Function Study Of PRKAG2 With A Novel Mutation G100S Responsible For PRKAG2 Cardiac Syndrome In Chinese

Background:Familial PRKAG2 cardiac syndrome is an autosomal dominant inherited disease.The phenotype of the family was characterized by cardiac hypertrophy, conduction abnormity and ventricular preexcitation.Nine mutations(Arg302Gln, His383Arg,Thr400Asn,Tyr487His,Asn488Ile,Gln506Lys,Arg531Gly,Ser548Pro,Ins Leu351) have been so far reported in PRKAG2 gene,but mainly in patients in seventeen families with apparently isolated cardiac phenotype,and occur in the Bateman domain region.The most frequent mutation is R302Q and have been identified in nine families and one sporadic patient.Kui Hong did not detect any novel mutation as well as R302Q in PRKAG2 gene in a Chinese family.Another Chinese family was studied by Doctor Jing Zhang and presented with cardiac hypertrophy(56%),preexcitation syndrome(46%),sinus node dysfunction,advanced atrioventricular block,even sudden death.Atrial arrhythmia, such as atrial fibrillation and atrial flutter also coud been observed in some family members.The clinical phenotype in this Chinese family with PRKAG2 cardiac syndrome corresponded to general characters of the foreign families.A previously undetected missense mutation,glycolamine(100) to serine,in exon 3 of PRKAG2 was identified and shown to be present in the living affected family members.The mutation results from guanine(G) substituted for adenine(A) at nucleotide 298.At present,noticeable performance on study of pathogenic mechanism of PRKAG2 gene has been achieved abroad,meanwhile,any study have not been reported in China. Some suggested that PRKAG2 mutations caused energy deficiency,whereas others suggested that abnormal membrane currents or abnormal gene expression/heart development caused cardiomyopathy with conduction abnormalities.However,subsequent studies indicated that PRKAG2 cardiomyopathy is a unique type of glycogen storage disease primarily involving the heart.More recently,analyses of accumulated carbohydrates in murine models and in the most severe forms of human disease demonstrated that glycogen accumulates in this cardiomyopathy.There is a 30-fold increase in cardiac glycogen in mice expressing AMPK carrying the PRKAG2 N488I missense mutation.Subsequent studies of mutationally altered AMPK activity explained the dominant mode of disease inheritance and the phenotypes of cardiac glycogen accumulation and preexcitation in the conduction system.To verify pathopoiesis of PRKAG2 with a novel missense mutation G100S responsible for Chinese PRKAG2 cardiac syndrome and promote pathogenic investigation of the PRKAG2 gene,we study the functional consequences of the mutation in the level of the cell and molecule by overexpressing mutants of PRKAG2 in CCL13 cells.Objective:To investigated the functional consequences of PRKAG2 with a novel missense mutation G100S responsible for Chinese familial ventricular hypertrophy with conduction system abnormalities and ventricular preexcitation in vitro.Methods:(1) Gene Splicing and Construction of PRKAG2 Gene TA CloningVector:The incomplete PRKAG2 eDNA clone(GenBank accession number BC068598), which presents N-end loss of 44 amino acids,was obtained by purchase.The PRKAG2 gene sequence was designed according to the known PRKAG2 gene sequence(GenBank accession number NM₀16203).The full-length PRKAG2 gene was acquired using the designed primers by PCR-based gene assembly method.The PCR product was cloned into TA cloning vector.(2) Construction and identification of the recombinant lentivirus vector carrying wide type and mutants of human PRKAG2 gene:The site-directed mutagenesis of PRKAG2 gene was made by PCR.Four sets of primers were designed according to the sequence of the PRKAG2 cDNA,and mismatches were introduced into primers.Mutagenesis was performed in a two-step PCR.The amplified fragments contained the mutation sites.The PCR products(GS and RQ) and normal PRKAG2 gene (WT) were cloned into the lentivirus vector.The recombination vectors were identified by different restriction endonuclease reactions and sequencing analysis.