Investigation On The Variants Of ABCA3 And MUTYH Genes And Genetic Basis Of Idiopathic Pulmonary Fibrosis In Chinese Population

Idiopathic pulmonary Fibrosis(IPF)is the most common form of interstitial lung disease(ILD)with high mortality rate.IPF is an aging-related disease that predominantly affects older adults over 60 years old.In addition,the incidence of IPF in male patients is higher than that in females.To date,the cause of IPF has not yet been clarified.In addition to lung transplantation,there are not effective treatment modalities and clinical interventions for IPF.Therefore,IPF is one of the most urgent clinical problems.The epidemiological data showed that approximately 20%of IPF patients had a familial form of the disease,which was known as familial pulmonary fibrosis(FPF).Until now,several gene variants and dysfunctional proteins were detected to be associated with the pathogenesis of FPF.Approximately 18%of FPF carried variations in two major telomerase components:telomerase reverse transcriptase(TERT)and telomerase RNA component(TERC).Mutations in the dyskeratosis congenita 1(DKC1)and regulator of telomere elongation helicase 1(RTEL1),that could induce telomere dysfunction,were also involved in IPF occurrence and phenotype development.Moreover,the mutations of surfactant protein C(SFTPC)and surfactant protein A2(SFTPA2)were found to be the basis of a significant portion of FPF.Both of the genes were associated with the alveolar stability.On the other hand,the mutations of TERT and TERC were only detected in 1%-3%of the sporadic IPF patients.The genetic predisposition of sporadic IPF remain to be elucidated.Oxidative damage during aging is one of the pathogenesis mechanisms of IPF.It has been known that the oxidative damage/apoptosis of alveolar epithelial cells(AECs)with aging is one of the pathogenesis mechanisms of IPF.The human MutY glycosylase homolog(MUTYH)is one of the base excision repair(BER)genes for repairing oxidative DNA damage and maintaining DNA replication fidelity.We previously reported an AluYb8 insertion in the MUTYH gene(AluYb8MUTYH)in Chinese population.This variant has been shown to cause the accumulation of oxidative damage in mitochondrial DNA(mtDNA)and serve as a risk factor for age-related diseases.To investigate the genetic basis of IPF,we carried out this study in two sections:(1)Screening for the predisposing gene of IPF and identifying the germline mutations of the targeted gene;(2)The functional significance of IPF related genetic variation in clinical phenotype development.Section 1:Study on the germline mutations of ABCA3 gene and the genetic susceptibility of idiopathic pulmonary fibrosis in Chinese populationABCA3 is a member of the ATP-binding cassette family of proteins that mediate the translocation of lipid,and highly expresses in the AECs.The homozygous mutations of ABCA3 have been associated with fatal respiratory distress syndrome and pediatric interstitial pneumonia.Whether the variants of ABCA3 gene have a role in the pathogenesis of adult ILD,including sporadic IPF remains to be addressed.Based on the results of exome sequencing in 10 patients with sporadic IPF in our laboratory,we used bioinformatic analysis to sort and filter susceptibility loci,and to make conformation with Sanger sequencing for the variants detected by high-throughput sequencing.We then took the ABCA3 gene as the target locus to have a further investigation on the relationship between the variants of ABCA3 gene and the genetic susceptibility for IPF.We firstly screened the germline mutations of the ABCA3 gene by exon sequencing among 30 patients with sporadic IPF and 30 matched healthy controls.Eleven missense mutations were found in 13 of the 30 IPF patients and all of these loci were heterozygous.The detection rate in IPF patients was significantly higher than that in control group,in that only two missense mutations were detected in 2/30 healthy controls.To characterize the ABCA3 mutations for the risk of IPF,we conducted bioinformatic analysis on all of the detected mutations and performed cohort analysis in four of them(p.L39V,p.S828F,p.V968M and p.G1205R)in ABCA3 gene.We randomly recruited 1,054 ILD patients,containing 250 IPF and 774 connective tissue disease-ILD(CTD-ILD)patients,and 1,084 healthy controls.Taqman probe technique was used to uncover the frequencies of the four missense mutations in the groups of the patients and healthy controls respectively.Our results showed that the allele frequency of p.G1205R in ABCA3 gene was significantly higher in ILD(including IPF)patients than in healthy controls,indicating that the variant p.G1205R could increase the risk of ILD(including IPF).The increased frequency of p.L39V was detected in IPF patients but not