Two novel de novo mutations of KRT6A and KRT16 genes in two Chinese pachyonychia congenita pedigrees with fissured tongue or diffuse plantar keratoderma

Mutations in the KRT6A or KRT16 gene cause pachyonychia congenita type 1 (PC-1), while mutations in KRT16 or KRT6C underlie focal palmoplantar keratoderma (FPPK). A new classification system of PC has been adopted based on the mutated gene. PC rarely presents the symptoms of diffuse plantar keratoderma. Mutation in the tail domain of keratins is rarely reported. PC combined with fissured tongue has never been described.

To investigate the genotype-phenotype correlations between clinical features and gene mutational sites in two unrelated southern Chinese PC pedigrees (one family presented with specific fissured tongue, the other with diffuse plantar keratoderma).

The whole coding regions of the KRT6A/KRT16/KRT17/KRT6B genes were amplified and directly sequenced to detect the mutation. To confirm the effect of the IVS8-2A>C mutation in KRT6A at the mRNA level, total RNA from the plantar lesion of a patient was extracted and reverse-transcribed to cDNA for sequence analysis.

Two novel de novo mutations, a splice acceptor site variant IVS8-2A>C (p.S487FfsX72) in KRT6A and a heterozygous substitution c.AA373_374GG (p.N125G) within exon 1 of KRT16, were found separately in the two PC families.

Genotype-phenotype correlations among PC patients with codon-125 mutation in KRT16 were established, while the phenotypes caused by the IVS8-2A>C mutation in KRT6A need further studies to confirm the rare feature of fissured tongue.

ARTICLE

ejd.2012.1773

Auteur(s) : Zhen-Fang Du^1,a, Chen-Ming Xu^1,2,a, Yan Zhao¹, Wen-Ting Liu¹, Xiao-Ling Chen¹, Chun-Yue Chen³, Hong Fang⁴, Hai-Ping Ke⁵, Xian-Ning Zhang¹ zhangxianning@zju.edu.cn

¹ Department of Biochemistry and Genetics, Zhejiang University School of Medicine, 866 Yuhangtang Road, Hangzhou 310058, China

² Women's Hospital, Zhejiang University School of Medicine, Hangzhou, China

³ Hangzhou Red Cross Hospital, Hangzhou, Zhejiang Province, China

⁴ Department of Dermatology, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

⁵ Department of Biology, Ningbo College of Health Sciences, Ningbo, Zhejiang Province, China

Reprints: X. Zhang

^a These authors contributed equally to this work.

Pachyonychia congenita (PC, OMIM: 167200) is a rare autosomal dominant genodermatosis with variable clinical findings: hyperhidrosis (79%), oral leukokeratosis (75%), follicular keratosis (65%), palmar keratoderma (60%), cutaneous cysts (35%), hoarseness or laryngeal involvement (16%), coarse or twisted hair (26%), early primary tooth loss (14%), and presence of natal/prenatal teeth (2%) [1]. More than 97% of PC cases exhibit toenail thickening, plantar keratoderma, and plantar pain [2]. Some rare clinical features of PC include the development of alopecia areata, skeletal abnormalities, painful oral and nipple lesions during breastfeeding, copious production of waxy material in the ears, inability to walk without an ambulatory aid (50%) and transgrediens involvement of the dorsal feet [1, 3-5]. Classically, based on the clinical presentation, patients with mutations in KRT6A and KRT16 have been grouped to PC-1 (Jadassohn-Lewandowski syndrome), and KRT6B and KRT17 to PC-2 (Jackson-Lawler syndrome) [6-8]. Patients with PC-1 are reported to have more prominent oral leukokeratosis, while patients with PC-2 classically are reported to have cysts and natal teeth, but this feature is not fully penetrant and its absence does not preclude the PC-2 phenotype [1, 9]. Recent studies showed that 45-52% of a PC kindred had KRT6A, 28-30% had KRT16, 17% had KRT17, and 3-8% had KRT6B mutations [2, 10]. The classic separation of PC into the two subtypes is not justified based on the genotyping and phenotyping of nearly 1,000 patients now available through the International Pachyonychia Congenita Research Registry (IPCRR) [11, 12]. The variability of clinical phenotypes in PC is further highlighted by reports of unique mutations within the same gene leading to different clinical symptoms [13, 14]. So a new molecular classification linking the mutant gene and clinical subtype has been adopted (PC-6a, PC-16, PC-17, PC-6b and PC-U for unknown pathogenic genes) [10, 11].

Focal palmoplantar keratoderma (FPPK, OMIM: 613000) is a rare autosomal dominant keratin disorder which presents as a separate clinical entity with subtle or absent nail changes [15]. Mutations in KRT16 or KRT6C underlie FPPK [14-17]. Hyperkeratosis and hyperhidrosis of the palms and soles are common in PC. This may be focal or widespread and may not develop until later in childhood. The phenotypes of PC-1 and FPPK are ambiguous for the involvement of nail changes. Fissured tongue has been described previously to be of familial transmission and associated with Melkersson-Rosenthal syndrome, but PC combined with fissured tongue has never been reported [18, 19].

