(Background)Genesis of novel gene regulatorymodules is largely responsible formorphological and functional evolution.New active cis-regulatory elements(CREs)can arise from transposition,promoter switching,co-option or de novo generation.Obviously,the last model,de novo genesis of CREs could be a rare event in genome evolution,due to the odds of creating totally novel CREs aremuch lower than that of altering existing CREs.It is only reported by a sole case in fishes.However,genetic variation drives either new genesis of CREs or phenotypic alteration has not been reported.Results: Here,we took advantage of a spontaneous hearing loss model found in pigs,and studied the mutation in a non-regulatory region of the melanocyte-specific promoter of microphthalmia-associated transcription factor(MITF)gene.The mutation generated a novel silencer and eliminated expression of MITF-M.This consequently caused the early degeneration of intermediate cells of the cochlear stria vascularis and profound hearing loss,similar to the typical phenotype of Waardenburgsyndrome in humans.We found that the mutation exclusively affected MITF-M,not other isoforms.The essential function of Mitf-m in hearing development was further validated using a knock-out mouse mode.l Conclusions:These results demonstrated that dysfunction in the MITF-M isoform alone was sufficient to cause deafness and depigmentation.To our knowledge,this study provides the first evidence of a systemic functional de novo CRE in mammals.