WO1999055909A1

WO1999055909A1 - A FREQUENT MUTATION IN THE CYSTATHIONINE β-SYNTHASE GENE INCREASES RISK FOR CORONARY ARTERY DISEASE

Info

Publication number: WO1999055909A1
Application number: PCT/NL1998/000234
Authority: WO
Inventors: Rendrik Franca Franco; Maria Dorothea Trip; Pieter Hendrik Reitsma
Original assignee: Amsterdam Molecular Therapeutics AMT BV
Current assignee: Amsterdam Molecular Therapeutics AMT BV
Priority date: 1998-04-27
Filing date: 1998-04-27
Publication date: 1999-11-04
Anticipated expiration: 2000-10-27
Also published as: AU7350598A

Abstract

A novel 844ins68 mutation in the cystathionine β-synthase (CBS) gene as a candidate risk factor for cardiovascular disease (CVD). In addition, we measured homocysteine levels, both basal and post-methionine loading (PM), and levels of plasma folate, vitamins B6 and B12 in patients only. For heterozygous and homozygous carriers combined, the odds ratio (O.R.) for CVD was 1.7 (95 % CI 1.1-2.7). The O.R. for heterozygous carriers was 1.5 (95 % CI 0.9-2.3), whereas for homozygotes it accrued to 14.0 (95 % CI 1.7-115). Both fasting and PM homocysteine and vitamin B6 and folate levels did not differ between carriers and non-carriers of the mutation. These results show that a common insertion in the CBS gene represents a novel risk factor for premature CVD, and exhibits a strong gene dosage effect. In addition elevated fasting or PM homocysteine levels are shown not to be a feature of this CBS insertion.

Description

A FREQUENT MUTATION IN THE CYSTATHIONINE β-SYNTHASE GENE INCREASES RISK FOR CORONARY ARTERY DISEASE

INTRODUCTION

Cystathionine β-synthase (CBS) is a critical enzyme in the transsulfuration pathway of homocysteine metabolism catalysing the conversion of homocysteine into cystathionine, with pyridoxine (vitamin B6) as a cofactor (1). Numerous molecular defects in the CBS gene have been shown to result in CBS enzyme deficiency and hyperhomocysteinemia, a risk factor for venous and arterial thrombotic disease (1-7). Classical homocystinuria, caused by homozygous CBS deficiency, is characterised by premature atherosclerotic vascular disease and venous thromboembolic events (7). To date, 29 different CBS mutations have been identified in homocystinuric patients (7).

Recently, a novel mutation in the CBS gene (844ins68; a 68-bp insertion in exon 8 of the coding region of the gene) was reported to occur with a high carrier frequency in the general population (8, 9, 19 ). This frequent mutation, when homozygous, does not however appear to result in severe enzyme deficiency or homocystinuria. Thus it also does not appear to be linked with a risk for CVD.

Reference 8 published in 1996 specifically states the 844ins68 mutation was found in homocystinuria patients and in non patients. In the latter they only found the heterozygous form and it was always associated with the T833C mutation on the same allele. c-DNA reverse transcriptase PCR on fibroblast cytoplasmic RNA by use of primers from exon 7 and exon 9 did not retain the insertion however using primers from the beginning of exon 8 and the same exon 9 primer on nuclear cDNA did. Sperandeo et al report there is no data on the final effect the 844ins68 mutation has on the CBS protein amount and on the homocysteine metabolism. They speculate that the combined homozygosity of the C677T mutation of 5, 10-methylenetetrahydro folate reductase and the heterozygosity for the 844ins68 might hamper the homocysteine metabolism. The link between homozygosity for the MTHFR mutation having been made previously (14) as has the link between homocysteinemia (3) and vascular disease.

