Patent Application
20080131879
This invention is in the field of coronary heart disease (“CHD”) risk detection and provides certain nucleic acid sequences encoding sterol transporters ABCG8 to identify subjects that are responsive for plant sterol modulating interventions, creating the possibility for individualized (drug) treatment.
Plant sterols have been postulated, like cholesterol, as a risk factor for coronary heart disease. The two most abundant plant sterols in nature are campesterol and sitosterol, which are both chemically closely related to cholesterol. Despite the fact that Western diets provide approximately the same amounts of plant sterols (160-360 mg/day) and cholesterol (300 mg/day), plasma concentrations of cholesterol are much higher. This is partly due to the very low absorption of plant sterols, which amounts for campesterol and sitosterol to 1.89 and 0.51%, respectively (1). In contrast, cholesterol absorption varies between 30 and 80% (2).
Two recently discovered ATP binding cassette (ABC) transporters—ABCG5 and ABCG8—play an important role in the regulation of intestinal plant sterol absorption by secreting already absorbed plant sterols out of the enterocytes back into the intestinal lumen (3). ABCG5 and G8 are half-transporters that function together as a heterodimer. Formation of a heterodimer is an absolute necessity to direct the ABCG5/G8 heterodimer from the endoplasmatic reticulum (ER) to the apical membrane (4). This feature explains earlier observations that mutations in only one of the half transporters already causes the rare inheritable autosomal recessive disease sitosterolemia (5-7).
Sitosterolemic patients are characterized by severely elevated serum plant sterol concentrations, normal to moderately increased serum cholesterol concentrations and a high risk to develop coronary heart disease already at a very young age (8, 9). Polymorphisms in the genes encoding ABCG5 and ABCG8 may therefore be related to differences in plant sterol metabolism between subjects. Although not generally accepted, several studies have suggested that elevated concentrations of plant sterols are a risk factor for premature atherosclerosis in sitosterolemic patients (10-12) and even in non-sitosterolemic subjects (13, 14).
Besides the various rare mutations in ABCG5 or ABCG8 as observed in sitosterolemic patients (15), more common sequence variations in both half-transporters—without the sitosterolemic phenotype—have been described. These polymorphisms in ABCG5 and ABCG8 are related to serum plant sterol concentrations (16).
There is, therefore, a need to further investigate whether the above-mentioned genetic variations are also associated to variations in serum plant sterol concentrations during interventions known to affect serum plant sterol metabolism, such as, for example, plant stanol esters which are known for their plant sterol-lowering properties.
In accordance with the present invention at least one of the genotypes selected from the group consisting of ABCG8 T400K and ABCG8 D19H is provided as a diagnostic tool for the detection of individuals at risk for CHD by a genotype evaluation based on an elevated sitosterol concentration in blood samples of said individuals.
These and other aspects of the present invention will be described hereinafter in more detail.
ABCG5 and ABCG8 play an important role in regulating intestinal plant sterol absorption, and therefore polymorphisms in these genes may be related to differences in plant sterol metabolism between subjects.
Surprisingly, it has now been found that in a number of 112 non-hypercholesterolemic healthy subjects cross-sectional cholesterol-standardized serum campesterol and sitosterol concentrations were significantly associated with ABCG8 T400K genotype. In addition, interactions between the ABCG8 T400K genotype with changes in serum plant sterol concentrations were shown after plant stanol consumption, which lower plant sterol levels. However, no significant associations with serum cholesterol concentrations, neither cross-sectional nor after plant stanol intervention, were found.
For the first time interactions between the ABCG8 T400K genotype with changes in serum plant sterol concentrations were evaluated after a daily consumption of 3.8-4.0 gram plant stanols, which lower plant sterol levels. The reduction of −57.1±38.3 102 μmol/mmol cholesterol for sitosterol in TT subjects was significantly greater as compared to that of −36.0±18.7 in subjects with the TK genotype (P=0.021; 95% CI −39.1 to −3.1 102×μmol/mmol cholesterol) and to that of −16.9±13.0 in subjects with the KK genotype (P=0.047; 95% CI −85.2 to 4.8 102×μmol/mmol cholesterol).
Therefore, genetic variation in ABCG8 not only explains cross-sectional differences in serum plant sterol concentrations, but also determines a subjects' responsiveness to changes in serum plant sterols during interventions known to affect plant sterol metabolism. These transporters are not related to changes in serum cholesterol concentrations suggesting that they are primarily involved in plant sterol metabolism and not in cholesterol metabolism.
