLA基因错义突变c.758TC(p.I253T),表现为经典型Fabry病,累及肾和神经系统。对其一家四代中的32个家系成员进行家系调查,发现2个女性杂合突变携带者和2个男性早发心脏性猝死。两例先证者经室间隔心肌切除术后梗阻解除,后者应用分子伴侣药物Migalastat治疗后肾功能稳定于31 ml/min。结论首次发现Fabry病在汉族HCM中并不少见,基因检测有助于早期鉴别诊断及筛查家系内突变携带者。GLA c.758TC为恶性基因型,男性突变携带者猝死风险高危,室间隔心肌切除术和Migalastat有助于改善预后。     

PRKAG2基因新突变致心肌肥厚和严重心力衰竭但不合并心律失常的心脏综合征

目的对1个肥厚型心肌病先证者及其四代家系成员进行致病基因筛查研究。方法收集先证者及其家系成员外周血,提取基因组DNA,应用二代测序法对先证者进行致病突变筛查。发现可疑致病位点后,进一步通过Sanger测序法在家系内其他成员及600个健康个体中进行验证。用PolyPhen-2,SIFT及Mutation Taster等生物信息学软件对发现的潜在致病突变进行致病性分析和功能预测。结果先证者及家系内多个成员均携带PRKAG2基因杂合突变,p.Ser98Asn (c.293GA)。但600例健康个体中并未发现该突变。生物信息学分析结果显示PRKAG2基因的p.Ser98Asn (c.293GA)杂合突变位于进化保守区域,并可能影响蛋白功能,为致病性突变。与携带该基因其他突变致PRKAG2心脏综合征的患者合并多种心律失常不同,携带该PRKAG2基因突变位点的家系成员有心肌肥厚和心衰等PRKAG2心脏综合征的表现,但不合并心律失常。结论我们首次报道一个新的PRKAG2基因致病性错义突变,该突变导致的PRKAG2心脏综合征包括心肌肥厚和严重心力衰竭,但不合并心律失常。     

JPH2基因p.R616C突变与家族性扩张型心肌病的相关性分析

目的:对1例家族性扩张型心肌病(dilated cardiomyopathy, DCM)家系进行致病基因筛查,分析其基因型与表型的相关性。方法:研究对象为1例DCM先证者及其家系成员,另选取体检中心健康成年人为对照。收集先证者家系的临床资料,对先证者及发病家系成员进行全基因组测序,筛选可疑致病基因,并通过Sanger测序法进行验证。对其他家系成员及对照组进行相关基因筛查。结果:对该家系筛查发现包括先证者在内的5例DCM患者,且基因检测发现均存在JPH2基因的c.1846C>T(p.Arg616Cys)突变,另有1例无临床表型的突变基因携带者。余家系成员及对照组相同位点未见异常。在随访过程中先证者出现恶性心律失常。结论:本研究发现JPH2基因的p.R616C突变可能导致家族性DCM,且该基因突变可能为恶性基因突变。     

肥厚型心肌病家系携带ACTN2、MYBPC3和TNNI3基因突变分析

目的:对收集的1例肥厚型心肌病家系的致病基因进行突变位点分析,阐明基因型与临床表型的关系。方法:利用目标外显子捕获技术和二代测序技术对先证者的与肥厚型心肌病有关的基因进行基因突变筛查,并使用Sanger测序法验证可疑突变位点,同时筛查患者家系成员4例和健康人100例,确定该家系患者的致病突变,并利用SIFT、Polyphen2和MutationTaster这3种软件进行突变基因功能检测。结果:该家系除先证者外,4例有血缘关系的研究对象中3例携带ACTN2基因c.1162TA错义突变(p.Trp388Arg),2例携带MYBPC3基因c.472GA错义突变(p.Val158Met),2例携带TNNI3基因c.235CT错义突变(p.Arg79Cys)。该家系的先证者同时携带上述3种突变基因。3种预测软件预测这3种突变均为有害突变。结论:在该患者家系中发现的基因突变位点可能是肥厚型心肌病的致病突变,携带多种突变的家系成员更易发生肥厚型心肌病,但其确切发病机制仍需进一步研究。     

