Indian Journal of Human Genetics Medknow Publications on behalf of Indian Society of Human Genetics ISSN: 0971-6866 EISSN: 1998-362x Vol. 10, Num. 2, 2004, pp. 46-52

Indian Journal of Human Genetics, Vol. 10, No. 2, July-December, 2004, pp. 46-52

Original Article

Family based analysis of quantitative changes of erythrocyte membrane proteins in essential hypertension

Ivanov Vladimir P., Polonikov Alexey V., Emeliyanova Oksana G., Solodilova Mariya A., Mandrik Irina A.

Medical Biology, Genetics and Ecology Department. Kursk State Medical University

Correspondence Address: Russian Federation 305041 Kursk. Karl Marx street, 3. Kursk State Medical University, Medical Biology, Genetics and Ecology Department, Russian Federation
medbiol@kursknet.ru

Code Number: hg04011

ABSTRACT

Keywords: Essential hypertension, genetic contribution, major gene effect, pleiotropy, erythrocyte membrane proteins, anion exchanger, glucose transporter, cytoskeletal proteins, quantitative variation

INTRODUCTION

Essential hypertension is known to be a multifactorial disease that develops as a result of complex interactions of genetic and environmental factors.[1],[2],[3],[4],[5] The recent advances in molecular genetics have provided a lot of candidate gene loci responsible for susceptibility to hypertension. However, widespread candidate gene approach remains challenging in part due to genetic heterogeneity within hypertensive populations and lack of unified insights on the general pathogenetic pathway of EH. Despite a success in human genome sequencing and QTL mapping the novel proteome technologies require that we should have returned to a classical quantitative genetic methodology as a first powerful tool to select target major genes having pleiotropic effects on hypertension-associated phenotypes. As has been shown by multiple studies,[6],[7],[8],[9],[10],[11],[12],[13] structural and functional characteristics of cell membranes can be considered as intermediate phenotypes directly involved in the pathogenesis of hypertension. Red blood cell membrane is well known to be useful object in the modeling of widespread membrane abnormalities found in EH.[7],[8],[9],[10],[14],[15],[16] Our previous study has shown that, compared with healthy individuals, patients with essential hypertension have significant quantitative changes in erythrocyte membrane proteins (EMP) such as anion exchanger, glucose transporter, actin, dematin, glyceraldehyde-3-phosphate dehydrogenase and protein band 4.1.[17] The purpose of the present study was to evaluate the contribution made by genetic and environmental factors to quantitative variability of erythrocyte membrane proteins in essential hypertension.

MATERIALS AND METHODS 

We studied 115 untreated patients with essential hypertension, 126 healthy subjects, 235 of their first-degree relatives and also 24 mono- and dizygotic twin pairs. The following intrafamilial pairs were studied: 92 proband/mother pairs, 61 proband/father pairs and 82 sibling pairs. Our investigations were carried out in keeping with the ethical principles of the Helsinki Declaration.

The precipitation of erythrocytes was performed by Beutler′s method[18] with non-significant modifications. The preparation of erythrocyte membranes was performed according to Dodge′s method.[19] One-dimensional SDS polyacrylamide gel electrophoresis was used to fractionate EMP according to Laemmli′s method.[20] Coomassie-stained gels were scanned by laser densitometer "Ultrascan XL" (LKB, Sweden). The identification and molecular weight calculations of individual membrane proteins was carried out as in previously reported study.[21] The erythrocyte membrane protein levels were quantified through known concentration of BSA (µg) by analytical package "One-Dscan" 1.3 ("Scanalytics", CSP Inc).

We used a variance decomposition approach, implemented in the statistical genetic software package GENE 1.0, (DOS-based software product, developed by professor Trubnikov V.I.)[22] according to standard methodology of quantitative genetics.[23],[24],[25],[26] The interclass correlations among parents and their offspring were calculated as simple linear Pearson correlations[24],[25] whereas those among siblings or twins were calculated by one-factor variance analysis.[22],[25],[26] The decomposition of total phenotypic variance for EMP was based on the "least squares method".[22],[23],[24],[25],[26] Standard clustering techniques were applied to evaluate both shared genetic (genes with pleiotropic effects) and environmental factors that participate in the control of EMP levels in hypertensive and normotensive individuals.[22]

RESULTS

Results of component genetic analysis in hypertensive and normotensive cohorts are shown in [Table - 1]. The total contribution of genetic factors to quantitative variability of EMP was greater in hypertensive patients than in normotensive subjects. In particular, variations in anion exchanger (band 3), protein 4.5.1 and dematin (band 4.9) in hypertensives were mainly controlled by polygenes whereas those in protein 4.1, pallidin (band 4.2) and glucose transporter (band 4.5) were attributed to major gene effects. Interestingly, characteristic differences in the genetic control of variability were obtained for proteins whose contents were changed in hypertensives in comparison with normotensives.[17] The significant interindividual variation in level of anion exchanger found in hypertensive patients was contributed extremely by additively interacting genes. Unlike an anion exchanger, quantitative variation in glucose transporter was depended only on the effect of genetic dominance. The decreased variation in cytoskeletal protein 4.1 found in hypertensives compared with normotensives appeared to depend largely on major gene effect. Comparable additive-polygenic and major gene effects largely affected variability of peripheral proteins such as actin and glyceraldehyde-3-phosphate dehydrogenase in hypertensives but not in normotensives.

