Mutation Detection Enhancement Gel Research Articles

Helicobacter pylori Infection Induces Genetic Instability of Nuclear and Mitochondrial DNA in Gastric Cells

Helicobacter pylori is a major cause of gastric carcinoma. To investigate a possible link between bacterial infection and genetic instability of the host genome, we examined the effect of H. pylori infection on known cellular repair pathways in vitro and in vivo. Moreover, various types of genetic instabilities in the nuclear and mitochondrial DNA (mtDNA) were examined. We observed the effects of H. pylori infection on a gastric cell line (AGS), on C57BL/6 mice, and on individuals with chronic gastritis. In AGS cells, the effect of H. pylori infection on base excision repair and mismatch repair (MMR) was analyzed by reverse transcription-PCR, Western blot, and activity assays. In mice, MMR expression was analyzed by reverse transcription-PCR and the CA repeat instabilities were examined by Mutation Detection Enhancement gel electrophoresis. Mutation spectra in AGS cells and chronic gastritis tissue were determined by PCR, single-stranded conformation polymorphism, and sequencing. H. pylori vacA and cagA genotyping was determined by multiplex PCR and reverse hybridization. Following H. pylori infection, the activity and expression of base excision repair and MMR are down-regulated both in vitro and in vivo. Moreover, H. pylori induces genomic instability in nuclear CA repeats in mice and in mtDNA of AGS cells and chronic gastritis tissue, and this effect in mtDNA is associated with bacterial virulence. Our results suggest that H. pylori impairs central DNA repair mechanisms, inducing a transient mutator phenotype, rendering gastric epithelial cells vulnerable to the accumulation of genetic instability and thus contributing to gastric carcinogenesis in infected individuals.

A Variant in XPNPEP2 Is Associated with Angioedema Induced by Angiotensin I–Converting Enzyme Inhibitors

Angiotensin I-converting enzyme inhibitors (ACEi), which are used to treat common cardiovascular diseases, are associated with a potentially life-threatening adverse reaction known as angioedema (AE-ACEi). We have previously documented a significant association between AE-ACEi and low plasma aminopeptidase P (APP) activity. With eight large pedigrees, we hereby demonstrate that this quantitative trait is partially regulated by genetic factors. We tested APP activity using a variance-component QTL analysis of a 10-cM genomewide microsatellite scan enriched with seven markers over two candidate regions. We found significant linkage (LOD = 3.75) to a locus that includes the XPNPEP2 candidate gene encoding membrane-bound APP. Mutation screening of this QTL identified a large coding deletion segregating in one pedigree and an upstream single-nucleotide polymorphism (C-2399A SNP), which segregates in the remaining seven pedigrees. Measured genotype analysis strongly suggests that the linkage signal for APP activity at this locus is accounted for predominantly by the SNP association. In a separate case-control study (20 cases and 60 controls), we found significant association of this SNP to ACEi-induced AE (P=.0364). In conclusion, our findings provide supporting evidence that the C-2399A variant in XPNPEP2 is associated with reduced APP activity and a higher incidence of AE-ACEi.

Molecular characterization of histidinemia: identification of four missense mutations in the histidase gene

Histidinemia (MIM235800) is characterized by elevated histidine in body fluids and decreased urocanic acid in blood and skin and results from histidase (histidine ammonia lyase, EC 4.3.1.3) deficiency. It is the most frequent inborn metabolic error in Japan. Although the original description included mental retardation and speech impairment, neonatal screening programs have identified the majority of histidinemic patients with normal intelligence. Molecular characteristics of histidase in histidinemia have not been determined, and cytogenetically visible deletions of 12q22-24.1 in which histidase gene resides have not been identified in histidinemic patients. In order to investigate whether individuals with this disorder have small deletions, additions, or point mutations in the histidase gene, we screened genomic DNA isolated from 50 histidinemic individuals who were discovered by the neonatal screening program. The methods employed included polymerase chain reaction (PCR) amplification of exons 1-21 of the histidase gene, followed by mutation detection enhancement gel electrophoresis and sequencing of the PCR products displaying heteroduplex bands. Four missense mutations (R322P, P259L, R206T, and R208L), two exonic polymorphisms (T141T c.423A-->T and P259P c.777A-->G), and two intronic polymorphisms (IVS6-5T-->C and IVS9+25A-->G) were identified. The frequencies of each polymorphism estimated either by dot blot allele-specific oligonucleotide hybridization, restriction enzyme digestion, or direct sequencing of the PCR products amplified from 50 unrelated normal individuals were 0.28, 0.30, 0.40, and less than 0.01, respectively. Mutation analysis of one family demonstrated that the patient inherited R322P from the mother and P259L from the father. This report describes the first mutations occurring in the coding region of the histidase structural gene in patients with histidinemia.

