A novel mutation in KRT12 associated with Meesmann's epithelial corneal dystrophy.

BACKGROUND: The molecular basis of Meesmann's epithelial corneal dystrophy (MECD) has recently been attributed to mutations in the cornea specific keratin genes KRT3 and KRT12. The mechanisms by which these mutations cause the Meesmann's phenotype are not clear. This study presents new data, examines clinical, histological, ultrastructural, and molecular aspects of MECD, and compares the features seen in this condition with those observed in other well studied keratin diseases such as epidermolysis bullosa simplex. METHODS: A two generation family with typical features of Meesmann's epithelial corneal dystrophy (MECD) was studied. All family members were examined under a slit lamp. Biopsy material from elective keratoplasty was studied by histopathological and ultrastructural analysis using standard techniques. Direct automated sequencing of genomic DNA was used for mutation detection, mutations were confirmed by restriction digest analysis. RESULTS: The abnormal corneal epithelium was acanthotic and contained numerous dyskeratotic cells and intraepithelial vesicles. By electron microscopy abnormally aggregated and clumped keratin filament bundles were detected in basal and suprabasal keratinocytes from the centre of the cornea. Direct sequencing of the patients' genomic DNA revealed a novel missense mutation (423T>G) in exon 1 of the cornea specific keratin 12 (KRT12) gene. This mutation predicts the amino acid change N133K within the helix initiation motif of the K12 polypeptide. Comparative studies with well established keratin disorders of other human epithelia underscore the pathogenic relevance of K3 and K12 gene mutations in Meesmann's epithelial corneal dystrophy. The morphological data presented here illustrate the disruptive effects of keratin gene mutations on the integrity of corneal keratinocytes. CONCLUSIONS: A clinical, histopathological, and ultrastructural study of a previously unreported family with MECD is presented. In this family the disease is ascribed to a novel mutation in KRT12. A molecular mechanism is proposed for MECD based on the comparison with other well characterised keratin diseases.

[Mutations in the keratin gene as a cause of Meesman-Wilke corneal dystrophy and autosomal dominant skin cornification disorders]. / Keratin-Gen-Mutationen als Ursache der Epitheldystrophie Meesmann-Wilke und autosomal dominanter Verhornungsstörungen der Haut.

BACKGROUND: Meesmann's corneal dystrophy (OMIM 122,100) is a rare autosomal dominant disorder of the corneal epithelium. It manifests in early childhood and affects both eyes. The disease is characterized by variable patterned dot-like corneal opacities and intraepithelial vesicles, which can be seen by slit-lamp examination and retro-illumination. Further signs include punctate erosions, lacrimation, photophobia, and blepharospasm. Vision is usually only slightly diminished. By histology, the corneal epithelium is irregularly thickened. It shows vacuolated epithelial cells and intraepithelial formation of vesicles. By electron microscopy fibrogranular aggregates are seen in the cytoplasm of epithelial cells. RESULTS: Linkage analyses in descendants of the family described by Meesmann and Wilke and other affected kinships showed that the putative genetic defect locates within the keratin type I gene cluster on chromosome 17 (17q12-21). Molecular genetic analyses in more than ten affected families showed that mutations in the cornea-specific keratin genes K3 and K12 represent the causative genetic defects of the disease phenotype. CONCLUSION: Comparative studies in autosomal dominant skin disorders of cornification suggest that the mutations identified in patients with Meesmann's corneal dystrophy exert dominant negative effects on keratin filament assembly. Disturbed filament formation results in intracellular keratin clumping, identifiable as fibrogranular aggregates. As a result the mechanical resilience of the affected cells and the epithelial tissue appears markedly reduced. Whether abnormalities of functionally related structural proteins, e.g. desmosomal components, could result in a phenotype similar to Meesmann and Wilke's corneal dystrophy remains to be seen.

A novel keratin 12 mutation in a German kindred with Meesmann's corneal dystrophy.

