Three point mutations in the CFTR gene in French cystic fibrosis patients: Identification by denaturing gradient gel electrophoresis

Summary The cystic fibrosis (CF) gene was recently identified as a gene spanning 250 kilobases (kbp) and coding for a 1480 amino acid protein, cystic fibrosis transmembrane conductance regulator (CFTR). Approximately 70% of CF mutations involve a three-base-pair deletion in CFTR exon 10, resulting in the loss of a phenylalanine at position 508 in the gene product (ΔF508). In order to screen for other molecular defects, we have used a strategy based on denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified gene segments. This method, which permits rapid detection of any sequence change in a given DNA stretch, was used successfully to analyse 61 non-ΔF508 CF chromosomes from French CF patients. A study of CFTR exons 10, 11, 14a, 15 and 20 detected three mutations located in exons 14a, 15 and 20, along with several nucleotide sequence polymorphisms. These nucleotide changes were identified by direct sequencing of PCR fragments displaying altered electrophoretic behaviour, together with some of the polymorphisms and mutations previously characterized by others. The strategy presented here constitutes a valuable tool for the development of carrier testing for individuals or couples with a family history of cystic fibrosis, and will contribute to deciphering the functionally important regions of the CFTR gene.     

Homozygosity for multiple contiguous single-nucleotide polymorphisms as an indicator of large heterozygous deletions: identification of a novel heterozygous 8-kb intragenic deletion (IVS7-19 to IVS15-17) in a patient with glycogen storage disease type II

Current methods for detection of mutations by polymerase chain reaction (PCR) and sequence analysis frequently are not able to detect heterozygous large deletions. We report the successful use of a novel approach to identify such deletions, based on detection of apparent homozygosity of contiguous single-nucleotide polymorphisms (SNPs). The sequence analysis of genomic DNA PCR products containing all coding exons and flanking introns identified only a single heterozygous mutation (IVS18+2t-->a) in a patient with classic infantile-onset autosomal recessive glycogen storage disease type II (GSDII). Apparent homozygosity for multiple contiguous SNPs detected by this sequencing suggested presence of a large deletion as the second mutation; primers flanking the region of homozygous SNPs permitted identification and characterization by PCR of a large genomic deletion (8.26 kb) extending from IVS7 to IVS15. The data clearly demonstrate the utility of SNPs as markers for large deletions in autosomal recessive diseases when only a single mutation is found, thus complementing currently standard DNA PCR sequence methods for identifying the molecular basis of disease.     

Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): major implications for genetic counselling

More than 900 mutations and more than 200 different polymorphisms have now been reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Ten years after the cloning of the CFTR gene, the complete scanning of the 27 exons to identify known and novel mutations remains challenging. Rapid accurate identification of mutated alleles is important for prenatal diagnosis, for cascade screening in families at risk of cystic fibrosis (CF) and for understanding the correlation between genotype and phenotype. In this study, we report the successful use of denaturing ion-pair reverse-phase high performance liquid chromatography (D-HPLC) to analyse rapidly the complete coding sequence of the CFTR gene. With 27 pairs of polymerase chain reaction primers, we optimised the temperature conditions required for the analysis of each amplicon and validated thetest conditions on samples from a panel of 1552 CF patients who came from France and other European countries and who had mutations and polymorphisms located in the various melting domains of the gene. D-HPLC identified 415 mutated alleles previously characterised by denaturing gradient gel electrophoresis and DNA sequencing, plus 74 novel mutations reported here.This new technique for screening DNA for sequence variation was extremely accurate (it identified 100% of the CFTR alleles tested so far) and rapid (the complete CFTR gene could be analysed in less than a week). Our approach should reduce the number of untyped CF alleles in populations and thus decrease the residual risk in couples at risk of CF. This technique may be important not only for CF,but also for many other genes with a high frequency of point mutations at a variety of sites.     

