Globalization of DNA-based prenatal diagnosis for recessive dystrophic epidermolysis bullosa.

Globalization of economies and improvements in international telecommunications has led to increased demand for better access to the latest developments in healthcare, wherever they may be available. In this report, we describe the first case from Thailand of DNA-based prenatal testing of a mother at risk for recurrence of severe recessive dystrophic epidermolysis bullosa (RDEB), whose affected child had died in early childhood. In the absence of previous access to prenatal diagnostic tests, the mother had undergone several terminations for fear of having another affected child. To prevent this happening again, DNA from the mother and her consanguineous partner was sent from Bangkok to a specialist laboratory at St John's Institute of Dermatology in London and screened for pathogenic mutations in the COL7A1 gene: both individuals were shown to be heterozygous carriers of a splice-site mutation, c.2440G --> C. In a subsequent pregnancy, amniocentesis was performed at 18 weeks' gestation in Bangkok, and fetal DNA was extracted and sent to London for analysis. Restriction endonuclease digestion of the amplified fetal DNA revealed the wild-type COL7A1 sequence only, and 5 months later, a clinically unaffected boy was born. This case represents the first example of DNA-based prenatal diagnosis for RDEB in Thailand and illustrates the benefits for patients in establishing international links with diagnostic centres with technological expertise that is not widely available in certain countries.

Unusual molecular findings in Kindler syndrome.

Kindler syndrome (KS) is a rare inherited skin disorder with blistering and poikiloderma as its main clinical features. It is caused by loss-of-function mutations in the C20orf42 (KIND1) gene which encodes kindlin-1, an actin cytoskeleton-focal contact-associated protein which is predominantly expressed in keratinocytes. We investigated the molecular basis of KS in a 16-year-old Indian boy who had additional clinical findings, including scleroatrophic changes of the hands and feet, pseudoainhum and early onset of squamous cell carcinoma on his foot. Immunostaining for kindlin-1 in the patient's skin was completely absent and sequencing of C20orf42 (KIND1) genomic DNA showed a homozygous splice-site mutation at the -6 position, IVS9-6T-->A. Amplification and sequencing of cDNA from the skin revealed aberrant splicing with either deletion of exon 10 or deletion of exons 9, 10 and 11, both of which involve loss of the pleckstrin homology domain of kindlin-1 that is thought to play a role in cytoskeletal attachment and integrin-mediated cell signalling. Pathogenic splice-site mutations at the -6 position are unusual and have rarely been reported for any genetic disorder. Collectively, these findings extend the spectrum of clinical and molecular abnormalities in this rare genodermatosis.

Functional redundancy of extracellular matrix protein 1 in epidermal differentiation.

BACKGROUND: Extracellular matrix protein 1 (ECM1) is a secreted protein expressed in skin. Its dermatological relevance has been highlighted by the discovery of loss-of-function mutations in ECM1 in patients with lipoid proteinosis (LiP). OBJECTIVES: To determine the role of ECM1 in epidermal differentiation by examining gene and protein expression of epidermal differentiation markers in individuals with LiP and histological assessment of transgenic mouse skin that overexpresses Ecm1a in basal or suprabasal epidermis. METHODS: Subconfluent, confluent and postconfluent LiP and control keratinocyte cultures were analysed by Northern and Western blotting for differences in expression of differentiation markers. Expression of these markers was analysed in skin of patients with LiP by immunohistochemistry. To study effects of Ecm1 overexpression on epidermal differentiation, transgenic mice were generated under control of either a keratin 14 or an involucrin promoter. RESULTS: No differential expression of the different markers analysed was observed in LiP keratinocytes compared with controls. No histological differences were found in Ecm1-overexpressing mouse skin compared with wild-type. CONCLUSIONS: Absence of ECM1 does not lead to differences in epidermal differentiation. Moreover, overexpression of Ecm1a in vivo does not exert dramatic effects on epidermal structure. Collectively, these findings suggest no role of ECM1 in epidermal differentiation.

Target proteins in inherited and acquired blistering skin disorders.

