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。     

家族性高胆固醇血症一家系成员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3代家系成员进行基因检测和系谱分析,探讨该FH患儿发病的分子病理基础.方法 收集FH先证者家系3代共29例血标本及临床资料.对先证者进行心电图和超声检查.对其家系成员进行血脂测定,改良苯酚-氯仿法提取患儿及家系成员基因组DNA并鉴定,应用多聚酶链反应-单链构象多态性(PCR-SSCP)分析结合DNA直接测序方法 ,检测其低密度脂蛋白受体(LDLR)基因全部18个外显子和启动子及载脂蛋白B100(ApoB100)R3500Q位点,核苷酸序列分析结果 与GenBank比对寻找突变.结果 1.血管超声示先证者右锁骨下动脉起始,双侧颈总动脉分叉处中段内-中膜轻度增厚,右侧颈总动脉中段后壁多发小钙化斑,主动脉瓣轻度关闭不全;2.该家系排除ApoB100基因R3500Q突变;3.核苷酸序列分析证实先证者及其父亲、祖父和叔叔等6人LDLR基因第10外显子发生W462X杂合突变,为色氨酸改变为终止密码子,使终止密码子在第462位提前出现;先证者及母亲和外祖母LDLR基因第13外显子发生A606T杂合突变,为第1 879位G→A碱基置换,导致丙氨酸改变为苏氨酸.结论 该FH先证者LDLR基因存在W462X/A606T复合杂合突变,分别来源于父系及母系遗传.这种复合杂合突变患者具有与FH纯合子相同的临床特征.     

家族性高胆固醇血症纯合表型患儿及家系致病基因突变分析

目的 对1例临床确诊为纯合型家族性高胆固醇血症(FH)患儿及其家系进行基因检测和系谱分析,探讨该家系可能存在的致病基因突变.方法 于先证者家中收集该家系3代共47例血标本及临床资料.对先证者及其家系成员进行血脂测定和心电图检查;酚氯仿法提取患者基因组DNA并鉴定,PCR扩增基因外显子和内含子,采用变性高效液相色谱(HPLC)法及扩增产物直接序列分析检测其低密度脂蛋白受体(LDL-R)基因的全部18个外显子和启动、载脂蛋白B(ApoB100)R3500Q位点、枯草溶菌素9(PCSK9)基因全部12个外显子有无点突变;用多重连接探针扩增法(MLPA)检测先证者有无LDL-R基因的长片段的缺失或插入突变;核苷酸序列分析结果与Gen Bank比对寻找突变.结果 先证者临床确诊为纯合子FH.根据血脂和临床表现,先证者家系47例中16例诊断为高胆固醇血症,其中临床确诊1例纯合子FH,6例杂合子FH;系谱分析该家系遗传方式符合常染色体显性遗传规律.先证者核苷酸序列分析排除LDL-R、ApoB100、PCSK9基因点突变;MLPA法排除了先证者LDL-R基因的长片段缺失.结论 该家系为遗传性高胆固醇血症家系,通过对该家系进行基因突变检测和家系分析排除了已知的致FH基因突变,认为先证者的FH样表型可能是一种未知的基因突变,尚需采用进一步的全基因扫描方法寻找其致病基因.     

家族性高胆固醇血症患者及其家系成员候选基因突变分析

目的对1例临床确诊为杂合子家族性高胆固醇血症先证者及其3代家系成员进行候选基因检测和系谱分析,探讨其发病机制。方法收集该家系3代共13例血标本及临床资料。对先证者进行心电图和超声检查。对其家系成员进行血脂测定。酚氯仿法提取患儿及家系成员基因组DNA并鉴定,应用多聚酶链反应-单链构象多态性(PCR-SSCP)分析结合DNA直接测序方法,检测其低密度脂蛋白受体(LDLR)基因的启动子及编码区,枯草溶菌素转化酶9(PCSK9)基因启动子及编码区,载脂蛋白B100(apoB100)26外显子,核苷酸序列分析结果与GenBank比对。结果1.先证者颈动脉中膜厚度增厚,局部可见强回声斑块;左房轻度增大,主动脉瓣、二尖瓣轻度返流,冠状动脉血流储备减低。2.该家系排除apoB100基因3500及附近位点突变。3.核苷酸序列分析证实先证者LDLR基因第10外显子发生W462X杂合突变,为色氨酸变为终止密码子;PCSK9基因第1外显子发生第158位碱基T取代C,63、64碱基间插入CTG,分别导致53位丙氨酸(A)变为缬氨酸(V)和21、22位氨基酸之间插入亮氨酸(L)。结论该先证者LDLR基因存在W462X杂合突变,可能为该家...     

