Rapid detection of exon 1 NRAS gene mutations using universal heteroduplex generator technology.

Specific NRAS oncogene missense mutations have been frequently found in some tumors and several hematological diseases, especially in those of myeloid origin. There is a wide range of PCR-based methods for screening and detection of NRAS exon 1 single-base substitutions. However, there are disadvantages and ambiguities associated with these techniques because all of them require either separate probes, separate PCR amplifications, or complicated post-PCR manipulations. This report describes a new approach for detection of NRAS gene mutations at codon 12 and 13 based on the DNA heteroduplex analysis method. The strategy relies upon differential electrophoretic behavior of induced heteroduplex molecules formed by cross-hybridization of two PCR-amplified species, the sample under analysis and the synthetic universal heteroduplex generator (UHG). The screening of a panel of all codon 12 and 13 NRAS mutant DNA variants indicated that this approach discriminates all 12 relevant mutations. The sensitivity of the method was estimated by a competitive assay where mutant alleles could be detected at a dilution level of 1 to 16 wild-type alleles. This UHG technology was tested on some clinical samples previously studied by PCR-ASO. This methodology is highly specific, sensitive, and achieves an appreciable reduction in workload and time because it requires one PCR amplification followed by polyacrylamide gel electrophoresis in standard conditions. We propose that this new approach may be applied as an alternative strategy for codon 12-13 NRAS mutations and it could be easily incorporated into the range of routine assays performed in oncology laboratories.

Frameshift mutations in the type VII collagen gene (COL7A1) in five Mexican cousins with recessive dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the type VII collagen gene (COL7A1). In this study, we assessed the molecular basis of recessive DEB in five affected individuals from two Mexican families. Both fathers of the affected children were first cousins. Genomic DNA was extracted from peripheral blood samples and assessed for COL7A1 mutations by polymerase chain reaction (PCR) amplification, heteroduplex analysis and direct automated sequencing of PCR products displaying heteroduplex bandshifts. In one family, we identified a homozygous 1 bp insertion of a G nucleotide in exon 19 of COL7A1, designated 2470insG, in three affected sisters. This mutation causes a frameshift and a premature termination codon on both alleles 178 bp downstream from the insertion; both parents were shown to be heterozygous carriers of this mutation. In the second family, the father of the other two affected children was also found to be a heterozygous carrier of this frameshift mutation. In addition, his unrelated partner was shown to be a heterozygous carrier of a different COL7A1 frameshift mutation, an insertion of a T nucleotide in exon 32, designated 3948insT. This mutation also results in a premature termination codon, 126 bp downstream from the insertion. Both affected children were compound heterozygotes for the 2470insG/3948insT mutations in COL7A1. Overall, these molecular findings offer a genetic explanation for the skin fragility in these related Mexican patients with recessive DEB. Immediate benefits from elucidation of the mutations include assessment of carrier status in other members of the family and the feasibility of DNA-based prenatal testing in subsequent pregnancies.

HIV-1 detection and subtyping by PCR and heteroduplex mobility assay in blood donors: can these tests help to elucidate conflicting serological results?

Testing blood donors for the human immunodeficiency viruses (HIV-1 and 2), requires serological tests that are frequently inconclusive. Peripheral blood mononuclear cells of 50 blood donors with the screening test (ELISA) reactive to HIV-1, but with indeterminate results in the first Western Blot (WB) performed, were submitted to the polymerase chain reaction (PCR) and heteroduplex mobility assay (HMA), a nonsequencing method that can distinguish between HIV-1 subtypes (A to I). PCR amplification of HIV-1 env gene regions has been obtained in 12 (24.0%) samples. HMA of the amplified DNA showed that 12 belonged to HIV-1 B subtype and one to F subtype. PCR testing of the amplified DNA helped to elucidate doubtful serological results and HMA proved to be a relatively simple and rapid way of subtyping HIV-1 for epidemiological purposes.

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.

PCR haplotypes for the human Y chromosome based on alphoid satellite DNA variants and heteroduplex analysis.

