变性高效液相色谱法筛查间隙连接蛋白2耳聋基因突变

目的　探讨变性高效液相色谱法 (DHPLC)在耳聋基因筛查中的应用价值。方法　应用DHPLC检测 6个家系的 4 8名个体 ,然后进行直接测序。结果　 6个家系中A ,B ,C ,E ,F家系有间隙连接蛋白 2 (GJB2 )耳聋基因的变化 ,F家系表现为 2 35delC突变 ,B家系表现为 2 35delC和 2 32G→A杂合性突变。 79G→A(V2 7I)、341A→G (E1 1 4G)杂合性改变分别为 1 9例和 1 6例 ;纯合性改变分别为 1 0例和 6例。 2种方法的检测结果均为阳性者 30例 ,均为阴性者 1 0例 ;DHPLC阳性 ,测序阴性 1例 ;DHPLC阴性 ,测序阳性 2例。DHPLC方法的敏感性为 95 % ,特异性为 91 %。结论　DHPLC是一种高效、经济、简便、灵敏的耳聋基因筛查技术     

变性高效液相色谱技术在家族性高胆固醇血症低密度脂蛋白受体基因突变检测中的应用

目的 以变性高效液相色谱(DHPLC)，分析检测家族性高胆固醇血症(FH)一汉族家系成员的低密度脂蛋白受体(LDLR)基因突变，以明确诊断。方法 收集临床诊断为家族性高胆固醇血症的汉族一个家系共37名成员，其中30人为一级和二级亲属，7名为亲属配偶作为对照，提取基因组DNA，聚合酶链反应(PCR)方法扩增LDLR基因包含启动子和全部基因编码区(1-18外显子)及临近的内含子序列共21个片段，琼脂糖凝胶电泳鉴定产物。采用DHPLC技术检测了LDLR基因，对洗脱曲线异常者进行核苷酸序列分析。结果 该家系中发现4处变异，其中1处经核苷酸序列测定明确了突变的性质为第3内含子的剪接突变，并在此家系5名成员中得到证实，而对照组中未检出。结论 成功地建立了以DHPLC筛查LDLR基因点突变的方法及技术参数，该方法简便，结果稳定，可作为大样本筛查突变位点的一种便捷可靠手段。     

4个具有相同突变的CADASIL家系临床特征分析

目的分析总结4个来自河南地区不同家系但具有相同突变的伴有皮质下梗死和白质脑病常染色体显性遗传性脑动脉病(cerebral autosomal dominantarteriopathy with subcortical infarcts and leukoencephalopathy,CADASIL)家系的影像学表现及临床特征,调查家系成员的发病情况。方法回顾性分析4个CADASIL家系先证者的影像学特征和临床特征,调查4个家系中其他成员的发病情况,对患者行Notch3基因3、4、11、18号外显子突变检测,并进行分析。结果4例先证者均以单侧肢体无力为始发症状,其中2例有高血压病史,1例合并糖尿病,1例身体健康;1例有高血压病患者影像学提示丘脑出血,患者无相关主诉;基因检测发现4例先证者及部分家系成员存在p.R607C突变位点。结论存在p.R607C突变位点的4例CADASIL患者临床上首发症状相似,但不同家系成员间存在个体化差异;高血压、糖尿病等脑血管病危险因素可存在于CADASIL患者。     

变性高效液相色谱技术在线粒体基因突变检测中的应用

目的以变性高效液相色谱(DHPLC)技术分析检测线粒体肌病患者线粒体基因突变,以明确诊断。方法收集4例临床诊断为线粒体肌病的患者,以50例健康体检者作为对照,提取患者肌肉组织及对照组外周血细胞DNA,用聚合酶链反应(po lym erase cha in reaction,PCR)扩增线粒体22个tRNA基因,利用变性高效液相色谱分析技术(denaturing h igh-per-form ance liqu id chrom atography,DHPLC)对PCR产物进行突变筛选,出现异常峰型的tRNA基因进行核苷酸序列测定,明确突变位点。结果4例线粒体肌病患者均检测出线粒体基因突变,突变分别为:例1tRNA-V a l基因发生A 1625G纯合突变,例2 tRNA-V a l基因发生A 1625G/A杂合突变,例3 RNA-A rg基因发生A 10411C/A杂合突变,例4 RNA-T rp基因发生T 5553C纯合突变。结论DHPLC能有效地用于线粒体基因突变的检测,该方法简便,结果稳定,可作为大样本筛查突变位点的一种便捷可靠手段。     

