Allele-specific PCR Amplification Research Articles

Direct analysis of R408W mutation in phenylalanine hydroxylase gene by allele-specific PCR amplification.

Journal Article Direct analysis of R408W mutation in phenylalanine hydroxylase gene by allele-specific PCR amplification Get access Dana Dvořáková, Dana Dvořáková * Department of Biochemical and Molecular Genetics, Pediatric Research InstituteČernopolní 9, CS-66262 Brno, Czechoslovakia *To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Lenka Fajkusová Lenka Fajkusová Department of Biochemical and Molecular Genetics, Pediatric Research InstituteČernopolní 9, CS-66262 Brno, Czechoslovakia Search for other works by this author on: Oxford Academic PubMed Google Scholar Human Molecular Genetics, Volume 2, Issue 3, March 1993, Page 323, https://doi.org/10.1093/hmg/2.3.323 Published: 01 March 1993 Article history Received: 23 November 1992 Revision received: 23 December 1992 Accepted: 23 December 1992 Published: 01 March 1993

Identification of a leucine-to-proline mutation in the keratin 5 gene in a family with the generalized Köbner type of epidermolysis bullosa simplex.

We have previously reported linkage of a large Finnish family with the generalized (Köbner) type of epidermolysis bullosa simplex to chromosome 12q in the region containing the type II keratin gene cluster (Ryynänen et al., Am J Human Genet 49:978-984, 1991). In this study, we examined the possibility that keratin 5, the type II keratin expressed in the basal keratinocytes, harbors the mutation in this family. Nucleotide sequencing revealed a T-to-C transition within exon 7 of the keratin 5 gene in the affected individuals of the family, while the unaffected individuals showed no evidence of C. The presence of the T-to-C transition in the affected individuals was confirmed by restriction enzyme digestion analysis with NciI endonuclease, as well as with PCR amplification of specific alleles (PASA) analysis. The PASA analysis also indicated that the mutated allele was not found among the 100 alleles tested within the general Finnish population indicating that the mutated allele is not a common polymorphism. Furthermore, the mutated allele was not present in nine individuals representing three different EBS families of Finnish origin. The T-to-C transition at the nucleotide level resulted in substitution of a leucine by a proline at the amino acid level, and the substitution affected a leucine residue which was invariant among eight different human keratins in a highly conserved segment at the carboxy-terminal region of the keratin 5 polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

A single-site mutation in the XPAC gene alters photoproduct recognition.

The XPAC (xeroderma pigmentosum group A complementing) gene, which is located on chromosome 9, carries a variety of point mutations in XP group A patients. We investigated the role of the XPAC gene product in excision repair by generating revertants of an XP group A cell line (XP12RO) that have increased resistance to ultraviolet light. One of these cell lines, XP129, can repair (6-4) pyrimidine-pyrimidone photoproducts normally but has reduced repair of cyclobutane dimers, as in XP12RO. Sequence analysis of cDNA from the XPAC gene indicated that XP12RO contains a termination codon at amino acid position 207, resulting in a reduced amount of mRNA and no detectable protein. In the revertant XP129 line, this termination codon has been mutated further and now encodes glycine in one allele instead of the wild-type arginine. The mRNA level detected by allele-specific polymerase chain reaction amplification was greater for the reverted sequence than for the chain-terminating sequence. These observations indicated that a point mutation resulting in a mis-sense mutation in the XPAC gene and altered expression of the XPAC protein can alter the substrate specificity of the excision repair system, and imply that the XPAC gene product plays an important role in photoproduct recognition.

Non-radioactive detection of the most common mutations in the cystic fibrosis transmembrane conductance regulator gene by multiplex allele-specific polymerase chain reaction.

A rapid, simple, nonradioactive method for detection of four common mutations causing cystic fibrosis (CF) has been developed combining multiplexing with allele-specific polymerase chain reaction amplification. This approach (MASPCR) provides an easy assay for direct genotyping of normal and mutant CF alleles in homozygotes and heterozygotes. The strategy involves multiplex PCR of exons 10, 11, and 21 within the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a single reaction containing three common oligoprimers and either the four normal or four mutant oligos corresponding to the delta F508, G551D, G542X, and N1303K mutations. Primers are chosen so that the size of the four PCR products differ, thereby facilitating detection on agarose gels following amplification in the same reaction. Patient samples are primed with either four normal or four mutant oligo mixtures, and PCR products run in parallel on gels to detect band presence or absence. This approach provides a simple and potentially automated method for cost-effective population screening.

