Missing links : weber cockayne keratin mutations implicate the L12 linker domain in effective cytoskeleton function. (1994 Feb)
missing links : weber cockayne keratin mutations implicate the L12 linker domain in effective cytoskeleton function . We have identified mutations in keratins K5 ( arg331cys ) and K14 ( val270met ) in two kinships affected by the dominantly inherited skin blistering disease , weber cockayne epidermolysis bullosa simplex ( EBS WC ) . linkage analysis , DNA sequencing and clinical and ultrastructural analysis are combined to provide the first detailed description of classical EBS WC . Both phenotypes show similar blistering on trauma , indicating that both mutations compromise the structural resilience of the basal keratinocytes by affecting the keratin cytoskeleton . The location of these mutations in the L12 linker , which bisects the alpha helical rod region of intermediate filament proteins , identifies another keratin mutation cluster leading to hereditary skin fragility syndromes .

Identification of a leucine to proline mutation in the keratin 5 gene in a family with the generalized köbner type … (1993 Aug)
identification of a leucine to proline mutation in the keratin 5 gene in a family with the generalized köbner type of epidermolysis bullosa simplex . We have previously reported linkage of a large finnish family with the generalized ( köbner ) type of epidermolysis bullosa simplex to chromosome 12q in the region containing the type II keratin gene cluster ( ryynänen et al . , Am J human genet 49 : 978 984 , 1991 ) . In this study , we examined the possibility that keratin 5 , the type II keratin expressed in the basal keratinocytes , harbors the mutation in this family . nucleotide sequencing revealed a T to C transition within exon 7 of the keratin 5 gene in the affected individuals of the family , while the unaffected individuals showed no evidence of C . The presence of the T to C transition in the affected individuals was confirmed by restriction enzyme digestion analysis with NciI endonuclease , as well as with PCR amplification of specific alleles ( PASA ) analysis . The PASA analysis also indicated that the mutated allele was not found among the 100 alleles tested within the general finnish population indicating that the mutated allele is not a common polymorphism . furthermore , the mutated allele was not present in nine individuals representing three different EBS families of finnish origin . The T to C transition at the nucleotide level resulted in substitution of a leucine by a proline at the …

Dominant and recessive compound heterozygous mutations in epidermolysis bullosa simplex demonstrate the role of the stutter region in keratin intermediate … (2002 Jun)
dominant and recessive compound heterozygous mutations in epidermolysis bullosa simplex demonstrate the role of the stutter region in keratin intermediate filament assembly . keratin intermediate filaments are important cytoskeletal structural proteins involved in maintaining cell shape and function . mutations in the epidermal keratin genes , keratin 5 or keratin 14 lead to the disruption of keratin filament assembly , resulting in an autosomal dominant inherited blistering skin disease , epidermolysis bullosa simplex ( EBS ) . We investigated a large EBS kindred who exhibited a markedly heterogeneous clinical presentation and detected two distinct keratin 5 mutations in the proband , the most severely affected . One missense mutation ( e170k ) in the highly conserved helix initiation peptide sequence of the 1A rod domain was found in all the affected family members . In contrast , the other missense mutation ( e418k ) was found only in the proband . The e418k mutation was located in the stutter region , an interruption in the heptad repeat regularity , whose function as yet remains unclear . We hypothesized that this mutated stutter allele was clinically silent when combined with the wild type allele but aggravates the clinical severity of EBS caused by the e170k mutation on the other allele . To confirm this in vitro , we transfected mutant keratin 5 cDNA into cultured cells . although only 12 . 7 of the cells transfected with the e170k mutation alone showed disrupted keratin filament aggregations , significantly more cells ( …

