Human keratin 8 mutations that disturb filament assembly observed in inflammatory bowel disease patients.

We have identified miss-sense mutations in keratin 8 in a subset of patients with inflammatory bowel disease (Crohn disease and ulcerative colitis). Inflammatory bowel diseases are a group of disorders that are polygenic in origin and involve intestinal epithelial breakdown. We investigated the possibility that these keratin mutations might contribute to the course of the disease by adversely affecting the keratin filament network that provides mechanical support to cells in epithelia. The mutations (Gly62 to Cys, Ile63 to Val and Lys464 to Asn) all lie outside the major mutation hotspots associated with severe disease in epidermal keratins, but using a combination of in vitro and cell culture assays we show that they all have detrimental effects on K8/K18 filament assembly in vitro and in cultured cells. The G62C mutation also gives rise to homodimer formation on oxidative stress to cultured intestinal epithelial cells, and homodimers are known to be polymerization incompetent. Impaired keratin assembly resulting from the K8 mutations found in some inflammatory bowel disease patients would be predicted to affect the maintenance and re-establishment of mechanical resilience in vivo, as required during keratin cytoskeleton remodeling in cell division and differentiation, which may lead to epithelial fragility in the gut. Simple epithelial keratins may thus be considered as candidates for genes contributing to a risk of inflammatory bowel disease.

Generation and characterization of epidermolysis bullosa simplex cell lines: scratch assays show faster migration with disruptive keratin mutations.

BACKGROUND: Epidermolysis bullosa simplex (EBS) is an inherited skin fragility disorder caused by mutations in keratin intermediate filament proteins. While discoveries of these mutations have increased understanding of the role of keratins and other intermediate filaments in epithelial tissues, progress towards the development of therapy for these disorders is much slower. OBJECTIVES: Cell culture model systems that display these structural defects are needed for analysis of the cellular consequences of the mutations and to enable possible therapeutic strategies to be developed. Our aim was to generate immortalized cell lines as such model systems for the study of EBS. METHODS: We generated a series of stable cell lines expressing EBS-associated keratin mutations, by immortalizing keratinocytes from EBS-affected skin biopsies with either simian virus 40 (SV40) T antigen or human papillomavirus 16 (HPV16) E6/E7, and assessed their keratin expression (by immunofluorescence), proliferation rates and migratory behaviour (in outgrowth and scratch wound assays). RESULTS: Clonal immortalized keratinocyte cell lines KEB-1, KEB-2, KEB-3 (using SV40 T antigen) and KEB-4, KEB-7 and NEB-1 (using HPV16 E6/E7) were established. These include two lines from a single individual with Weber-Cockayne EBS (i.e. KEB-3 and KEB-4, mutation K14 V270M), and three cell lines from a second family, two from siblings carrying the same mutation (KEB-1, KEB-2 lines from Dowling-Meara EBS, mutation K5 E475G) and one from an unaffected relative (NEB-1). The sixth cell line (KEB-7), with a previously unreported severe mutation (K14 R125P), was the only one to show keratin aggregates in resting conditions. Despite variations in the immortalization procedure, there was no significant difference between cell lines in keratin expression, outgrowth capabilities or response to transient heat shock. However, cell migration, as measured by speed of scratch wound closure, was significantly faster in cells with severe EBS mutations. CONCLUSIONS: These cell lines provide useful culture systems in which to assess aspects of EBS-induced cell changes. The faster migration after scratch wounding of the EBS keratinocytes may be a consequence of the known upregulation of stress-activated kinase pathways in these cells.

Diagnosis and confirmation of epidermolytic palmoplantar keratoderma by the identification of mutations in keratin 9 using denaturing high-performance liquid chromatography.