A novel connexin 26 compound heterozygous mutation results in deafness.

Mutations of the gap junction beta 2 (GJB2) gene coding for the protein connexin 26 account for up to 50% of nonsyndromic sensorineural hearing loss (NSHL), with specific mutations associated with distinct ethnic groups. A biracial family with nonsyndromic sensorineural deafness consistent with autosomal recessive inheritance was examined for connexin 26 (Cx26) mutations. Prospective observational study. A family consisting of a Caucasian mother and a Chinese father with two of six children affected by NSHL was examined for Cx26 mutations. Peripheral blood lymphocyte DNA was used to amplify by polymerase chain reaction the Cx26 coding region, followed by mutation detection enhancement gel screening and complete sequencing. Phenotypic characterization using audiometric testing was completed for all children and both parents. The two affected children were found to be compound heterozygotes for Cx26 mutations, displaying a previously unreported combination of 35delG and 235delC. The parents were each unaffected heterozygotes consistent with their ethnic heritage, specifically, the Caucasian mother a 35delG heterozygote and the Chinese father a 235delC heterozygote. Connexin 26 mutations account for a significant proportion of NSHL worldwide, with specific mutations linked to distinct ethnic groups. Genetic analysis of a biracial family with NSHL revealed a novel 35delG/235delC compound heterozygous state in phenotypically affected children. These results highlight the usefulness of Cx26 mutation screening for genetic counseling and suggest that the 235delC mutation is present in China as it is in Japan and Korea.

Rapid Detection of FGFR Mutations in Syndromic Craniosynostosis by Temporal Temperature Gradient Gel Electrophoresis

Several autosomal dominant craniosynostosis syndromes, including Crouzon, Pfeiffer, Apert, and Jackson-Weiss syndromes, are caused by mutations in the fibroblast growth factor receptor (FGFR) gene family (1). Mutations are not uniformly dispersed among these genes, and most mutations described to date are in exons IIIa and IIIc of the FGFR2 gene (2)(3). The advent of molecular analysis of these genes has allowed confirmatory testing in ambiguous cases and for those families desiring prenatal diagnosis. However, molecular analysis is difficult at present, especially for Crouzon syndrome, where current methods rely on the sequencing of the aforementioned exons. We report the rapid and inexpensive detection of FGFR mutations by a relatively novel screening assay, temporal temperature gradient gel electrophoresis (TTGE). TTGE is a heteroduplex detection method similar to denaturing gradient gel electrophoresis (DGGE). Like DGGE, but often unlike single-strand conformation polymorphism and heteroduplex analysis using mutation detection enhancement gels, TTGE is highly sensitive (4)(5)(6). However, TTGE is simpler than DGGE in avoiding the use of a chemical denaturing gradient gel and GC clamps (4). In TTGE, a homogeneous gel is bathed in a tank of buffer in which the temperature of the entire unit increases linearly throughout the electrophoretic run (5). After PCR, the products are heated and allowed to cool gradually, which leads to the formation of heteroduplex DNA if sequence heterogeneity is present. In dominant diseases, such as FGFR mutations that cause craniosynostosis syndromes, the sequence difference between the two alleles in the heteroduplex causes a physical bulge at the site of the sequence mismatch, …

Genotyping Taenia tapeworms by single-strand conformation polymorphism of mitochondrial DNA.

To overcome limitations in identifying tapeworms of the genus Taenia by traditional approaches, we have established a single-strand conformation polymorphism (SSCP) method utilizing two different regions of mitochondrial (mt) DNA as targets. The NADH dehydrogenase 1 and the cytochrome c oxidase subunit I genes were amplified from genomic DNA by polymerase chain reaction (PCR), denatured and subjected to electrophoresis in mutation detection enhancement gels. SSCP analysis achieved delineation among eight different species of Taenia from different hosts based on characteristic profiles and enabled the detection of intraspecific variability in profiles for some taxa. This SSCP-based typing method has important implications for taxonomy, diagnosis and for studying the genetic structure of Taenia populations.