Mutation analysis of the entire keratin 5 and 14 genes in patients with epidermolysis bullosa simplex and identification of novel … (2003 Mar)
mutation analysis of the entire keratin 5 and 14 genes in patients with epidermolysis bullosa simplex and identification of novel mutations . epidermolysis bullosa simplex is a group of blistering skin disorders caused by defects in one of the keratin genes , KRT5 and krt14 . previously reported KRT5 and krt14 mutations are clustered in several hotspots , namely the rod ends of the 1A and 2B domains and in the non helical linker region L12 . therefore , genomic KRT5 and krt14 mutation analysis was initially limited to these hotspots . In this study we describe the screening of nine EBS patients for mutations in the hotspots . In two patients , with the koebner and the weber cockayne subtypes of epidermolysis bullosa simplex respectively , we could , however , not identify any mutation in one of the hotspot domains of KRT5 and krt14 . therefore , it appeared to be necessary to screen the entire genes for mutations . For KRT5 , a complete genomic mutation detection system was previously described . We now developed a complete genomic mutation detection system for krt14 . For the amplification of the krt14 genes , we make use of restriction sites to exempt the keratin 14 pseudogene sequence from polymerase chain reaction amplification . using the complete genomic mutation detection system for both KRT5 and krt14 , we identified four novel KRT5 mutations ( IVS1 1G C , k404e , a438d , e475k ) , two of which are outside …

Identification of novel and known mutations in the genes for keratin 5 and 14 in danish patients with epidermolysis bullosa … (1999 Feb)
identification of novel and known mutations in the genes for keratin 5 and 14 in danish patients with epidermolysis bullosa simplex : correlation between genotype and phenotype . epidermolysis bullosa simplex ( EBS ) is a group of autosomal dominant inherited skin diseases caused by mutations in either the keratin 5 ( K5 ) or the keratin 14 ( K14 ) genes and characterized by development of intraepidermal skin blisters . The three major subtypes of EBS are weber cockayne , koebner , and dowling meara , of which the dowling meara form is the most severe . We have investigated five large danish families with EBS and two sporadic patients with the dowling meara form of EBS . In the sporadic dowling meara EBS patients , a novel K14 mutation ( n123s ) and a previously published K5 mutation ( n176s ) were identified , respectively . A novel K14 mutation ( k116n ) was found in three seemingly unrelated families , whereas another family harbored a different novel K14 mutation ( l143p ) . The last family harbored a novel K5 mutation ( l325p ) . The identified mutations were not present in more than 100 normal chromosomes . Six polymorphisms were identified in the K14 gene and their frequencies were determined in normal controls . these polymorphisms were used to show that the K14 k116n mutation was located in chromosomes with the same haplotype in all three families , suggesting a common ancestor . We observed a …

A mutation ( Met Arg ) in the type I keratin ( K14 ) gene responsible for autosomal dominant epidermolysis … (1993 May)
A mutation ( Met Arg ) in the type I keratin ( K14 ) gene responsible for autosomal dominant epidermolysis bullosa simplex . We have identified a single base change in exon 4 of the type I keratin gene which results in the replacement of a methionine for an arginine residue at codon 272 in an irish family displaying an autosomal dominant simplex ( koebner ) form of epidermolysis bullosa ( EB ) . This family had previously provided tentative evidence for linkage to genetic markers on chromosome 1q . The mutation cosegregates with the disease , producing a lod score of 4 . 8 at theta 0 .

The genetic basis of weber cockayne epidermolysis bullosa simplex. (1993 Sep)
The genetic basis of weber cockayne epidermolysis bullosa simplex . epidermolysis bullosa simplex ( EBS ) is a group of autosomal dominant skin diseases characterized by blistering , due to mechanical stress induced degeneration of basal epidermal cells . recently , it was discovered that the more severe types , dowling meara and koebner , are genetic disorders of the basal epidermal keratins , keratin 5 ( K5 ) and keratin 14 ( K14 ) . Here , we show that the mildest type of EBS , weber cockayne , is also a disorder of these keratins . affected members of two unrelated families with weber cockayne EBS had a T G point mutation in the second base position of codon 161 of one of two K5 alleles , leading to an Ile Ser mutation . This mutation was not present in unaffected members or in 156 alleles from normal individuals . linkage analyses mapped the defect to the type II keratin gene cluster on chromosome 12q11 q13 ( peak logarithm of odds score at theta 0 of 3 . 0 ) , providing strong additional evidence that this mutation is responsible for the weber cockayne EBS phenotype . conserved among type II keratins , Ile 161 is in the nonhelical head domain of K5 , a region previously shown to be important for 10 nm filament assembly . The mutation generates a potential substrate site for protein kinase C , which could influence intermediate filament architecture , perhaps leading …

