Keratin K6irs is specific to the inner root sheath of hair follicles in mice and humans.

BACKGROUND: Keratins are a multigene family of intermediate filament proteins that are differentially expressed in specific epithelial tissues. To date, no type II keratins specific for the inner root sheath of the human hair follicle have been identified. OBJECTIVES: To characterize a novel type II keratin in mice and humans. METHODS: Gene sequences were aligned and compared by BLAST analysis. Genomic DNA and mRNA sequences were amplified by polymerase chain reaction (PCR) and confirmed by direct sequencing. Gene expression was analysed by reverse transcription (RT)-PCR in mouse and human tissues. A rabbit polyclonal antiserum was raised against a C-terminal peptide derived from the mouse K6irs protein. Protein expression in murine tissues was examined by immunoblotting and immunofluorescence. RESULTS: Analysis of human expressed sequence tag (EST) data generated by the Human Genome Project revealed a fragment of a novel cytokeratin mRNA with characteristic amino acid substitutions in the 2B domain. No further human ESTs were found in the database; however, the complete human gene was identified in the draft genome sequence and several mouse ESTs were identified, allowing assembly of the murine mRNA. Both species' mRNA sequences and the human gene were confirmed experimentally by PCR and direct sequencing. The human gene spans more than 16 kb of genomic DNA and is located in the type II keratin cluster on chromosome 12q. A comprehensive immunohistochemical survey of expression in the adult mouse by immunofluorescence revealed that this novel keratin is expressed only in the inner root sheath of the hair follicle. Immunoblotting of murine epidermal keratin extracts revealed that this protein is specific to the anagen phase of the hair cycle, as one would expect of an inner root sheath marker. In humans, expression of this keratin was confirmed by RT-PCR using mRNA derived from plucked anagen hairs and epidermal biopsy material. By this means, strong expression was detected in human hair follicles from scalp and eyebrow. Expression was also readily detected in human palmoplantar epidermis; however, no expression was detected in face skin despite the presence of fine hairs histologically. CONCLUSIONS: This new keratin, designated K6irs, is a valuable histological marker for the inner root sheath of hair follicles in mice and humans. In addition, this keratin represents a new candidate gene for inherited structural hair defects such as loose anagen syndrome.

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.

Cyclic ichthyosis with epidermolytic hyperkeratosis: A phenotype conferred by mutations in the 2B domain of keratin K1.

Bullous congenital ichthyosiform erythroderma (BCIE) is characterized by blistering and erythroderma in infancy and by erythroderma and ichthyosis thereafter. Epidermolytic hyperkeratosis is a hallmark feature of light and electron microscopy. Here we report on four individuals from two families with a unique clinical disorder with histological findings of epidermolytic hyperkeratosis. Manifesting erythema and superficial erosions at birth, which improved during the first few months of life, affected individuals later developed palmoplantar hyperkeratosis with patchy erythema and scale elsewhere on the body. Three affected individuals exhibit dramatic episodic flares of annular, polycyclic erythematous plaques with scale, which coalesce to involve most of the body surface. The flares last weeks to months. In the interim periods the skin may be normal, except for palmoplantar hyperkeratosis. Abnormal keratin-filament aggregates were observed in suprabasal keratinocytes from both probands, suggesting that the causative mutation might reside in keratin K1 or keratin K10. In one proband, sequencing of K1 revealed a heterozygous mutation, 1436T-->C, predicting a change of isoleucine to threonine in the highly conserved helix-termination motif. In the second family, a heterozygous mutation, 1435A-->T, was found in K1, predicting an isoleucine-to-phenylalanine substitution in the same codon. Both mutations were excluded in both a control population and all unaffected family members tested. These findings reveal that a clinical phenotype distinct from classic BCIE but with similar histology can result from K1 mutations and that mutations at this codon give rise to a clinically unique condition.

Homozygous nonsense mutation in helix 2 of K14 causes severe recessive epidermolysis bullosa simplex.

We have studied a consanguineous family containing two children with severe, generalized epidermolysis bullosa simplex (EBS). Electron microscopy of skin biopsies from the affected individuals showed that basal keratinocytes were devoid of tonofilament bundles, although some single intermediate filament were visible. Genetic linkage analysis with the microsatellite probe D12S96 excluded the type II keratin gene cluster in this family. However, homozygosity by descent was observed with the polymorphic probes KRT9, KRT10 Ava II, and D17S1787 in both affected children, consistent with a recessive defect in a type I keratin. Immunoreactivity to keratin K5 and K15 was normal, but monoclonal antibodies LL001 and RCK107 against K14 showed no staining, suggesting a deficiency of K14 in these individuals. mRNA extracted from biopsy material was amplified by RT-PCR to obtain full-length K14 cDNA. Direct automated sequencing identified a homozygous nonsense mutation, W305X. A Hinf I restriction enzyme site is created by this nucleotide transition, which was used to confirm the presence of the mutation in this kindred and exclude it from 100 normal chromosomes. This is the fourth kindred with severe recessive EBS for whom a mutation has been found in the K14 gene. In this instance, the premature termination codon is the farthest downstream of the reported cases, occurring in the helix 2 domain and so giving a much longer translation product. Nevertheless, the heterozygous carriers are unaffected by the disease and display no epidermal fragility. We postulate that translation of the potentially dominant-negative truncated K14 might be down-regulated due to instability of the mutant mRNA, as observed in previous cases with similar mutations.

