Careful clinical study unable to identify any features of Ectodermal dysplasia or mental retardation, as mentioned in available literatures in any of the affected family members including proband. Initially the pattern of inheritance was seems to be autosomal dominant type but sequence of all the coding exons and exon-intron boundaries of two major candidate genes viz. PAX9 and MSX1 failed to reveal any pathogenic mutation, leading to possible association of novel candidate gene/s with the disease phenotype. Whole genome sequence analysis by next generation or third generation sequencing techniques has become a powerful tool for identification of novel gene or mutation underlying disease condition. Two of the affected family members selected for whole genome sequencing showed 119 novel non-synonymous DNA sequence variants compared to the unaffected and those were distributed in 117 different genes across the genome. However, among those 119 sequence variants a c.956G.T mutation in X-linked EDA1 was the unique one among six candidate genes associated with non-syndromic tooth Epirubicin HCl agenesis known so far. Functional EDA1 is a type-II trimeric transmembrane Diiodohydroxyquinoline protein belonging to TNF ligand superfamily. It has four distinct functional parts; an N-terminal intracellular domain, a collagen like repeat domain, a furin cleavage site and a C-terminal TNF homology domain. The C-terminal domain subsequently cleaved off at the furin cleavage site and binds with EDAR and subsequently regulates cellular physiology by NF-kB pathway. The amino acid variation observed in DEN12 family is located with in the TNF homology domain which shows 100% amino acid sequence similarity with mouse Eda. Several studies recognize TNF homology domain along with collagen sub-domain as a mutational hotspot for EDA protein. Sequence homology modeling of EDA protein using PDB entries showed that TNF homology domain has two distinct regions, one consists of seven trimer forming residues and another consists of five receptor binding residues. So far 74 mutations have been identified out of which 47 are located in conserved TNF homology domain. 13 mutations among those 47 are responsible for nonsyndromic tooth agenesis.