Missense mutation of keratin 9 (c.487C>T (p.R163W) in southern Chinese patients with epidermolytic palmoplantar keratoderma

ARTICLE

Auteur(s) : Chen-Ming Xu^1,2, Xiao-Ling Chen², Chun-Yue Chen², Xian-Ning Zhang²

¹Department of Reproductive Endocrinology, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, China
²Institute of Cell Biology and Department of Biochemistry & Genetics, National Education Base for Basic Medical Sciences, Zhejiang University School of Medicine, Hangzhou, China

Epidermolytic palmoplantar keratoderma (EPPK, OMIM: 144200) is an autosomal dominant (AD) genodermatosis characterized by diffuse hyperkeratosis confined to the palmar and plantar epidermis. Keratin 9 (K9) is expressed in a very specific manner limited to palms and soles, making this a prime candidate for EPPK [1, 2]. There are 18 different mutations reported in the 1A domain and 2 mutations in the 2B domain of K9 (Human Intermediate Filament Mutation Database, www.interfil.org).

We present both a kindred and a sporadic case from Fujian and Guangdong province of southern China, respectively, diagnosed with EPPK, based on the clinical and histological examinations. The same KRT9 missense mutation (p.R163W) in the 1A domain was confirmed. This is the first report of a p.R163W mutation of EPPK on the Chinese mainland.

The pedigree is a four-generation family including 9 affected and 22 unaffected individuals, exhibiting AD inheritance (figure 1A). All the affected family members showed severe epidermolytic hyperkeratosis, surrounded by a characteristic erythematous border on the palmoplantar surface. No other body sites were involved. The isolated case is a 9-year-old girl without any siblings, her growth and development have been normal to date. Her parents described the presentation of erythema and peeling of skin confined to the palms and soles soon after birth, with progressive symmetric hyperkeratosis which appeared 3-4 months later (figure 1B). The severity of the skin lesions runs in cycles of improvement and worsening with seasonal variations. After obtaining informed consent, 21 blood samples from the four-generation family (7 affected and 14 unaffected individuals) and 5 samples from the sporadic case were collected. Molecular genetic analysis, including polymerase chain reaction (PCR), denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing for the 7 encoding exons of KRT9 was undertaken, guided by our previous work on Chinese EPPK [2].

After a 436-bp fragment of exon 1 encoding part of the head domain and helix 1A of K9 was amplified from affected and unaffected individuals and subjected to hetero-duplex analysis using DHPLC, different profiles were observed (figure 1C). The normal profile exhibited a single peak, while profiles of all affected members showed four distinct peaks, which were observed under partial denaturing conditions (at 59.8°C). This indicated a sequence variation within the fragment analyzed. Sequence analysis identified a C-to-T transition at nucleotide 487 that resulted in an arginine (CGG) to tryptophan (TGG) substitution at position 163 in all affected individuals, as compared with the normal sequence (figure 1C). There was also a homozygous single nucleotide polymorphism (c. 429A>C transversion) in all affected and unaffected members, which did not result in amino acid changes (data not shown) [3].

The majority of keratin mutations are usually in the helix initiation (1A) or termination (2B) motifs [4]. Mutations in the boundary peptides are highly disruptive to filament assembly, cause tonofilament aggregation, and generally lead to more severe phenotypes. In the case of K9, the 1A hotspot mutations are p.R163W and p.R163Q which arise commonly because of a CpG methylation site within this codon, leading to chemical instability [3, 5]. It is believed the major form of DNA modification in the human genome involves methylation of cytosine residues (to form 5-methylcytosine), specifically when they are located immediately 5’ to a guanine (i.e., as the dinucleotide 5’-CG-3’). Spontaneous deamination of 5-methylcytosine to thymidine in the CG doublet gives rise to C>T or G>A transitions (depending on the strand of DNA in which the 5-methylcytosine is mutated). More than 30% of all single nucleotide substitutions are of this type, and they occur at a rate 25 times greater than any other random mutation occurs [5]. Thus, the CG doublet represents a true “hotspot” for mutation, especially for spontaneous, de novo mutation in human genetic disease genes. Here, substitution of arginine by virtually any other amino acid (with the sole possible exception of lysine) will prevent the formation of H-bonds which are required to stabilize the coiled-coil, resulting in loss of 1A stability. Our report of a spontaneous p.R163W mutation in a southern Chinese patient, together with the previous reports in Taiwan provide supporting evidence for the hypothesis that codon 163 of KRT9 is fatal for keratin filament assembly and an important hotspot for mutation in EPPK, regardless of race or ethnicity [3, 6].

Acknowledgements

References

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