家族性肥厚型心肌病致病突变检测及基因型表型关联分析

目的研究中国人家族性肥厚型心肌病的致病基因突变位点,分析其基因型与表型的关系。方法利用目标区域重测序技术,对一个中国肥厚型心肌病家系先证者的64个与遗传性心肌病相关的基因进行筛查。Sanger测序验证可疑突变位点并筛查家系成员8例和正常个体100例,同时分析家系突变携带者4例的临床表型特点。结果本家系中包括先证者在内的4例存活家系成员携带MYBPC3基因c.1377del C突变,在100例正常对照中未发现此突变。携带者中有2例确诊为肥厚型心肌病,均表现为室间隔肥厚,另外2例为无症状携带者,心电图和超声心动图均未见异常。家系中有3例猝死,4例有晕厥史,3例发病年龄小于40岁,2例在40岁后发病。结论基因检测在肥厚型心肌病鉴别诊断和家系筛查中有重要的临床意义。     

人钠/碘转运体基因G395R与先天性甲状腺功能减退症相关

先天性甲状腺功能减退症 5 2例和 10 6例健康婴幼儿 ,采用PCR RFLP技术对钠 /碘同向转运体 (hNIS)基因 3 95位点进行基因突变筛查。研究表明hNIS基因G3 95R突变可能不是青岛地区先天性甲状腺功能减退症发病的主要原因。     

家族性肥厚型心肌病一家系临床资料及健康管理策略分析

目的分析家族性肥厚型心肌病(FHCM)一家系的临床资料,并对其提出合理的健康管理策略。方法采用高通量基因测序技术对该FHCM家系成员进行基因测序分析,记录其相关临床资料。对该家系成年人、女性携带者及青少年给予遗传咨询,并针对性地制定合理的健康管理策略。结果该家系共10例成员,先证者为女性,生育3个儿子,其中大儿子有2个子女、二儿子有3个子女、三儿子有1个子女。先证者MYH7基因存在c.1063G> A(编码区第1 063号核苷酸由G变为A)的杂合核苷酸变异,导致第355号氨基酸由Ala变为Thr (p.Ala355Thr),属于错义突变。先证者后代筛查结果显示,Ⅱ1、Ⅱ2、Ⅲ2、Ⅲ3、Ⅲ4、Ⅲ5均为MYH7基因突变位点携带者。先证者心电图检查结果显示,ST-T异常,完全性左束支传导阻滞,左心室肥厚,快速型心房颤动;超声心动图检查结果提示肥厚型心肌病改变(非对称梗阻型),入院后行改良Morrow手术进行治疗。针对该家系MYH7基因突变位点携带者,成年携带者建议进行营养管理、药物治疗、介入治疗及手术治疗等;生育期女性携带者建议尽早生育、严重家族史者可采取第三代试管婴儿技术筛选健康胚胎...     

Congenital hypothyroidism caused by new mutations in the thyroid oxidase 2 (THOX2) gene

OBJECTIVE: Congenital primary hypothyroidism (CH) occurs in one of 4000 births and in 20% of the cases CH is due to a defect in thyroid hormonogenesis. Candidate genes were examined to determine the precise aetiology of suspected dyshormonogenesis in CH. DESIGN: The genes that code for thyroid peroxidase (TPO), pendrin (PDS), sodium iodide symporter (NIS) and thyroid oxidase 2 (THOX2) were sequenced directly from genomic DNA. PATIENTS: Two girls found to have CH in the neonatal screening programme and suspected of having thyroid dyshormonogenesis were investigated to identify their molecular defect. RESULTS: Patient A had a novel heterozygous 1 bp insertion in the THOX2 gene (ins602g). This insertion results in a frameshift that predicts a premature stop at codon 300. Analysis of cDNA, transcribed from lymphocyte RNA, showed that this mutation causes skipping of exon 5, resulting in a frameshift and a premature stop at codon 254. The euthyroid mother was also a heterozygous carrier of the mutation whereas the father was homozygous for the wild-type THOX2 gene. In patient B, compound heterozygous mutations (ins602g-->fsX300 and D506N) were identified. D506N was present in one allele of the clinically unaffected mother and in a brother, whereas the euthyroid father was heterozygous for ins602g. Sixty normal individuals did not harbour the mutations. Sequencing of the TPO, PDS and NIS genes revealed no mutations. CONCLUSIONS: The identified THOX2 mutations, which have not been described previously, are the probable causes of CH in the patients. Mutations in the THOX2 gene should be considered as the molecular cause of CH in young patients with thyroid dyshormonogenesis.     

