Gender differences in a Type 2 (non-insulin-dependent) diabetic population with respect to apolipoprotein E phenotype frequencies

Diabetologia, Mar 1993

Summary Apolipoprotein E polymorphism was examined in an Italian population of Type 2 (non-insulin-dependent) diabetic patients. There were significant differences (p < 0.05) in allele frequencies between male and female patients due to an under-representation of the E4 allele in the female group. No differences in allele frequencies were noted when non-diabetic male and female control subjects were compared. Both control groups exhibited similar allele distributions to that of male diabetic patients, but were significantly different (p < 0.05) from female diabetic patients. A closer examination of the female diabetic population revealed that under-representation of the E4 allele was principally confined to patients aged 60 years or older. This sub-group showed a significantly different (p < 0.05) allele frequency profile from control subjects (both men and women) and diabetic men, whereas this was not observed in the younger diabetic women (≤ 59 years). The results are consistent with the suggestion that the E4 allele may be a particular risk factor for female diabetic patients.

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Gender differences in a Type 2 (non-insulin-dependent) diabetic population with respect to apolipoprotein E phenotype frequencies

Gender differences in a Type 2 (non-insulin-dependent) diabetic population with respect to apolipoprotein E phenotype frequencies M. B o e m i t 0 1 R. W. J a m e s 0 1 E R o m a g n o l i t 0 1 P. G e r b e r 0 1 D . P o m e t t a 0 1 P. F u m e l l i 0 1 0 1Division of Diabetology, INRCA , Ancona , Italy , and 2Division of Diabetology, University Hospital , Geneva , Switzerland 1 Dr. R. W. James Division of Diabetology Department of Medicine University Hospital CH-1211 Geneva 14 Switzerland Summary. Apolipoprotein E polymorphism was examined in an Italian population of Type 2 (non-insulin-dependent) diabetic patients. There were significant differences (p < 0.05) in allele frequencies between male and female patients due to an under-representation of the E4 allele in the female group. No differences in allele frequencies were noted when non-diabetic male and female control subjects were compared. Both control groups exhibited similar allele distributions to that of male diabetic patients, but were significantly different (p < 0.05) from female diabetic patients. A closer examination of the female diabetic population revealed that under-representation of the E4 allele was princi- Type 2 (non-insulin-dependent) diabetes mellitus; apo E polymorphism; plasma lipids; lipoproteins; cardiovascular risk factor - 9 Springer-Verlag 1993 Cardiovascular disease is one of the principal complications of diabetes mellitus and a major cause of morbidity and mortality [ 1 ]. Female diabetic patients are notably at increased risk as their natural protection from atherosclerotic disease is compromised by the diabetic condition [ 2, 3 ]. Lipid abnormalities are a c o m m o n occurrence in poorly-controlled diabetic subjects [ 1, 4-6 ]. It is logical to consider them as a primary source of subsequent cardiovascular complications given their status as leading risk factors in the non-diabetic population. Studies over the last decade [ 7-10 ] have clarified the importance of apolipoprotein (apo) E to blood lipid/lipoprotein metabolism. It mediates hepatic elimination of potentially atherogenic remnant particles generated by the lipolysis of triglyceride-rich lipoproteins (chylomicrons, V L D L ) . O f equal clinical significance are the consequences of apo E polymorphism. T h r e e major isoforms are recognised: apo E3, the ancestral protein, and two mutants, apo E2 and apo E4, derived from the f o r m e r by point mutations [11]. B o t h mutations can pathologically influence blood levels of triglycerides and cholesterol [ 7, 8, 12 ]. Indeed, for a single gene locus, apo E polymorphism would appear to have an astonishingly