Reference 9 published in 1996 specifically states the presence of the 68base pair insertion is not associated with hyperhomocysteinemia. Assays of CBS activity in transformed lymphocytes from individuals who were homozygous or heterozygous for this mutation showed normal activity. Lack of post methionine load hyperhomocysteinemia in individuals with the insertion sugests the insertion does not introduce a premature termination codon leading to a non functional CBS protein. Furthermore reverse transcription PCR showed the individuals carrying this mutation to have normal sized mRNA thus the mutation appears to generate both quantitatively normal and qualitatively normal mRNA and CBS enzyme. Furthermore the prevalence of the mutation in patients with premature coronary artery disease is disclosed as not being statistically significant. In addition reference 9 discloses that individuals carrying the insertion mutation also carry a T->C mutation at nucleotide 833 on the same allele that includes the 68 base pair insertion. The authors of reference 9 conclude the insertion is most likely a benign mutation for which the biological significance if any is unknown.

Reference 19 published in 1997 also specifically states "this common 844ins68 variant is a neutral insertion variant".

Description of the invention

Unexpectedly it has now been determined there is a relationship between this CBS insertion variant and cardiovascular disease (CVD) notwithstanding the absence of homocysteinuria and the statements of others skilled in the art regarding the irrelevance of the mutation.The 833 T -> C mutation is not relevant to the risk factor. The risk factor is present both in absence and presence of the 833T -> C mutation.

Our results show that the 844ins68 mutation is indeed a common risk factor for arterial disease. In addition it was ascertained that although it exhibits a gene dosage effect for CVD risk, both fasting and PM homocysteine levels are not elevated in the presence of this mutation. Apart from assessing that the 68-bp insertion in the CBS gene is a risk factor for premature CVD it has also now been ascertained that homozygous carriers have a particularly high risk of arterial disease. In fact this risk is 5-10 times above the already 50% increased risk of heterozygous individuals.

In addition the risk associated with this CBS insertion depends also on other factors. In particular dyslipidemia, suggestive of gene-gene or gene-environment interaction has been found to increase the risk. Surprisingly in view of the increased CVD risk determined the homocysteine levels (both fasting PM) were similar in carriers and non-carriers. This indicates that the CBS 844ins68 gene does not underly hyperhomocysteinemia, which is in agreement with the previous results of in vitro assays of enzyme activity (9 and 19). The latter results do not for certain entail that the insertion does not influence the homocysteine pathway. Contradictory messenger RNA data (8) suggested that the allele carrying the insertion is poorly transcribed and therefore subtle abnormalities in the methionine pathway could be present that escape our laboratory methods for detection.

Both cohorts used in the Examples of the subject description were previously analysed for the 677 C— »T mutation in the methylenetetrahydrofolatereductase (MTHFR) gene, which is associated with higher plasma homocysteine levels (13). Indeed, a significant relationship between MTHFR genotype and homocysteine or vitamin levels was found, in concordance with previous studies (14, 15). Notwithstanding this however the MTHFR mutation did not increase the odds ratio for premature CVD, again in concordance with previous reports (16, 17, 18). This leaves us with the paradoxical situation that a common CBS variant, not associated with plasma homocysteine levels, is a risk factor for premature CVD, while a MTHFR mutation that is associated with elevated homocysteine levels does not confer a risk for atherosclerosis. We currently have no good explanation for these observations.

In conclusion, these data represent the first extensive analysis of the 844ins68 mutation in the CBS gene in patients with premature CVD, and its relationship with homocysteine and vitamin levels. Although not associated with increased homocysteine levels, the CBS insertion increased the odds for CVD relative to controls, indicating that this mutation may represent a novel genetic risk factor for cardiovascular disease.