Mutation analysis of thyroid peroxidase gene in Chinese patients with total iodide organification defect: identification of five novel mutations

Total iodide organification defect (TIOD), where the iodide in the thyroid gland cannot be oxidized and/or bound to the protein, is caused by a defect in the thyroid peroxidase (TPO) gene. Single strand conformation polymorphism analysis was used to screen for mutations in the TPO gene from five unrelated TIOD patients in Taiwan, and five novel mutations were detected. Three of these were frameshift mutations: a single T insertion between nucleotide position 2268 and 2269 (c.2268-2269 insT) in exon 13 and two single C deletions at nucleotide positions 843 (c.843 delC) and 2413 (c.2413 delC) in exon 8 and 14 respectively. The other two were single nucleotide substitutions (c.G1477>A and c.G2386>T) located in exons 9 and 13 respectively. While the former would result in amino acid substitution (Gly493Ser) in the highly conserved region of the TPO polypeptide, the latter would result in either amino acid substitution (Asp796Tyr) or alternative splicing. Of those identified TPO mutations, c.2268-2269 insT was most prevalent and was detected as heterozygous in all but one TIOD patients. All five TIOD patients investigated in this study were compound heterozygous. The method presented in this study could be used for carrier assessment and mutation analysis of newly identified TIOD patients.     

Loss of heterozygocity at the thyroid peroxidase gene locus in solitary cold thyroid nodules.

Germline mutations in both alleles of the thyroid peroxidase (TPO) gene have been reported as a frequent cause of congenital hypothyroidism resulting from a total iodide organification defect (TIOD). Because TPO mutations have a prevalence of 1 in 66,000 newborns and is inherited in an autosomal recessive mode the frequency of a heterozygous germline mutation in the TPO gene should reach about 1 in 260 in the population. A somatic TPO mutation coinciding with a somatic loss of one of the TPO alleles or a TPO germline mutation could lead to somatic loss of TPO activity with impairment of thyroid hormone synthesis and decrease of growth control. The latter would lead to increased thyroid epithelial cell proliferation and the subsequent development of a scintigraphically cold thyroid nodule (CTN). To test this hypothesis we studied 40 CTN for the presence of mutations or loss of heterozygosity (LOH) in the TPO gene. For comparisons we also studied LOH in 17 autonomously functioning thyroid nodules (AFTN). Genomic DNA was extracted from nodular and surrounding tissue, polymerase chain reaction (PCR) amplified, sequenced, and analyzed for LOH. In 6 CTNs of 37 informative cases we detected LOH using the genomic markers sRA, D2S2268, and D2S319 within or near the TPO gene locus (2p24-25). In contrast, a genomic marker closer to the centromer (D2S144, 2p24-21) shows LOH in only 1 CTN. We did not detect LOH in AFTN. In none of the cases a germline or somatic mutation in the TPO gene was detectable in the TPO gene. LOH in 6 of 37 CTNs suggests that genetic defects at the TPO or the chromosomal locus 2p24-25 might play a role in the etiology of CTNs. However, we did not find the combination of LOH with a somatic mutation in the TPO gene. It is therefore likely that a gene defect near the TPO locus is part of the neoplastic process in a subgroup of CTNs.     