AIM: To study a kindred with Meesmann's corneal dystrophy (MCD) to determine if a mutation within the cornea specific K3 or K12 genes is responsible for the disease phenotype. METHODS: Slit lamp examination of the cornea in four members of the kindred was carried out to confirm the diagnosis of MCD. The region encoding the helix initiation motif (HIM) of the K12 polypeptide was polymerase chain reaction (PCR) amplified from genomic DNA derived from affected individuals in the kindred. PCR products generated were subjected to direct automated sequencing. Restriction enzyme analysis employing Ban I was used to confirm the presence of the mutation in affected individuals of the family. RESULTS: Sequencing of the K12 gene in an affected individual from the family revealed a novel heterozygous missense mutation (413A-->C), predicting the substitution of a proline for a glutamine at codon 130 (Q130P) in the HIM of the K12 protein. The mutation was excluded from 50 normal, unaffected individuals by restriction enyzme analysis and was therefore unlikely to be a common polymorphism. CONCLUSION: A novel missense mutation in the K12 gene leads to MCD in a German kindred. Missense mutations have now been identified within the region encoding the helix initiation motif of the K12 protein in eight of 11 MCD kindreds analysed at the molecular level.

Molecular genetics of Meesmann's corneal dystrophy: ancestral and novel mutations in keratin 12 (K12) and complete sequence of the human KRT12 gene.

Recently, we identified the first mutations in corneal keratins K3 and K12 in families with Meesmann's corneal dystrophy (MCD). Here, we sequenced all regions of the human K12 gene, to enable mutation detection for all exons using genomic DNA as a template. The human K12 genomic sequence spans 5919 bp and consists of eight exons. A microsatellite dinucleotide repeat was identified within intron 3, which was highly polymorphic and which we developed for use in genotype analysis. In addition, two mutations in the helix initiation motif of K12 were found in families with MCD. A novel mutation was detected in an American kindred, 410T-->C, which predicts the amino acid substitution M129T. In a German family, mutation 428G-->C was identified, predicting amino acid change R135T. The latter mutation was identical to that which we identified in the original kindred described by Meesmann. Using the intragenic microsatellite polymorphism in K12 and additional flanking markers, we were able to show that this family shares a common haplotype with the original Meesmann kindred. These results strongly imply that R135T represents an ancestral mutation in the German population. Both mutations occur in the highly conserved helix initiation motif of the K12 polypeptide. A total of eight mutations have now been reported in the K12 gene.

Moderation of phenotypic severity in dystrophic and junctional forms of epidermolysis bullosa through in-frame skipping of exons containing non-sense or frameshift mutations.

Non-sense mutations on both alleles of either the type VII collagen gene (COL7A1) or the genes encoding laminin 5 (LAMA3, LAMB3, or LAMC2) usually result in clinically severe forms of recessive dystrophic or junctional epidermolysis bullosa, respectively. In this study we assessed two unrelated families whose mutations in genomic DNA predicted severe recessive dystrophic epidermolysis bullosa or junctional epidermolysis bullosa phenotypes but in whom the manifestations were milder than expected. The recessive dystrophic epidermolysis bullosa patients had a homozygous single base-pair frameshift mutation in exon 19 of COL7A1 (2470insG). Clinically, there was generalized blistering but only mild scarring. Skin biopsy revealed positive type VII collagen immunoreactivity and recognizable anchoring fibrils. The junctional epidermolysis bullosa patients were compound heterozygotes for a frameshift/non-sense combination of mutations in exons 3 and 17 of LAMB3 (29insC/Q834X). These patients did not have the lethal form of junctional epidermolysis bullosa but, as adults, displayed the milder generalized atrophic benign epidermolysis bullosa variant. There was undetectable laminin 5 staining at the dermal-epidermal junction using an antibody to the beta3 chain, but faintly positive alpha3 and gamma2 chain labeling, and there was variable hypoplasia of hemidesmosomes. To explain the milder recessive dystrophic epidermolysis bullosa and junctional epidermolysis bullosa phenotypes in these families, reverse transcription-polymerase chain reaction, using RNA extracted from frozen skin, was able to provide evidence for some rescue of mutant mRNA transcripts with restoration of the open- reading frame. In the recessive dystrophic epidermolysis bullosa patients, transcripts containing in-frame skipping of exon 19 of COL7A1 in the cDNA were detected, and in the junctional epidermolysis bullosa patients transcripts with in-frame skipping of exon 17 of LAMB3 were identified. The truncated proteins encoded by these transcripts are expected to lack certain critical domains involved in cell-matrix attachment, but may still be able to contribute to adhesion thereby moderating the severity of the skin blistering. This study shows the limitations in predicting phenotype in epidermolysis bullosa solely based on mutation analysis of genomic DNA and emphasizes the importance of immunohistochemistry, electron microscopy, and mRNA assessment as parallel investigations.

