Detection of virtually all mutations-SSCP (DOVAM-S): a rapid method for mutation scanning with virtually 100% sensitivity.

Dideoxy fingerprinting (ddF) was used as a tool to search for a generic set of conditions with sufficient power to detect virtually all mutations. For each condition tested, a very large sample of mutation-containing, single-stranded segments (about 1500) were analyzed with ddF. Correlation coefficients identified pairs of conditions in which single-strand conformation polymorphism (SSCP) mobilities were poorly correlated. The data strongly suggest that tertiary structure (e.g., base-sugar and sugar-sugar interactions) rather than secondary structure is the predominant determinant of mobility shifts by SSCP. Five conditions were selected with sufficient redundancy to detect all the mutations. The sensitivity of detection of virtually all mutations-SSCP (DOVAM-S) was determined by blinded analyses on samples containing additional mutations scattered throughout the eight exons and splice junctions in the factor IX gene. The factor IX gene sequence (2.5 kb) was scanned in one lane by 15 PCR-amplified segments (125 kb of sequence scanned per gel). All of the 84 single-base substitutions were detected in the blinded analyses, the first consisting of 50 hemizygous mutant and wild-type (WT) samples and the second consisting of 50 heterozygous mutant and WT samples. DOVAM-S is estimated to be five times faster than fluorescent DNA sequencing for the detection of virtually all mutations when the five conditions are applied.     

Candidate gene analyses by scanning or brute force fluorescent sequencing: a comparison of DOVAM-S with gel-based and capillary-based sequencing

For epidemiological and diagnostic applications, detection of virtually all mutations is desired. Herein, blinded analyses of DOVAM-S (Detection Of Virtually All Mutations-SSCP), a robotically enhanced multiplex SSCP method, demonstrate that all of 525 mutations (391 unique) are detected by the method. In addition, the costs of DOVAM-S, gel-based fluorescent sequencing and capillary-based fluorescent sequencing are compared. The relative cost effectiveness of gel-based and capillary-based sequence analysis depends on throughput and whether depreciation and service are considered. DOVAM-S reduces the cost of candidate gene analyses relative to brute force sequencing by about threefold.     

Toward a fluorescent single-strand conformation polymorphism technique that detects all mutations: F-DOVAM-S

Although DOVAM-S (detection of virtually all mutations-SSCP) in effect detects all mutations and is less costly than direct sequencing, the technique currently requires the use of radioactivity. F-DOVAM-S (fluorescent DOVAM-S) was developed to replace the isotopic label with fluorescence and to increase throughput via dye color multiplexing. As proof of principle, two multitemperature slab gel electrophoresis conditions were evaluated through the blinded analysis of mutations in the factor IX (FIX) genes of 88 hemophilia B (HB) patients and 7 wild-type controls. Using only two conditions, it was determined that F-DOVAM-S had a detection sensitivity of 97%. It is anticipated that when three or four optimized conditions are employed, F-DOVAM-S will detect all mutations. Three patient samples were multiplexed per well using three different fluorescent dyes (6FAM, VIC, and NED), demonstrating that it is possible to analyze up to 44 kb of diploid, color-coded amplification product per gel lane. This value corresponds to a throughput of approximately 4 Mb of DNA analyzed per 96-well gel, which is approximately triple that of conventional radiolabeled DOVAM-S. Throughput is further enhanced by the rapidity at which the fluorescent signal can be captured and the resultant multicolor chromatograms analyzed. Given these data, F-DOVAM-S has the potential to be a particularly powerful technology for clinical diagnosis because it allows the mutation analysis of multiple patients to be performed within 24h.     

Multiplex dosage pyrophosphorolysis-activated polymerization: application to the detection of heterozygous deletions

Large heterozygous chromosomal deletions and gene duplications are important classes of mutations that are generally missed by standard PCR amplification and sequencing. Multiplex dosage pyrophosphorolysis-activated polymerization (MD-PAP), a derivative of PAP, was utilized to detect these types of mutations. PAP is a method for nucleic acid amplification in which 3' blocked oligonucleotides (P*) are activated by pyrophosphorolysis when annealed to the target template and subsequently extended. A key advantage to this technology is that PAP reactions produce little or no primer-dimer or false priming. As a result of this enhanced specificity, MD-PAP is easy to optimize. Herein, we utilize MD-PAP to determine gene dosage of each exon of the human factor IX gene by comparison with one endogenous internal control from the ATM gene. Estimated dosage is proportional to the actual template copy number over a minimum dynamic range from 1 to 16 copies. A blinded analysis detected 100% of 43 heterozygous deletions of exons in the human factor IX gene.     

