Exome Sequencing Identified Causal Gene EPS8L3for Marie Unna Hereditary Hypotrichosis

Background Marie Unna hereditary hypotrichosis(MUHH, MIM146550/612841) is a rare autosomal dominant inheritance congenital hair disease. MUHH was first reported by Marie Unna in1925.The incidence of men and women was alike, but the clinical manifestations of male patient is more serious than female patient. MUHH is characterized by normal, sparse or absent hair at birth, then develop to coarse, twisted and wiry hair during childhood and progress during puberty to an almost complete alopecia. Eyebrows, eyelashes, armpit hair, pubic hair and beard hair are also markedly diminished or absent. But no other ectodermal abnormalities are observed. The physical and intelligence of all patients are normal. The histopathological examination showed that hair follicle is surrounded by a small amount of inflammatory cell infiltration or fibrosis, but the number of mature hair follicles is a significant reduction and hair follicles were shrink. Optical microscopy showed that the hair was flat, rough and irregular-shaped distortions.Scanning electron microscopic studies display an irregularly twisted hair, longitudinal fractures, longitudinal split, irregular cross section, mild peeling and irregular hair shafts in MUHH. Many genetic linkage studies have mapped the MUHH locus to chromosome8p21, but no genes responsible for MUHH were identified, including HR (Human hairless gene) gene. In2009, Professor Zhang Xue found13different disease-causing mutations in the U2HR region of HR gene and established MUHH genetic mutation spectrum through international cooperation in19different ethnic MUHH families. Followed by multiple research groups had found the pathogenic mutation of U2HR.In2004, Yan et al excluded a four-generation MUHH pedigree by microsatellite markers, which indicated that MUHH is a heterogeneous disorder. In2005, Yang et al found a locus for MUHH on chromosome1p21.1-1q21.3to a17.5cM region between markers D1S248and D1S2345in this family. With the rapid development of next-generation sequencing technology, whole genome exome sequencing strategy has been successfully applied to identify causing gene of Mendel disease, susceptibility genes of cancer and complex disease, as well as helped for clinical diagnosis which difficult to diagnose cases.Objective We used whole genome exome sequencing strategy and combined with the result of genome-wide linkage study to identify the causative gene of this family.Methods (1) We conducted exome sequencing in two affected individuals and one unaffected individual from this MUHH family.(2) We got the candidate gene set which filter the mutations of exited in dbSNP database(version135) and unaffected individua step by step and located in location region.(3) To identify complete co-segregation between the mutation and MUHH phenotype, we sequenced the mutation of the candidate gene set in the extended pedigree of this family.(4) We sequenced all coding exons and flanking introns of the candidate genes in one family and four sporadic patients in order to discovery new mutations.(5) We sequenced the U2HR in one pedigree and four sporadic patients with MUHH. We also searched for the mutation reports of U2HR through Chinese Biology Medicine (CBM) disk and Pubmed, and find the relationship between genotype and phenotype. Results (1) We obtained SNP and indels of three samples by whole genome exome sequencing.(2) We focused our analyses primarily on nonsynonymous variants (NS), splice acceptor/donor site mutations (SS) and coding insertions/deletions (indels) that are more likely to be pathogenic mutations. In addition, we predicted that variants underlying MUHH are rare and thus unlikely identified previously. We therefore selected the variants that were absent from most updated dbSNP database (version135) for further analysis. Assuming a dominant model, we found91novel NS/SS/indels that were shared by the two affected individuals but absent in the unaffected individual in this family. Of the91variants, only one heterozygous mutation in EPS8L3(c.22G>A [p.Ala8Thr]) was found to be located within the linkage region on1p21.1-1q21.3established in our previous linkage study. The mutation was predicted to be "damaging" by ANNOVAR program and affect a conserved amino-acid residue by PhastCons software.(3) We analyzed the mutation in all the available affected and unaffected individuals (including three samples performed with exome sequencing) of this family by Sanger sequencing. All the eight affected individuals carried this heterozygous mutation in EPS8L3(c.22G>A [p.Ala8Thr]) which was absent in the three unaffected family members, suggesting complete co-segregation between the mutation and MUHH phenotype.(4) This mutation was not detected in additional exome sequencing data of676unrelated, ethnically and geographically matched controls. In addition, we also checked the exome sequencing data of781unrelated, ethnically and geographically matched patients of other disease and did not found the mutation. All these results suggested that this mutation is a causal variant for MUHH, instead of a rare polymorphism.(5) We did not find mutation of EPS8L3gene in the other family2and four sporadic patients.(6) We identified one missense mutation c.73C G (p.pro25ala) in U2HR of proband and his mother from family2, but the mutation was not detected in the patient’s unaffected father.(7) There are16species mutations of U2HR which had been reported, including5initiation codon mutations, two nonsense mutations, seven missense mutations, two stop codon mutations. And we did not observe obvious genotype-phenotype correlation in MUHH patients.Conclusion (1) Our study identified EPS8L3as a novel disease gene for MUHH by combining exome sequencing with previously established linkage information in a large multi-generation MUHH family of Chinese population. Our study has also demonstrated the effectiveness of exome sequencing, combined with linkage analysis, in discovering disease genes for Mendelian disorders.(2) We had found a missense mutation (c.73C>G, p.pro25ala) of U2HR which had been reported in family2. This study contributes to lay the foundation for future genetic counseling, prenatal diagnosis, gene therapy and suggested that MUHH is a genetically heterogeneous disorder.