先天性甲状腺功能减低症汉族儿童的促甲状腺素受体基因失活突变

目的探讨汉族儿童促甲状腺素受体（TSHR）基因失活突变与先天性甲状腺功能减低症（CH）的相关性。方法（1）选择79例CH汉族儿童（亚临床甲减14例,年龄1～5.5岁,男8例,女6例;甲减65例,年龄1.5～6岁,男27例,女38例）为研究对象;100名正常儿童（男40例,女60例,年龄1～8岁）作为对照组。（2）采用PCR和DNA测序技术检测TSHR基因失活突变。结果（1）79例CH患儿中有1例发生复合杂合子突变,其突变位点为（Pro52Thr／Val689Gly）。1例发生杂合子突变,其突变位点为（Gly245Ser）。30例患儿在第10外显子2181位核苷酸处发生C-G转换（GAC→GAG）,使727位密码子天冬氨酸被谷氨酸代替（Asp727Glu）。47例患儿在第7外显子561位核苷酸处发生T-c转换（AAT→AAc）,相应的187位氨基酸（Asn）不发生改变。（2）33例正常对照儿童在第10外显子2181位核苷酸处发生C-G转换;50例正常对照儿童在第7外显子561位核苷酸处发生T-c转换（AAT→AAc）。结论浙江汉族CH患儿TSHR基因有3个杂合子突变位点：（Pr052Thr）、（Gly245Ser）、（Val689Gly）,第10外显子2181位核苷酸处（GAc→GAG）及第7外显子561位核苷酸处（AAT→AAC）存在TSHR基因多态性。     

山东地区儿童先天性甲状腺功能减低症伴甲状腺发育不全TUBB1基因突变的研究

目的 探讨山东地区先天性甲状腺功能减低症（CH）伴甲状腺发育不全（TD）患儿TUBB1基因突变的类型和特点。方法 对山东地区289例确诊CH伴TD患儿进行TUBB1基因全编码区突变研究。提取患儿外周血全基因组DNA，PCR扩增TUBB1基因全编码区，对扩增产物进行Sanger测序，并进行生物信息学分析。结果 289例CH伴TD患儿中发现4例（1.4%） TUBB1基因存在c.952C > T （p.R318W）杂合变异，导致TUBB1蛋白第318位色氨酸变成精氨酸，根据美国医学遗传学与基因组学学会遗传变异分类标准与指南，该变异评级为"可能致病的"。结论 在山东地区CH伴TD患儿中发现了新的TUBB1基因变异，提示TUBB1基因可能是CH伴TD的候选致病基因。     

Congenital hypothyroidism caused by a unique thyroid peroxidase allele containing two mutations, C1708T and C2737T

Thyroid peroxidase (TPO) is a key enzyme of thyroid hormone biosynthesis. TPO abnormality is considered to be a major cause of congenital hypothyroidism (CH) with total iodide organification defect. In the present study, we examined the TPO gene of three siblings, 3 and 2 year-old brothers and a newborn sister, with severe CH. All 17 exons and the promoter region in the TPO gene were directly sequenced using genomic DNA. Two homozygous mutations, C1708T and C2737T, were found in all three patients. The C1708T mutation introduces a premature terminal codon, which is suggested to be a cause of CH. The other mutation, C2737T, and 13 single nucleotide polymorphisms in the patients' TPO genes were also detected as homozygous. We suspect that the mutated alleles were inherited from a single, common ancestor. The haplotype including the two mutations was conserved in a narrow region between D2S2268 and D2S323 microsatellite markers on the end of chromosome 2.     