激素依赖型肾病综合征患儿NPHS2基因突变

目的 分析中国激素依赖型肾病综合征(SDNS)汉族儿童NPHS2基因突变及其特点.方法 分别取1例SDNS患儿及其父母、弟弟(汉族)及50例尿检正常成年人外周静脉血各3 mL,枸橼酸钠抗凝,提取基因组DNA.应用PCR方法扩增NPHS2基因的全部8个外显子及其启动子全长,对PCR扩增产物直接进行DNA测序.结果 在SDNS患儿NPHS2基因外显子5上检测到1个杂合错义突变-596AT;在NPHS2基因外显子8上检测到1个杂合同义突变 -954TC.在该患儿NPHS2基因启动子区检测到-116CT纯合变异,其母亲和弟弟NPHS2基因上也检测到596AT的杂合错义突变和954TC杂合同义突变.在其父亲和弟弟NPHS2基因启动子区检测到-116CT杂合变异.但患儿的父母和弟弟尿检均正常.596AT为新发现的突变,导致NPHS2基因的编码蛋白podocin的第199位的天冬氨酸(位于stomatin蛋白家族的高度保守区)变为异亮氨酸,即N199I.分析对照人群100条染色体,未发现NPHS2基因596AT突变;但检测到954TC同义突变和-116CT变异.结论 汉族肾病综合征患儿对激素依赖可能与NPHS2基因突变有关.     

三个家族性高胆固醇血症样表型纯合子家系遗传异质性及其系谱分析

目的对3个纯合子型家族性高胆固醇血症（FH）样表型家系进行基因检测及系谱分析，初步探讨FH异质性的分子基础。方法对3个先证者及家系成员进行血脂测定、心电图、心脏及大血管彩色多普勒超声检查，采用聚合酶链反应（PCR）-变性高效液相色谱（DHPLC）法结合扩增产物直接序列分析检测低密度脂蛋白受体（LDL-R）、载脂蛋白（apoB）、枯草溶菌素9（PCSK9）基因，结果与GenBank比对，并进行家系分析。结果先证者3例初诊时年龄均〈16岁，血浆胆固醇水平〉258mg／dL，出生时即有黄色瘤，之后多处出现黄色瘤并有角膜环，可确诊为纯合子型FH。1例出现早发冠心病，多次发生心绞痛。LDL-R基因发现313＋1G→A纯合突变。检查3家系3代共86例，根据血脂和临床表现确诊17例杂合子FH患者，系谱分析家系1、2遗传方式符合常染色体显性遗传规律，家系3符合常染色体隐性遗传规律。结论证实3个家族性高胆固醇血症样表型系谱，系谱分析可初步明确FH遗传方式，结合基因检测可对FH异质性的诊断和治疗提供依据。     

儿童散发Rett综合征临床特征、基因突变与遗传咨询

目的 寻找散发典型Rett综合征患儿甲基化CpG结合蛋白2基因(MECP2基因)突变,探讨基因型和表型之间的关系,并为遗传咨询提供帮助.方法 提取患者及其父母静脉血白细胞基因组DNA,使用MECP2基因外显子特异引物进行PCR扩增和DNA测序检测.结果 2例患者MECP2基因外显子1、2、3未发现突变,外显子4分别存在一种杂合错义突变,核苷酸变化分别为c.C473T和c.C397T,导致相应的氨基酸变化p.T158M和p.R133C;其父母均无相应突变；c.C397T患者的临床表型较c.C473T轻.结论 典型Rett综合征患者多数存在MECP2基因突变,应行基因检测,阳性结果患者应检测其父母相应变化,为遗传咨询提供依据.     

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。     

白介素7受体α基因缺陷导致严重联合免疫缺陷病一例

目的 探讨我国首例白介素7受体α(IL-7Rα)基因缺陷患儿的临床特征和基因突变类型.方法 患儿为男性,5月龄,生后15 d开始反复出现发热、咳嗽、腹泻,伴卡介苗接种处破溃、流脓和左腋下包块.采用PCR方法扩增患儿及父母IL-7Rα基因组DNA.采用RT-PCR扩增患儿及父母IL-7Rα mRNA.PCR产物直接进行双向序列测定.结果 患儿的免疫球蛋白IgG 6867 mg/L,IgA249 mg/L,IgM 206 mg/L,IgE 2.3 U/ml.淋巴细胞分类T淋巴细胞(CD3~+)0,B淋巴细胞(CD19~+)58%,NK细胞(CD16~+CD56~+)42%.基因分析患儿为IL-7Rα基因的复合杂合突变.在第4内含子剪接位点+1位发生突变[intron4(+1)GA],患儿父亲为此突变基因的携带者;第5外显子638位核苷酸发生无义突变(638 CT,R206X),患儿母亲为此突变基因的携带者.第4内含子剪接位点突变[intron4(+1)GA]为首次报道的突变类型.RT-PCR检测发现患儿IL-7Rα mRNA表达明显降低.患儿IL-7Rα cDNA经巢式PCR扩增并T-A克隆,测序发现外显子4出现64个核苷酸缺失(496-559del,K158fs160X).结论 通过临床筛查和基因分析,鉴定出我国首例IL-7Rα基因复合杂合突变[intron4(+1)GA和638 CT]患儿.     

