Genetic Analysis of KRT9 Gene Revealed Previously Known Mutations and Genotype-Phenotype Correlations in Epidermolytic Palmoplantar Keratoderma

Epidermolytic palmoplantar keratoderma (EPPK, OMIM 144200) is an autosomal dominant inherited disease, clinically characterized by diffuse yellowish thickening of the skin on the palms and soles, usually with erythematous borders developing during the first weeks or months after birth. Pathogenesis of EPPK is determined by mutations in the keratin gene (KRT9). Thirty three mutations in the KRT9 gene from 100 EPPK families have been identified. Among these, 23 mutations are located in the 1A region (a mutation hot spot region), 7 are located in the 2B region, and the remaining 3 are synonymous mutations. In this study, three heterozygous mutations (p.N161S, p.R163W, and p.R163Q), located in regions of the gene encoding the conserved central a-helix rod domain, were detected in the KRT9 gene of the three large Chinese families. This study confirms that codon 163 (48 of 100 cases) is a hot spot mutation site for KRT9. Additional findings identified p.N161S (4%) and p.R163W (4%) as potential hot spot mutations for EPPK associated with knuckle pads, and p.R163Q (15 of 100 cases) as the hot spot mutation of EPPK not occurring in combination with knuckle pads. In conjunction with future studies, this research may help lay the foundation for genetics counseling, prenatal diagnosis and clinical treatment of EPPK.

Keratin 9 is composed of the functional head domain, the functional α-helical domain and the functional tail domain, and is expressed only in the suprabasal layers of the palmoplantar epidermis (Uitto et al., 2007). To date, domestic and foreign scholars have found 33 KRT9 gene mutations in 100 EPPK families, of which 15 cases are associated with knuckle pads.
There is no report making a detailed summary and analysis. In this study, cases of EPPK were analyzed to look for genotypephenotype correlations by searching the database and consulting the literatures, providing a theoretical basis for the prenatal diagnosis of, genetic counseling for, and clinical treatment of EPPK.

COMPLIANCE WITH ETHICAL STANDARDS
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed written consents were obtained from all individual participants or their legal representatives (parents) included in the study. The study was approved without restrictions by the Medical Ethics Committee of the First Affiliated Hospital of Anhui Medical University. The probands and their family members provided written informed consent for the publication of this case report.

CASE PRESENTATION
Three unrelated Chinese EPPK pedigrees from Shandong and Anhui Province were investigated. All patients exhibited typical EPPK features. There were no close relatives bettwen these families. Family 1 was a six generational EPPK pedigree with 17 affected members, including 11 males  and 6 females. The minimum age of onset is 1 year of age. The proband was a 42-year-old man who presented with diffuse thickening and hyperkeratosis on palms with the nails being normal, and combined knuckle pads, hyperhidrosis and camptodactyly (Figures 1A, 2A). There is no evidence that the proband was associated with other diseases. Family 2 was a 4 generational EPPK pedigree with 11 affected members, and the 20-year-old female proband presented with diffuse thickening and hyperkeratosis on palms and soles (Figures 1B, 2B). Family 3 was a 5 generational EPPK pedigree with 10 affected members, and the proband was a 42-year-old man who demonstrated hyperkeratosis of both palmar and plantar skin within 1 year of birth (Figures 1C, 2C).

LABORATORY INVESTIGATIONS Genetic Testing and Confirmation of Mutation
Peripheral blood samples were collected from members of the three families and 100 healthy unrelated Chinese individuals. Genomic DNA was extracted using a Flexi Gene DNA Kit (250). The primers were amplified by polymerase chain reaction (PCR) and PCR products were directly sequenced by an ABI3730 DNA Sequencer (ABI, USA). The sequence was analyzed by Chromas 2.0 software.

