Exome sequencing identifies a KRT9 pathogenic variant in a Chinese pedigree with epidermolytic palmoplantar keratoderma

Abstract Background Epidermolytic palmoplantar keratoderma (EPPK) is a rare skin disorder and its pathogenesis and inheritability are unknown. Objective To investigate the inheritance and pathogenesis of EPPK. Methods Two EPPK cases occurred in a three‐generation Chinese family. Patient–parents trio EPPK was carried out and the identified candidate variants were confirmed by Sanger sequencing. Results A heterozygous missense pathogenic variant, c.488G > A (p.Arg163Gln), in the keratin (KRT) 9 gene was detected in the proband and his son via targeted exome sequencing, and then validated by Sanger sequencing. This pathogenic variant cosegregated with the EPPK in extended family members, and was predicted to be pathogenic by SIFT, PolyPhen2, PROVEAN, and Mutation Taster. This heterozygous variation was not evident in 100 healthy controls. Conclusion This report describes a KRT9 c.488G > A (p.Arg163Gln) variant causing a diffuse phenotype of Chinese EPPK. The current results broaden the spectrum of KRT9 pathogenic variants responsible for EPPK and have important implications for molecular diagnosis, treatment, and genetic counseling for this family.


| Ethics statement
Human samples used in this study were obtained in accordance with the principles expressed in the Declaration of Helsinki, and were approved by the Institutional Review Board from Nanfang Hospital, Southern Medical University. Blood and skin biopsy sample collection procedures used were in accordance with the institutional and national ethical standards of human experimentation and all participants provided written informed consent.

| Clinical material
The proband (II1) (Figure 1a) was a 35-year-old man, who was presented with yellow thickening of the palms and soles with an erythematous margin for 35 years. At 1 month of age, he developed symmetrical erythematous on the palms and soles. At the age of 2 years, hyperkeratotic plaques appeared. Cutaneous examination showed hyperkeratosis yellow thickening plaques on the palms and soles (Figure 1b,c). Oral mucosa, teeth, and nails remained unchanged. The histological examination of the proband's lesion revealed epidermal basket-weave hyperkeratosis, acanthosis, a large number of keratohyalin granules in the upper part of the spinous cell layer, and superficial ectatic capillaries and perivascular infiltration with a few lymphocytes in the dermal layer ( Figure  1d). His elder (III1) (Figure 1a) son had similar symptoms on the palms and soles (Figure 1e,f). The electron microscopy revealed that the epidermis was acanthotic and thickened, and the compact stratum corneum. The stratum corneum was compact and regular distribution. The keratin filaments were remarkably clumped or aggregated and irregularly distributed in the stratum corneum.

| Exome sequencing and sequence analysis
Exome sequencing was then performed. The data for this study were generated by Illumina Hiseq2500 (Illumina, USA). The sequence reads were aligned to the NCBI reference genome (hg19/GRCh37). We used various tools including Inheritance State Consistency Analysis (ISCA) for sequence alignment analysis, ANNOVAR for singlenucleotide polymorphism (SNP) annotation, and GERP for substitution analysis to analyze the genome sequencing results. After annotation of variants, we focused only on nonsynonymous variants (NSVs), splice acceptor-site or donor-site mutations (SSMs), and insertions/deletions (indels) that were more likely to be pathogenic than other variants.

Sanger sequencing
The Sanger sequencing approach was used to validate the EPPK pathogenic variant in the KRT9 gene. PCR was performed around the candidate pathogenic variant positions according to standard techniques. The PCR products were purified using QIAquick PCR purification kit from Qiagen (Germantown, MD) and sequenced by ABI Dye Terminator Cycle Sequencing on an ABI 3730XL DNA analyzer (Life Technologies, Carlsbad, CA). Sequence trace files were analyzed by Sequencer software version 4.5 (GeneCodes, Ann Arbor, MI).

