The Contribution of Genetic and Environmental Factors to Quantitative Variability of Erythrocyte Membrane Proteins in Primary Hypotension

doi: 10.1046/j.1529-8817.2004.00102.x

The Contribution of Genetic and Environmental Factorsto Quantitative Variability of Erythrocyte MembraneProteins in Primary Hypotension

V. P. Ivanov1, A. V. Polonikov2 and M. A. Solodilova3

1Medical Biology, Genetics and Ecology Department. Kursk State Medical University. Russian Federation. 305041 Kursk. KarlMarx street, 32Medical Biology, Genetics and Ecology Department. Kursk State Medical University3Medical Biology, Genetics and Ecology Department. Kursk State Medical University. Russian Federation. 305041 Kursk. KarlMarx street, 3

Summary

Our previous studies have shown that, compared with healthy individuals, patients with primary arterial hypoten-sion (PAH) have significant quantitative changes in erythrocyte membrane proteins. The purpose of the presentstudy was to evaluate the contribution made by genetic and environmental factors to quantitative variation oferythrocyte membrane proteins in PAH. We studied 109 hypotensive patients, 124 normotensive subjects, 222 oftheir first-degree relatives and 24 twin pairs by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis.The decomposition of total phenotypic variance of erythrocyte membrane proteins to genetic and environmentalcomponents was performed on the basis of correlations among first-degree relatives by the least squares method.The genetic dominance and shared environmental factors were found to influence the variability of cytoskeletalmembrane proteins whose contents were changed in PAH. Furthermore, variations in α-spectrin, actin and anionexchanger in hypotensives were substantially influenced by major gene and maternal effects. Ankyrin 2.1 and actincontent was under the control of common underlying genes. Variations in membrane-associated glutathione-S-transferase and tropomyosin were predominantly affected by polygenes. These findings suggest that the putativemajor genes with pleiotropic effects appear to be involved in the control of quantitative disorders of erythrocytemembrane proteins in primary hypotension.

Keywords: arterial hypotension, genetic factors, environmental factors, membrane proteins, cytoskeletal proteins,quantitative variation, erythrocytes

Introduction

The primary arterial hypotension (PAH) known aschronic idiopathic hypotension is one of the widespreadand largely uninvestigated multifactorial cardiovasculardiseases of the young (Owens et al. 2000; Nadezhd-ina, 1990; Kaloeva (1993). As well as being an essentialhypertension, PAH may be considered as a risk fac-tor of atherosclerosis and essential hypertension (Kapset al. 1982; Perova et al. 1990; Rozanov et al. 1990;

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Rose et al. 2000). Relatively few studies have ex-amined the role of genetic factors in the pathogene-sis of hypotension (Robertson 1994; Iwai et al. 2001;Rankinen et al. 2000). Indeed, both the genetic na-ture and molecular mechanisms of PAH are still un-known, with the exception of orthostatic hypotension(DeStefano et al. 1998). Investigation into the geneticcontribution to intermediate biochemical phenotypesassociated with susceptibility to PAH may provide ad-ditional insights into the general regulatory pathwaysinvolved in the continuum from hypotension to hyper-tension. As reported previously, great progress in theunderstanding of the molecular and genetic mecha-nisms of primary hypertension was achieved by multiple

C© University College London 2004 Annals of Human Genetics (2005) 69,25–35 25

V. P. Ivanov et al.

studies on cell membranes (Postnov et al. 1984;Motulsky et al. 1987; O’Byrne et al. 1998; Ambler et al.1999; Orlov et al. 1999; Bianchi et al. 2003). Unfor-tunately, no analogous studies for primary hypotensionhave been carried out. The structural proteins of theplasma membrane are known to be involved in the regu-lation of transmembrane permeability for various cations(Nelson et al. 1987; Fossel et al. 1981; Baskin et al. 1981;Kazennov et al. 1991). Our previous studies have shownquantitative changes both in integral and peripheral pro-teins of erythrocytes in hypotensive patients comparedwith normotensive controls (Ivanov et al. 2000, 1999).The increased levels of α-spectrin, 2.1 ankyrin and pro-tein band 3, and decreased levels of actin, tropomyosinand glutathione-S-transferase, were found in hypoten-sives in comparison with normotensives. Moreover, thesubstantial quantitative variances of erythrocyte mem-brane proteins were characteristic for patients with PAHbut not for healthy subjects. The interrelation of foundquantitative changes in erythrocyte membrane proteinswith liability to PAH has been proved by statistical ge-netic analyses (Ivanov et al. 1999). The purpose of thepresent study was to evaluate the contribution of geneticand environmental factors to quantitative variationsof erythrocyte membrane proteins in primary arterialhypotension.