Molecular Epidemiology and Diagnosis of PBG Deaminase Gene Defects in Acute Intermittent Porphyria

Acute intermittent porphyria (AIP) is the major autosomal dominant form of acute hepatic porphyrias. The disease is due to mutations in the gene encoding for porphobilinogen (PBG) deaminase and is characterized by life-threatening neurovisceral attacks, often precipitated by drugs, fasting, cyclical hormonal changes, or infectious diseases. This report describes a prospective study on the molecular epidemiology of PBG deaminase gene defects in AIP. It uses a sensitive, reliable, and easy-to-handle method for routine AIP molecular diagnosis and family study based on an exon-by-exon denaturing gradient gel electrophoresis (DGGE) strategy followed by direct sequencing. Fifteen genomic DNA fragments, including all the coding sequence and covering 3.35 kb of the PBG deaminase gene, were investigated in 405 subjects from 121 unrelated French Caucasian AIP families who had not been screened previously at the DNA level. PBG deaminase gene mutations were identified in 109 families, but only 78 were of different type, and each of them had a prevalence rate < 5%. Among these mutations, 33 had not been published previously. Sixty percent of these 78 mutations were located in only three exons (exons 10, 12, and 14), 44% were missense, 18% were splice defect, 19% were frameshift, and 16% were nonsense. In addition, two de novo mutational events were characterized. The evaluation of the efficiency of the standard PBG deaminase enzymatic screening method for gene-carrier detection indicated 95% of concordancy with the molecular-based diagnosis.     

Mutation screening using fluorescence multiplex denaturing gradient gel electrophoresis (FMD): detecting mutations in the BRCA1 gene

Fluorescent multiplex denaturing gradient gel electrophoresis (FMD) is a mutation screening technique designed to detect unknown as well as previously identified mutations. FMD constitutes a recent modification of the standard denaturing gradient gel electrophoresis (DGGE) technique, which combines multiplex PCR amplification of target DNA using fluorescently labeled primers with DGGE separation of the amplicon mixture, allowing immediate identification of sequence variants by wet gel scanning. FMD permits the simultaneous detection of small insertions, deletions and single nucleotide substitutions among multiple DNA fragments (up to 480 fragments) from 96 samples in parallel for each run. It increases output and reduces cost dramatically compared with classical DGGE, without sacrificing sensitivity and accuracy in detecting mutations. This protocol details an accurate, fast, nonradioactive and cost-effective way to screen the BRCA1 gene for mutations with high sensitivity, providing easily interpreted results. It may also be adapted to screen other target genes and/or used in large-scale epidemiological studies.     

Genomic organization and amplification of the human epidermal type II keratin genes K1 and K5

Keratins are a family of structurally related proteins that form the intermediate filament cytoskeleton in epithelial cells. Mutations in K1 and K5 result in the autosomal dominant disorders epidermolytic hyperkeratosis/bullous congenital ichthyosiform erythroderma and epidermolysis bullosa simplex, respectively. Most disease-associated mutations are within exons encoding protein domains involved in keratin filament assembly. However, some mutations occur outside the mutation hot-spots and may perturb intermolecular interactions between keratins and other proteins, usually with milder clinical consequences. To screen the entire keratin 1 and keratin 5 genes we have characterized their intron-exon organization. The keratin 1 gene comprises 9 exons spanning approximately 5.6 kb on 12q, and the keratin 5 gene comprises 9 exons spanning approximately 6.1 kb on 12q. We have also developed a comprehensive PCR-based mutation detection strategy using primers placed on flanking introns followed by direct sequencing of the PCR products.     

