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.     

Epidermolysis bullosa simplex with mottled pigmentation due to de novo P25L mutation in keratin 5 in an Italian patient

Epidermolysis bullosa simplex with mottled pigmentation (EBS-MP) is an autosomal dominant inherited disorder of the skin, which manifests as recurrent blistering, punctate palmo-plantar hyperkeratoses, and mottled pigmentation of the trunk and extremities. Previous reports have identified the P25L mutation within the non-helical V1 domain of keratin 5 as the unique cause of the disease. We found this mutation in the first Italian case of EBS-MP. The proband was heterozygous for the P25L mutation, which occurred de novo as this change was not detected in the peripheral blood DNA of the healthy parents. Our report extends the limited number of EBS-MP cases and gives further evidence that mutation P25L is responsible for this unusual phenotype.     

Epidermolysis bullosa simplex with mottled pigmentation - mutation analysis proved the diagnosis in a four-generation pedigree

Epidermolysis bullosa simplex with mottled pigmentation (EBS-MP) is a rare variant of the basal form of EBS, characterized by mild intraepidermal blistering due to lysis of basal keratinocytes and with a progressive reticular hyperpigmentation on the trunk and extremities. A?limited number of cases - to date twenty unrelated families - have been published from all over the world, including thirteen reports from Europe. We here?report the first Hungarian case in a four generation pedigree with EBS-MP symptoms and prove the diagnosis by mutation analysis. A?heterozygous p.Pro25Leu mutation in the first exon of KRT5, together with the heterozygous polymorphism p.Gly138Glu, was identified in all the five affected family members studied. Our report extends the limited number of EBS-MP cases and gives further evidence that KRT5 mutations are responsible for this rare phenotype.     

Clinical heterogeneity of 1649delG mutation in the tail domain of keratin 5: a Japanese family with epidermolysis bullosa simplex with mottled pigmentation

Twenty-five- and 22-y-old Japanese women, who are cousins, presented with distal skin fragility, widespread small, pigmented macules, and toenail deformity. Blisters occurred between the epidermis and the dermis with degeneration of the basal cells, suggesting epidermolysis bullosa simplex with mottled pigmentation (EBS-MP). Electron microscopy of the pigmented spots demonstrated vacuolization of basal cells as well as disturbed junctional structures and incontinence of pigmentation. Gene analysis resulted in detection of a heterozygous deletion of a guanine nucleotide in exon 9 at position 1649. P25L mutation was not detected in either case. It is possible that EBS-MP occurs not only based on the P25L mutation of the keratin 5 molecule, but also because of other types of mutations of epidermal keratin genes.     

Novel keratin 5 and 14 gene mutations in patients with epidermolysis bullosa simplex from Poland

Mutation analysis in keratins 5/14 (K5/14) had been performed in five Polish families with epidermolysis bullosa simplex (EBS) to extend genotype-phenotype correlation and to add to the mutation database. All the patients had been clinically classified into two subtypes of EBS; Weber-Cockayne (EBS-WC) and Dowling-Meara (EBS-DM) as well as one case of EBS with mottled pigmentation (EBS-MP). DNA from patients and their family members was assessed for mutations in K5 or 14 using polymerase chain reaction amplification and subsequent direct sequencing. We identified four different missense mutations in K5 and one missense mutation in K14. Three of these are novel. Mutations associated EBS-DM resided in the highly conserved 20 amino acids end of the 1A domain in K5. Direct nucleotide sequencing of a case of EBS-MP revealed a heterozygous P25L mutation in K5. However, no genotype-phenotype correlation was identified in families with EBS-WC. The present study demonstrates the first series of molecular genetic data in EBS from Poland.     

