[Darier disease]. / Morbus Darier.

Darier disease (Darier-White disease, dyskeratosis follicularis) is a rare autosomal dominant genodermatosis with regional differences in prevalence. The responsible mutations have been identified on chromosome 12q23-24.1. The gene encodes a calcium-ATPase type 2 in the sarco-/endoplasmic reticulum (SERCA2), which belongs to the large family of P-type cation pumps. This pump couples ATP hydrolysis to the transport of cations across membranes and thus plays a significant role in intracellular calcium signaling. Neuropsychiatric disorders are often associated with Darier disease. However, these diseases are not due to mutations in the gene ATP2A2 but to a susceptibility locus in a 6.5 Mb region near this gene. Currently, the treatment is strictly limited to the relief of symptoms. In severe cases, oral retinoids (acitretin: initial 10-20 mg/Tag and isotretinoin: 0.5-1 mg/kg/day) lead to a response in 90% of cases. However, side effects often prevent long-term use of vitamin A derivatives.

Seven novel mutations in the ATP2A2 gene of Austrian patients with Darier's disease.

Darier's disease (DD, OMIM 124200) is an autosomal dominant inherited genodermatosis characterized by warty papules and plaques in seborrheic areas, and loss of adhesion between suprabasal epidermal keratinocytes (acantholysis) and abnormal keratinisation (dyskeratosis). Till date, more than 150 pathogenic mutations in the ATP2A2 (SERCA2) gene, which encodes the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase isoform 2, have been identified as the genetic basis of DD. Our report of eight DD patients from Austria add seven novel variants (L32P, 149-158del10 each in two different non-consanguineous patients, S72Y, F73S, K460X, 2734delC, T982 M) to the repertoire of ATP2A2 mutations in the DD database which is in line with previous reports that most mutations are related to the 5'- and the 3'-end of the gene.

[Hereditary blistering diseases. Symptoms, diagnosis and treatment of epidermolysis bullosa]. / Erbliche blasenbildende Erkrankungen. Klinik, Diagnostik und Therapie der Epidermolyis bullosa.

Hereditary epidermolysis bullosa (EB) is a term for a heterogeneous group of rare genetic disorders characterized by marked fragility of the skin and mucous membranes following minor trauma. Significant progress has been made in understanding the molecular basis of EB, which has far-reaching implications for an improved classification with consequences for prognosis, genetic counseling, DNA-based prenatal and preimplantation testing, and the development of future treatments including gene therapy. Besides mucocutaneous changes, EB leads to a number of systemic manifestations whose management requires multidisciplinary access. Extracutaneous complications include ophthalmologic, dental, gastrointestinal, pulmonary, urogenital, hematologic, and nutritional problems. This article reviews the progress that has been made in the understanding of the molecular basis of EB, clinical aspects of major EB subtypes, and the management of patients suffering from EB, and it gives an outlook on molecular therapy projects such as gene, cell, vector, and protein therapies.

Introducing a fast and simple PCR-RFLP analysis for the detection of mutant thiopurine S-methyltransferase alleles TPMT*3A and TPMT*3C.

BACKGROUND: Azathioprine, in combination with corticosteroids, is the first-line therapy of severe forms of pemphigus vulgaris. Patients with an impaired thiopurine S-methyltransferase (TPMT) activity are at risk of developing severe myelo-suppression upon treatment with thiopurines such as azathioprine. Analysis of the TPMT status prior to drug administration is therefore highly recommended. However, because of the limited availability of TPMT testing outside of specialized centres, pre-emptive TPMT testing is not widespread. To avoid laborious biochemical and sequencing assays, we evaluated a new restriction fragment length polymorphism (RFLP) analysis. METHODS: We designed a rapid genetic polymerase chain reaction (PCR)-RFLP screen for the most prevalent mutant TPMT*3A and TPMT*3C alleles that are known to result in reduced TPMT enzyme activity. RESULTS: Validating our fast system on 871 Caucasian DNA samples, we observed that 8.61% of our probands carried the TPMT*3A allele and 0.23% were heterozygous for the TPMT*3C allele, which is in concordance with previously reported allele frequencies. CONCLUSION: This simple and low-cost PCR-RFLP TPMT polymorphism testing approach can be performed in a standard laboratory. It should be applied to all patients prior to receiving thiopurine drug therapy to avoid the severe, but predictable, haematopoietic side-effects of thiopurine drug administration.

Characteristic immunohistochemical and ultrastructural findings indicate that Kindler's syndrome is an apoptotic skin disorder.

BACKGROUND: Kindler's syndrome is a rare genodermatosis mainly characterized by the onset of skin blistering in early childhood, web formation of fingers and toes, photosensitivity, and progressive poikiloderma. There is still debate whether this disease represents a distinctive entity in the spectrum of congenital bullous poikilodermas or a variant of dystrophic epidermolysis bullosa. OBJECTIVE: To evaluate the recently proposed and debated characteristic immunohistochemical and ultrastructural features of Kindler's syndrome. PATIENT/METHODS: Immunofluorescence (IF) antigen mapping and transmission electron microscopy (TEM) were performed on a skin specimen from non-sun-exposed inner aspect of the upper arm of a 49-year-old patient with characteristic clinical features of Kindler's syndrome. RESULTS: IF studies revealed focally an extensively broadened, partly reticular staining pattern in the dermoepidermal basement membrane zone (BMZ) with antibodies against laminin-5 and type IV as well as type VII collagen. Anti-alpha6 and beta4 integrin staining revealed small gaps in the linear reactivity in the BMZ. Abundant keratin bodies, as detected by anti-immunoglobulin M (IgM) staining, were focally present in the dermis, indicating prominent epidermal apoptosis. This was verified by a histochemical apoptosis stain [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) reaction]. Transmission electron microscopic examination showed manifold reduplications of the lamina densa (with attached anchoring fibrils) as well as a keratin body surrounded by a fibroblast in the upper dermis. CONCLUSION: We present characteristic immunohistochemical and ultrastructural features of Kindler's syndrome identical to those described by Shimizu et al. and provide evidence that Kindler's syndrome might primarily be an apoptotic disorder of basal keratinocytes.

