Function Study Of PRKAG2with A Novel Mutation G100S Responsible For PRKAG2Cardiac Syndrome In Chinese

Background: PRKAG PRKAG2cardiac syndrome is a rare autosomal dominantdisease caused by genetic defects of PRKAG2gene which codes adenosinemonophosphate activated protein kinase γ2subunit, its typical performance is ventricularpreexcitation, progressive conduction system disease and cardiac hypertrophy. It was firstreported that PRKAG2gene mutation associated with familial pre-excitation syndrome,conduction system disease and cardiac hypertrophy by Gollob in2001. Thereafter,PRKAG2gene mutation associated with similar pedigree was reported in succession. Sofar, there are at least11kinds of mutations of the PRKAG2gene reported in20familialconduction system abnormalities with ventricular pre-excitation and cardiac hypertrophyfamilies at home and abroad. A Chinese family was reported by doctor Jing Zhang in2007,the clinical phenotype of which was found to be in line with the common foreign pedigreereported. After direct DNA sequencing was used, glycolamine (100) to serine(PRKAG2G100S), in exon3of PRKAG2was identified and shown to be present in the livingaffected family members. The mutation has never been reported before and could be thecause of PRKAG2cardiac syndrome in Chinese. From the recent studies,the PRKAG2cardiac syndrome is a cardiac metabolic diseases that caused by a perturbation in theactivity of AMPK lead to glycogen deposition in the heart. The PRKAG2gene mutationsreported abroad were all within the cystathionine β-synthase (CBS) domains,whichprompted the Bateman domain binding AMP capacity decreased resulting in AMPKactivity changes. In this Chinese family, the G100S mutation was not within thecystathionine β-synthase (CBS) domains of the PRKAG2gene,and the Mutation sitespreviously reported abroad sequenced completely normal. The pathogenic mechanism ofthe G100S mutation in the family with myocardial hypertrophy is unclear. To verify thepathogenic role of PRKAG2G100S mutation in the Chinese PRKAG2cardiac syndromefamily, we over-expressed PRKAG2G100S in zebrafish to research gene expression,AMPK activity, glycogen metabolism,and cardiac hypertrophy,to study the new mutationfunction and confirm the functional significance of the mutation in vivo, and to deepenunderstanding of the non-CBS domains of the PRKAG2gene, to clearify preliminaryly thepathogenic mechanism of the PRKAG2cardiac syndrome.Objective: To investigate the functional consequences of PRKAG2gene with a novelmissense mutation G100S responsible for Chinese familial ventricular hypertrophy with conduction system abnormalities and ventricular preexcitation in vivo.Methods:(1) Cloning and functional identification of the zebrafishcardiac-specific promoter vmhc: Zebrafish genomic DNA was extracted from zebrafishtissues. A1952bp gene fragment of zebrafish cardiac-specific myosin heavy chain(ventricular myosin heavy chain, vmhc) gene promoter was amplified by PCR fromzebrafish. The recombinant vector of pEGFP-vmhc was constructed,from which the genefragment contain vmhc promoter sequences,enhanced green fluorescent protein gene(EGFP) sequences and3'UTR sequences was amplified by PCR.The purifiedvmhc-EGFP gene fragment was injected into fertilized eggs of zebrafish by microinjectionto observe the characteristics and specific expression of the promoter in zebrafish cardiac.(2) Construction of the recombinant vector of HA-vmhc-WT,HA-vmhc-G100S andHA-vmhc-R302Q: Healthy human whole blood mRNA was extracted by Trizol methodand the human wild-type PRKAG2gene was cloned by RT-PCR. The recombinant vectorof pEGFP-PRKAG2was constructed.The recombinant vector of pEGFP-G100S andpEGFP-R302Q was constructed by PCR site-directed mutagenesis. The recombinantvector of pEGFP-vmhc-WT, pEGFP-vmhc-G100S and pEGFP-vmhc-R302Q wereconstructed by digested connection and then HA tag was inserted by the bridge PCR.