Lamellar ichthyosis: further narrowing, physical and expression mapping of the chromosome 2 candidate locus. (1/62)

Lamellar ichthyosis (LI) is an autosomal recessive genodermatosis which has been shown to be both clinically and genetically heterogeneous. Keratinocyte transglutaminase (or transglutaminase 1: TGM1) has been demonstrated to be the disease-causing gene in some families, whilst in others, a second unidentified LI gene was mapped to chromosome 2q33-35 (ICR2B locus). In this study, we present a physical map that encompasses the ICR2B locus, including the mapping of new microsatellite markers. Based on this new map, genotyping additional families highly suggests a reduction in size of the candidate interval. The final interval is covered by a single yeast artificial chromosome (937-H-3) which is 2.2Mb in length. Fine mapping of potential candidate transcripts was also focused on this region.  (+info)

Clinical and morphological correlations for transglutaminase 1 gene mutations in autosomal recessive congenital ichthyosis. (2/62)

Autosomal recessive congenital ichthyosis (ARCI) is a group of inherited disorders of cornification in which progress has recently been made in the identification of pathogenic mechanisms causing the disease. Transglutaminase 1 (TGM1) has been found as a defective gene in a large fraction of patients with lamellar ichthyosis (LI), a severe inherited scaling disorder of the skin. We have previously performed molecular genetic studies of 38Finnish ARCI families and found six different mutations in 13 families of 38 (34%). In this study we compared the molecular genetic alterations with clinical and electron microscopic findings of these patients. Families were classified by electron microscopy in ichthyosis congenita (IC) types I, II, III, IV and a non-defined group. TGM 1 gene mutation was found in all of the IC type II and 1/3 of the IC type 1 families. Although electron microscopy is not always used to classify ARCI patients, it can distinguish groups which are parallel with molecular genetic findings. This finding might be useful in the classification of ARCI patients for further linkage studies. Clinically typical phenotype of the TGM1 mutation carrier includes large, thick, brownish scales, but ichthyosis of some of these patients tends to be milder.  (+info)

Efficient in vitro transfection of human keratinocytes with an adenovirus-enhanced receptor-mediated system. (3/62)

Efficient gene delivery to the skin is important for gene therapy of skin diseases and in-depth biologic studies of epidermis. In this report, we investigated three nonviral transfection systems for gene transfer in cultured human keratinocytes and organotypic cultures. SuperFect is a highly branched polycationic transfection reagent, PrimeFector a polycationic liposome compound, and the AVET (adenovirus-enhanced transferrin-mediated) system consists of a ternary complex of biotinylated chemically inactivated adenovirus noncovalently complexed with plasmid DNA and polylysine-transferrin. After AVET transfection of cultured keratinocytes with pCIbetagal, a CMV/beta-galactosidase reporter plasmid, 28.8% +/- 1.4% of the cells were stained blue. SuperFect was about 2-fold less efficient, whereas Primefector did not transfect keratinocytes. Similar results were obtained when transfection efficiencies were measured by enzyme assays. Addition of holotransferrin to the culture medium or replacement of polylysine-transferrin by polylysine in the ternary complex did not affect the transfection efficiency. Using AVET complexes without adenovirus, however, strongly diminished gene delivery. This indicates that the AVET complex is taken up by an adenovirus receptor. Separation of AVET/pCIbetagal transfected keratinocytes by adhesion to collagen IV into two fractions (rapidly and slowly adhering cells) showed that the latter were transfected at a 3-fold higher level. Therefore, it seems that putative stem cells adhering rapidly to collagen IV are not efficiently transfected by AVET. AVET-transfected keratinocytes derived from keratinocyte trans- glutaminase negative lamellar ichthyosis patients with a CMV-TGK expression plasmid showed that it is possible to reach a level of total enzyme activity similar to that found in cultured keratinocytes from normal individuals. In organotypic cultures from outer root sheath cells AVET transfection was not successful, which might be due to the presence of the cornified layer or inaccessibility of the adenovirus receptor. In summary, the AVET system provides a powerful tool for transient in vitro transfection of keratinocytes.  (+info)

Role of Sp1 response element in transcription of the human transglutaminase 1 gene. (4/62)

This study addresses the contribution of an Sp1 response element in the proximal promoter of the transglutaminase 1 gene to transcription in normal epidermis and in a case of lamellar ichthyosis lacking transglutaminase 1 activity. The latter exhibited an Sp1 promoter mutation previously hypothesized to suppress transcription. In this study, several experiments indicated that the native Sp1 response element was functional, but it had only a small influence on transcription, and the previously observed mutation had no effect. These experiments involved mobility shift assays and transfections of promoter constructs in which the Sp1 site was mutated or lacking altogether. In addition the proximal 1.6 kb of the promoter from the affected individual was as active in transfections as the promoter from unaffected individuals. A search for sequence alterations in mRNA transcribed in keratinocytes from the patient revealed a novel single base mutation in codon 661 of the transglutaminase coding region predicted to result in premature termination of protein translation. The presence of this mutation in parental genomic DNA was confirmed by restriction digestion. Thus the lamellar ichthyosis phenotype in this case is likely attributable to a novel non-sense mutation in the coding region leading to reduced transglutaminase 1 mRNA levels rather than mutation of the Sp1 site.  (+info)