Detection of a new polymorphism of the human prothrombin (F2) gene by combination of PASA and mutated primer-mediated PCR-RFLP.

A new polymorphism of the human prothrombin (F2) gene was detected by a combination of polymerase chain reaction (PCR) amplification of specific alleles (PASA) and mutated primer-mediated PCR restriction fragment length polymorphism (PCR-RFLP). The method is simple and useful for detecting polymorphisms and mutations. The new polymorphism of C1 and C2 examined by this method is highly heterozygous and serves as a good human DNA marker.

Allele-specific and asymmetric polymerase chain reaction amplification in combination: A one step polymerase chain reaction protocol for rapid diagnosis of familial defective apolipoprotein B-100

We have combined the asymetric polymerase chain reaction (PCR) with allele-specific PCR to detect a single point mutation. A set of two priming oligonucleotides and a third allele-specific primer were used to identify heterozygotes for a G to A mutation at nucleotide 10,708 in the apolipoprotein B (apo B) gene. The system requires neither restriction enzyme digestion nor allele-specific oligonucleotides as conventionally applied for allele-specific hybridization of slot blots. This method clearly allows for the detection of the mutant allele by inspection, after agarose gel electrophoresis of a single PCR reaction. DNA from 40 patients with familial defective apo B-100 due to the G to A mutation at nucleotide 10,708 in the apo B gene and their normal relatives was analyzed. Complete agreement with allele-specific hybridization of slot blots confirms supposition that the system is effective to screen a larger population.

PCR Amplification of Specific Alleles

PCR Amplification of Specific Alleles

Analysis of the CYP2D6 gene in relation to debrisoquin and desipramine hydroxylation in a Swedish population.

The molecular basis of polymorphic debrisoquin hydroxylation was studied in 223 Swedish white subjects, 187 extensive metabolizers and 36 poor metabolizers phenotyped with debrisoquin and desipramine. Restriction fragment length polymorphism (RFLP) analysis of the CYP2D6 gene revealed that 52% of unrelated poor metabolizers were homozygous for Xba I 29 kb fragment, and only 8% had two mutant alleles detected with RFLP. Allele-specific polymerase chain reaction (PCR)-based DNA amplification, however, revealed that all but one of the poor metabolizers had two mutant alleles of the CYP2D6A or CYP2D6B type or both. Extensive metabolizers who were heterozygous for wild-type and CYP2D6B genes had metabolic ratios for debrisoquin and desipramine that were higher than those of subjects who were homozygous for the wild-type gene. The 16 + 9 kb Xba I RFLP pattern was associated with the poor metabolizer phenotype and CYP2D6B mutations. Three extremely rapid metabolizers of debrisoquin had a 44 kb Xba I fragment that did not carry either CYP2D6A or CYP2D6B mutations. In conclusion, in the Swedish population studied, allele-specific PCR amplification allowed prediction of the debrisoquin hydroxylation phenotype with 99% accuracy.

Polymorphisms of N-acetyltransferase genes.

1. A genetic polymorphism of human liver arylamine N-acetyltransferase (NAT) enzyme activity leads to wide variation in the disposition of many drugs and potential carcinogens, resulting in differential susceptibility to chemical-induced toxicity. 2. During studies to determine the biochemical and molecular mechanisms underlying this pharmacogenetic defect, we cloned two human genes, NAT1 and NAT2, which encode the functional acetylating enzymes NAT1 and NAT2. 3. NAT1 and NAT2 are both expressed in human liver cytosol, the latter as two closely related isoforms NAT2A and NAT2B. 4. NAT2 gene locus is the site of the human acetylation polymorphism, because its products NAT2A and NAT2B selectively acetylate 'polymorphic' arylamine substrates (e.g. sulphamethazine), and since the liver content of these isozymes is markedly reduced in genetically slow acetylator subjects. 5. NAT1 shows marked kinetic selectivity for 'monomorphic' substrates (e.g. p-aminobenzoic acid) whose in vivo acetylation rates do not correlate with the acetylation polymorphism. 6. Despite the drastic reduction in NAT2A/B proteins in livers from phenotypically slow acetylators, levels of the NAT2 gene transcript are not altered. 7. Three common mutant alleles at the NAT2 gene locus have so far been identified, which may be detected by restriction fragment length polymorphism (RFLP) analysis on Southern blots or by allele-specific polymerase chain reaction amplification.