The invention will be further illustrated and elucidated in the Examples. Thus, the invention covers a method of diagnosing increased risk of arterosclerosis comprising ascertaining whether an individual carries the 68 base pair insertion mutation at nucleotide 844 of the cystathionine β-synthase gene in a manner known per se for ascertaining the presence of a mutation in the genome of an individual, wherein the presence of the mutation indicates an increased risk. Upon further differentiating whether the individual is homozygotic or heterozygotic for the mutation the risk can be further precicised. The homozygotic individual for the insertion mutation exhibits an even more increased risk. The risk can be assessed even better when the individual is tested also for further risk factors of coronary disease. Such further risk factors can be the presence of LDL level above 4,5, the presence of HDL level below 0,9 and the presence of triglycerides at a level above 2,3 in serum, another contributory risk factor is the exhibition of diabetes mellitus. Notwithstanding the fact that an individual does not exhibit homocysteinemia that individual can exhibit increased risk of CVD, thus the method according to the invention can lead to earlier diagnosis of risk i.e. before clinical symptoms arise. Also the existing methods for determining deficient CBS have illustrated that the individuals exhibiting increased risk of CVD do not have to exhibit deficient CBS as determined in a CBS activity assay to have an increased risk of CVD if they possess the insertion mutation. The invention not only comprises the diagnosis method but also a kit for diagnosing increased risk of arterosclerosis with which the method according to the invention can be carried out. Such a kit in a suitable embodiment comprises a nucleic acid sequence capable of discerning whether a sample comprises the 68 base pair insertion mutation at nucleotide 844 of the cystathionine β-synthase gene in a manner known per se for ascertaining the presence of a mutation in genomic nucleic acid, wherein the presence of the mutation indicates an increased risk. The kit can also comprise instructions or a brochure illustrating the relevant sequence details and/or the risk details and instructions for applying the kit. The nucleic acid sequence in the kit can suitably be a nucleic acid probe specific for the insertion. Alternatively the kit comprises at least two nucleic acid sequences capable of serving as nucleic acid primers for a fragment of nucleic acid surrounding the 844 nucleotide of the cystathionine β-synthase gene. It can also comprise both a nucleic acid probe specific for the insertion and at least two nucleic acid sequences as nucleic acid primers for a fragment of nucleic acid surrounding the 844 nucleotide of the cystathionine β-synthase gene. The length and location of the sequences to be used as probes or primers will be apparent to the person skilled in the art. THe rate of amplification must be sufficient for detection. The specificity must be sufficiently high to eliminate false positives. The amplification conditions and length of hybridising sequences contribute to this and will be selected accordingly in a manner known in the art. The selection of optimal conditions lies within the reach of the skilled person. The kit can suitably comprise a further nucleic acid sequence suitable as probe and/or primer specific for ascertaining the presence or absence of sequences characteristic for increased risk of arterosclerosis. It can also comprise other compounds suitable for detecting such risk factors, the kit can also comprise a questionnaire aimed at ascertaining whether additional risk factors are present e.g. factors associated with life style as well as other previously diagnosed risk factors. In another suitable embodiment the kit can comprise a further nucleic acid sequence suitable as probe and/or primer specific for ascertaining the presence or absence of sequences characteristic for increased risk of a debilitating or fatal hereditary disease. Thus providing a test for CVD risk in combinaton with one or more debilitating or hereditary diseases. This could for example be useful for insurance companies or personnel screening programmes.

EXAMPLES

SUBJECTS AND METHODS Subjects

The patient group included 229 referred patients (183 men, mean age 40, range 25-50 years; and 46 women, mean age 40, range 21-50 years) with premature cardiovascular disease (coronary and/or peripheral) proven by angiography. 183 patients had clinical manifestations of coronary disease, 35 of peripheral disease, and 11 suffered from a combination of both. Controls were healthy volunteers (n=421) without a history of cardiovascular disease, mostly recruited among the employees of the Academic Medical Center in Amsterdam. Although unmatched for age and sex, patients and controls shared the same ethnic background, i.e. Caucasians from the Netherlands. All individuals gave their informed consent for the study.

Methods

CBS Genotyping Genomic DNA was obtained from peripheral blood leukocytes employing standard methods. DNA analysis was carried out by PCR amplification of a 184-bp DNA fragment containing exon 8 of the CBS gene, as previously described (9). The methodology is incorporated by reference herein. Obviously with the knowledge of the sequence data of the cysthathione gene and the sequence data of the 68 base pair insert numerous probes and/or primers can be applied to ascertain the presence and/or absence of the 68 base pair mutation at nucleotide 844. The sequence information can be derived from references 8 and 9 and is to be considered incorporated herein. The method can comprise amplification of the specific inserted nucleic acid with a primer specific for the 6 insert and/or amplification of nucleic acid surrounding the 844 nucleotide and determination of fragment size of the amplified product. The conditions being selected such that the presence or absence of the 68 base pair fragment can be made visible. This falls within routine experimentation for the person skilled in the art having the sequence knowledge of the exon surrounding nucleotide 844 and the insert, amplificatin of genomic nucleic acid from human samples being described in numerous handbooks and kits. PCR and NASBA are well known examples well established in the art for which commercial kits are available.