[Pachyonychia congenita. Keratin gene mutations with pleiotropic effect]. / Pachyonychia congenita. Keratingen-Mutationen mit pleiotroper Wirkung.

Pachyonychia congenita (PC) comprises a heterogeneous group of autosomal dominantly inherited conditions showing characteristic nail thickening and associated signs such as palmoplantar keratoderma, follicular keratoses, and mucosal leukokeratoses. Less frequently epidermal cysts, hairshaft abnormalities, natal teeth and laryngeal involvement may be seen. Phenotypically and genetically two major forms of PC are recognized, pachyonychia congenita Jadassohn-Lewandowsky/PC type I (Medelian inheritance in man-MIM-167200) and pachyonychia congenita Jackson-Lawler/PC type II (MIM 167210). Both conditions show nail deformities, focal palmoplantar keratoderma, and follicular hyperkeratoses. Diagnostically relevant are leukokeratoses of the oral mucosa in patients with PC type I. In contrast individuals affected with PC type II show premature dentition and multiple pilosebaceous cysts predominantly affecting the upper trunk. The latter closely resemble eruptive vellus hair cysts and steatocystoma multiplex. By mutational analysis keratin K6a and K16 gene mutations have been detected in patients with PC type I, and keratin K6b and K17 gene mutations have been shown to be the underlying genetic defect in patients with PC type II.

Association of lymphocytic colitis with linear IgA dermatosis.

The case of a 66-year-old female patient is presented, who suffered from chronic watery diarrhea. In addition, she developed linear IgA dermatosis after oral treatment of a presumed yeast infection with nystatin. To evaluate the reason for her diarrhea, colonoscopy was performed. The macroscopic aspect of the colon mucosa was described as normal with no specific alterations for chronic inflammatory bowel disease or for bacterial infections. In contrast, the histologic examination revealed the typical characteristics of lymphocytic colitis. This disease is thought to be caused by immunological reactions against as yet unknown luminal antigens. After treatment with steroids and dapsone the diarrhea as well as the skin disease disappeared. To our knowledge, the present report describes for the first time the association of linear IgA dermatosis with lymphocytic colitis after oral treatment with nystatin. A possible causative link between these two disease entities is discussed.

Comèl-Netherton syndrome complicated by papillomatous skin lesions containing human papillomaviruses 51 and 52 and plane warts containing human papillomavirus 16.

We describe a 28-year-old woman with characteristic clinical signs of Comèl-Netherton syndrome (CNS) who showed numerous plane warts on her face and forearms and papillomatous skin tumours affecting her groins and genitoanal skin. Using human papillomavirus (HPV) type-specific primers for cutaneous and mucosal HPV types we identified HPV 16-specific sequences in plane warts and HPV 51- and HPV 52-specific DNA in papillomatous skin from the patient's groins, suggesting a pathogenetic role (cofactor) for HPV in the development of verrucous skin lesions in patients with CNS. Whether the susceptibility to HPV infections is due to decreased cellular immunity or epidermal defence mechanisms remains to be seen.

Linear IgA disease associated with lymphocytic colitis.

