Analysis Of The Clinical Phenotype And Investigation Of Pathogenic Gene In A Chinese Han Family With Punctate Palmoplantar Keratoderma

Background Palmoplantar Keratodermas?PPKs?are a group of keratiniazation disorders characterized by the excessive formation of keratin on the palms and soles.Four clinical types of PPKs can be classified by the patterns of lesions:diffuse,focal,striate and punctuate.Punctate Palmoplantar Keratoderma?PPPK??OMIM#148600?,also known as keratosis punctate palmoplantaris Buschke-Fischer-Brauer,is a rare autosomal dominant skin disorder characterized by the progressive development of discrete areas of hyperkeratosis on the palms and soles,followed by more extensive diffuse hyperkerotasis on the pressure-bearing areas of palmoplantar skin.To date,a total of 38 AAGAB gene mutations and 1 COL14A1 gene mutation have been reported in families with PPPK.AAGAB gene encodes a-and y-adaptin binding protein p34 which is involved in clathrin-coated vesicle trafficking.p34 deficiency may impair endocytic recycling of epidermal growth factor receptors,leading to increased signaling and proliferation and causing PPPK.Further investigation is required to confirm the role of COL14A1 gene in PPPK.The whole-exome sequencing technology has been widely used in detecting causative genes for mendelian diseases.It has many merits such as few samples consumption,short cycle,wide coverage and high precision,which had developed as a most efficient method in discovering molecular mechanisms for mendelian diseases.Objectives?1?To analyze clinical phenotype characters of a Chinese Han family with Punctate Palmoplantar Keratoderma.?2?To identify the causative gene of a Chinese Han family with Punctate Palmoplantar Keratoderma and offer genetic and disease counseling.Methods?1?We investigated a four-generation family composed of a total number of 34 individuals?6 affected and 28 unaffected?with PPPK from Tongling Anhui China.Clinical characteristics and inheritance of the family were evaluated,and pedigree was drawn based on medical history investigation.?2?Ten exons of AAGAB gene of all affected individuals from the family were scanned by PCR amplification and DNA direct sequencing.?3?Two serious affected individuals??2 and ?5?from the family were investigated by the whole exome sequencing and additional 2 cases and all controls of the second and third generations were evaluated in the validation analysis.?The purified DNA samples were randomly fragmented with length of 200-300bp.?Extracted DNA was amplified by ligation-mediated PCR?LM-PCR?,purified,and hybridized to the exome array for enrichment.?Captured LM-PCR products were subjected to Agilent 2100 Bioanalyzer and quantitative PCR to estimate the magnitude of enrichment.Each qualified captured library was then loaded on Illumina HiSeq 4000 platforms?BGI,ShenZhen,China?and we performed high-throughput sequencing for each captured library to ensure that each sample met the desired average sequencing coverage.?Raw image filewere recognized by Illumina basecalling software ?We used Burrows-Wheeler Aligner software to map reads onto the the human reference genome?GRCh37/HG19?to detect all the variants.? All genomic variations,including SNPs and InDels were detected by the state-of-the-art software,such as HaplotypeCaller of GATK?v3.3.0?.? After high-confident SNPs and InDels were identified,the SnpEfftool was applied to perform:?a?To identify whether SNPs or InDels cause protein coding changes and the aminoacids that are affected.?b?To identify variants that are reported in dbSNP v141or in the 1000 Genome Project or identify the subset of variants with MAF>=1%or many other annotations on specific mutations.?Further Sanger DNAsequencing was performed for validating the candidate variants.The additional 3 cases and all controls of the second and third generations were evaluated in the validation analysis.Results?1?The PPPK patients in the family characterized by keratotic papules on the palms and soles,which gradually increased in size and number with age and coalesced with each other especially over the pressure part of the palms and soles.?2?A four-generation family composed of a total 34 individuals,including 6 patients?3 males and 3 females?with punctate palmoplanter keratoderma.PPPK in this family was mode of autosomal dominant inheritance.?3?No mutation was found in the 10 exons of AAGAB gene of the affected individuals in the family.?4?Data shows the coverage of target region in 2 samples both reached to at least 98%.Further data processing by filtering out common variants and case-common selection established collection variants of 40 SNVs and 60 InDels.Finally we identified 3 candidate variants,NM₀02016.1:c.1333₁334insCCCA ?p.Leu445Arg446,NM₀01009931.2:c.8292₈293insCT? p.Ala2764Gly2765,NM₀01971.5:c.419delC? p.Ala140Val,in FLG,HRNR and CELA 1 in the PPPK family.A heterozygous frameshift variant c.419delC in the 5th exon of CELA1 gene was identified in the affected individuals by Sanger sequencing,but not found in unaffected individuals.Conclusions?1?The severity of the skin lesions in the PPPK family was associated with the patient' age and laboring intensity.?2?PPPK in this family was mode of autosomal dominant inheritance.?3?There was no evidence to indicate the correlation between this PPPK pedigree and coding sequences mutations of AAGAB gene.?4?A heterozygous frameshift variant c.419delC in CELA1 gene could be the causative gene of the family with PPPK.