Specific detection of Flt3 point mutations by highly sensitive real-time polymerase chain reaction in acute myeloid leukemia

Among activating class III receptor tyrosine kinase (Flt3) mutations, internal tandem duplications of Flt3 (Flt3-ITD) are detected in about 25% of patients with acute myeloid leukemia (AML). In contrast, mutations within the tyrosine kinase domain of Flt3 (Flt3-TKD mutations) are less frequent (approximately 7%), and there are only limited data on the frequency of recently demonstrated activating Flt3 point mutation at codon 592 (Flt3-V592A mutation). We evaluated a new approach for rapid screening of Flt3-TKD and Flt3-V592A mutations using the fluorescence resonance energy transfer (FRET) principle in a group of 122 patients. Based on individual Flt3-TKD mutations, we designed patient-specific primers to perform a highly sensitive polymerase chain reaction (PCR) assay for rapid detection of minimal residual disease (MRD). We also used a model system with MonoMac-6 cells carrying the Flt3-V592A mutation to establish a mutation-specific real-time PCR approach also for this molecular aberration. We identified 9 cases (8%) of Flt3-TKD mutations (5 cases of mutation D835Y, 3 cases of mutation D835H, and 1 case of mutation Del836), and no cases of Flt3-V592A mutation. Screening for Flt3-TKD mutations with fluorescent probes is equivalent to conventional screening using standard PCR followed by EcoRV restriction. We present a real-time PCR protocol that can be used for MRD analyses based on individual Flt3-TKD mutations. Examples of MRD analyses are presented for all 3 subtypes of Flt3-TKD mutation identified in this study. In summary, we demonstrate new methodological approaches for rapid screening of Flt3 point mutations and for detection of MRD based on patient-specific Flt3-TKD mutations.     

A seminested PCR test for simultaneous detection of two common mutations (35delG and 167delT) in the connexin-26 gene.

BACKGROUND: Several mutations described in the connexin-26 gene cause nonsyndromic autosomal recessive deafness (NARD). The prevalence of two frame-shift mutations, known as 35delG and 167delT, was relatively high in patients with NARD from different populations. METHODS AND RESULTS: A seminested PCR test has been developed for simultaneous detection of two common mutations in the connexin-26 gene. The test is based on PCR amplification of a 285-bp DNA fragment that covers both the 35delG and 167delT mutations. The latter mutation destroys a Pst I site and is easily detected by Pst I digestion of the 285-bp DNA fragment. However, the 35delG mutation does not destroy or create a restriction site. To create a site, we designed a mismatched primer that generated an EcoN I site in an 87-bp DNA fragment, but only if the 35delG mutation was present. The test was validated using five DNA samples previously characterized for the connexin-26 mutations. After validation, we screened 45 unrelated patients with NARD and 280 healthy Omani subjects for the presence or absence of the 35delG and 167delT mutations. Neither mutation was found to be present in patients or control subjects. CONCLUSION: We developed a seminested PCR test for the simultaneous detection of both common mutations in the connexin-26 gene. In our analysis of 45 patients and 280 control subjects, the 35delG and 167delT mutations were absent in both groups.     

Rapid, nonradioactive detection of mutations in the human genome by allele-specific amplification

A simple, rapid, nonradioactive method has been developed to facilitate the direct detection of point mutations that cause genetic disease. The method operates on the basis of the specific amplification of a target allele by the polymerase chain reaction with extension primers designed such that their 3' end is placed at the mutation site. When this base is complementary to that of the specific allele, the DNA segment is amplified; when it is not complementary, the polymerase chain reaction cannot proceed. When alpha 1-antitrypsin (alpha 1AT) deficiency was used as a model, the technique of allele-specific amplification was capable of selective detection of five different mutations that cause the alpha 1AT deficiency state, including three different naturally occurring single-base substitution mutations (alleles Z, S, and Nullbellingham), an insertion mutation (Nullmattawa), and a deletion mutation (Nullgranite falls). Double-blind evaluation of 47 samples of genomic DNA demonstrated 100% accuracy of the method. The technique of allele-specific amplification is rapid, simple, and does not require the existence of a convenient restriction endonuclease site or the use of radioactive materials, and thus should have broad applicability for the detection of known genetic diseases in a highly sensitive and specific fashion.     