DHPLC在检测Miyoshi肌病家系致病基因突变中的应用

目的检测Miyoshi肌病家系成员的DYSF基因突变,探讨变性高效液相色谱分析法(DHPLC)在基因研究中的效能。方法用RT-PCR技术扩增DYSF基因的编码序列,利用变性高效液相色谱分析技术(DHPLC)对PCR产物进行突变筛选,出现异常峰型的片段进行核苷酸序列测定,明确突变位点。结果患者DYSF基因存在6429delG突变。结论DHPLC能有效地用于基因突变的检测,该方法快速高效,可作为大样本筛查突变位点的一种便捷可靠手段。     

二个无虹膜症家系致病基因的鉴定

目的:明确2个呈常染色体显性遗传的无虹膜症家系的临床特征,并找出导致该病的基因突变。方法:2个家系无虹膜症患者接受遗传咨询及眼科检查,并采用候选基因法分别对2个家系成员的人类配对盒基因6(human paired box gene 6,PAX6)进行突变筛查,对未检出突变的家系进成员行外显子捕获及全外显子组测序。结果:家系1患者完全符合无虹膜症的临床特征,家系2患者还出现上睑下垂的表型。突变筛查发现,家系1的PAX6基因存在一个已知的基因突变c.718CT(p.Arg240~*),但家系2未筛查到任何已知的基因突变。结论:家系1中发现一个PAX6基因突变(p.Arg240~*),但在家系2中尚未筛查到致病基因,提示无虹膜症尚存在新的遗传异质性。     

遗传性凝血因子Ⅶ缺陷症患者的基因诊断与表型分析

目的:探讨3例遗传性凝血因子Ⅶ缺陷症患者的基因突变类型及其临床特征。方法:检测先证者及其家系成员凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、纤维蛋白原(FIB)、凝血酶时间(TT)、FⅦ活性(FⅦ:C)及FⅦ抗原(FⅦ:Ag)等进行表型诊断;用DNA直接测序法分析先证者F7基因的全部外显子、侧翼、5'和3'非翻译区及家系成员相应的突变位点区域,用反向测序证实所发生的突变。结果:在3例患者及其家系成员中发现5种基因突变,包括4种错义突变和1种剪切位点突变。在3例遗传性FⅦ缺陷症患者中有2例为双杂合突变、1例为纯合突变。患者1为p.His408Gln和p.Arg413Gln双杂合突变,其家系成员中有一位为p.His408Gln和p.Arg413Gln双杂合突变,1例为His408Gln突变的杂合子,其相应FⅦ:C分别为5.0%、3.0%和75.0%。;患者2为p.Arg364Gln和p.His408Gln双杂合突变,其家系成员为p.Arg364Gln和IVS6-1G A双杂合突变,其相应FⅦ:C分别为2.0%、2.0%;患者3为p.Arg337Cys纯合突变, FⅦ:C为3%。结论:在3例遗传性FⅦ缺陷症患者及其家系成员中发现5种基因突变,其中p.His408Gln突变较为常见,而临床表现及出血程度与FⅦ:C及FⅦ:Ag无相关性。     

变性高效液相色谱技术在β地中海贫血分型诊断中的应用

目的 探讨变性高效液相色谱技术(DHPLC)在快速诊断β地中海贫血及分型中的应用价值.方法 采用外周血红细胞平均体积(MCV)、红细胞分布宽度(RDW)、红细胞脆性和Hb电泳4项指标相结合的方法筛选地中海贫血可疑标本226份.运用PCR反向斑点杂交技术(PCR-RDB)和DHPLC对226份可疑标本进行基因分型确诊.结果 226份可疑静脉血标本中,经PCR-RDB和DHPLC确诊的β地中海贫血为69份,两种方法检测缺失和突变的基因型别完全一致,占地中海贫血筛查总人数的30.5%.其中CD41/CD42(-TCTT)移码突变37例(54%);IVS-Ⅱ-654(C→T)插入序列突变12例(17%);TATA-28(A→G)转录突变10例(15%);CD17(A→T)无义突变5例(7%);CD71/CD72(+A)移码突变5例(7%).结论 DHPLC可快速、高效和准确地对β地中海贫血进行基因分型诊断.     