先天性甲状腺功能减退症患儿DUOX2基因突变的研究

目的 研究先天性甲状腺功能减退症(congenital hypothyroidism, CH)患儿DUOX2 基因突变类型和特点,并初步探讨基因型- 表现型的关系,为CH 患儿的基因诊断和基因治疗提供理论依据.方法 从10例CH 伴甲状腺肿大患儿外周血白细胞中提取基因组DNA,采用PCR 扩增和直接测序的方法对DUOX2 全部外显子进行基因突变检测.结果 在1 例患儿中发现DUOX2 基因第28 外显子cDNA 的3632 位点发生了G>A 的突变(c.G3632A),导致第1 211 密码子的精氨酸变为组氨酸(p.R1211H).在3 例患儿中发现DUOX2 基因第17 外显子cDNA 的2 033 位点发生了T>C 的突变(c.T2033C),导致第678 密码子的组氨酸变为精氨酸(p.H678R).此两种突变均为杂合型的错义突变.结论 CH 患儿存在DUOX2 基因杂合突变,该杂合突变可能引起蛋白质功能的改变从而导致CH;基因型与表现型的关系尚不明确,需要进一步的研究.     

Genetic Analysis in Children with Transient Thyroid Dysfunction or Subclinical Hypothyroidism Detected on Neonatal Screening

About 30% of children with elevated TSH levels during neonatal screening have a transient form of disorder. On the other hand, it has been reported that subclinical hypothyroidism persists in late childhood in about 30% of children found to be false-positive during neonatal screening. The aim of this study was to determine whether transient thyroid dysfunction and subclinical hypothyroidism detected during neonatal screening are influenced by genetic background. The TSH receptor (TSHR), thyroid peroxidase (TPO) and dual oxidase 2 (DUOX2) genes, for which it has been reported that heterozygous defects cause neonatal transient thyroid dysfunction, were analyzed. Nine children with transient thyroid dysfunction or subclinical hypothyroidism detected during neonatal screening were studied. One child was heterozygous for a TSHR gene mutation (R450H), and another child was heterozygous for a TPO gene mutation (P883S). No children with mutation of the DUOX2 gene were identified. Genetic background may contribute to development of transient thyroid dysfunction and subclinical hypothyroidism detected during neonatal screening.     

A Novel Missense Mutation in the Thyroid Peroxidase Gene,R175Q,Resulting in Insufficient Cell Surface Enzyme in Two Siblings

Thyroid peroxidase (TPO) abnormality is one of the causes of congenital hypothyroidism. Two missense mutations were found as a compound heterozygous mutation in two siblings with congenital goitrous hypothyroidism. One of these mutations, G614A (R175Q), was a novel mutation. Characterization of the novel mutation and a cotransfection experiment with two mutated TPO mRNAs were carried out. G614A-mRNA introduced into CHO-K1 cells expressed TPO protein with the same molecular weight as that of wild-type mRNA. The R175Q-TPO was thought to possess enzyme activity. In terms of localization, a very small amount of mutated TPO was expressed on the plasma membrane of CHO-K1 cells. This plasma membrane expression of R175Q-TPO was insufficient to perform thyroid hormone synthesis, but was markedly different from R665W-TPO. When G614A- and C2083T-mRNAs were cotransfected, cell surface TPO-positive cells were only 13.1% in contrast to 54.4% for wild-type mRNA. The low positivity and intensity of cell surface TPO suggested that in the patients’ thyroids thyroid hormone synthesis was hardly performed. The congenital hypothyroidism of the patients was thought to be a result of the mutations of the TPO gene (G614A/C2083T).     

先天性甲状腺功能减低症35例甲状腺过氧化物酶基因突变检测

目的 检测35例先天性甲状腺功能减低症(CH)患儿甲状腺过氧化物酶(TPO)基因突变.方法 抽取35例先天性甲状腺功能减低症患儿外周血并提取DNA,用PCR扩增患儿TPO基因所有17个外显子、外显子-内含子交界区以及3'端和5'端非翻译区,用DNA测序技术和限制性内切酶检测基因突变,并对发现突变的CH患儿父母进行对照分析.结果 5例CH患儿存在TPO基因突变:1例为c.961A＞G和c.2422delT突变复合杂合子,1例为c.2268insT和c.1477G＞A突变复合杂合子,3例为c.2268insT突变纯合子.其中c.961 A＞G[p.Thr321 Ala]为未见文献报道的突变.结论 在35例先天性甲状腺功能减低症检测到4种TPO基因突变.