A 66-year-old woman presented with a bullous skin eruption and chronic diarrhoea. Lesional skin showed subepidermal blistering, and direct immunofluorescence of perilesional skin revealed linear deposits of IgA at the dermoepidermal junction, establishing a diagnosis of linear IgA disease (LAD). Chronic watery diarrhoea complicated by substantial loss of body weight preceded the skin eruption for several months. On endoscopy, the colon appeared macroscopically normal. On histology, the colon mucosa showed increased numbers of intraepithelial lymphocytes and infiltrates of mononuclear cells in the lamina propria, indicative of lymphocytic colitis. Treatment with methylprednisolone and dapsone led to complete clearing of the bullous skin eruption and marked improvement of the patient's diarrhoea. Gastrointestinal disorders such as lymphocytic colitis have rarely been reported in patients with LAD. Whether the simultaneous occurrence of these two diseases is coincidental or due to related pathogenetic mechanisms remains to be seen.

[Progressive disseminated essential telangiectasia with conjunctival involvement]. / Progressive disseminierte essentielle Teleangiektasien mit konjunktivaler Beteiligung.

BACKGROUND: Widespread idiopathic telangiectasia (generalized essential telangiectasia) is a rare skin disorder characterized by the development and gradual spreading of telangiectases. The condition tends to affect women in their midthirties. For no apparent reason telangiectases start to appear to the lower extremities and progress steadily to involve the skin of the trunk, the arms, and the face. General health is not affected by the condition and standard laboratory tests consistently yield normal results. CASE REPORT: In February 1997 a 78-year-old lady was admitted for treatment of cataracta corticonuclearis of her left eye. Complete ophthalmological and dermatological examinations were performed. She presented marked conjunctival telangiectases of both eyes and widespread cutaneous telangiectases involving her face, trunk, arms, and legs. Complete blanching of lesional skin was observed on diascopy. The Rumpel-Leede-test was normal. Cutaneous and conjunctival changes appeared not to be associated with internal disease or bleeding abnormalities. DISCUSSION: The patient presented here shows widespread idiopathic telangiectasia with marked conjunctival involvement. Ocular changes rarely have been reported in patients with generalized essential telangiectasia to date. Concomittant conjunctival and cutaneous telangiectases may be seen in other conditions such as hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber disease) and ataxia telangiectasia (Louis-Bar syndrome). The former shows an associated bleeding abnormality and is transmitted autosomal dominantly. The latter presents associated neurological signs such as cerebellar ataxia, strabism, nystagmus, apraxia, and mental retardation.

Generalized atrophic benign epidermolysis bullosa in 2 siblings complicated by multiple squamous cell carcinomas.

BACKGROUND: Generalized atrophic benign epidermolysis bullosa is a form of junctional epidermolysis bullosa characterized by skin fragility; atrophic alopecia; sparse eyebrows, eyelashes, and axillary and pubic hair; dystrophic fingernails and toenails; and enamel defects in decidual and permanent teeth. Substantial progress was recently made elucidating the genetic defects underlying this disorder. In affected persons, pathogenetic mutations were identified in the genes encoding the beta 3 chain of laminin 5 (LAMB3) or the 180-kd bullous pemphigoid antigen (BPAG2/COL17A1). OBSERVATIONS: Two brothers, aged 39 and 32 years, had characteristic clinical features of generalized atrophic benign epidermolysis bullosa. By electron microscopy, dermoepidermal separation was seen at the level of the lamina lucida, establishing a diagnosis of junctional epidermolysis bullosa. Lesional and clinically unaffected skin showed basal keratinocytes with hypoplastic hemidesmosomes, possibly indicating a defect of hemidesmosomal or associated proteins. Both patients presented with multiple fungating tumors on atrophic and scarred skin on their lower legs; 2 tumors in the older sibling and 4 tumors in the younger sibling were diagnosed as well-differentiated squamous cell carcinomas. Tumor staging elicited no evidence of regional lymph node involvement or systemic disease. Treatment was by microscopically controlled surgery. All wounds were allowed to heal by secondary intention. In both patients, wound healing was markedly delayed and characterized by the formation of abundant granulation tissue and poor re-epithelialization. CONCLUSIONS: In the absence of other apparent risk factors for the development of squamous cell carcinomas, chronic wounding resulting from recurrent skin blistering probably provided an important prerequisite for tumor promotion in these patients. The 2 cases presented herein provide evidence that the development of malignant skin tumors in patients with epidermolysis bullosa is not confined to the dystrophic forms but also may occur in some variants of junctional epidermolysis bullosa, such as generalized atrophic benign epidermolysis bullosa.