单纯型大疱性表皮松解症Weber-Cockayne亚型一家系角蛋白5基因突变的检测

鉴定单纯型大疱性表皮松解症Weber-Cockayne亚型一家系中的基因突变位点。方法:应用聚合酶链反应(PCR)及DNA直接测序的方法,检测患者角蛋白5(KRT5)及角蛋白14(KRT14)基因的全部编码序列;针对所发现的突变,以限制性内切酶片段长度多态性(RFLP)分析加以验证。结果:该家系患者存在KRT5基因突变:第2外显子第596位碱基由腺嘌呤突变为胞嘧啶,导致第199位氨基酸由赖氨酸变为苏氨酸(K199T),而正常对照无此替代。结论:KRT5K199T为导致此家系中患者临床表现的特异突变,此突变位点为国内外首次报道。     

A new keratin 5 mutation (K199T) in a family with Weber-Cockayne epidermolysis bullosa simplex

A new missense mutation in the keratin 5 gene (KRT5) in a Chinese family with Weber-Cockayne type epidermolysis bullosa simplex is reported. Direct sequencing identified a heterozygous A --> C substitution at nucleotide 596 altering codon 199 of KRT5 from lysine to threonine in all affected family members, but not in the unaffected family members or in 50 unrelated control samples. The mutation is designated K199T. This mutated lysine residue is sited within the 1A domain of keratin 5 and is highly conserved among all type II keratins. The mutation may perturb the alignment of tonofilaments and, as a consequence, result in skin fragility and blistering.     

A new mutation in the linker 12 domain of keratin 5 in a Chinese family with Weber-Cockayne epidermolysis bullosa simplex

A previously undescribed missense mutation was detected in the L12 domain of keratin 5 (K5) in a Chinese family with Weber-Cockayne epidermolysis bullosa simplex. Direct sequencing of the PCR products identified a single base substitution (983A-->G) that changes the aspartic acid residue at codon 328 to glycine in all affected family members, while no mutation was observed either in the healthy individual or 50 unrelated control samples. Asp328 of K5 is remarkably conserved among all type II keratins. D328G is the fourth mutation found to affect this residue in K5-related epidermolysis bullosa simplex, indicating the importance of Asp328 for K5 structure and the dramatic effect that fine changes can have on keratin intermediate filament integrity.     

Analysis of the KRT9 Gene in a Mexican Family with Epidermolytic Palmoplantar Keratoderma

Epidermolytic palmoplantar keratoderma (EPPK), an autosomal‐dominant genodermatosis, is the most frequently occurring hereditary palmoplantar keratoderma. EPPK is characterized by hyperkeratosis of the palms and soles. Approximately 90% of patients present with mutations in the KRT9 gene, which encodes for keratin 9. Many of these mutations are located within the highly conserved coil 1A region of the alpha‐helical rod domain of keratin 9, an important domain for keratin heterodimerization. The objective was to assess the clinical and molecular characteristics of a Mexican family with EPPK. The clinical characteristics of members of this family were analyzed. The KRT9 gene of affected members was polymerase chain reaction amplified from genomic DNA and sequenced. All affected members of the family had hyperkeratosis of the palms and soles with knuckle pads. The R163W mutation in the KRT9 gene was present in all affected individuals who were tested. Although R163W is the most frequent KRT9 mutation in patients with EPPK, only two families have been reported with knuckle pads associated with this mutation. Our findings indicate that knuckle pads can be associated with EPPK and the R163W mutation in a family with a genetic background different from that described here.     

Weber-Cockayne亚型单纯型大疱性表皮松解症一家系报告

目的:研究Weber-Cockayne亚型单纯型大疱性表皮松解症(EBS-WC)一家系的基因突变,并进行产前诊断。方法:应用PCR及DNA直接测序方法明确突变位点,针对所发现的突变以限制性内切酶片段长度多态性(RFLP)分析加以验证,在此基础上于妊娠24周时对从胎儿羊水所提取的DNA进行测序及酶切验证。结果:该家系患者存在角蛋白(keratin,KRT)5基因突变:第7外显子第1388位碱基由胸腺嘌呤突变为胞嘧啶,导致第463位氨基酸由亮氨酸变为脯氨酸(L463P)。50名健康对照者不存在此突变。羊水细胞DNA不存在此突变的胎儿,出生后未患大疱性表皮松解症。结论:KRT5第7外显子的突变是引起该家系临床症状的特异性突变。     