Serum plant sterol concentrations vary widely between individuals (14, 25), but 16 are within subjects rather stable over time (16, 26). This suggests a strong effect of genetic background on plant sterol metabolism. Indeed, heritability may account for more than 80% of the variability in serum plant sterol concentrations between individuals (16).
It has now been found in accordance with the present invention that genetic variation in ABCG5 and ABCG8 not only explains cross-sectional serum plant sterol concentrations, but in addition that subjects with high plant sterol concentrations (ABCG8 exon 8, TT genotype) had the largest reduction in serum plant sterol concentrations during plant stanol ester treatment. In particular ABCG8 T400K showed an allele dose dependent relation. See also
These findings indicate that the functionality of the ABCG5/G8 heterodimer is reduced in subjects with the ABCG8 TT genotype. Although the inventors do not wish to be bound to any theory, it is believed that this may result in a lowered transport of plant sterols out of the enterocytes back into the intestinal lumen or from the hepatocytes into bile, and ultimately in increased serum plant sterol concentrations. Why these subjects also showed the largest decrease in serum plant sterol concentrations after consumption of plant stanols is more difficult to explain. It is known that serum plant sterol concentrations are lowered by consumption of plant stanol esters (27-29), probably since plant stanols compete with sterols for incorporation into mixed micelles and hence for uptake into the enterocytes. As plant sterol uptake is not mediated by ABCG5/G8, the lower flux of plant sterols into the enterocyte during plant stanol ester consumption does not depend on ABCG8 genotype. However, if ABCG8 transports the same proportion of plant sterols out of the enterocytes and hepatocytes into the lumen and bile respectively, as before plant stanol ester consumption, it can be calculated that the circulating concentrations of plant sterols in subjects with the TT genotype (with a lower ABCG8 activity) may decrease to a greater degree than in other subjects.
Concerning the potential functionality of the different genotypes it should be remarked that the polymorphic sites of the ABCG8 T400K and D19H polymorphisms are located in a coding region, but are predicted not to contain a transmembrane domain, a signature, or a walker motif (15).
As none of the polymorphisms showed any relationship with (changes in) serum lipid or lipoprotein concentrations, the data according to the present invention do suggest that ABCG8 and ABCG5 do not determine the serum lipoprotein profile. This agrees with observations in mice that disruption of the ABCG8 and ABCG5 genes increased the fractional absorption of dietary plant sterols, but not of cholesterol (3, 18, 19). As a result, serum and hepatic plant sterol concentrations were dramatically increased in ABCG5(−/−)/G8 (−/−) (3, 18) and ABCG5 (−/−) (19) mice, whereas serum cholesterol concentrations were not changed. Cholesterol concentrations in the liver were however increased, which was due to a disturbed biliary cholesterol secretion. Unfortunately, biliary plant sterol secretion was not measured in that study, but in view of the increased hepatic plant sterols levels, it is likely that biliary plant sterol secretion was disturbed as well. In line with these findings, Yu et al (17) reported that over-expression of ABCG8 and ABCG5 significantly lowered serum plant sterol concentrations, whereas serum cholesterol concentrations were again not different between transgenic and wild-type mice. In contrast to what could be expected from the studies with the ABCG5 (−/−)/G8 (−/−) mice, it was measured that these transgenic mice showed a 50% reduction in dietary cholesterol absorption. As discussed however by the authors, measurements of dietary cholesterol absorption were not accurate due to an increase in the intestinal cholesterol pool by the large amount of biliary cholesterol entering the intestinal lumen. In addition, biliary cholesterol levels increased five to seven-fold, while hepatic cholesterol concentration remained unchanged. The unchanged hepatic and serum cholesterol concentrations could be explained by a counteractive two to four-fold increase in hepatic cholesterol synthesis.
Therefore, results in ABCG5/G8 knock-out and over-expressing mice showed that ABCG5 and G8 affects serum plant sterol concentrations, but not those of cholesterol. In addition it seems that ABCG8 and ABCG5 play a role in the intestinal absorption of plant sterols, whereas hepatic ABCG8 and ABCG5 are involved in excretion of cholesterol into bile and possibly also of plant sterols.
Weggemans et al (30) found that subjects with the ABCG5 Q604E EE genotype had higher serum cholesterol concentrations than carriers with the Q allele. Responses to dietary treatments were however not related to this genotype. Other studies however did not observe any associations between these polymorphisms with serum lipid concentrations (16, 31), while we found a significantly higher serum LDL cholesterol concentration in subjects with the QQ genotype as compared to carriers of the E allele. In a recent study, a statistically significant association between the ABCG8 D19H polymorphism and the proportional reduction in LDL cholesterol during atorvastatin treatment was observed (31). As statin treatment may increase plasma plant sterol concentrations (10-12), it would have been interesting to know if changes in serum plant sterol concentrations were also related to this polymorphism in the ABCG8 gene. In relation to this, Gylling and coworkers (33) recently showed that the higher efficacy of atorvaststin treatment in DH/HH subjects might be related to a higher endogenous cholesterol synthesis and a lower cholesterol absorption as compared to DD subjects. Taken together, these studies have not revealed a consistent relationship between ABCG5 or ABCG8 genotypes with serum lipid or lipoprotein concentrations.