Materials and Methods

Study Subjects

We studied 109 hypotensive patients, 124 normotensivesubjects, 222 first-degree relatives, and also 24 pairs ofmonozygotic and dizygotic twins. First degree relatives(from both hypotensive and healthy families) consistedof the following intrafamilial pairs: 89 proband/motherpairs, 61 proband/father pairs and 72 sibling pairs. Ve-nous blood samples were taken for all of the investigatedgroups including first degree relatives and twins. Our in-vestigations were carried out in keeping with the ethicalprinciples of the Helsinki Declaration.

Precipitation and Purification of Erythrocytes

The precipitation of red blood cells was performed bythe method of Beutler et al. (1976) with non-significantmodifications. Heparinized blood samples were precipi-tated two times in isotonic sodium phosphate buffer (pH

7.4) containing 3% dextran T-500 for 30 min. Erythro-cytes were filtered through a glass column with micro-granular cellulose and centrifuged at 3000g for 10 minwith isotonic sodium phosphate buffer.

Preparation of Erythrocyte Ghosts

Erythrocyte membranes (ghosts) were prepared at 4◦Caccording to a modified method of Dodge et al. (1963).The washed erythrocytes were haemolysed in 20 vol-umes of 10 mmol/l Na-phosphate buffer (pH 7.4) andsedimented twice at 8000 g for 15 min. The final sed-imentation of membrane ghosts was performed using5 mmol/l Na-phosphate buffer (pH 7.4) under thesame conditions for centrifugation. Washed ghosts werelyophilized and frozen at −20◦C.

Fractionation and Detection of ErythrocyteMembrane Proteins

One-dimensional SDS-electrophoresis of solubilizedghosts in polyacrylamide gel (gradient 5-25%) was car-ried out by Laemmli’s method Laemmli 1970. Gelswere stained with Coomassie R-250 according to Fair-banks’s method (Fairbanks et al. 1971). Coomassie-stained gels were scanned by laser densitometer “Ul-trascan XL” (LKB, Sweden). The identification andmolecular weight calculations of erythrocyte membraneproteins were performed using protein marker stan-dards such as human gamma globulin (Mr = 160kD),bovine serum albumin (Mr = 67kD), ovalbumin(Mr = 45kD), chymotrypsinogen A (Mr = 24kD),myoglobin (Mr = 18kD) and cytochrome C (Mr =13kD). The detection of individual membrane proteinswas carried out using the well-characterized locationsof interest proteins in one-dimensional polyacrylamidegels (molecular weights points, Rf-values etc.) by im-munological methods (precipitation to monoclonal an-tibodies) as described by Steck T. (Fairbanks et al. 1971)and other researchers (Steck 1974; Bjerrum et al. 1975;De Angelis et al. 1996). The electrophoretogram oferythrocyte membrane proteins is shown in Figure 1.The erythrocyte membrane protein levels were quanti-fied using known concentrations (µg) of bovine serumalbumin, with the analytical package “One-Dscan”1.3 (Windows-based software product distributed by“Scanalytics”). The gene expression levels of erythro-cyte membrane proteins were considered indirectly,

26 Annals of Human Genetics (2005) 69,25–35 C© University College London 2004

Quantitative Genetics of Membrane Protein Disorders in Hypotension

Figure 1 The pattern of SDS one-dimensionalpolyacrylamide gel electrophoresis of erythrocyte ghosts isperformed according to Laemmli’s method (Laemmli 1970).

The numeration of protein bands are designated on theright: band 1 – α-subunit of spectrin (Mw = 240kD); band 2– β-subunit of spectrin (Mw = 220kD); band 2.1 – 2.1ankyrin (Mw = 200kD); band 2.2 – 2.2 ankyrin (Mw =l86kD); band 2.3 – 2.3 ankyrin (Mw = l66kD); band 3 –anion exchanger (Mw = l00kD); band 4.1 – protein band 4.1(Mw = 78kD); band 4.2 – pallidin (Mw = 72kD); band 4.5.1(it is not designated) – unidentified protein (Mw = 60kD);band 4.5 – glucose transporter (Mw = 55kD); band 4.9 –dematin (Mw = 48kD); band 5 – actin (Mw = 42kD); band 6– glycolytic enzyme glyceraldehyde-3-phosphatedehydrogenase (Mw = 35kD); band 7 – tropomyosin (Mw =27kD); band 8 – enzyme glutathione-S-transferase (Mw =19kD); Hb – membrane-associated hemoglobin.