Keratins are abundant proteins in epithelial cells where they form a network that acts as a resilient, pliable scaffold that enables epithelial cells to sustain mechanical and non-mechanical stresses to maintain tissue integrity [20, 21]. Inherited mutations in keratin genes affect the keratin intermediate filament (IF) network and result in epithelial fragility disorders such as PC. Similar to several other keratin disorders, the vast majority of causative mutations in the PC-related keratins are heterozygous missense mutations or small insertion/deletion mutations that occur in the helix boundary motifs and disrupt cytoskeletal function via dominant-negative interference and lead to epithelial cell fragility [22]. In PC, this is manifest as cytolysis and hyperkeratosis in the subset of differentiated epithelial tissues in which K6A, K6B, K16, and K17 are predominantly expressed, specifically the palmoplantar epidermis, nail bed, mucosae, and the pilosebaceous unit. Thus, the cardinal phenotypic features of PC are palmoplantar (predominantly plantar) keratoderma, hypertrophic nail dystrophy, oral leukokeratosis and a variety of cysts arising from hyperkeratosis of the pilosebaceous apparatus. Cytokeratins usually form obligatory heterodimers [23]. For example, the K6/K16 heterodimer is involved in wound healing and hyperproliferation [24, 25]. Mutations in these two genes cause a similar phenotype (PC-1, traditional subtype), but a recent study indicated that PC patients with KRT6A and KRT16 mutations have distinct phenotypic differences [12].

Here, we described two Chinese PC pedigrees: PC-6a with a de novo splice acceptor site variant IVS8-2A>C (p.S487FfsX72) within the exon-intron junction of exon 9 in KRT6A, and PC-16 harboring a heterozygous substitution c.AA373_374GG (p.N125G) within exon 1 of KRT16.

Materials and methods

Subjects

Two unrelated southern Chinese PC families each with two affected members were investigated (figure 1A). In Family A (FA), the proband (FA-II-1) was a 32-year-old woman with toenail thickening, persistent hyperkeratosis with fissures on both soles and plantar pain, while both palms had several mild focal persistent hyperkeratoses without fingernail change and palm pain. Local oral leukokeratosis, chapped lips and fissured tongue were observed (figures 1B-H). Histopathology from the plantar lesion showed hyperkeratosis, acanthosis, and a moderate increase in the granular layer with minimal lymphocytic infiltrate in the upper dermis (figure 1I). Her 7-year-old daughter (FA-III-1) only had focal plantar hyperkeratosis, fissured tongue and gingivitis without toenail and hand involvement.

In Family B (FB), the proband (FB-II-2) was a 36-year-old female with severe hypertrophic nail dystrophy (HND), diffuse keratoderma on both soles and plantar pain, while both hands presented only with several subtle focal persistent hyperkeratoses on the palms, with palm pain (figures 1J-N). Her 5-year-old daughter (FB-III-2) showed focal hyperkeratosis on both plantar points of contact without toenail and hand involvement.

All patients complained of hyperhidrosis of the hands and feet. No hoarseness, natal teeth, pilosebaceous cysts, hair or other organ abnormalities were noted. There was no consanguinity, and no siblings or other family members had similar lesions.

Mutation detection

Genomic DNA was isolated from the peripheral blood of all family members and 100 unrelated healthy controls. The coding regions of KRT6A, KRT16, KRT17 and KRT6B were amplified using primers specific to the respective functional genes to avoid amplification of KRT6C or pseudogenes as described previously [10]. For each gene, there are two primer sets for the mutation hotspot exons to overcome the potential problem of very rare or as-yet unidentified single nucleotide polymorphisms in primers designed to amplify these regions, followed by direct sequencing [10, 26]. If the hotspot region scanning did not reveal the mutated allele, the gene testing was extended to the entire coding sequence.

Splice site variant confirmation

To confirm the effect of the IVS8-2A>C mutation in KRT6A at the mRNA level, total RNA from the plantar lesion of FA-II-1 was extracted and reverse-transcribed. cDNA was amplified using KRT6A-specific primers (forward: 5’- AGT GCA GGC TGA ATG GCG AA-3’, reverse: 5’- TTG AGA GCC AGT GGA AAG T-3’), then the PCR product was sequenced by Sanger clonal sequencing.

This study was conducted in conformity with the Helsinki Declaration and approved by the Zhejiang University Review Board. Written informed consent was given by all subjects.

Results

Two novel de novo mutations were found. In FA, there was a splice-site variant IVS8-2A>C of KRT6A, which resulted in an 11-bp deletion (c.1460_1470delCTGTGGTGCAG) at the beginning of exon 9 due to activation of a downstream splice acceptor site ( AGTCCACCGTCT) (figures 2A-B). This deletion is predicted to remove 4 amino acids and cause a subsequent frameshift p.S487FfsX72 (figures 2C-E). In FB, a heterozygous substitution c.AA373_374GG within exon 1 of KRT16 occurred, i.e., p.N125G within the 1A rod domain of keratin 16 (figure 2F). Neither normal family members nor controls revealed the mutated allele.