important effect on plasma cholesterol concentrations of the general population, being responsible for some 16 % of genetic variation (8 % of overall variation [ 13, 14 ]). pally confined to patients aged 60 years or older. This subgroup showed a significantly different (p < 0.05) allele frequency profile from control subjects (both men and women) and diabetic men, whereas this was not observed in the younger diabetic women ( <_59 years). The results are consistent with the suggestion that the E4 allele may be a particular risk factor for female diabetic patients. Given the propensity of diabetic patients to develop lipid disorders, notably hypertriglyceridaemia, an obvious consideration is whether apo E polymorphism is a particular risk factor in this population. Only a limited n u m b e r of studies have b e e n p e r f o r m e d concerning this topic [ 15-19 ] and have t e n d e d to use small groups of patients. O u r intention was to address the question in a large, clinically well-documented population. During the course of the study, we observed male-female differences in apo E polymorphism of the diabetic populations. This is the first occasion that such gender differences have b e e n noted and could have important implications for apo E polymorphism as a risk factor in female diabetic patients. Subjects and m e t h o d s Studypopulations Diabetic patients were recruited from those consecutively attending the Diabetes Centre of Ancona Hospital for their routine visits (every 1-2 months). A total of 517 patients were enrolled, of which 81 were Type 1 (insulin-dependent) diabetic subjects.These subjects were excluded from the present study, leaving 436 Type 2 (non-insulin-dependent) diabetic patients, 211 men and 225 women. A total of 73 patients (23 men, 50 women) were taking lipid lowering medication (which was not discontinued) and 123 patients (52men, 71 women) were receiving insulin. Diagnosis of Type 2 diabetes was based on World Health Organ177 diagnostic criteria, supplemented where necessary by measurements of plasma and urinary C-peptide. Non-diabetic control subjects were recruited from those attending the blood donor centre in Ancona. A total of 365 subjects were enlisted, 296 men (43.2  10.6 years; range 20-64 years) and 69 women (42.0  14.3 years; range 21-64 years) which is a representative ratio of subjects attending the centre. Fasting blood samples were centrifuged to obtain plasma which was rapidly frozen and stored at -70~ The frozen samples were transferred on dry ice to Geneva every 1-2 months for apo E phenotyping and measurement of blood lipid and apolipoprotein levels. Apolipoprotein E phenotyping A p o E was phenotyped using a modified version of the procedure of Menzel et al. [ 20 ]. The modifications [ 21 ] entailed the use of whole plasma, instead of very low density lipoprotein, as the source of apo E, and visual177 of apo E using a high affinity, pan apo E monoclonal antibody produced in our laboratory. The procedure facilitates differentiation of sialylated and non-sialylated forms of apo E e. g. apo E3 from sialylated apo E4 and apo E2 from sialylated E3, as the non-sialylated forms focus in front of the sialylated derivatives [2Ol. Lipid and apolipoprotein assays Plasma cholesterol, triglycerides and H D L cholesterol (after precipitation of lower density lipoproteins with phosphotungstate) were assayed enzymatically and apos A-I and B by electroimmunoassay, as described previously [ 22 ]. LDL-cholesterol was calculated as (total cholesterol- H D L - c h o l e s t e r o l - (trig[ycerides/5)) for triglycerides less than 5.0 mmol/1. Five diabetic patients (three with E3/3, one with E3/2 and one with E4/3) were excluded from this analysis due to triglyceride levels above 5.0 retool/1. Statistical analysis Statistical analysis of phenotype distributions was performed with the chi-square test. Other comparisons were made using the unpaired Student's t-test. Results V a r i o u s c l i n i c a l p a r a m e t e r s o f t h e m a l e a n d f e m a l e , T y p e 2 d i a b e t i c p o p u l a t i o n s a r e s h o w n in T a b l e 1. B o t h p o p u l a t i o n s h a d g o o d m e t a b o l i c c o n t r o l as t h e y w e r e w i t h i n t h e u p p e r n o r m a l r a n g e ( l i m i t 8 . 