Whole-exome sequencing improves mutation detection in a diagnostic epidermolysis bullosa laboratory.

BACKGROUND: Subtypes of inherited epidermolysis bullosa (EB) vary significantly in their clinical presentation and prognosis. Establishing an accurate diagnosis is important for genetic counselling and patient management. Current approaches in EB diagnostics involve skin biopsy for immunohistochemistry and transmission electron microscopy, and Sanger sequencing of candidate genes. Although informative in most cases, this approach can be expensive and laborious and may fail to identify pathogenic mutations in ~15% of cases. OBJECTIVES: Next-generation DNA sequencing (NGS) technologies offer a fast and efficient complementary diagnostic strategy, but the value of NGS in EB diagnostics has yet to be explored. The aim of this study was to undertake whole-exome sequencing (WES) in nine cases of EB in which established diagnostic methods failed to make a genetic diagnosis. METHODS: Whole-exome capture was performed using genomic DNA from each case of EB, followed by massively parallel sequencing. Resulting reads were mapped to the human genome reference hg19. Potentially pathogenic mutations were subsequently confirmed by Sanger sequencing. RESULTS: Analysis of WES data disclosed biallelic pathogenic mutations in each case, with all mutations occurring in known EB genes (LAMB3, PLEC, FERMT1 and COL7A1). This study demonstrates that NGS can improve diagnostic sensitivity in EB compared with current laboratory practice. CONCLUSIONS: With appropriate diagnostic platforms and bioinformatics support, WES is likely to increase mutation detection in cases of EB and improve EB diagnostic services, although skin biopsy remains an important diagnostic investigation in current clinical practice.

Under-recognition of acral peeling skin syndrome: 59 new cases with 15 novel mutations.

BACKGROUND: Acral peeling skin syndrome (APSS) is a rare skin fragility disorder usually caused by mutations in the transglutaminase 5 gene (TGM5). METHODS: We investigated the mutation spectrum of APSS in the U.K., Germany and Poland. RESULTS: We identified 59 children with APSS from 52 families. The phenotype was readily recognizable, with some variation in severity both within and between families. Most cases had been misdiagnosed as the localized form of epidermolysis bullosa simplex (EBS-loc). Eighteen different TGM5 mutations were identified, 15 of which were novel. Eight mutations were unique to a single family, nine each occurred in two families, while the common p.Gly113Cys mutation linked to a second missense variant p.Thr109Met occurred in 47 of the 52 families and was homozygous in 28. Most patients were of nonconsanguineous white European origin. CONCLUSIONS: We propose that APSS is under-reported and widely misdiagnosed as EBS-loc, with significant counselling implications as APSS is autosomal recessive while EBS-loc is dominant. We recommend screening for TGM5 mutations when EBS-loc is suspected but not confirmed by mutations in KRT5 or KRT14. Our report trebles the number of known TGM5 mutations. It provides further evidence that p.Gly113Cys is a founder mutation in the European population. This is consistent with the striking ethnic distribution of APSS in U.K., where the majority of patients are of nonconsanguineous white European origin, in contrast to the pattern of other recessive skin disorders.

A new homozygous nonsense mutation in LAMA3A underlying laryngo-onycho-cutaneous syndrome.

Laryngo-onycho-cutaneous (LOC) syndrome is a subtype of autosomal recessive junctional epidermolysis bullosa in which there is prominent skin and mucosal granulation tissue that can lead to delayed wound healing, laryngeal obstruction and blindness. Thus far, all cases are of Punjabi ancestry and have been shown to result from a founder mutation in the LAMA3 gene, notably involving a single nucleotide insertion mutation in exon 39, which is specific to the LAMA3A (designated exon 1 of LAMA3A) and not the LAMA3B1 or LAMA3B2 isoforms. Here, we describe a new pedigree with LOC syndrome. Affected individuals (from Iran) have the characteristic clinicopathological and molecular features of LOC syndrome: prominent granulation tissue (especially affecting the eyes), normal intensity laminin-332 immunostaining at the dermal-epidermal junction, and autosomal recessive mutations in the LAMA3A-specific exon. The pathogenic mutation is a homozygous nonsense mutation, designated p.Gln57X, which just affects the laminin-α3a transcript. These findings therefore expand the molecular basis of LOC syndrome.

Absence of detectable measles virus genome sequence in blood of autistic children who have had their MMR vaccination during the routine childhood immunization schedule of UK.

Leukocyte preparations from children with documented evidence of MMR vaccination and confirmed diagnosis of autism were examined by several assays designed to target multiple regions of the measles virus genome sequence. No sample was found positive by any method. The assays applied were highly sensitive, specific and robust in nature, and were based on the amplification of measles virus RNA transcripts by real-time quantitative RT-PCR (QRT-PCR) as well as by conventional RT-PCR-nested PCR. The assays applied were potentially able to detect measles virus RNA down to single figure copy numbers per reaction. The amount of total nucleic acid extract of leukocytes subjected to various measles virus-specific investigations was several fold higher than minimally required of a sample where measles virus persistence is well documented. This study failed to substantiate reports of the persistence of measles virus in autistic children with development regression.

Comparative evaluation of measles virus specific TaqMan PCR and conventional PCR using synthetic and natural RNA templates.

Comparative evaluation of TaqMan RT-polymerase chain reaction (PCR) methodology developed during this study with the conventional RT-PCR-nested PCR methodology developed earlier, using measles virus RNA templates derived from synthetic and natural sources against a number of primer sets belonging to various regions of the genome, revealed the existence of similar assay thresholds for both methods. An exception to this finding was, however, noted using primer sets of the N and M genes regions with RNA templates extracted from the wild type measles virus strain where the nested PCR method proved to be 10- to 100-fold more sensitive than the end points established with the N gene specific TaqMan RT-PCR method with synthetic RNA templates. These differences were not evident when the same primer sets were evaluated with RNA templates extracted from a brain sample of SSPE patient. These findings indicate that the genetic make up of measles virus strain in any given clinical specimen, in relation to the amplifying primers/probe sequences, can have impact on the overall sensitivity and specificity of the methodology applied. Both methods are equally suitable for the molecular detection of measles virus sequences in clinical specimens, although the TaqMan RT-PCR method may be preferred due to its advantages of contamination control, automation, and real-time product quantitation.