Seven tracks of erythrocyte membrane proteins are present ingiven electrophoretogram. 5th and 7th tracks are correspondedto hypotensive patients. 1–4th and 6th tracks are correspondedto healthy subjects. Visually lowered level of protein band 8and 7 occurred in hypotensives (5th and 7th tracks) comparedwith controls (1–4th and 6th tracks) as has been previouslyreported (Ivanov et al. 2000; Ivanov et al. 1999).

through levels of the protein products of the correspond-ing genes.

Statistical Genetic Analyses

In our analyses, we assumed that variation in erythrocytemembrane proteins was jointly influenced by genes andenvironment. We used a variance decomposition ap-proach, implemented in the statistical genetic softwarepackage GENE 1.0 (DOS-based software product, de-veloped by professor Trubnikov V.I.) (Trubnikov et al.1992), to estimate the effects of genes, selected envi-

ronmental covariates, and unmeasured non-genetic fac-tors, on quantitative variances in erythrocyte membraneproteins. This approach was performed according tostandard methodology of quantitative genetics (Falconer1960; Nance 1982; Lynch et al. 1998; Trubnikov et al.1981). The interclass correlations among first-degreerelatives (between parents and their offspring) were cal-culated as simple linear Pearson correlations (Lynch et al.1998; Trubnikov et al. 1981). Otherwise, the intraclassfamilial correlations (between siblings or twins) werecalculated by one-factor variance analysis (Trubnikov1992, 1981; Nance 1976). The decomposition of to-tal phenotypic variance for erythrocyte membrane pro-teins, based on the “least squares method”, allowed us toobtain the following genetic and non-genetic compo-nents: Ga – additive genetic factors; Gd – genetic dom-inance; Ec - common (shared) environmental factors;Ew - random environmental factors; and ME – mater-nal effects. Maternal effect was considered as a sum ofthe effects attributed to cytoplasmic, prenatal and othermaternal factors (Trubnikov 1992, 1981). The values ofgenetic correlations were interpreted as contributionsof additively interacting genes (Lynch et al. 1998; Trub-nikov 1981; Nance et al. 1976). The diagonal values ofgenetic correlation matrices were considered as coef-ficients of genetic determination of erythrocyte mem-brane protein contents (Trubnikov 1992, 1981). Envi-ronmental correlations were attributed not only to en-vironmental effects but also to non-linear genetic effectssuch as dominance or epistasis (Trubnikov 1992; Nance1982). Standard clustering techniques were applied toevaluate both shared genetic (genes with pleiotropiceffects) and environmental factors that participate inthe control of erythrocyte membrane protein levels inhealthy and hypotensive individuals (Trubnikov 1992).

Results

The Role of Genetic and EnvironmentalFactors in the Quantitative Variabilityof Erythrocyte Membrane Proteins in HealthySubjects and Patients with PrimaryArterial Hypotension

Based on intrafamilial correlations, a component ge-netic analysis was performed to evaluate the contri-bution made by genetic and environmental factors to


V. P. Ivanov et al.

Table 1 Contribution of genetic and environmental factors to quantitative variability of erythrocyte membrane proteins in healthysubjects

Genetic components, % Environmental components, % Maternal effect, %

Membrane protein bands Ga Gd Ec Ew ME

1. Band 1 0.32 44.43 38.23 5.07 11.952. Band 2 - 46.86 20.77 8.42 23.963. Band 2.1 21.59 4.11 44.26 30.04 -4. Band 2.2 3.59 22.75 24.20 34.70 14.765. Band 2.3 52.37 6.33 10.23 31.07 -6. Band 3 26.32 30.78 28.88 14.02 -7. Band 4.1 23.84 14.05 45.95 8.15 8.018. Band 4.2 - 49.07 34.93 7.53 8.479. Band 4.5.1 18.11 19.09 33.59 29.21 -10. Band 4.5 - 48.48 26.77 5.86 18.8911. Band 4.9 29.10 26.13 13.23 17.27 14.2712. Band 5 3.21 39.58 54.23 2.47 0.5113. Band 6 23.51 29.85 32.30 9.40 4.9414. Band 7 4.63 42.47 48.62 4.28 -15. Band 8 18.59 34.71 39.36 7.34 -