Development of spliceosome-mediated RNA trans-splicing (SMaRT) for the correction of inherited skin diseases.

Gene therapy of large genes (e.g. plectin and collagen genes) is hampered by size limitations for insertions of the currently used viral vectors. To reduce the size of these insertions spliceosome-mediated RNA trans-splicing (SMaRT), which provides intron-specific gene-correction at the pre-RNA level, can be an alternative approach. To test its applicability in skin gene therapy, SMaRT was used in the context of the 4003delTC mutation in the collagen XVII gene (COL17A1) causing generalized atrophic benign junctional epidermolysis bullosa. A beta-galactosidase (beta-gal) trans-splicing assay system was established using intron 51 of COL17A1 as the target for trans-splicing. In this system, intron 51 is flanked by the 5'exon and the 3'exon of the beta-gal gene, the latter containing two in-frame stop codons. Cotransfection of a pre-trans-splicing molecule consisting of the binding domain of intron 51 and the 3'exon of beta-gal without the stop codons resulted in a 300-fold increase of beta-gal activity compared to controls. A 2-3-fold increase in efficiency was obtained through an elongation of the binding domains. Replacement of the complete 3'end of the COL17A1 gene was shown using a collagen XVII mini-gene construct. The beta-gal assay was used in human keratinocytes to evaluate the influence of a keratinocyte-specific spliceosome background. Reverse transcription polymerase chain reaction and beta-gal activity assay showed functional correction of the stop-codons in cultured human keratinocytes and in an immortalized GABEB cell line harbouring the 4003delTC mutation. These results demonstrate that SMaRT is feasible in a keratinocyte-specific context and therefore may be applied in skin gene therapy.

A novel homozygous nonsense deletion/insertion mutation in the keratin 14 gene (Y248X; 744delC/insAG) causes recessive epidermolysis bullosa simplex type Köbner.

We report the sixth case of a human keratin 14 'knockout' mutation resulting in recessive epidermolysis bullosa simplex (EBS). A novel, homozygous nonsense mutation resulting from a deletion/insertion mutation (744delC/insAG) leads to a premature termination codon in the KRT14 gene (Y248X). The patient suffers from generalized cutaneous blistering since birth, mild nail dystrophy, involvement of mucous membranes and multiple epidermolysis bullosa naevi. The clinical variability noted in K14-deficient EBS patients suggests phenotypic modulation by additional genetic and/or epigenetic factors.

A compound heterozygous one amino-acid insertion/nonsense mutation in the plectin gene causes epidermolysis bullosa simplex with plectin deficiency.

Plectin is a cytoskeleton linker protein expressed in a variety of tissues including skin, muscle, and nerves. Mutations in its gene are associated with epidermolysis bullosa simplex with late-onset muscular dystrophy. Whereas in most of these patients the pathogenic events are mediated by nonsense-mediated mRNA decay, the consequences of an in-frame mutation are less clear. We analyzed a patient with compound heterozygosity for a 3-bp insertion at position 1287 leading to the insertion of leucine as well as the missense mutation Q1518X leading to a stop codon. The presence of plectin mRNA was demonstrated by a RNase protection assay. However, a marked reduction of plectin protein was found using immunofluorescence microscopy of the patient's skin and Western blot analysis of the patient's cultured keratinocytes. The loss of plectin protein was associated with morphological alterations in plectin-containing structures of the dermo-epidermal junction, in skeletal muscle, and in nerves as detected by electron microscopy. In an in vitro overlay assay using recombinant plectin peptides spanning exons 2 to 15 the insertion of leucine resulted in markedly increased self-aggregation of plectin peptides. These results describe for the first time the functional consequences of an in-frame insertion mutation in humans.

Simplified PCR set-up using a frozen preformulated mix for the detection of cytomegalovirus.

A simple PCR set-up for the detection of cytomegalovirus in clinical specimen was developed. All components of the PCR master mix including Taq DNA polymerase, uracil N-glycosilase, and primers were preformulated and stored frozen in aliquots. After thawing the master mix aliquots, the PCR was immediately started after the addition of sample DNA. This method gave excellent reproducible PCR-results without loss of enzyme activities following storage at -20 degrees C for at least 4 months.

Identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza C virus and bovine coronavirus.

We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substrates p-nitrophenyl acetate, alpha-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse alpha1 macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.

Gram type-specific broad-range PCR amplification for rapid detection of 62 pathogenic bacteria.

Broad-range PCR has proven to be useful for the detection of bacteria. A set of broad-range PCR primers directed against conserved regions in the 16S rRNA gene was designed to specifically amplify either gram-positive or gram-negative bacteria. The gram type-specific broad-range PCR correctly classified all 62 pathogenic species tested.