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 ( …

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 .

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 …

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 …

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 …

A keratin K5 mutation ( Leu 463 Pro ) in a family with the weber cockayne type of epidermolysis bullosa … (1997 Sep)
A keratin K5 mutation ( Leu 463 Pro ) in a family with the weber cockayne type of epidermolysis bullosa simplex .

Long range polymerase chain reaction for specific full length amplification of the human keratin 14 gene and novel keratin 14 … (2003 Feb)
Long range polymerase chain reaction for specific full length amplification of the human keratin 14 gene and novel keratin 14 mutations in epidermolysis bullosa simplex patients .

Arginine in the beginning of the 1A rod domain of the keratin 10 gene is the hot spot for the … (1999 May)
arginine in the beginning of the 1A rod domain of the keratin 10 gene is the hot spot for the mutation in epidermolytic hyperkeratosis . keratin intermediate filaments are expressed in specific type I / type II pairs in the stage of differentiation of keratinocytes . The mutations in the keratin genes expressed in the epidermis are etiologically responsible for several epidermal genetic skin diseases , such as epidermolysis bullosa simplex , epidermolytic hyperkeratosis ( EHK ) , ichthyosis bullosa of siemens , palmoplantar keratoderma , pachyonchia congenita and white sponge nevus . The mutations of keratins 1 / 10 which are expressed in spinous and granular layers are confirmed to cause EHK . there are several trials to correlate between the clinical phenotypes and sites of mutations of the keratin genes . One of these is that EHK is divided into two groups : the palms and soles involvement ( PS ) group and the non palms and soles ( NPS ) group . So far the PS group had the mutations in the keratin 1 and the NPS group in keratin 10 . Most of the mutations of the NPS group were reported in the beginning of the 1A rod domain and over 2 / 3 of the mutations in the 1A rod domain were the base pair substitution of arginine . Here we find two different mutations in two unrelated korean kindreds classified as NPS group r156c and r156h in the 1A rod domain of keratin 10 …

Temperature sensitivity of the keratin cytoskeleton and delayed spreading of keratinocyte lines derived from EBS patients. (1996 Mar)
temperature sensitivity of the keratin cytoskeleton and delayed spreading of keratinocyte lines derived from EBS patients . point mutations in the keratin intermediate filament genes for keratin 5 or keratin 14 are known to cause hereditary skin blistering disorders such as epidermolysis bullosa simplex , in which epidermal keratinocytes are extremely fragile and the skin blisters on mild trauma . We show that in 2 phenotypically diverse cases of epidermolysis bullosa simplex , the keratin mutations result in a thermoinstability of the intermediate filament cytoskeleton which can be reproducibly demonstrated even in the presence of tissue culture induced keratins and in conditions where filament fragility is not otherwise obvious . SV40 T antigen and hpv16 ( E6 E7 ) immortalised keratinocyte cell lines were examined , established from control and epidermolysis bullosa simplex affected individuals with either severe ( dowling meara ) or mild ( weber cockayne ) forms of the disease . In standard tissue culture conditions no significant and consistent abnormality of the keratin cytoskeleton could be demonstrated . however after thermal stress a reduced stability of the keratin filaments was demonstrable in the epidermolysis bullosa simplex cell lines , with filaments breaking into aggregates similar to those seen in skin from EBS patients . these aggregates were maximal at 15 minutes after heat shock and the filament network structure was substantially reversed by 60 minutes . differences were also seen in the cells during respreading after replating : cells containing mutant keratins were slower to respread than …

Mapping of epidermolysis bullosa simplex mutation to chromosome 12. (1991 Nov)
mapping of epidermolysis bullosa simplex mutation to chromosome 12 . epidermolysis bullosa simplex ( EBS ) is a dominantly inherited genodermatosis characterized by intraepidermal blister formation . recent reports have suggested that EBS mutations may relate to keratin abnormalities . In this study , we conducted RFLP analyses to test the hypothesis that EBS is linked to one of the keratin gene clusters on chromosome 12 or chromosome 17 . although these keratin gene loci are not defined by rflps , several mapped rflps in the same chromosomal regions could be tested for linkage . A large EBS family with 14 affected and 12 unaffected individuals in three generations was analyzed for RFLP inheritance . within this family there was no evidence for linkage of the EBS mutation to markers on chromosome 17q . however , there was evidence for close linkage to d12s17 located on chromosome 12q , with a maximum LOD score of 5 . 55 at theta 0 . mapping of this mutation to chromosome 12 defines an EBS locus distinct from both EBS1 ( Ogna ) and EBS2 ( koebner ) , which are on chromosomes 8 and 1 , respectively . further mapping will determine whether this EBS locus on chromosome 12 resides within the keratin gene cluster at 12q11 q13 .

A premature stop codon mutation in the 2B helix termination peptide of keratin 5 in a german epidermolysis bullosa simplex … (1999 Jul)
A premature stop codon mutation in the 2B helix termination peptide of keratin 5 in a german epidermolysis bullosa simplex dowling meara case . epidermolysis bullosa simplex ( EBS ) is caused by defective assembly of keratin intermediate filaments in basal keratinocytes and recent studies indicated causal mutations in the keratin KRT5 and krt14 genes . In this study , we describe a novel KRT5 mutation in a german sporadic case of EBS dowling meara . transition of G to T ( nucleotide position 2334 ) leads to a premature stop codon ( e477stop , residue 93 of the 2B helix ) in the last residue of the highly conserved helix termination peptide K / llege of the 2B rod domain of keratin K5 . This represents the first premature stop codon mutation identified within the K / llege motif of any disorder reported so far that is caused by keratin mutations .

Novel mutation ( asp158val ) in H1 domain of keratin 5 gene in a japanese patient with köbner type epidermolysis … (2006 Oct)
novel mutation ( asp158val ) in H1 domain of keratin 5 gene in a japanese patient with köbner type epidermolysis bullosa simplex . We describe a 19 year old japanese male with köbner type epidermolysis bullosa simplex ( EBS KB ) with a novel keratin gene mutation . The patient developed blisters on the feet , palms , elbows and knees soon after birth . His father is similarly affected with blistering , but his mother and younger brother are not affected . histological examination revealed that the primary separation in the blister occurred within the basal cell layer . sequence analysis demonstrated an A to T transition at the second position of codon 158 in the keratin 5 ( K5 ) gene . The amino acid at codon 158 was deduced to have changed from asparagine to valine . We identified a novel mutation ( asp158val ) in the H1 domain of the K5 gene in this japanese patient with EBS KB . This is the first gene mutation report of EBS KB in the H1 domain of the K5 gene .