Rapid identification of mutations in the glucocerebrosidase gene of Gaucher disease patients by analysis of single-strand conformation polymorphisms

To detect mutations in the glucocerebrosidase gene in Gaucher disease patients, we used the recently described technique of single-strand conformation polymorphism (SSCP) analysis in combination with selective amplification. We analyzed exon 8, 9, 10 and 11 of the glucocerebrosidase gene; these exons were sequentially amplified using the selectively amplified products as templates. We found variant SSCP patterns corresponding to the presence or absence of the 6433C mutation, which was detected by NciI digestion analysis, in exon 10. Furthermore, we detected four variant SSCP patterns in exon 8, 10 and 11. Sequencing analysis consistently revealed four single-base substitutions in the corresponding exons, three novel missense mutations (5409A, 6375G and 6682T) and one silent polymorphism (6594A). These mutations were found only in one patient; therefore, these findings have confirmed the marked genetic heterogeneity of Gaucher disease. SSCP analysis in combination with selective amplification is a rapid and sensitive procedure for the screening of the mutations in the glucocerebrosidase gene of patients with Gaucher disease.     

Validation of fluorescent SSCP analysis for sensitive detection of p53 mutations

We have developed a fluorescence-based single strand conformation polymorphism (SSCP) method that offers fast and sensitive screening for mutations in exons 5-8 of the human p53 gene. The method uses an ABI 377 DNA sequencer for unique color detection of each strand, plus accurate alignment of lanes for better detection of mobility shifts. To validate the method, 21 cell lines with reported mutations in p53 exons 5-8 were analyzed by SSCP using various gel conditions. The sensitivity for mutation detection was 95% for all cell lines studied, and no false positives were seen in 10 normal DNA samples for all four exons. Experiments mixing known amounts of tumor and normal DNA showed that mutations were detected even when tumor DNA was mixed with 80% normal DNA. Fluorescent SSCP analysis using the ABI sequencer is a useful tool in cancer research, where screening large numbers of samples for p53 mutations is desired.     

A novel mutant (transthyretin Ile-50) related to amyloid polyneuropathy. Single-strand conformation polymorphism as a new genetic marker.

DNA sequence polymorphisms in transthyretin (TTR) genes were investigated by single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction products. The amplified DNA fragments that encode each exon of the normal TTR gene showed two bands, representing the two complementary single strands of DNA. In one patient with amyloid polyneuropathy, the exon 3 DNA showed a unique, aberrant migration pattern. Direct sequencing analysis of the amplified exon 3 revealed a single base change (G-to-T), resulting in a novel amino acid substitution (Ser-50----Ile). We also present the SSCP patterns for five known Japanese TTR variants.     

'Cold SSCP': a simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses.

A rapid (< 2.5 hrs) method for single-strand conformation polymorphism (SSCP) analysis of PCR products that allows the use of ethidium bromide staining is described. PCR products ranging in size from 117 to 256 bp were evaluated for point mutations and polymorphisms by 'cold SSCP' in commercially available pre-cast polyacrylamide mini-gels. Several electrophoretic parameters (running temperature, buffers, denaturants, DNA concentration, and gel polyacrylamide concentration) were found to influence the degree of strand separation and appeared to be PCR fragment specific. Use of the 'cold' SSCP technique and the mini-gel format allowed us to readily optimize the electrophoretic conditions for each PCR fragment. This greatly increased our ability to detect polymorphisms compared to conventional, radioisotope-labeled 'hot' SSCP, typically run under two standard temperature conditions. Excellent results have been obtained in resolving mutant PCR fragments from human p53 exons 5 through 8, human HLA-DQA, human K-ras exons 1 and 2, and rat K-ras exon 3. Polymorphisms could be detected when mutant DNA comprised as little as 3% of the total gene copies in a PCR mixture. Compared to standard 'hot' SSCP, this novel non-isotopic method has additional advantages of dramatically increased speed, precise temperature control, reproducibility, and easily and inexpensively obtainable reagents and equipment. This new method also lacks the safety and hazardous waste management concerns associated with radioactive methods.     

