中国南方汉族人3个家族性激素耐药型肾病综合征家系CD2AP和NPHS1基因突变分析

目的 分析中国南方汉族人家族性激素耐药型肾病综合征(SRNS)家系CD2AP和NPHS1基因突变及其特点.方法 研究对象为A、B、C 3个南方汉族SRNS家系先证者及其父母,A、B 2个家系先证者的姐姐和50例尿检正常的南方汉族成年人.取所有研究对象外周静脉血,提取基因组DNA,PCR方法扩增CD2AP基因全部18个外显子和NPHS1基因全部29个外显子及其周围的部分内含子,对PCR产物直接进行DNA序列测定.结果 在3个SRNS家系先证者未检测出CD2AP基因致病突变.在B家系的先证者检测出NPHS1基因2398C＞T(R800C)杂合突变,先证者父亲亦携带此杂合突变,但先证者母亲及姐姐未发现该突变.在50例对照人群中未发现2398C＞T突变.此外,在3个先证者及50例对照人群还检测出9种已报道的CD2AP基因多态性--IVS4-25G＞A、IVS8-95G＞A、IVS10+36C＞A、IVS10-153A＞T、IVS10-110A＞G、IVS11+82T＞C、1204C＞T、IVS16+24G＞A、IVS17-66T＞C和4种已报道的NPHS1基因多态性--349G＞A、IVS24+36C＞T、3315G＞A和IVS27+45C＞T.结论 在1个中国南方汉族SRNS家系先证者检测出NPHS1基因突变--2398C＞T,证实中国南方汉族人家族性SRNS儿童存在NPHS1基因突变,提示对其需进行NPHS1基因突变分析.     

散发性激素耐药型肾病综合征患儿30例足细胞基因突变分析

目的分析散发性激素耐药型肾病综合征(SRNS)儿童足细胞基因突变及其特点。方法研究对象为30例散发性SRNS患儿和50例尿检正常的健康志愿者。采用PCR扩增NPHS1、NPHS2和CD2AP基因全部外显子及其周围的部分内含子,WT1基因外显子8和9及其周围的部分内含子;应用DNA序列直接测定法对其PCR产物进行测序。结果在10例应用激素和免疫抑制剂治疗肾病无缓解的SRNS患儿中,发现1例携带WT1基因杂合突变——1180C>T(R394W),1例携带NPHS1基因复合杂合突变——2677A>G(T893A)和*142T>C,1例携带CD2AP基因杂合突变IVS13-137G>A。在20例应用激素或免疫抑制剂治疗肾病缓解的SRNS患儿中,发现4例患儿携带NPHS1基因单杂合突变——928G>A、IVS8+30C>T、IVS21+14G>A和IVS25-23C>T,1例患儿携带CD2AP基因单杂合突变(IVS7-135G>A)。结论对激素和免疫抑制剂均耐药的SRNS患儿需进行足细胞基因突变分析。     

Novel and recurrent mutations of ITGA2B and ITGB3 genes in Korean patients with Glanzmann thrombasthenia

Glanzmann thrombasthenia (GT) is an autosomal recessive bleeding disorder caused by defective glycoprotein, αIIb and β3, encoded by ITGA2B and ITGB3 genes, respectively. We herein describe four unrelated Korean patients with genetically confirmed GT. Two patients were homozygous for c.1913+5G>T (IVS11+5G>T) mutation of ITGB3 with a signature of founder effect. The other two patients were compound heterozygous for two mutations of ITGA2B: c.[2333A>C];[2975delA] (p.[Q778P];[E992Gfs*30]) and c.[1750C>T];[2333A>C] (p.[R584X];[Q778P]). The c.2975delA mutation was a novel frameshift mutation of ITGA2B. Although from a limited number of patients, these results suggests c.1913+5G>T of ITGB3 is a recurrent mutation in Korean patients with GT.     