We have developed a system for revealing informative and useful haplotypes for the human Y chromosome using PCR. Variant alphoid satellite DNA subunits were amplified and analysed by digestion with HindIII to score a restriction site polymorphism, or on polyacrylamide gels to reveal 13 heteroduplex haplotypes. Heteroduplexes are double-stranded DNA molecules containing mismatches; the haplotype is the combination of alleles on the same chromosome. Structural studies showed that the heteroduplexes analysed here were formed from loci at the left (short arm) and right (long arm) edges of the centromeric alphoid array which differed by a 4-bp insertion/deletion and several point mutations. Consequently, many haplotypes may have arisen only once and are useful for evolutionary studies.

Rapid genetic characterization of HIV type 1 strains from four World Health Organization-sponsored vaccine evaluation sites using a heteroduplex mobility assay. WHO Network for HIV Isolation and Characterization.

To assist in the preparation for the testing of vaccines against human immunodeficiency virus (HIV) we, as part of the World Health Organization Network for HIV Isolation and Characterization (WHO-NHIC), evaluated the genotypic variation of HIV-1 in cohorts from Brazil, Rwanda, Thailand, and Uganda. Here we report the results from a pilot study of 65 HIV-1-infected individuals. In all cases in which viral envelope gene fragments could be amplified by polymerase chain reaction, subtypes could be assigned using a heteroduplex mobility assay (HMA)1 by comparison with HIV-1 strains representing six HIV-1 envelope subtypes. All subtype classifications matched those found by envelope gene sequencing. Phylogenetic relationships were further clarified by heteroduplex formation between samples within each subtype. A relatively homogeneous subtype E virus population predominated over subtype B viruses in the sample set from Thailand. Viruses from the other countries were also limited to one or two subtypes but were more divergent within each subtype. All samples from Rwanda (13/13) and some from Uganda (3/16) were of subtype A; all Brazilian samples were of subtype B, except for one belonging to subtype C; most samples from Uganda (13/16) clustered with the subtype D. Analysis by HMA is therefore applicable for screening of HIV-1 genotypes in countries under consideration for large-scale vaccine trials. It should be generally useful when samples containing at least one variable genetic locus need to be rapidly classified by genotype and/or analyzed for epidemiological clustering.

Genetic analysis of HIV-1 isolates from Brazil reveals presence of two distinct genetic subtypes.

The spread of the human immunodeficiency virus type 1 (HIV-1) is by now virtually worldwide. An understanding of the genetic, biological, and immunological differences among isolates collected in different geographic locales is crucial for the development of globally effective vaccines. Here we report the genetic characteristics of 21 HIV-1 isolates from Brazil. The isolates were initially characterized using a heteroduplex mobility assay. The majority (17 of 21) were related to North American/European reference isolates of genetic subtype B. Four isolates belonged to a more recently identified genotype, termed subtype F. The subtype F sequences from Brazil are distinguishable in both gag and env from five other genetic subtypes of HIV-1 currently recognized. Like many locales, Brazil harbors more than one HIV-1 subtype.

PIP: The spread of the human immunodeficiency virus type 1 (HIV-1) is by now virtually worldwide. An understanding of the genetic, biological, and immunological differences among isolates collected in different geographic locales is crucial for the development of globally effective vaccines. The genetic characteristics of HIV-1 isolates from whole blood samples of 21 HIV-1-seropositive Brazilian patients collected during 1989 and 1990 are reported. Virus was isolated by cocultivation of patient peripheral blood mononuclear cells (PBMCs) with phytohemagglutinin (PHA)-stimulated donor PBMCs. The isolates were initially characterized using a heteroduplex mobility assay, which estimates the DNA sequence homology of a selected genomic region from different HIV-1 isolated from the migration of heteroduplexes in polyacrylamide gels. Five distinct HIV-1 envelope subtypes were identified, and a sixth subtype, termed F, was identified using isolates from Brazil and Romania. One Brazilian isolate was identified as subtype B by the more rapid relative migration of heteroduplexes. The majority (17 of 21) were related to North American/European reference isolates of genetic subtype B, whereas 4 isolates belonged to subtype F, a more recently identified genotype. The gag and env genes of several Brazilian isolates belonging to subtypes B and F were cloned and sequenced to allow a detailed analysis of their phylogenic relationships. This further established the existence of two distinct and well-separated genetic subtypes among Brazilian HIV-1 isolates. The subtype F sequences from Brazil are distinguishable in both gag and env from 5 other genetic subtypes of HIV-1 currently recognized. Like many locales, Brazil harbors more than one HIV-1 subtype.