A simple method for non-radioactive PCR-SSCP using MDE gel solution and a midi gel format: application for the detection of variants in the GLUT1 and CTLA-4 genes

The need to identify disease-causing mutations and DNA polymorphisms has increased with the continuing identification of new candidate genes. PCR single-strand conformation polymorphism (PCR-SSCP) is one of the techniques most widely used to identify a mutant sequence or a polymorphism in a known gene. However, the original SSCP protocols using the incorporation of radioactive label and polyacrylamide gel electrophoresis on sequencing gels for detection were labour intensive and time-consuming. Here we describe a simple SSCP protocol using MDE gel solution and a midi gel format to detect SSCP variations in the glucose transporter gene GLUT1, that we have previously analysed with the standard radioactive SSCP protocol, and we have also tested this method on the previously described point mutation (A/G transition in exon 1) of the CTLA-4 (cytotoxic T lymphocyte associated-4) gene. All known variants were detected. Based on the results, this technique appears to be simple, with no use of radioactive labels and with easy handling of the gel. Furthermore, it needs little optimisation, is relatively rapid and highly sensitive. We propose this method for the first screening for candidate gene variants.     

Gene analysis of the N-terminal region of the estrogen receptor alpha in preeclampsia

Alterations in the NH(2)-terminal region of the estrogen receptor alpha (ERalpha) gene expressed in placental bed tissue may be implicated in the development of preeclampsia, the pathogenesis of which involves the spiral arteries. Therefore, mutations and polymorphisms on exons 1 and 2 of the gene encoding ERalpha were studied. Placental bed biopsies were taken from 20 healthy, normotensive pregnant women and 16 preeclamptic patients. DNA was extracted from the tissue and exon 1 and exon 2 were amplified by PCR prior to denaturing gradient gel electrophoresis analysis or to single stranded conformational polymorphism analysis. In exon 1, a codon 10 polymorphism, either homozygous for the wild type gene, homozygous for the mutant type gene, or heterozygous, was revealed in both patients and healthy individuals. A codon 87 polymorphism, homozygous for the wild type gene, was detected in both groups. No mutations or polymorphisms were found in exon 2. The allele distribution for either codon 10 or 87 between patients and healthy individuals showed no significant differences. In conclusion, genetic alterations in the NH(2)-terminal region of the ERalpha molecule are not correlated with preeclampsia.     

Screening of the c-kit gene missense mutation in invasive ductal carcinoma of breast among north Indian population

Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer deaths among females across the world, accounting for 23 % (1.38 million) of total new cancer cases and 14 % (0.45 million) of the total cancer deaths in 2008. c-kit is expressed in mast cell growth factor, cellular migration, proliferation, melanoblasts, haematopoietic progenitors and germ cells. We have designed our study with aim to explore the c-kit gene mutations in invasive ductal carcinoma (IDC) breast. To ascertain the range of mutations in exon 11, 13 and 17 of c-kit gene in 53 cases of IDC breast, we carried out PCR-SSCP followed by DNA sequencing. The mutation frequency of c-kit gene in exon 11, 13 and 17 were 9.43 % (5/53), 1.88 % (1/53) and 3.77 % (2/53), respectively. During our mutational analysis, we have detected five missense mutations in exon 11 (Pro551Leu, Glu562Val, Leu576Phe, His580Tyr and Phe584Leu), one missense mutation in exon 13 (Ser639Pro) and two missense mutations in exon 17 (Arg796Gly and Asn822Ser). It seems that c-kit mutations might participate in breast cancer pathogenesis and may be utilized as predictive marker, since the loss of c-kit positivity is generally linked with different types of breast cancer. Further molecular studies are necessary to validate the association of c-kit gene mutation in IDC breast pathogenesis.     

Mutation screening of the FSH receptor gene in infertile men.

