Two cases of KRT1 mutation-associated epidermolytic ichthyosis without typical epidermolytic hyperkeratosis in the neonatal skin lesions

Epidermolytic ichthyosis (EI) is a rare genetic disorder of keratinization caused by mutations in either KRT1 or KRT10. Histopathologically, epidermolytic hyperkeratosis (EHK) is a hallmark of EI. Here, we report two EI cases in which KRT1 mutation was confirmed by molecular study, but without typical EHK present on skin biopsies performed within 1 week of age. Our cases demonstrate that EHK may not be evident in EI if skin biopsy is performed during the neonatal period.     

A novel mutation of keratin 9 in a large Chinese family with epidermolytic palmoplantar keratoderma

BACKGROUND: Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant inherited skin disorder characterized by diffuse yellow thickening of the skin of the palms and soles, sharply bordered with erythematous margins. Histologically and ultrastructurally, EPPK presents cytolysis of keratinocytes and abnormal aggregation of tonofilaments in the suprabasal layers of the epidermis. To date, 15 different mutations of the keratin 9 gene (KRT9) have been demonstrated to cause most cases of EPPK. OBJECTIVES: To identify the KRT9 mutation in a large Chinese family with EPPK. METHODS: Denaturing high-performance liquid chromatography (DHPLC), DNA sequencing and allele-specific polymerase chain reaction (AS-PCR) were used to screen exon 1 of the KRT9 gene for sequence variations. RESULTS: The DHPLC elution profiles of the DNA fragments amplified from the affected samples differed from those obtained from unaffected individuals, indicating that a sequence variation existed within the analysed fragment of KRT9. DNA sequencing revealed a novel insertion-deletion mutation in the exon 1 of KRT9, 497delAinsGGCT, resulting in the change of tyrosine(166) to tryptophan and leucine (Y166delinsWL). AS-PCR confirmed the mutation was not a common polymorphism. CONCLUSIONS: The results suggest the molecular basis of EPPK in this Chinese family and provide further evidence that mutations in the helix initiation motif of keratin 9 underlie Chinese EPPK.     

Epidermolytic palmoplantar keratoderma due to a novel type of keratin mutation, a 3-bp insertion in the keratin 9 helix termination motif

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis characterized by diffuse keratoderma, typically with an erythematous border. Histologically, palmoplantar epidermis shows suprabasal cytolysis and ultrastructurally, tonofilament aggregation with overlying epidermolytic hyperkeratosis. Mutations in the KRT9 gene, encoding keratin 9 (K9), a cytoskeletal protein expressed exclusively in suprabasal keratinocytes of palmoplantar epidermis, have been reported to cause EPPK. To date, all KRT9 defects reported in EPPK have been missense mutations in exon 1, which encodes the start of the alpha-helical rod domain. However, based on studies of other keratin disorders, it was postulated that mutations at the other end of the rod domain might also produce the EPPK phenotype. Here, we report the first mutation in the 2B domain of KRT9, 1362ins3, leading to an insertion of histidine in the helix termination motif of the K9 polypeptide. Insertional mutations have not been previously described in keratins. The phenotype of this case is similar to EPPK caused by 1A domain mutations, demonstrating that mutations in either of the helix boundary motif sequences of K9 are detrimental to keratin function and keratinocyte structure.     

Mutations in the keratin 9 gene in Pakistani families with epidermolytic palmoplantar keratoderma