海南省妇女儿童医学中心5246名女性新生儿葡萄糖-6-磷酸脱氢酶基因型与酶活性分析

目的 分析海南省妇女儿童医学中心女性新生儿葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症的基因突变特点,探讨基因型与酶活性的关系。方法 以2017—2019年在海南省妇女儿童医学中心新生儿疾病筛查中心完成G6PD初筛的女性新生儿血样本为研究对象,收集G6PD酶缺乏的干血斑样本2153份,同时为提高女性杂合子的检出率,随机抽取酶活性正常的干血斑样本3093份,共计5246份样本纳入研究。提取脱氧核糖核酸（DNA）,采用多色探针荧光聚合酶链反应（PCR）熔解曲线法（MMCA法）检测G6PD基因突变。结果 在5246份女性新生儿样本中共检出基因突变2801份,总检出率为53.39%(2801/5246),其中酶活性正常者占26.24%(735/2801)。发现13个突变位点的变异,有32种突变基因型,包括杂合突变型12种,纯合突变型6种和复合突变型14种。前7位最常见的突变位点依次是c.1376G>T、c.1388G>A、c.871G>A、c.95A>G、c.1024C>T、c.392G>T、c.519C>T,其中c.871G>A的检出率高于c.95...     

Citrin 缺陷导致的新生儿肝内胆汁淤积症 SLC25A13 基因分析

目的：Citrin 缺陷导致的新生儿肝内胆汁淤积症（NICCD）是一种由SLC25A13基因突变引起的常染色体隐性遗传病,临床可表现为肝内胆汁淤积性黄疸、低出生体重、生长迟缓和低蛋白血症等。本研究通过DNA测序技术探讨中国NICCD患儿 SLC25A13 基因突变类型。方法：针对 SLC25A13 基因的 18 个外显子及其侧翼区碱基序列设计引物,应用 PCR 技术扩增目的片度。PCR 扩增、纯化后直接测序,确定其突变类型。IVS16ins3kb 突变则采用巢式 PCR 和 RT-PCR 进行检测。结果：发现7种SLC25A13基因突变类型,包括851del4、1638ins23、IVS16ins3kb、IVS6+5G>A、c.775C>T（p.Q259X)、c.1505C>T（p.P502L） 和 c.1311C>T（p.C437C）;并确认一种复合突变类型［1638ins23+IVS16ins3kb］。其中c.775C>T（p.Q259X）、c.1505C>T（p.P502L）和 c.1311C>T（p.C437C）为新发现的基因突变类型。在20例NICCD患儿中,6 例为 851del4 纯合突变,7 例为杂合突变,另有 7 例为单一突变类型的杂合子。突变类型以 851del4 为主,占所有突变类型64%;其次为 1638ins23、IVS16ins3kb 和 IVS6+5G>A（分别占15%、12% 和 6%）。结论：851del4突变在 NICCD 患儿中最为常见。     

IVS6+5G>A found in Wiskott-Aldrich syndrome and X-linked thrombocytopenia in a Korean family

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) are caused by a mutation in the WAS gene on Xp11.22. We report two patients with IVS6+5G>A of WAS in a Korean family. The proband presented with classic WAS, whereas his maternal cousin had symptoms limited to XLT. Their mothers were proved to be carriers. The IVS6+5G>A mutation was reported to result in incomplete splicing of the donor site and typically associated with mild form of disease, XLT. Our observation of the intrafamilial variability of clinical manifestations of WAS further expands the genotype-phenotype correlations and suggests the presence of modifying genetic factors.     