A novel mutation in the second half of the keratin 17 1A domain in a large pedigree with delayed-onset pachyonychia congenita type 2

Pachyonychia congenita type 2 (PC-2), also known as Jackson-Lawler type PC, is an autosomal dominant disorder characterized by hypertrophic nail dystrophy associated with focal keratoderma and multiple pilosebaceous cysts. We report a large Chinese pedigree of typical delayed-onset PC-2 that includes 19 affected members. Direct sequencing of PCR products revealed a novel heterozygous 325A-->G mutation in the affected members. This mutation predicts the substitution of asparagine by aspartic acid in codon 109 (N109D) located in the second half of the keratin 17 1A domain, where similar mutation in keratin 5 is associated with the mild Weber-Cockayne form of epidermolysis bullosa simplex.     

Keratin 17 mutation in pachyonychia congenita type 2 with early onset sebaceous cysts

BACKGROUND: Pachyonychia congenita (PC) is a group of autosomal dominant ectodermal dysplasias caused by mutations in four differentiation-specific keratin genes. Two major clinical subtypes of PC have been generally recognized. Symmetrically thickened fingernails and toenails are the defining characteristic of PC type 2 (PC-2) with onset at infancy. Pilosebaceous cysts are the best hallmark of PC-2, but they usually occur at puberty. OBJECTIVES: To report a Chinese pedigree of PC-2 with unusually early onset sebaceous cysts and to explore the genetic mutation and its phenotype. METHODS: Exon 1 of keratin 17 was amplified by polymerase chain reaction (PCR) from genomic DNA from the three patients in the pedigree, the proband, his half-sister and his younger son, two unaffected members in the pedigree and 50 unrelated and unaffected people. PCR products were directly sequenced to detect the mutation. RESULTS: Direct sequencing of the PCR products revealed a heterozygous 275A-->G mutation in all three affected members. This mutation predicts the substitution of asparagine by serine in codon 92 (N92S) located in the 1A domain of keratin 17. CONCLUSIONS: Mutation in the 1A domain of keratin 17 underlies the affected members' phenotype, PC-2 with early onset sebaceous cysts and late-onset thickened fingernails and toenails. The onset of the cysts is very early in some people within this family and the age at onset of thickened fingernails and toenails is variable within the family, implying the existence of modifying factors.     

A novel mutation and large size polymorphism affecting the V2 domain of keratin 1 in an African-American family with severe, diffuse palmoplantar keratoderma of the ichthyosis hystrix Curth-Macklin type

Keratin gene mutations affecting nonhelical head and tail domains are not usually associated with prominent skin blistering and keratin filament clumping. Instead, they have been associated with several distinct clinical phenotypes, such as epidermolysis bullosa simplex with mottled pigmentation (mutation P25L in the V1 domain of keratin 5), epidermolysis bullosa simplex with migratory circinate erythema (frameshift mutation c1649delG in the V2 domain of keratin 5), striate palmoplantar keratoderma (PPK), and ichthyosis hystrix Curth-Macklin (different frameshift mutations in the V2 domain of keratin 1 (K1)). We have studied a family with severe, diffuse, nonepidermolytic PPK and verrucous hyperkeratotic plaques over the joints and in flexures and identified a new KRT1 gene mutation that is predicted to completely alter the K1 tail domain. In addition, a new K1 size polymorphism has been detected, which is especially prevalent among the African-American population. These results further emphasize the functional importance of the nonhelical tail domain in keratin molecules despite the obvious variability in the number of glycine loop motifs and underscore the broad phenotypic spectrum of disorders due to dominant keratin tail mutations.     