Background. Keratins are heteropolymeric proteins that form the intermediate filament cytoskeleton in epithelial cells. The common basic structure of all keratins is organized in a central α‐helical rod domain flanked by nonhelical, variable head and tail regions. Most mutations in keratins are found in the central α‐helical rod domain. Keratin 9 (K9) is expressed only in the suprabasal layers of palmoplantar epidermis. Mutations in the keratin 9 gene (KRT9) have been shown to cause epidermolytic palmoplantar keratoderma (EPPK; OMIM 144200), an autosomal dominant genodermatosis characterized clinically by diffuse hyperkeratosis limited to the palms and soles, and histologically by epidermolysis in suprabasal layers of the epidermis. Aim. To elucidate the genetic basis of EPPK in five Pakistani families. Methods. Using microsatellite markers localized to the areas around the type I keratin gene cluster on chromosome 17q21, genotyping of these families was performed, followed by sequencing of the KRT9 gene. Results. The analysis resulted in the identification of two novel (p.M157K and p.Y454H) and two recurrent (p.M157T and p.R163Q) mutations in the KRT9 of all five families. All mutations occurred within the highly conserved helix initiation or termination motif of K9. Conclusions. The affected members of all five families possess mutations in the KRT9 gene that severely affect heterodimer formation with the type II keratin partner. The results of our study further underscore the crucial role of K9 protein in the palmoplantar epidermis.     

Atypical epidermolytic palmoplantar keratoderma presentation associated with a mutation in the keratin 1 gene

BACKGROUND: Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant genodermatosis characterized by epidermolytic hyperkeratosis strictly confined to the palms and soles, and usually associated with mutations in the keratin K9 gene (KRT9). Mutations in the keratin K1 gene (KRT1) have been shown to underlie a variety of phenotypes typically involving generalized epidermolytic hyperkeratosis, but in some cases the phenotype can be more regionally restricted. OBJECTIVES: To identify the genetic defect in two unrelated families initially presenting with EPPK but where careful examination revealed hyperkeratosis extending on to the proximal wrist flexure. Methods Linkage analysis and DNA sequencing. RESULTS: We found that this phenotype is caused by a heterozygous missense mutation in the K1 gene, designated I479T. This mutation lies in the highly conserved helix termination motif of K1, previously shown to be important for keratin assembly and filament formation. In general, mutations in this region of keratins are associated with more severe disease phenotypes. However, K1 mutations in this region and the I479T mutation in particular have previously been associated with both severe and mild bullous congenital ichthyosiform erythroderma phenotypes. When further clinical enquiries were made, several affected individuals in the families studied here were found to have had transient flexural peeling and hyperkeratosis in the neonatal period. CONCLUSIONS: K1 mutations may underlie a phenotype closely resembling EPPK. A history of transient flexural peeling and hyperkeratosis in childhood and palmoplantar keratoderma which extends beyond the boundary of the palmoplantar margins may indicate a K1 mutation rather than a K9 defect. As K1 mutations are also associated with severe widespread phenotypes, with important implications for prognostic and genetic counselling, whole body examination is recommended for patients presenting with EPPK.     

R156C mutation of keratin 10 causes mild form of epidermolytic hyperkeratosis

A 37-year-old Japanese male presented to us with persistent asteatotic skin with mild erythema on the trunk and extremities. Skin biopsy from the left knee showed marked epidermal acanthosis and hyperkeratosis, and milder granular degeneration. Ultrastructural analysis revealed clumping of the keratin filaments within suprabasal keratinocytes of the epidermis. Following direct sequencing, we found a single nucleotide substitution in one allele at the residue position 466 of the 1A rod domain segment (CGC to TGC, arginine to cysteine; R156C) in keratin 10. Clinical manifestations and molecular analysis indicated that R156C mutation in keratin 10 gene (KRT10) causes a mild form of epidermolytic hyperkeratosis (EHK) in the presented case.     