青岛市苯丙氨酸羟化酶缺乏症患儿基因突变分析

目的探讨青岛市苯丙氨酸羟化酶(phenylalanine hydroxylase,PAH)缺乏症患儿的基因突变特点,为青岛市PAH缺乏症的产前诊断、治疗提供科学参考依据。方法对经青岛市新生儿疾病筛查确诊的44例PAH缺乏症患儿,应用第二代高通量测序及多重连接酶探针依赖扩增(multi-ligase probe dependent amplification,MLPA)技术进行基因分析,检测患儿基因突变位点,应用Sanger测序对其父母的PAH基因相应突变位点进行检测并验证。根据患儿血苯丙氨酸浓度,分为经典型苯丙酮尿症、轻度苯丙酮尿症和轻度高苯丙氨酸血症。结果①44例PAH缺乏症患儿PAH基因中均检测到2个突变位点,其中2例为纯合突变,纯合突变的频率为4.6%,所有突变在患儿父母相应突变位点处均能检测到。②44例PAH缺乏症患儿共检测到突变36种,其中c.728G>A突变频率最高(15.9%,14/88),其次是c.1068C>A(10.2%,9/88),再次为c.158G>A(9.1%,8/88)。③21例经典型苯丙酮尿症患儿PAH基因突变19种,其中c.1068C>A突变频率最高(21.4%,9/42),其次是c.728G>A(19.0%,8/42)。10例轻度苯丙酮尿症患儿PAH基因突变14种,其中c.721C>T/722delG突变频率最高(15.0%,3/20),其次为c.1197A>T、c.1301C>A、c.721C>T、c.728G>A(均为10.0%,2/20)。13例轻度高苯丙氨酸血症患儿PAH基因突变17种,其中c.158G>A突变频率最高(26.9%,7/26),其次为c.728G>A(15.4%,4/26)。结论青岛市PAH缺乏症患儿PAH基因突变以复合杂合突变为主,具有明显热点突变(c.728G>A、c.1068C>A、c.158G>A),经典型苯丙酮尿症患儿以c.1068C>A、c.728G>A为主,轻度苯丙酮尿症患儿以c.721C>T/722delG为主,轻度高苯丙氨酸血症患儿以c.158G>A为主。本研究明确了青岛市PAH缺乏症患儿基因的突变类型与特点,为深入开展PAH缺乏症的诊断以及进一步的基因治疗奠定了基础。     

非典型溶血尿毒综合征患儿14例临床表型与基因型分析

目的：对非典型溶血尿毒综合征（aHUS）临床表型和基因型分析了解与预后的关系。方法：纳入临床表现符合血栓性微血管病（TMA）的患儿,通过ELISA法行ADAMTS13酶活性的检测,对ADAMTS13酶活性＞10%,且排除其他类型HUS,行肾脏267个基因panel（包括aHUS已知的与发病相关的C3、C4、C5、CFH、CFB、CFI、MCP、CFHR1、CFHR3、CFHR5、THBD、PLG、DGKE）二代测序,对测序结果阳性的基因行sanger验证。提取纳入的aHUS患儿的随访资料,比较基因突变与未突变患儿预后,系统检索文献,行aHUS临床表型与基因文献复习。结果：14例aHUS患儿进入本文分析,男6例,女8例,5例累及肾脏以外系统。基因检测阳性和阴性者SCr分别为335.3和247.8,eGFR分别为28.8和21.2,C3降低分别为3例和4例,随访时间1~67个月,末次随访结局：基因检测阳性和阴性死亡分别为2例和0例,复发分别为2例和0例,终末期肾病（ESRD）分别为3例和0例,eGFR分别为48和103.7。检索Pubmed数据库,对包括本文病例在内的成人（148例）和儿童（154例）aHUS病例行临床特征和基因型分析,男性病例儿童多于成人,差异有统计学意义;前驱感染（消化和呼吸道）儿童较成人比例高,差异有统计学意义;除神经系统受累、肺泡内出血及高血压外,儿童较成人更易累及其他系统（肝炎、胰腺炎等多器官受损）;成人Scr异常高值大于儿童,差异有统计学意义。CFH突变率欧洲人群高于亚洲,差异有统计学意义。结论：儿童和成人CFH突变均较未突变的aHUS病例预后差,儿童较成人aHUS病例更易累及肾脏、神经以外的系统。     