Simultaneous detection of size and sequence polymorphisms in the transcribed trinucleotide repeat D2S196E (EST00493).

Long expansions of transcribed trinucleotide microsatellites have been etiologically associated with some neurological diseases. The investigation of such novel polymorphisms has thus become a subject of great interest. We searched the expressed sequence tag databank for reiterated trinucleotides and selected EST00493 (D2S196E) with 14 tandem ACA triplets as a potentially polymorphic locus. Size variation was readily detected, with four common alleles containing 12-15 repeats. In addition, we observed distinct heteroduplexes in amplifications from individuals with identical ACA genotypes. Sequencing of their polymerase chain reaction (PCR) products revealed a G-->A transition immediately preceding the trinucleotide repeats, hence defining 8 distinct haplotypes and 36 possible genotypes. Indeed, mutation detection enhancement gel electrophoresis of mixed PCR products from cloned haplotypes revealed 24 distinct heteroduplex patterns for the six possible trinucleotide heterozygotes. The observation of heteroduplex patterns in non-denaturing polyacrylamide gel electrophoresis (instead of the more commonly used denaturing gels) can thus be utilized to increase the informativeness of microsatellite polymorphisms by unraveling otherwise cryptic sequence variation. The D2S196E polymorphism has proved useful for demonstrating microsatellite instability and loss of heterozygosity in colorectal tumors.

Rapid detection of sequence polymorphisms in the human mitochondrial DNA control region by polymerase chain reaction and single-strand conformation analysis in mutation detection enhancement gels.

The article describes a rapid approach for the detection of sequence polymorphisms in the mitochondrial (mt)DNA control region that involves enzymatic amplification of each entire mtDNA control region (HV1 and HV2) and the subsequent analysis of the PCR products by single-strand conformation analysis (SSCA) in mutation detection enhancement (MDE) gels, followed by silver stain detection. HV1 and HV2 SSC reference ladders were developed to standardize the classification of the different mtDNA types. Twenty-five mtDNA types were observed among the 45 Spanish individuals analyzed: 11 types were observed in the HV1 region as compared with 10 types in the HV2 region. This mutation scanning strategy could be a promising method of potential use not only in forensic genetics but also in population and evolutionary studies.

Single Base Polymorphism Linked to the Ataxia-Telangiectasia Locus Is Detected by Mismatch PCR

A polymorphic site at D11S384, which shows zero recombination with the ataxia-telangiectasia (A-T) locus, was originally detected by Mutation Detection Enhancement Gel and Denaturing Gradient Gel Electrophoresis. In order to increase the throughput and decrease the complexity of the assays, we developed a site directed mutagenesis using primers with mismatched 3′ends, followed by restriction digestion, as a rapid, nonradioactive method for detecting polymorphisms/mutations.

Prenatal diagnosis in a family with mitochondrial acetoacetyl-coenzyme A thiolase deficiency with the use of the polymerase chain reaction followed by the heteroduplex detection method.

Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase deficiency is an organic aciduria which affects isoleucine and ketone body catabolism. GK16 (the index patient) was affected with this disorder and previous studies had revealed that GK16 was a compound heterozygote with IVS8(+1) gt to tt and A301P mutations. In a subsequent pregnancy, prenatal diagnosis was performed and the fetus's amniocytes were analysed by the polymerase chain reaction (PCR) followed by the heteroduplex detection method on a Mutation Detection Enhancement gel. The fetus was identified as a carrier of the IVS8(+1) mutation. We confirmed the diagnosis by immunoblot analysis of extracted amniocytes and gene analysis with blood filter paper after delivery. This is the first report of prenatal diagnosis of this disorder at the gene level.

The electrocardiogram is a more sensitive indicator than echocardiography of hypertrophic cardiomyopathy in families with a mutation in the MYH7 gene.

