Homozygosity Mapping and Mutational Analysis of Rare Human Hereditary Skin Disorders

Abstract

Background: Genetic skin diseases, or genodermatoses, are a diverse group of heritable (Mendelian) diseases that make about one-third of all monogenic diseases, with over 1,000 genes currently being associated with cutaneous manifestations. About 50% of genetic skin diseases are inherited as autosomal recessive (AR) disorders. The phenotypic impacts of genodermatoses are diverse, including epidermolysis bullosa (keratinization disorder), albinism (pigmentary disorder), ichthyosis (hereditary cornification disorders), hypotrichosis (hair loss disorder), and dysplasias of ectoderm etc. The routine diagnosis of genetic skin diseases is complicated due to phenotypic and genetic heterogeneity. Consanguineous marriages have become a common practice in a culturally rich and religious country like Pakistan, which become the main driver of disease risk like monogenic disorder (autosomal recessive) in the upcoming generations. It has been predicted that almost 82.5% of Pakistanis have consanguineous marriages due to which Pakistani population is considered as a goldmine in the field of genetic skin research. Method: This study is basically concerned with genetic mutation analysis of fifteen families (A-O) affected with four major rare hereditary skin disorders which includes Epidermolysis bullosa (A, B, C, D, E, F, G), Ichthyosis (H, I, J, K), hypotrichosis (L, M) and oculocutaneous albinism (N, O). Linkage analysis and Whole Exome Sequencing (WES) approach was used to identify the pathogenic sequence variants in probands. The segregation of these variants in other participants was confirmed by Sanger sequencing. Results: The study results in detection of 13 novel and 2 known gene variants in 15 families (A-O) affected with four rare genetic skin disorders included in the dissertation. These mutations includes seven novel variants in five disease causing genes of epidermolysis bullosa i-e nonsense mutation in COL17A1: NM_000494.3: c.4041T>G: p.Tyr1347Ter in family A, a nonsense mutation in COL17A1:NM_000494.4: c.3067C>T: p.Gln1023Ter in family B, a deletion mutation in PLEC: NM_201380.3:c.1284_1286delGCT: p.L429Sfs*30 in family C, a missense variant in COL7A1: NM_000094.3: c.7034G>A, p.Gly2345Asp in family D, a frame shift mutation in COL7A1: NM_000094.3: c.385del: p.His129MetfsTer18 in family E, a splice region variant in LAMA3 NM_198129.2: vi c.8043G>A:p.Ser2681 in family F, and a frame shift variant in LAMB3 NM_000228.3:c.561delG: p.Lys188Argfs*9 in family G, four novel gene variants in four different genes of ichthyosis that includes a splice donor site variant in TGM1 NM_000359.2: c.2088 + 1G > A in family H, a missense mutation in SULT2B1 NM_177973.1: c.419C>T: p. Ala140Val in family I, a splice site variant in SPINK5 NM_001127698.1:c.882+1G>T in family J ,and a nonsense variant in FLG NM_002016.1:c.6109C > T: p. Arg2037Ter in family K, one known and one novel mutation in two genes of hypotrichosis including a missense variant in LARP6 NM_001162497.1: c.436G>A: p.Gly146Arg in family L and splice site deletion mutation DCAF1 NM_025000.3: c.1423-1_1425delGACA in family M and one known and one novel mutation in two different genes of oculocutaneous albinism including a missense variant in OCA2 NM_000275.2:c.2458 T>C (p.Ser820Pro) in family N and a novel splice site variant in TYRP1 NM_000550.2: c.1408+5 G>C) in family O. Conclusion: This study unravels the molecular basis of the fifteen families of four rare human skin disorder of Pakistani origin and validates the involvement of COL17A1, PLEC, COL7A1, LAMA3, LAMB3, TGM1, SULT2B1, SPINK5 , FLG, DCAF17, LPAR6, TYRP1 and OCA2 in the aetiology of these diseases. To the best of our knowledge, all the new mutations identified in this study are first of their kind to demonstrate novel gene variants causing various forms of EB, ichthyosis, hypotrichosis and albinism in homozygous state both globally and in Pakistani population. The identified variants confirm the vital role of these genes in the morphogenesis of skin and its associated appendages. Various bioinformatics tools like Polyphen-2 programme, MutationTaster, PROVEAN, SIFT, I-Mutant 3.0, PhD-SNP, MutPred2, and Varsome, etc were used to predict the likely impact of these mutations on protein structure and function which predict them to be pathogenic and disease causing in most of the cases. These findings will expand the mutation spectrum of associated diseases, help in confirmation of the diagnosis, and allow a more accurate prognosis and a proper genetic counseling of the patients and their family members. Furthermore, it will also help in the development of the latest diagnostic testing and gene editing technologies which will have the potential to improve the diagnosis and therapeutics in inherited disorder. Keywords: Epidermolysis bullosa; Ichthyosis; hypotrichosis; albinism; Whole Exome Sequencing; autosomal recessive.