Epidermolytic palmoplantar keratoderma of Vörner: re-evaluation of Vörner's original family and identification of a novel keratin 9 mutation

In 1901, Hans V?rner observed a family with a diffuse non-transgredient palmoplantar keratoderma of autosomal dominant inheritance. Histopathologically, he found epidermolytic hyperkeratosis as a characteristic sign and diagnostic criterion of this disorder. We performed a follow-up study of the family originally seen by V?rner in 1901 with clinical, histopathological, and molecular investigations. Clinically, affected family members showed the typical diffuse keratoses over the entire surface of the palms and soles sharply bordered by red margins. A mycotic infection was additionally found in two patients examined. Histopathological investigations confirmed epidermolytic hyperkeratosis. Molecular studies revealed a novel mutation in keratin 9, N160I, in patients from the family. The mutation in the coil-1A domain is thought to have a dominant negative effect on the assembly of keratin intermediate filaments, explaining the dominant inheritance of the phenotype. These findings give further evidence that palmoplantar keratoderma of V?rner represents the same entity as palmoplantar keratoderma of Thost, which was recently re-evaluated in Thost's original family and shown to be caused by a similar mutation, R162 W, in the same segment of keratin 9.     

Laryngeal involvement in the Dowling-Meara variant of epidermolysis bullosa simplex with keratin mutations of severely disruptive potential

The clinical features of the Dowling-Meara variant of epidermolysis bullosa simplex (EBS-DM) can, in an infant, be indistinguishable from other severe forms of epidermolysis bullosa (EB). Two unrelated infants with no family history of skin disease are described who, within hours of birth, developed extensive blistering of skin and oral mucosae and who both subsequently developed hoarse cries. Despite this superficial resemblance to other forms of EB, electron microscopy revealed a basal cell rupture and keratin aggregates characteristic of EBS-DM in the skin of both infants and in the vocal cord epithelium of one. Molecular analysis confirmed the diagnosis by identification of mis-sense point mutations in basal cell keratin genes in both cases. One patient carries a point mutation in keratin 14 (converting arginine at position 125 to histidine) and the other has a novel point mutation in keratin 5 (converting serine at position 181 to proline). Hoarseness is not a well documented feature of EBS-DM and is usually associated with junctional EB. These two patients demonstrate that the presence of a hoarse cry in an infant affected by severe EB does not necessarily indicate a poor prognosis.     

Pure hair-nail ectodermal dysplasia maps to chromosome 12p11.1-q21.1 in a consanguineous Pakistani family

Summary Ectodermal dysplasias (ED) are developmental disorders affecting tissues of ectodermal origin including hair, nail, teeth and sweat glands. To date, four different types of ectodermal dysplasias involving only hair and nails have been described. In an effort to understand the molecular basis of ED of hair and nails, a Pakistani family with multiple affected individuals was studied. Linkage analysis was carried out by genotyping eight members of the family (five normal and three affected) using microsatellite markers linked to the related phenotype. The diseased phenotype was mapped to chromosome 12p11.1–q21.1 (Z_max = 3.1). DNA sequence analysis of the coding exons and splice sites of six hair keratin genes, located in the linkage interval, failed to detect any pathogenic mutation in the affected individuals of the family. Failure to detect a mutation in the epithelial keratin genes suggests that the mutation lies either in the regulatory region of one of the keratin genes or in another unknown gene, located in the linkage interval, with a possible role in the development of ectodermal appendages.     

