Direct detection of point mutations by mismatch analysis: application to haemophilia B

Montandon, A. J.; Green, P.M.; Giannelli, F.; Bently, D.R.

doi:10.1093/nar/17.9.3347

Cited by 153 publications

(59 citation statements)

References 32 publications

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“…Recently, an allele-specific PCR (ASPCR) amplification technique to diagnose point mutations has also been introduced (18). Some of the above techniques do not detect all mutations that involve single nucleotides and are technically quite demanding (12)(13)(14)(15)(16). Others require optimization of conditions that allows specific hybridization of the ASO probe (9) or specific amplification of the selected allele by ASPCR (19).…”

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confidence: 99%

Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes.

Kuppuswamy

Hoffmann

Kasper

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

153

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In this report, we describe an approach to detect the presence of abnormal alleles in those genetic diseases in which frequency of occurrence of the same mutation is high (e.g., cystic fibrosis and sickle cell disease), and in others in which multiple mutations cause the disease and the sequence variation in an affected member of a given family is known (e.g., hemophilia B). Initially, from each subject, the DNA fragment containing the putative mutation site is amplified by the polymerase chain reaction. For each fragment two reaction mixtures are then prepared. Each contains the amplified fragment, a primer (18-mer or longer) whose sequence is identical to the coding sequence of the normal gene immediately flanking the 5' end of the mutation site, and either an a-32P-labeled nucleotide corresponding to the normal coding sequence at the mutation site or an a-32P-labeled nucleotide corresponding to the mutant sequence. Single nucleotide primer extensions are then carried out and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. As predicted by the Watson-Crick base-pair rule, in the wild type only the normal base, in an affected member only the mutant base, and in carriers both the normal and the mutant base are incorporated into the primer. Thus, an essential feature of the present methodology is that the base immediately 3' to the template-bound primer is one of those altered in the mutant, since in this way an extension of the primer by a single base will give an extended molecule characteristic of either the mutant or the wild type. The method is rapid and should be useful in carrier detection and prenatal diagnosis of every genetic disease with a known sequence variation.One goal of molecular biology is to identify the mutations that cause genetic diseases and to develop strategies and related technologies to diagnose them. Toward this end, in the past decade or so many methodological advances have been made to detect the human genetic abnormalities at the DNA level. These include indirect methods such as linkage analysis by the Southern blotting technique (1) in which the inheritance of a disorder is associated with the presence of a restriction fragment length polymorphism (RFLP)-e.g., Duchenne muscular dystrophy (2). Other indirect methods include RNase A cleavage at mismatches in probe RNA-sample DNA duplexes or denaturing gradient gel electrophoresis for mismatches in probe DNA-sample DNA duplexes-e.g., 3-thalassemia (3, 4). The direct methods include detection with the restriction enzymes or with the allele-specific oligonucleotide (ASO) probes-e.g., the sickle cell mutation (5, 6).A majority of the above approaches have now been combined with the polymerase chain reaction (PCR) for diagnosis of the sequence variations (7,8). Initially, the target DNA is amplified by PCR followed by analysis of the sequence variation by ASO hybridization (e.g., the sickle cell mutation; ref. 9), restriction enzyme analysis (e.g., the sickle cell mutation and a hemophilia B mutation;...

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confidence: 99%

Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes.

Kuppuswamy

Hoffmann

Kasper

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

153

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“…Internal radioactive labeling was used in these studies, thus preventing precise localization of the mutation. A wild-type double-stranded probe, radioactively labeled at both ends, was used in other studies to reduce the number of chemical reactions (12). Our procedure differs in important ways from these and other previous approaches.…”

Section: Discussionmentioning

confidence: 99%

Efficient detection of point mutations on color-codedstrands of target DNA.

Verpy

Biasotto

Meo

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Presently available methods for screening large genetic regions for unknown point mutations are neither flawless nor particularly efficient. We describe an approach, especially well suited to identiyng mutations present in the heterozygous state, that combines several improvements in a protocol called fluorescence-assisted mmath analysis (FAMA). Appropriate gene regions of the wild-type and the putative mutant allele are simultaneously amplified from genomic DNA by using the polymerase chain reaction, and large DNA fragments, so far up to 800 bp, are end labeled with strand-specific fluorophores. Aliquots are denatured and reannealed to form heteroduplexes and subjected to conventional cytosne-and thymine-specific modfifications. Cleavages occuring on opposite strands are detected by denatring gel electrophoresis using an automated DNA sequencer. Since the DNA a nts derived from the mutant allele are also end labeled, the number of informative mispaired bases is doubled compared to conventional searches using wild-type probes. The sensiivty of detection is also increased, because differential fluorescent end labeling allows the identification and measurement ofstand-specific background cleavages at matched cytosine or thymine residues. Automatic superimposition of tracings from different subjects allows mia detection at sites that, because of the nature of the bases involved and of the neighboring sequence, are known to be less susceptible to cleavage. The effects of the latter parameters have been studied quantitatively on a series of point mutations found in the human Cl-inhibitor gene in patents affected by hereditary angioedema. Dilution experiments have demonstrated that most mutations are detected even when the mutant chromosome is diluted 10-fold or more compared with the normal one.The identification of loci responsible for a large number of monogenic diseases and of a growing number of genes involved in tumor predisposition and multifactorial diseases has rapidly expanded the range of application of DNA diagnosis. Since the majority of inherited disorders are heterogeneous at the gene level and are more often due to subtle changes affecting one or several nucleotides than to large deletions or duplications, considerable efforts have been made to develop methods able to detect not only mutations previously identified in probands or known to be prevalent in a given population but also as yet unknown mutations (for reviews, see refs. 1-3). Each of the methods has, however, disadvantages that render its use problematic when searching unknown point mutations within large DNA regions. Denaturing gradient gel electrophoresis (4-6) requires, for example, computer-assisted optimization of the target region and of the electrophoresis conditions, depending on the sequence content of the DNA fragment. The single-strand conformation polymorphism technique (7), in spite of its experimental simplicity, has shown limited sensitivity for the detection of mutations in DNA fragments longer than 200 bp (8, 9).Neith...