The data according to the present invention indicate that ABCG8 genotype can predict to what extent serum plant sterol concentrations change after interventions. Plasma plant sterol concentrations were found to decrease, though increases in cholesterol-standardized serum plant sterol concentrations are possible after treatment with statins (10) and consumption of functional foods enriched with plant sterol esters (27, 32).
Whether these increases also relate to ABCG8 genotype may be relevant to know. In the Scandinavian Simvastatin Survival Study (4S) study, for example, it was found that changes in plasma plant sterol concentrations after 5-year treatment were positively related to baseline values. More importantly, however, recurrence of coronary events during simvastatin treatment was not reduced in subjects from the highest quartile of plasma sterol concentrations at baseline, despite the fact that reductions in serum total cholesterol concentrations were comparable in all quartiles (10, 12). These observations do of course not prove that increased plant sterol concentrations counteract the beneficial effects of statins on cardiovascular risk.
In conclusion, serum cholesterol-standardized campesterol and sitosterol concentrations, as well as their changes after consumption of plant stanol enriched foods, are related to a variation in the ABCG8 gene, which is present in about 70% of the population. No relations with serum lipid and lipoprotein concentrations were observed. This suggests that changes in the functionality of the ABCG5/G8 heterodimer, mainly affects plasma sterol concentrations, but not those of cholesterol. Whether these findings should have consequences for a patients' optimal cholesterol-lowering drug treatment, warrants further investigation.
Evaluation of ABCG8 and ABCG5 nucleic acid sequences, such as for example the ABCG8 T400K and D19H polymorphisms can be used as a biomarker for the detection of future coronary events. This predictive characteristic originates from its association with cholesterol-standardized serum plant sterol concentrations. In particular, the genotypes ABCG8 T400K (“TT”) and ABCG8 D19H (“DD”) show a strong cross-sectional association with cholesterol-standardized serum plant sterol concentrations and in addition TT subjects show the largest responsiveness towards plant sterol modulating interventions. This enlarged responsiveness has not yet been shown for DD subjects but is likely to be present. Therefore, knowledge regarding a subjects ABCG8 T400K and probably also someone's ABCG8 D19H genotype can be used to predict which subjects will respond with a large increase in serum plant sterols upon treatment using strategies known to elevate cholesterol-standardized serum plant sterol concentrations.
An example of such a therapeutic strategy is the treatment of hyper-cholesterolemic patients with HMGCoA reductase inhibitors (i.e. statins), plant sterol enriched functional foods, or a combination of both. Before starting these types of treatments it would be of great importance to determine a subject's ABCG8 T400K or D19H genotype since in particular TT and DD subjects may respond with a strong increase in cholesterol-standardized serum plant sterol concentrations. Alternatively these patients should be treated with serum cholesterol lowering drugs or other interventions from which it is known that they do not elevate serum plant sterol concentrations. Therefore evaluation of a subjects ABCG8 and ABCG5 genetic make up can be used as a diagnostic tool in the prediction of future coronary events, particularly when optimal drug treatment is considered.