Total Homocysteine and Vitamin Assays

Only patients were subjected to a methionine-loading test (11). Homocysteine concentrations before and after loading were measured according to Ubbink et al (12) with minor modifications. Folic acid and vitamin B12 concentrations were measured in heparinized plasma, and vitamin B6 concentration was determined in heparinized whole blood.

Statistics

Genotype prevalence differences between patient and controls were assessed by calculating the χ² from a two by two contingency table. Differences in homocysteine and vitamin levels between CBS genotype groups were analysed by Students t-test. Odds ratios were calculated in the standard unmatched fashion. A 95% confidence interval was calculated using standard methods. The confidence interval of the odds ratio under assumption of Hardy- Weinberg equilibrium (vide infra) was calculated as described before (10).

RESULTS

Twelve percent of the 421 control individuals were carrier of the 844ins68 mutation in the CBS gene (51 heterozygotes and 1 homozygote). In 229 patients, 38 heterozygotes and 7 homozygotes for the CBS insertion were observed (20% carriers). The allele frequency of 844ins68 was therefore almost double in patients compared to controls (0.11 versus 0.06, χ²=13, p< 0.01).

Fasting and post-methionine total homocysteine levels were not statistically different between the genotypes; 14±6 and 44±21 μmol/L in non-carriers of the mutation, 16±11 and 47+24 in heterozygous carriers, and 16+7 and 37±15 in homozygous carriers respectively. Plasma folate levels (in nmol/L) were similar for non- carriers (8+4) and carriers (7+2 in heterozygotes and 9±4 in homozygotes). Vitamin B₁₂ levels were marginally but significantly (P<0.03) higher among carriers of the 68bp insertion; 397+182 pmol/L in non-carriers, 478±243 pmol/L in heterozygous carriers, and 477±205 pmol/L in homozygotes, respectively. Heterozygotes for the CBS insertion had lower vitamin B₆ levels (55±15 nmol/L) in comparison with non-carriers of the mutation (65±43 nmol/L). However, this effect was not significant and also not seen in homozygotes who exhibited a mean level of 77± 31 nmol/L. When heterozygous and homozygous carriers were combined the odds ratio for CVD came to 1.7 (95% CI 1.1-2.7). More specifically heterozygosity was associated with a 50 % increased risk (odds ratio 1.5, 95% CI 0.9-2.4), whereas the risk for homozygous carriers of the 844ins68 mutation was fourteen-fold increased (odds ratio 14, 95% CI 1.7-114). The number of homozygotes found was low and therefore the confidence limits (CI) of the risk estimates were wide. As an alternative, under the assumption of Hardy- Weinberg equilibrium, the expected number of homozygous individuals in the control population can be calculated from the allele frequency (10). For the current allele frequency of 0.062945, Hardy- Weinberg equilibrium predicts 1.67 homozygous individuals in a cohort of 421. Using this number in the risk estimate yields a lower odds ratio for arterial disease of 8.4 but with a narrower 95%> CI of 2,8-18.

Table 1 tabulates general characteristics and CV risk factors for the three CBS genotypes. The data show that, as expected, major risk factors play an important role in each of the three groups. Table 2 lists the odds ratios that can be calculated while taking classical risk factors into account. The data show that dyslipidemia increases the risk associated with the mutation considerably, suggesting gene-gene or gene- environment interaction. Diabetes mellitus also contributes to a marked increased risk. o

REFERENCES

1. Mudd SH, Levy HL, Skovby B. Disorders of transsulfuration. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds). "The metabolic and molecular bases of inherited disease". 7th edition, New York, McGraw-Hill, 1995; 1279-1327.