1 % ) f o r H b A l c c o n c e n t r a t i o n s , a l t h o u g h f e m a l e s h a d s i g n i f i c a n t l y h i g h e r l e v e l s t h a n m e n . T h e t w o g r o u p s d i d n o t s h o w s i g n i f i c a n t d i f f e r e n c e s w i t h r e s p e c t t o B M I o r d u r a t i o n o f d i a b e t e s . A s r e g a r d s l i p i d p a r a m e t e r s , t r i g l y c e r i d e l e v e l s w e r e s i m i l a r in b o t h g r o u p s , w h e r e a s p l a s m a c h o l e s t e r o l c o n c e n t r a t i o n s w e r e s i g n i f i c a n t l y l o w e r in m a l e s . Plasma concentrations (mean + SD) of lipids (mmol/1) and apolipoproteins (g/l) in diabetic patients as a function of apo E polymorphism. Statistical comparisons were made only for phenotypes E3/2, E3/3 and E4/3 using the unpaired Student's t-test, ap<0.02; Up < 0.005; ~ < 0.001 vs corresponding phenotype of female patients. Other comparisons were not significant atp _<0.05. For LDLcholesterol of male patients, n = 139 for E3/3 and 30 for E3/2; for LDL-cholesterol of female patients, n - 173 for E3/3 and n = 19 for E4/3 2/2 3/2 3/3 4/3 4/4 4/2 Relative phenotypic frequencies for diabetic (D) and control (C) subjects were calculated from the absolute frequency distributions which are the values given in parentheses Relative allele frequencies for the populations were calculated from the phenotypes given in Table 3 Table 2 gives the lipid and apolipoprotein levels in the diabetic populations as a function of apo E phenotype. Statistical comparisons b e t w e e n male and female patients were limited to E3/2, E3/3 and E4/3 phenotypes. Significantly higher plasma and LDL-cholesterol concentrations were n o t e d in diabetic women with E3/2 or E3/3 phenotypes, but not those with the E4/3 phenotype. Similar results were obtained when subjects taking lipid lowering medication were eliminated from the subgroups. W h e n comparisons were made within genders, w o m e n had significantly lower total and L D L cholesterol levels in the E3/2 subgroup as c o m p a r e d to E3/3 and E4/3 phenotypes; the latter subgroups did not differ significantly. In men, E3/2 phenotypes had significantly lower levels of these lipids than E3/3 and E4/3 phenotypes; the E3/3 subgroup was also significantly lower than E4/3 subgroup for these parameters. T h e r e were no significant differences in triglyceride levels. T h e apo E phenotypic and allele frequencies of the male and female populations are shown in Tables 3 and 4. W h e n the distribution for the major apo E phenotypes (E3/2, E3/3 and E4/3) were compared, they were found to be significantly different between the two populations (2a = 6.35,p < 0.05). This difference was also evident when allele frequencies were c o m p a r e d (22 = 6.46,p < 0.05). Closer examination of the apo E frequency distribution indicated that gender differences resided mainly within the E4 isoform: the relative frequency of this allele in the female population was one-half that observed in the male population. T h e relative frequencies of the other apo E mutant, E2, were similar in the two groups. To determine whether these observations reflected inherent differences b e t w e e n Italian male and female populations, apo E polymorphism in non-diabetic control populations was analysed. As shown in Table 4, no significant, gender-based differences in allele frequencies were evident (;(2=0.9). Neither the non-diabetic nor the diabetic m e n demonstrated differences in allele frequencies (see Table 4; Za = 0.9). T h e r e were, however, significant differences (p < 0.05) b e t w e e n female non-diabetic and diabetic subjects. Correspondingly, the combined male/female control population was found to be in HardyWeinberg equilibrium with the male diabetic population, but not with the female diabetic population (p < 0.05). In comparison