BACKGROUND: Epidermolytic palmoplantar keratoderma (EPPK) is one of a number of disorders characterized by diffuse thickening of palm and sole skin. Although EPPK is not a life-threatening condition, palmoplantar keratoderma can be associated with cancer and heart disease and therefore differential diagnosis is important so that adequate surveillance can be provided for the more serious conditions. Most cases of EPPK are caused by mutations in the gene encoding the palm- and sole-specific keratin 9 (K9), and this provides an option for molecular diagnosis of this condition. OBJECTIVES: To identify the molecular basis of diffuse palmoplantar keratoderma in four British families. METHODS: Denaturing high-performance liquid chromatography (dHPLC) and DNA sequencing were used to screen exon 1 of the k9 gene for sequence variations. RESULTS: The dHPLC profiles obtained from individuals with EPPK differed from control samples, indicating sequence variations within the fragment analysed. The profiles varied between families, suggesting that underlying mutations were different for each family; this was confirmed by DNA sequencing. In three cases previously reported mutations were found that resulted in the change of methionine156 to valine and arginine162 to either tryptophan or glutamine. A novel mutation was identified in a fourth family that changed valine170 to methionine. dHPLC was used to screen control samples for this sequence variation and confirmed that it was not a common polymorphism. CONCLUSIONS: These results confirm the diagnosis of EPPK in these families and underline the usefulness of dHPLC as a method of screening samples for heterozygous mutations.

Multiple epidermal connexins are expressed in different keratinocyte subpopulations including connexin 31.

Recent genetic studies have demonstrated the importance of epidermal gap junctions with mutations in four beta-connexins associated with autosomal dominant epidermal disease. One of these disorders, erythrokeratoderma variabilis, is associated with germline mutations in the genes encoding connexins (Cx) Cx31 and Cx30.3. Towards understanding the functional mechanism of Cx31 mutations in epidermal disease, we have developed and characterized a polyclonal antibody raised against human Cx31. Using this antibody to immunostain normal epidermis, Cx31 protein was found to be expressed predominately in the stratum granulosum with a punctate pattern of staining at the plasma membrane. In addition, we used reverse transcriptase polymerase chain reaction and, where reagents were available, immunocytochemistry to investigate which other connexins are expressed in the epidermis. Surprisingly, this analysis revealed that there are at least 10 connexins expressed with an overlapping distribution and localization to distinct keratinocyte subpopulations. These data provide additional evidence for multiple gap junction channel types in the human epidermis. Elucidation of this complexity of channel types with respect to specific permeabilities and function of each wildtype and mutant channel type in epidermal biology will require further investigations.

A novel mutation in the keratin 13 gene causing oral white sponge nevus.

White sponge nevus (WSN) is an autosomal-dominantly inherited form of mucosal leukokeratosis. Defects in keratins, proteins that form the stress-bearing cytoskeleton in epithelia, have been shown to cause several epithelial fragility disorders. Recently, mutations in the genes encoding mucosal-specific keratins K4 and K13 were shown to be the underlying cause of WSN. We have studied a large Scottish family with 19 persons affected by WSN in four generations. The K4 locus was excluded by genetic linkage analysis; however, genetic linkage consistent with a K13 defect was obtained. Subsequently, a heterozygous missense mutation 335A>G was detected in exon 1 of the KRT13 gene, predicting the amino acid change N112S in the 1A domain of the K13 polypeptide. The mutation was confirmed in affected family members and was excluded from 50 unaffected people by restriction enzyme analysis. These results confirm that mucosal keratin defects are the cause of WSN.

Molecular confirmation of the unique phenotype of epidermolysis bullosa simplex with mottled pigmentation.

BACKGROUND: A distinctive subtype of epidermolysis bullosa simplex, with the additional feature of mottled pigmentation (EBS-MP), was initially characterized in a Swedish family in 1979, and seven further families have been reported. Features of EBS-MP that are observed in most affected patients include acral blistering early in childhood, mottled pigmentation distributed in a number of sites, focal punctate hyperkeratoses of the palms and soles, and dystrophic, thickened nails. The genetic basis of EBS-MP has been ascribed in five unrelated families to a heterozygous point mutation, P25L, in the non-helical V1 domain of K5. OBJECTIVES: We report a clinical, ultrastructural and molecular study of two of the earliest families to be clinically characterized as EBS-MP. METHODS: The P25L mutation was identified in all affected members of each of these families, bringing the total number of EBS-MP families with this mutation to seven. RESULTS: This unusual recurrent mutation may uniquely cause EBS-MP. CONCLUSIONS: While the exact molecular mechanisms by which this mutation causes epidermolysis, palmoplantar keratoderma and pigmentation remain elusive, we suggest possible molecular mechanisms through which the P25L substitution could cause this unusual phenotype.