Abstract：

Objective To identify thyroid peroxidase (TPO) gene mutations in 35 patients with congenital hypothyroidism. Method Genomic DNA was isolated from peripheral blood samples of 35 patients with congenital hypothyroidism. All of the 17 exons and flanking introns of TPO gene were amplified by PCR, then the PCR products were sequenced bi-directionally and were analyzed by restriction endonucleases. Result One patient had compound heterozygous mutations c. 961A ＞ G/c. 2422delT, one was c. 2268insT/c. 1477G ＞ A, and three was homozygous mutation c. 2268insT. The TPO gene mutation c.961A ＞ G [p. Thr321Ala] was one novel mutation. Conclusion High frequency mutation in TPO gene was detected in patients with congenital hypothyroidism.     

先天性甲状腺功能减退症患儿GNAS和THRA基因突变分析

目的 对70例先天性甲状腺功能减退症（CH）患儿的刺激性G蛋白α亚基（GNAS）基因和甲状腺素受体α（THRA）基因进行二代测序分析,并初步探讨GNAS和THRA基因突变型与CH患儿的临床表现型之间的关系。方法 选取70例通过新生儿筛查确诊为CH的患儿,采集外周血并进行DNA样本提取,利用二代测序技术对GNAS和THRA基因进行突变筛查,利用生物信息学软件分析基因突变的致病性。结果 70例CH患儿中,3例患儿（4%）检出9种GNAS基因的错义突变（包括3种已知基因突变和6种新突变）,4例患儿检出同1种THRA基因多态c.508A > G（p.I170V）。经过生物信息学软件预测和ACMG/AMP指南分析发现2种GNAS基因突变[c.301C > T（p.R101C）、c.334G > A（p.E112K）]致病的可能性大。3例携带GNAS基因突变的患儿存在不同程度的甲状腺功能低下表现。结论 GNAS基因突变与CH的发病有关,患儿的临床表现存在较大的异质性;THRA基因突变可能与CH的发病无相关性。     

甲状腺球蛋白增高的先天性甲状腺功能减退症1例报告并文献复习

目的 探讨甲状腺球蛋白(TG)增高的先天性甲状腺功能减退症(CH)家系的临床特征及TG基因变异特征.方法 回顾分析1个TG增高的CH家系的临床及TG基因检测结果,并复习相关国内外文献.结果 先证者,女,45日龄,生后黄疸消褪延迟伴便秘.甲状腺功能检测提示为CH,同时发现TG水平增高.基因检测结果显示患儿TG基因存在c....     

线粒体相关肾病2例病例报告及文献复习

目的 总结2例线粒体相关肾病患儿临床特征及基因突变的特点,提高对该病的认识。方法 收集2例线粒体相关肾病患儿的病史特点、肾脏病理、相关实验室检查和家族史等资料。采用外显子捕获的方法对4 000种人类单基因病的相关致病基因进行高通量测序,包括线粒体DNA A3243G等37个基因和ADCK4等13个参与辅酶Q10生物合成的基因,利用生物信息学对测序结果进行分析,用Sanger法对高通量测序结果进行验证,并在家系中进行突变分析。并进行相关文献复习。结果 2例患儿男女各1例。女性患儿11.7岁起病,主要临床表现为蛋白尿和肾功能异常,无肾外症状,肾脏病理为局灶节段性肾小球硬化(FSGS),检测到NPHS1基因已报道的p.E447K和p.G601A杂合突变,ADCK4基因纯合p.D209H错义突变,为新发现的突变。家系突变分析发现,NPHS1基因p.E447K和p.G601A杂合突变均来自父亲,其哥哥也有相同的基因型,其母亲不携带该2个突变;患儿父母和哥哥分别携带p.D209H杂合突变。男性患儿出生后起病,多个系统受累,表现为精神、运动发育落后,心脏和大血管多发畸形,肾病综合征。检测到COQ6基因的纯合p.R360W错义突变,为新发现的突变。家系突变分析显示,患儿父母分别携带杂合p.R360W错义突变。ADCK4基因p.D209H错义突变和COQ6基因p.R360W错义突变经在线软件PolyPhen和SIFT预测为有害性突变,经多物种蛋白序列比对,2个突变位点均具有保守性。结论 2例患儿肾脏表型分别由辅酶Q10合成基因ADCK4和COQ6突变引起的线粒体相关肾病。新发现p.D209H和p.R360W突变分别丰富了ADCK4和COQ6基因突变谱。     