∗Ga – additive componentGd – dominance componentEc – common (shared) environmental factorsEw – random environmental factorsME – maternal effect 0The quantitative variation for some cytoskeletal proteins such as 2.1 and 2.3 ankyrins, and protein band 4.1, in healthy subjects was sub-stantially dependant upon polygenes. Variations in α and β-spectrins, 2.2 ankyrin, pallidin, actin, tropomyosin, glutathione-S-transferaseand a glucose transporter were largely influenced by genetic dominance. Moreover, a major gene effect plus additive-polygenicinfluences were responsible for variances in an anion exchanger, protein band 4.5.1, dematin and glyceraldehyde-3-phosphatedehydrogenase. Variances of ankyrins, protein 4.5.1 and dematin were markedly sensitive to random environmental factors. At the sametime, variances of spectrins, 2.2 ankyrins, proteins 4.1 and 4.2, glucose transporter, actin, dematin and glyceraldehyde-3-phosphatedehydrogenase were also influenced by maternal effects.

the variability of erythrocyte membrane proteins inhealthy subjects (Table 1). As can seen from Table 1,genetic factors play a significant role in quantitative vari-ances of erythrocyte membrane proteins in healthy sub-jects. The proportion of total phenotypic variability oferythrocyte membrane proteins accounted for by en-vironmental effects tended to be considerably smallerthan that accounted for by genetic effects. Importantly,the variations in membrane proteins were attributableto both single gene and additive genetic effects. Fur-thermore, shared environmental factors influenced onquantitative variances of the majority of erythrocytemembrane proteins in healthy subjects. The contri-bution of genetic and environmental components toquantitative variability of erythrocyte membrane pro-teins in hypotensive patients is given in Table 2. Ge-netic dominance appeared to account for the majorityof erythrocyte membrane protein variances in hypoten-sive subjects. Special attention was focused on quanti-

tative variation in membrane proteins whose contentswere changed in hypotensives in comparison with nor-motensives. Almost 70% of tropomyosin (band 7) varia-tion was attributed to additive-polygenic gene effects.At the same time, environmental factors responsiblefor tropomyosin variation were largely contributed byshared factors. The quantitative variation of glutathione-S-transferase (band 8) explained by the major geneand polygenic effects was 7% and 48%, respectively;shared environmental factors contributed almost 37% ofthe variation in this membrane-associated enzyme. Thequantitative variations in cytoskeletal proteins such asspectrins (bands 1 and 2), actin (band 5) and protein 4.1(band 4.1) were largely affected by genetic dominanceand shared environmental factors. Whereas, variabilityof 2.1 ankyrin (band 2.1) was influenced by compara-ble levels of genetic dominance and various environ-mental factors. Furthermore, α-spectrin, actin and an-ion exchanger variances in hypotensives compared with



Table 2 Contribution of genetic and environmental factors to quantitative variability of erythrocyte membrane proteins in patientswith primary arterial hypotension

Genetic components, % Environmental components, % Maternal effect, %

Membrane protein bands Ga Gd Ec Ew ME

1. Band 1 - 37.96 34.65 7.88 19.522. Band 2 0.58 57.30 29.60 6.60 5.923. Band 2.1 9.80 33.20 27.28 29.72 -4. Band 2.2 46.60 - 22.67 30.73 -5. Band 2.3 - 39.60 27.33 33.07 -6. Band 3 - 44.47 26.05 9.73 19.757. Band 4.1 13.24 22.00 36.99 5.61 22.168. Band 4.2 29.48 16.80 34.48 15.72 3.529. Band 4.5.1 25.76 5.60 25.51 24.89 18.2410. Band 4.5 1.40 42.60 35.45 4.25 16.3011. Band 4.9 - 42.38 15.65 13.54 28.4312. Band 5 - 53.91 24.15 5.66 16.2813. Band 6 - 62.80 32.30 4.65 0.2514. Band 7 69.44 - 21.99 3.11 5.4615. Band 8 48.10 7.40 37.25 7.25 -

∗Ga – additive componentGd – dominance componentEc – common (shared) environmental factorsEw – random environmental factorsME – maternal effect54% of Actin variation in hypotensive patients was due to genetic dominance. The environmental factors responsible for actinvariation were presented by shared environmental effects (24%). The interindividual variation in level of protein 4.1 was attributed tocomparable effects of total genetic (35%) and common environmental (37%) factors. Variability in ankyrin levels was largely influencedby random environmental factors. Variation of 2.1 ankyrin was attributed to major gene effect (33%) and additively interacting genes(10%). Variability in anion exchanger level was explained by major gene effects (45%) and shared environment (26%). The maternaleffects were found to influence on the variability of α-spectrin (20%), actin (16%), protein 4.1 (22%) and anion exchanger (20%).

normotensives were substantially influenced by maternaleffects.