K15 expression implies lateral differentiation within stratified epithelial basal cells.

Keratins are intermediate filament proteins whose expression in epithelial tissues is closely linked to their differentiated state. The greatest complexity of this expression is seen in the epidermis and associated structures. The critical basal (proliferative) cell layer expresses the major keratin pair, K5 and K14, but it also expresses an additional type I keratin, K15, about which far less is known. We have compared the expression of K15 with K14 in normal, pathological, and tissue culture contexts; distinct differences in their expression patterns have been observed that imply different regulation and function for these two genes. K15 appears to be preferentially expressed in stable or slowly turning over basal cells. In steady-state epidermis, K15 is present in higher amounts in basal cells of thin skin but in lower amounts in the rapidly turning over thick plantar skin. Although remaining high in basal cell carcinomas (noninvasive) it is suppressed in squamous cell carcinomas (which frequently metastasize). Wounding-stimulated epidermis loses K15 expression, whereas K14 is unchanged. In cultured keratinocytes, K15 levels are suppressed until the culture stratifies, whereas K14 is constitutively expressed throughout. Therefore, unlike K14, which appears to be a fundamental component of all keratinocytes, K15 expression appears to be more tightly coupled to a mature basal keratinocyte phenotype.

A keratin 14 'knockout' mutation in recessive epidermolysis bullosa simplex resulting in less severe disease.

Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused in most cases by mis-sense mutations in genes encoding the basal epidermal keratin (K) 5 and K14. The inheritance is usually autosomal dominant and the mutant keratin proteins appear to exert a dominant negative effect on the keratin intermediate filament cytoskeleton in basal keratinocytes. We report a child with a homozygous K14 mutation resulting in the complete absence of K14 protein in the epidermis; remarkably, he only had mild to moderate disease. Electron microscopy of a skin biopsy showed a marked reduction in numbers of keratin intermediate filaments in the basal keratinocytes. Immunofluorescence microscopy using monoclonal antibody LL001 against K14 showed no staining, suggesting a functional knockout of K14. Sequence analysis of genomic DNA revealed a homozygous mutation in codon 31 of K14 that resulted in a premature stop codon further downstream in exon 1. The child's mother, who is unaffected by the disease, is heterozygous for the mutation. The consanguineous father was unaffected and unavailable for testing. The resulting mRNA is predicted to encode a protein of 116 amino acids, of which the first 30 are identical to the normal K14 sequence, and the remaining 86 residues are mis-sense sequence. Four previously reported cases of autosomal recessive EBS with functional knockout of K14 were severely affected by blistering, in contrast to our patient in whom the predicted protein has only the first 30 amino acids of K14 and is therefore the closest to a true knockout of K14 protein yet identified.

DNA based prenatal testing for the skin blistering disorder epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is a skin fragility disorder in which mild physical trauma leads to blistering. The phenotype of the disorder is variable, from relatively mild affecting only the hands and/or feet, to very severe with widespread blistering. For the severest forms of EBS there is a demand for prenatal diagnosis which until now has involved a fetal skin biopsy in the second trimester. The identification of mutations in the genes encoding keratins K5 and K14 as the cause of EBS opens up the possibility of much earlier diagnosis of the disease. We report here four cases in which prenatal testing was performed. In three of the cases the genetic lesions were unknown at the start of the pregnancy, requiring the identification of the causative mutation prior to testing fetal DNA. In two of the four cases novel mutations were identified in K14 and in the two remaining families, a previously identified type of mutation was found. Fetal DNA, obtained by chorionic villus sampling or amniocentesis, was analysed for the identified mutations. Three of the DNA samples were found to be normal; a mutant K14 allele was identified in the fourth case and the pregnancy was terminated. These results demonstrate the feasibility of DNA-based prenatal testing for EBS in families where causative mutations can be found.

Identification of two novel mutations in keratin 13 as the cause of white sponge naevus.