表皮松解性角化过度型鱼鳞病两家系基因突变研究

目的 探讨两个表皮松解性角化过度型鱼鳞病（EHK）家系的基因突变情况。 方法 收集两个EHK家系的临床资料,提取外周血DNA,通过PCR扩增角蛋白基因KRT1和KRT10编码区的全部外显子及其侧翼序列并测序,以表型正常家系成员及50例健康人作为对照。结果 发现两个家系中患者均存在KRT10基因突变,分别为KRT10的剪接位点突变c.1030-2A > G和错义突变c.467G > A,在家系中健康人及健康对照者未发现上述突变。结论 剪接位点突变c.1030-2A > G和错义突变c.467G > A,可能分别是导致这两个家系临床表型的原因。     

一个表皮松解性掌跖角化病家系的KRT1基因突变分析

目的 探讨一个中国汉族人表皮松解性掌跖角化病(EPPK)家系的角蛋白基因KRT1、KRT9、KRT10突变情况.方法 收集1个EPPK家系的临床资料,提取外周血DNA,通过PCR扩增角蛋白KRT1、KRT9、KRT10基因编码区的全部外显子及其侧翼序列并测序,以表型正常家系成员及50例健康人为正常对照.结果 发现家系内6例患者均存在KRT1基因错义突变c.1436T>C,导致第479位的异亮氨酸被苏氨酸取代(I479T),在家系中6例正常人及50例对照者未发现上述突变.结论 错义突变KRTI的c.1436T>C可能为导致该家系临床表型的主要原因.本例为国内首次发现的KRT1突变引起的EPPK家系.

Abstract：

Objective To analyze the mutations in keratin 1 (KRT1), KRT9 and KRT10 genes in a Chinese family with epidermolytic palmoplantar keratoderma (EPPK). Methods Clinical data were collected from a family with EPPK. Genomic DNA was extracted from the peripheral blood of 12 family members, including 6 patients and 6 unaffected members, as well as from 50 unrelated normal human controls. PCR was performed to amplify all the exons and flanking sequences of KRT1, KRT9 and KRT10 genes followed by DNA sequencing.Results A missense mutation C.1436T > C was found in the highly conserved helix termination motif of KRT1 gene of all the patients, resulting in a substitution of isoleucine by threonine at position 479 of the KRT1 protein. No mutation was found in the unaffected members or unrelated controls. Conclusions The missense mutation C.1436T > C in K.RT1 gene is likely to be the main cause of the phenotype of EPPK in this family.This is the first report of a pedigree with KRT1 gene mutation-induced EPPK in China.     

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.     

Splice site and deletion mutations in keratin (KRT1 and KRT10) genes: unusual phenotypic alterations in Scandinavian patients with epidermolytic hyperkeratosis

Epidermolytic hyperkeratosis is a rare autosomal dominant inherited skin disorder caused by keratin 1 or keratin 10 mutations. Keratins are major structural proteins of the epidermis, and in keratinocytes committed to terminal differentiation the intermediate filaments are composed of keratin 1 and keratin 10 heterodimers. The majority of reported mutations (86.6%) are heterozygous single point mutations and most of these are located in the 1A and 2B regions of the highly conserved keratin alpha-helical rod domain. We have studied eight Scandinavian families with epidermolytic hyperkeratosis and identified three point mutations, two codon deletions, two splice site mutations, and a complex deletion/insertion. Two of the point mutations were in the KRT1 gene (F191C and K177N) and the other was in KRT10 (L453P). All three patients had associated palmoplantar keratoderma. The splice site mutations in KRT1 both caused a large deletion removing 22 codons (delta176-197) from the 1A helical domain. Codon deletions were found in KRT1 (delta170-173) and in KRT10 (delta161-162) in two patients with a severe phenotype. A final patient had a more complex mutation with a large deletion (442 bp) together with a large insertion (214 bp) of unknown origin that caused deletion of exon 6 in KRT1. In conclusion, we have found eight novel keratin mutations that cause epidermolytic hyperkeratosis with differing phenotypes. Even when a large part of keratin 1 (46 amino acids) is deleted, surprisingly mild phenotypes can result, suggesting that genotype-phenotype relationships in epidermolytic hyperkeratosis are complex and do not solely depend on the type of mutation but also depend on interactions between the behavior of the mutant protein and the cellular environment.     

