Relationship Between Apolipoprotein Gene Polymorphism And Dyslipidemia In Children Undergoing Primary Nephrotic Syndrome

Background and objectivePrimary nephrotic syndrome(PNS)is a common chronic disorder, characterized by alterations of permselectivity at the glomerular capillary wall,resulting in its inability to restrict the urinary loss of protein. Dyslipidemia is not only the clinical manifestation and diagnostic evidence of PNS,but also involved in the pathologic processes of renal lesion.Hypoproteinemia and low plasma colloid osmotic pressure stimulate an increased lipoprotein synthesis by the liver,the compositions with higher molecular weight are hardly discharged from kidneys,which may be the main mechanism inducing hyperlipidemia(HLP)secondary to PNS.Furthermore,many researches have demonstrated that hereditary basis contributed to the onset of PNS in the recent years.There are individual variations in the degree of proteinuria and HLP.Therefore,it is very important to comprehend the characteristics and probe the genetic mechanism of dyslipidemia in PNS children for blocking the progression of chronic kidney disease,removing the risk factors of cardiovascular disease(CVD),elucidating susceptibility,performing individualized treatment,and ameliorating prognosis.Apolipoprotein E(apoE),the ligand of low-density lipoprotein(LDL) receptor,not only transports the cholesterol(CH)from periphery to liver, but also elevates the sulfate-proteoglycan in glomerular filtration membrane,and inhibits the proliferation of mesengial cell.However,it remains controversial whether the genetic variance of apoE deteriorates the glomerular lesion,proteinuria and dyslipidemia.Apolipoprotein B(apoB)is the major component of chylomicra, very low density lipoprotein,intermediated-density lipoprotein and LDL。Other than helping serum lipids' transportation and metabolism,apoB can stimulate the CH esterification in macrophages,encourage the formation of foam cells,and finally result in AS.Therefore,apoB gene as one of candidates for CVD has been identified by many scholars.However,no report about the relationship between apoB gene polymorphism and dyslipidemia in children with PNS has been found to date.On the foundation of comprehending the characteristics and clinical significances of lipid abnormalities secondary to PNS,we analyzed apoE and apoB gene polymorphism through polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technique combined with gene sequence determination,in order to probe their effects on the metabolisms of serum lipids.Methods1.Blood samples were taken after a fasting period of 12 hours from 150 PNS and 100 healthy children,24 hours urine samples were obtained from all the patients.The fasting serum levels of five major lipids and two apolipoproteins,total cholesterol(TC),triglyceride(TG),lipoprotein a(LPa),high density lipoprotein-cholesterol(HDL-C),LDL-C, apolipoprotein A1(apoA1)and apoB,were measured.TC,TG and LPa were determined by enzymatic methods;HDL-C was measured by direct method using polyethylene-glycol-pretreated enzymes;LDL-C was determined by the liquid double reagent assay;non high density lipoprotein-cholesterol(nonHDL-C)was calculated by subtracting HDL-C from TC;apoA1 and apoB were measured by immunoturbidimetric methods.Total protein(TP)in serum was determined by biuret;albumin(AlB)and globulin(GLO)were determined by bromocresol green.24 hours urine protein(24hUPr)was measured biuret colorimetric method.2.ApoE and apoB gene polymorphism were analyzed through PCR-RFLP technique combined with gene sequence determination, alleles frequency were calculated by balancing methods.Results1.Serum levels of TP,AlB,albumin/globulin(A/G),and apoA1/B were obviously lower in PNS children than that in control group(P＜0.01);however,serum levels of LPa,TC,TG,HDL-C,nonHDL-C, LDL-C,LDL-C/HDL-C,and apoB were siginificantly increased in all of the nephritic children(Pz＜0.05).2.Negtive correlations were obsvered between 24hUPr and serum TP, AlB,GLO in PNS children repectively(P＜0.05).3.There were outstanding positive correlations between 24hUPr and serum Lpa,HDL-C,apoA1/B(P＜0.05);negative correlations between