Genetic Contribution to Content ofErythrocyte Membrane Proteins in HealthySubjects and Patients with PrimaryArterial Hypotension

To evaluate the role of genetic factors responsible for thecontrol of contents of erythrocyte membrane proteins,both in healthy and hypotensive individuals, a geneticcorrelation analysis was performed. The matrix of av-eraged genetic correlations of erythrocyte membraneproteins in normotensives is given in Table 3. As canbe seen from Table 3, the contribution of genetic fac-tors to the control of levels of membrane proteins inhealthy subjects varied between 21% and 63%. Levelsof α-spectrin, actin, tropomyosin, protein bands 4.1, 8,6 and ankyrins 2.1 and 2.2 were under strong genetic

control. The matrix of averaged genetic correlationsfor erythrocyte membrane protein levels in hypoten-sive subjects is given in Table 4. As well as in healthysubjects, the majority of membrane proteins in patientswith PAH was under strong genetic control. Impor-tantly, erythrocyte membrane proteins whose levels dif-fered significantly between hypotensives and normoten-sives were most genetically influenced. Tropomyosinand glutathione-S-transferase content in hypotensivescompared with normotensives appeared to be tightlycontrolled by genetic factors. The genetic contributionto anion exchanger content was greater in hypotensivepatients than in healthy controls. The level of geneticfactors under α-spectrin content in hypotensives did notdiffer from that in normotensives. However, the influ-ence of genetic factors likely controlling expression ofcytoskeletal proteins such as actin and 2.1 ankyrin in pa-tients with PAH was substantially lower than in healthysubjects.


V. P. Ivanov et al.

Table 3 Matrix of genetic correlations of levels of erythrocyte membrane proteins in healthy subjects (p < 0.05)

bands 1 2 2.1 2.2 2.3 3 4.1 4.2 4.5.1 4.5 4.9 5 6 7 8

1 0.4962 0.555 0.3562.1 − .062 0.255 0.5642.2 0.341 −.201 0.548 0.4222.3 0.313 −.431 0.530 0.358 0.3673 − .104 −.431 0.093 − .018 0.240 0.3584.1 − .040 −.443 − .143 −.200 0.049 − .058 0.5944.2 0.030 −.415 − .194 0.215 − .068 −.230 0.414 0.3964.5.1 −.424 −.297 −.523 −.208 −.374 −.226 0.157 0.300 0.2714.5 0.016 − .149 −.315 − .023 0.286 − .054 0.052 0.176 0.135 0.3604.9 −.472 − .152 0.243 − .139 0.175 0.537 − .057 0.298 −.390 −.438 0.2135 −.377 0.050 −.365 −.273 −.253 −.461 − .077 0.204 0.459 0.007 − .136 0.6316 0.071 − .015 −.195 0.159 −.213 −.646 − .081 0.300 0.404 −.563 −.219 0.205 0.4157 −.537 − .178 −.453 − .139 −.292 − .060 − .058 −.389 0.628 − .106 − .061 0.269 0.475 0.4558 − .106 −.332 −.484 − .098 −.321 − .092 0.184 − .106 0.332 − .078 −.191 − .040 0.123 0.460 0.449

∗ The significant correlations are designated by bold face.Coefficients of genetic determination (diagonal values) of erythrocyte membrane proteins in normotensive individuals varied from21% to 63%. The majority of genetic correlations was of substantial magnitude and statistical significance. Contents of the maincytoskeletal proteins such as α-spectrin, 2.1 ankyrin, protein 4.1 and actin were under strong genetic control.

Table 4 Matrix of genetic correlations of levels of erythrocyte membrane proteins in patients with primary arterial hypotension (p <

0.05)