Follicle stimulating hormone (FSH) is important for controlling spermatogenesis through binding with its receptor. However, little information is available on mutations of the FSH and its receptor gene in infertile men. To study the genetic defects, which caused problems in spermatogenesis, we screened the point mutations of the FSH receptor gene in infertile men with high serum FSH concentrations. Seventy male infertile patients with high FHS levels (> 12 mIU/ml) were screened for mutations in each of the 10 exons of the FSH receptor gene, using genomic DNA PCR and a single-strand conformation polymorphism (SSCP) analysis. From this study, three shifted bands were detected by SSCP. The first shifted band was found in the PCR product of exon 4, including the exon-intron boundary sequence in only one patient. The sequence analysis revealed a nucleotide A to T substitution in intron 3 (IVS3-4A-->T). The second shifted band was detected in exon 10 with high frequency (33%). A nucleotide A to G substitution was found at the position of the 994th nucleotide, predicting a Thr to Ala substitution at the position of the 307th amino acid (Thr307Ala). The third shifted band in the 3' region of exon 10 was detected frequently in infertile patient and normal groups. It was tightly linked to the Thr307Ala variant. Thus, all of the abnormalities represent neutral polymorphisms, and not pathological mutations of the FSH receptor gene. In conclusion, we did not confirm that the genomic mutation of the FSH receptor is a major genetic cause in Korean infertile patients with high FSH levels.     

BRCA1 mutations and polymorphisms in a hospital-based consecutive series of breast cancer patients from Apulia, Italy

INTRODUCTION: Hereditary breast cancer has been partly attributed to germline mutations in the BRCA1 gene that are deleterious for BRCA1 protein activity. This paper analyzes the incidence and characteristics of detectable BRCA1 mutations and polymorphisms in a hospital-based consecutive series of breast cancer patients from southern Italy to investigate the incidence and the association of these molecular alterations with breast cancer biology and family history. METHODS: One hundred cases with familial characteristics were selected from a consecutive series of 511 patients with a first diagnosis of breast cancer. DNA from peripheral blood was screened for whole BRCA1 gene mutations utilizing dHPLC as a pre-screening analysis and automatic DNA sequencing for the identification of specific alterations. RESULTS: In the overall series of 511 patients, 100 had a family history of breast cancer and were investigated for BRCA1 mutations. Two types of BRCA1 mutations were identified, 5382insC in six cases and 4566delA in one case. The 5382insC mutation was present in two out of six cases with ovarian cancer while 4566delA in one case of male cancer. The most frequent missense polymorphisms were E1038G, P871L, K1183R in exon 11, S1613G, M1652I in exon 16 and D1778G in exon 22. Confirming what found in previous studies, patients in whom pathological BRCA1 mutations were detected had early-onset breast cancer (p=0.05), positive nodal status (p=0.05), lower ER (p=0.02) and PgR (p=0.01) content. Interestingly, the K1183R polymorphism and, less strongly, S1613G polymorphism were associated to mutational risk (K1183R: OR 0.1 p=0.03; S1613G: OR 2.7 p=0.08). CONCLUSION: Mutations in the BRCA1 gene are frequent also in our consecutive series of patients from southern Italy. An association between two detected single nucleotide polymorphisms (SNPs) and BRCA1 mutational risk was ascertained. Finally, we confirm the fact that peculiar clinical-pathological features seem to characterize patients with a family history of breast cancer and BRCA1 alterations.     

Four new nucleotide sequence polymorphisms in the LDL receptor gene detected by SSCP analysis

Seven nucleotide sequence polymorphisms were detected within exons of the low-density lipoprotein (LDL) receptor gene using single-strand conformation polymorphism (SSCP) analysis followed by direct sequence analysis on amplified DNA. Four nucleotide changes at nucleotide positions 1617, 1725, 2232, and 2635 were new nucleotide sequence polymorphisms not previously described. The remaining three nucleotide changes were identical with restriction fragment length polymorphisms and a previously reported nucleotide sequence polymorphism. These nucleotide sequence polymorphisms, detectable by SSCP analysis, are useful genetic markers for linkage analysis of the LDL receptor gene and familial hypercholesterolemia.     

Molecular and genetic analysis of a compound heterozygote for dysprothrombinemia of prothrombin Tokushima and hypoprothrombinemia.