家族性高胆固醇血症低密度脂蛋白受体基因突变的研究

目的 分析中国儿童家族性高胆固醇血症低密度脂蛋白受体基因突变,并建立筛查该基因突变的方法。方法 以一家两患儿及其父母的基因组DNA为模板,用聚合酶链反应（PCR）及增该基因的启动子和全部18个外显子。用温度梯度凝胶电泳（TGGE）分析检测PCR产物,对电泳结果异常者进行DNA测序。结果 平行TGGE发现,两患儿第13外显子存在一纯合突变,其父母此外显存在杂合突变。DNA测充证实两患儿第13外显子发生Ala^606→Thr纯合错义突变,其父母均为此Ala^606→Thr杂合突变。结论 此家系家族性高胆固醇血症患者的LDL受体 基因存在Ala^606→Thr突变。TGGE结合DNA测序法的建立可用本症的基因诊断。     

Rett综合征的临床特点及MECP2基因突变分析

目的 分析典型的Rett综合征患者的临床特点,并对患儿甲基化CpG结合蛋白-2（MECP2）基因进行突变分析。方法 使用PCR扩增和测序的方法 对近期诊断的9例RETT综合征患儿及其父母MECP2基因的3个外显子进行检测。结果 在9例患儿中发现5例存在MECP2基因的杂合突变,突变率超过50%,其中1例为碱基插入导致的移码突变（c.913insT）;其余4例为点突变,分别为位于外显子3的c.316C>T（R106W）,位于外显子4的c.502C>T（R168X）、c.808C>T（R270X）和c.1126C>T（P376S）,其中c.913insT为首次发现的突变,这些患儿父母均未检测到突变。2例患儿MECP2基因突变位于转录抑制区域,与其他患儿相比,这2个患儿语言功能几乎丧失,且发育明显落后。结论 该研究确诊的5例Rett综合征的患儿存在MECP2基因突变,且多数位于外显子4上;MECP2蛋白转录抑制区域突变可能影响患儿语言功能及发育明显落后。     

Apert 综合征 1 例临床特征与基因类型分析

目的分析Apert综合征(AS)临床特征与基因类型。方法回顾1例AS患儿的临床资料及患儿和其父亲FGFR2基因测序结果,并复习相关文献。结果男性患儿,1岁1个月,扁头,突眼,眼距宽,耳位低,下颌小,高腭弓,无腭裂,双手五指并指并挛缩,双足五趾并趾。FGFR2基因外显子7 c.758CG,p.P253R杂合变异,父亲未检测到相关基因突变,支持Apert综合征诊断。文献检索到AS个案24例,22例明显颅面部畸形,1例轻微畸形,1例无畸形;均有手足并指/趾畸形。基因类型为S252W 13例,P253R 3例,Alu元件插入3例,基因缺失2例,杂合突变2例,序列变异1例。结论 AS患儿颅面部畸形及手足并指/趾明显,FGFR2基因以S252W、P253R突变为主。     

Differential diagnosis between Pendred and pseudo-Pendred syndromes: clinical, radiologic, and molecular studies