Phenotypic/genotypic correlations in patients with epidermolytic hyperkeratosis and the effects of retinoid therapy on keratin expression

Dominant-negative mutations in the KRT1 and KRT10 genes cause epidermolytic hyperkeratosis, a rare form of ichthyosis sometimes associated with palmoplantar keratoderma. Although there is no permanent cure, some patients improve on retinoid therapy. More knowledge is needed, however, about the mechanism of action of retinoids and the genotypic/phenotypic correlations in this disease. Thirteen patients from 10 families with generalized disease and 2 sporadic patients with nevoid lesions were studied, probably representing most of the patients in Sweden and Norway. All patients, except one nevoid case, were known to have KRT1 or KRT10 mutations. Those with mutated keratin 1 (K1) invariably had associated keratoderma (n=6). In contrast, only 1 of 7 patients with K10 mutations had this problem (p = 0.0047). Five out of 6 patients with KRT10 mutations benefited from treatment with oral acitretin (5-25mg/day) or topical tretinoin/tazarotene, but none of the patients with KRT1 mutations derived any benefit. Quantitative analysis of K1 and K10 mRNA in skin biopsies obtained before and after retinoid therapy (n=8) showed no consistent down-regulation of mutated keratin that would explain the therapeutic outcome. Instead, the mRNA expression of K2e (a normal constituent of the upper epidermis) diminished especially in nonresponders. In contrast, K4 mRNA and protein (marker of retinoid bioactivity in normal epidermis) increased in almost all retinoid-treated patients. In conclusion, our study confirms a strong association between KRT1 mutations and palmoplantar keratoderma. Retinoid therapy is particularly effective in patients with KRT10 mutations possibly because they are less vulnerable to a down-regulation of K2e, potentially functioning as a substitute for the mutated protein in patients with KRT1 mutations.     

Keratin 1 gene mutation detected in epidermal nevus with epidermolytic hyperkeratosis

Since 1994, four cases of epidermal nevus with epidermolytic hyperkeratosis (EH) caused by keratin 10 gene mutations have been reported, although no keratin 1 (K1) gene mutation has yet been reported. We detected a K1 gene (KRT1) mutation in epidermal nevus with EH in a 10-year-old Japanese male. The patient showed well-demarcated verrucous, hyperkeratotic plaques mainly on the trunk, covering 15% of the entire body surface. No hyperkeratosis was seen on the palms or soles. He had no family history of skin disorders. His lesional skin showed typical granular degeneration and, ultrastructurally, clumped keratin filaments were observed in the upper epidermis. Direct sequence analysis of genomic DNA extracted from lesional skin revealed a heterozygous 5' donor splice site mutation c.591+2T>A in KRT1. This mutation was not detected in genomic DNA samples from the patient's peripheral blood leukocytes or those of other family members. The identical splice mutation was previously reported in a family with palmoplantar keratoderma and mild ichthyosis, and was demonstrated to result in a 22 amino-acid deletion p.Val175_Lys196del in the H1 and 1A domains of K1. To our knowledge, the present patient is the first reported case of epidermal nevus associated with EH caused by a K1 gene mutation in a mosaic pattern.     

A novel keratin 9 gene mutation (Asn160His) in a Taiwanese family with epidermolytic palmoplantar keratoderma

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant inherited disorder of keratinization. Recent molecular studies have shown that EPPK is caused by mutations in keratin 9 gene (KRT9). We report a Taiwanese family with EPPK with a novel mutation with an A-->C transition at the first nucleotide of codon 160 in KRT9. The mutation is predicted to result in an asparagine to histidine substitution (N160H) at the beginning of the alpha-helical 1A domain of keratin 9. Mutations in this region could disrupt keratin filament assembly, leading to degeneration or cytolysis of keratinocytes. Our mutation analysis confirms that codon 160 in KRT9 is one of the mutation hot spots in EPPK.     