CYP3A4基因多态性与晚期胃癌患者接受紫杉醇/奥沙利铂多线化疗、化疗周期数及不良反应的相关性

目的:应用变性高效液相色谱技术(denaturing performance liquid chromatography,DHPLC)高通量的实验平台,探讨晚期胃癌患者细胞色素P450代谢酶CYP3A4基因的多态性与患者接受多线化疗、化疗周期数及不良反应的相关性。方法:福建省肿瘤医院住院的接受含紫杉醇和/或奥沙利铂联合姑息化疗晚期胃癌患者外周血53份,进行全血DNA分离、提取,经PCR,DHPLC分析筛查以及DNA测序鉴定基因型,观察并评价患者接受多线化疗、化疗周期数及不良反应与CYP3A4多态性的相关性。结果:53例进展期胃癌患者CYP3A4经DHPLC筛查,单峰者(野生型)32例,双峰者(突变型)21例。测序结果显示双峰者CYP3A4第10号外显子上27位C的缺失突变。单峰组接受一线治疗15例,二线治疗10例,二线以上治疗8例,其中3例为三线以上治疗;平均化疗周期数为8.2周期,中位总生存时间为13.0个月(95%CI:7.092~18.908个月)。双峰组接受一线治疗10例,二线治疗8例,二线以上治疗3例,平均化疗8.7周期,中位总生存时间为14.0个月(95%CI:7.555~20.445个月)。两组中位总生存时间比较差异无统计学意义(P=0.326)。野生型组较突变型组不良反应发生率低,在3~4级毒副作用方面较为明显,结论:CYP3A4突变型者第10号外显子上第27位C缺失;该基因多态性与化疗不良反应相关,尚不能作为疗效的预测指标。     

牙本质生长不全Ⅱ型家系DSPP基因内含子2的mRNA剪接位点新的缺失突变

目的报告1例牙本质生长不全Ⅱ型家系(dentinogenesis imperfecta typeⅡ,DGIⅡ)牙本质唾液酸焦磷酸蛋白(dentin sialophosphoprotein,DSPP)内含子2的mRNA剪接位点新的缺失突变。方法在对疾病基因进行连锁分析的基础上,对其候选基因DSPP的调控序列、前4个外显子、外显子/内含子交界处进行序列分析,对突变的序列进行变性高效液相色譜(denaturing high performance liquid chromatography,DHPLC)证实。结果发现DSPP内含子2的3′端上游第3→25位置上的23个碱基缺失,使该内含子受体的剪接位点由CAG→AAG,可能还使得分支点序列功能消失。该突变是杂合突变,见于家系所有患者中,非患病成员及50名健康人对照均未发现相同突变。结论该突变是家系牙本质生长不全Ⅱ型疾病的分子原因。DSPP内含子2的缺失突变是首次报告。     

A novel mutation (C271F) in the Notch3 gene in a Chinese man with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult-onset hereditary condition caused by mutations in the Notch3 gene. A Chinese man was studied. METHOD: Electronic microscopy examination of skin biopsy. The Notch3 gene was screened for mutations by polymerase chain reaction and direct DNA sequencing. RESULTS: Electronic microscopy showed the presence of deposits of granular osmiophilic material in dermal capillaries in the index patient. A novel heterozygous C271F in exon 6 was detected in the index patient. This heterozygous C271F mutation was also detected in the asymptomatic elder son but was not detected in the asymptomatic wife of the patient. Allele specific amplification showed that C271F was not detected in 100 normal subjects. CONCLUSION: We established the molecular basis of CADASIL in a Chinese man. Mutation detection assay provides a reliable method for confirming the diagnosis of CADASIL.     

GENETICS

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary cerebrovascular disease leading to accumulating neurologic deficits and dementia. CADASIL has been linked to nucleotide substitutions and deletions in the Notch3 gene. All the mutations described until now lead to unpaired cysteine residue in the epidermal growth factor-like repeats. The authors report a family with CADASIL carrying a deletion in the Notch3 gene that did not involve a cysteine residue.
Comment: At least 400 families from countries around the world have been described with CADASIL. The four classic features are migraine with aura, stroke, dementia, and psychiatric disturbances. Migraine with aura, especially familial hemiplegic migraine and migraine with prolonged aura, is linked genetically to CADASIL on chromosome 19, occurs in up to 30% of patients, and when present is usually the earliest symptom. Definitive diagnosis is by skin biopsy, with characteristic depositions surrounding degenerating smooth muscle cells in the media. —Stewart J. Tepper, MD     

