Whole Exome Sequencing Searchs For The Causative Gene Of Familial Progressive Hyper- And Hypopigmentation In One Chinese Family

Background Familial progressive hyper- and hypopigmentation(FPHH) is a rare autosomal dominant genodermatosis presenting with dyschromatosis at birth or in the first year. This disorder is characterized by diffuse and progressive hyperpigmentation and hypopigmentation. The café-au-lait macules or ash-leaf like white macules were also present. It usually involved the face, neck, trunk and limbs. Hyperpigmentation increased in extent and confluence with increasing age. Mental deficiency and growth retardation were also present in some patients. Amyere firstly proposed the concept of FPHH in 2011, they performed a genome-wide linkage analysis in seven families with FPHH(5 cases of Germany,1 case of the United States, 1 case of Danish pedigrees), and mapped the pathogenic gene on the chromosome 12q21.12-q22 by using the whole genome of parametric and nonparametric multipoint linkage analysis and haplotype analysis. Three mutations in KITLG gene were detected in 4 cases in a family(c.107A>G(p.Asn36Ser)、c.98T>C( p.Val33Ala)、c.100A>C(p.Thr34Pro)). Objective(1) We made genetic survey and clinical study on this FPHH family to explore the clinical features of FPHH.(2)Mutation analysis of KITLG gene is performed to explore whether this family was caused by the mutation of KITLG.(3) Gene typing and linkage analysis were performed by Zhong Hua chip.(4)We performed whole exome sequencing in one patient and two normal controls from this family, then to determine the possible pathogenic SNP combined with Zhong Hua-chip linkage analysis. Sanger sequencing was used to demonstrate candidate SNPs.Methods(1) We made detailed genetic survey and clinical examination on all available affected and unaffected members in the family.(2) We collected blood samples from 14 members in this family( 8 affected and 6 unaffected) and 200 normal healthy controls, and used Flexi Gene DNA kits to extract Genomic DNA;(3) Primers were designed by Primer Premier 5.0 software to amplify all coding exons and the respective exon-intron boundaries of the KITLG gene. A touchdown polymerase chain reaction(PCR) was performed to amplificate the purified products,then sequenced on an ABI Prism3730 XL.(4) Linkage analysis was performed by Illumina Zhong Hua-Chip to locate linkage region of FPHH.(5) We performed whole exome sequencing in one affected and two unaffected individuals, then selected possible pathogenic SNPs in the linkage region.(6) If no variant was found in the linkage intervals, we would expand the selection of chromosomes and determine candidate SNPs by using SIFT and Polyphen software, then demonstrate causative gene by Sanger sequencing.Results(1) Familial progressive hyper- and hypopigmentation(FPHH) is a rare autosomal dominant genodermatosis presenting dyschromatosis at birth or in the first year. This disorder is characterized by diffuse and progressive hyperpigmentation and hypopigmentation. The café-au-lait macules or ash-leaf like white macules were also present.(2) Sanger sequencing showed no pathogenic mutation in KITLG gene.(3) Whole exome sequencing and sanger sequencing revealed no causative gene.Conclusion(1) The mutation of KITLG gene is not responsible for this family, suggesting the genetic heterogeneity in this disorder.(2)We didn’t obtain any significant linkage signal, and considered Zhong Hua-Chip is suitable for linkage analysis in a small pedigree.(3)We didn’t find any pathogenic gene by using whole exome sequencing and sanger sequencing. New strategy is needed to search for the causative gene in this FPHH family.