Functional Study Of A Novel Mutation In ABCD1 Identified In A Family With Adrenomyeloneuropathy

Background and objectiveAdrenomyeloneuropathy(AMN)is the late-onset form of the X-linked adrenoleukodystrophy(X-ALD)which is the most common peroxisomal disorder.X-ALD is caused by mutations in the ABCD1 gene which results in defective peroxisomal ?-oxidation and the accumulation in all tissues of very long-chain fatty acids(VLCFA,?C22).Adrenoleukodystrophy protein(ALDP),encoded by ABCD1 and with a length of 745 amino acids,is a perisomal membrane protein belonging to the human protein family of ATP-binding cassette(ABC)transporters.The phenotypes include Addison-only,cerebral ALD(childhood,adolescent and adult),AMN with or without cerebral demyelination,and symptomatic or asymptomatic carriers.The adrenal cortex and the nervous system is influenced primarily,the pathology in the nervous system might be either an inflammatory demyelination or a more slowly progressive axonopathy affecting the ascending and descending tracts of the spinal cord.An mRNA of 4.3 kb is encoded by ABCD1,which maps to Xq28 and consists of 10 exons.More than 775 unique mutations have been identified,large intragenic deletions,frameshifts,premature terminations and heterogeneous missense mutations have been detected throughout the gene.Mutation analysis of ABCD1 gene for each new ALD family is necessary because there is no relationship between the genotype and phenotype.As we all know,autophagy is the major cellular process responsible for protein turnover in eukaryotic cells.Research has shown autophagic flux was impaired in spinal cords of the ABCD1-mouse model,and excess VLCFAs down-regulated autophagy in human fibroblasts.We diagnosed A family with AMN.Clinical and neuroimaging examination,target region capture and high-throughput sequencing were performed in the proband and carriers,the stability and pathogenic mechanism of the mutant ABCD1 Protein was also included.Part 1 A novel mutation in ABCD1 identified in a family with AdrenomyeloneuropathyObjectiveIn this research,We collected clinical and MR imaging findings of the patient,performed gene mutation screening for ABCD1 gene in the family pedigrees,and predicted the protein function of the mutant gene.MethodsAn AMN family was diagnosed in the First Affiliated Hospital of Zhengzhou University.Clinical datas including clinical symptoms,imaging findings,electrophysiological test results,VLFCA levels in plasm and others were performed in the proband.We filed blood samples and extracted DNA of the patient and his consanguinity.After PCR amplification,the mutation analysis of ABCD1 gene has been performed by Sanger sequencing.Phyre2 was used for the tertiary structures simulation of ALDP with native Val 111 residue and Gly mutation,also for the protein functional region,transmembrane helical region prediction.Homology on ALDP amino acid sequence in different species has been analyzed.Online programs of PolyPhen2,Align GVGD?PROVEAN?Mutation Taster and SIFT were used to predict the pathogenicity and the effect on protein function of the single nucleotide missense mutation.Result1.Clinical results:The patients' clinical symptoms,imaging findings,electrophysiological parameters,and VLFCA levels fully met the typical AMN diagnostic criteria.2.Sequencing resultsThe mutation screening of the ABCD1 gene in proband revealed a novel missense mutation c.332T>G occurring in exon 1 and changing a valine to glycine at the position 111 of ALDP.This mutant gene also presented in carriers,the proband's mother and his two daughters.3.Bioinformatics evaluationResults of homology analysis revealed that valine amino acid at the position 111 is 100%conserved across all analyzed species.Tertiary structures of ALDP with native Val 111 residue and Gly mutation were generated by Phyre2 prediction program.There is a slight structural change in mutant protein.Analyzing by molecular biology softwares such as PolyPhen,Align GVGD,Mutation Taster,PROVEAN,and SIFT,V111G mutation was harmful to the ALDP protein function.Conclusion1.A novel ABCD1 missense point mutation was identified in this research,c.332T>G,p.V111G.2.V111G mutation is a pathogenicity mutation.Part 2 Protein expression of ABCD1 mutant gene and relationship on autophagyObjectiveTo investigate the effect of mutant ABCD1 gene on protein expression and the pathogenic mechanism of ALDP(V111G).MethodsConstructed the wild type and the mutant ABCD1 gene(p.V111G)recombinant eukaryotic expression vectors and transfected them into HEK-293T cells.We investigated the expression level and subcellular localization of GFP-ALDP and GFP-ALDP V111G by immunofluorescence and immunoblotting analysis.Iimmunofluorescence and immunoblotting analysis were applied in the research of expression level and subcellular localization of ALDP-GFP and ALDP-GFP V111G.Immunoblotting was also used to detect the expression of LC3-? and Beclin-1 in order to study autophagy.Results1.Construction of plasmidsWe successfully constructed pEGFP-N1-ABCD1 and pEGFP-N1-ABCD1-V111G plasmids identified by enzyme digestion and sequencing.2.ImmunofluorescenceCells transfected with mutant type GFP-ALDP show downregulating fluorescence intensity compared with the cells transfected with wild type GFP-ALDP.The green fluorescence of GFP-ALDP was distributed in lumps,while the green fluorescence of GFP-ALDP(V111G)was scattered in punctate.Then we observed the cytoskeleton and nucleus by a fluorescence microscope.It was found that the wild type GFP-ALDP is presence of mass lesions,and displayed the correct peroxisomal localization in HEK-293T.However,the mutant GFP-ALDP(V111G)was diffuse in the cytosol.3.Western blotBoth the cells transfected with the wild and the mutant type recombinant plasmid successfully expressed GFP-ALDP,and the expression levels of GFP-ALDP in cells transfected with wild type were significantly higher than that of transfected with mutant type(P<0.01).We also assessed expression levels of LC3-II/LC3-1 and Beclin-1.Both of them were downregulated in the mutant GFP-ALDP(V111G)than in the GFP-ALDP.Conclusion1.The expression of GFP-ALDP(V111G)was down-regulated than GFP-ALDP and the combination of ALDP and peroxidase may be reduced.2.The expression of GFP-ALDP(V111G)induced down-regulation of LC3-II/LC3-I and beclin-1,and inhibited macroautophagy level of cells.