Summary Background Epidermolytic palmoplantar keratoderma (EPPK) is one of a number of disorders characterized by diffuse thickening of palm and sole skin. Although EPPK is not a life‐threatening ...condition, palmoplantar keratoderma can be associated with cancer and heart disease and therefore differential diagnosis is important so that adequate surveillance can be provided for the more serious conditions. Most cases of EPPK are caused by mutations in the gene encoding the palm‐ and sole‐specific keratin 9 (K9), and this provides an option for molecular diagnosis of this condition.
Objectives To identify the molecular basis of diffuse palmoplantar keratoderma in four British families.
Methods Denaturing high‐performance liquid chromatography (dHPLC) and DNA sequencing were used to screen exon 1 of the k9 gene for sequence variations.
Results The dHPLC profiles obtained from individuals with EPPK differed from control samples, indicating sequence variations within the fragment analysed. The profiles varied between families, suggesting that underlying mutations were different for each family; this was confirmed by DNA sequencing. In three cases previously reported mutations were found that resulted in the change of methionine156 to valine and arginine162 to either tryptophan or glutamine. A novel mutation was identified in a fourth family that changed valine170 to methionine. dHPLC was used to screen control samples for this sequence variation and confirmed that it was not a common polymorphism.
Conclusions These results confirm the diagnosis of EPPK in these families and underline the usefulness of dHPLC as a method of screening samples for heterozygous mutations.
ST7 is a candidate tumour suppressor gene at human chromosome locus 7q31.1. We have performed mutational analysis of ST7 in a wide-range of cell lines and primary epithelial cancers and detected only ...one missense change in a breast cancer cell line. Other mutations previously found in cell lines and primary tumours were not evident in our analysis. These results imply that another tumour suppressor gene at this locus may be more important than ST7 in carcinogenesis.
Abstract Objective Recently, several genes have been reported with mutations or variants that underlie a number of syndromic and non-syndromic forms of oligodontia including MSX1, PAX9, AXIN2, EDA ...and WNT10A. This study aimed to identify the causal mutations in a consanguineous Pakistan family with oligodontia and microdontia. Design Exome sequencing was performed in two of affected members of the Pakistan family. Results The exome sequencing data revealed that the affected individuals were homozygous with a novel mutation in exon 8 of the SMOC2 gene, c.681T>A (p.C227X). Conclusions This is the second report describing SMOC2 mutations with oligodontia and microdontia underlining the key role for this signalling molecule in tooth development.
We report a missense mutation in the gap junction protein β-3 (encoding Connexin 31), which was detected in only the affected members of a family in which the autosomal dominant skin disease ...erythrokeratoderma variabilis was segregating. The nucleotide change results in an arginine to proline substitution in codon 42. This residue is positioned on the first transmembrane/first extracellular domain of the gap junction protein with the mutation replacing a negatively charged residue with a nonpolar residue. This change may disrupt the conformation of the protein and voltage gating polarity leading to impaired channel function.
There is a need to investigate the mechanistic basis of the human skin irritation response if relevant in vitro test systems for the predictive identification of skin irritation hazards are to be ...developed. Recent progress in genomics technologies mean that tools for the identification and investigation of important biochemical events in the processes of skin irritation are now available. The aim of this work was to identify genes (for further mechanistic investigation) which may be regulated in response to skin irritation, following exposure of the EpiDerm™ skin model to the known skin irritant sodium lauryl sulphate (SLS). EpiDerm cultures were treated in triplicate with a non-cytotoxic dose of SLS (0.1 mg/ml, as determined by the MTT assay and histological examination) for 15 min, 30 min, 1 h, 2 h, 3 h, 4 h and 24 h. Total RNA was extracted from the pooled EpiDerm cultures and used to probe Atlas™ human arrays (Clontech) covering approximately 3600 genes. Preliminary data indicated an up-regulation at early time points (15–30 min) of a number of genes involved in transportation (e.g. the sodium and chloride dependent taurine transporter) and receptors (e.g. ZAP70 and protocadherin 42 precursor). The gene encoding the UV excision repair protein and other DNA repair genes (e.g. DNA-directed RNA polymerase II) were up-regulated after 1–3 h, along with TGFβ3 and other tumour suppressors, which play a role in cellular development and wound healing. At the later time points of 4–24 h, genes involved in protein translation (e.g. Cathepsin D receptor) and metabolism (e.g. CYP27A) were up-regulated. In addition, a number of genes were down-regulated in response to treatment with SLS, although these followed less of a time dependent pattern. These results indicate the differential regulation of a number of genes in response to treatment with SLS, some of which may provide additional clues to the molecular events underpinning the irritation response to this particular surfactant and possibly to other chemical irritants.