| Exome sequencing
Family members, I1, I2, Ⅱ1, and III1 underwent sequencing to examine a genetic cause for the disorder independently. An average of 10.8 billion bases of sequence was produced per individual as paired-end, 90-bp reads. After mapping to the human reference genome (NCBI Build 36.3, hg19), we achieved ~5.5 Gb of map able, targeted exome sequences with a mean coverage of 107.7-fold. On average, 99.2% of the exomes were covered at least fourfold. Under the assumption of autosomal dominant inheritance with full penetrance, heterozygous variants that were present in all the affected individuals, but not in unaffected participants, were selected as possible candidates. As a result, we detected 21 NSVs, SSMs, or indels in 20 candidate genes. We then used ANNOVAR and GERP to assess these variants for their probable functional impact. We focused on one heterozygous variant that we considered to be a likely candidate of the 21 possible variants. This was in KRT9 (c.488G > A) located within chromosome 17. Therefore, we selected KRT9 as a EPPK candidate gene for further studies.

| Sequencing KRT9
To investigate whether the pathogenic variant was inherited, all nine family members underwent sequencing of KRT9. A missense mutation encoding a c.488G > A alteration (p.Arg163Gln) in the first exon of the KRT9 was found in two affected individuals (Figure 2a), and was absent in the seven unaffected family members ( Figure  2b). We first tested for cosegregation of the pathogenic variant (c.488G > A) with the EPPK phenotype in the extended pedigree of the family. We further sequenced KRT9 (c.488G > A) in 100 healthy controls and did not find a KRT9 pathogenic variant.

| DISCUSSION
PPK comprise a diverse group of acquired and hereditary disorders marked by excessive thickening of the epidermis of palms and soles (Has & Technau-Hafsi, 2016). While PPKs may represent the sole or dominant clinical feature, they may also be associated with other ectodermal defects or extracutaneous manifestations (Has & Technau-Hafsi, 2016). The exact morphological evaluation of keratoderma-diffuse, striate, focal, and punctate-is diagnostically helpful (Austin, 2016;Guo, Shi, & Tan, 2014;Has & Technau-Hafsi, 2016;Ke et al., 2014;Mao et al., 2018). The degree of severity may vary in the same family or among individuals bearing similar pathogenic variants. The proband and his son were presented with yellowish, compact hyperkeratoses, surrounded by erythematous margins, and cover the entire surface of the palms and soles. Histopathology of the proband showed epidermolytic hyperkeratosis. The clinical feature and histopathology confirmed to the diagnosis of EPPK. Early onset and positive family history of the proband and his son suggest a genetic cause. Previous studies showed that similar clinical pictures of PPK may result from pathogenic variants in different genes. Therefore, pathogenic variant analysis was required to determine the precise type of these patients. By exome sequencing, a heterozygous missense pathogenic variant, c.488G > A (p.Arg163Gln), in the KRT9 gene was detected in the proband and his son, and then validated by Sanger sequencing. Usually caused by pathogenic variants in the KRT9 gene, this is the most common form of EPPK. KRT9c.488G > A (p.Arg163Gln) pathogenic variant showed autosomal dominant inheritance and perfectly cosegregated with the EPPK phenotype in all family members. This heterozygous variation was not evident in the proband's parents and 100 healthy controls, which suggested that KRT9 p.Arg163Gln pathogenic variant was a de novo pathogenic variant in the proband and the pathogenic variant of his elder son was inherited from the proband. Keratin 9, expressed in suprabasal cells of the epidermis of palm and sole, where it contributes to reinforcing the skin in these regions to greater stress, is the source of several pathogenic variants causing EPPK (Austin et al., 2016).
In conclusion, we have identified a pathogenic variant in KRT9 gene in two patients from one family. Our finding expands the mutant spectrum of KRT9 gene. Identification of more individuals carrying new variants in KRT9 gene is needed to fully characterize the full phenotypic spectrum associated with EPPK.

IRB STATEMENT
Human samples used in this study were obtained in accordance with the principles expressed in the Declaration of Helsinki, and were approved by the Institutional Review Board from Nanfang Hospital, Southern Medical University. Blood and skin biopsy sample collection procedures used were in accordance with institutional and national ethical standards of human experimentation and all participants provided written informed consent.