Mutations in the cardiac beta myosin heavy chain gene causing hypertrophic cardiomyopathy have been identified, and to assist both diagnosis and prediction of outcome attempts have been made to correlate phenotype and genotype. Two new mutations in codon 403 of the gene in three unrelated families are described and attention drawn to variable or even absent phenotypic expression in different family members. The polymerase chain reaction and heteroduplex analysis on Mutation Detection Enhancement gels were used to search for mutations in the globular head of the beta myosin heavy chain gene in families with hypertrophic cardiomyopathy. Two mutations were found in exon 13 (codon 403) of the gene. In two unrelated Polish families the mutation resulted in the conversion of arginine to tryptophan (CGG: >TGG). A second mutation, found in a British family, converted the same arginine to leucine (CGG: >CTG). These mutations were detected in family members who had electrocardiographic and echocardiographic features typical of hypertrophic cardiomyopathy; however, they were also detected in 7 other adult relatives with an abnormal electrocardiogram but a normal echocardiogram. Two unrelated adult relatives had completely normal clinical findings but carried the gene mutation. Identification of a specific mutation gives no guide to the clinical phenotype. There is considerable variability in the phenotypic expression of hypertrophic cardiomyopathy. Mutations were detected in adults previously regarded as normal or in whom the diagnosis was questionable. The fact that the clinical significance of the mutation in these people is still unknown emphasises the dilemma facing screening programmes. Isolated, unexplained electrocardiographic abnormalities in first degree relatives in a family with a definitive diagnosis of hypertrophic cardiomyopathy should be regarded as evidence of a carrier state.

Identification of a Nonsense Mutation in ALD Protein cDNA from a Patient with Adrenoleukodystrophy

The molecular basis of X-linked adrenoleukodystrophy (ALD) was investigated. Six (A to F) fragments of cDNA for ALD protein (Mosser et al. Nature 361 : 726-730, 1993) from an adult patient with adrenomyeloneuropathy were amplified by PCR and mutations were screened by Mutation Detection Enhancement gel electrophoresis. A single base substitution (2154 C → T), which resulted in the formation of a termination codon for glutamine (Q590STOP) and deletes Pst I site (CTGCAG → CTGTAG), was detected. Eight other ALD patients did not have this mutation. A family study revealed the presence of both the mutant and normal alleles in the mother, a sister and a niece, indicating that these individuals were carriers. A nephew with childhood ALD who died 10 years earlier had the same mutant allele as detected by Pst I restriction assay. This report is the first description of a mutant allele for ALD, at the cDNA level, and presents confirmatory evidence of ALD protein as the primary etiology of ALD

Mutations of the chloramphenicol acetyl transferase transgene driven by the immunoglobulin promoter and intron enhancer.

Transgenic mice containing the chloramphenicol acetyl transferase (CAT) gene driven by the immunoglobulin (Ig) VH promoter and the heavy chain intron enhancer were prepared to increase our understanding of the mechanism responsible for somatic mutation. The transgene showed similar tissue specificity in terms of expression as endogenous Ig genes. Hybridomas were prepared after multiple immunization of a transgenic mouse with (4-hydroxy-3-nitrophenyl)acetyl-chicken gammaglobulin, a thymus-dependent antigen. We analyzed mutations in the CAT coding region as well as in the region 5' upstream of the promoter after amplification of DNA using the PCR followed by sequencing of cloned DNA. Mutation detection enhancement gel electrophoresis was also used to detect mutations. Only a single band was observed in PCR products from the region 5' upstream of the promoter and from the enhancer, whereas in those from the CAT coding region, three out of 11 hybridomas showed multiple bands. In DNA sequences of the CAT coding region from these three hybridomas, a total of six nucleotide substitutions were observed, but none in the region 5' to the promoter. The results of DNA sequencing and the electrophoresis were in good agreement, suggesting that mutation occurred only in the CAT gene but not in the region 5' to the promoter or the enhancer. The rate of mutation in the CAT gene was estimated to be 1.6 x 10(-5) per base pair per cell division, which was a lower limit of the rate for somatic mutation reported for Ig genes. Location and distribution of mutations were similar to that of the Ig gene, suggesting that (i) mutation in the CAT gene was induced by a mechanism similar to that occurring in the Ig gene and (ii) the mutator mechanism can operate in a non-Ig gene provided it is flanked by the VH promoter and heavy chain intron enhancer.