The disease gene for Pendred syndrome has been recently characterized and named PDS. It codes for a transmembrane protein called pendrin, which is highly expressed at the apical surface of the thyroid cell and functions as a transporter of chloride and iodide. Pendrin is also expressed at the inner ear level, where it appears to be involved in the maintenance of the endolymph homeostasis in the membranous labyrinth, and in the kidney, where it mediates chloride-formate exchange and bicarbonate secretion. Mutations in the PDS gene and the consequent impaired function of pendrin leads to the classic phenotype of Pendred syndrome, i.e. dyshormonogenic goiter and congenital sensorineural hearing loss. In the present study, we performed a detailed clinical, radiologic, and molecular analysis of six families presenting with clinical diagnosis of Pendred syndrome. In two families a homozygous pattern for PDS mutations was found, whereas the affected members of the other four families were compound heterozygotes. One family did not harbor PDS mutations. Among the four novel mutations described, one is a transversion in exon 2 (84C>A), leading to the substitution S28R. Two other novel mutations lie in exon 4 (398T>A) and in exon 16 (1790T>C), leading to the substitutions S133T and L597S, respectively. The fourth novel mutation (1614+1G>A) is located in the first base pair of intron 14, probably affecting the splicing of the PDS gene. Clinically, all patients had goiter with positive perchlorate test, hypothyroidism, and severe or profound sensorineural hearing loss. In all the individuals harboring PDS mutations, but not in the family without PDS mutations, inner ear malformations, such as enlargement of the vestibular aqueduct and of the endolymphatic duct and sac, were documented. The pseudo-Pendred phenotype exhibited by the family without PDS mutations is likely caused by an autoimmune thyroid disease associated with a sensorineural hearing loss of different origin.     

Low TSH congenital hypothyroidism: identification of a novel mutation of the TSH beta-subunit gene in one sporadic case (C85R) and of mutation Q49stop in two siblings with congenital hypothyroidism

Isolated congenital hypothyroidism resulting from mutation of the TSH beta-subunit gene, has rarely been reported. In the present article, we report a new mutation (C85R) in exon 3 of the TSH beta-subunit gene in one sporadic case and the mutation Q49stop in two siblings with congenital hypothyroidism. The novel mutation is a T to C transition at codon 85, resulting in a change of cysteine to arginine (C85R) of the ss-subunit. Because the cysteine residues of all glycoproteins are highly conserved, this mutation is expected to result in conformational changes of the ss-subunit, rendering it incapable to form a functional heterodimer with the alpha-subunit. The second mutation described is a C to T transition resulting in a premature stop at codon 49 (Q49stop), leading to the formation of a truncated protein. Although the two siblings reported herein carried the same mutation, they had slightly modified clinical and biochemical phenotype. The mutation C85R and the previously described E11stop have, thus far, exclusively been detected in Greek patients. The Q49stop mutation initially detected in Greek patients was subsequently identified in an Egyptian girl and most recently in two Turkish siblings. These three reports possibly indicate the presence of a mutational hot spot on the TSH beta-subunit gene. Hence, with the novel mutation herein reported, a total of five mutations of the TSH beta-subunit gene are recognized as a cause of low-TSH congenital hypothyroidism worldwide.     

Congenital hypothyroidism caused by a novel homozygous mutation in the thyroglobulin gene

Congenital hypothyroidism (CH) due to thyroglobulin (TG) deficit is an autosomal recessive disease (OMIM #274700) characterized by hypothyroidism, goiter, low serum TG, and a negative perchlorate discharge test. The aim of this study was to perform the genetic analysis of the TG gene in two sisters born from consanguineus parents and affected by CH and low serum TG levels. The index patient and her sister were identified at neonatal screening for CH and treated with L-thyroxine (L-T4). After discontinuation of L-T4 therapy, hypothyroidism was confirmed, serum TG was undetectable, and no organification defect after ¹²³I scintigraphy and perchlorate test was shown; thyroid ultrasound showed a eutopic gland of normal size. DNA was extracted from peripheral white blood cells of the two sisters and the father. All 48 exons of TG gene were amplified by polymerase chain reaction and subjected to direct sequencing. A novel homozygous point mutation in exon 10 of TG gene was identified in the patient and her sister. The mutation determined a stop codon at position 768 (R768X) resulting in an early truncated protein or in the complete absence of the protein. The father (euthyroid) was heterozygous carrier of the mutation. Conclusion: Genetic analysis of TG gene was performed in two sisters affected by CH. A novel point mutation of the TG gene determining a stop codon at position 768 of the protein was identified. The early truncated nonfunctioning protein or the absence of the protein due to the premature degradation of abnormal mRNA may be responsible of the observed phenotype.     