Identification of Three Distinct Mutations in the KRT9 Gene of Three Large Chinese Families
Patients of family 1 had a heterozygous mutation c.482A>G (P.Asn161His) in KRT9 gene ( Figure 3A). Patients of family 2 had a heterozygous mutation c.487C>T (p.Arg163Trp) in KRT9 gene ( Figure 3B). Patients of family 3 had a heterozygous mutation c.488G>A (p.Arg163Gln) in KRT9 gene ( Figure 3C).These mutations were not found in normal members of three families and in 100 healthy controls.  Table 1). Disease causing mutations are as follows: of which 23 are located in the 1A region (hot spot mutation region), 7 in the 2B region, and the remaining 3 are synonymous mutations. Missense mutations at amino acid 163 (48% of all mutations) is indeed a hot spot mutation site for KRT9. We also found that the mutations of EPPK associated with knuckle pads (15% of 100 cases) are p.N161S (4%), p.R163W (4%), p.L168S (3%), p.M157T (1%), p.L160F (1%), p.C406R (1%), and p.L458p (1%). Since these mutations are the most prevalent we can suggest that p.N161S and p.R163W are potential hot spot mutations of EPPK associated with knuckle pads. The hot spot mutation of EPPK not associated with knuckle pads is p.R163Q (15 of 100 cases).

DISCUSSION
Epidermolytic palmar hyperkeratosis (EPPK) is rare in clinical settings with a prevalence of ∼4.4/100,000. In mild cases, only the epidermis of the palmoplantar is rough, and severe horny thickening plaques appear in the palmoplantar region. It may even spread to the lateral edge of the palmoplantar skin or the hands and feet and may be accompanied by knuckle pads and finger-toe flexion deformities. The keratin mutations that have been found so far are concentrated in the 1A helix region and the 2B helix region, namely, the KRT9 gene mutation hot spot (Guo et al., 2014;Liang et al., 2014), especially the 1A region, which affects the formation of keratinous network structures leading to severe clinical manifestations.
Researchers at home and abroad have found that the 163rd amino acid is the hotspot mutation region of the KRT9 gene (48 of 100 cases) (Rugg et al., 2002). EPPK combined with knuckle pads maybe associated with mutations in many KRT9 genes, such as p. Met157Thr, p.Leu160Phe, p.Asn161Ser,p.Arg163Trp, p.Leu168Ser, p.Cys406Arg, and p.Leu458Pro (Escobar et al., 2007;Codispoti et al., 2009;Li et al., 2009;Wang et al., 2010;Du et al., 2011;Mao et al., 2018;Xiao et al., 2018). In this study, we studied three large Chinese families with EPPK and found 3 heterozygous gene mutations of KRT9: c.482A>G (p.Asn161Ser), c.487C>T (p.Arg163Trp) and c.488G>A (p.Arg163Gln). These mutations were not found in normal members of three families and in 100 healthy controls, indicating that the mutations detected in the families were the pathogenic mutations. The mutation p.Asn161Ser has been reported several times in relation to the typical hyperkeratotic manifestations of palmoplantar skin in patients with EPPK, but it has not been associated with other clinical phenotypes. In 2005, Japanese scholars reported a case of EPPK in a 13-yearold patient with knuckle pads, and the genetic test results were found to be associated with the mutation p.Asn161Ser. In this study, the pathogenic mutation of family 1 was p.Asn161Ser, and all patients in the family showed knuckle pads, consistent with previous reports. This study found that 33 KRT9 gene mutations in 100 EPPK families have been reported by domestic and foreign scholars, and the mutations of EPPK associated with knuckle pads (15 of 100 cases) are p.N161S (4%), p.R163W (4%), p.L168S (3%), p.M157T (1%), p.L160F (1%), p.C406R (1%), and p.L458p (1%), suggesting p.N161S and p.R163W are potential hot spot mutations of EPPK associated with knuckle pads, and p.R163Q (15 of 100 cases) as the hot spot mutation of EPPK not occurring in combination with knuckle pads.Based on the above studies, this case reveals knuckle pads may also be one of the less common clinical phenotypes of EPPK, and we think this study should help lay the foundation for genetics counseling, prenatal diagnosis and clinic treatment of EPPK.

AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

ACKNOWLEDGMENTS
We would like to thank the individuals and their families who participated in this project. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.