Comparative mutation detection screening of the type VII collagen gene (COL7A1) using the protein truncation test, fluorescent chemical cleavage of mismatch, and conformation sensitive gel electrophoresis.

Department of Cell and Molecular Pathology, St John's Institute of Dermatology, St Thomas' Hospitals' Medical School, London, UK.
Journal of Investigative Dermatology (Impact Factor: 6.37). 11/1999; 113(4):673-86. DOI: 10.1046/j.1523-1747.1999.00732.x
Source: PubMed

ABSTRACT Mutations in the type VII collagen gene, COL7A1, give rise to the blistering skin disease, dystrophic epidermolysis bullosa. We have developed two new mutation detection strategies for the screening of COL7A1 mutations in patients with dystrophic epidermolysis bullosa and compared them with an established protocol using conformational sensitive gel electrophoresis. The first strategy consisted of an RNA based protein truncation test that amplified the entire coding region in only four overlapping nested reverse transcriptase-polymerase chain reaction assays. These fragments were transcribed and translated in vitro and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have used the protein truncation test procedure to characterize 15 truncating mutations in 13 patients with severe recessive dystrophic epidermolysis bullosa yielding a detection sensitivity of 58%. The second strategy was a DNA-based fluorescent chemical cleavage of mismatch (fl-CCM) procedure that amplified the COL7A1 gene in 21 polymerase chain reaction assays. Mismatches, formed between patient and control DNA, were identified using chemical modification and cleavage of the DNA. We have compared fl-CCM with conformational sensitive gel electrophoresis by screening a total of 50 dominant and recessive dystrophic epidermolysis bullosa patients. The detection sensitivity for fl-CCM was 81% compared with 75% for conformational sensitive gel electrophoresis (p = 0.37 chi2-test). Using a combination of the three techniques we have screened 93 dystrophic epidermolysis bullosa patients yielding an overall sensitivity of 87%, detecting 79 different mutations, 57 of which have not been reported previously. Comparing all three approaches, we believe that no single method is consistently better than the others, but that the fl-CCM procedure is a sensitive, semiautomated, high throughput system that can be recommended for COL7A1 mutation detection.

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    ABSTRACT: Direct nucleotide sequencing is the reference standard critical to all molecular biology whether it is used for the elucidation of an entire genome or the characterization of a specific mutation. Sequencing protocols were initially developed using either dideoxy nucleotides (1) or chemicals (2), although the latter became less attractive because of the toxic nature of the chemicals used. It is obvious that if sequencing was less expensive, then there would be no need for mutation-scanning techniques. However, although fluorescent technology is improving and capillary electrophoresis is replacing polyacrylamide gel electrophoresis, the cost of consumables is rising and laboratories are, therefore, forced to use other techniques to detect mutations. Mutation-scanning techniques need to detect new mutations within the entire coding region of a gene and there are several methods available to scan for sequence changes in either cellular RNA or genomic DNA. These include denaturing gradient gel electrophoresis (DGGE) (3) (see Chapter 8), chemical cleavage of mismatch (CCM) (4), enzyme mismatch cleavage (EMC) (5,6), single-stranded conformation polymorphism (SSCP) (see Chapter 7) (CR7), heteroduplex analysis (HA) (8), conformation-sensitive gel electrophoresis (CSGE) (9), the protein truncation test (PTT) (10); and, more recently, denaturing high-performance liquid chromatography (DHPLC) (11,CR12) (see Chapter24). The most critical factor that determines the success of any gene screening protocol is the sensitivity of the detection technique. The sensitivities of these methods vary greatly depending on the size of DNA/RNA template screened. For example, SSCP has a sensitivity of >95‰ for fragments of 155 bp, but this is reduced to only 3‰ for 600 bp (13). Once optimised, DGGE has a sensitivity of approx 99‰ for fragments of up to 500 bp (14), and CSGE has a sensitivity of 90–100‰ for fragments of up to 450 bp (15). CCM and EMC, on the other hand, have sensitivities of 95–100‰ for fragments >1.5 kb in size ( 16,17) and are ideal for screening compact genes where more than one exon can be amplified together using genomic DNA as the template. All of these techniques detect sequence changes such as nonsense, frame shift, splice site, and missense mutations, as well as polymorphisms, however, the PTT screens only for truncating mutations and is predicted to have a sensitivity of >95‰ and can be used for RNA or DNA fragments in excess of 3 kb.
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    ABSTRACT: Individuals with inherited skin diseases often pose one of the most difficult diagnostic challenges in dermatology. The hunt for the underlying molecular pathology may involve candidate gene screening or linkage analysis, which is usually determined by the initial history, the physical findings and laboratory tests. Recent technical advances in DNA sequencing, however, are shifting the diagnostic paradigm. Notably, next-generation sequencing allows a more comprehensive approach to diagnosing inherited diseases, with potential savings of both time and money. In the setting of a paediatric dermatology genetics clinic in Kuwait, we therefore performed whole-exome sequencing on seven individuals without a priori detailed knowledge of the patients' disorders: from these sequencing data, we diagnosed X-linked hypohidrotic ectodermal dysplasia (two cases), acrodermatitis enteropathica, recessive erythropoietic protoporphyria (two siblings) and localized recessive dystrophic epidermolysis bullosa (two siblings). All these groups of disorders are clinically and genetically heterogeneous, but the sequencing data proved inherently useful in improving patient care and avoiding unnecessary investigations. Our observations highlight the value of whole-exome sequencing, in combination with robust bioinformatics analysis, in determining the precise molecular pathology and clinical diagnosis in patients with genetic skin disorders, notably at an early stage in the clinical evaluation of these often complex disorders and thereby support a new paradigm for future diagnostics.
    Experimental Dermatology 12/2013; 22(12):825-31. DOI:10.1111/exd.12276 · 4.12 Impact Factor
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    ABSTRACT: Background Dystrophic epidermolysis bullosa (DEB) is a heterogeneous inherited blistering skin disorder. The mode of inheritance may be autosomal dominant or recessive but all forms of DEB result from mutations in the gene encoding the anchoring fibril protein, type VII collagen, COL7A1. Consequently, in spite of careful clinical and skin biopsy examination, it may be difficult to distinguish mild recessive cases from de novo dominant disease in families with clinically normal parents and no other affected siblings; this distinction has significant implications for the accuracy of genetic counselling.Objectives To assess whether COL7A1 mutation analysis might help determine mode of inheritance in mild to moderate DEB.Methods We performed COL7A1 screening using heteroduplex analysis and direct nucleotide sequencing in four individuals with mild to moderate ‘sporadic’ DEB and clinically unaffected parents.Results In each patient, we identified a heterozygous glycine substitution within the type VII collagen triple helix. However, in two cases these mutations had been inherited in trans with a non-sense mutation on the other allele (i.e. autosomal recessive DEB). In the other two cases, no additional mutation was identified and neither mutation was present in parental DNA (i.e. de novo dominant disease).Conclusions This study highlights the usefulness of DNA sequencing in determining the inherited basis of some sporadic cases of DEB. However, delineation of glycine substitutions should prompt comprehensive COL7A1 gene sequencing in the affected individual, as well as clinical assessment of parents and mutation screening in parental DNA, if the true mode of inheritance is to be established correctly.
    British Journal of Dermatology 09/2003; 149(4):810 - 818. DOI:10.1046/j.1365-2133.2003.05315.x · 4.10 Impact Factor

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