Identification And Pathogenic Mechanism Of New GLA Gene Mutation In Fabry Disease

Fabry disease(OMIM #301500,FD)is a rare X-linked recessive hereditary systemic disorder of glycosphingolipid metabolism,caused by total or partial decreased activity of alpha-galactosidase A.Symptoms depending on where the substrate accumulates in the patient.Male patients often have severe symptoms,and female patients are generally carriers with mild symptoms.In order to further study the molecular mechanism of FD caused by GLA gene mutation,a cell model of GLA wild type or mutant was constructed,and the pathogenicity of the novel mutation site was verified,and the structure and function of the mutants were further studied.1.The experiment was performed on the GLA gene generation of 5 FD probands,and the mutation sites were determined as follows: c.119C>A,c.101 A > G,c.680 G > C,c.801+1G>A and c.280T>C.We found that c.801+1G>A and c.280T>C are novel mutations of GLA.Therefore,this study will focuse on the pathogenicity of these two mutations.2.The m RNA expression of GLA in the patients' blood was detected by q RT-PCR.Compared with healthy person,the GLA expression in patients are down-regulated and patient carry c.801+1G>A mutation has lowest gene expression.Since the mutation c.801+1G>A is located in the first position of intron 5,it may affect the GLA splicing.The transcripts of the GLA gene in the patients are detected by RT-PCR.The result showed that there is one extra transcript which is bigger than the normal in the patient.Sequencing results showed that this transcript contains a 36 bp sequence of intron 5 between Exon 5 and Exon 6;to investigate whether the alternative splicing was due to the c.801+1G > A mutation,we constructed a minigene containing entire exon 5,exon 6,and intron 5 sequence with G or A at c.801+1 of GLA.Both m RNA and protein expressions confirmed the single nucleotide substitution(c.801+1G > A)is the main determinant of alternative splicing.3.Through sequence alignment,we found the c.801+1G > A mutation caused a translational frameshift,and the premature stop codon appeared at codon 272(p.L268 Ifs X3),which caused a 161-amino-acid-residue change and partial loss of C-terminal domain.Evolutionary conservation analysis of amino acid residues showed that these impaired amino acid residues in the truncated protein were most highly evolutionary conserved among GLA proteins from different species.This mutation results in a truncated protein lacking the C-terminal end containing part of the first domain(residues269–330)and the whole second domain(residues 331–429).Molecular modeling showed that the impaired amino acid residues C94 was involved in the formation of disulfide bound with C52 and close to the enzyme activity site(D92,D93).The missense mutation(c.280T>C,p.C94R)disturbs the formation of disulfide bridge between C52 and C94 and might affect the enzyme activity.4.We introduced the mutation into wild type GFP-tagged full length GLA and expressed the corresponding proteins in He La cells.In GFP alone transfected cells,GFP signal was detected in both nucleus and cytoplasm.In GFP-GLA-WT transfected cells,the GLA fusions were uniformly expressed in the cytoplasm.Surprisingly,puncta structures were observed in the cytoplasm of GLA c.801+1G?A mutant transfected cells.In GLA c.280 T>C transfected cells,nuclear shrinkage was observed.Further,GLA c.801+1G?A mutation increased senescence of transfected cells.The enzyme activity of HEK293 T cells transfected with both mutant GLA constructs were significantly lower compared to that of GLA wild type transfected cells.In summary,two novel GLA mutations were identified from two Fabry patients by Sanger sequencing.By using bioinformatics,molecular biology,biochemistry and cell biology approach,we confirmed these mutations are pathogenetic.Data in this study enrich Fabry mutation database and provide a FD causative mutation for accurate molecular diagnosis as well as scientific information.