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.

Missense mutations in keratin 17 cause either pachyonychia congenita type 2 or a phenotype resembling steatocystoma multiplex.

Pachyonychia congenita (PC) is a group of autosomal dominant ectodermal dysplasias in which the main phenotypic characteristic is hypertrophic nail dystrophy. In the Jackson-Lawler form (PC-2), pachyonychia is accompanied by multiple pilosebaceous cysts, natal teeth, and hair abnormalities. By direct sequencing of genomic PCR products, we report heterozygous K17 missense mutations in the same conserved protein motif in a further five PC-2 families (K17 N92S in one familial and three sporadic cases; K17 Y98D in one familial case) confirming that mutations in this gene are a common cause of PC-2. We also show heterozygous missense mutations in K17 (N92H and R94H) in two families diagnosed as steatocystoma multiplex. Mild nail defects were observed in some but not all of these patients on clinical re-evaluation of these families. All the K17 mutations reported here were shown to co-segregate with the disease in the pedigrees analyzed and were excluded from 100 unaffected, unrelated chromosomes by restriction enzyme analysis of K17 genomic PCR products. We conclude that phenotypic variation is observed with K17 mutations, as is the case with other keratin disorders.

Human keratin diseases: hereditary fragility of specific epithelial tissues.

Keratins are heteropolymeric proteins which form the intermediate filament cytoskeleton in epithelial cells. Since 1991, mutations in several keratin genes have been found to cause a variety of human diseases affecting the epidermis and other epithelial structures. Epidermolysis bullosa simplex (EBS) was the first mechanobullous disease for which the underlying genetic lesion was found, with mutations in both the K5 and K14 genes rendering basal epidermal keratinocytes less resilient to trauma, resulting in skin fragility. The site of mutation in the keratin protein correlates with phenotypic severity in this disorder. Since mutations were identified in the basal cell keratins, the total number of keratin genes associated with diseases has risen to eleven. The rod domains of suprabasal keratins K1 and K10 are mutated in bullous congenital ichthyosiform erythroderma (BCIE; also called epidermolytic hyperkeratosis, EH) and mosaicism for K1/K10 mutations results in a nevoid distribution of EH. An unusual mutation in the VI domain of K1 has also been found to cause diffuse non-epidermolytic palmoplantar keratoderma (DNEPPK). Mutations in palmoplantar specific keratin K9 cause epidermolytic palmoplantar keratoderma (EPPK) and mutations in the late differentiation suprabasal keratin K2e cause ichthyosis bullosa of Siemens (IBS). In the last year or so, mutations were discovered in differentiation specific keratins K6a and K16 causing pachyonychia congenita type 1 and K17 mutations occur in pachyonychia congenita type 2. K16 and K17 mutations have also been reported to produce phenotypes with little or no nail changes: K16 mutations can present as focal non-epidermolytic palmoplantar keratoderma (NEPPK) and K17 mutations can result in a phenotype resembling steatocystoma multiplex. Recently, mutation of mucosal keratin pair K4 and K13 has been shown to underlie white sponge nevus (WSN). This year, the first mutations in a keratin-associated protein, plectin, were shown to cause a variant of epidermolysis bullosa associated with late-onset muscular dystrophy (MD-EBS). An unusual mutation has been identified in K5 which is responsible for EBS with mottled pigmentation and genetic linkage analysis suggests that the hair disorder monilethrix is likely to be due to a mutation in a hair keratin. The study of keratin diseases has led to a better understanding of the importance of the intermediate filament cytoskeleton and associated connector molecules in maintaining the structural integrity of the epidermis and other high stress epithelial tissues, as well as allowing diagnosis at the molecular level thus facilitating prenatal testing for this heterogeneous group of genodermatoses.

Effects of keratin 14 ablation on the clinical and cellular phenotype in a kindred with recessive epidermolysis bullosa simplex.

We studied a kindred with recessive epidermolysis bullosa simplex in which the affected members lacked expression of the basal cell keratin 14. The patients had severe generalized skin blistering that improved slightly with age. The basal cells of the patients did not express keratin 14 and contained no keratin intermediate filaments. The expression of keratin 5, the obligate copolymer of keratin 14, was slightly reduced. The expression of keratin 15, the alternative basal cell keratin, was increased, suggesting upregulation or stabilization to compensate for the lack of keratin 14. The expression of keratin 16, keratin 17, and keratin 19 in the patient's skin was not different from controls. Immunoelectron microscopy showed a loose network of keratin 5/keratin 15 protofilaments in the basal cells. Keratin 15 filaments did not aggregate into higher order bundles. Sequence analysis of genomic DNA revealed a homozygous mutation in the 3'-acceptor splice site of intron 1 (1840 A-->C) in the affected individuals. This mutation led to the skipping of exon 2 in 24% of the KRT14 transcripts and to the use of a cryptic splice site in 76% of the transcripts. Premature termination codons were generated in all transcripts (codons 175+1 or 175+29), leading to a truncated keratin 14 protein within the helical 1B rod domain. The disorder was associated with circumscribed hyperkeratotic lesions with the histology of epidermolytic hyperkeratosis. The prognosis of keratin 14 ablation is much better in the human than in the mouse.