Maintenance of an intact epidermis depends on secure adhesion between adjacent keratinocytes, and between basal keratinocytes and the underlying epidermal basement membrane. The major adhesion units that achieve this are the hemidesmosomes and desmosomes, but when these structures are disrupted, e.g., by gene mutations or autoantibodies, the resilience of the epidermis is lost and blisters develop. Recently, there have been considerable advances in our knowledge of the proteins and glycoproteins that contribute to maintaining keratinocyte adhesion via hemidesmosomes and desmosomes, as well as new insights into the molecular pathogenesis of several inherited and autoimmune blistering skin diseases. These new basic scientific data are clinically relevant, helping to improve patient management and to provide a rationale for developing better and more specific treatments for patients with inherited or acquired blistering skin diseases. In addition, there have also been improvements in our understanding of the organization and assembly of these adhesion structures, and their involvement in signalling pathways, intricately linked to skin development, wound healing and tumour invasion. This review provides an update on the structure and organization of hemidesmosomes and desmosomes, and on the molecular pathology of their various components that result in bullous skin diseases.

Transient bullous dermolysis of the newborn in three generations.

Transient bullous dermolysis of the newborn (TBDN) is a rare form of dystrophic epidermolysis bullosa (DEB) that presents with neonatal skin blistering but which usually improves markedly during early life or even remits completely. Skin biopsies reveal abnormal intraepidermal accumulation of type VII collagen which results in poorly constructed anchoring fibrils and a sublamina densa plane of blister formation. The reason for the spontaneous clinical improvement is not known, but there is a gradual recovery in type VII collagen secretion from basal keratinocytes to the dermal-epidermal junction, with subsequent improvement or correction of anchoring fibril morphology. In this report, we describe TBDN occurring in three generations of the same family. Blistering occurred only during the first few months after birth, and all affected individuals were found to have a heterozygous glycine substitution mutation in exon 45 of the type VII collagen gene, COL7A1, designated G1522E. This mutation represents the third report of a pathogenic COL7A1 mutation in TBDN. Despite limited understanding of the disease mechanism in TBDN, this distinct form of DEB is important to recognize as it typically has a benign and self-limiting course. However, not all cases of DEB associated with intraepidermal type VII collagen are 'transient'. Genetic counselling in such patients therefore should be guarded until the pathophysiology of TBDN is better understood.

Molecular abnormalities of the desmosomal protein desmoplakin in human disease.

Desmoplakin is the principal plaque protein of desmosomes, specialized adhesion junctions found in various tissues including skin, heart and meninges. It is an entirely intracellular protein and in keratinocytes desmoplakin binds to other structural components of desmosomes such as cadherins and armadillo proteins, as well as to keratin filaments. Clues to the biological significance of desmoplakin have recently emerged from a number of naturally occurring human desmoplakin gene mutations. Both autosomal dominant and autosomal recessive disorders have been reported. The spectrum of clinical features includes varying degrees of keratoderma, blisters, nail dystrophy, woolly hair and, in some cases, cardiomyopathy. This review provides an update on genotype-phenotype correlation for human desmoplakin mutations as well as an overview of desmoplakin abnormalities in other conditions, including autoimmune blistering diseases, epithelial malignancies and blood vessel morphogenesis.

Complete paternal uniparental isodisomy of chromosome 1 resulting in Herlitz junctional epidermolysis bullosa.

Herlitz junctional epidermolysis bullosa (JEB) is an autosomal recessive mechanobullous disorder that results from loss-of-function mutations in the genes encoding the basement membrane component, laminin 5. Typically, there are frameshift, splice site or nonsense mutations on both alleles of either the LAMA3, LAMB3 or LAMC2 genes, with affected individuals inheriting one mutated allele from each parent. In this report, we describe a patient with Herlitz JEB in whom DNA analysis revealed homozygosity for the recurrent nonsense mutation R635X in LAMB3, located on chromosome 1q32.2. However, screening of parental DNA showed that although the patient's father was a heterozygous carrier of this mutation, the mother's DNA showed only wild-type sequence. Subsequent genotype analysis using 13 microsatellite markers spanning chromosome 1 revealed that the affected child was homozygous for the entire series of markers tested and that all of the alleles originated from the father. These results indicate that the Herlitz JEB phenotype in this patient is due to complete paternal isodisomy of chromosome 1 and reduction to homozygosity of the mutant LAMB3 gene locus. This is the fourth case of uniparental disomy to be described in Herlitz JEB, but it represents the first example of complete paternal isodisomy for chromosome 1 with a pathogenic mutation in the LAMB3 gene. These findings have important implications for mutation screening in JEB and for genetic counselling.