BACKGROUND: White sponge naevus (WSN) is a rare autosomal dominant condition which is characterised by benign, white spongy plaques (oral leukokeratoses) affecting non-cornifying, wet mucosa. WSN shares several ultrastructural characteristics (eg, epithelial thickening, acanthosis, keratin filament aggregation) with a number of epithelial disorders caused by mutations in keratin genes and to-date two mutations, one in each of the mucosal specific keratins, K4 and K13, have been identified as the molecular basis of the disorder. OBJECTIVES: To identify the molecular basis of WSN in two families with a history of the disease. RESULTS: Two novel mutations were identified in helix initiation motif of K13. A T-to-C transition was found in the affected members of one family which is predicted to change leucine115 to proline. In the second family, a similar T-to-C transition was found in codon 108 which is predicted to change methionine to threonine in the protein sequence. These changes were not found in 50 unrelated, unaffected individuals. CONCLUSIONS: The mutations in the helix initiation motif of K13 are the cause of WSN in these families. These cases confirm mutations in the mucosal specific keratins as a significant cause of the disorder.

Donor splice site mutation in keratin 5 causes in-frame removal of 22 amino acids of H1 and 1A rod domains in Dowling-Meara epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) arises from mutations within the keratin 5 and 14 (K5 and K14) genes which alter the integrity of basal keratinocytes cytoskeleton. The majority of these defects are missense mutations in the rod domain, whose locations influence the disease severity. We investigated a large family dominantly affected with the Dowling-Meara form of EBS (EBS-DM). Sequencing of amplified and cloned K5 cDNA from cultured keratinocytes revealed a 66 nucleotide deletion in one allele corresponding to the last 22 amino acid residues encoded by exon 1 (Val164 to Lys185). Sequencing of amplified genomic DNA spanning the mutant region revealed a heterozygous G-to-A transition at +1 position of the consensus GT donor splice site of intron 1 of K5. This mutation leads to the use of an exonic GT cryptic donor splice site, located 66 nucleotides upstream from the normal donor splice site of intron 1. The corresponding peptide deletion includes the last five amino acids of the H1 head domain and the first 17 amino acids of the conserved amino terminal end of the 1A rod domain, including the first two heptad repeats and the helix initiation peptide. The shortened polypeptide is expressed in cultured keratinocytes at levels which are comparable to the normal K5 protein. This is the first splice site mutation to be reported as a cause of EBS-DM. Owing to the functional importance of the removed region, our data strongly suggest that shortened keratin polypeptide can impair keratin filament assembly in a dominant manner and causes EBS-DM.

cDNA cloning, expression, and assembly characteristics of mouse keratin 16.

There has been speculation as to the existence of the mouse equivalent of human type I keratin 16 (K16). The function of this keratin is particularly intriguing because, in normal epidermis, it is usually confined to hair follicles and only becomes expressed in the suprabasal intrafollicular regions when the epidermis is traumatized. Previous studies suggested that K16 is highly expressed in the skin of mice carrying a truncated K10 gene. We therefore used the skin of heterozygous and homozygous mice to create a cDNA library, and we report here the successful cloning and sequencing of mouse K16. Recent in vitro studies suggested that filaments formed by human K16 are shorter than those formed by other type I keratins. One hypothesis put forward was that a proline residue in the 1B subdomain of the helical domain was responsible. The data presented here demonstrate that this proline is not conserved between mouse and human, casting doubt on the proposed function of this proline residue in filament assembly. In vitro assembly studies showed that mouse K16 produced long filaments in vitro. Also, in contrast to previous observations, transfection studies of PtK2 cells showed that mouse K16 (without the proline) and also human K16 (with the proline) can incorporate into the endogenous K8/K18 network without detrimental effect. In addition, K16 from both species can form filaments de novo when transfected with human K5 into immortalized human lens epithelial cells, which do not express keratins. These results suggest that reduced assembly capabilities due to unusual sequence characteristics in helix 1B are not the key to the unique function of K16. Rather, these data implicate the tail domain of K16 as the more likely protein domain that determines the unique functions.

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.

Plectin deficiency results in muscular dystrophy with epidermolysis bullosa.