The proportion of total phenotypic variability of EMP accounted for by environmental effects tended to be considerably smaller than that accounted for by the genetic effects. Shared environmental factors influenced on quantitative variances of the majority of membrane proteins in hypertensives, as well as in normotensives. Furthermore, anion exchanger, glucose transporter, 2.1 ankyrin, dematin and glyceraldehyde-3-phosphate dehydrogenase variances in hypertensive patients compared with healthy controls were substantially influenced by maternal effects.

To estimate the genetic contribution to EMP levels a genetic correlation analysis was performed. The matrix of averaged genetic correlations of EMP in hypertensives is given in [Table - 2]. Importantly, EMP whose levels differed significantly between hypertensives and normotensives were most genetically influenced. The genetic contribution to anion exchanger and glucose transporter levels was greater in hypertensives than in normotensives [Table - 3]. Content of cytoskeletal proteins such as bands 4.1, 4.9 and 6 in hypertensives compared with normotensives appeared to be tightly controlled by genetic factors. However, the influence of genetic factors likely controlling expression of actin in hypertensive patients was substantially lower than in healthy individuals.

A cluster analysis of genetic correlations was applied to evaluate the common genetic factors influencing on the levels of erythrocyte membrane proteins [Figure - 1]. Three independent clusters of genetic factors were found to be involved in the control of EMP content in hypertensive individuals [Figure - 2]A. On the other hand, at least five clusters of genetic factors were found to influence EMP content in healthy individuals [Figure - 2]B. Characteristically, functionally related EMP such as spectrins or ankyrins were influenced by common underlying genetic factors in healthy subjects but not in hypertensives. Importantly, the levels of anion exchanger, glucose transporter, proteins bands 4.1, 4.9 and actin were at least partially controlled by common underlying genes. The marked pleiotropic effects of shared genes (r=0.642-0.928, P< 0.05) have been found within IInd cluster described above. The inverse genetic correlation between bands 3 and 4.1 found in hypertensives was strongest (r=0.928, P< 0.05), suggesting that genes in common contribute to increases in the level of anion exchanger and decreases in the level of cytoskeletal protein 4.1 [Table - 2].

As has been estimated by cluster analyses of environmental correlations, at least three independent groups of environmental factors were found to affect the levels of EMP in hypertensives [Figure - 2]. Shared environmental factors were responsible for variation in anion transporter and dematin levels in hypertensives. Meanwhile, actin, glyceraldehyde-3-phosphate dehydrogenase and glucose transporter levels were attributed to common underlying environmental effects.

DISCUSSION

In the present study, genetic and environmental factors have been found to play a different role in the control of levels of erythrocyte membrane proteins both in hypertensive and normotensive individuals. As a whole, genetic contribution to quantitative variability of majority of EMPs was greater in hypertensive patients than in healthy subjects, suggesting an essential role of genetic factors in determining of these traits in essential hypertension. Furthermore, EMPs whose levels were significantly changed in hypertensives compared with normotensives were strongly influenced by genetic factors. Special attention has been focused on transmembrane proteins such as anion exchanger and glucose transporter, taking into account the direct involvement of integral proteins into ion transport abnormalities in EH. Genetic contribution to variation in anion exchanger explained by additively interacting genes was stronger in hypertensive patients (88%) than in normotensive subjects (36%). Unlike hypertensives, major gene (31%) plus polygenic effects (26%) controlled variation in anion exchanger in normotensives.

The major gene effects, rather than interactions between multiple loci, underlay the genetic basis of interindividual variation of glucose transporter in healthy subjects as well as in hypertensive patients. The increased expression of glucose transporter found in hypertensive patients compared with healthy subjects appeared to be attributed to substantial effect of genetic factors (74%). Importantly, the levels of glucose transporter and anion exchanger were strongly controlled by common underlying genes in hypertensive patients (r=0.921, P< 0.05). This suggests the existence of a number of common genes having precise pleiotropic effects on expression of integral membrane proteins in EH. As reported previously,[17] quantitative changes in the levels of integral membrane proteins were closely linked to changes in the levels of cytoskeletal proteins such as protein band 4.1, actin and dematin found in hypertensive patients compared with healthy subjects. The inverse genetic correlation (r= -0.928, P< 0.05) between anion exchange protein and protein 4.1 was strong, suggesting that genes in common contribute to decreases in the protein 4.1 level (epistatic effect) and increases in anion exchanger level in patients with EH. The substantial influence of genetic dominance on the variation of cytoskeletal protein 4.1 and glucose transporter seems to be reflect major gene effect found previously in studies on high blood pressure variation.[28],[29],[30],[31] Interestingly, same common underlying genes with pleiotropic effects were found to influence the levels of actin, dematin and anion exchanger in hypertensives but not in normotensives in our study.

The putative major genes with pleiotropic effects were found to be involved in the control of quantitative disorders of erythrocyte membrane proteins in EH. These findings suggest that pleiotropic effects of multiple genes responsible for variation in membrane protein content may play a role in etiology of essential hypertension. The action of these genes on hypertensive phenotype may be mediated through the widespread membrane abnormalities that are not limited only by red blood cell membranes.[6],[8] The effects of shared genes, modifying the levels of membrane proteins in hypertensives, seem to be associated with existence of common biochemical catalyst (catalysts) during protein biosynthesis.

On the basis of obtained estimates, it is reasonable to start looking for genomic regions and major genes that are responsible for pleiotropic effects on intermediate quantitative phenotypes such as erythrocyte membrane protein levels. Results that are obtained due to quantitative genetic approach would not only benefit our understanding of the main principles of functioning of gene networks, but also substantially facilitate the precise identifying of candidate genes responsible for complex phenotype such as blood pressure, avoiding large-scale association and transmission disequilibrium studies.

REFERENCES

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