2. Rees MM, Rodgers GM. Homocysteinemia: association of a metabolic disorder with vascular disease and thrombosis. Thromb Res 1993; 71 : 337-359.

3. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Graham I. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 1991; 324: 1149-1155.

4. Den Heijer M, Koster T, Blom HJ, Bos GMJ, Briet E, Reitsma P, Vandenbroucke JP, Rosendaal FR. Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. N Engl J Med 1996; 334: 759-762.

5. De Stefano V, Finazzi G, Mannucci PM. Inherited thrombophilia: pathogenesis, clinical syndromes, and management. Blood 1996; 87: 3531-3544.

6. Boers GHJ. Hyperhomocysteinemia as a risk factor for arterial and venous disease. A review of evidence and relevance. Thromb Haemost 1997; 78: 520-522.

7. D'Angelo A, Selhub J. Homocysteine and thrombotic disease. Blood 1997; 90: 1-11. 8. Sperandeo MP, de Franchis R, Andria G, Sebastio G. A 68-bp insertion found in a homocystinuric patient is a common variant and is skipped by alternative splicing of the cystathionine β-synthase m RNA. Am J Hum Genet 1996; 59: 1391-1393.

9. Tsai MY, Bignell M, Schwichtenberg K, Hanson NQ. High prevalence of a mutation in the cystathionine β-synthase gene. Am J Hum Genet 1996; 59: 1262-1267.

10. Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH. High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance). Blood 1995; 85: 1504-1508.

11. Boers GH. Fowler B. Smals AG. Trijbels FJ. Leermakers AL Kleijer WJ. Kloppenborg PW. Improved identification of heterozygotes for homocystinuria due to cystathionine synthase deficiency by the combination of methionine loading and enzyme determination in cultured fibroblasts. Hum Genet 1985: 69: 164-169.

12. Ubbink JB, Vermaak WJH, Bissbort S. Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum. J Chromatogr 1991; 565:441-446.

13. Verhoeff, BJ, Trip, MD, Prins, MH, Kastelein, JJP, Reitsma, PH. The effect of a common methylenetetrahydrofolate reductase mutation on levels of homocysteine, folate, vitamin B6 and B12 and on the risk of premature atherosclerosis. Submitted for publication.

14. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP, Rozen R. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nature Genet 1995; 10: 111-113.

15. Harmon DL, Woodside JV, Yarnell JW, McMaster D, Young IS, McCrum EE, Gey KF, Whitehead AS, Evans AE. The common 'thermolabile' variant of methylenetetrahydrofolate reductase is a major determinant of mild hyperhomocysteinaemia. QJM 1996; 89: 571-577. 16. Ma J, Stampfer MJ, Hennekens CH, Frosst P, Selhub J, Horsford J,

Malinow MR, Willett WC, Rozen R. Methylenetetrahydrofolate reductase polymorphism, plasma folate, homocysteine, and risk of myocardial infarction in US physicians. Circulation 1996; 94: 2410-2416.

17. Christensen B, Frosst P, Lussier-Cacan S, Selhub J, Goyette P, Rosenblatt DS, Genest J Jr, Rozen R. Correlation of a common mutation in the methylenetetrahydrofolate reductase gene with plasma homocysteine in patients with premature coronary artery disease. Arteriosclerosis, Thromb Vase Biol 1997; 17: 569- 573.

18. Schwartz SM, Siscovick DS, Malinow MR, Rosendaal FR, Beverly RK, Hess DL, Psaty BM, Longstreth WT Jr, Koepsell TD, Raghunathan TE, Reitsma PH.

Myocardial infarction in young women in relation to plasma total homocysteine, folate, and a common variant in the methylenetetrahydrofolate reductase gene. Circulation 1997; 96: 412-417.