The molecular and genetic basis of a compound heterozygote for dys- and hypoprothrombinemia was analyzed. Abnormal nucleotide sequences of the human prothrombin gene were screened by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion and mutated primer-mediated PCR-RFLP. A single nucleotide substitution responsible for dysprothrombinemia of prothrombin Tokushima was detected, as were three polymorphisms. The mutation for hypoprothrombinemia was detected by PCR-single-strand conformation polymorphism (PCR-SSCP) with endonuclease digestion in exon 6, near MboII-RFLP and NcoI-RFLP. Sequencing of PCR-amplified genomic DNA revealed a single base insertion of thymine (T) at position 4177. The resulting frameshift mutation caused both an altered amino acid sequence from codon 114 and a premature termination codon (i.e., TGA) at codon 174 in exon 7. Because exon 7 encodes the kringle 2 domain preceding the thrombin sequence, this frameshift leads to the null prothrombin phenotype. The inheritance of the hypoprothrombinemia gene from the father to the proband was proved by PCR-SSCP with endonuclease digestion and mutated primer-mediated PCR-RFLP.     

Sequence of DNA flanking the exons of the HEXA gene, and identification of mutations in Tay-Sachs disease.

The rapid identification of mutations causing Tay-Sachs disease requires the capacity to readily screen the regions of the HEXA gene most likely to be affected by mutation. We have sequenced the portions of the introns flanking each of the 14 HEXA exons in order to specify oligonucleotide primers for the PCR-dependent amplification of each exon and splice-junction sequence. The amplified products were analyzed, by electrophoresis in nondenaturing polyacrylamide gels, for the presence of either heteroduplexes, derived from the annealing of normal and mutant DNA strands, or single-strand conformational polymorphisms (SSCP), derived from the renaturation of single-stranded DNA. Five novel mutations from Tay-Sachs disease patients were detected: a 5-bp deletion of TCTCC in IVS-9; a 2-bp deletion of TG in exon 5; G78 to A, giving a stop codon in exon 1; G533 to T in exon 5, producing the third amino acid substitution detected at this site; and G to C at position 1 of IVS-2, expected to produce abnormal splicing. In addition, two mutations, (G1496 to A in exon 13 and a 4-bp insertion in exon 11) that have previously been reported were identified.     

Single-strand conformational polymorphism (SSCP)-detected p53 gene mutations are a less sensitive marker of malignancy in pleural fluids than p53 immunostaining

p53 immunostaining has been advocated as a marker of malignancy in pleural biopsies and serous fluids. The object of this study was to compare the sensitivity and specificity of p53 immunostaining for the detection of malignant cells in pleural fluids with a technique designed to detect p53 gene mutations in exons 5, 6, 7 and 8 by SSCP and nucleotide sequencing. Five out of eight pleural fluids containing adenocarcinoma showed p53 immunostaining and two of these also showed polymorphisms on SSCP and a mutation on sequencing. None of the 10 benign pleural fluids showed immunostaining for p53 or polymorphisms on SSCP. We believe that the poor sensitivity of p53 gene mutation by SSCP is mainly due to DNA from the background reactive cells 'swamping' the mutant DNA. We do not advocate its use as a diagnostic aid.     

Detection of polymorphisms at exons 3 (Tyr113-->His) and 4 (His139-->Arg) of the microsomal epoxide hydrolase gene using fluorescence PCR method combined with melting curves analysis