to other apo E isoforms, it has b e e n reported that the E4 p h e n o t y p e is associated with cardiovascular disease at a significantly y o u n g e r age [ 23, 24 ]. Thus, subjects possessing the E4 isoform m a y potentially be lost earlier from such populations. We analysed w h e t h e r the E4 allele frequency differed b e t w e e n younger ( < 60 years) and older ( > 60 years) diabetic patients. Neither of the male diabetic subgroups showed significant differences (Z2= 1.66 and 0.17, respectively for y o u n g e r (n = 74) and older (n = 137) patients) in allele frequency when c o m p a r e d to non-diabetic control subjects. In contrast, older female patients (n = 1 6 5 ) h a d a significantly different allele distribution from that of female control subjects ( ~ = 6.96,p < 0.05). This was again attributable to a reduced E4 allele frequency (4.8 % vs 11.6 % ). T h e apo E allele frequency of younger diabetic women (n = 60) did not differ significantly from that of control subjects (Z2: 1.94). Discussion This is the first report of a gender difference in the apo E allelic distribution profile of a diabetic population. It is due to a lower E4 allele representation in diabetic women, a group where the natural female resistance to cardiovascular disease is severely compromised. In addition, there may be an age factor, as diabetic women over 60 years of age had a significantly different frequency from nondiabetic control subjects, whilst this was not the case for younger female diabetic patients. In contrast to these observations in diabetic women, apo E phenotype/allele frequencies were not significantly different between male diabetic and control subjects. Although we offer no hard data, it appears logical to link our observations to cardiovascular disease, given the role of apo E in lipid metabolism. As mentioned earlier, apo E4 is associated with pathological modifications of blood lipid levels, notably increases in plasma cholesterol concentrations [ 7-10 ] and deleterious influences on triglyceride and H D L cholesterol levels [25]. This may be reflected in the tendency for E4 subjects to develop coronary heart disease at a younger age, at least in m e n [ 23, 24 ]. Such results have led Davignon et al. [ 7 ] to suggest that age may have a major influence on the impact of apo E on cardiovascular disease. These same authors also concluded that the E4 allele in particular may confer a genetic predisposition to atherosclerotic disease in subjects placed in an unfavourable environment. This eventuality, taken together with our observations that the female diabetic group, particularly older patients, has a reduced E4 allele frequency, raises s o m e interesting questions. Although the underlying mechanisms have yet to be elucidated, it is well d o c u m e n t e d [ 2, 3 ] that diabetes particularly augments the female susceptibility to atherosclerotic disease. Could the diabetic condition p r o v i d e an unfavourable e n v i r o n m e n t wherein the E4 i s o f o r m has a m o r e deleterious influence in female patients? If so, a corollary to this question is why the male diabetic population does not also show a reduced E4 allele representation. C o m parisons of m a l e and female diabetic patients according to p h e n o t y p e offered no indications as to the causes of the gender differences in the E4 allele frequency. Neither cholesterol nor triglycerides were significantly different between patients with the E4/3 p h e n o t y p e (even w h e n patients taking lipid lowering medication were eliminated from the subgroups). O t h e r comparisons, including H b A l c , B M I and duration of diabetes, also failed to reveal differences b e t w e e n m a l e and f e m a l e patients with the E4 isoform. However, it is difficult to extrapolate f r o m such comparisons as they will be influenced by the a p p a r e n t preferential loss of female patients with the E4 isoform. T h e majority of studies p e r f o r m e d to date has failed to reveal gender differences in apo E p o l y m o r p h i s m [ 13, 20, 23, 