Denaturing high-performance liquid chromatography for the detection of mutations and polymorphisms in UBE3A

Angelman syndrome (AS) is caused by maternal deficiency of UBE3A, the gene encoding E6-AP ubiquitin-protein ligase. Our objectives were to develop conditions for denaturing high-performance liquid chromatography (dHPLC) analysis of UBE3A and to compare the sensitivity to direct genomic sequencing. Genomic DNA was obtained from 17 Angelman patients with known mutations and from 120 normal controls. DNA was amplified for the 10 coding exons and 6 upstream noncoding exons of UBE3A. Using dHPLC, the mutations previously identified in 17 Angelman patients were all easily detected using a single dHPLC condition for most exon-containing fragments. An analysis of all 16 exons in 120 normal controls identified 15 other DNA alterations of varying frequency, all of which are assumed to be benign. We conclude that dHPLC is a reliable and convenient method for detecting mutations in UBE3A causing Angelman syndrome. No disease-causing mutations were found in the noncoding exons.     

Detection of sequence variations in the adenomatous polyposis coli (APC) gene using denaturing high-performance liquid chromatography

We have evaluated the usefulness of denaturing high performance liquid chromatography (dHPLC) for scanning the adenomatous polyposis coli (APC) gene for point mutations, small deletions, and insertions. Our assay consists of 28 sets of primers to amplify the 15 exons of the APC gene. All PCR reactions were amplified simultaneously using the same reaction conditions in a 96-well format and then analyzed by dHPLC, using empirically determined optimum temperatures for partial fragment denaturation. Previously studied DNA specimens from 47 familial adenomatous polyposis (FAP) patients were analyzed by dHPLC and all mutations were correctly identified and confirmed by sequence analysis. This approach identified a single-base substitution in exon 6 and a 2-bp insertion in exon 15 that initially had not been detected by single-strand conformational polymorphism (SSCP) analysis. A novel mutation in exon 15 of the APC gene, 2065delG (codon 689) that had previously been undetected by the protein truncation test (PTT) was also identified by dHPLC. We present our validation studies of dHPLC technology for APC gene analysis in terms of sensitivity and specificity and compare it to current standard scanning technologies including PTT, SSCP, and conformational sensitive gel electrophoresis (CSGE).     

A fluorescent multiplex-DGGE screening test for mutations in the BRCA1 gene

Screening for mutations in the BRCA1 gene is challenging because of the wide spectrum of mutations found in this large gene. As the extensive exon 11 is commonly screened by the protein truncation test (PTT), here a fluorescent multiplex denaturing gradient gel electrophoresis (FMD) mutation screening technique was developed to test the remaining numerous small exons and splice sites of the gene. The method is based upon the use of an efficient multiplex polymerase chain reaction (PCR) amplification of the target regions, followed by denaturing gradient gel electrophoresis (DGGE) separation of the amplicon mixture, and the immediate achievement of results by wet gel scanning. The technique was applied to screen 16 samples with different BRCA1 sequence variants distributed over 12 exons. All variants were detected. In addition, 188 DNA samples from ovarian cancer patients were screened, identifying 22 new sequence variants (11.7% of the samples) and 243 common polymorphisms in the BRCA1 locus. Variants included 16 single nucleotide substitutions, 3 deletions of 2 nucleotides, 1 deletion of 4 nucleotides, and 2 insertions of 1 nucleotide. The FMD test provides an accurate, fast, nonradioactive and cost-efficient way to scan the BRCA1 gene with high sensitivity and an ease of result interpretation. This technique may prove to be a useful research tool for the detection of mutations and polymorphisms in the BRCA1 gene and for large-scale epidemiologic studies.     

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     

Genomic organization and amplification of the human keratin 15 and keratin 19 genes

Keratin intermediate filaments are the major components of the cytoskeleton in epithelial cells. Mutations in keratin genes have been documented in many disorders of the skin, nails, hair, and mucous membranes. Although no mutations have been described in either keratin 15 or keratin 19, they are good candidates for other as yet uncharacterized genetic disorders of keratinization, particularly as the skin, nails, hair, and conjunctiva are sites of keratin 15 and 19 expression. To facilitate future mutation detection analyses, we have therefore characterized the intron-exon organization of the human keratin 15 and keratin 19 genes. The keratin 15 gene comprises 8 exons spanning approximately 5.1 kb on 17q21, and the keratin 19 gene consists of 6 exons covering approximately 4.7 kb on 17q21. We have also developed a PCR-based mutation detection strategy using primers placed on flanking introns followed by direct sequencing of the PCR products.     