家族性高胆固醇血症6例临床与生化分析

目的 探讨家族性高胆固醇血症的临床、生化特点与诊断、治疗方法。方法 对6例患儿进行回顾性分析。结果 就诊年龄2～16岁。均可见不同程度的皮肤黄色瘤，血清总胆固醇6．38～18．53mmol／L，LDLC 4．71～16．35mmol／L，甘油三酯浓度正常：4例心电图ST-T段下降。2例分别合并腔隙性脑梗死和眼底动脉硬化。例5及其父母接受了LDL-R基因分析，证实例5及其父亲4A外显子444位碱基存在杂合突变(T→A)。经低胆固醇饮食和降脂药物治疗，5例患儿血脂逐渐下降，全身情况改善。结论 家族性高胆固醇血症严重损害患儿心血管系统和皮肤，通过低胆固醇饮食和降脂药物治疗可有效地改善预后，早期诊断至关重要。     

家族性载脂蛋白B_(100)缺陷第3500位氨基酸突变检测方法比较及初步应用

为比较和探讨家族性载脂蛋白Ｂ１００缺陷患者ａｐｏＢ－３５００位突变的检测方法，对４个高胆固醇血症家族的１９名成员，采用聚合酶链反应（ＰＣＲ）结合酶切法、等位基因特异寡核苷酸（ＡＳＯ）探针杂交法及ＰＣＲ产物序列分析法检测此点突变，并通过定点突变法构建一人工突变体作为阳性对照，以确保试验方法可靠。结果：４个家族１９名成员通过ＰＣＲ结合酶切及ＡＳＯ探针杂交两种方法皆没有检测到ａｐｏＢ－３５００位突变，与测序结果一致；测序结果验证人工构建突变体的１０６５８位核苷酸为Ａ。结论：比较而言，ＰＣＲ结合酶切法是检测ａｐｏＢ－３５００位突变的一种简便而可靠方法；通过设计适当引物在ＰＣＲ产物中引进酶切位点及构建人工阳性对照的方法，是检测点突变疾病的新思路；ａｐｏＢ－３５００位突变不是引起此４个家族成员胆固醇升高的原因。     

A molecular approach to galactosemia

Classical galactosemia (G/G) is caused by the lack of galactose-1-phosphate uridyltransferase (GALT) activity. A more common clinical variant, Duarte/Classical (D/G) produces partial enzymatic impairment. Although neonatal death due to G/G galactosemia has been largely eliminated by populationbased screening and intervention, long-term outcome in some is associated with impaired growth, ovarian failure, dyspraxic speech and neurologic deficits. At least 32 variants in the nucleotide sequence of the GALT gene have been identified and 9 have transferred impaired GALT activity to transformed cells in transfection experiments. We here define the prevalence and biochemical phenotype of two mutations. An A to G transition in exon 6 of the GALT gene converts a predicted glutamine at codon 188 to an arginine (Q188R), and introduces a new HpaII cut site into the gene which enables population screening by polymerase chain reaction. An A to G transition in exon 10 in the GALT gene produces a codon change converting an asparagine to aspartic acid at codon 314 (N314D) and adds an AVA II cut site. We screened a large population for the Q188R and N314D sequence changes to investigate the prevalence of Q188R in G/G galactosemia, the effect of homozygosity for Q188R on outcome, and the prevalence and biochemical phenotype of the N314D sequence change. We found that the Q188R mutation has a prevalence of 62% in a predominately Caucasian population of 107 patients with G/G galactosemia. homozygosity for Q188R was associated with a poor clinical outcome in a subgroup of these patients. The N314D mutation is associated with the Duarte biochemical phenotype with extraordinary concordance.     

反复发热伴MEFV基因突变患儿10例临床分析及文献复习

目的　探讨在非高危人群中家族性地中海热（FMF）的诊断要点及其临床表现与基因型的关系。方法　回顾性分析重庆医科大学附属儿童医院2016-07-27—2017-11-15收治的10例反复发热伴MEFV基因突变患儿的基因检测报告及临床表现,结合国内外FMF相关文献,对诊断要点及其临床表现与基因型进行总结归纳。结果　10例患儿中共发现4个MEFV基因突变,分别为G304R、E148Q、P369S、R408Q,其中纯合突变4例（40.0%）,杂合突变2例（20.0%）,复杂突变4例（40.0%）。10例患儿中9例有反复发热,1例有反复化脓性扁桃体炎。该10例患儿根据Tel Hashomer标准,有1例患儿疑似诊断FMF,有8例患儿临床怀疑FMF;若根据Turkish儿科标准,则有7例患儿可临床诊断FMF。结论　FMF主要依靠临床诊断,可以通过基因检测来支持但不一定排除。携带M694V纯合型突变的FMF患者比其他突变的患者起病年龄更早,临床表现很可能更严重;而E148Q的致病性存在争议,其作为惟一突变时不支持FMF诊断。中国儿童中FMF发病率可能被低估,尚需纳入更多的研究对象,进一步研究。     