中国人多种羧化酶缺陷症患儿4例及其父母基因突变分析

目的研究中国人多种羧化酶缺陷症（multiple carboxylase deficiency,MCD）患儿及其父母基因突变情况。方法应用聚合酶链反应-直接测序的方法,对4例临床确诊MCD的中国患儿的生物素酶（BT）基因和全羧化酶合成酶（HLCS）基因的各个外显子及其两侧侧翼序列进行突变的检测,并对其父母进行相应突变基因检测。结果4例患儿皆为HLCS基因突变,没有发现BT基因突变。共发现1个缺失突变：780de1G,3个错义突变：1522C〉T（R508W）、1367A〉G（Y456C）和1900G〉A（I）634N）。例1为HLCS基因的第11号外显子上的1522C〉T的错义突变,为纯合突变;例2为第11号外显子的1522C〉T和第9号外显子的1367A〉G的复合性杂合突变;例3为第11号外显子的1522C〉T和第13号外显子的1900G〉A的复合性杂合突变;例4为第11号外显子的1522C〉T和第7号外显子的780de1G的复合性杂合突变;4例患儿的父母均是突变基因携带者。结论R508W突变可能是中国MCD中HLCS缺陷患儿较常见的突变。     

LHCGR基因突变(Asp578His)致家族性男性性早熟1例临床特点及基因分析

该文报道1例由LHCGR基因激活性杂合突变导致的家族性男性性早熟(FMPP)患儿的临床特点及基因分析。患儿为男性,婴儿时期出现了外生殖器增长过快、生长加速,伴阴毛及阴茎勃起,结合患儿性征检查、促性腺激素释放激素兴奋试验、血清睾酮检测及骨龄检查等结果,临床诊断为外周性性早熟。随后对患儿及其父母的性早熟相关基因进行检测。基因测序结果提示患儿黄体生成素/绒毛膜促性腺激素受体(LHCGR)基因第11外显子1732GC突变,导致578位氨基酸由天冬氨酸变为组氨酸,该突变是1个未见文献报道的新突变,且父母基因检测结果未见异常。联用第3代芳香化酶抑制剂来曲唑和抗雄激素制剂螺内酯治疗半年后,患儿症状得到控制。该研究结果扩展了LHCGR基因突变谱,为FMPP病因诊断及家系的遗传咨询和产前诊断提供了分子依据。     

Pseudohypoparathyroidism type 1a with congenital hypothyroidism

Pseudohypoparathyroidism type la (PHP-1a) is an uncommon disorder that results from an inactivating mutation in the GNAS gene. It can present with resistance to several hormones, in addition to parathyroid hormone (PTH). Patients may have the classic Albright's hereditary osteodystrophy (AHO) phenotype and can develop resistance to thyroid stimulating hormone (TSH), gonadotropins, growth hormone releasing hormone (GHRH), and other hormones that rely on the Gsalpha protein to regulate signal transmission at their receptors. We report two siblings with PHP-1a and congenital hypothyroidism. The patients were found to have a heterozygous mutation at nucleotide 305 in exon 4 (c305C-->A) of the GNAS gene, which has not been previously linked to congenital hypothyroidism.     

17α羟化酶/17,20碳链裂解酶缺陷症一家系CYP17A1基因突变分析

目的 对一儿童17α羟化酶/17,20碳链裂解酶缺陷症(17OHD)先证者及其主要家系成员进行CYP17A1基因突变分析,旨在提高认识和引起重视.方法分别获取先证者及其父母、双胎姐姐的基因组DNA,应用PCR和DNA直接测序方法,进行CYP17A1基因突变检测.结果 先证者存在CYP17A1基因复合型点突变,即Exl的nt186delC和Ex6的nt1085G>A,使17α羟化酶完全失活,从而导致170HD.并在其父Exl、其母和其双胎姐姐Ex6上均发现同类型基因突变.其中nt186delC为新发现突变位点,健康对照者未发现类似基因突变.结论 异卵双胎先证者分别在CYP17A1基因的Exl和Ex6存在复合点突变,其父母和双胎姐姐均为突变携带者.开展基因诊断有助于提高临床诊治水平.