腓骨肌萎缩综合征患者MFN2基因突变检测的研究

目的 对1个腓骨肌萎缩综合征核心家系的致病突变进行鉴定和遗传学分析.方法 采用全外显子组测序技术筛选先证者中的致病突变,用Sanger测序技术检测核心家系中致病突变位点的基因型,并对其进行生物信息学分析.结果 先证者MFN2基因存在单碱基杂合突变c.1090C>T p.R364W,母亲与妹妹均为该位点单碱基杂合突变,且...     

Six novel variants in the PKLR gene associated with pyruvate kinase deficiency in Argentinian patients

BackgroundPyruvate kinase deficiency (PKD) is a rare recessive congenital hemolytic anemia caused by mutations in the PKLR gene. The disease shows a marked variability in clinical expression. We studied the molecular features of nine unrelated Argentinian patients with congenital hemolytic anemia associated with erythrocyte pyruvate kinase deficiency. Design and Methods: Routine hematologic investigations were performed to rule out other causes of chronic hemolytic anemia. Sanger sequencing and in-sílico analysis were carried out to identify and characterize the genetics variants. Results: Six different novel missense variants were detected among the 18 studied alleles: c.661 G > C (Asp221His), c.956 G > T (Gly319Val), c.1595 G > C (Arg532Pro), c.347 G > A (Arg116Gln), c.1232 G > T (Gly411Val), c.1021G > A (Gly341Ser). Structural implications of amino-acid substitutions were correlated with the clinical phenotypes seen in the probands. Conclusions: This is the first comprehensive report on molecular characterization of pyruvate kinase deficiency in Argentina and the second from South America that would contribute to our knowledge on the distribution and frequency of PKLR variants in our population but also offer new insights into the interpretation of the effect of PKLR variants and phenotype.     

Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). This deficiency of TP leads to increased circulating levels of thymidine (deoxythymidine, dThd) and deoxyuridine (dUrd) and has been associated with multiple deletions and depletion of mitochondrial DNA (mtDNA). Here we describe 36 point mutations in mtDNA of tissues and cultured cells from MNGIE patients. Thirty-one mtDNA point mutations (86%) were T-to-C transitions, and of these, 25 were preceded by 5'-AA sequences. In addition, we identified a single base-pair mtDNA deletion and a TT-to-AA mutation. Next-nucleotide effects and dislocation mutagenesis may contribute to the formation of these mutations. These results provide the first demonstration that alterations of nucleoside metabolism can induce multiple sequence-specific point mutations in humans. We hypothesize that, in patients with TP deficiency, increased levels of dThd and dUrd cause mitochondrial nucleotide pool imbalances, which, in turn, lead to mtDNA abnormalities including site-specific point mutations.     

肝小静脉闭塞病的影像学分析(附4例报道)

目的 探讨肝小静脉闭塞病(hepatic veno-occlusive disease,HVOD)的影像学特征.方法 回顾性分析4例HVOD的临床及影像资料.4例行MRI三期增强扫描及超声检查,其中3例同时接受CT增强三期扫描.重点观察肝实质及肝血管的动态扫描变化并与平扫时进行比较.结果 2例经穿刺活检确诊,另外2例...     

非小细胞肺癌患者外周血游离DNA中检测EGFR基因突变

目的检测非小细胞肺癌患者外周血游离DNA中表皮生长因子受体(EGFR)基因19和21外显子的突变情况,并与相应的肿瘤组织检测结果进行比较,探讨非小细胞肺癌患者应用外周血游离DNA检测EGFR突变的临床意义。方法应用实时荧光聚合酶链反应(PCR)技术检测32例非小细胞肺癌患者术后肿瘤组织和术前外周血游离DNA中EGFR基因19和21外显子的突变,所有扩增标本均经基因测序法验证。结果 32份外周血标本中,共检测到13份EGFR基因突变,突变率达40.6%,肿瘤组织中有16份EGFR基因突变,突变率达50.0%,外周血和肿瘤组织EGFR基因同时突变的有13份,两者突变一致性达到81.2%,两者间差异无统计学意义(P0.05)。结论外周血游离DNA代替肿瘤组织进行EGFR基因突变检测具有可行性,为无法取得肿瘤组织的非小细胞肺癌患者提供一种更快捷、简便的EGFR基因突变检测方法,其检测结果可为临床选择靶向药物治疗提供依据。     

Detection of the most common mutation of adenine phosphoribosyltransferase deficiency among Japanese by a non-radioactive method.