Ultimate prove concerning the true predictive value of these new biomarkers can be obtained from large prospective intervention trials in which a large group of patients are followed for 5 to 10 years (while registrating characteristics of morbidity and mortality) during treatment with placebo or statins (known to elevate cholesterol-standardized serum plant sterol concentrations). After ending the follow-up period ABCG5 and ABCG8 nucleic acid sequences should be determined and related to morbidity and mortality rates in the placebo and statin treated groups. In case there is a clear association between mortality rates and for example ABCG8 T400K TT and/or D19H DD genotype, this is highly indicative that our assumption regarding the predictive value of this genotype is correct. In addition, evaluation of changes in serum plant sterol concentrations during the statin treatment period can be supportive. In the past numerous of these trials have been carried out. One example is the famous 4S trial in which 4444 hypercholesterolemic patients with coronary heart disease were treated with simvastatin for a period of 5.4 years, simvastatin was found to significantly lower morbidity and mortality rates. Relative risk of death in the simvastain group was 0.70 and relative risk of coronary death was 0.58. Although the number of coronary events were lower in the simvastatin group (n=431) as compared to the placebo group (n=622) resulting in a 0.66 relative risk, there were still 431 coronary events and 111 coronary deaths in the simvastatin group. In 2000 regarding the Finnish subcohort of the 4S trial (8868 patients), Miettinen and coworkers (10) reported that the difference between statin treated patients that did not survive 5 years follow up on statin treatment showed a significant higher increase in serum campesterol concentrations as compared to the patients that survived the 5 years follow-up period on statin treatment (Miettinen ATVB 2000). Since the reduction in serum LDL cholesterol was similar in both groups, it was concluded that the elevation in serum campesterol was one of the possible explanations for the observed difference in survival. One of the opportunities to prove the value of our new biomarker will be evaluation of ABCG8 nucleic acid sequences in this 4S cohort and show that the died patients were more often carriers of the TT and/or DD genotype as compared to the surviving patients. Besides the 4S trial, there are many potentially interesting statin trials—with a similar design however another statin—from which evaluation of ABCG8 nucleic acid sequences in relation to mobidity and mortality rates could be confirmative.
Analysis of known ABCG5 and ABCG8 SNPs, for example ABCG8 T400K or D19H, can be carried out, for example, on a Taqman realtime PCR using allelic discrimination methodology and corresponding software as developed by Applied Biosystems. By using this approach we are able to determine for example ABCG8 T400K genotype of approximately 300 patients a day.
The following Examples illustrate the invention and are in no way intended to limit the scope of the invention in any respect.
Details of the study have been described before (20, 21). In brief, 112 healthy non-hypercholesterolemic volunteers (41 males and 71 females) from Maastricht and surrounding areas were asked to replace during a four-week run-in period their habitual margarines and baking fats for a low erucic acid rapeseed oil (LEAR) based margarine and shortening. For the next eight weeks subjects were randomly allocated, stratified for gender and age, to one of the three intervention groups. The control group (N=42) continued to use the rapeseed-oil based margarine and shortening, while the second and the third group used the same margarine and shortening to which a vegetable-oil based (N=36) or a wood-based (N=34) plant stanol ester mixture was added. The compositions of the experimental products have been described in detail before (22). The margarine was used at breakfast and lunch, and the shortening at diner. During the intervention period, daily intake of plant stanols in the vegetable-oil based group was 3.8±0.6 g (means ±SD) and in the wood-based group 4.0±1.8 g. Plant stanols were esterified with fatty acids from rapeseed oil. All experimental products were prepared by the RAISIO GROUP, Raisio, Finland. Energy intake and the proportions of energy from carbohydrates, fatty acids, and protein, as well as cholesterol and fibre intake did not change during the study (20).
At the start of the study (day 1) blood was sampled in a 10 ml EDTA tube (Monoject sterile, Sherwood Medical, Ballymoney, North Ireland), which was used for DNA isolation. At the end of the run-in period (weeks 3 and 4) and at the end of the experimental period (weeks 11 and 12), blood was sampled in a 10 mL clotting tube (CORVAC, integrated serum separator tube, Sherwood Medical Company, St Louis USA) after an overnight fast. Subjects abstained from drinking alcohol the day preceeding, and from smoking on the morning of blood sampling. Serum was prepared by centrifugation at 2000×g for thirty minutes at 4° C., minimally one hour after venipuncture, and aliquots were stored directly at −80° C. for analysis of serum lipids, lipoproteins, plant sterols, plant stanols and cholesterol precursor concentrations at the end of the study (20, 21).
Genotyping of exons 2, 8, and 13 from ABCG8 and of exon 13 from ABCG5 was performed by analysis of restriction fragment length polymorphisms (RFLPs), as described (15). In brief, PCR amplifications were performed in 20 μL volumes containing 350 ng genomic DNA, 25 pmol of each nucleotide primer (Sigma Genosys, Cambridge, UK), 1 unit Taq polymerase (Pharmacia Biotech, Roosendaal, The Netherlands), 0.2 mM of each dNTP (Pharmacia Biotech, Roosendaal, The Netherlands), and 1.5 mM MgCl2. Before amplification, each sample was denaturated for 5 minutes at 95° C. For ABCG8 genotyping each of the following thirty cycles consisted of 15 seconds at 96° C. (denaturation), 15 seconds at 60° C. (primer annealing), and 30 seconds at 72° C. (extension), followed by 10 minutes at 72° C. (elongation). For ABCG5 genotyping the program was slightly modified in that annealing occurred for 30 seconds at 60° C. and no elongation step was used. PCR products were digested with specific restriction enzymes and the DNA fragments obtained were electrophoresed for 1.5 hours at 125V on a 2.5% agarose gel containing gelstar (Sanvertech, Heerhugowaard, The Netherlands). DNA fragments were visualized by UV at 312 nm using a Wratten gelatine filter on a VDS Imagemaster (Pharmacia Biotech, San Francisco, USA). Sequences of the primers have been described (15). The restriction enzymes (New England Biolabs, Beverly Mass., USA) used were SexAI, MseI, and NcoI for ABCG8 exon 2, 8, and 13 respectively, and XmnI for ABCG5 exon 13.