Novel mutations of the transglutaminase 1 gene in lamellar ichthyosis. (5/62)

Lamellar ichthyosis, one form of congenital autosomal recessive ichthyosis, is caused by mutations in the gene (TGM1) encoding the transglutaminase 1 enzyme. Mutations, deletions, or insertion of TGM1 have been reported so far. Here we report that three novel mutations of TGM1, D101V, N288T, and R306W, cause lamellar ichthyosis in two different families. The patient in family LI-KD has N288T and R306W mutations, and the patient in family LI-LK has D101V and R306W mutations. The activity of the transglutaminase 1 enzyme of the patient in family LI-LK was only about 15% of normal. Also, three-dimensional structural prediction analyses revealed that the N288T and R306W mutations, and possibly the D101V mutation, cause misfolding in the central catalytic core domain of the transglutaminase 1 enzyme that would probably result in reduced enzyme activity. Our data suggest that the greatly reduced transglutaminase 1 activities are due to disruptions of the native folding of transglutaminase 1, and that these mutations may play a critical role in the pathology of lamellar ichthyosis.  (+info)

Aberrant lipid organization in stratum corneum of patients with atopic dermatitis and lamellar ichthyosis. (6/62)

There are several skin diseases in which the lipid composition in the intercellular matrix of the stratum corneum is different from that of healthy human skin. It has been shown that patients suffering from atopic dermatitis have a reduced ceramide content in the stratum corneum, whereas in the stratum corneum of lamellar ichthyosis patients, the amount of free fatty acids is decreased and the ceramide profile is altered. Both patient groups also show elevated levels of transepidermal water loss indicative of an impaired barrier function. As ceramides and free fatty acids are essential for a proper barrier function, we hypothesized that changes in the composition of these lipids would be reflected in the lipid organization in stratum corneum of atopic dermatitis and lamellar ichthyosis patients. We investigated the lateral lipid packing using electron diffraction and the lamellar organization using freeze fracture electron microscopy. In atopic dermatitis stratum corneum, we found that, in comparison with healthy stratum corneum, the presence of the hexagonal lattice (gel phase) is increased with respect to the orthorhombic packing (crystalline phase). In lamellar ichthyosis stratum corneum, the hexagonal packing was predominantly present, whereas the orthorhombic packing was observed only occasionally. This is in good agreement with studies on stratum corneum lipid models that show that the presence of long-chain free fatty acids is involved in the formation of the orthorhombic packing. The results of this study also suggest that the ceramide composition is important for the lateral lipid packing. Finally, using freeze fracture electron microscopy, changes in the lamellar organization in stratum corneum of both patient groups could be observed.  (+info)

Harlequin fetus: three-dimensional sonographic findings and new diagnostic approach. (7/62)

Congenital ichthyosis, otherwise known as harlequin fetus, is a severe, generally lethal, anomaly. Prenatal diagnosis is usually possible in families at risk but requires invasive fetoscopy for skin biopsy. The application of three-dimensional ultrasound enables a greatly improved analysis of the facial morphology and thus provides an important contribution to prenatal diagnosis. Although such three-dimensional diagnostic procedures are performed in specialized centers, sonographers should be aware of the signs observed at routine two-dimensional ultrasound examination in order to ensure appropriate referral for diagnosis. We describe two consecutive pregnancies of the same parents in which two- and three-dimensional ultrasound were used in the prenatal diagnosis of harlequin fetus.  (+info)

A null mutation in the cystatin M/E gene of ichq mice causes juvenile lethality and defects in epidermal cornification. (8/62)

Cystatin M/E (CST6 ), a new member of the cystatin gene family, has a restricted expression pattern in humans, which is largely limited to cutaneous epithelia. Although cystatin M/E possesses two distinct biochemical properties, being a cysteine proteinase inhibitor and a substrate for transglutaminase, its physiological function is unknown. Here we report the isolation and characterization of the mouse Cst6 orthologue and the assignment of the chromosomal localization to the proximal end of mouse chromosome 19. This region corresponds to the locus of the spontaneous harlequin ichthyosis (ichq) mouse mutation, for which no causative gene has been identified so far. We found a nonsense mutation in the Cst6 gene of BALB/cJ-ichq/+ mice, which precludes the synthesis of functional protein. Immunohistochemistry confirmed the absence of cystatin M/E at the protein level in ichq/ichq mice. Mice that are homozygous for two null alleles display a hyperplastic, hyperkeratotic epidermis and abnormal hair follicles, and die between 5 and 12 days of age. In wild-type mice, cystatin M/E was found in the stratum granulosum and in the infundibulum of the hair follicle indicating that the anatomical site in the skin where cystatin M/E is normally expressed correlates with the abnormalities at the tissue level in ichq/ichq mice. Our data provide evidence that cystatin M/E is required for viability and for correct formation of cornified layers in the epidermis and hair follicles. The ichq mouse mutation may serve as a model for human type 2 harlequin ichthyosis.  (+info)