应用PCR-RFLP技术检测阿德福韦耐药相关性HBV变异

目的建立一种简便、快速、实用的检测阿德福韦(ADV)耐药相关性HBV变异(rtA181V/T/S和rtN236T变异)的方法。方法根据GenBank收录的HBV基因全序设计套式-PCR引物,使野毒株的PCR产物上游末端引入BglI的酶切位点,使rt236变异株的PCR产物下游末端引入BseDI(SecI)的酶切位点,建立PCR-限制性片段长度多态性(RFLP)方法。用此方法分别检测含rt181变异株、rt236变异株及野毒株质粒和3例ADV耐药慢性乙型肝炎患者血清标本。将变异株和野毒株配成不同比例,再用此方法检测,以了解该方法的敏感性。结果本研究建立的PCR—RFLP方法可同时检测rt181变异和rt236变异;用此方法检测血清标本,结果与测序及克隆分析一致;此方法可检测出标本中10%的变异株。结论应用PCR- RFLP方法可同时检测rtA181V/T/S和rtN236T变异,具有较高的敏感性,可用于ADV耐药变异的早期诊断。     

Rapid detection of 3500Q and 3531 mutations and MspI polymorphism in exon 26 at the apolipoprotein B gene

Several environmental and genetic factors are associated with high levels of cholesterol. Hypercholesterolemia is the main phenotype of Familial Defective Apolipoprotein B and Familial Hypercholesterolemia that are caused by mutations at the apolipoprotein (apo) B and LDL receptor genes, respectively. Identification of the specific genetic alteration associated with hypercholesterolemia is an important issue in clinical diagnosis of high risk for CAD. Apo B gene mutations and polymorphisms are usually screened by SSCP, DGGE, and heteroduplex, which must be confirmed by DNA sequencing or by direct detection using PCR techniques. In this study, we have optimized a PCR-RFLP procedure for identification of 3500Q and 3531 mutations and MspI polymorphism at the apo B gene. The technique can be performed in a single reaction, using the restriction endonuclease MspI for simultaneous detection of 3500Q mutation and MspI polymorphism, and NsiI for detection of 3531 mutation. The procedure was validated by analysis of control DNA samples from individuals carrying these mutations. Screening of 186 Brazilian hypercholesterolemic individuals showed that the frequency of the M-allele (7.8%) of MspI polymorphism was similar to that found in other individuals with CAD. However, neither 3500Q nor 3531 mutations were detected in this group. In conclusion, this procedure is simple and rapid, being easily introduced in clinical laboratories for direct detection of the more frequent mutations at the apo B gene associated with hypercholesterolemia.     

Gene probes: application to prenatal and postnatal diagnosis of genetic disease

Gene probes can now be used to detect a variety of mutations that produce single-gene disorders. In present clinical practice, restriction endonuclease analysis is used for the prenatal diagnosis of sickle cell anemia, alpha-thalassemia, and beta-thalassemia. Direct detection of the mutation is possible in alpha-thalassemia, where a deletion has usually occurred, and in sickle cell anemia, where the mutation alters the recognition sequence of the restriction endonuclease, Mst II. Indirect detection of beta-thalassemia is based on using normal variations in DNA (DNA polymorphisms) to track normal and affected beta-globin genes in families. This latter kind of analysis is also useful in detecting the phenylalanine hydroxylase genes affected in phenylketonuria and will often be used in disorders where the mutations are unknown. In cases where the mutation is known, direct analysis by use of oligonucleotide probes is a new and important advance. An example of this type of gene detection in a family with classical hemophilia is presented. In addition, with chorion villus biopsy, detection of these inherited diseases is feasible by the 12th week of pregnancy.     

Allelic drop-out in the LDLR gene affects mutation detection in familial hypercholesterolemia

OBJECTIVES: Familial hypercholesterolemia is a monogenic disorder caused by mutations in the LDL receptor (LDLR) gene. We observed allelic drop-out during LDLR genotyping and aimed at redesigning mutation detection. DESIGN AND METHODS: The NanoChip microelectronic array technology and PCR restriction fragment length polymorphism analysis were used. RESULTS: Allele drop-out caused false homozygous diagnoses and was overcome using PCR primers without polymorphisms in the primer binding site. CONCLUSIONS: This report presents the importance of allele drop-out in LDLR genotyping.     