Altered expression of L-arginine metabolism pathway genes in chronic wounds in recessive dystrophic epidermolysis bullosa.

Individuals with the severe, mutilating Hallopeau-Siemens form of recessive dystrophic epidermolysis bullosa (HS-RDEB) have trauma-induced blisters and skin erosions which often progress to wounds that are slow to heal. These chronic wounds cause considerable morbidity and there is an increased risk of squamous cell carcinoma arising in the wound margins. Currently, little is known about the keratinocyte cell biology in these wounds. Therefore, we compared the gene expression profiles of wound edge with nonwounded skin from two individuals with HS-RDEB. Trauma-induced wound sites had been present in both patients for more than 3 months. Hybridizations using DermArray gene expression filters showed relative differences in gene expression between wounded and unwounded skin. Notably, there was a fivefold increase in expression of arginase-1 (ARG1) in the chronic wound samples. Expression of seven other genes relevant to L-arginine metabolism also showed differences greater than twofold. L-arginine is known to have a critical role in the synthesis of nitric oxide as part of normal tissue repair. Although alterations in arginase isoenzymes have been detected previously in other chronic wounds (human and animal models), this is the first study to demonstrate differences in several components of the L-arginine metabolism pathway in chronic wounds, and the first to examine chronic wounds in HS-RDEB. The data show that the cascade of L-arginine metabolites is altered in HS-RDEB and the findings may provide new insight into the pathology of chronic wounds in this genodermatosis.

Dermatological applications of DNA array technology.

The Human Genome Project and other large scale sequencing consortia continue to generate huge amounts of DNA sequence data. Despite the identification of specific disease-related genes and genetic markers, we still appear to know little about how diverse gene products actually interact with each other or respond to other chemical or biological stimuli. Such information is of course fundamental to understanding complex disease pathways and biochemical processes and, as such, has spawned new fields of investigative genetics, that of functional genomics and proteomics. DNA array technology is emerging as a powerful, high-throughput and versatile tool that can be applied to the study of functional genomics. This article reviews the methodology involved in array analysis and provides insight into how, as an investigative tool, DNA arrays are becoming increasingly useful in understanding fundamental abnormalities in dermatological disease and also in refining the management of patients with certain skin disorders.

EEC (Ectrodactyly, Ectodermal dysplasia, Clefting) syndrome: heterozygous mutation in the p63 gene (R279H) and DNA-based prenatal diagnosis.

BACKGROUND: Germline mis-sense mutations in the DNA-binding domain of the p63 gene have recently been established as the molecular basis for the autosomal dominant EEC (Ectrodactyly, Ectodermal dysplasia, Clefting) syndrome. OBJECTIVES: To examine genomic DNA from a 36-year-old woman, her 58-year-old father and her 11-year-old son, all with the EEC syndrome, to determine the inherent p63 mutation and, after genetic counselling, to use knowledge of the mutation to undertake a first-trimester DNA-based prenatal diagnosis in a subsequent pregnancy. METHODS: Fetal DNA was extracted from chorionic villi and used to amplify exon 7 of p63 containing the potential mutation. Direct sequencing and restriction endonuclease digestion (loss of AciI site on mutant allele) were used for DNA-based prenatal diagnosis. RESULTS: We identified a heterozygous arginine to histidine p63 mutation, R279H, in all three affected individuals. Prenatal diagnosis demonstrated a homozygous wild-type sequence predicting an unaffected child: a healthy boy was subsequently born at full-term. CONCLUSIONS: These data expand the p63 gene mutation database and provide the first example of a DNA-based prenatal test in this ectodermal dysplasia syndrome.

Three cases of de novo dominant dystrophic epidermolysis bullosa associated with the mutation G2043R in COL7A1.

