Mutations in CSTA, encoding Cystatin A, underlie exfoliative ichthyosis and reveal a role for this protease inhibitor in cell-cell adhesion

Autosomal-recessive exfoliative ichthyosis presents shortly after birth as dry, scaly skin over most of the body with coarse peeling of nonerythematous skin on the palms and soles, which is exacerbated by excessive moisture and minor trauma. Using whole-genome homozygosity mapping, candidate-gene analysis and deep sequencing, we have identified loss-of-function mutations in the gene for protease inhibitor cystatin A (CSTA) as the underlying genetic cause of exfoliative ichthyosis. We found two homozygous mutations, a splice-site and a nonsense mutation, in two consanguineous families of Bedouin and Turkish origin. Electron microscopy of skin biopsies from affected individuals revealed that the level of detachment occurs in the basal and lower suprabasal layers. In addition, in vitro modeling suggests that in the absence of cystatin A protein, there is a cell-cell adhesion defect in human keratinocytes that is particularly prominent when cells are subject to mechanical stress. We show here evidence of a key role for a protease inhibitor in epidermal adhesion within the lower layers of the human epidermis.     

Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Mal de Meleda (MDM) caused by mutations in the gene for SLURP-1 in patients from Germany,Turkey, Palestine,and the United Arab Emirates

Mal de Meleda (MDM) or keratosis palmoplantaris transgrediens of Siemens is an autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma (PPK) and transgressive keratosis with an onset in early infancy. There is no associated involvement of other organs; however, a spectrum of clinical presentations with optional and variable features has been described. Mutations in the ARS (component B)-81/s gene ( LY6LS) on chromosome 8q24-qter, which encodes SLURP-1, have recently been identified in patients with MDM. Here, we have analyzed four MDM families for mutations in SLURP-1. In a large Palestinian pedigree with multiple consanguinity, patients are homozygous for a new mutation that substitutes an arginine for a conserved glycine residue at position 86. A different mutation in Turkish patients results in the same amino acid exchange. Some remarkable similarities are seen in the clinical picture of patients from both families. Patients of an Emirati Bedouin family have a homozygous alteration of the translation initiation codon. In a German family with no known consanguinity, we have shown pseudodominant inheritance. Three affected children and their affected mother are homozygous for the missense mutation W15R. Our findings indicate that the MDM type of transgressive PPK is caused by SLURP-1 mutations in patients from various origins and demonstrate allelic heterogeneity for mutations in SLURP-1.     

Localisation of a gene for Papillon-Lefèvre syndrome to chromosome 11q14–q21 by homozygosity mapping

Papillon-Lefèvre syndrome is an autosomal recessively inherited palmoplantar keratoderma of unknown aetiology associated with severe periodontitis leading to premature loss of dentition. Three consanguineous families, two of Turkish and one of German origin, and three multiplex families, one of Ethiopian and two of German origin, with 11 affected and 6 unaffected siblings in all were studied. A targeted genome search was initially attempted to several candidate gene regions but failed to demonstrate linkage. Therefore a genome-wide linkage scan using a combination of homozygosity mapping and traditional linkage analysis was undertaken. Linkage was obtained with marker D11S937 with a maximum two-point lod score of Z _max = 6.1 at recombination fraction θ = 0.00 on chromosome 11q14–q21 near the metalloproteinase gene cluster. Multipoint likelihood calculations gave a maximum lod score of 7.35 between D11S901 and D11S1358. A 9.2-cM region homozygous by descent in the affected members of the three consanguineous families lies between markers D11S1989 and D11S4176 harbouring the as yet unknown Papillon-Lefèvre syndrome gene. Haplotype analyses in all the families studied support this localisation. This study has identified a further locus harbouring a gene for palmoplantar keratoderma and one possibly involved in periodontitis. Received: 19 July 1997 / Accepted: 22 August 1997     

Genotype/phenotype correlation in autosomal recessive lamellar ichthyosis.