(3)Experimental study of human PRKAG2wild-type and mutant genes in the zebrafish:The vmhc-WT/G100S/R302Q-HA gene fragment were injected into fertilized eggs ofzebrafish by microinjection to verify the PRKAG2gene expression by real-time PCR,PRKAG2protein expression by western blot, to detect myocardial glycogen content inzebrafish heart by PAS staining and myocardial AMPK activity by colorimetricquantitative measurement, respectively. In this study, PRKAG2R302Q was used as apositive control, wild-type PRKAG2WT as a negative control, the zebrafish embryoswithout microinjection of exogenous genes as a blank control.Results:(1) Vmhc promoter gene fragment sequences (1952bp) were cloned correctly.Green fluorescence was seen in the cardiac region of the zebrafish injected with genefragment containing vmhc promoter seguence, EGFP seguence and3' UTR seguence bythe fluorescence microscope. The successful expression of the exogenous EGFP gene andprotein in zebrafish were verified by RT-PCR and western blot, respectively.(2) A humanwild-type PRKAG2gene was cloned correctly. The recombinant vector HA-vmhc-WT,HA-vmhc-G100S and HA-vmhc-R302Q were constructed successfully.(3) There wasPRKAG2gene expression by real-time PCR in the zebrafish injected with vmhc-WT/G100S/R302Q-HA gene fragments, while not in the blank control group. Itindicated that vmhc promoter can drive the WT, GS, RQ gene expressed successfully inzebrafish. Analyzing the CT value of the PRKAG2gene and internal reference actin, it wasfound that the levels of three exogenous gene expression were as follows: WT> GS> RQ.HA protein expression was detected by western blot in the zebrafish injected withvmhc-WT/G100S/R302Q-HA gene fragment, while not in the blank control group. TheHA protein expression of the RQ group was the lowest in all groups,and the HA proteinexpression of GS group was weaker than that of the WT group. It was suggested that vmhcpromoter can drive the WT, GS, RQ protein expresse successfully in zebrafish. G100Smutation and R302Q mutation hindered the normal expression of PRKAG2protein inzebrafish.The effect of G100S mutation on expression of PRKAG2protein was weakerthan that of the R302Q mutation. The wall thickness of the zebrafish heart was comparedamong the groups. It was found that the wall thickness of GS group (14.71±1.97μm) wassignificantly thicker than that of the blank control group(11.74±1.86μm)(P=0.021)andWT group(11.81±2.09μm)(P=0.024), the wall thickness of RQ group(17.94±2.30μm)was significantly thicker than that of GS group(P=0.014). These results suggest thatG100S mutation and R302Q mutation may cause cardiac hypertrophy in zebrafish. PASstain of the zebrafish heart slides of the different groups showed that glycogen depositionsin the zebrafish heart wall of WT, GS, RQ group were more than that of the blank controlgroup. The AMPK activities of GS group (0.046213±0.001501μmol NADH/min/mg) andRQ group (0.022037±0.001228μmol NADH/min/mg) were significantly lower than thatof the blank control group (0.06257±0.000935μmol NADH/min/mg)(P=0.000) andWT group (0.084877±0.001879μmol NADH/min/mg)(P=0.000). The AMPK activityof GS group was significantly higher than that of RQ group (P=0.000). The AMPKactivity of WT group was significantly higher than that of the blank control group(P=0.000).Conclusions:(1) The novel missense mutation G100S alters the biological functionof PRKAG2gene and is responsible for PRKAG2cardiac syndrome in Chinese.This studyconfirms that the pathogenesis of the Chinese PRKAG2cardiac syndrome pedigree is thatPRKAG2G100S mutation impairs AMPK activity and leads to glycogen accumulation.(2)The gene biological functions caused by G100S mutation within the non-CBS domains ofthe PRKAG2gene are similar to R302Q mutation within the CBS domains of thePRKAG2gene. The AMPK activity regulation mechanism caused by G100S mutation may be consistent with that of R302Q mutation. G100S mutation may also have effect on theBateman domain, and damage the ability of the Bateman domain to bind AMP. Genebiological effects caused by the G100S mutation within the non-CBS domains of thePRKAG2gene are weaker than those of the mutations within the CBS domains. The role ofnon-CBS domains of the PRKAG2gene may be weaker than that of CBS domains.