Specialized keratin expression pattern in human ridged skin as an adaptation to high physical stress.

We have analysed the expression of keratins in the epidermis of normal human palm and sole skin (ridged skin) using immunohistochemistry and in situ hybridization. The epidermis of human ridged skin expresses a more complex pattern of keratins than thin skin, which is probably due to the greater stress that ridged skin has to withstand. In addition to keratin K9, we document specific expression patterns of keratins K6, K16 and K17 which are suggestive of regional adaptations of this epidermis to a high cell turnover rate. In particular, the sequestered location of nests of K17-positive cells at the bottom of the deep primary epidermal ridges supports the notion of functional heterogeneity of basal cells and suggests that the K17-positive sites may include stem cells. Expression of K6 and K16 in some basal and most suprabasal keratinocytes is compatible with a constitutively high proliferative activity of normal ridged epidermis, but may also reflect different physical properties of the suprabasal cells, in contrast with regions expressing K9. The distinct labelling patterns observed in primary and secondary epidermal ridges as well as epidermal layers above dermal papillae suggest the existence of local microenvironmental niches leading to differences in keratinocyte differentiation.

Mutations in cornea-specific keratin K3 or K12 genes cause Meesmann's corneal dystrophy.

The intermediate filament cytoskeleton of corneal epithelial cells is composed of cornea-specific keratins K3 and K12 (refs 1,2). Meesmann's corneal dystrophy (MCD) is an autosomal dominant disorder causing fragility of the anterior corneal epithelium, where K3 and K12 are specifically expressed. We postulated that dominant-negative mutations in these keratins might be the cause of MCD. K3 was mapped to the type-II keratin gene cluster on 12q; and K12 to the type-I keratin cluster on 17q using radiation hybrids. We obtained linkage to the K12 locus in Meesmann's original German kindred (Zmax = 7.53; theta = 0) and we also showed that the phenotype segregated with either the K12 or the K3 locus in two Northern Irish pedigrees. Heterozygous missense mutations in K3 (E509K) and in K12 (V143L; R135T) completely co-segregated with MCD in the families and were not found in 100 normal unrelated chromosomes. All mutations occur in the highly conserved keratin helix boundary motifs, where dominant mutations in other keratins have been found to severely compromise cytoskeletal function, leading to keratinocyte fragility phenotypes. Our results demonstrate for the first time the molecular basis of Meesmann's corneal dystrophy.

Gene targeting at the mouse cytokeratin 10 locus: severe skin fragility and changes of cytokeratin expression in the epidermis.

Bullous congenital ichthyosiform erythroderma (BCIE) is a dominantly inherited blistering skin disorder caused by point mutations in the suprabasal cytokeratins 1 or 10. Targeting the murine cytokeratin 10 gene in ES cells resulted in mice with different phenotypes in the homozygotes and heterozygotes; both of which exhibit similarities to specific clinical characteristics of BCIE. Homozygotes suffered from severe skin fragility and died shortly after birth. Heterozygotes were apparently unaffected at birth, but developed hyperkeratosis with age. In both genotypes, aggregation of cytokeratin intermediate filaments, changes in cytokeratin expression, and alterations in the program of epidermal differentiation were observed. In addition we demonstrate, for the first time, the existence of the murine equivalent of human cytokeratin 16.

Morphology of the keratin filament network in palm and sole skin: evidence for site-dependent features based on stereological analysis.