浙江地区新生儿葡萄糖-6-磷酸脱氢酶缺乏症的遗传学分析

目的　分析浙江地区新生儿葡萄糖-6-磷酸脱氢酶（glucose-6-phosphate dehydrogenase，G6PD）缺乏症的基因突变特点，探讨其遗传多样性。方法　以2015年3月至2017年9月在浙江地区出生、经浙江省新生儿遗传代谢筛查中心G6PD缺乏症筛查发现的2242例患儿为对象，收集其新生儿筛查的G6PD活性值与剩余干滤纸血斑，并提取其血斑的基因组DNA。采用MassARRAY技术检测35个G6PD突变位点。采用SPSS 22.0软件统计分析基因型与G6PD活性的关系，P＜0.05为差异有统计学意义。结果　2163例检出突变，总检出率为96.47%，其中男性为96.51%（1995/2067），女性为96%（168/175）。共检出21种突变位点，44种变异基因型，其中男性半合子19型，女性杂合子14型，女性纯合子3型，女性复合杂合8型。95.93%的G6PD突变位于12、9、2、5外显子，其中c.1376G＞T、c.1388G＞A、c.1024C＞T、c.95A＞G、c.871G＞A、c.392G＞T占92.96%。c.1376G＞T、c.1388G＞A、c.1024C＞T、c.95A＞G四种基因型的G6PD活性差异有统计学意义（P＜0.0001）。结论　浙江地区G6PD缺乏症存在基因突变热点，c.1024 C＞T的突变频率具有明显地域特征，MassARRAY技术检测特定G6PD突变位点可推荐为G6PD缺乏症的二级筛查方法之一。     

浙江地区新生儿葡萄糖-6-磷酸脱氢酶缺乏症的遗传学分析

目的　分析浙江地区新生儿葡萄糖-6-磷酸脱氢酶（glucose-6-phosphate dehydrogenase，G6PD）缺乏症的基因突变特点，探讨其遗传多样性。方法　以2015年3月至2017年9月在浙江地区出生、经浙江省新生儿遗传代谢筛查中心G6PD缺乏症筛查发现的2242例患儿为对象，收集其新生儿筛查的G6PD活性值与剩余干滤纸血斑，并提取其血斑的基因组DNA。采用MassARRAY技术检测35个G6PD突变位点。采用SPSS 22.0软件统计分析基因型与G6PD活性的关系，P＜0.05为差异有统计学意义。结果　2163例检出突变，总检出率为96.47%，其中男性为96.51%（1995/2067），女性为96%（168/175）。共检出21种突变位点，44种变异基因型，其中男性半合子19型，女性杂合子14型，女性纯合子3型，女性复合杂合8型。95.93%的G6PD突变位于12、9、2、5外显子，其中c.1376G＞T、c.1388G＞A、c.1024C＞T、c.95A＞G、c.871G＞A、c.392G＞T占92.96%。c.1376G＞T、c.1388G＞A、c.1024C＞T、c.95A＞G四种基因型的G6PD活性差异有统计学意义（P＜0.0001）。结论　浙江地区G6PD缺乏症存在基因突变热点，c.1024 C＞T的突变频率具有明显地域特征，MassARRAY技术检测特定G6PD突变位点可推荐为G6PD缺乏症的二级筛查方法之一。     

Severe hepatic Wilson's disease in preschool-aged children

A 3-year-old girl presented with hemolytic anemia, hepatosplenomegaly, ascites, and evidence of decompensated chronic liver disease. Genotypic DNA analysis revealed that the patient was homozygous for a splice site mutation now designated IVS4-1:G>C, expected to destroy completely the functional gene product of ATP7B, the gene responsible for Wilson's disease. We suggest that this severe mutation caused very early liver disease. Wilson's disease should be considered in the differential diagnosis of established liver disease in the preschool-aged child.     

Homozygous point mutations in platelet glycoprotein ITGA2B gene as cause of Glanzmann thrombasthenia in 2 families

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder characterized by quantitative and/or qualitative defects of the platelet glycoprotein (GP) IIb/IIIa complex. Physiologically, the integrin GPIIb/IIIa binds Von Willebrand factor and fibrinogen on activated platelets. GT is caused by genetic alterations in ITGA2B or ITGB3 (genes encoding GPIIb and GPIIIa).This study describes 2 siblings diagnosed with GT type I associated with homozygous point mutations in ITGA2B. All patients presented with typical bleeding disorder including moderate hematomas, petechiae, and mucocutaneous bleedings.Both siblings showed severely reduced platelet aggregation especially after stimulation with collagen and adenosine diphosphate. Absence of platelet GPIIb/GPIIIa complex was determined using flow cytometry. Molecular genetic analysis revealed 2 distinct homozygous point mutations in exon 18 of ITGA2B. Family 1 was identified with c.1878G>C and family 2 with c.1787T>C substitution. While the c.1787T>C mutation causes a single amino acid substitution p.I565T, the c.1878G>C mutation (p.Q595H) is predicted to induce a mRNA splicing anomaly.These mutations were identified as cause of GT type I in the described patients. Patients with GT should be documented in a prospective register to verify the correlation between the severity of bleeding symptoms and the pathogenic mutation. This can have effects on therapeutic decisions.     