An association between exon 3 polymorphisms of the gene encoding microsomal epoxide hydrolase (mEH) and susceptibility to the development of chronic obstructive pulmonary disease (COPD) has been described. We have developed two methods for detecting polymorphisms at exons 3 (Tyr113-->His) and 4 (His139-->Arg) of the mEH gene based on different melting temperatures (T(m)) of fluorescent-labeled oligonucleotide hybridization probes using single-step assays that combine fluorescence PCR and melting curve analysis (LightCycler methodology). DNA was extracted from blood in 79 COPD patients and 146 healthy controls. Results were compared with those obtained by restriction fragment length polymorphism (RFLP) analysis to detect Tyr113His variants and a single-strand conformation polymorphism (SSCP) assay for His139Arg detection. The T(m) of the exon 3 polymorphisms were 61.3 degrees C for Tyr113 (wild type) and 67.5 degrees C for His113 (mutant). The T(m) values of the exon 4 polymorphisms were 67.5 degrees C for His139 (wild type) and 59.2 degrees C for Arg139 (mutant). The within- and between-run melting peaks for the same allele differed by less than 0.5 degrees C for both the exon 3 and the exon 4 polymorphisms. Thus, melting analysis allowed easy and unambiguous assignment of genotyping by means of the respective melting curves. The proportion of individuals who were homozygous mutant for exon 3 was significantly higher in the COPD group than in the control group (p=0.004). LightCycler fluorescence genotyping of exon 4 polymorphisms correlated perfectly with SSCP results. RFLP assay classified 2 patients as homozygous mutant while LightCycler analysis genotyped them as heterozygous. DNA analysis by PCR and sequencing confirmed the LightCycler result. These high-speed (about 40 min for 32 samples), highly sensitive, and specific small-volume assays with low labor requirements hold great promise as tools for rapid detection of COPD susceptibility.     

中国广东汉族人群核苷酸修复基因hMTH1 遗传多态性研究

为研究中国南方汉族人群核苷酸修复基因hMTH1遗传多态性,应用聚合酶链反应-单链构象多态性技术检测172名健康人外周血白细胞hMTH1基因启动子及全部5个外显子多态性,并进行DNA测序。结果发现hMTH1基因启动子及外显子1序列保守,未见突变;外显子2第73位碱基存在T→C杂合型突变,基因型TT和TC频率分别为93.02%、6.98%,等位基因T和C频率分别为96.51%、3.49％;外显子3第45位遗传密码存在T→C杂合型突变,基因型TT和TC频率分别为95.35%、4.65%,等位基因T和C频率分别为97.67%、2.33%,该多态性为首次发现;外显子4第83位遗传密码存在G→A杂合型突变,基因型GG和GA频率分别为89.53%、10.47%,等位基因G和A频率分别为94.77%、5.23％;外显子5第119位氨基酸遗传密码存在C→T杂合型突变,基因型CC和CT频率分别为95.93%、4.07%,等位基因C和T频率分别为97.97%、2.03％。Abstract: In order to study the genetic polymorphisms of nucleotide repair gene hMTH1 in southern Chinese Han population, the polymorphisms of the gene’s promoter and its five exons among peripheral blood lymphocytes of 172 Chinese Han people were analyzed with polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing. The sequences of the promoter and exon 1 of hMTH1 gene were conserved. A T to C polymorphism was detected at the 73th base in exon2. The genotype frequencies of TT and TC were 93.02% and 6.98%, respectively. The allelic frequencies of T and C were 96.51% and 3.49％, respectively. A T to C polymorphism was detected at codon 45 in exon3, which was first reported. The genotype frequencies of TT and TC were 95.35% and 4.65%, respectively. The allelic frequencies of T and C were 97.67% and 2.33%, respectively. A G to A polymorphism was detected at codon 83 in exon4. The genotype frequencies of GG and GA were 89.53% and 10.47%, respectively. The allelic frequencies of G and A were 94.77% and 5.23％, respectively. A C to T polymorphism was detected at codon 119 in exon5. The genotype frequencies of CC and CT were 95.93% and 4.07%, respectively. The allelic frequencies of C and T were 97.97% and 2.03％, respectively.     