铜绿假单胞菌临床分离株gyrA基因突变与喹诺酮类耐药关系研究

目的：了解铜绿假单胞菌临床分离株gyrA基因突变与喹诺酮类药物耐药的关系。研究其临床意义。方法：通过PCR HinfⅠ酶切分析,PCR－单链构象多态性分析（SSCP）等方法检测铜绿假单胞菌ATCC27853及36株铜绿假单胞菌临床分离株的gyrA基因突变情况。结果HinfⅠ酶切结果显示,所有13株环丙沙星耐药株,3株环丙沙星中介株中的2株,20株环丙沙星敏感株中的1株存在gyrA基因HinfⅠ酶切酶点突;SSCP分析结果显示,所有耐药菌株和中介菌株的gyrA基因基因HinfⅠ酶切位点突变密切相关,但gyrA基因突变不局限于该位点,在常规药敏试验的基础上开展gyrA基因突变检测有重要的临床意义。     

95例耐多药结核菌株rpoB基因突变的分子特征

目的:探讨广东地区MDR-TB菌株rpoB基因突变的分子特征.方法:对95例MDR-TB菌株rpoB基因453-564位密码子片段进行PCR-直接测序.结果:95例MDR-TB菌株rpoB基因突变率91.58％.86例为点突变,1例插入突变,未发现缺失.常见位点为531 (63.22％)、526(20.69％)、516(9.20％).其中:单位点突变69例(80.23％),双位点突变16例(18.60％),三位点突变1例(1.17％).511位点突变常同时伴有其他位点突变(57.14％).结论:主要突变位点与国内外报道基本相同,但各位点所占比例具有地域差异;联合突变率较高,占19.54％.512位点插入(AGGAGC)突变可能为新突变类型.     

Restriction site generating-polymerase chain reaction (RG-PCR) for the probeless detection of hidden genetic variation: application to the study of some common cystic fibrosis mutations.

In this report we describe the use of a DNA amplification technique in which modified primers introduce a base substitution adjacent to the codon of interest and create an artificial restriction site for the detection of mutations which do not produce or modify a naturally occurring restriction site (restriction site generating-polymerase chain reaction, RG-PCR). RG-PCR was developed and applied to the screening in an Italian population sample of several relatively common cystic fibrosis mutations which are not amenable to analysis with a known restriction endonuclease: G542X, 2869insG, Y913C, N1303K, and 1717-1GA. This method, which allows the identification of virtually any single base change by restriction enzyme analysis and without the need for molecular probes, is rapid and easy to perform. The combined use of RG-PCR for several different CF mutations in multiplex tests further expands the advantages of this approach.     

高通量检测技术检测乙型肝炎病毒变异的临床意义

目的探讨基因芯片技术检测乙型肝炎病毒(HBV)6位点变异的临床意义。方法对91例乙型肝炎患者采用基因芯片技术,检测HBV 6位点的自然变异。结果91例乙型肝炎患者HBV变异率98.9%(90/91),其中BCP区1762位点变异率最高(61.5%)。重度肝炎和肝硬化中双位点变异显著高于轻中度肝炎(P<0.01)。29例进行过拉米呋啶抗病毒治疗的病例中P区552位点变异率为96.6%(28/29)。结论基因芯片技术检测HBV变异具有高通量、高灵敏等特点,对观察HBV基因突变有很高的临床应用价值。     

Detection of a R173W Mutation in the Porphobilinogen Deaminase Gene in the Nova Scotian “Foreign Protestant” Population with Acute Intermittent Porphyria: a Founder Effect

Objectives: Acute intermittent porphyria (AIP) is caused by mutations in the porphobilinogen deaminase (PBGD) gene that disrupt the function of the enzyme. Many mutations that lead to decreased PBGD activity have been described. An Arg to Trp substitution at codon 173 (CGG→TGG in exon 10) and designated R173W, which leads to a CRIM-negative phenotype, has been reported in Swedish, Finnish, Scottish, and South African kindreds, and in a Nova Scotian proband with fatal AIP. In this work, we investigated the presence of this mutation in a Nova Scotian patient population presenting with AIP.

Design and Methods: Single-strand conformation polymorphism analysis and DNA sequencing by TA cloning and Sanger’s dideoxy chain termination method, were used to confirm the maternal transmission of this mutation to the proband. The mutation also eliminates an NciI (also MspI) endonuclease restriction site, which allows for detection of the mutant allele by polymerase chain reaction amplification and restriction enzyme digestion.

Results: The family of the Nova Scotian proband and four other AIP kindreds showed the presence of the same mutation. These five families are descendants of German, Swiss, and French immigrants historically known as the “Foreign Protestants,” who were recruited to Nova Scotia in the 1750s.