Mouse mutants have a key role in discerning mammalian gene function and modelling human disease; however, at present mutants exist for only 1-2% of all mouse genes. In order to address this phenotype ...gap, we have embarked on a genome-wide, phenotype-driven, large-scale N-ethyl-N--nitrosourea (ENU) mutagenesis screen for dominant mutations of clinical and pharmacological interest in the mouse. Here we describe the identification of two similar neurological phenotypes and determination of the underlying mutations using a novel rapid mapping strategy incorporating speed back-crosses and high throughput genotyping. Two mutant mice were identified with marked resting tremor and further characterized using the SHIRPA behavioural and functional assessment protocol. Back-cross animals were generated using in vitro fertilization and genome scans performed utilizing DNA pools derived from multiple mutant mice. Both mutants were mapped to a region on chromosome 11 containing the peripheral myelin protein 22 gene (Pmp22). Sequence analysis revealed novel point mutations in Pmp22 in both lines. The first mutation, H12R, alters the same amino acid as in the severe human peripheral neuropathy Dejerine Sottas syndrome and Y153TER in the other mutant truncates the Pmp22 protein by seven amino acids. Histological analysis of both lines revealed hypo-myelination of peripheral nerves. This is the first report of the generation of a clinically relevant neurological mutant and its rapid genetic characterization from a large-scale mutagenesis screen for dominant phenotypes in the mouse, and validates the use of large-scale screens to generate desired clinical phenotypes in mice.
We have assembled a panel of monochromosomal somatic cell hybrids for use in gene mapping. DNA from each individual hybrid was used as a probe on normal human metaphases to identify the human ...chromosome and any fragments by reverse painting. To test the efficiency of the panel PCR amplification of DNA from the monochromosomal somatic cell hybrid panel was used in combination with human specific oligonucleotide primers to assign alpha-catenin (CTNNA1) and p21/WAF1 to chromosomes 5 and 6 respectively. These genes were localized further using hybrids containing specific translocations to 5q11-qter and 6p21 respectively. We also developed primers to enable us to assign 17 ESTs sequenced by the HGMP Resource Centre. The hybrid panel was developed with support of the UK HGMP and the DNA is available to all registered users.
Summary
Fluorescence resonance energy transfer (FRET) by acceptor photobleaching is a simple but effective tool for measurements of protein–protein interactions. Until recently, it has been ...restricted to qualitative or relative assessments owing to the spectral bleed‐through contamination resulting from fluorescence overlap between the donor and the acceptor. In this paper, we report a quantitative algorithm that combines the spectral unmixing technique with FRET by acceptor photobleaching. By spectrally unmixing the emissions before and after photobleaching, it is possible to resolve the spectral bleed‐through and retrieve the FRET efficiency/interaction distance quantitatively. Using a human keratinocyte cell line transfected with cyan fluorescent protein (CFP)‐ and yellow fluorescent protein (YFP)‐tagged Cx26 connexins as an example, FRET information at homotypic gap junctions is measured and compared with well‐established methods. Results indicate that the new approach is sensitive, flexible, instrument independent and solely FRET dependent. It can achieve FRET estimations similar to that from a sensitized emission FRET method. This approach has a great advantage in providing the relative concentrations of the donor and the acceptor; this is, for example, very important in the comparative study of cell populations with variable expression levels.
Focal nonepidermolytic palmoplantar keratoderma (NEPPK), or tylosis, is an autosomal, dominantly inherited disorder of the skin that manifests as focal thickening of the palmar and plantar surfaces. ...In three families studied, the skin disorder cosegregates with esophageal cancer and oral lesions. New haplotype analysis, presented here, places the tylosis esophageal cancer (TOC) locus between D17S1839 and D17S785. Envoplakin (EVPL) is a protein component of desmosomes and the cornified envelope that is expressed in epidermal and esophageal keratinocytes and has been localized to the TOC region. Mutation analysis of EVPL in the three affected families failed to show tylosis-specific mutations, and haplotype analysis of three intragenic sequence polymorphisms of the EVPL gene placed it proximal to D17S1839. Confirmation of the exclusion of EVPL as the TOC gene by location was obtained by integration of the genetic and physical mapping data using radiation hybrid, YAC, BAC, and PAC clones. This new physical map will allow further identification of candidate genes underlying NEPPK associated with esophageal cancer, which may also be implicated in the development of sporadic squamous cell esophageal carcinoma and Barrett's adenocarcinoma.
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