bands 1 2 2.1 2.2 2.3 3 4.1 4.2 4.5.1 4.5 4.9 5 6 7 8

1 0.5182 0.431 0.3762.1 0.284 0.325 0.4722.2 −.335 −.431 0.391 0.6012.3 0.655 −.338 0.335 − .097 0.1803 0.044 0.285 − .019 − .111 0.370 0.4454.1 −.292 − .123 − .152 0.458 −.524 −.638 0.5624.2 −.398 − .039 −.349 0.396 −.365 0.251 −.213 0.4194.5.1 −.242 −.214 −.377 − .142 −.366 −.480 0.194 0.082 0.5654.5 −.403 −.448 −.281 0.405 − .069 −.239 0.368 −.339 0.198 0.4354.9 −.221 − .032 0.103 0.079 0.241 − .022 − .153 − .027 −.293 − .073 0.4735 − .034 0.007 −.592 −.304 − .081 −.429 0.215 0.094 0.140 0.052 − .001 0.4216 −.416 −.281 −.201 0.011 −.539 −.461 0.261 0.304 0.409 − .022 − .082 0.074 0.3627 −.417 −.567 −.270 − .128 −.515 −.416 0.214 0.274 0.571 − .012 − .168 0.118 0.571 0.6598 −.394 −.479 −.582 − .174 −.351 −.508 0.247 0.101 0.778 0.447 − .123 0.369 0.333 0.577 0.713

∗The significant correlations are designated by bold face.Coefficients of genetic determination (diagonal values) of erythrocyte membrane proteins in hypotensives varied widely from 18% to71%. The genetic contribution to α-spectrin level in patients with PAH did not differ from that in healthy subjects (52% and 50%,respectively). On the one hand, the genetic control of cytoskeletal proteins such as actin and 2.1 ankyrin were lower in hypotensivesthan in normotensives. On the other hand, the contents of tropomyosin, glutathione-S-transferase and anion exchanger were understrong genetic control.

The Influence of Shared Genes on the Levelsof Erythrocyte Membrane Proteins in HealthySubjects and Patients with PrimaryArterial Hypotension

A cluster analysis of genetic correlations was appliedto evaluate the common underlying genetic factors in-

fluencing on the levels of erythrocyte membrane pro-teins (Figure 2). At least five independent clusters ofgenetic factors were found to be involved in the con-trol of erythrocyte membrane protein content in healthyindividuals (see Figure 2A). It was characteristic thatfunctionally related erythrocyte membrane proteinswere clustered by common underlying genetic factors.



Figure 2 Dendrograms of genetic correlations of erythrocyte membrane protein content inhealthy subjects (A) and patients with primary arterial hypotension (B).

A. The levels of erythrocyte membrane proteins in healthy subjects were under control of fiverelatively independent genetic factors. These clusters were following: 1st – bands 1 and 2; 2nd –2.1, 2.2 and 2.3 bands; 3rd – 4.5.1, 7 and 8 bands; 4th – 3, 6, 4.5 and 4.9 bands; 5th – 4.1 and 4.2bands. Actin (band 5) content was under control of independent genes.

B. Five clusters of shared genes appear to be involved in the control of levels of erythrocytemembrane proteins in hypotensive patients. These clusters were following: 1st – bands 1, 2.3 and6; 2nd – 2, 4.5.1, 7 and 8 bands; 3rd – 3 and 4.1 bands; 4th – 2.2, 4.2 and 4.5 bands: 5th – 2.1 and 5bands. Dematin (band 4.9) content was under control of the independent genes described above.

The content of α and β spectrin (bands 1 and 2), as wellas ankyrins (bands 2.1, 2.2 and 2.3), were at least partiallycontrolled by common underlying genes. The negativegenetic correlation between protein band 3 and proteinband 6 was strong, suggesting that genes in commoncontribute to increases in the level of anion exchangerand decreases in the level of glyceraldehyde-3-phosphatedehydrogenase. Shared genes were found to influencethe contents of cytoskeletal proteins 4.1 and 4.2. As canbe seen from Figure 2 (B), the clusters of genetic cor-relations in hypotensives differed markedly from thosein normotensives. The characteristic features in the ge-netic control of content of erythrocyte membrane pro-teins occurred in hypotensive patients compared withhealthy subjects. Interestingly, the quantitative changesfor individual membrane proteins found in hypoten-sives were influenced by different genetic factors. Inparticular, 2.1 ankyrin (band 2.1) and actin (band 5)contents appeared to be controlled by the same under-lying genes. Meanwhile, the levels of α-spectrin (band1), 2.3 ankyrin (band 2.3) and protein band 6, but notβ-spectrin, were controlled by shared genetic factors.

Furthermore, genetic correlations between levels of an-ion exchanger (band 3) and protein 4.1 occurred inhypotensives but not in normotensives, due to com-mon underlying genes. It was found that shared ge-netic factors were involved in the control of the levelsof tropomyosin (band 7) and glutathione-S-transferase(band 8) in healthy subjects, as well as in hypotensivepatients.