(3)Effects of the lentivirus vector carrying wide type and mutants of the PRKAG2 gene in vitro:The 293T cells were cotransfected with the three plasmids(PAX,PMD,and pWPT-PRKAG2).The CCL13 cell lines overexpressing the PRKAG2 gene were obtained. PRKAG2 R302Q and PRKAG2 WT were used as positive and negative control respectively.The CCL13 cells was used as blank control.The infection capacity of the lentiviral particle was tested by FACS and the expression of PRKAG2 was detected by Western blot.Subcellular localization of PRKAG2 was detected by immunofluorescence. The proliferation of CCL13 cells and the kinase activity of AMPK was measured using MTT assay and ELISA respectively.At last,glycogen contents in CCL13 cells were detected by PAS.The functional consequences of PRKAG2 with the mutation G100S in CCL13 cells were studied.Results:(1) The full-length PRKAG2 gene was obtained.The TA cloning vector was identified being PRKAG2 fragment,with the restriction enzyme and sequencing methods. (2) The sequencing analysis showed that the mutated sites were correct.Mutation from A to C 904 and G to A 905 sites of PRKAG2 cDNA were found.Additionally,mutation from G to A 298 site of PRKAG2 cDNA were found.The sites of 302 and 100 were changed from Arg to Gln,Gly to Ser respectively,suggesting that the expression vectors were constructed successfully.(3) The CCL13 cell lines(CCL13/WT,CCL13/GS,CCL13/RQ) overexpressing the normal or mutational PRKAG2 gene were obtained.FACS showed that CCL13 cells were successfully infected by the lentiviral particle.Western blot analysis using whole cell.lysates revealed that both mutants as well as WT were expressed in CCL13 cells at comparable levels.The PRKAG2 protein expressed in both mutational groups less than CCL13/WT and in CCL13/GS more than CCL13/RQ.Immunofluoresce-nce using anti-PRKAG2 antibody and tetramethylrhodamine isothiocyanate-labeled antibody revealed that transfected both mutants and WT were localized in the cytoplasm and nucleus in CCL13 cells.MTT assay using CCK-8 showed that AICAR had no obvious influence on proliferation of CCL13 cells between the mutants(CCL13/GS:30min: 2.8157±0.0325,90min:2.7223±0.0740,24h:2.5520±0.0110;CCL13/RQ:30min: 2.8230±0.0251,90min:2.6193±0.0560,24h:2.5467±0.0045) and WT(30min: 2.7200±0.0556;90min:2.7010±0.0030;24h.2.5597±0.0071).PAS using dyeing kit revealed that more glycogen were diffusely distributed in the cytoplasm and nucleus in both mutants,however,less glycogen in WT and blank control.The level of AMPK in every group was in dose-dependent manner with respect to AICAR concentration,which hinted that AMPK activity was corresponding to AMPK concentration.Compared with CCL13/WT group(no AICAR:25.4903±0.8947;0.5Mm AICAR:31.5990±0.6950;2mM AICAR:34.7823±0.2081) and CCL13 group(no AICAR:4.9363±1.0655;0.5mM AICAR: 6.4203±0.9160;2mM AICAR:7.3463±0.9451),AMPK activity was more lower in the mutants(CCL13/GS group:no AICAR:13.6240±0.1310,0.5mM AICAR:19.5847±0.4711, 2mM AICAR:22.4957±0.9460;CCL13/RQ group:no AICAR:12.6133±0.1158,0.5mM AICAR:17.3713±0.9644,2mM AICAR:19.3697±0.4264)(P<0.01).AMPK activity was lower in CCL13/RQ than CCL13/GS,and statistical difference was significant(P<0.05).Conclusions:(1) The novel missense mutation G100S alters the biological function of PRKAG2 gene and is responsible for PRKAG2 cardiac syndrome in Chinese.This study confirmed that PRKAG2 cardiac syndrome is a glycogen storage disease and PRKAG2 G100S mutation impairs AMPK activity.(2) Both the mutation G100S in exon 3 and R302Q in Bateman domain interfere with the expression of PRKAG2 protein and activation of AMPK and the effect is more remarkable in the latter than the former. Consistent with these findings,the study reveals that the mutation G100S exerts an essential function in the influence of Bateman domain and indicates that the two mutations maybe have similar regulative mechanism to AMPK activity and the pathogenesis of PRKAG2 cadiac syndromes does not have racial diversify.