Molecular Characteristics Of GBA Mutations And Functional Analysis Of The Novel Mutations Identified In 22 Chinese Patients With Gaucher Disease

BackgroundGaucher disease?GD,MIM 230800?is a common lysosomal storage disorder.It is caused by the deficiency of the lysosomal enzyme glucocerebrosidase?GBA,EC 3.2.1.45?,which catalyzes the breakdown of the glucosylceramide to glucose and ceramide.The disease occurs at a frequency of 1/50,000 to 1/100,000 in the general population.Three main clinical forms of GD have been described: Type 1 non-neuronopathic,Type 2 acute neuronopathic,and Type 3 subacute neuronopathic.Type 1 GD is the most common form and accounted for 95% of Jewish and non-Jewish Caucasian cases of GD,but the neuronopathic forms?type 2 and type 3?comprise over half of GD patients in Asia?61.1% in Korean,56.5% in Japanese and 42.0-57.9% in Chinese?.Gaucher disease is mainly caused by the mutations in the glucocerebrosidase gene?GBA,MIM No.606463?.The GBA gene,spans ⁷.5kb of genomic DNA,is located on chromosome 1q21 and composed of 11 exons.A highly homologous ⁵.5kb pseudogene?GBAP,MIM No.606463?,which shares 96% exonic sequence homology,is located 16 kb downstream from the functional gene.To date,at least 437 different mutations causing GD have been identified?HGMD Professional 2016.1?.The mutation spectrum of GBA in Asians is quite different from that in Ashkenazi Jewish patients and non-Jewish patients,where N370 S,84insG,L444 P and IVS2+1G>A mutations account for about 97% and 75% of the mutant alleles,respectively.In Asians,L444 P,F213I and RecNciI mutations are the most common mutants.There are few clear genotype/phenotype correlations in GD: N370 S and N188 S mutations are related with type 1 GD;L444P/Rec NciI phenotype is associated with type 2 GD.Therapeutic approaches for GD include enzyme replacement therapy?ERT?,substrate reduction therapy?SRT?,pharmacological chaperone therapy?PCT?and gene therapy.Although ERT and SRT can improve the symptoms significantly,they are not effective in type 2 and 3 GD.PCT is a promising alternative therapeutic approach for its ability to penetrate the blood-brain barrier to treat the neuronopathic forms.Recently,ambroxol was described as an effective chaperone on fibroblasts with L444P/L444 P and L444P/Rec NciI genotype.In addition,ambroxol was confirmed to be effective in the GD patients with N188 S,F213I or Rec NciI heterozygote.ObjectiveWe intended to explore the molecular characteristics of GBA mutations and to further analyze the relationship between genotype and phenotype of GD in Chinese and to construct a transient expression system of GBA gene for functional verification.MethodsTwenty-two unrelated GD patients from China?Guangdong,Hunan,Jiangxi and Guizhou Province?were enrolled in this study.Fifteen were male and seven were female.The diagnosis of GD was based on clinical findings and confirmed by the deficiency of GBA activity in peripheral leukocytes measured by Guangzhou Women and Children's Medical Center from February 2010 to March 2015.Genomic DNA was extracted from peripheral blood samples.To explore the molecular characteristics of GD patients,GBA gene were analyzed by nest PCR and direct Sanger-sequencing.Once variants were identified,the bioinformatic analysis of the amino acid conservation in 12 different species was used to predict the possible impact on GBA protein.Pathogenicity of novel missense variants were analyzed using SIFT and Polyphen-2 software.To investigate the pathogenicity of identified novel mutations,we constructed plasmid expression system pcDNA3.1-GBA-EGFP carrying the wild type of human GBA gene,and then the mutant plasmid expression systems were constructed by Site-Directed Mutagenesis.COS-7 cells were transfected by pcDNA3.1-EGFP vector,GBA wild-type and seven mutants,respectively,using Lipofectamine 2000.After 72 h transfection,cells were harvested for GBA enzyme assays and Western blotting.Results1.The GBA activities in leukocytes of GD patients were significantly low?2.05±1.49nmol/mg/h vs.normal reference range 10²5nmol/mg/h?.Among the 22 GD patients,19 patients were classified as type 1 and three as Type 2.In Type 1 patients,63%?12/19?of them had onset at less than 2 years old and presented severe hepatosplenomegaly and hematological complications?anemia,thrombocytopenia,epistaxis?.About 26%?5/19?of them were died before 3 years old.The three Type 2 patients had onset before 6 months and quickly progressed to present opisthotonus,dystonia,ophthalmoplegia,psychomotor retardation and severe hepatosplenomegaly.All of them died before 2 years old.Six patients were L444 P homozygote with early onset and severe manifestations.Six patients were L444 P heterozygote with manifestations of weight difference.L444P/ RecNci I genotype was found in two Type 2 GD patients.2.We successfully identified 44 GBA mutant alleles from 22 unrelated GD patients?100% identification?.There were 18 different mutations,including 14 reported mutations?F37V,L96 P,R120W,M123 V,L264I,R359 Q,Y363C,V375 L,D409H,L444 P,R496C,RecNci I,IVS2+1G>A,IVS6-1G>C?and four novel mutations?Y22C,F109 L,L149F and c.983₉90delCCCACTGG?.L444 P mutation accounted for 47.7% of the mutant alleles.The other mutations with less frequency in our patients were RecNciI?6.8%?.SIFT and Polyphen-2 software illustrated that the novel missense mutations were possibly pathogenic.Y22 C mutation may affect the glycosylation.L96 P,F109L,L149 F mutations may destroy the catalytic domain.3.Novel mutations Y22 C,F109L,L149 F expressed low enzyme activities in COS-7 cell and retained reduced amounts of GBA protein compared to wild-type,suggesting a decrease in the total amount of GBA protein in these mutants.Conclusion1.We identified 18 different mutations including 14 previously reported mutations?F37V,L96 P,R120W,M123 V,L264I,R359 Q,Y363C,V375 L,D409H,L444 P,R496C,Rec NciI,IVS2+1G>A,IVS6-1G>C?and four novel mutations?Y22C,F109 L,L149F and c.983₉90delCCCACTGG?in 22 Chinese patients with GD.The L444 P was the most frequent mutation accounting for 47.7% of the mutant alleles and followed by RecNciI.2.Novel mutations Y22 C,F109L and L149 F were verified to be pathogenic mutation in the expression study.3.L444 P homozygote genotype was associated with severe Type 1 GD.The phenotype of L444 P heterozygote depends largely on the severity of another mutation.L444P/ RecNciI genotype was associated with Type 2 GD.