Exome Sequencing Identified Causal Gene COL14A1for Punctate Palmoplantar Keratoderma

Background Punctate palmoplantar keratoderma (PPPK)(MIM number:148600) is an rare autosomal dominantly inherited skin disorder characterized by numerous hyperkeratotic papules distributed on the palms and soles. Buschke and Fischer first described it in1910, and Brauer confirmed the heredity of this dermatosis in1913. Hence, the disease is often referred to as keratosis punctata palmoplantaris Buschke-Fischer-Brauer. PPPK is clinically characterized by multiple tiny punctuate keratoses over the entire palmoplantar surfaces, coalescence of the punctate keratoses into a more diffuse pattern over the pressure points of the soles, and variable nail changes. The lesions usually start to develop in late childhood to adolescence, but may also start to appear up to the fifth decade in life:the previously reported ages at onset of the disease range from12to33years. Lesions of the disease were generally more severe on the soles than on the palms, probably due to exposure of higher pressure to the soles. In addition, PPPK has been reported to be associated with malignancies such as breast cancer, prostatic carcinoma, Hodgkin’s disease, colonic adenocarcinomas and metastatic non-small-cell carcinoma of the lung. The majority of cases are diagnosed incidentally since most patients do not complain about symptoms.Martinez-Mir et al. performed a genome-wide search in three families with PPPK and mapped the chromosome location to a9.98-cM interval flanked by markersD15S534and D15S818on15q22-15q24.1. Zhang et al. also performed a genome-wide search in two Chinese families with PPPK, but they located the disease gene within a9.20-cM region between markers D8S1804and D8S1720. These results indicate that PPPK is genetically heterogeneous. Gao et al. confirmed the first disease locus and positioned the PPPK locus distal to D15S651and proximal to D15S988. This region overlaps by5.06cM with the previously reported PPPK region. El Amri I et al confirmed genetic factor with the suspected locus on chromosome15q. This region is flanked by markers D15S987and D15S153. However, the causal genes for this disease remain yet elusive. Exome sequencing as a new powerful approach to uncover genetic causality in rare Mendelian disorders has been successfully demonstrated by several impressive applications, especially in combination with linkage analysis.Objective (1) In order to identify causal gene by means of exome sequencing in combination with genome-wide linkage analysis.(2) The aim of this study was to characterize the clinical and genetic features of PPPK in Chinese Han families and to gain a better understanding on PPPK.Methods (1) Four affected (Ⅰ1,Ⅱ2, Ⅲ2and Ⅲ5) and two unaffected (Ⅰ2and Ⅲ6) individuals from PPPK703were studied using exome sequencing.(2) Validation for the mutation was performed by Sanger sequencing. The primer of all the47coding exon and intron-exon boundaries of the COL14A1were designed using the web-based version of the Primer3.0program.(3) we conducted a systematic literature search using databases such as CBMdisc and Pubmed, and found9PPPK families reported in Chinese Han population in the past20years since1991. Combining all the data and comparing them with PPPK in other populations, we tried to investigate the overall clinical and genetic features of PPPK in Chinese Han populaion and explore the disease characters within the general populations Statistical analyses were performed using SPSS13.0.Results (1) A novel variant was defined as one that exclusively existed in all patients but did not appear in the controls in the family as well as in the databases including dbSNP129, eight HapMap and1000Genomes. Assuming the pathogenic mutations would have dominant effects and be close to the linkage region we previously reported, we identified a missense mutation in exon37of COL14A1(c.4505C->T [p.Pro1502Leu]) that is located at the3.4Mb upstream of the linkage region at chromosome8q24.13-8q24.21. And then we selected COL14A1as the most important candidate causal gene for PPPK.(2) We tested another4affected and6unaffected family members from PPPK703, and identified the same missense mutation in all these patients, but not in unaffected individuals of the kindred.(3) We screened this mutation in additional676unrelated, ethnically and geographically matched controls and781patients with other disease. Our finding highlighted that this mutation was a causal variant rather than one polymorphism for PPPK since it did not exist in all the unaffected individuals within PPPK703as well as676unrelated population controls and781unrelated subjects of other disease. This mutation was predicted to be "damaging" with99.7%confidence by ANNOVAR program and to be "probably damaging" by the PolyPhen program, respectively. The shared COL14.41mutation, p.Pro1502Leu, is a missense substitution at a highly conserved amino acid residue across multiple species.(4) We also sequenced all coding exons and intron-exon boundaries of COL14A1in the proband from the kindred PPPK712. We found no more additional potential pathogenic mutation.(5) All patients presented the typical punctate keratoses on the palms and soles. Disease in these pedigrees showed a continuous genetic phenomenon, no atavism was found. The distribution of lesion between affected men and women showed no difference (p>0.05, Student’s t-test). The lesions usually started to develop in late childhood to adolescence, but in family4they also started to appear in the fifth decade in life. Next generation was younger than the former generations in terms of age of onset, which was observed in family2,3,4,5,6. In these families the oldest affected members’s symptoms was the most serious among all patients and the symptoms of the younger patient was milder than the older. Complicating symptoms in4families were found, patients within2families complained with colonic adenocarcinomas, lung carcinoma. Two affected members with PPPKwithin2pedigrees also suffered from psoriasis. In family3and6affected members suffered from hyperhidrosis. One affected member in family4showed fissures.Conclusions (1) We identified a heterozygous mutation in underlying causal gene COL14A1(c.4505C->T [p.Prol502Leu]) in a PPPK family by means of exome sequencing in combination with genome-wide linkage analysis.(2) The power of combining exome sequencing and linkage analysis in the study of genetics of autosomal dominant disorders, even in simplex cases, has been demonstrated.(3) There existed the genetic heterogeneity in PPPK.(4) Our study reveals that PPPK generally follows an autosomal dominant pattern of inheritance with high penetrance. Most of families existed obvious anticipation in Chinese Han population. The older the patient, the heavier the symptoms in a family. There was interfamilial but also intrafamilial variation in phenotype and the severity of disease manifestations of PPPK in Chinese Han population. It will help us to gain a better understanding on PPPK both on the clinical and genetic aspects.