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“…The method used was based on that of Cotton et al [23] as modified by Montandon et al [24]. Briefly, the PCR product (5 ng) from control cells was end-labelled with [ 32 P]dCTP and mixed with a fivefold or tenfold excess of unlabelled PCR product from the proband.…”

Section: Methodsmentioning

confidence: 99%

“…Heteroduplexes were formed by heating to 100°C for 10 min, then at 65°C for 2 h in 0.3 M NaCl, 0.1 M Tris, pH 8.0, and were recovered by ethanol precipitation. Base mismatches in the heteroduplexes were detected by chemical modification with hydroxylamine or osmium tetroxide and cleavage with piperidine, exactly as described by Montandon et al [24] followed by analysis on a denaturing 5% polyacrylamide gel and autoradiography.…”

Section: Methodsmentioning

confidence: 99%

Preferential pre‐mRNA utilisation of an upstream cryptic 5′ splice site created by a single base deletion mutation in exon 37 of the FBN‐1 gene

Gibson

Ellis

Adès

et al. 1998

European Journal of Biochemistry

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A heterozygous deletion of a single base (A4704) from exon 37 of the fibrillin-1 gene was defined in a patient with Marfan syndrome and subsequently in his previously undiagnosed father. The deletion created a cryptic 5′ splice site in exon 37 which was utilised in preference to the normal 5′ splice site during pre-mRNA processing in skin fibroblasts cultured from the proband. The mutant mRNA showed a 48-bp deletion from the 3′ end of exon 37 which was predicted to restore the reading frame in the mutant mRNA and result in the deletion of a 16-amino-acid sequence from a central eight-cysteine repeat motif of the fibrillin-1 molecule. Interestingly, the cryptic 5′ splice site in exon 37 and the normal 5′ splice site had equally strong consensuses for splice-site selection. The preferential utilisation of the cryptic site is discussed in relation to current theories on the mechanisms involved in pre-mRNA splicing. Analysis by reverse-transcription PCR indicated that, in the patients skin fibroblasts, the steady-state level of the mis-spliced mutant mRNA was close to that from the normal allele. In addition, evidence from immunoblotting and pulse-chase biosynthetic labelling indicated that close to normal amounts of fibrillin-1 were being synthesised and secreted by the cells. However, in contrast to control cells cultured from an unaffected individual, little fibrillin-1 was detected, either biosynthetically or by immunofluorescence, in the extracellular matrix produced by the proband's fibroblasts. Thus, the slightly shorter mutant fibrillin-1 molecules appeared to be exerting a powerful dominant-negative effect on the incorporation of normal fibrillin-1 molecules into microfibrils in this culture system. This severe inhibition of microfibril synthesis in cell culture contrasts with the 'classic' phenotype of the proband, suggesting that factors influencing microfibril formation may differ greatly between in vivo and in vitro environments.Keywords : cryptic splicing ; exon mutation; fibrillin; Marfan syndrome. FBN-1, the gene for fibrillin-1, a 350-kDa glycoprotein component of extracellular microfibrils [1Ϫ3], have been causally linked to the congenital disorder, Marfan syndrome and related phenotypes [4Ϫ6]. Marfan syndrome is a common (about 1 in 10 000 live births) autosomal dominant condition of connective tissue which affects the cardiovascular, skeletal and ocular systems. The syndrome is characterised by a range of abnormalities including overgrowth of the ribs and long bones, distension of the ascending aorta, heart valve defects, arachnodactyly, and lens dislocation. Although penetrance is complete, the severity and range of clinical features are variable, often within the same family. Even individuals with mild skeletal abnormalities can die prematurely, usually from dissecting aneurysm of the aorta [4]. The rate of detection of fibrillin-1 gene (FBN-1) mutations in Marfan patients is currently about 20% of those screened, although higher detection rates have been reported using heteroduplex analysis of...

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Direct detection of point mutations by mismatch analysis: application to haemophilia B

Cited by 153 publications

References 32 publications

Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes.

Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes.

Efficient detection of point mutations on color-codedstrands of target DNA.

Preferential pre‐mRNA utilisation of an upstream cryptic 5′ splice site created by a single base deletion mutation in exon 37 of the FBN‐1 gene

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