TP and TC,nonHDL-C,LDL-C,LDL-C/HDL-C,apoA1/B(P＜0.05); negative correlations between AlB and Lpa,TC,nonHDL-C,LDL-C, LDL-C/HDL-C,apoB,apoA1/B(P＜0.05);negative correlations between GLO and TC,nonHDL-C,LDL-C(P＜0.05);negative correlations between A/G and LPa,TC,nonHDL-C,LDL-C, LDL-C/HDL-C,apoB,apoA1/B in PNS children repectively(P＜0.05).4.Six genotypes of apoE were determined in 150 PNS children, including homozygotes E2/2(4 subjects,2.67%),E3/3(117,78.00%)and E4/4(5,3.33%),heterozygotes E3/2(13,8.67%),E4/2(5,3.33%),E4/3 (6,4.00%);the alleles frequencies of e2,e3 and e4 were 8.67%,84.33% and 7.00%repectively.Five genotypes were determined in 100 healthy children,including homozygotes E3/3(73,73.00%)and E4/4(5,5.00%), heterozygotes E3/2(15,15.00%),E4/2(1,1.00%),E4/3(6,6.00%);the allelic frequencies of e2,e3 and e4 were 8.00%,83.50%and 8.50% repectively.The apoE genotypic and allelic distributions of the whole PNS group were not significantly different when compared with the healthy controls(P＞0.05).5.There were no obvious differences in serum lipids among apoE genotypes and alleles of both healthy and PNS children(P＞0.05).6.The Xba I genotypes of apoB in 150 PNS children were X-/X-(132 subjects,88.00%),X+/X-(17,11.33%)and X+/X+(1,0.67%);the allelic frequencies of X- and X+ were 93.67%and 6.33%respectively. The Xba I genotypes of apoB in 100 healthy children were X-/X-(95, 95.00%)and X+/X-(5,5.00%);the allelic frequencies of X- and X+ were 97.50%and 2.50%repectively.The Xba I genotypic and allelic distributions of the whole PNS group were not significantly different when compared with the healthy controls(P＞0.05).7.The EcoR I genotypes of apoB in 150 PNS children were E-/E-(15 subjects,10.00%),E+/E-(17,11.33%)and E+/E+(118,78.67%);the allelic frequencies of E-and E+ were 15.67%and 84.33%respectively. The EcoR I genotypes of apoB in 100 healthy children were E-/E-(8, 8.00%),E+/E-(13,13.00%)and E+/E+(79,79.00%);the allelic frequencies of E-and E+ were 14.50%and 85.50%respectively.The EcoR I genotypic and allelic distributions of the whole PNS group were not significantly different when compared with the healthy controls (P＞O.05).8.The Msp I genotypes of apoB in 150 PNS children were M-/M-(1 subjects,0.67%),M+/M-(12,8.00%)and M+/M+(137,91.33%);the allelic frequencies of M-and M+were 4.67%and 95.33%respectively. The Msp I genotypes of apoB in 100 healthy children were M+/M-(2, 2.00%)and M+/M+(98,98.00%);the allelic frequencies of M-and M+ were 1.00%and 99.00%respectively.The Msp I genotypic and allelic distributions of the whole PNS group were not statistically different when compared with the healthy controls(P＞0.05).9.Individuals carrying X+allele in both control and PNS group exhibited significantly higher serum levels of LPa,TC,nonHDL-C, LDL-C,LDL-C/HDL-C and apoB than did the X- subjects(P＜0.05), whereas for apoA1/B the opposite was observed(P＜0.01).10.E-/E- carriers had significantly higher LPa,TC,HDL-C,and apoA1 concentrations than did E+/E- or E+/E+ carriers in control group (P＜0.05).Healthy children carrying the rare EcoR I allele had higher mean LPa,TC,and HDL-C levels than homozygotes for E+(P＜0.05). Higher LPa serum concentrations were observed in patients with E- allele (P＜0.05).11.Children undergoing PNS had no significance for serum lipids between both alleles of Msp I locus(P＞0.05).Conclusions1.PNS is characterized by dyslipidemia.Mass proteinuira may be the main trigger for the dyslipidemia in children with PNS.2.Geographical difference exists in the frequencies of apoE and apoB Xba I,EcoR,Msp I genotypes and alleles.3.No significant differences in genotypic and allelic frequencies are exhibited for apoE in PNS children as compared to controls.Lipid profiles are not influenced by the polymorphism of apoE gene in PNS children.4.No significant differences in genotypic and allelic frequencies are exhibited for apoB Xba I,EcoR and Msp I restriction sites in PNS children as compared to controls.X+ and E- allele of apoB gene may increase the susceptibility to HLP in children undergoing PNS.Lipids monitoring and dietary modification should be undertaken as early as possible in patients with X+ and/or E- allele.