In the absence of a positive family history, it is often difficult to determine whether a single case of mild-to-moderately severe dystrophic epidermolysis bullosa (DEB) represents autosomal recessive or de novo dominant disease. Recent molecular analyses of the type VII collagen gene, COL7A1, have established that the vast majority of such cases are recessive in nature. Nevertheless, a small number of de novo dominant patients have been documented. In this report, we describe three further examples of de novo dominant disease. In each case the COL7A1 mutation comprised the same glycine substitution, G2043R. This mutation has previously been reported in both dominant DEB pedigrees and as a de novo phenomenon and is the most common COL7A1 mutation in dominant DEB throughout the world. These cases emphasize the importance of molecular analysis in providing accurate genetic counselling in this genodermatosis.

Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63.

Hay-Wells syndrome, also known as ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome (OMIM 106260), is a rare autosomal dominant disorder characterized by congenital ectodermal dysplasia, including alopecia, scalp infections, dystrophic nails, hypodontia, ankyloblepharon and cleft lip and/or cleft palate. This constellation of clinical signs is unique, but some overlap can be recognized with other ectodermal dysplasia syndromes, for example ectrodactyly--ectodermal dysplasia--cleft lip/palate (EEC; OMIM 604292), limb--mammary syndrome (LMS; OMIM 603543), acro-dermato-ungual-lacrimal-tooth syndrome (ADULT; OMIM 103285) and recessive cleft lip/palate--ectodermal dysplasia (CLPED1; OMIM 225060). We have recently demonstrated that heterozygous mutations in the p63 gene are the major cause of EEC syndrome. Linkage studies suggest that the related LMS and ADULT syndromes are also caused by mutations in the p63 gene. Thus, it appears that p63 gene mutations have highly pleiotropic effects. We have analysed p63 in AEC syndrome patients and identified missense mutations in eight families. All mutations give rise to amino acid substitutions in the sterile alpha motif (SAM) domain, and are predicted to affect protein--protein interactions. In contrast, the vast majority of the mutations found in EEC syndrome are amino acid substitutions in the DNA-binding domain. Thus, a clear genotype--phenotype correlation can be recognized for EEC and AEC syndromes.

Heterozygous germline missense mutation in the p63 gene underlying EEC syndrome.

Mutations in the p63 gene have recently been delineated as the molecular basis for some cases of the ectrodactyly, ectodermal dysplasia and cleft lip/palate (EEC) syndrome, an autosomal dominant disorder (MIM 129900). In this report, we describe a 35-year-old woman with EEC syndrome and document a heterozygous germline missense mutation, R304W, in exon 8 of the p63 gene. As with most other p63 mutations in EEC syndrome, this mutation has arisen de novo and is located within the core DNA-binding domain of p63. Identification of this mutation has implications for genetic counselling and the feasibility of future DNA-based prenatal diagnosis in this individual.

The association of skin diseases with human herpesvirus 8 infection in HIV carriers.

The seroprevalence to Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus type 8 (HHV-8) was surveyed in human immunodeficiency virus type 1 (HIV-1) carriers with or without skin diseases, and also in HIV-1 negative individuals in Thailand. Using an immunofluorescence assay, the seropositive rates to lytic antigens of HHV-8 in HIV-1 carriers with or without skin diseases were 25% and 7.4%, respectively, but none of HIV-1 negative individuals had antibody. The seroprevalence to HHV-8 antigens was high in HIV positive individuals with low CD4/CD8 ratio, suggesting that HHV-8 is reactivated during the immunosuppressive state. Several polypeptides with apparent molecular weights of 34-38,000 and 40,000, which were specific to HHV-8, were identified by the immunoprecipitation test using the seropositive sera. Our results suggested that HHV-8 co-existed with HIV in HIV-1 carriers and the existence of HHV-8 may be associated with clinical features in the skin.

Clinical effect of dynorphin on heroin addicts.

This study assessed the clinical effectiveness, physiological responses and also adverse effects of dynorphin in heroin dependent detoxication. The subjects were randomly assigned into three groups and received intravenous injections of dynorphin at the dose of 180 (6 patients) or 60 (7 patients) or 0 (8 patients) micrograms/kg three times a day for 6 days. There was no statistical difference in withdrawal syndrome between each group. However, the drug craving feeling in the dynorphin groups was reduced when compared to the placebo group. There was no report of any adverse effects of the drug or abnormal laboratory results from the subjects during the study period.