Epidermolytic hyperkeratosis with polycyclic psoriasiform plaques resulting from a mutation in the keratin 1 gene

Epidermolytic hyperkeratosis (EHK) is a genodermatosis caused by mutations in either the keratin 1 (K1) or keratin 10 (K10) genes, and characterized by erythroderma and blistering at birth, with development of a ribbed, ichthyotic hyperkeratosis and palmoplantar keratoderma. A wide variety of mutations within the highly conserved helix termination motifs of the central rod domains of the K1 or K10 genes correlate with the highly variable phenotypic severity observed in EHK. We report a unique EHK-like phenotype exhibiting autosomal dominant inheritance with variable expressivity in four affected individuals in a single family. Clinically, affected individuals manifest transient blistering at birth followed by chronic diffuse palmoplantar keratoderma without transgradiens. Intermittent flares of non-migratory polycylic erythematous psoriasiform plaques which worsen and abate in severity were present in all affected individuals, but showed immense individual variation in both severity and duration, ranging from weeks to months. Histopathologic examination of the psoriasiform plaques demonstrated the characteristic features of EHK. Sequencing of the K1 gene in affected family members revealed a heterozygous A-to-T transversion at nucleotide 1435 within exon 7, converting isoleucine (ATT) to phenylalanine (TTT), (I479F). The mutation resides within the highly conserved helix termination motif of the helix 2B segment of the K1 gene. This unique clinical phenotype and the associated K1 mutation have not been previously described, and it is referred to here as EHK with polycyclic, psoriasiform plaques (EHK/PPP).     

Identification of the keratin K9 R162W mutation in patients of Italian origin with epidermolytic palmoplantar keratoderma

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant skin disorder characterized by hyperkeratosis of the palms and soles associated with histologic findings of hyperkeratosis and epidermolysis. Ultrastructurally, there is vacuolization of the cytoplasm and abnormal keratin filament network with tonofilament clumping. EPPK is caused by mutations in the keratin 9 gene (KRT9), which is expressed exclusively in suprabasal keratinocytes of palmoplantar epidermis. The mutation R162W is the most frequent keratin 9 alteration reported in patients from different geographical areas. We present three unrelated Italian families affected by EPPK in which we confirmed the presence of the R162W mutation, by RT-PCR analysis followed by sequencing of the KRT9 gene, in all affected members. The finding of the same mutation in all patients, together with the previous reports of the disease, strongly suggest that position 162 of the KRT9 gene represents a mutation "hot-spot", probably due to the peculiarity of the sequence.     

Identification of the keratin 9 (KRT9) N161S mutation in a Chinese kindred with epidermolytic palmoplantar keratoderma

We present a family from Northeast China affected by epidermolytic palmoplantar keratoderma (EPPK) in which we confirmed the presence of the N161S mutation as the result of a 548A>G transition in exon1 of the keratin 9 gene. Genomic DNA from peripheral blood of all available members in this family was used for amplification of exon 1 of KRT9 by polymerase chain reaction. The mutation was detected by direct sequence analysis and identified by restriction endonuclease DdeI digestion. The finding of the same mutation in all available patients, together with the previous reports of the disease, strongly suggested that position 161 of the KRT9 gene also represents a mutation "hotspot" for EPPK. Our result is an important contribution to the investigation of the genotype/phenotype correlation and affords molecular genetic knowledge for future clinical diagnosis and gene therapy of EPPK.     

A usual frameshift and delayed termination codon mutation in keratin 5 causes a novel type of epidermolysis bullosa simplex with migratory circinate erythema

We report here two unrelated families in Japan and Korea having patients with a unique type of epidermolysis bullosa simplex and a novel mutation in the keratin gene KRT5, i.e., a frameshift and delayed stop codon inconsistent with any subtype described before. The patients showed migratory circinate erythema and multiple vesicles on the circular belt-like areas affected by erythema. Electron microscopy of skin biopsies showed a reduction in the number of keratin intermediate filaments in the basal cells without tonofilament clumping. We identified a novel heterozygous deletion mutation (1649delG of KRT5) in both cases. This deletion is predicted to produce a mutant keratin 5 protein with a frameshift of its terminal 41 amino acids and 35 amino acids longer than the wild-type keratin 5 protein due to a delayed termination codon. As the same abnormal elongated mutant KRT5 gene was found in the independent families, the predicted abnormal elongated keratin protein is likely to lead to an atypical clinical phenotype that has never been reported, possibly by interfering with the functional interaction between keratin and its associated proteins.