About 79% of all the Japanese patients with adenine phosphoribosyltransferase (APRT) deficiency have been estimated to possess at least one APRT*J allele with a substitution of ACG for ATG at codon 136. We developed a non-radioactive method for diagnosing genotypes of this disease. Part of the genomic DNA including the mutation site of the APRT*J allele was amplified using polymerase chain reaction and the amplified product was dot-blotted onto nylon membranes and then hybridized with either APRT*J-specific or non-APRT*J-specific synthetic oligonucleotides labelled at the 5' termini with biotin in the presence of non-labelled competitive synthetic sequences. The temperature was gradually decreased during the hybridization. When competitive sequences were omitted, difference in the intensity of the hybridization between APRT*J-containing and non-containing samples was not sufficiently clear to differentiate the genotypes. When an excess amount of competitive sequences was added in addition to biotin-labelled oligonucleotides, this method effectively differentiated samples containing only APRT*J alleles from those containing only non-APRT*J alleles. The present method was also useful to differentiate samples with both APRT*J and non-APRT*J alleles from those having only either of the alleles. An equivalent procedure using competitive sequence for hybridization and gradually decreasing the temperature will be useful for detecting point mutations in other genes.     

Presenilin-2 mutation and polymorphism in Japanese Alzheimer disease patients.

The Asn141Ile mutation of the presenilin 2 gene is responsible for familial early-onset Alzheimer disease found in Volga-German kindreds. However, the genetic influence of presenilin 2 gene on sporadic Alzheimer disease remains unknown. In this study, the frequency of the mutation and genetic association with the presenilin 2 locus were investigated in Japanese sporadic cases. The Asn141Ile mutation was not found in 88 cases of sporadic Alzheimer disease or 13 unrelated cases of familial Alzheimer disease. Fifty cases of late onset sporadic Alzheimer disease and 50 age-matched controls indicated no association with an exon 3 polymorphism of the presenilin 2 gene. These results indicate that the presenilin 2 mutation is not a major cause of Alzheimer disease.     

AVPR2基因突变所致先天性肾型尿崩伴生长激素缺乏症的临床特征

目的探讨1例X连锁隐性遗传的先天性尿崩伴生长激素缺乏患者的临床特征。方法收集患者的临床资料,抽取外周静脉血,提取基因组DNA,聚合酶链反应（PCR）扩增AVPR2基因的全部外显子区,产物经纯化后测序明确突变情况,并结合文献进行分析。结果该患者以多尿、多饮、身材矮小为主要临床表现,禁水加压试验诊断肾型尿崩症,IGF-1低,骨龄落后,低血糖生长激素兴奋试验确诊完全性生长激素缺乏症,基因检测提示AVPR2第二外显子g．636C〉T突变。结论先天性肾型尿崩症患者常合并身材矮小,生长激素缺乏是其原因之一。结合文献随诊研究表明,在控制尿崩症后大部分患者终身高仍小于平均水平。     

白血病患者FLT3基因第二酪氨酸激酶结构域点突变分析

目的研究FLT3基因酪氨酸激酶结构域(TKD)点突变与急性白血病的关系及临床意义。方法采用PCR结合限制性内切酶酶切及序列测定,检测143例急性髓系白血病(AML)、25例急性淋巴细胞白血病(ALL)、2例急性杂合细胞白血病(AHL)、17例骨髓增生异常综合征(MDS)和7例慢性粒细胞白血病急变期(CMLBC)患者骨髓单个核细胞中FLT3基因外显子20中的TKD点突变,分析其临床相关性。结果143例AML患者中9例(6.3%)存在FLT3TKD点突变(FLT3TKD+),阳性率显著低于FLT3基因内部串联重复(ITD)突变(25.9%,P<0.01)。FLT3TKD+存在于AMLM2(3/53)、M3(3/40)、M5(2/23)、M6(1/2)亚型中。2例FLT3-TKD+患者同时存在FLT3-ITD突变,M2、M3各1例。在25例ALL、2例AHL、17例MDS和7例CMLBC患者中未检测到FLT3TKD+。测序分析显示TKD点突变累及密码子D835,未改变FLT3的开放式阅读框架,为错义突变。D835的第1个核苷酸G被T替换,即D835Y。FLT3-TKD+组和FLT3-TKD组在性别、年龄组成、白细胞计数、骨髓原始细胞比例及化疗缓解率方面差异无统计学意义。结论AML患者中FLT3-TKD点突变的阳性率显著低于FLT3ITD。TKD点突变累及密码子D835,为错义突变。TKD点突变可单独存在,也可与ITD同时存在。与FLT3-ITD相比,TKD点突变与高白细胞无关。     