26, 27 ], although a study in octogenarians suggested under-representation of the E4 isoform notably in w o m e n [ 28 ]. O t h e r reports suggest that apo E isomers have consistent effects on plasma cholesterol levels irrespective of the population source or the average cholesterol concentration of that population [ 7 ]. However, Xhignesse et al. [ 27 ], comparing non-diabetic male and female p o p u l a tions, have concluded that there m a y be gender differences in the degree to which apo E p o l y m o r p h i s m affects plasma lipid levels, the influence of the E4 allele on L D L m e t a b o l i s m being of greater magnitude in women. Also, in a report which a p p e a r e d after completion of the present investigation, L a a k s o et al. [ 29 ] concluded that the apo E4 isoform was an i m p o r t a n t risk indicator for coronary heart disease in non-insulin-dependent diabetes (although they were unable to offer an explanation for the m e c h a n i s m underlying the effect). Unfortunately, the study only considered m a l e patients. In the light of these reports, our observations are consistent with the proposal that the combination of diabetes and the E4 allele could confer a particular disadvantage on the female population. T h e r e is at present no obvious explanation as to why female diabetic patients possessing the E4 allele should be at a disadvantage in comparison to their male diabetic counterparts, but it m a y have a bearing on their greatly increased risk of developing cardiovascular disease, for which an explanation is similarly lacking. T h e small number of studies of a p o E p o l y m o r p h i s m in diabetic p o p u l a tions has revealed no gender differences, although it should be emphasised that females were not specifically targeted in these studies. Further studies are m e r i t e d to determine w h e t h e r our observations can be extrapolated to other populations, or are specific to the Italian p o p u l a tion, and to test our suggestion that the E4 allele m a y be a particular risk factor in the female diabetic population. Acknowledgements. The authors are very grateful for the technical expertise of Ms B.Wojtek, M.-C. Brulhart, F.Ruinard and Dr. R. Ricciotti. The study was supported by grants from the Fonds National de la Recherche Scientifique (No. 32-30782.91) and from the Fondation suisse du Diabbte. 1. PyOrfil~iK, Laakso M , Uusitupa M ( 1987 ) Diabetes and atherosclerosis: an epidemiological view . Diab Metab Rev 3 : 463 - 524 2. Barrett-Connor E , Wingard DL ( 1983 ) Sex differential in ischaemic heart disease mortality in diabetics: a prospective population-based study . Am J Epidemio1118: 489 - 496 3. Krowlewski AS , Warram JH , Valsania R Martin BC , Laffel LMB , Christlieb AR ( 1991 ) Evolving natural history of coronary artery disease in diabetes mellitus . Am J Med 90 [Suppl 2A]: 56S-61S 4. Barrett-Connor E , Grundy S , Holdbrook MJ ( 1982 ) Plasma lipids and diabetes mellitus in an adult community . Am J Epidemio1115: 65 % 663 5. Assmann G , Schulte H ( 1987 ) The prospective cardiovascular Mt~nster (PROCAM) study: prevalence of hyperlipidemia in persons with hypertension and/or diabetes mellitus and the relationship to coronary heart disease . Am Heart J 116 : 1713 - 1724 6. Stern MR Patterson JK , Haffner SM , Hazuda HR Mitchell BD ( 1989 ) Lack of awareness and treatment of hyperlipidemia in type II diabetes in a community survey . JAMA 262 : 360 - 364 7. Davignon J , Gregg RE , Sing CF ( 1988 ) Apolipoprotein E polymorphism and atherosclerosis . Arteriosclerosis 8 : 1 - 21 8. Utermann G ( 1987 ) Apolipoprotein E polymorphism in health and disease . Am Heart J 113 : 433 - 440 9. Mahley RW ( 1988 ) Apolipoprotein E: cholesterol transport protein with expanding role in cell biology . Science 240 : 622 - 630 10. Breslow JL ( 1985 ) Human apolipoprotein molecular biology and genetic variation . Annu Rev Biochem 54 : 699 - 727 11. Zannis VI , Breslow JL , Utermann G e t al. ( 1982 ) Proposed nomenclature of apo E isoproteins, apo E genotypes and phenotypes . J Lipid