Identification of mutations in two families with sporadic hemophilia A

Direct sequencing of segments of the factor VIII gene in 30 hemophiliacs with sporadic disease (32+ kb of sequence in total) revealed two missense transitions: glutamate 1704 to lysine (E1704----K) in a patient with severe hemophilia A and proline 2300 to serine (P2300----S) in a patient with mild hemophilia. Both transitions are likely to be causative mutations because the amino acids affected were evolutionarily conserved. Haplotype and sequence analysis of the mother and grandparents of patient HA12 (E1704----K) indicate that the mutation arose in the grandfather who was 27 years old when his daughter was conceived. The origin of mutation in patient HA39 (P2300----S) could not be determined. As mutations that cause mild disease can be found in seemingly unrelated families, 96 unrelated hemophiliacs were screened rapidly for the P2300----S mutation with polymerase chain reaction (PCR) amplification of specific alleles (PASA). None of these patients had the mutation. PASA was also used to conveniently assess a polymorphic site in intron 7. The polymorphism is estimated to be informative in 13% of Korean females and in 23% of Western European females.

Identification of genetic differences in drug metabolism: Prediction of individual risk of toxicity or cancer Gough AC, Miles JS, Spurr NK, Moss JE, Gaedigk A, Eichelbaum M, Wolf CR. Identification of the primary gene defect at the cytochrome P450 CYP2D locus. Nature 1990;347:773–776. Heim M, Meyer UA. Genotyping of poor metabolisers of debrisoquine by allele-specific PCR amplification. Lancet 1990;336:529–532

Identification of genetic differences in drug metabolism: Prediction of individual risk of toxicity or cancer Gough AC, Miles JS, Spurr NK, Moss JE, Gaedigk A, Eichelbaum M, Wolf CR. Identification of the primary gene defect at the cytochrome P450 CYP2D locus. Nature 1990;347:773–776. Heim M, Meyer UA. Genotyping of poor metabolisers of debrisoquine by allele-specific PCR amplification. Lancet 1990;336:529–532

T296?M, a common mutation causing mild hemophilia B in the Amish and others: founder effect, variability in factor IX activity assays, and rapid carrier detection

By direct genomic sequencing, we have delineated the causative mutation in 64 families of European decent with hemophilia B. Six (9%) had a C----T transition at base 31008, which substitutes methionine for threonine 296 (T296----M) in the catalytic domain of factor IX. Five of the patients had the same haplotype (frequency of 16% in the northern European population). These individuals are of Amish/German descent and they are likely to share a common ancestor. The sixth patient had a different haplotype, which indicates that his mutation had an independent origin. The data highlight the importance of clinical criteria for the classification of hemophilia B. All six patients had clinically mild disease and their factor IX coagulant activities were in the range of 3%-6% when tested simultaneously in one laboratory, yet the factor IX activities provided with patient records varied 40-fold. Due to the high frequency of this mutation, we have utilized the technique of polymerase chain reaction amplification of specific alleles (PASA) to perform rapid and inexpensive carrier diagnoses in the families with this mutation. This is of particular importance for the Amish since the mutation should account for much of, if not all, the mild hemophilia B that is commonly found in this population.

Two‐tiered DNA‐based diagnosis of transthyretin amyloidosis reveals two novel point mutations

We analyzed 11 consecutive unrelated cases of polyneuropathy due to transthyretin amyloidosis. Direct sequencing of the promoter region, exons, and splice junctions revealed that each patient was heterozygous for a mutation: six patients had valine 30 substituted by methionine (V30----M; Portuguese-Japanese type), one had threonine 60 substituted by alanine (T60----A; Appalachian type), and two had serine 77 substituted by tyrosine (S77----Y; Illinois type). In addition, two patients had previously undescribed mutation: phenylalanine 33 substituted by leucine (F33----L) and phenylalanine 64 substituted by leucine (F64----L). From present information, the probands of these novel mutations do not exhibit any pathology that clearly distinguishes them from individuals with the other mutations. The mutations extend the range of mutations associated with amyloidotic polyneuropathy. In our 11 patients, the different mutations did not seem to correlate with distinct clinical phenotypes. We developed PASA assays (PCR amplification of specific alleles) for each of the five mutations. PASA can be used by any diagnostic laboratory that can perform PCR to rapidly detect any of the known mutations. The minority of samples with an undescribed mutation can be sent to a specialty laboratory for delineation of the mutation by direct genomic sequencing. The presently described combination of methods may have widespread utility in the diagnosis of genetic disease.