表皮松解性掌跖角皮症一家系KRT9基因新突变

目的:检测表皮松解性掌跖角皮症一家系患者角蛋白9(KRT9)基因突变。方法:收集家系成员的临床资料和血样,提取家系中4例患者和3名正常人及50名与本家系无关的正常对照外周血DNA,采用PCR技术扩增KRT9基因所有编码区并进行测序,分别检测家系中的突变情况。结果:该家系中所有患者均存在KRT9基因错义突变(c.484TC),导致第162位密码子由TCT(丝氨酸)转变为CCT(脯氨酸)(p.S162P),家系中3名正常个体和50名健康对照均未发现上述突变。结论:KRT9基因c.484TC错义突变是导致该家系发生表皮松解性掌跖角皮症的遗传基础。     

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. Our results are compatible with the above classification and suggest that the arginine in the beginning of the 1A rod domain is the hot spot for the mutation of the keratin 10 gene.     

表皮松解性掌跖角化病二家系基因突变检测

目的：检测表皮松解性掌跖角化病二家系患者致病基因。方法：收集二家系资料,提取二家系成员及100名（无亲缘关系）正常对照血样DNA,采取聚合酶链反应技术对KRT1、KRT9和KRT16基因进行扩增,并对其产物进行测序。结果：家系1先证者中检测到 KRT1基因突变c.598T>C（p.F200L）。家系2三例患者中检测到KRT9基因含杂合突变c.488G>A（p.R163Q）。而家系正常成员及家系外无亲缘关系的100名正常对照中均不存在以上突变。结论：本研究表皮松解性掌跖角化病二家系发病与KRT1、KRT9基因突变有关,且KRT1基因突变p.F200L为国内首次报道。     

表皮松解性掌跖角化病研究进展

表皮松解性掌跖角化病是一种常染色体显性遗传性单基因病,以掌跖部对称性弥漫性角化过度为主要特征,其组织学特点为表皮松解性角化过度.目前已从分子水平上阐明表皮松解性掌跖角化病由角蛋白9及角蛋白1的基因突变引起.此外,环境及药物卡培他滨也可能为其致病因素.表皮松解性掌跖角化病主要以对症治疗为主,小干扰RNA的研究逐步成为热点,为表皮松解性掌跖角化病的基因治疗提供一定的理论基础.随着对此病分子基础的研究,产前诊断的技术正不断发展.     

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.     

Selective involvement of keratins K1 and K10 in the cytoskeletal abnormality of epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma).

Aggregation of tonofilaments within epidermal keratinocytes is a characteristic histologic feature of epidermolytic hyperkeratosis including the generalized form known as bullous congenital ichthyosiform erythroderma. The histologic distribution and the keratin composition of the altered tonofilaments were investigated to determine whether the aggregation was specific to any particular keratin(s). Skin samples from seven patients and one mid-trimester fetus with generalized epidermolytic hyperkeratosis, and from one patient with a localized or "nevoid" form of epidermolytic hyperkeratosis, were analyzed by using various microscopical and immunocytochemical methods. A conjunctival sample and cultured epidermal keratinocytes from one patient with generalized epidermolytic hyperkeratosis were also examined by electron microscopy and immunocytochemistry. Ultrastructurally, tonofilament aggregates were distributed within the suprabasal stratified epithelial cell layers of the epidermis, of the infundibular part of outer root sheaths, and of the sebaceous ducts and sweat ducts, selectively following the known distribution pattern of keratins K1 and K10. The abnormal tonofilaments were not found in any other cutaneous epithelia, in conjunctival epithelium, or in cultured keratinocytes, where K1 and K10 are absent or only minimally expressed. Immunoelectron microscopy showed that among the keratins detected in suprabasal epidermolytic hyperkeratosis epidermis (K1/K5/K10/K14/K16), the aggregated tonofilaments predominantly expressed K1 and K10 rather than other keratins. These results suggest that the keratin filament abnormality in epidermolytic hyperkeratosis principally involves K1 and K10 and raise the question whether epidermolytic hyperkeratosis might be primarily a disorder of one or both of these keratins.