Primers for exon specific amplification of the KRT5 gene : identification of novel and recurrent mutations in epidermolysis bullosa simplex … (1997 Mar)
primers for exon specific amplification of the KRT5 gene : identification of novel and recurrent mutations in epidermolysis bullosa simplex patients . The KRT5 and krt14 genes encode the proteins keratin 5 and 14 , respectively , which are the primary structural components of the 10 nm intermediate filaments of the mitotic epidermal basal cells . A single mutation in either gene can disrupt the keratin intermediate filament cytoskeleton , resulting in the skin fragility and blistering that is characteristic of the group of inherited disorders known as epidermolysis bullosa simplex . We have established a mutation detection system that facilitates KRT5 gene analysis from leukocyte genomic DNA , obviating the need for a skin sample or keratinocyte culture for cDNA synthesis . KRT5 intronic regions that flanked each exon were sequenced and sets of facing intronic primers were designed for specific amplification of each of the nine KRT5 exons . direct sequencing of KRT5 amplified exons identified three novel missense mutations . One mutation recurred in two unrelated patients with sporadic EBS . This glutamate to lysine substitution ( e477k ) , located in the highly conserved kllege motif at the end of the central rod domain , is the third recurrent mutation identified in dominant epidermolysis bullosa simplex disease . The corresponding glutamate in keratin 2e was previously reported to be frequently mutated in ichthyosis bullosa of siemens , suggesting that this highly conserved residue may be a potential mutational hot spot in other type II keratins or …

Epidermolysis bullosa simplex dowling meara due to an arginine to cysteine substitution in exon 1 of keratin 14. (2002 Feb)
epidermolysis bullosa simplex dowling meara due to an arginine to cysteine substitution in exon 1 of keratin 14 . epidermolysis bullosa simplex ( EBS ) is a blistering disorder affecting the basal layer of the epidermis usually inherited in an autosomal dominant fashion . Most cases are caused by mutations in the genes encoding keratin 5 ( K5 ) and keratin 14 ( K14 ) and are characterized by cytolysis within the basal layer of the epidermis . We report a patient manifesting the dowling meara variant of EBS in whom we characterized a cytosine to thymine transition at codon 125 ( r125c ) in K14 . This missense mutation is located at the amino terminus of the helical rod domain of the keratin 14 molecule , resulting in defective pairing with K5 , thereby disrupting keratin tonofibril integrity .

A novel recessive missense mutation in krt14 reveals striking phenotypic heterogeneity in epidermolysis bullosa simplex. (2005 Jan)
A novel recessive missense mutation in krt14 reveals striking phenotypic heterogeneity in epidermolysis bullosa simplex .

Do the ends justify the mean ? proline mutations at the ends of the keratin coiled coil rod segment are … (1992 Mar)
Do the ends justify the mean ? proline mutations at the ends of the keratin coiled coil rod segment are more disruptive than internal mutations . intermediate filament ( IF ) assembly is remarkable , in that it appears to be self driven by the primary sequence of IF proteins , a family ( 40 220 kd ) with diverse sequences , but similar secondary structures . Each IF polypeptide has a central 310 amino acid residue alpha helical rod domain , involved in coiled coil dinner formation . Two short ( approximately 10 amino acid residue ) stretches at the ends of this rod are more highly conserved than the rest , although the molecular basis for this is unknown . In addition , the rod is segmented by three short nonhelical linkers of conserved location , but not sequence . To examine the degree to which different conserved helical and nonhelical rod sequences contribute to dimer , tetramer , and higher ordered interactions , we introduced proline mutations in residues throughout the rod of a type I keratin , and we removed existing proline residues from the linker regions . To further probe the role of the rod ends , we introduced more subtle mutations near the COOH terminus . We examined the consequences of these mutations on ( a ) IF network formation in vivo , and ( b ) 10 nm filament assembly in vitro . surprisingly , all proline mutations located deep in the coiled …