Conclusion: In all these families, descent from one couple that settled in Nova Scotia in 1751 has been identified by genealogy research, consistent with a founder effect within this population. This is the first identified mutation in PBGD causing AIP that has been linked to a founder effect in descendants of an immigrant population to North America, and which could be traced to such a distant background, similar to the South African variegate porphyria mutation.     

A novel mutation which represents the fifth non-pathogenic polymorphism in the coding sequence of the Arylsulfatase A gene

A novel mutation, a C→T transition at nucleotide 455 of the coding sequence of the ARSA gene, was found in a control individual during the search for metachromatic leukodystrophy mutations. Its distribution in three different populations was examined. The frequency of the T allele was 0·058, 0·025 and 0·033, in Italian, German and Greek populations, respectively. The mutation results in no amino acid substitution and can be identified as it creates a polymorphic site for the restriction endonucleaseNlaIII.     

药物性耳聋相关线粒体12S rRNA突变

  目的  探讨极重度非综合征性耳聋患者线粒体DNA(mitochondrial DNA, mtDNA)1555A > G和1494C > T突变情况。  方法  选取黑龙江省佳木斯地区聋哑学校学生和北京协和医院门诊散发的极重度感音神经性耳聋患者共208例作为研究对象, 使用基因芯片方法和限制性内切酶法对其mtDNA 1555和1494两个位点进行检测, 并用直接测序的方法进行验证。  结果  208例患者中共发现1555A > G突变者10例, 该突变的携带率为4.81%;未发现1494C > T突变。  结论  mtDNA 1555A > G突变在极重度非综合征性耳聋患者中阳性率较高, 而在汉族人群中1494C > T突变较为罕见。     

遗传性凝血因子Ⅶ缺陷症家系中R152Q及IVS6+1G→T突变的鉴定

目的检测1个遗传性凝血因子Ⅶ缺陷症家系中因子Ⅶ基因的突变。方法应用PCR结合直接测序的方法对先证者的因子Ⅶ基因9个外显子及其侧翼序列进行分析，鉴别其中可能存在的基因变异。应用PCR产物反向测序法或PCR限制性内切酶分析技术对突变位点进行确证。随机选取100名健康体检者作为正常对照。结果先证者FⅦ基因第6外显子和第6内含子分别存在R152Q和IVS6＋1G→T杂合突变，家系分析表明这2个突变是双重杂合子型，前者遗传自父亲，后者遗传自母亲。100名健康对照者均未发现R152Q错义突变。结论FⅦ基因第6外显子R152Q错义突变和第6内含子供体剪接位点IVS6＋1G→T突变可能是先证者因子Ⅶ先天性缺陷的原因。     

DNA微阵列芯片法检测遗传性耳聋基因

目的 应用基因芯片技术对临床散发性耳聋患者进行基因检测,评价在临床检测中的应用价值。方法 抽取患者静脉血,EDTA抗凝,在万级洁净间内进行DNA提取和PCR扩增杂交,对中国人常见的4个耳聋基因的9个突变位点进行检测。结果 24例患者中,共检出突变7例,阳性率为29.17%,检出GJB2基因突变4例(16.67%),其中176 del 16位点杂合突变型1例,235 del C位点纯合突变型1例,299 del AT位点杂合突变型2例。1例(4.17%)SLC26A4基因IVS7-2A>G位点杂合突变型; 2例(8.33%)线粒体12SrRNA基因1555A>G位点均质突变型,未检出GJB3基因突变。结论 遗传性耳聋基因芯片技术可快速、高通量检出耳聋相关突变位点,满足临床耳聋基因检测需求。     

ARMS法检测肺腺癌EGFR基因突变及临床意义

目的 探讨扩增阻滞突变系统(ARMS)法在肺腺癌中表皮生长因子受体(EGFR)基因突变检测的应用及其临床意义。方法 收集2015年1月～2016年8月西安交通大学第一附属医院病理科的肺腺癌标本566例作为研究对象。其中,胸腔积液细胞块标本34例,肺活检标本401例,手术切除标本131例,采用ARMS法进行石蜡标本EGFR基因突变检测,分析EGFR基因突变与肺腺癌患者临床资料的相关性。结果 肺腺癌标本566例中,吸烟腺癌患者239例,非吸烟腺癌患者327例。吸烟患者中,EGFR突变率与患者年龄、性别、手术方式等无明显关联(P>0.05),而与肺癌原发部位关系密切(P<0.05); 非吸烟患者中,EGFR突变率与性别、年龄、标本类型及肺癌原发病灶部位无明显相关性(P>0.05)。结论 ARMS法可有效应用于肺腺癌临床病理石蜡标本中EGFR基因突变检测。吸烟是EGFR突变率的重要影响因子,且对左、右肺均有影响,但是对右肺的影响较大。     

A frequent polymorphism in the coding exon of the human cannabinoid receptor (CNR1) gene.