Genetik und Klinik der Hämophilie A und B

Haemophilia A and B are caused by various mutations in the factor VIII (FVIII) and factor IX (FIX) genes, respectively. The clinical course of the disease is variable, dependent on the severity of the molecular defect. Nowadays, haemophilia patients can excellently be treated by plasma-derived or recombinant clotting factor concentrates. Thus, bleeding and its consequences can be almost completely prevented with nearly normal quality of life and life expectancy. The most severe complication of this treatment is the formation of antibodies (inhibitors) against the substituted clotting factor. The risk of inhibitor formation correlates significantly with specific mutation types that preclude endogenous factor VIII/IX protein synthesis and can be as high as 20–50%. The information on the expected clinical course is at present the most important indication for FVIII/IX gene analysis. Knowledge of the underlying FVIII/IX gene mutation further allows a reliable and fast carrier diagnosis in female relatives of patients with haemophilia.     

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.     

The use of SSCP analysis in the assessment of phytoplasma gene variability

Single-strand conformation polymorphism (SSCP) analysis is a broadly used technique for detecting mutations. The aim of this work was to assess the applicability of SSCP as a new tool for the detection of the molecular variability of uncultivable mollicutes - phytoplasmas. Three phytoplasma regions were investigated: 16S rDNA, tuf gene, and dnaB gene. Fragments amplified by PCR were subjected to SSCP under conditions optimized for each fragment length. In all of the analyzed regions, SSCP revealed the presence of polymorphism undetected by routine RFLP analyses. Reliability of the method was confirmed by the multiple alignments and phylogenetic analyses of representative sequences showing different SSCP profiles.     

Dideoxy fingerprinting (ddE): a rapid and efficient screen for the presence of mutations.

We describe dideoxy fingerprinting (ddF), a hybrid between dideoxy sequencing and SSCP that can detect the presence of single base and other sequence changes in PCR-amplified segments. As implemented herein, ddF involves a Sanger sequencing reaction with one dideoxynucleotide followed by nondenaturing gel electrophoresis. When ddF was used to examine segments of the human factor IX gene, 84 of 84 different mutations were detected with a very low rate of false positive signals. The approximate locations of the sequence changes could be determined from the ddF pattern and samples with different sequence changes had different fingerprints. In addition, large segments could be amplified and rapidly screened by ddF in multiple smaller subsegments. The patterns observed with ddF are instructive in that they suggest an inherent limitation in the detection of certain mutations by SSCP.     

Diagnosis of haemophilia B using the polymerase chain reaction

The polymerase chain reaction (PCR) was used to amplify specific DNA sequences within the factor IX gene of haemophilia B patients and their relatives. Three of the amplified fragments contain polymorphic sites, which can be used as markers in segregation analyses. These restriction fragment length polymorphisms (RFLPs) were until recently detected by Southern blotting after digestion with the restriction enzymes Taq I, Dde I and Xmn I. All three RFLP's are located in introns of the factor IX gene and together are informative in approximately 70% of all cases. Each of the polymorphisms was successfully used in carrier detection studies after amplification of the relevant fragments. This method is also suitable for rapid antenatal diagnosis. Additionally we were able to amplify all eight exons of the factor IX gene including the splice junctions and a part of the 5'-region. Large deletions or insertions can be detected without further analysis. Several possibilities for the rapid detection of point mutations after DNA amplification have been described recently. The complete amplification of all functional parts of the Factor IX gene in combination with these new techniques should enable us to detect the majority of mutations leading to haemophilia B.     

Mutations in the catalytic domain of human coagulation factor IX: rapid characterization by direct genomic sequencing of DNA fragments displaying an altered melting behavior

Deficiency in coagulation factor IX, a plasma glycoprotein constituent of the clotting cascade, results in hemophilia B, an inherited recessive X-linked bleeding disorder. Some affected individuals, referred to as antigen positive or CRM+, express an inactive factor IX gene product at normal levels and are expected to have natural mutations altering domains of the molecule that are critical for its correct function. The serine protease catalytic domain of activated factor IX, encoded by exons VII and VIII of the gene, is a possible target for such mutations. We designed a strategy allowing rapid analysis of this region through enzymatic amplification of genomic DNA, analysis of the amplification products by denaturing gradient gel electrophoresis, and direct sequencing of the fragments displaying an altered melting behavior. This procedure permitted us to characterize two previously undescribed mutations. Factor IX Angers is a G-to-A substitution generating an Arg in place of a Gly at amino acid 396 of the mature factor IX protein. Factor IX Bordeaux is an A-to-T substitution introducing a nonsense codon in place of the normal codon for Lys at position 411. Moreover, the already described factor IX Vancouver defect was found in three apparently independent families. These results provide further insight into the molecular heterogeneity of hemophilia B. In addition, we demonstrate the usefulness of this rapid screening procedure, which has broad applications in human genetics and can be used as an alternative to RFLP analysis in carrier detection or prenatal diagnosis studies.     