Supplementation of a mutant keratin by stable expression of desmin in cultured human EBS keratinocytes. (2001 Jan)
supplementation of a mutant keratin by stable expression of desmin in cultured human EBS keratinocytes . mutations in keratin genes give rise to a number of inherited skin fragility disorders , demonstrating that the intermediate filament cytoskeleton has an essential function in maintaining the structural integrity of epidermis and its appendages . epidermolysis bullosa simplex ( EBS ) is an autosomal dominant disorder caused by mutations in keratins K5 or K14 , which are expressed in the basal layer of stratified epithelia . using a keratinocyte cell line established from an EBS patient , we investigated whether the muscle specific intermediate filament protein desmin would be able to functionally complement a mutant keratin 14 in cultured keratinocytes . We show that in stably transfected EBS cells , desmin forms an extended keratin independent cytoskeleton . immunogold EM analysis demonstrated that in the presence of numerous keratin filaments attached to desmosomes , desmin could nevertheless interact with desmosomes in the same cell , indicating the dynamic nature of the filament desmosome association . When desmin transfected cells were subjected to heat shock , the mutant keratin filaments showed a transient collapse while desmin filaments were maintained . Thus the defective keratin filaments and the wild type desmin filaments appear to coexist in cells without interference . expression of a type III intermediate filament protein like desmin may offer a strategy for the treatment of patients suffering from epidermal keratin mutations .

A hot spot mutation alters the mechanical properties of keratin filament networks. (2001 May)
A hot spot mutation alters the mechanical properties of keratin filament networks . keratins 5 and 14 polymerize to form the intermediate filament network in the progenitor basal cells of many stratified epithelia including epidermis , where it provides crucial mechanical support . inherited mutations in K5 or K14 result in epidermolysis bullosa simplex ( EBS ) , a skin fragility disorder . The impact that such mutations exert on the intrinsic mechanical properties of K5 / K14 filaments is unknown . Here we show , by using differential interference contrast microscopy , that a hot spot mutation in K14 greatly reduces the ability of reconstituted mutant filaments to bundle under crosslinking conditions . rheological assays measure similar small deformation mechanical responses for crosslinked solutions of wild type and mutant keratins . The mutation , however , markedly reduces the resilience of crosslinked networks against large deformations . single particle tracking , which probes the local organization of filament networks , shows that the mutant polymer exhibits highly heterogeneous structures compared to those of wild type filaments . Our results indicate that the fragility of epithelial cells expressing mutant keratin may result from an impaired ability of keratin polymers to be crosslinked into a functional network .

The genetic basis of epidermolytic hyperkeratosis : a disorder of differentiation specific epidermal keratin genes. (1992 Oct)
The genetic basis of epidermolytic hyperkeratosis : a disorder of differentiation specific epidermal keratin genes . epidermolytic hyperkeratosis ( EH ) is a skin disease characterized by keratin filament clumping and degeneration in terminally differentiating epidermal cells . We have discovered that the genetic basis for EH resides in mutations in differentiation specific keratins . Two of six distinct incidences of EH had a keratin 10 ( K10 ) point mutation in a highly conserved arginine . remarkably , this same residue is mutated in the basal epidermal K14 in three incidences of another skin disease , epidermolysis bullosa simplex ( EBS ) . By genetic engineering , gene transfection , and 10 nm filament assembly , we show that this mutation is functionally responsible for the keratin filament clumping that occurs in basal ( EBS ) or suprabasal ( EH ) cells . these studies strengthen the link between filament perturbations , cell fragility , and degeneration first established with EBS . They also suggest a correlation between filament disorganization and either cytokinesis or nuclear shape , giving rise to the seemingly binucleate cells typical of EH .