The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients

Mutations in Notch3 cause CADASIL (cerebral autosomal dominant adult onset arteriopathy), which leads to stroke and dementia in humans. CADASIL arteriopathy is characterized by major alterations of vascular smooth muscle cells and the presence of specific granular osmiophilic deposits. Patients carry highly stereotyped mutations that lead to an odd number of cysteine residues within EGF-like repeats of the Notch3 receptor extracellular domain. Such mutations may alter the processing or the trafficking of this receptor, or may favor its oligomerization. In this study, we examined the Notch3 expression pattern in normal tissues and investigated the consequences of mutations on Notch3 expression in transfected cells and CADASIL brains. In normal tissues, Notch3 expression is restricted to vascular smooth muscle cells. Notch3 undergoes a proteolytic cleavage leading to a 210-kDa extracellular fragment and a 97-kDa intracellular fragment. In CADASIL brains, we found evidence of a dramatic and selective accumulation of the 210-kDa Notch3 cleavage product. Notch3 accumulates at the cytoplasmic membrane of vascular smooth muscle cells, in close vicinity to but not within the granular osmiophilic material. These results strongly suggest that CADASIL mutations specifically impair the clearance of the Notch3 ectodomain, but not the cytosolic domain, from the cell surface.     

Investigating the association between Notch3 polymorphism and migraine

OBJECTIVE: The aim of the present study was to evaluate whether the functional Notch3 polymorphism T6746C, which is not causative for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), might be a risk factor for migraine. BACKGROUND: It has been recently demonstrated that migraine is characterized by subclinical brain infarctions and white matter lesions. Several genetic risk factors have been associated with migraine, but no study has unraveled a possible relationship between migraine and Notch3, which is involved in vascular damage. Mutations in Notch3 gene have been demonstrated to be pathogenetic for CADASIL, a small vessel disease of the brain characterized by migraine. METHODS: A total of 156 migraine patients and 128 nonheadache healthy volunteers entered the study. Demographic and clinical characteristics were carefully recorded, and a neurological work-up was performed. Moreover, each subject underwent a blood sampling for Notch3 genotype determination. RESULTS: Notch3 genotypes as well as allele frequencies did not differ in migraine patients compared to controls, even adjusting for the presence of possible confounds. No difference has been found either in migraine patients with aura or in those without aura. CONCLUSIONS: These findings support the view that functional polymorphism T6746C in Notch3 gene is not involved in increasing the risk of migraine or migraine subtypes.     

Use of denaturing HPLC to provide efficient detection of mutations causing familial hypercholesterolemia

BACKGROUND: Autosomal dominant familial hypercholesterolemia (FH) attributable to mutations in the LDL receptor (LDLR) gene is one of the most common genetic disorders associated with significant morbidity and mortality. Definitive diagnosis would help to initiate appropriate treatment to prevent premature cardiovascular disease. Currently, clinical diagnosis of FH is imprecise, and molecular diagnosis is labor-intensive and expensive because of the size of the LDLR gene and number of coding exons. METHODS: We used PCR to amplify all exons, including exon/intron boundaries, and the promoter of the LDLR gene. Nine individuals from five families with typical findings for a clinical diagnosis of heterozygous FH, 2 heterozygous FH cell lines, and 50 control individuals were screened for mutations by denaturing HPLC (DHPLC) followed by direct sequencing of aberrantly migrating fragments. RESULTS: Mutations that were previously reported to be disease causing were identified in eight of nine individuals with FH and both cell lines (V502M, C146X, E207X, C660X, C646Y, and delG197), but none were found in controls. The one individual with FH in whom no mutation was found had a previously unreported change in the 5'-untranslated region of unknown significance. In addition, we identified several previously reported polymorphism both in controls and individuals with FH. CONCLUSIONS: DHPLC can be used to detect mutations causing FH. On the basis of our current experience with DHPLC, this method combined with confirmatory DNA sequencing is likely to be sensitive and efficient.     

Denaturing HPLC profiling of the ABCA4 gene for reliable detection of allelic variations

BACKGROUND: Mutations in the retina-specific ABC transporter (ABCA4) gene have been associated with several forms of macular degenerations. Because the high complexity of the molecular genotype makes scanning of the ABCA4 gene cumbersome, we describe here the first use of denaturing HPLC (DHPLC) to screen for ABCA4 mutations. METHODS: Temperature conditions were designed for all 50 exons based on effective separation of 83 samples carrying 86 sequence variations and 19 mutagenized controls. For validation, samples from 23 previously characterized Stargardt patients were subjected to DHPLC profiling. Subsequently, samples from a cohort of 30 patients affected by various forms of macular degeneration were subjected to DHPLC scanning under the same conditions. RESULTS: DHPLC profiling not only identified all 132 sequence alterations previously detected by double-gradient denaturing gradient gel electrophoresis but also identified 5 sequence alterations that this approach had missed. Moreover, DHPLC scanning of an additional panel of 30 previously untested patients led to the identification of 26 different mutations and 29 polymorphisms, accounting for 203 sequence variations on 29 of the 30 patients screened. In total, the DHPLC approach allowed us to identify 16 mutations that had never been reported before. CONCLUSIONS: These results provide strong support for the use of DHPLC for molecular characterization of the ABCA4 gene.     