Missense mutations and evolutionary conservation of amino acids: evidence that many of the amino acids in factor IX function as "spacer" elements.

We report 31 point mutations in the factor IX gene and explore the relationship between the level of evolutionary conservation of an amino acid and the probability of a mutation causing hemophilia B. From our total sample of 125 hemophiliacs and from those reported by others, we identify 95 independent missense mutations, 94 of which occur at amino acids that are evolutionarily conserved in the available mammalian factor IX sequences. The likelihood of a missense mutation causing hemophilia B depends on whether the residue is also conserved in the factor IX-related proteases: factor VII, factor X, and protein C. Most of the possible missense mutations in generically conserved residues (i.e., those conserved in factor IX and in all the related proteases) should cause disease. In contrast, missense mutations in factor IX-specific residues (i.e., those conserved in human, cow, dog, and mouse factor IX but not in the related proteases) are sixfold less likely to cause disease. Missense mutations at nonconserved residues are 33-fold less likely to cause disease. At least three models are compatible with these observations. A comparison of sequence alignments from four and nine species of factor IX and an examination of the missense mutations occurring at CpG residues suggest a model in which most residues fall on opposite ends of a spectrum. In about 40% of residues, virtually any missense mutation in a minority of the residues will cause disease, while virtually no missense mutations will cause disease in most of the remaining residues. Thus, many of the residues in factor IX are spacers; that is, the main chains are presumably necessary to keep other amino acid interactions in register, but the nature of the side chain is unimportant.     

Strand-separating conformational polymorphism analysis: efficacy of detection of point mutations in the human ornithine delta-aminotransferase gene.

We tested the use of a modified method of single-strand conformational polymorphism (SSCP) analysis for the detection of point mutations in the human ornithine-delta-aminotransferase gene. Using a combination of three different electrophoretic conditions, we detected 20/20 known mutations. In a prospective study of 24 previously uncharacterized mutant OAT genes, we found 13 different mutations accounting for 19 (79%) of the 24. We conclude that SSCP is an efficient technique with high sensitivity and specificity.     

Factor IXPortland: a nonsense mutation (CGA to TGA) resulting in hemophilia B

Male siblings with severe hemophilia b were studied for the molecular defect responsible for their disorder. To define the precise DNA alteration, a 362-bp fragment in the first part of exon VIII of the factor IX gene was amplified and sequenced. A single-base-pair substitution of C----T at the nucleotide sequence 30875 was found which resulted in a nonsense mutation (TGA) and terminated the protein synthesis of factor IX at amino acid residue 252. The single-base change occurred as a classic CG dinucleotide alteration to TG (or CA), a common mechanism for point mutations in mammals.     

应用4种方法检测结核分枝杆菌临床分离株对链霉素的耐受性

通过DNA测序、SSCP、RFLP和反向斑点杂交技术分析167株结核分枝杆菌临床分离株的耐药基因型,评价结核分枝杆菌rpsL或rrs基因突变与链霉素（SM）耐受性之间的关系,比较4种分子方法检测SM耐受性的临床价值。98株耐SM分离株中,78株（79．6％）rpsL 43位或88位密码子错义突变导致赖氨酸置换为精氨酸,6株（6．1％）rrs 513位碱基A突变为C或T或516位C突变为T,14株（14．3％）未发现突变;69株SM敏感的分离株未发现这两个基因突变。应用SSCP、RFLP和RDBH方法分析上述突变和野生序列的结果与DNA测序完全一致,RDBH方法可从98株耐SM分离株中正确鉴定出84株（85．7％）分离株的5种突变基因型。结果表明,应用分子技术分析rpsL和rrs基因突变可快速检测大多数结核分枝杆菌对SM的耐受性,反向斑点杂交方法是一个快速、简便和可靠地检测药物耐受性的分子方法。     

Factor VIII gene mutations found by a comparative study of SSCP, DGGE and CMC and their analysis on a molecular model of factor VIII protein

Screening of the factor VIII (FVIII) gene which spans 186 kb and codes for 26 exons, was originally hampered by its size but is now feasible because rapid DNA scanning methodologies have been developed. The present study for the first time directly compares the three most widely applied screening methods, denaturing gradient gel electrophoresis (DGGE), single-stranded conformational polymorphism (SSCP) and chemical mismatch cleavage (CMC) for their sensitivity of mutation detection in a selected group of ten haemophilia A patients. Nine of these patients are known to be cross-reacting material positive and eight exhibited a mild to moderate phenotype. Of the ten patients screened, we identified mutations in nine by all three screening methods. Of the mutations characterised, two are previously unpublished. T to C (S373P) and G to A (D525N). In one mildly affected haemophiliac, we identified a second T to C sequence change in the 5′ untranslated region at –601 bp, probably having no effect on FVIII gene expression. Modelling studies were performed on those mutations lying within the A domains of FVIII (D525N, R527W, I566T) to study the possible effect of these mutations on structure and/or function. When the three methods are performing optimally and have been standardised, our experience is that CMC and DGGE are equally efficient at sequence variation detection while SSCP is slightly less sensitive. Received: 20 June 1997 / Accepted: 20 August 1997     