Screening for Pax8 mutations in patients with congenital hypothyroidism in South-West Germany

AIMS: To study the frequency of mutations in the Pax8 gene in a cohort of patients with congenital hypothyroidism (CH) in South West Germany. METHODS: A cohort of 95 patients with CH (60 females, 35 males), identified in our newborn screening program, was analyzed for mutations in Pax8 by single-stranded conformational polymorphism (SSCP) and DNA sequencing. RESULTS: SSCP analysis and direct sequencing of exon 3 of a female patient with a hypoplastic thyroid gland revealed two heterozygous mutations in Pax8 resulting in a transition of T to C (codon 34) and G to A (codon 35), replacing isoleucine by threonine and valine by isoleucine. Using allele-specific PCR we could demonstrate that both mutations are located on the same allele. Furthermore, a polymorphism was documented in 24 patients with thyroid hypoplasia in intron 6 at nucleotide +51 (CC, GG, CG). Comparison of the polymorphisms between hypothyroid patients and controls revealed no significant differences suggesting that this polymorphism does not play a role in the pathogenesis of hypothyroidism. No further mutations or polymorphisms were found in the cohort. CONCLUSIONS: These findings confirm the contribution of mutations in the Pax8 gene to the etiology of thyroid dysgenesis with a variable penetrance, but also demonstrate the rare overall incidence in CH.     

结核分枝杆菌rpoB基因突变的检测(简报)

结核病主要是由结核分枝杆菌(Mycobacterium tuberculosis)引起的一种慢性传染性疾病。利福平是结核病化疗方案中一个关键性的药物,它在结核病的短程化疗中起着重要的作用。但是,在我国结核菌对利福平的耐药发生率呈上升局势,而通过传统的依赖生物生长的药敏试验方法进行结核菌对利福平耐药性检测所需时间较长(4-8周),不能满足临床早期开展有效化疗的需要,所以迫切需要建     

Autosomal dominant retinal dystrophy (Rdy) in Abyssinian cats: exclusion of PDE6G and ROM1 and likely exclusion of Rhodopsin as candidate genes

Retinal dystrophy (Rdy) is an autosomal dominant photoreceptor dysplasia of Abyssinian cats and a model for autosomal dominant retinitis pigmentosa (ADRP) in man. We have pursued a candidate gene approach in the search for the causal mutation in Rdy. The genes RHO (encoding rhodopsin), ROM1 (encoding the structural retinal outer-membrane protein-1) and PDE6G (encoding the gamma subunit of the visual transduction protein cyclic guanosine monophosphate-phosphodiesterase) were polymerase chain reaction-amplified from normal feline genomic DNA. Leader, coding and 3' untranslated regions of each gene, and parts of introns were sequenced. Single-stranded conformation polymorphism (SSCP) analysis of Rdy-affected and normal cats was used to identify intragenic polymorphisms within ROM1 and PDE6G. DNA sequencing of all three genes in Rdy-affected cats was used to confirm results from SSCP. For both ROM1 and PDE6G polymorphisms identified by SSCP and sequencing showed disconcordance between the polymorphism and the disease phenotype within an Rdy disease pedigree. SSCP analysis of RHO performed across the 5' untranslated region, the entire coding sequence and the intron/exon boundaries in Rdy-affected and control cats failed to identify any intragenic polymorphisms that could be used for linkage analysis. DNA sequencing of these regions showed no differences between Rdy-affected and control cats. Mutations in ROM1 or in PDE6G are not causative of feline Rdy. The absence of potentially pathogenic polymorphisms in sequenced portions of the RHO gene makes it unlikely that a mutation in this gene is the cause of Rdy.     

Genomic organization and mutational analysis of KVLQT1, a gene responsible for familial long QT syndrome

To elucidate the role of the KVLQT1 gene in the pathogenesis of long QT syndrome (LQTS), we have established a screening system for detecting KVLQT1 mutations by the polymerase chain reaction-single strand conformation polymorphism technique (PCR-SSCP). We first determined exon/intron boundaries and flanking intronic sequences, and found that the KVLQT1 gene consists of 17 coding exons. Subsequently, we synthesized oligonucleotide primers to cover the coding region and the flanking intronic sequences, and searched for mutations in 31 Japanese LQTS families. When genomic DNA from each proband was examined by PCR-SSCP followed by direct DNA sequencing, mutations were detected in five families; two independent families carried the same mutation and three of the four were novel. Each mutation was present in affected relatives of the respective proband. This work will enable us to search more thoroughly for LQTS mutations associated with KVLQT1, and eventually will help us in finding genotype/phenotype relationships. Received: 20 March 1998 / Accepted: 30 April 1998