Autosomal recessive lamellar ichthyosis is a severe congenital disorder of keratinization, characterized by variable erythema of the whole body surface and by different scaling patterns. Recently, mutations have been identified in patients with lamellar ichthyosis in the TGM1 gene coding for keratinocyte transglutaminase, and a second locus has been mapped to chromosome 2. We have now analyzed the genotype/phenotype correlation in a total of 14 families with lamellar ichthyosis. Linkage analyses using microsatellites in the region of the TGM1 gene confirmed genetic heterogeneity. In patients not linked to the TGM1 gene, the second region identified on chromosome 2 and a further candidate region on chromosome 20 were excluded, confirming as well the existence of at least three loci for lamellar ichthyosis. Sequence analyses of the TGM1 gene in families compatible with linkage to this locus revealed seven different missense mutations, five of these unpublished so far, and one splice mutation. No genotype/phenotype correlation for mutations in the TGM1 gene was found in this group of patients, which included two unrelated patients homozygous for the same mutation. Similarly, no clear difference in the clinical picture was seen between patients with TGM1 mutations and those unlinked to the TGM1 locus. Comparison of genetic and clinical classifications for patients with lamellar ichthyosis shows no consistency and thus indicates that clinical criteria currently in use cannot discriminate between the molecularly different forms of the disease.     

Medullary cystic kidney disease type 1: mutational analysis in 37 genes based on haplotype sharing

Medullary cystic kidney disease type 1 (MCKD1) is an autosomal dominant, tubulo-interstitial nephropathy that causes renal salt wasting and end-stage renal failure in the fourth to seventh decade of life. MCKD1 was localized to chromosome 1q21. We demonstrated haplotype sharing and confirmed the telomeric border by a recombination of D1S2624 in a Belgian kindred. Since the causative gene has been elusive, high resolution haplotype analysis was performed in 16 kindreds. Clinical data and blood samples of 257 individuals (including 75 affected individuals) from 26 different kindreds were collected. Within the defined critical region mutational analysis of 37 genes (374 exons) in 23 MCKD1 patients was performed. In addition, for nine kindreds RT-PCR analysis for the sequenced genes was done to screen for mutations activating cryptic splice sites. We found consistency with the haplotype sharing hypothesis in an additional nine kindreds, detecting three different haplotype subsets shared within a region of 1.19 Mb. Mutational analysis of all 37 positional candidate genes revealed sequence variations in 3 different genes, AK000210, CCT3, and SCAMP3, that were segregating in each affected kindred and were not found in 96 healthy individuals, indicating, that a single responsible gene causing MCKD1 remains elusive. This may point to involvement of different genes within the MCKD1 critical region.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Living with myotonic dystrophy; what can be learned from couples? a qualitative study

Background  

Myotonic dystrophy type 1 (MD1) is one of the most prevalent neuromuscular diseases, yet very little is known about how MD1 affects the lives of couples and how they themselves manage individually and together. To better match health care to their problems, concerns and needs, it is important to understand their perspective of living with this hereditary, systemic disease.     

Free‐living nematodes as prey for higher trophic levels of forest soil food webs

Nematodes are the most abundant invertebrates in soils and are key prey in soil food webs. Uncovering their contribution to predator nutrition is essential for understanding the structure of soil food webs and the way energy channels through soil systems. Molecular gut content analysis of consumers of nematodes, such as soil microarthropods, using specific DNA markers is a novel approach for studying predator–prey interactions in soil. We designed new specific primer pairs (partial 18S rDNA) for individual soil‐living bacterial‐feeding nematode taxa (Acrobeloides buetschlii, Panagrellus redivivus, Plectus velox and Plectus minimus). Primer specificity was tested against more than 100 non‐target soil organisms. Further, we determined how long nematode DNA can be traced in the gut of predators. Potential predators were identified in laboratory experiments including nine soil mite (Oribatida, Gamasina and Uropodina) and ten springtail species (Collembola). Finally, the approach was tested under field conditions by analyzing five mite and three collembola species for feeding on the three target nematode species. The results proved the three primer sets to specifically amplify DNA of the respective nematode taxa. Detection time of nematode DNA in predators varied with time of prey exposure. Further, consumption of nematodes in the laboratory varied with microarthropod species. Our field study is the first definitive proof that free‐living nematodes are important prey for a wide range of soil microarthropods including those commonly regarded as detritivores. Overall, the results highlight the eminent role of nematodes as prey in soil food webs and for channelling bacterial carbon to higher trophic levels.     

Initial steps towards a production platform for DNA sequence analysis on the grid

Background  

Bioinformatics is confronted with a new data explosion due to the availability of high throughput DNA sequencers. Data storage and analysis becomes a problem on local servers, and therefore it is needed to switch to other IT infrastructures. Grid and workflow technology can help to handle the data more efficiently, as well as facilitate collaborations. However, interfaces to grids are often unfriendly to novice users.