The Correlation Between CYP26A1 Gene Mutation And Congenital Microtia

BackgroundCongenital microtia is a developmental malformation caused by abnormal development of the first and second branchial arches during embryonic development.This disease is primarily characterized by an abnormal auricle,stenosis or atresia of the external auditory canal,middle ear dysplasia and conductive hearing impairment.The incidence of congenital microtia varies by race and region.According to a neonatal epidemiological survey in China,the incidence of microtia is approximately 3.06/10,000,and the incidence is increasing each year.The aesthetic concerns of this anomaly can lead to severe psychological sequelae for both patients and their parents.Conductive hearing impairment caused by malformations of the outer and middle ear has a certain impact on the language development of patients with microtia.In addition,the complexity of auricle repair or reconstruction surgery and the corresponding costs impose heavy mental and economic burdens on patients and their families.Therefore,it is of great significance to identify the etiology and explore the pathogenesis of microtia.The etiology of congenital microtia has not been fully elucidated to date,and previous studies have reported that environmental factors have a strong correlation with its incidence.Lack of nutrients during pregnancy,exposure to drugs,maternal virus infection or suffering from certain chronic diseases are all risk factors for microtia.Genetic factors are also of great importance for the onset of microtia.Copy number variation or gene locus variation in specific regions of the genome are the focus of research on the etiology of microtia.Whole exome sequencing technology focuses on exon regions that can be transcribed and translated into proteins and perform specific biological functions.It has been widely used in the study of genetic information due to its high efficiency,high accuracy and low cost.In this study,whole exome sequencing technology was used to explore the suspected pathogenic genes in a microtia family with autosomal dominant inheritance.The influence of the gene mutation on the protein structure and function was further verified to analyze the possible mechanism of the pathogenesis of microtia.It is hoped that this study can provide a basis for the future exploration of the exact mechanism of this gene and provide a new idea for the study of the genetic etiology of microtia.Methods1.Basic clinical information,imaging data and elbow venous blood samples of a third-generation family with microtia were collected for whole exome sequencing.Bioinformatic analysis of the sequencing results showed that the cytochrome P450 family 26 subfamily A member 1(CYP26A1)was initially screened as a suspected pathogenic gene.The gene was further verified in sporadic microtia patients.2.The evolutionary conservation of the mutant site was detected by comparing the mutation sites among multiple species.The three-dimensional structures of wild-type and mutant proteins were simulated using software to evaluate and analyze the influence of mutation sites on the protein structure.The secondary structure diagram of the coding protein was drawn to explore the position of the mutant site in CYP26A1.3.Plasmids carrying wild-type and mutant CYP26A1 sequences were constructed and transfected into HEK-293T cells to observe the subcellular localization.qPCR and Western blotting technology were used to detect the difference in mRNA and protein expression levels between wild-type and mutant plasmids.Results1.The results from the whole exome sequencing and bioinforrmatic analysis showed that a single nucleotide nonsense mutation in exon 7 of the CYP26A1 gene(NM₀00783:c.C1252T:p.R418X,NM₀57157:c_C1045T:p.R349X)was identified as the pathogenic mutation in this family.The site was found in all included patients with microtia,and did not exist in unaffected members.Sequencing results from 20 patients with sporadic microtia showed that one of them carried a missense mutation in exon 2 of the CYP26A1 gene(NM₀00783:c.G276C:p.M92I,NM₀57157:c.G69C:p.M23I).The above sites were not found in three genetic databases,and the prediction software analyzed the effect of these sites as harmful mutations.2.The analysis of the evolutionary conservation among species showed that the site was highly conserved among multiple species.In addition,most of the 79 amino acids downstream of this nonsense mutation site were highly conserved.The computer simulation results of the three-dimensional structure of the wild-type and mutant proteins showed that the length of the protein was significantly shortened after truncation,and the overall structure of the protein,especially the terminal structure,was significantly changed.The analysis of CYP26A1 gene and amino acid sequences showed that 70 amino acid sequences in the mutant and subsequent deletion sites were located in the conserved domain of cytochrome P450.3.Immunofluorescence localization detection showed that the subcellular localization of the mutant protein was significantly different from that of the wild-type transfected group;qPCR and Western blotting analysis showed that the mRNA and protein expression levels were significantly decreased in the mutant transfected group.ConclusionsWe used whole exome sequencing to preliminarily screened a rare nonsense mutation of the CYP26A1 gene in a microtia family;this gene is suspected to be the pathogenic gene in this family.A series of bioinformatic analyses and experimental results showed that the mutation in the CYP26A1 gene affected the protein structure,subcellular localization,and mRNA and protein expression levels,which may affect the concentration gradient distribution of retinoic acid during embryonic development and subsequently lead to the occurrence of microtia.