Genetic variants of the NOTCH3 gene in migraine--a mutation analysis and association study

Mutations in the NOTCH3 gene cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Exons 3 and 4 are mutation hotspots. Migraine is a clinical hallmark of CADASIL. The objective of this study was to investigate whether genetic variants in exons 3 and 4 of the NOTCH3 gene are associated with migraine. Exons 3 and 4 of the NOTCH3 were analysed for mutations and polymorphisms by direct DNA sequencing in 97 migraineurs and the same number of control individuals. No mutations in exons 3 and 4 of the NOTCH3 gene were found in 97 patients with migraine. However, association analysis revealed significant association of the single nucleotide polymorphism (SNP) rs1043994 with migraine.     

Identification of new mutations of the HFE, hepcidin, and transferrin receptor 2 genes by denaturing HPLC analysis of individuals with biochemical indications of iron overload

BACKGROUND: Hereditary hemochromatosis is a recessive disorder characterized by iron accumulation in parenchymal cells, followed by organ damage and failure. The disorder is mainly attributable to the C282Y and H63D mutations in the HFE gene, but additional mutations in the HFE, transferrin receptor 2 (TfR2), and hepcidin genes have been reported. The copresence of mutations in different genes may explain the phenotypic heterogeneity of the disorder and its variable penetrance. METHODS: We used denaturing HPLC (DHPLC) for rapid DNA scanning of the HFE (exons 2, 3, and 4), hepcidin, and TfR2 (exons 2, 4 and 6) genes in a cohort of 657 individuals with altered indicators of iron status. RESULTS: DHPLC identification of C282Y and H63D HFE alleles was in perfect agreement with the restriction endonuclease assay. Fourteen DNA samples were heterozygous for the HFE S65C mutation. In addition, we found novel mutations: two in HFE (R66C in exon 2 and R224G in exon 4), one in the hepcidin gene (G71D), and one in TfR2 (V22I), plus several intronic or silent substitutions. Six of the seven individuals with hepcidin or TfR2 coding mutations carried also HFE C282Y or S65C mutations. CONCLUSION: DHPLC is an efficient method for mutational screening for the genes involved in hereditary hemochromatosis and for the study of their copresence.     

Denaturing HPLC-based approach for detecting RYR2 mutations involved in malignant arrhythmias

BACKGROUND: Mutations in the RYR2 gene, which encodes the cardiac ryanodine receptor, have been reported in patients showing either arrhythmogenic right ventricular cardiomyopathy, type 2, or stress-induced polymorphic ventricular tachycardia. Both clinical phenotypes are characterized by a high risk of sudden death. Detection of RYR2 mutations is particularly important because beta-blocker treatment has been shown to be effective in preventing fatal arrhythmias in affected patients. METHODS: We used denaturing HPLC (DHPLC) to identify mutations in the human RYR2 gene. Fifty-three single exons, possibly targeted by mutations, were identified by comparison with the distribution of pathogenic mutations of the RYR1 gene, the skeletal muscle counterpart of RYR2. PCR primers for amplification of the entire coding sequence (116 amplicons, corresponding to 105 exons) were tested, and optimal DHPLC conditions were established. DHPLC analysis of critical exons was performed on 22 unrelated patients with effort-induced polymorphic ventricular arrhythmias but lacking a precise diagnosis. RESULTS: We identified four novel missense mutations among 22 patients. Their pathogenic role was related to present knowledge of the structure and function of RyR2 protein. CONCLUSIONS: Under optimized conditions, DHPLC is a cost-effective, highly sensitive, rapid, and efficient method for mutation screenings. A four-step approach is proposed for mutation screening of the RYR2 gene: (a) DHPLC analysis of 48 critical exons (2-4, 6-15, 17-20, 39-49, 83, 84, 87-97, and 99-105); (b) DNA sequencing of 5 critical exons unsuitable for DHPLC; then, in case of negative results, (c) DHPLC analysis of the remaining 39 exons and (d) DNA sequencing of the last 13 amplicons unsuitable for DHPLC analysis.