Ridged or glabrous skin of palms and soles has a specialized function and can be preferentially involved in various disorders of keratinization. To better define the morphological features of ridged skin, we carried out a qualitative and quantitative (stereological) analysis of normal epidermis from the palm and sole of four subjects. Skin from the upper arm was examined for control purposes. The study focused on the appearance and arrangement of the keratin filament network in relation to epidermal differentiation. Whereas palm and sole epidermis was essentially similar both qualitatively and quantitatively, it differed markedly from the epidermis from the arm. The volume density of keratin filaments was significantly higher (P < 0.03) in all subcorneal layers of the palm and sole compared with the arm. The volume density of the keratin filaments increased markedly from the basal to the upper spinous layer of ridged skin and they formed denser aggregates in the upper spinous and granular layers, providing an extensive matrix for the deposition of keratohyalin. The presence of dense keratin aggregates appeared to be a distinct ultrastructural feature of human ridged skin. Such keratin aggregates have not been described in normal skin from other sites, but showed some resemblance to the keratin clumps seen in non-ridged skin of patients with the Dowling-Meara form of epidermolysis bullosa simplex.

Keratin 16 and keratin 17 mutations cause pachyonychia congenita.

Pachyonychia congenita (PC) is a group of autosomal dominant disorders characterized by dystrophic nails and other ectodermal aberrations. A gene for Jackson-Lawler PC was recently mapped to the type I keratin cluster on 17q. Here, we show that a heterozygous missense mutation in the helix initiation motif of K17 (Asn92Asp) co-segregates with the disease in this kindred. We also show that Jadassohn-Lewandowsky PC is caused by a heterozygous missense mutation in the helix initiation peptide of K16 (Leu130Pro). The known expression patterns of these keratins in epidermal structures correlates with the specific abnormalities observed in each form of PC.

Ultrastructural changes resulting from keratin-9 gene mutations in two families with epidermolytic palmoplantar keratoderma.

Palmoplantar keratoderma of Voerner type (or epidermolytic palmoplantar keratoderma) is an autosomal dominant inherited disorder of keratinization with histologic features of epidermolytic hyperkeratosis. We studied members of two large unrelated kindreds with epidermolytic palmoplantar keratoderma, and biopsy specimens of lesional palmar skin from both families confirmed the histologic changes of epidermolytic hyperkeratosis. Whorls of abnormally aggregated keratin filaments were seen ultrastructurally to be associated with signs of cellular disintegration in spinous and granular cells. Direct sequencing of genomic DNA samples obtained from several members of each family established the substitution of a highly conserved arginine by tryptophan (R162W) in the 1A region of the alpha-helical rod domain of keratin 9. This arginine residue in a highly conserved region of keratins 1 and 10 is affected by disruptive missense point mutations in many patients with bullous ichthyosiform erythroderma. An equivalent position in the sole and palm restricted keratin 9 appears to be the mutation hot spot in epidermolytic palmoplantar keratoderma. To date, R162W is the most prevalent genetic defect reported in this genodermatosis.

Analysis of a terminal Xp22.3 deletion in a patient with six monogenic disorders: implications for the mapping of X linked ocular albinism.

The molecular characterisation of chromosomal aberrations in Xp22.3 has established the map position of several genes with mutations resulting in diverse phenotypes such as short stature (SS), chondrodysplasia punctata (CDPX), mental retardation (MRX), ichthyosis (XLI), and Kallmann syndrome (KAL). We describe the clinical symptoms of a patient with a complex syndrome compatible with all these conditions plus ocular albinism (OA1). He has a terminal Xp deletion of at least 10 Mb of DNA. Both the mother and sister of the patient are carriers of the deletion and show a number of traits seen in Turner's syndrome. The diagnosis of ocular albinism was confirmed in the patient and his mother, who shows iris translucency, patches and streaks of hypopigmentation in the fundus, and macromelanosomes in epidermal melanocytes. By comparative deletion mapping we can define a deletion interval, which locates the OA1 gene proximal to DXS143 and distal to DXS85, with the breakpoints providing valuable starting points for cloning strategies.