Sporadic congenital nonautoimmune hyperthyroidism caused by P639S mutation in thyrotropin receptor gene

Germline mutations of thyrotropin receptor (TSHR) gene determining a constitutive activation of the receptor were identified as a molecular cause of familial or sporadic congenital nonautoimmune hyperthyroidism (OMIM: 609152) (Nat Genet 7:396-401, 1994; N Engl J Med 332:150-154, 1995; Acta Endocrinol (Copenh) 100:512-518, 1982). We report the case of an Italian child subjected to the first clinical investigation at 24?months for an increased growth velocity; biochemical investigation showed high FT4 and FT3 serum values and undetectable thyrotropin in the absence of anti-thyroid antibodies; the thyroid gland was normal at ultrasound examination. Treatment with methimazole was started at the age of 30?months when her growth velocity was high and the bone age was advanced. DNA was extracted from her parents', brother's, and the patient's blood. Exons 9 and 10 of the TSHR gene were amplified by polymerase chain reaction and subjected to direct sequencing. In proband, a heterozygous substitution of cytosine to thymine determining a proline to serine change at position 639 (P639S) of the TSHR was detected while the parents and brothers of the propositus, all euthyroid, showed only the wild-type sequence of the TSHR gene. This mutation was previously described as somatic in patients affected by hyperfunctioning thyroid nodules and as germline in a single Chinese family affected by thyrotoxicosis and mitral valve prolapse. This constitutively activating mutation is able to activate both the cyclic AMP and the inositol phosphate metabolic pathways when expressed in a heterologous system. In conclusion, we describe the first case of sporadic congenital nonautoimmune hyperthyroidism caused by de novo germinal P639S mutation of TSHR.     

Rapid detection of phenylketonuria mutations by non-radioactive single-strand conformation polymorphism analysis

A non-radioactive single-strand conformation polymorphism (SSCP) method was used to detect various phenylketonuria (PKU) mutations in Japanese and Chinese patients. Arginine⁴¹³-to-proline (R413P) mutation in exon 12 of the phenylalanine hydroxylase gene was identified in a Japanese patient by this method. The segregation of the R413P mutation in the proband's family was clearly demonstrated and the carrier status of each family member was determined. Analysis of DNA fragments containing exon 7 originated from Chinese patients revealed two mutations, arginine²⁴³-to-glutamine (R243Q) and arginine²⁶¹-to-glutamine (R261Q), and a polymorphism, valine²⁴⁵-to-valine (V245V). Although R261Q has been identified previously among Caucasian subjects, this report is the first to describe this mutation among Orientals. Since the non-radioactive SSCP method employs pre-cast acrylamide gels and pre-made gel buffer strips combined with semi-automated temperature-controlled electrophoresis, it can be performed without much expertise in molecular biological techniques. The ability of this method to detect various mutations as demonstrated in this study and its ease of use make it feasible to detect PKU mutations in a routine DNA diagnostic laboratory.     

KCNJ11 activating mutations cause both transient and permanent neonatal diabetes mellitus in Cypriot patients

Heterozygous mutations of the KCNJ11 gene encoding the Kir6.2 subunit of the ATP-sensitive potassium channel (K(ATP) channel) of the pancreatic β-cell cause diabetes in about 30-60% of all permanent neonatal diabetes mellitus cases diagnosed before 6 months of age. The K(ATP) channel plays an essential role in the regulation of the electrical status of the membrane through which the secretion of insulin is activated. Transient neonatal diabetes mellitus due to KCNJ11 mutations is less frequent than abnormalities affecting the imprinted region of chromosome 6q24. We studied the genetic basis of two Cypriot patients who developed diabetes before 6 months of age. They both carried mutations of the KCNJ11 gene. The R201H mutation was identified in a patient who developed hyperglycemia and ketoacidosis at the age of 40 d and was successfully transferred to sulphonylureas which activate the channel through an ATP independent route. The R50Q mutation was identified in a child diagnosed at day 45 after birth with remission of his diabetes at 9 months of age. The same defect was identified both in his asymptomatic mother and the recently diagnosed 'type 2' diabetic maternal grandmother. The remission-relapse mechanism in cases of transient neonatal diabetes is not known. Nevertheless, it is possible that the residue of the mutation within the Kir6.2 molecule is associated with the sensitivity to ATP reflecting to the severity of the diabetic phenotype.