石家庄市苯丙氨酸羟化酶缺乏症的基因突变研究

目的 分析石家庄市新生儿疾病筛查诊治中心确诊的苯丙氨酸羟化酶缺乏症(PAHD)患儿中PAH基因的突变规律及特点,为石家庄市PAHD的产前诊断、治疗提供有力的科学依据.方法 收集在石家庄市新生儿疾病筛查诊治中心确诊的PAHD患儿67例作为研究对象,提取患儿及其父母外周血DNA标本进行二代测序,测序范围包括13个外显子和外...     

A genomic approach to mutation analysis of holocarboxylase synthetase gene in three Chinese patients with late-onset holocarboxylase synthetase deficiency

OBJECTIVE: Multiple carboxylase deficiency (MCD, MIM:253270) is a common organic aciduria and caused by deficiency of either biotinidase or holocarboxylase synthetase (HLCS; EC 6.3.4.10). Patients commonly present during early infancy with acute metabolic derangements and severe metabolic acidosis. Recently, a late onset form of HLCS deficiency was also described. The different phenotypes (early and late presenting) may be related to a spectrum of mutations in HLCS gene. Applications of mutation analysis in HLCS had been limited previously by the requirement of cDNA from living tissue for study. We described here a genomic approach for molecular diagnosis of HLCS deficiency which we have used to detect mutations in Chinese patients who had the late-onset form of HLCS deficiency. In addition, a fibroblast cell line with MCD from Coriell Cell repositories was also studied. DESIGN AND METHODS: Three Chinese patients with late onset HLCS deficiency were studied. The genomic sequence of HLCS was retrieved and newly designed primers were used to cover all coding sequences of the gene. PCR products were analyzed by direct sequencing. Population allelic frequencies of mutations detected were determined by genotyping of control samples by restriction fragment length polymorphism. RESULTS: We found a recurrent mutation, R508W, in the three unrelated Chinese patients. Two were homozygous for this mutation. The other patient was a compound heterozygote of R508W and a novel mutation, D634N. The results suggest that R508W may be an important and relatively prevalent disease-causing mutation in Chinese MCD patients. A fibroblast cell-line from an African patient revealed an additional novel mutation, R565X and a known mutation, V550M. CONCLUSION: R508W is a recurrent mutation in Chinese MCD patients which is associated with the late onset phenotype. This new genomic approach for mutation analysis of HLCS gene provides new opportunities in studies of MCD.     

应用多色探针熔解曲线分析法检测湘潭地区G6PD缺乏症基因突变

目的 探讨多色探针熔解曲线分析(MMCA)法检测湘潭地区葡萄糖-6-磷酸脱氢酶(G6PD)缺乏症基因突变的性能,为临床诊断提供参考.方法 纳入2017年1月至2020年9月于该院进行G6PD缺乏症筛查的90221例新生儿,以其中454例同时进行G6PD酶活性法检测和基因检测[MMCA法、外显子基因测序(Sanger测序)法]的G6PD缺乏症初筛阳性新生儿作为研究对象,以Sanger测序法的检测结果为"金标准",对酶活性法和MMCA法进行方法学比较.结果 酶活性法诊断男性新生儿G6PD缺乏症的灵敏度为97.6％,特异度为100.0％;诊断女性新生儿G6PD缺乏症的灵敏度为59.5％,特异度为100.0％.酶活性法与Sanger测序法诊断男性新生儿G6PD缺乏症的结果一致性较好(Kappa=0.974,P<0.05),诊断女性新生儿G6PD缺乏症的结果一致性一般(Kappa=0.676,P<0.05).MMCA法诊断男性新生儿G6PD缺乏症的灵敏度为97.0％,特异度为100.0％;诊断女性新生儿G6PD缺乏症的灵敏度、特异度均为100.0％.MMCA法与San-ger测序法诊断男性新生儿G6PD缺乏症的结果一致性较好(Kappa=0.967,P<0.05),诊断女性新生儿G6PD缺乏症的结果完全吻合(Kappa=1.000,P<0.05).结论 与传统的酶活性法比较,MMCA法具有灵敏度、特异度高的优点,是一种快速、准确、适用于临床诊断G6PD缺乏症的方法.     