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

Rapid detection of point mutations in genomic DNA has been achieved by chemical mismatch analysis of heteroduplexes formed between amplified wild-type and target sequences in the human factor IX gene. Amplification and mismatch detection (AMD) analysis of DNA from relatives of haemophilia B patients permitted carrier diagnosis by direct identification of the presence or absence of the mutation in all cases, thus eliminating the need for the informative segregation of polymorphic markers. This extends diagnostic capability to virtually all haemophilia B families. AMD analysis permits detection of all sequence variations in genomic DNA and is therefore applicable to direct diagnosis of X-linked and autosomal diseases and for identification of new polymorphisms for genetic mapping.     

Blood clotting factor IX BM Nagoya. Substitution of arginine 180 by tryptophan and its activation by alpha-chymotrypsin and rat mast cell chymase

Factor IX BM Nagoya (IX Nagoya) is a natural mutant of factor IX responsible for severe hemophilia B. A patient with this mutant is characterized by a markedly prolonged ox brain prothrombin time. IX Nagoya was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that treatment of IX Nagoya with factor XIa/Ca2+ resulted in cleavage only at the Arg145-Ala146 bond. Reversed-phase high performance liquid chromatography of a trypsin digest of IX Nagoya showed an aberrant peptide, which was further digested with proteinase Asp-N. Primary structure analysis of one of the Asp-N peptides revealed that Arg180 is replaced by Trp. An essentially complete (99%) amino acid sequence of IX Nagoya was obtained by sequencing fragments derived from a lysyl endopeptidase digest in which no other substitutions in the catalytic triad or substrate binding site were found. We also found that IX Nagoya is activated by alpha-chymotrypsin or rat mast cell chymase by monitoring the rate of factor X activation using a fluorogenic peptide substrate in the presence of factor VIII, phospholipids, and Ca2+. These results indicate that the substitution of Arg180 by Trp impairs the cleavage by factor XIa required for activation of this zymogen and that the substitution causes hemophilia BM.     

Characterization of mutations in patients with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene, on chromosome 11q13, has recently been cloned, and mutations have been identified. We have characterized such MEN1 mutations, assessed the reliability of SSCP analysis for the detection of these mutations, and estimated the age-related penetrance for MEN1. Sixty-three unrelated MEN1 kindreds (195 affected and 396 unaffected members) were investigated for mutations in the 2,790-bp coding region and splice sites, by SSCP and DNA sequence analysis. We identified 47 mutations (12 nonsense mutations, 21 deletions, 7 insertions, 1 donor splice-site mutation, and 6 missense mutations), that were scattered throughout the coding region, together with six polymorphisms that had heterozygosity frequencies of 2%-44%. More than 10% of the mutations arose de novo, and four mutation hot spots accounted for >25% of the mutations. SSCP was found to be a sensitive and specific mutational screening method that detected >85% of the mutations. Two hundred and one MEN1 mutant-gene carriers (155 affected and 46 unaffected) were identified, and these helped to define the age-related penetrance of MEN1 as 7%, 52%, 87%, 98%, 99%, and 100% at 10, 20, 30, 40, 50, and 60 years of age, respectively. These results provide the basis for a molecular-genetic screening approach that will supplement the clinical evaluation and genetic counseling of members of MEN1 families.     

Novel stop and frameshifting mutations in the autosomal dominant polycystic kidney disease 2 (PKD2) gene

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most frequent inherited disorders. The majority of cases are due to mutation of the PKD1 gene, on 16p13.3, while in most of the remainder the disease maps to the PKD2 locus, at chromosome 4q21-q23. Recently, the PKD2 gene has been positionally cloned and three nonsense mutations within the coding sequence of the gene identified. Here we report a systematic mutation screening of all 15 exons of the PKD2 gene in chromosome 4-linked ADPKD families, using heteroduplex and SSCP analyses. We have identified and characterized seven novel mutations, with a detection rate of approximately 90% in the population studied. All of the mutations result in the premature stop of translation: four nonsense changes and three deletions. The deletions are all frameshifting, of four T nucleotides in one case and one G nucleotide in the other two. All mutations are unique and are distributed throughout the gene without evidence of clustering. Comparison of specific mutations with the clinical profile in ADPKD2 families shows no clear correlation. Received: 5 April 1997 / Accepted: 31 July 1997