The Influences of Environmental Factors onthe Levels of Erythrocyte Membrane Proteinsin Healthy Subjects and Patients with PrimaryArterial Hypotension

As we have shown that variability of erythrocyte mem-brane proteins in both hypotensives and normotensivesis highly dependant on a number of non-genetic factors,the environmental correlations were estimated (matri-ces of environmental correlations are not shown). Toevaluate the common environmental factors influenc-ing the levels of erythrocyte membrane proteins, a clus-ter analysis of matrices of environmental correlations


V. P. Ivanov et al.

Figure 3 Dendrograms of environmental correlations of erythrocyte membrane proteincontent in healthy subjects (A) and patients with primary arterial hypotension (B).

A. At least five clusters of environmental factors were found to influence the erythrocytemembrane protein content in normotensives: 1st – bands 1, 4.5, 2.2 and 6; 2nd – 2 and 2.3bands; 3rd – 3, 4.9 and 8 bands; 4th – 4.5.1 and 7 bands; 5th – 2.1, 4.2 and 4.1 bands. Thelevel of protein band 5 was influenced by independent environmental factors.

B. Predominantly two groups (clusters) independent environmental factors appear toparticipate in the control of erythrocyte membrane protein content in hypotensives. Theseclusters were following: 1st – 1, 5, 4.1, 2.2, 4.5, 2.3, 3 and 4.2 bands; 2nd – 2, 4.5.1, 7, 8, 6,2.1 and 4.9 bands.

was applied in each cohort (Figure 3). The clusters ofenvironmental correlations of erythrocyte membraneproteins in healthy subjects are given in Figure 3A.At least five independent clusters of environmentalfactors may be involved in the regulation of expres-sion of erythrocyte membrane proteins in normotensiveindividuals. Interestingly, as compared with genetic fac-tors, common environmental factors affected the levelsof functionally unrelated membrane proteins in healthyindividuals. The clusters of environmental correlationsdiffered markedly between normotensives and hypoten-sives. At least two independent clusters of environmen-tal factors were found to affect the levels of erythrocytemembrane proteins in patients with PAH (Figure 3B).Importantly, erythrocyte membrane proteins whosecontent differed between hypotensives and normoten-sives were attributed to common underlying environ-mental effects. These features were characteristic espe-cially for the following membrane proteins: tropomyosin(band 7) and glutathione-S-transferase (band 8),α-spectrin (band 1) and actin (band 5).

Discussion

Our previous studies have shown quantitative changesin protein composition of erythrocyte membranesin PAH (Ivanov et al. 1999, 2000). However, thegenetic nature of these changes is still unknown. Inthe present study, genetic factors have been foundto play a significant role in the control of levels oferythrocyte membrane proteins both in hypotensiveand healthy subjects. The genetic contribution toquantitative variability of individual erythrocytemembrane proteins differed among hypotensives aswell as normotensives. Interactions between multipleloci, rather than variants of a single gene, underlaythe genetic basis of interindividual variation of themajority of erythrocyte membrane proteins in healthysubjects. The substantial effect of genetic dominanceon the variation of major cytoskeletal proteins appearedto demonstrate the important role of these proteinsin the membrane architecture both in normotensivesand hypotensives. Also, the strongest major gene effects



were found to affect the majority of variances oferythrocyte membrane proteins in hypotensive patients.Special attention has been focused on membraneproteins whose contents were substantially changed inhypotensive patients in comparison to healthy controls.These were peripheral proteins that constituted thespectrin-actin network at the inner side of the plasmamembrane of erythrocytes. Importantly, functionallyrelated erythrocyte membrane proteins such as spectrinsor actins were controlled by common underlying genesin healthy subjects. This suggests the existence of anumber of common genes having pleiotropic effectson cytoskeletal proteins. Series of such genes areconsidered today as gene networks that are involvedin the control of functionally related traits (specificinformation is available online at the GeneNet web-site:http://wwwmgs.bionet.nsc.ru/mgs/systems/genenet/).It is known that spectrin is a markedly abundantcytoskeletal protein of the erythrocyte membrane(Harris et al. 1980; Bennett 1990). The contributionof genetic and environmental factors to interindi-vidual variability of α-spectrin did not differ amonghypotensive patients and healthy subjects. Therefore,the increased level of α-spectrin in patients withPAH could not be explained by estimated geneticor environmental effects. Actin is one of the mainstructural cytoskeletal components known to organizethe spectrin-actin complexes as major junctions in thecell membrane of erythrocytes (Pinder et al. 1983;Cohen et al. 1984). Interestingly, the actin content inhypotensives tended to be under less genetic controlthan in normotensives. However, the interindividualvariation in actin levels in hypotensive patients was un-der strong genetic control, and was slightly dependanton shared environment. Interestingly, almost 77% ofcommon underlying environmental factors could beresponsible for the increased level of α-spectrin, andthe decreased level of actin, in hypotensives. Moreover,no common underlying genes that influenced bothactin and α-spectrin content were found. At the sametime, actin content in healthy subjects was under thecontrol of both independent genetic and environmentalfactors. The contribution of genetic and environmentalfactors to ankyrin levels (2.1, 2.2 and 2.3 bands)differed between hypotensive and healthy individuals.Ankyrin is known to facilitate the attachment of the