Fibroblast growth factor receptor 2 (FGFR2): genomic sequence and variations

Fibroblast growth factor receptors (FGFRs) play an important role in development and tumorigenesis. Mutations in FGFR2 cause more than five craniosynostosis syndromes. The FGFR2 genomic structure is the largest of the FGFR family. We have refined and extended the genomic organization of the FGFR2 gene by sequencing more than 119 kb of PACs, cosmids, and PCR products and assembling a region of approximately 175 kb. Although the gene structure has been reported to include only 20 exons, we have verified the presence of at least 22 exons, some of which are alternatively spliced. The sizes of six exons differed from those reported previously. Comparison of our sequence and those in the NCBI database detected more than 300 potential single nucleotide polymorphisms (SNPs). However, sequencing regions containing 52 of these potential SNPs verified only 14 in PCR products generated from 16 CEPH alleles. In contrast, direct sequencing of the CEPH DNAs revealed 21 other polymorphisms. Only one SNP was found in the 2,926 bp of coding sequence. Twenty-seven SNPs, two insertion polymorphisms and five microsatellite polymorphisms are contained in approximately 16.6 kb of non-coding sequence. These data yield an average of one polymorphism for approximately 488 bp of non-coding sequence examined. This collection of SNP, insertion, and repeat polymorphisms will aid future association studies between the FGFR2 gene and human disease and will enhance mutation detection.     

Targeted recovery of mutations in Drosophila

Reverse genetic techniques will be necessary to take full advantage of the genomic sequence data for Drosophila and other experimental organisms. To develop a method for the targeted recovery of mutations, we combined an EMS chemical mutagenesis regimen with mutation detection by denaturing high performance liquid chromatography (DHPLC). We recovered mutant strains at the high rate of approximately 4.8 mutations/kb for every 1000 mutagenized chromosomes from a screen for new mutations in the Drosophila awd gene. Furthermore, we observed that the EMS mutational spectrum in Drosophila germ cells shows a strong preference for 5'-PuG-3' sites, and for G/C within a stretch of three or more G/C base pairs. Our method should prove useful for targeted mutagenesis screens in Drosophila and other genetically tractable organisms and for more precise studies of mutagenesis and DNA repair mechanisms.     

Fluorescence-assisted mismatch analysis (FAMA) for exhaustive screening of the α-galactosidase A gene and detection of carriers in Fabry disease

We used the fluorescence-assisted mismatch analysis (FAMA) method to screen rapidly the α-galactosidase A gene in patients with Fabry disease in order to identify unknown mutations and help define genotype-phenotype correlations in this X-linked lysosomal storage disorder. Chemical cleavage at mismatches on heteroduplex DNA end-labeled with strand-specific fluorescent dyes, reliably detects sequence changes in DNA fragments of up to 1.5 kb and locates them precisely. Exhaustive scanning of the α-galactosidase gene was accomplished on four polymerase chain reaction-generated amplicons, covering all seven exons, the exon-intron boundaries, and 700 bp of 5′-flanking sequence. Mutations were identified in each of the 15 patients studied from nine unrelated kindreds. Among the seven previously undescribed sequence changes, three are obviously pathogenic because they lead to premature protein termination. The other four, a splice-site mutation and three missense mutations, were the only changes found upon complete scanning of the gene and its promoter. In addition, FAMA also detects female heterozygous carriers more dependably than direct sequencing, and thus provides a valuable diagnostic test. In Fabry disease, this molecular criterion is especially important for genetic counseling since heterozygotes can be asymptomatic and their enzymatic values within the normal range. Received: 9 April 1996 / Revised: 8 July 1996     