The central cannabinoid receptor (CB1) mediates the pharmacological activities of cannabis, the endogenous agonist anandamide and several synthetic agonists. The cloning of the human cannabinoid receptor (CNR1) gene facilitates molecular genetic studies in disorders like Gilles de la Tourette syndrome (GTS), obsessive compulsive disorder (OCD), Parkinsons disease, Alzheimers disease or other neuro psychiatric or neurological diseases, which may be predisposed or influenced by mutations or variants in the CNR1 gene. We detected a frequent silent mutation (1359G-->A) in codon 453 (Thr) of the CNR1 gene that turned out to be a common polymorphism in the German population. Allele frequencies of this polymorphism are 0.76 and 0.24, respectively. We developed a simple and rapid polymerase chain reaction (PCR)-based assay by artificial creation of a Msp I restriction site in amplified wild-type DNA (G-allele), which is destroyed by the silent mutation (A-allele). The intragenic CNR1 polymorphism 1359(G/A) should be useful for association studies in neuro psychiatric disorders which may be related to anandamide metabolism disturbances.     

原发性开角型青光眼家系的MYOC基因突变研究

目的 对来自重庆地区的1个原发性开角型青光眼(POAG)家系进行MYOC基因突变筛查,研究POAG与MYOC基因突变的相关性,探讨MYOC基因突变在中国人POAG发病中的作用.方法 1个4代共39例的青光眼家系,8例为已确诊患者,健康对照者100名.用单链构象多态性分析(SSCP)、PCR.限制性片段长度多态性(RFLP)和基因测序的方法筛选MYOC基因的突变(包括G34C、C136T、G144T、G227A、C624G、G736A、C1009G、A1036G、C1081T、G1099A、G1138A、A1139C、T1430A、C1441A和C1442T等),同时对检测到的突变结果进行生物信息学分析.结果 在该家系中,发现1个G227A(Arg76Lys)突变,该突变存在2例已确诊的POAG患者和1例家系表型正常者中,健康对照者中未榆出.发现1个缺失突变(C1009del),该突变存在于家系中所有发病患者和1例4岁的子代亲属,健康对照者中未检出.未发现其他突变.由于C1009del突变是首次发现,据此我们申请了GenBank号,已发表的GenBank序列号为FJ237047,对应的蛋白序列号为ACI62293.结论 G227A(Arg76Lys)突变为已报道的多态性位点,与该家系青光眼的发病无相关性.移码突变C1009del突变与青光眼的发病密切相关,也可由此推测青光眼患者亲属的发病率较正常人高.     

Evaluation of temperature gradient capillary electrophoresis for detection of the Factor V Leiden mutation: coincident identification of a novel polymorphism in Factor V.

AIM: The Factor V Leiden mutation (G1691A) is a clinically important polymorphism that results in an increased risk of thrombosis. The goal of this study was to compare a temperature gradient capillary electrophoresis (TGCE) platform for the detection of Factor V gene mutations to a conventional restriction fragment length polymorphism (RFLP) assay. METHODS: Three hundred and four samples were analyzed by both TGCE and a common clinical Mnl I/RFLP assay. Concordance of results between the two assays was observed for 302/304 (99.3%) of the samples. RESULTS: All of the Leiden mutants (23/23, 100%) were identified by TGCE. Of the two discrepant results, one was caused by low peak heights in the TGCE output data and was easily rectified by the addition of a minimum peak height threshold. The second discrepancy resulted from the presence of a G-->A transition 95 bp downstream of the Leiden mutation site. This polymorphism represents a previously unreported alteration of the Factor V gene. CONCLUSIONS: The TGCE assay is less labor-intensive and has a higher throughput capacity than the Mnl I/RFLP assay. TGCE is a less specific assay than the Mnl I/RFLP assay that allows for the detection of novel polymorphisms, but also creates the need for all positive TGCE results to be confirmed by an alternate method such as sequencing. Our results demonstrate that TGCE is a highly sensitive method for mutation detection and has utility for mutation discovery analysis.