cytoskeleton to the cytoplasmic domain of the anionexchange protein (Cianci et al. 1988; Mohler et al.2002). Furthermore, the active form of ankyrin (band2.2) was found to influence the mobility of the anionexchanger in the phospholipid matrix, and to regulateanion permeability across the plasma membrane (Wyattet al. 1992). The phenotypic effects of genetic factorson ankyrin variation were very similar to those on actinvariation. As with actin levels, 2.1 ankyrin levels inhypotensive patients were under lower genetic controlthan normotensive controls. As is evident from thegenetic correlations, 2.1 ankyrin had almost 60% ofgenetic factors in common with actin. These findingssuggest that the major genes involved could havepleiotropic effects on the content of these cytoskeletalproteins, and could be responsible for their quantitativechanges in hypotensive patients. The inverse geneticcorrelation (r = −0.64) between protein 4.1 and anionexchange protein was strong, suggesting that genes incommon contribute to decreases in the protein 4.1level (this tendency has been found in hypotensives)and increases in anion exchanger level in patients withPAH. Furthermore, the strong influence of geneticdominance on the variation of the anion exchangerseems to be explained by pleiotropic effects of one ofthe major genes likely playing a role in the geneticcontrol of primary hypotension. Interestingly, thequantitative variations of α-spectrin, actin and the an-ion exchanger in hypotensive patients were significantlyaffected by maternal effects. It suggests that a numberof factors capable of affecting fetal metabolism (forexample, biosynthesis of membrane proteins) duringa pregnancy could be responsible for quantitativechanges in erythrocyte membrane proteins of futurehypotensive patients. The strongest genetic effectshave been found to influence glutathione-S-transferasevariation in hypotensive patients. Moreover, almost58% of the quantitative variations in both glutathione-S-transferase and tropomyosin were under the controlof common underlying genes. In comparison withthe peripheral membrane proteins described above theinterindividual variation in glutathione-S-transferaseand tropomyosin content was predominantly due topolygenes. The marked contribution of the additive-polygenic component to glutathione-S-transferasevariability appears to be mirrored by the involvement of


V. P. Ivanov et al.

this enzyme in multiple metabolic pathways controlledby different gene networks (White et al. 1983; Haradaet al. 1987; Mizukami et al. 1984). In particular, adecreased level of glutathione-S-transferase may beconsidered as an indirect marker of lower intracellularcalcium concentration (White et al. 1983), whichlikely occurs in PAH. Moreover, some of the genesinfluencing glutathione-S-transferase variation could beresponsible for epistatic gene effects on the expressionof tropomyosin in hypotensive patients.

Thus, the quantitative variability of erythrocytemembrane proteins in hypotensive patients is influencedby a number of genetic and environmental factors. Thequantitative changes in both major cytoskeletal proteinsand an anion transporter in hypotensives are predom-inantly attributed to major gene and maternal effects.The phenotypic variances of glutathione-S-transferaseand tropomyosin are affected strongly by polygenes. Theputative major genes with pleiotropic effects were foundto be involved in the control of quantitative disordersof erythrocyte membrane proteins in primary arterialhypotension. On the basis of obtained estimates, it isreasonable to start looking for genomic regions and in-dividual genes, or quantitative trait loci (QTLs), thatare responsible for the genetic effects on intermediatequantitative phenotypes such as erythrocyte membraneprotein levels. Identifying these QTLs would not onlybenefit our understanding of the main principles of hu-man genome functioning, but also contribute to ourunderstanding of particular molecular-genetic mecha-nisms involving in the regulation of gene expression.This information would very likely also shed light onthe genetic nature of membrane mechanisms under-lying primary arterial hypotension as well as essentialhypertension.

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Received: 5 November 2003Accepted: 19 January 2004


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The Contribution of Genetic and Environmental Factors to Quantitative Variability of Erythrocyte Membrane Proteins in Primary Hypotension