Res 23 : 911 - 914 12. Brewer HB , Zech LA , Gregg RE , Schwartz D , Schaefer EJ ( 1983 ) Type III hyperlipoproteinemia: diagnosis, molecular defects, pathology and treatment . Ann Int Med 98 : 623 ~ 540 13. Boerwinkle E , Visvikis S , Welsh D , Steinmetz MS , Hanash SM , Sing CF ( 1987 ) The use of measured genotype information in the analysis of quantitative phenotypes in man: II. The role of the apolipoprotein E polymorphism in determining levels, variability and co-variability of cholesterol, beta-lipoprotein and triglycerides in a sample of unrelated individuals . Am J Med Genet 27 : 567 - 582 14. Boerwinkle E , Utermann G ( 1987 ) Simultaneous effects of the apolipoprotein E polymorphism on apolipoprotein E, apolipoprotein B and cholesterol metabolism . Am J Hum Genet 42 : 104 - 112 15. Eto E , Watanabe K , Iwashima Y , Morikawa A et al. ( 1986 ) Apolipoprotein E polymorphism and hyperlipemia in type II diabetics . Diabetes 35 : 1374 - 1382 16. James RW , Voliotis C , Grab B , Pometta D ( 1987 ) Phdnotypes de I'apoprot6ine E (apo E) et lipides s6riques des diabetiques . Schweiz Med Wschr t17: 2021 - 2023 17. Imari Y , Koga S , Ibayashi H ( 1988 ) Phenotype of apolipoprotein E and abnormalities in lipid metabolism in patients with non-insulin dependent diabetes mellitus . Metabolism 37 : 1134 - 1138 18. Winocour PH , Tetlow L , Durrington PN , Ishola M , Hillier V , Anderson VD ( 1989 ) Apolipoprotein E polymorphism in insulin treated diabetes mellitus . Atherosclerosis 75 : 167 - 173 19. Shriver MD , Boerwinkle E , Hewett-Emmett D , Hanis CL ( 1991 ) Frequency and effects of apolipoprotein E polymorphism in Mexican-American NIDDM subjects . Diabetes 40 : 334 - 337 20. Menzel HJ , Kladetzky RG , Assmann G ( 1983 ) Apolipoprotein E polymorphism and coronary artery disease . Arteriosclerosis3 : 310 - 315 21. Borghini I , James RW , Pometta D ( 1989 ) Maladie des remnants (remnants disease) associ6e fi l'apoprot6ine El; importance clinique des ph6notypes de l'apoprot6ine E (apo E) . Schweiz Med Wschr 119 : 1821 - 1824 22. James RW , Pometta D ( 1990 ) Differences in lipoprotein subfraction composition and distribution between type I diabetic men and control subjects . Diabetes 39 : 1158 - 1164 23. Cumming AM , Robertson F ( 1984 ) Polymorphism at the apo E locus in relation to the risk of coronary disease . Clin Genet 25 : 310 - 313 24. Lenzen H J , Assmann G , Buchwalsky R , Schulte H ( 1986 ) Association of apolipoprotein E polymorphism, low density lipoprotein cholesterol and coronary artery disease . Clin Chem 32 : 778 - 781 25. DallongevilteJ, Lussier-- Cacan S , Davignon J ( 1992 ) Modulation of plasma triglyceride levels by apo E phenotype: a meta-analysis . J Lipid Res 33 : 447 - 454 26. Ordovas JM , Litwack-Klein L , Wilson PWF , Schaefer MM , Schaefer EJ ( 1987 ) Apolipoprotein E isoform phenotyping methodology and population frequency with identification of apo E1 and apo E5 isoforms . J Lipid Res 28 : 371 - 380 27. Xhignesse M , Lussier-Cacan S , Sing CE Kessling AM , D avignon J ( 1991 ) Influences of common variants of apolipoprotein E on measures of lipid metabolism in a sample selected for health . Arterioscler and Thrombosis 11 : 1100 - 1109 28. Davignon J , Sing CF , Lussier-Cacan S , Nestruck AC , Bouthillier D ( 1986 ) Importance of apolipoprotein E polymorphism in determining plasma lipid levels and atherosclerosis . In: Fidge NH , Nestel PJ (eds) Atherosclerosis VII. Excerpta Medica , Amsterdam, pp 171 - 175 29. Laakso M , Kes/iniemi A, Kervinen K , Jauhiainen M , Py6r /~l~iK ( 1991 ) Relation of coronary heart disease and apolipoprotein E phenotype in patients with non-insulin dependent diabetes . Br Med J 303 : 1159 - 1162 Received: 24 June 1992 and in revised form: 13 October 1992


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M. Boemi, R. W. James, F. Romagnoli, P. Gerber, D. Pometta, P. Fumelli. Gender differences in a Type 2 (non-insulin-dependent) diabetic population with respect to apolipoprotein E phenotype frequencies, Diabetologia, 1993, 229-233, DOI: 10.1007/BF00399955