Due to the limited number of subjects, heterozygous and homozygous carriers of the genetic variants (ABCG8 Y54C; [YC+YY], ABCG8 T400K; [TK+KK], ABCG8 A632V; [VV+VA], ABCG5 Q604E; [QE+EE]) were combined before data analysis. Excluding the homozygous carriers did, however, not affect the conclusions.
At the end of the run-in period cross-sectional differences in metabolic parameters between genotype groups were compared with an unpaired t-test. When significant, the three genotype groups were also compared using ANOVA with Bonferroni correction to examine if relations were dose allele-dependent. In view of their well-known relationships with lipid metabolism, BMI, gender and age were considered as potential confounders. However, these parameters were not significantly different between the various genotype groups (results not shown) and were therefore not included into the models.
In search for gene-diet interactions, changes in metabolic parameters were calculated for each subject as differences between values of the experimental period and run-in period. Since responses in serum lipoprotein, plant sterol and stanol concentrations were not different between the two experimental groups (20, 21) results of the vegetable-oil based and the wood-based plant stanol esters groups were combined (N=70). Effects of genotype on these responses were examined with an unpaired t-test. Again, allele dose-dependent relationships were examined by ANOVA plus Bonferroni correction. All statistical analyses were performed with Statview 4.5 (24).
Frequency distributions for the genotypes in exons 2 (Y54C), 8 (T400K), and 13 (A632V) of ABCG8 and in exon 13 (Q604E) of ABCG5 are shown in Table 1.
Except for ABCG8 Y54C, all genotype frequency distributions were in Hardy-Weinberg equilibrium. The various polymorphisms were not related. At the end of the four-week run-in period, serum concentrations of LDL cholesterol, HDL cholesterol, and triacylglycerol in all subjects (N=112) were 2.95±0.78, 1.59±0.38, and 0.92±0.52 mmol/L, respectively. Serum concentrations of plant sterols, plant stanols, and lathosterol at the end of the run-in period are shown in Table 2.
93.2 ± 35.0c
aABCG8 Y54C: CC versus YC/YY subjects (p < 0.01)
bABCG8 T400K: TT versus TK/KK subjects (p < 0.001)
cABCG8 T400K: TT versus TK/KK subjects (p = 0.053)
Both ABCG8 Y54C and ABCG8 T400K polymorphisms were significantly related with cholesterol-standardized serum campesterol concentrations, while cholesterol-standardized serum sitosterol concentrations were only associated with ABCG8 T400K genotype (Table 2). The relationship between the ABCG8 T400K polymorphism with cholesterol-standardized serum campesterol or sitosterol concentrations was allele-dependent (
3.04 ± 0.75a
aABCG5 Q604E: QQ versus QE/EE subjects (p < 0.05)
In the 70 subjects that consumed the plant stanol ester enriched margarines, cholesterol-standardized serum campesterol and sitosterol concentrations decreased, while those of lathosterol increased as compared to the control group (Table 4).
aABCG8 T400K, TT versus TK/KK subjects (P < 0.05)
bABCG8 T400K, TT versus TK/KK subjects (P < 0.01)
These changes indicated that plant stanol ester consumption lowered cholesterol absorption and increased cholesterol synthesis, which is in agreement with the known underlying mechanism. As a consequence LDL cholesterol concentrations were significantly lowered. HDL and triacylglycerol concentrations were not affected (Table 5).
Changes in cholesterol-standardized serum campesterol and sitosterol concentrations were significantly associated with the ABCG8 T400K polymorphism (table 4,
Finally, results indicate that subjects with the genotype associated with high cross-sectional serum plant sterol concentrations and a large reduction in serum plant sterol concentrations upon plant stanol ester intervention (ABCG8 TT and ABCG5 EE) are characterized by a higher increase in cholesterol standardized plant stanol concentrations during plant stanol ester enriched margarine consumption.
Each of the above cited publications is herein incorporated by reference to the extent to which it is relied on herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 04076589.3 | May 2004 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP05/05851 | 5/30/2005 | WO | 00 | 9/18/2007 |