in CTD-ILD patients,suggesting that this missense mutations was only associated with the risk of IPF.However,no additional subject carrying the variant p.S828F or p.V968M was detected in the cohort analysis,neither in ILD patients nor in healthy controls,suggesting that the two missense mutations could be rare variants and may be related to the risk of IPF.These results indicate that the heterozygous mutation of ABCA3 gene could contribute to the genetic susceptibility of ILD,including IPF,in the Chinese population.Section 2:Study on the relationship between AluYb8 insertion in the MUTYH gene and mtDNA instability and clinical prognosis in patients with idiopathic pulmonary fibrosisIn previous studies,we described a polymorphism AluYb8 insertion in the fifteenth intron of the MUTYH gene,AluYb8MUTYH.The three genotypes of MUTYH were observed for this variant in Chinese populations as homozygous AluYb8MUTYH present(present/present,P/P),homozygous AluYb8MUTYH absent(absent/absent,A/A)and heterozygous(absent/present,A/P).This variant was associated with reduced expression of the MUTYH 1 isoforms,which is localized in the mitochondria,and affected the repair of mtDNA oxidative damage and mtDNA maintain.The AluYb8MUTYH has been shown to be a risk factor for the age-related diseases.IPF is a chronic lung diseas characterized by progressively worsening pulmonary function and its pathological development is related to aging.The mitochondrial functional impairment could be involved in the phenotypic development and clinical prognosis of IPF patients.To understand the relationship between this variant,AluYb8MUTYH,and mtDNA instability and clinical prognosis in patients with IPF,we compared the mtDNA content and integrity,and the expressions of mtDNA genes and the mitochondrial biogenesis-/dynamics-related gens among the IPF patients and healthy controls with different genotypes of the MUTYH gene.The works that we performed as following:① The mtDNA contents in the peripheral blood mononuclear cells were evaluated by quantitative real-time PCR(qRT-PCR)among 206 IPF patients with different genotypes of AluYb8MUTYH and among 206 matched healthy controls;②The mtDNA integrity was assessed with long range quantitative PCR amomg 105 IPF patients and 105 healthy controls with different genotypes of AluYb8MUTYH;③ The expressions of mtDNA coding genes,ATP6 and COX2,and the mitochondrial biogenesis-/dynamics-related genes,POLE,MFN2 and ATG7,were analysed among 87 IPF patients and 87 healthy controls with different genotypes of AluYb8MUTYH,respectively;④ The relationship between AluYb8MUTYH genotypes and clinical prognosis of IPF was analysed on the long-term follow-up data of 212 IPF patients.Results:① In the healthy controls group,the individuals with P/P genotype of AluYb8MUTYH had a decreased level of mtDNA content in their blood cells when compared to whom with A/A or A/P genotypes.It indicated that the homozygous insertion of AluYb8 impaired the maintenance of mtDNA in the carrier’s cells.However,the levels of mtDNA content in IPF patients with P/P genotype was increased,comparing with the IPF patients with A/A or A/P genotypes.With the results of the expressions of the genes related with mtDNA replication,mitochondrial fusion and autophagy,POLG,MFN2 and ATG7,we considered that the increasing of mtDNA content in IPF with P/P genotype could be due to the failure of oxidative damage repair and the accumulation of impaired mtDNA.② The assay of long range quantitative showed that the relative amplification of nuclear genome(nDNA)in IPF patients was significantly lower than that in healthy controls.It suggested that the oxidative stress enhanced the level of the oxidative damage of nDNA in IPF patients.A similar trend was also observed with long range quantitative PCR between the IPF patients and healthy controls with the same genotype of the AluYb8 insertion in MUTYH gene.However,the relative amplification of mtDNA in the subjects with P/P genotype was higher than that in the subjects with A/A or A/P genotypes,no matter the healthy control individuals or the IPF patients.It is suggested that the variant AluYb8MUTYH can selectively injure the repair of mtDNA oxidative damage.The increased relative amplification of mtDNA in P/P genotype cells was due to a reduction of the transient single strand break of mtDNA,that appeared in the process of the base excision repair.③ The follow-up data of the IPF patients showed that,compared to the patients with A/A genotype,the ages of onset and at death were lower in IPF patients with P/P genotype.These results suggest that the AluYb8 insertion in MUTYH gene would affect the mtDNA stability in patients with IPF and be involved in the clinical development and prognosis of IPF patients.