A novel SGCE gene mutation causing myoclonus dystonia in a family with an unusual phenotype

Background: Myoclonus dystonia is an autosomal dominant dystonia‐plus syndrome, characterized by symptom variability within families. Most often is the myoclonus the most debilitating symptom, and many patients report myoclonus reduction after alcohol intake. In several families, mutations in the SGCE gene have been identified. Method: We report of a three‐generation family with myoclonus dystonia displaying a varied phenotype and maternal imprinting. Additionally, this family displays some unusual clinical presentations including alcohol‐induced dystonia in an adult man, which will be discussed. Results: A novel mutation c.386T>C [p.I129T] was found within exon 3 of the SGCE gene in all three affected family members. In addition, two additional mutations [c.305G>A and IVS3+15G>A], judged to be polymorphisms in the SGCE gene, were found in two affected and one healthy family member. Conclusions: This report presents a novel mutation in the SGCE gene causing myoclonus dystonia and extends the phenotype of myoclonus dystonia to also include alcohol‐induced dystonia.     

生长激素受体基因突变与非生长激素缺乏性矮身材的相关研究

目的了解生长激素受体（GHR）基因突变与非生长激素缺乏性矮小的相关性,以及GHR基因突变患儿的临床特点。方法（1）选择47例（男33例,女14例,年龄2—16岁）非生长激素缺乏而又显著矮小的患儿作为研究对象;80例身高正常的儿童（男49例,女31例,年龄1—17岁）作为对照组。（2）应用PCR-SSCP和基因测序技术检测GHR突变。（3）通过家系成员和正常对照人群的基因分析以及氨基酸同源序列分析等,推测突变基因的性质。结果（1）在47例矮小患儿中有5例、4种不同的GHR基因突变：H56R、G148E、IVS6-30,-31CA〉TG和IVS8＋10G〉C。5例患儿均为杂合突变,杂合突变个体的检出频率为10．6％（5／47）。（2）对照组的基因分析显示这些突变非多态性改变,初步认为H56R和G148E突变可能对蛋白功能产生影响。（3）确定了1种多态性突变：G168G（GGA〉GGG）。该位点基因频率的分布在矮小儿童组和正常对照组之间的差异无统计学意义,但与西方白种人之间的差异有统计学意义。提示该突变可能是单核苷酸多态性改变,与身高没有相关关系。但该位点的等位基因存在人种的差异。结论非生长激素缺乏性矮身材患儿存在GHR基因杂合突变。     

成都市54025例早产儿葡萄糖-6-磷酸脱氢酶缺乏症筛查结果及分子遗传学特征分析

目的 分析成都市早产儿葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G6PD)缺乏症筛查结果及基因突变分布情况,探讨早产儿人群G6PD筛查流程改进方案.方法 采用干血斑G6PD荧光分析法,对成都市2015年1月1日至2019年12月31日出生的54025例早产儿足跟血样本进...     

Variations in IBD (ACAD8) in children with elevated C4-carnitine detected by tandem mass spectrometry newborn screening

The isobutyryl-CoA dehydrogenase (IBD) enzyme is involved in the degradation of valine. IBD deficiency was first reported in 1998 and subsequent genetic investigations identified acyl-CoA dehydrogenase (ACAD) 8, now IBD, as the gene responsible for IBD deficiency. Only three individuals homozygous or compound heterozygous for variations in the IBD gene have been reported. We present IBD deficiency in an additional four newborns with elevated C(4)-carnitine identified by tandem mass spectrometry (MS/MS) screening in Denmark and the United States. Three showed urinary excretions of isobutyryl-glycine, and in vitro probe analysis of fibroblasts from two newborns indicated enzymatic IBD defect. Molecular genetic analysis revealed seven new rare variations in the IBD gene (c.348C>A, c.400G>T, c.409G>A, c.455T>C, c.958G>A, c.1000C>T and c.1154G>A). Furthermore, sequence analysis of the short-chain acyl-CoA dehydrogenase (SCAD) gene revealed heterozygosity for the prevalent c.625G>A susceptibility variation in all newborns and in the first reported IBD patient. Functional studies in isolated mitochondria demonstrated that the IBD variations present in the Danish newborn (c.409G>A and c.958G>A) together with a previously published IBD variation (c.905G>A) disturbed protein folding and reduced the levels of correctly folded IBD tetramers. Accordingly, low/no IBD residual enzyme activity was detectable when the variant IBD proteins were overexpressed in Chang cells.     