Expression of muscle-type phosphorylase in innervated and aneural cultured muscle of patients with myophosphorylase deficiency.

Patients with McArdle's myopathy lack muscle glycogen phosphorylase (M-GP) activity. Regenerating and cultured muscle of patients with McArdle's myopathy presents a glycogen phosphorylase (GP) activity, but it is not firmly established whether M-GP or non-M-GP isoforms are expressed. We have cultured myoblasts from biopsy specimen of five patients with McArdle's myopathy. Skeletal muscle was cultured aneurally or was innervated by coculture with fetal rat spinal cord explants. In the patients' muscle biopsies and in their cultured innervated and aneural muscle we studied total GP activity, isoenzymatic pattern, reactivity with anti-M-GP antiserum, and presence of M-GP mRNA. There was no detectable enzymatic activity, no immunoreactivity with anti-M-GP antiserum, and no M-GP mRNA in the muscle biopsy of all patients. GP activity, M-GP isozyme, and anti-M-GP antiserum reactivity were present in patients' aneural cultures, increased after innervation, and were undistinguishable from control. M-GP mRNA was demonstrated in both aneural and innervated cultures of patients and control by primer extension and PCR amplification of total RNA. Our studies indicate that the M-GP gene is normally transcribed and translated in cultured muscle of patients with myophosphorylase deficiency.     

用套式基因扩增技术检测乙型肝炎病毒P基因区YMDD变异

目的了解套式基因扩增(nested PCR)技术在检测HBV P基因区酪氨酸-蛋氨酸-天冬氨酸-天冬氨酸(YMDD)变异中的敏感性和特异性.方法用套式基因扩增技术对3组共85例慢性乙型肝炎患者血清进行YMDD变异检测,检测结果经琼脂糖凝胶电泳判断.第1组40例未经拉米夫定治疗,HBV DNA>5×105拷贝/ml;第2组24例经拉米夫定治疗6～18个月,HBV DNA≤5×105拷贝/ml;第3组21例经拉米夫定治疗6～18个月,HBV DNA>5×105拷贝/ml.结果第1组检出YVDD 2例、YVDD+YIDD 1例,检出率为7.5%;第2组检出YVDD、YIDD各1例,检出率为8.3%;第3组检出YVDD 4例、YIDD 2例、YVDD+YIDD 3例,检出率为42.9%.结论套式基因扩增技术检测YMDD变异具有较高的敏感性和特异性,适合变异株快速检测,尤其适合检测处于弱势的YMDD变异株.     

用套式基因扩增技术检测乙型肝炎病毒P基因区YMDD变异

目的　了解套式基因扩增 (nestedPCR)技术在检测HBVP基因区酪氨酸 -蛋氨酸 -天冬氨酸 -天冬氨酸 (YMDD)变异中的敏感性和特异性。方法　用套式基因扩增技术对 3组共 85例慢性乙型肝炎患者血清进行YMDD变异检测 ,检测结果经琼脂糖凝胶电泳判断。第 1组 :4 0例未经拉米夫定治疗 ,HBVDNA >5× 10 5拷贝 /ml;第 2组 :2 4例经拉米夫定治疗 6～ 18个月 ,HBVDNA≤ 5× 10 5拷贝 /ml;第 3组 :2 1例经拉米夫定治疗 6～ 18个月 ,HBVDNA >5× 10 5拷贝 /ml。结果　第 1组检出YVDD 2例、YVDD YIDD 1例 ,检出率为 7.5 % ;第 2组检出YVDD、YIDD各 1例 ,检出率为 8.3% ;第 3组检出YVDD 4例、YIDD 2例、YVDD YIDD 3例 ,检出率为 4 2 .9%。结论　套式基因扩增技术检测YMDD变异具有较高的敏感性和特异性 ,适合变异株快速检测 ,尤其适合检测处于弱势的YMDD变异株