Mutational and genetic origin of LDL receptor gene mutations detected in both Belgian and Dutch familial hypercholesterolemics

DNA samples from 100 unrelated Belgian patients with familial hypercholesterolemia (FH) were screened for the presence of specific low-density lipoprotein receptor (LDLR) gene mutations, previously shown to be prevalent in related populations. Two point mutations, viz., P664L and a G to A splicing defect at position 1359–1, were detected in single Flemish-speaking families. A long-distance polymerase chain reaction (PCR) assay, used to screen for the 4-kb and 2.5-kb deletions previously identified by Southern blot analyses in different parts of The Netherlands, revealed a 3-kb deletion in two Belgian patients. Comparison of PCR product length showed that both Dutch deletions of exons 7–8 are identical to that found in Belgians, but different from the 2.5-kb deletion previously described in South Africans of mixed ancestry. The Belgian patients probably share a common ancestor, for each mutation identified, with FH patients from The Netherlands, since all three mutations were associated with the same LDLR gene haplotype as described for the Dutch population. Analysis of the deletion junctions demonstrated the role of a 31-bp repetitive sequence in the generation of large rearrangements involving exons 7 and 8 of the LDLR gene. The finding that only 4 out of 100 analyzed Belgian hypercholesterolemics carry a known LDLR mutation that is prevalent in related populations suggests that the Belgian FH population has its own spectrum of mutations. Received: 4 December 1996 / Accepted: 6 March 1997     

Mutation detection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene

The ability to sequence a large number of DNA samples rapidly and accurately for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequencing() is a non-electrophoretic real-time DNA sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DNA samples from skin-cancer patients were used. Two forms of nucleotide dispensation strategy were used, cyclic and programmed. Bi-directional pyrosequencing was performed and the overlapping sequence data produced were assembled to determine the sequence of the gene. Reliable sequencing data were obtained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequencing for detection of p53 mutations and allele distribution was demonstrated.     

Exon concatenation to increase the efficiency of mutation screening by DGGE

For genes that have a substantial number of exons and long intronic sequences, mutation screening by denaturing gradient gel electrophoresis (DGGE) requires the amplification of each exon from genomic DNA by PCR. This results in a high number of fragments to be analyzed by DGGE so that the analysis of large sample sets becomes labor intensive and time consuming. To address this problem, we have developed a new strategy for mutation analysis, lexon-DGGE, which combines the joining of different exons by PCR (also known as lexons) with a highly sensitive technique such as DGGE to screen for mutations. The lexon technique is based on the concatenation of several exons, adjacent or not, from genomic DNA into a single DNA fragment so that this approach could simultaneously be used to check the mutational status of several small genes. To show the feasibility of the approach, we have used the lexon-DGGE technique to analyze all coding exons, intron-exon junctions, noncoding exon 1, and part of the noncoding region of exon 11 of the TP53 gene. The validity and performance of the technique were confirmed by using negative and positive controls for each of the DNAfragments analyzed.     

A method for clone sequence confirmation using a mismatch-specific DNA endonuclease

Site-directed mutagenesis and polymerase chain reaction (PCR)-based cloning are well-established methods carried out routinely in most modern molecular biology laboratories. Application of these methods requires confirmation of the DNA sequence of the target gene by sequencing of DNA purified from multiple colonies, a laborious process. We have developed an alternative approach to screen DNA amplified directly from colony DNA for both desired and undesired mutations. This approach is based on the use of a plant mismatch DNA endonuclease, Surveyor Nuclease, to directly screen clones derived by site-directed mutagenesis. We have also used this approach to identify error-free clones of three genes from celery cDNA produced by PCR and TOPO cloning. Sequence confirmation using Surveyor Nuclease provides a fast and simple approach to obtain desired clones from site-directed mutagenesis and PCR-based cloning methods without the necessity of sequencing DNAs purified from multiple clones.