The Missense Alteration A5T of the Thyroid Peroxidase Gene is Pathogenic and Associated with Mild Congenital Hypothyroidism

Congenital hypothyroidism (CH) occurs with a prevalence of approximately 1:4000 live births. Defects of thyroid hormone synthesis account for 15-20% of these cases. Thyroid peroxidase (TPO) gene is the most common cause for dyshormonogenesis. So far, more than 60 mutations in the TPO gene have been described, resulting in a variable decrease in TPO bioactivity. We present an 8-day-old male with mild CH who was identified to have a G to A transition in the fifth codon of the TPO gene (c.13G>A; p.Ala5Thr). The unaffected family members were heterozygous carriers of the mutation, whereas 400 healthy individuals of the same ethnic background did not have the mutation. Mutation analysis of 11 known causative CH genes and 4 of our own strong candidate genes with next-generation sequencing revealed no mutations in the patient nor in any other family members. The results of in silico functional analyses indicated partial loss-of-function (LOF) in the resulting enzyme molecule due to mutation. The patient’s clinical finding s were consistent with the effect of this partial LOF of the mutation. In conclusion, we strongly believe that A5T alteration in the TPO gene is actually pathogenic and suggest that it should be classified as a mutation.     

Monoallelic expression of mutant thyroid peroxidase allele causing total iodide organification defect

Mutations in the thyroid peroxidase (TPO) gene lead to severe congenital hypothyroidism due to total iodide organification defect (TIOD). According to the recessive mode of inheritance, patients are homozygous or compound heterozygous for gene mutations. However, about 17% of cases with typical phenotype harbor a single TPO-mutated allele. We present a TIOD family in which the three affected siblings had a single genomic TPO mutation (R693W) inherited from the unaffected father. Other mutations were not found, although all TPO coding exons and exon/intron boundaries were sequenced. Eleven different polymorphisms were found in hetero- or homozygosity in all family members. On the contrary, using retrotranscribed thyroid tissue RNA, all heterozygous polymorphisms and the mutation were homozygous. The distribution of the polymorphisms indicated that only the mutant paternal allele is transcribed at the thyroid tissue level. We excluded the presence of major deletions involving the maternal chromosome at 2p25 and of maternal imprinting or mutations in part of the regulatory regions of the gene. In summary, we report one family with TIOD due to monoallelic expression of a mutant TPO allele in the thyroid. This mechanism might be generally involved in TIOD cases with a single TPO-mutated allele.     

Novel mutations of the thyroid peroxidase gene in patients with permanent congenital hypothyroidism.

OBJECTIVE: It is suggested that iodide organification defects account for 10% of all cases with congenital hypothyroidism (CH). One candidate gene for these defects is the thyroid peroxidase (TPO) gene. DESIGN: Exons 2, 8-10 and 14 of the TPO gene were examined in 30 patients with permanent CH without a family history of CH. This group was characterized by the presence of an orthotopic thyroid gland and elevated TSH levels. METHODS: The mutational screening was performed by single-strand conformational polymorphism followed by sequence analysis of fragments with abnormal migration patterns and by restriction enzyme analysis. RESULTS: In four patients we were able to identify mutations on both alleles which have not been described so far. One patient was a carrier of a new homozygous point mutation in exon 9 resulting in an exchange from Leu to Pro at codon 458. Another patient was found to be compound heterozygous for two mutations, a 20 bp duplication in exon 2 and a new mutation in exon 9 (Arg491His). Two brothers of consanguineous parents showed a homozygous T deletion in exon 14 at position 2512. CONCLUSIONS: Our findings confirm the genetic heterogeneity of TPO defects and support the suggested prevalence of organification defects.     