We report that mutation in the gene for plectin, a cytoskeleton-membrane anchorage protein, is a cause of autosomal recessive muscular dystrophy associated with skin blistering (epidermolysis bullosa simplex). The evidence comes from absence of plectin by antibody staining in affected individuals from four families, supportive genetic analysis (localization of the human plectin gene to chromosome 8q24.13-qter and evidence for disease segregation with markers in this region) and finally the identification of a homozygous frameshift mutation detected in plectin cDNA. Absence of the large multifunctional cytoskeleton protein plectin can simultaneously account for structural failure in both muscle and skin.

Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organization.

Plectin is a widely expressed high molecular weight protein that is involved in cytoskeleton-membrane attachment in epithelial cells, muscle, and other tissues. The human autosomal recessive disorder epidermolysis bullosa with muscular dystrophy (MD-EBS) shows epidermal blister formation at the level of the hemidesmosome and is associated with a myopathy of unknown etiology. Here, plectin was found to be absent in skin and cultured keratinocytes from an MD-EBS patient by immunofluorescence and immunoprecipitation, suggesting that plectin is a candidate gene/protein system for MD-EBS mutation. The 14800-bp human plectin cDNA was cloned and sequenced. The predicted 518-kD polypeptide has homology to the actin-binding domain of the dystrophin family at the amino terminus, a central rod domain, and homology to the intermediate filament-associated protein desmoplakin at the carboxyl terminus. The corresponding human gene (PLEC1), consisting of 33 exons spanning >26 kb of genomic DNA was cloned, sequenced, and mapped to chromosomal band 8q24. Homozygosity by descent was observed in the consanguineous MD-EBS family with intragenic plectin polymorphisms. Direct sequencing of PCR-amplified plectin cDNA from the patient's keratinocytes revealed a homozygous 8-bp deletion in exon 32 causing a frameshift and a premature termination codon 42 bp downstream. The clinically unaffected parents of the proband were found to be heterozygous carriers of the mutation. These results establish the molecular basis of MD-EBS in this family and clearly demonstrate the important structural role for plectin in cytoskeleton-membrane adherence in both skin and muscle.

A mutation in the mucosal keratin K4 is associated with oral white sponge nevus.

White sponge nevus (WSN) is a benign autosomal dominant disorder which affects non-cornifying stratified squamous epithelia (MIM 193900) (ref. 1). Phenotypically it presents as white 'spongy' plaques (oral leukokeratoses), most commonly in the mouth but also reported in the esophagus and anogenital mucosa. Histologically, the plaques show evidence of hyperproliferation, acanthosis and tonofilament aggregation. These types of pathogenic changes are characteristic of many of the epidermal keratin disorders. Keratins are expressed in pairs by epithelial cells in a tissue and cell specific manner. The major differentiation specific keratins of the buccal mucosa, nasal, esophageal and anogenital epithelia are K4 and K13 (ref. 7). The tissue distribution and nature of the lesions in patients affected by WSN suggested that mutations in K4 and/or K13 might be responsible for this disorder. We have now confirmed this hypothesis and report here a three base-pair (bp) deletion in the helix initiation peptide of K4 in affected members from two families with this condition.

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.

A functional "knockout" of human keratin 14.

The importance of keratins and other intermediate filaments in the maintenance of tissue structure is emphasized by the discovery that many hereditary skin-blistering diseases are caused by mutations in keratin genes. Here, we describe a situation in which keratin 14 (K14) is missing altogether in the epidermis: A homozygous 2-nucleotide deletion in exon I of the K14 gene causes premature termination of the mRNA transcripts upstream from the start of the rod domain and results in a K14 null phenotype. In this individual no keratin intermediate filaments are visible in basal epidermal cells, although filaments are present in the upper layers of the epidermis. No compensating keratin expression is detected in vivo, and K14 mRNA is down-regulated. The individual, diagnosed as Köbner (generalized) EBS, suffers from severe widespread keratinocyte fragility and blistering at many body sites, but although the phenotype is severe, it is not lethal. This K14-/- phenotype confirms that only one K14 gene is expressed in human epidermis and provides an important model system for examining the interdependence of different keratin filament systems and their associated structures in the skin.