Three novel and six common mutations in 11 patients with methylmalonic acidemia

BACKGROUND: Patients with a defect in methylmalonyl-coenzyme A mutase (MCM) are classified as having methylmalonic acidemia, which is divided into two subclasses: mut(0) and mut(-). Fifty-five disease-causing mutations have been identified. Although most are private mutations, only three (E117X, G717V, and N219Y) are reportedly common in Japanese, Black, and Caucasian populations, respectively. Here we identified mutations in 11 Japanese patients with MCM deficiency. METHODS: Mutational analysis was performed in 11 unrelated Japanese patients with MCM deficiency using polymerase chain reaction and direct sequencing. RESULTS: Three novel (L494X, R727X, and 449_461del) and six previously reported (R93H, E117X, N219Y, R369H, G648D and IVS2 + 5G>A) mutations were identified. The L494X mutation was found in three unrelated patients, and the R93H, E117X, R369H, G648D, and IVS2 + 5G>A mutations occurred more than once. Two of the patients were classified as mut(-) phenotype because of residual [(14)C]-propionate incorporation in the presence of a high concentration of hydroxocobalamin. The two mut(-) patients were heterozygous for the G648D mutation and presented with lethargy and metabolic acidosis after 2 years of life. Their psychomotor development has been documented as normal. The patients with the R727X or c.374_385del [corrected] mutations clinically exhibited mut(0) phenotype. Two patients with mut(0) phenotype died in infancy. One presented early in the neonatal period; the other was symptomatic in the late infantile period. CONCLUSIONS: The L494X, R93H, E117X, R369H, G648D, and IVS2 + 5G>A mutations are found in more than two unrelated families in the Japanese population. The short-term outcome was generally poor in patients with mut(0), and therefore alternative treatments should be considered.     

新生儿呼吸窘迫综合征ABCA3基因遗传缺陷的研究

目的 分析ATP结合盒式蛋白转运子亚单位基因ABCA3遗传缺陷与新生儿呼吸窘迫综合征(NRDS)的关系,从中探讨汉族人群NRDS发病的遗传机制.方法 收集在新生儿重症监护病房住院的11例重症NRDS患儿的临床资料,采集患儿和97例无关正常对照的血样,采用PCR扩增、DNA直接测序技术对11例患儿进行ABCA3基因序列分析,对于新发现的ABCA3错义突变在97例健康对照中进行单链构象多态性检测.对1例生后13 h死亡的NRDS患儿进行肺组织光镜和电镜检测.结果 在11例患儿中发现了3个ABCA3基因外显子遗传变异、1个剪切位点碱基变异和几个国外报道及尚未报道的ABCA3单核苷酸多态性(SNP).3个ABCA3基因外显子遗传变异分别为c.2169 G＞A (p.M723I)、c.1010 T＞G (p.V337G)、c.4972 A＞G(p.S1658G),1个剪切位点变异为Exon 30+2 T/G,发现的SNP位点包括未报道过的213 C ＞T(p.F71F)、Exon 21+ 34C/T和已报道的c.1059C＞T(p.F353F)等.1例生后12 h死亡的患儿携带c.2169G＞A纯合变异,该变异位于第17号外显子,碱基的变异导致第723位氨基酸由异亮氨酸代替蛋氨酸,97例健康对照者中未发现此变异.肺组织电镜检测显示该例肺泡Ⅱ型上皮细胞的板层小体变小、浓缩,电子致密物边集.结论 ABCA3基因突变可能是部分临床不能解释的重症NRDS患儿的遗传学病因或遗传背景,识别NRDS患儿ABCA3基因变异情况,有助于治疗措施的选择及评价,并为开展NRDS遗传咨询和早期预防性干预提供依据.