High prevalence of a novel mutation (2268 insT) of the thyroid peroxidase gene in Taiwanese patients with total iodide organification defect,and evidence for a founder effect

The mutation of the thyroid peroxidase (TPO) gene that causes the total iodide organification defect (TIOD) is a common and severe condition leading to dyshormonogenesis of the thyroid gland in Caucasians. However, the role of TIOD in Chinese patients with thyroid dyshormonogenesis is unknown. In this study we followed 16 patients from 16 unrelated families in Taiwan and performed perchlorate discharge examination. Seven patients had TIOD and 2 had the partial iodine organification defect (PIOD) among the 16 families. These 9 patients underwent screening in search of TPO gene mutations. Three new mutations (2268 insT, 2243 delT, and G157C) were detected in the 7 patients with TIOD, whereas no mutation in the TPO gene was found in the 2 patients with PIOD. The 2268 insT mutation was noted to be the most common among these TIOD patients (12 of 14 studied alleles, 86%). With 3 intragenic polymorphic markers, we found that the alleles carrying the 2268 insT mutation in Taiwan Chinese TIOD patients were tightly linked to a specific haplotype. The allele frequencies of this haplotype in the 8 patients with homozygous 2268 insT (5 unrelated families, 10 studied alleles) and in 49 normal individuals (98 studied alleles) were 1.00 and 0.02, respectively (P < 0.0001). This indicates that this common novel mutation among Taiwanese patients with TIOD is due to a founder effect.     

Congenital goitrous hypothyroidism: mutation analysis in the thyroid peroxidase gene

Background Iodide organification defect (IOD) is characterized by a reduced ability of the thyroid gland to retain iodide resulting in hypothyroidism. Mutations in thyroid peroxidase (TPO) gene appear to be the most common cause of IOD and are commonly inherited in an autosomal recessive fashion. The TPO gene is located on the chromosome 2p25. It comprises 17 exons, covers approximately 150 kb of genomic DNA and codes 933 amino acids. Objetives In this study, we characterize the clinical and molecular basis of seven patients from four unrelated families with congenital hypothyroidism (CH) because of IOD. Design and Methods All patients underwent clinical, biochemical and imaging evaluation. The promoter and the complete coding regions of the human TPO along with the flanking intronic regions were analysed by single‐strand conformation polymorphism analysis and direct DNA sequencing. Segregation analysis of mutations was carried out, and the effect of the novel missense identified mutations was investigated by ‘in silico’ studies. Results All subjects had congenital and persistent primary hypothyroidism. Three novel mutations: c.796C>T [p.Q266X], c.1784G>A [p.R595K] and c.2000G>A [p.G667D] and a previously reported mutation: c.1186_1187insGGCC [p.R396fsX472] have been identified. Four patients were compound heterozygous for p.R396fsX472/p.R595K mutations, two patients were homozygous for p.R595K, and the remaining patient was a compound heterozygous for p.Q266X/p.G667D. Conclusions Our findings confirm the genetic heterogeneity of TPO defects and the importance of the implementation of molecular studies to determinate the aetiology of the CH with dyshormonogenesis.     

High prevalence of thyroid peroxidase gene mutations in patients with thyroid dyshormonogenesis

OBJECTIVE: Thyroid dyshormonogenesis is a genetically heterogeneous group of inherited disorders in the enzymatic cascade of thyroid hormone synthesis that result in congenital hypothyroidism (CH). Thyroid peroxidase gene (TPO) mutations are one of the most common causes of thyroid dyshormonogenesis. The aim of this study was to identify TPO gene defects in a cohort of patients with thyroid dyshormonogenesis from Slovenia, Bosnia, and Slovakia. DESIGN AND METHODS: Forty-three patients with permanent CH and orthoptic thyroid glands from 39 unrelated families participated in the study. Mutational analysis of the TPO gene and part of its promoter consisted of single-stranded conformation polymorphism analysis, sequencing, and restriction fragment length polymorphism (RFLP) analysis. Results: TPO gene mutations were identified in 46% of participants. Seven different mutations were identified, four mutations of these being novel, namely 613C > T (R175X), 1519_1539del (A477_N483del), 2089G > A (G667S), and 2669G > A (G860R). Only a single allele mutation was identified in 65% of the TPO mutation carriers. CONCLUSIONS: The results showed a higher prevalence of TPO gene mutations in thyroid dyshormonogenesis when compared with published studies. The high percentage of single allele mutations implied possible intronic or regulatory TPO gene mutations or monoallelic expression.