Discussion

In FA, we identified a rare splice acceptor site mutation within the exon-intron junction of exon 9 in KRT6A. A corresponding mutation, IVS8-1G>A in KRT74, was reported to cause autosomal dominant woolly hair/hypotrichosis in a Pakistani family [27]. Protein-protein BLAST analysis (http://blast.ncbi.nlm.nih.gov/) showed that the mutant peptide sequence has no significant similarity to any human protein (data not shown). Keratin mutations are rarely located in the tail domain of IF proteins, which play important roles in the width control of filament formation and the regulation of IF dynamics and other post-translational modifications [28]. Notably, both patients presented typical features of PC except for an unreported symptom: fissured tongue. Kullaa-Mikkonen considered fissuring with normal papillary structure a variation of normal anatomy, whereas fissured and geographic tongue is a clinical and etiologic disease entity [18]. Smeets et al. described a female with Melkersson-Rosenthal syndrome with scrotal tongue since childhood who had a de novo t(9;21) (p11;p11) translocation, and suggested that the gene is located at 9p11 [19]. With the concomitant symptom of PC, in contrast to Melkersson-Rosenthal syndrome, we cannot conclude here that fissured tongue is caused by IVS8-2A>C of KRT6A, due to the limited sample, and the pathogenic mechanism needs further study. The mutation was located in a non-hotspot region where mutation is rarely reported [29]. Our case highlights the importance of extending gene testing to the entire coding region when the initial testing of a patient with clinical signs of PC fails to reveal any hot-spot mutations or when genotype-phenotype discrepancies emerge.

In FB, the patients exhibited severe toenail thickening and diffuse plantar hyperkeratosis, which may be due to the substitution of asparagine (N) to glycine within keratin 16. The vast majority of KRT16 mutations are single-base pair changes (89%) with 4% being deletions and 9% insertions, but a dinucleotide change has only rarely been described [12]. The asparagine residue of codon-125 in keratin 16 is located in the amino-terminal end of coil 1A [28]. This domain is extremely well-conserved for IFs and is nearly identical, particularly over the absolutely conserved sequence Leu- Asn-Asp-Arg [28]. Thus, converting asparagine (N) to glycine (G) significantly alters the character of the consensus motif, which may consequently interfere with the spatial organization of the keratin cytoskeleton [28]. Here, patients with the p.N125G variant presented features different from other reported codon-125 mutations of KRT16. Of the six p.N125S cases described, five exhibited mild nail changes and were finally diagnosed as FPPK [14, 16, 17]; one proband had typical HND and was diagnosed as PC-1 [17]. The p.N125D family had a more typical PC-1 presentation with severe HND affecting both hands and feet [17]. A recent study indicated that patients with p.N125D exhibit more severe disease than those carrying p.N125S [13]. Based on these studies, p.N125S is predisposed to be associated with FPPK as to the subtle change in the nails, while p.N125D correlates with PC-1 due to the severe HND. Here, the subtle focal hyperkeratoses in the proband's hands were less mild than both the p.N125S and p.N125D described previously, but the severe diffuse plantar keratoderma was clearly distinct from p.N125D and p.N125S patients who presented with FPPK [13, 14, 17]. The daughter (FB-III-2) had milder clinical features, possibly due to her age. Besides, the codon-125 mutation in KRT16 presented more severe FPPK symptoms than the KRT6C mutations reported previously [15, 30]. Therefore, together with previous studies, we propose that the clinical symptoms of PC depend on the type of amino-acid substitution.

A diagnostic triad of toenail thickening, plantar keratoderma, and plantar pain was confirmed in both of the probands. These symptoms are consistent with several recent studies [2, 10, 11]. However, the onset and severity of clinical symptoms varied considerably between our patients. A recent study concluded that PC-6a patients have more extensive nail involvement with 8-10 thickened toenails than their PC-16 counterparts [12]. But in our study, the patient with the KRT6A mutation (FA II-1) had 10 toenails affected, but much milder changes than the one with the KRT16 mutation (FB II-2). The proband with the KRT16 mutation had considerably more severe plantar keratoderma and pain than the one with the KRT6A mutation, which is consistent with the conclusion by the IPCRR [12]. And the daughter with the KRT6A mutation had all normal toenails, that is, she may have a later onset than the reported average of 0.35 years [2].

These two well-characterized and mutation-confirmed pedigrees offer the opportunity to supplement valid conclusions regarding genotype-phenotype relationships. The phenotype differences between the KRT6A and KRT16 mutations here support the adoption of a new classification system based on the mutant gene (PC-6a, PC-16, etc.). Meanwhile, our data support the previous conclusion that the variations in phenotypes caused by different substitutions at the same mutation site suggest a genotype-phenotype correlation.

Disclosure

Acknowledgments: We thank all the patients and their families who agreed to participate in this study. Financial support: This work was supported by the National Natural Science Foundation of China (30972644 and 30672250). Conflicts of interest: None.

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