Oligonucleotide typing of HLA DR by allele-specific PCR amplification: [formula omitted], L Geer, Y Wang, MS Park, and PI Terasaki, UCLA Tissue Typing Laboratory, UCLA School of Medicine, Los Angeles, CA

Oligonucleotide typing of HLA DR by allele-specific PCR amplification: [formula omitted], L Geer, Y Wang, MS Park, and PI Terasaki, UCLA Tissue Typing Laboratory, UCLA School of Medicine, Los Angeles, CA

Genotyping of poor metabolisers of debrisoquine by allele-specific PCR amplification

A method for genotyping poor metabolisers of debrisoquine is based on specific polymerase chain reaction (PCR) amplification of parts of mutant genes for hepatic cytochrome P450IID6. Analysis by restriction fragment length polymorphism allowed identification of only 25% of poor metabolisers, but when it was combined with allele-specific PCR over 95% of poor metabolisers could be identified. The PCR method also allowed the identification of heterozygous carriers of mutant alleles.

A past mutation at Isoleucine397 is now a common cause of moderate/mild haemophilia B

Of the factor IX sequence changes that we have identified in 65 consecutive males with haemophilia B, 11 (17%) are the same mutation. This mutation is a T----C transition at base 31311 which substitutes threonine for isoleucine397 (ile397) in the factor IX molecule. The 11 patients are of Western European descent and have the same haplotype: Hinf1 (-), Xmn1 (-), Taq1 (-), BamH1 (+), Malmö allele = thr148. The frequency of this haplotype was estimated and the probability of the same mutation occurring independently 11 times in this haplotype was miniscule. We conclude that these patients have a common ancestor despite the lack of overlapping pedigrees. The clinical symptoms of the disease were consistently moderate/mild in these 11 patients, whereas factor IX coagulation values obtained from the medical records varied more than sixfold between individuals. However, when plasma from five individuals was assayed by the same laboratory concurrently, the values varied less than twofold. Thus, in routine practice, clinical severity may correlate better with the presence of a given mutation than the factor IX coagulant activity. The high frequency of the mutation at ile397 indicates that carrier testing in families of Northern European descent with moderate/mild haemophilia B can be expedited by first determining whether this particular mutation is present. We demonstrate here that the technique of polymerase chain reaction (PCR) amplification of specific alleles (PASA) can be used to rapidly perform carrier testing in families with the ile397 mutation.

Specific HLA-DQB and HLA-DRB1 alleles confer susceptibility to pemphigus vulgaris.

The autoimmune dermatologic disease pemphigus vulgaris (PV) is associated with the serotypes HLA-DR4 and HLA-DRw6. Based on nucleotide sequence and oligonucleotide probe analysis of enzymatically amplified DNA encoding HLA-DR beta chain (HLA-DRB) and HLA-DQ beta chain (HLA-DQB; henceforth HLA is omitted from designations), we showed previously that the DR4 susceptibility was associated with the Dw10 DRB1 allele [encoding the mixed lymphocyte culture (MLC)-defined Dw10 specificity]. The DRw6 susceptibility similarly was shown to be associated with a rare DQB allele (DQB1.3), which differed from another nonsusceptible allele by only a valine-to-aspartic acid substitution at position 57. Given the linkage disequilibrium that characterizes HLA haplotypes, it is difficult to assign disease susceptibility to a specific locus rather than to a closely linked gene(s) on the same haplotype. To address this problem, we have analyzed all of the polymorphic loci of the class II HLA region (DRB1, DRB3, DQA, DQB, and DPB) on the DRw6 haplotypes in patients and controls. In 22 PV patients, 4 different DRw6 haplotypes were found that encode the same DQ beta chain (DQB1.3) but contained silent nucleotide differences at the DQB locus as well as coding sequence differences in the DQA and DRB loci. These results, obtained by using a method for allele-specific polymerase chain reaction amplification, strongly support the hypothesis that the allele DQB1.3 confers susceptibility. This DQB allele is correlated with the MLC-defined Dw9 specificity and is associated with two different DRB1 alleles (the common "6A" associated with DRw13 and the rare "6B" associated with DRw14). Since 86% (19 of 22) of DRw6+ patients contain the DQB1.3 allele (vs. 3% of controls), whereas 64% (14 of 22) contain the DRB1 allele 6B (vs. 6% of the controls), we conclude that most of the DRw6 susceptibility to PV can be accounted for by the DQ beta chain.