A human keratin 14 knockout : the absence of K14 leads to severe epidermolysis bullosa simplex and a function for … (1994 Dec)
A human keratin 14 knockout : the absence of K14 leads to severe epidermolysis bullosa simplex and a function for an intermediate filament protein . since their discovery , the function of intermediate filaments ( IFs ) has remained obscure . In skin , epidermal cells have extensive cytoskeletal architectures of IFs , composed of type I and type II keratin heterodimers . clues to possible functions of these proteins have come from recent studies showing that several autosomal dominant , blistering skin disorders are caused by defects in genes that encode epidermal keratins . these diseases all exhibit cell degeneration and keratin network perturbations in cells that express the particular mutant keratin gene . however , it is not clear from these studies whether cytolysis arises from the presence of large insoluble keratin aggregates that compromise cellular physiology or from the absence of an extensive keratin filament network , which jeopardizes mechanical integrity . We report here the analysis of an extremely rare case of severe recessive epidermolysis bullosa simplex ( EBS ) , where the patient lacks a discernible keratin filament network in basal epidermal cells . genetic analyses revealed a homozygous point mutation that yielded a premature termination codon in the major basal type I keratin gene and caused complete ablation of K14 . The consanguineous parents were normal , each harboring one copy of the null K14 mutation . analysis of cultured keratinocytes enabled us to document that the loss of K14 is not compensated for …

Epidermolysis bullosa simplex dowling meara : troublesome blistering and pruritus in an adult patient. (1993 Mar)
epidermolysis bullosa simplex dowling meara : troublesome blistering and pruritus in an adult patient . A 46 year old woman with the dowling meara variant of epidermolysis bullosa simplex ( DM EBS ) presented with worsening recurrent , pruritic , circinate crops of clear and haemorrhagic herpetiform blisters affecting her trunk and limbs . electron microscopy showed tonofilament aggregation and an intra epidermal level of blister formation confirming a diagnosis of DM EBS rather than an acquired immunobullous disorder . antihistamines failed to control the intense pruritus , but dapsone ( up to 150 mg daily ) was beneficial . mutations of basal cell keratin genes ( K14 and K5 ) are thought to be of aetiopathological significance in this form of epidermolysis bullosa , but the underlying cellular mechanisms responsible for the clinical deterioration and severe itching in this adult patient are not yet clear .

DNA based prenatal testing for the skin blistering disorder epidermolysis bullosa simplex. (2000 Jun)
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 …

Homozygous nonsense mutation in helix 2 of K14 causes severe recessive epidermolysis bullosa simplex. (1998 Jun)
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 …

Novel keratin 14 mutations in patients with severe recessive epidermolysis bullosa simplex. (2006 Jul)
novel keratin 14 mutations in patients with severe recessive epidermolysis bullosa simplex .

Partial revertant mosaicism of keratin 14 in a patient with recessive epidermolysis bullosa simplex. (2002 Mar)
partial revertant mosaicism of keratin 14 in a patient with recessive epidermolysis bullosa simplex . A patient with recessive epidermolysis bullosa simplex due to a previously described homozygous krt14 1842 2A C splice site mutation was re examined , because we unexpectedly found signs of revertant mosaicism . The germline mutation resulted in different aberrant transcripts containing premature termination codons , all leading to truncated keratin 14 proteins . basal keratinocytes in skin and in culture completely lacked keratin 14 and intermediate filaments . From this keratin 14 / patient we started cultures from a new skin biopsy and here , we serendipitously found keratinocytes that spontaneously expressed keratin 14 . This biopsy had been taken from an area of skin that was clinically affected , because blisters could simply be evoked by gentle rubbing . immunofluorescence and electron microscopy of additional biopsies from this skin area revealed a mosaic expression of keratin 14 and reappearance of intermediate filaments in basal keratinocytes . immunoblotting showed a revertant keratin 14 polypeptide with seemingly normal molecular weight . DNA analysis of exon 2 and its flanking intron borders showed no additional mutations in the genomic krt14 sequence . analysis of mRNA isolated from mosaic skin keratinocytes revealed an additional in frame transcript ( 1844t G , 1845delta6 ) that codes for an abnormal keratin 14 polypeptide with a two residue deletion and one amino acid change . The re expression of a revertant , albeit abnormal , keratin 14 polypeptide , so …