19. Kluijtmans LAJ, Boers GHJ, Trijbels FJM, van Lith-Zanders HMA, van den Heuvel LPWJ and Blom HJ. A common 844INS68 insertion variant in the cysthathonine β-synthase gene. Biomedical and Molecular Medicine 1997; 82: 23-35. 10

Table 1: General characteristics and risk factors for the three CBS genotypes in patients with premature CVD

Category CBS Genotype

+/+ +/ins ins/ins

Overall 184 38 7

Male 145 32 6

Female 39 6 1

Age (years) 40.1±0.5 40.4+0.9 42.2±2.2

Myocardial infarction 121 (66%) 25 (63%) 4 (57%)

Cholesterol 6.110.1 6.1+0.2 6.1+0.5

LDL 4.110.1 4.1+0.2 4.7+0.4

Diabetes mellitus 14 (8%) 2 (5%) 1 (14%)

Smokers 141 (77%) 30 (79%) 7 (100%)

Hypertension 40 (22%) 8 (21%) 2 (29%)

11

Table 2: Odds ratios

Odds ratios

(95% CI)

+/ins ins/ins*

Overall 1.5 (0.9-2.3) 8.4 (2.8-18.1)

Male 1.6 (1.0-2.6) 9.1 (2.4-21.4)

Female 1.1 (0.4-2.8) 5.8 (0.9-55.8)

Myocardial infarction 1.5 (0.9-2.5) 7.3 (2.3-22.2)

LDL >4.5 2.2 (1.2-4.1) 16.1 (2.2-22.6)

HDL <0.9 1.5 (0.7-3.1) 9.0 (1.6-32.4)

Triglycerides >2.3 1.6 (0.8-3.3) 8.3 (1.6-32.4)

Body mass index >30 2.0 (0.9-4.3)

Diabetes mellitus 1.0 (0.2-4.6) 15.8 (0.9-58.2)

Smoking 1.5 (0.9-2.5) 10.9 (2.8-18.1)

Hypertension 1.4 (0.6-3.3) 11.0 (1.5-32.4)

* Odds ratios and 95% CI for ins/ins genotype were calculated using an estimated 1.67 homozygotes in 369 controls according to Hardy Weinberg expectations

Claims

1. A method of diagnosing increased risk of arterosclerosis comprising ascertaining whether an individual carries the 68 base pair insertion mutation at nucleotide 844 of the cystathionine β-synthase gene in a manner known per se for ascertaining the presence of a mutation in the genome of an individual, wherein the presence of the mutation indicates an increased risk.

2. A method according to claim 1 further comprising differentiating whether the individual is homozygotic or heterozygotic for the mutation, in a manner known per se for distinguishing homozygous from heterozygous, wherein the homozygotic individual exhibits an even more increased risk.

3. A method according to claim 1 or 2, wherein the individual is tested also for a further risk factor of coronary disease.

4. A method according to any of the preceding claims, wherein the individual is further tested for the presence of LDL level above 4,5.

5. A method according to any of the preceding claims, wherein the individual is further tested for the presence of HDL level below 0,9.

6. A method according to any of the preceding claims, wherein the individual is further tested for the presence of triglycerides at a level above 2,3.

7. A method according to any of the preceding claims, wherein the individual is further tested for exhibiting diabetes mellitus.

8. A method according to any of the preceding claims, wherein the individual tests negative for homocysteinemia.

9. A method according to any of the preceding claims, wherein said individual tests negative for deficient CBS as determined in a CBS activity assay.

10. Kit for diagnosing increased risk of arterosclerosis in a manner according to any of the preceding claims, said kit comprising a nucleic acid sequence for discerning whether a sample comprises the 68 base pair insertion mutation at nucleotide 844 of the cystathionine β-synthase gene.

11. Kit according to claim 10 wherein said nucleic acid sequence is a nucleic acid probe specific for the insertion.

12. Kit according to claim 10 or 11 wherein the kit comprises at least two nucleic acid sequences capable of serving as nucleic acid primers for amplification of a fragment of nucleic acid surrounding the 844 nucleotide of the cystathionine β-synthase gene.

13. Kit according to any of claims 10-12 wherein the kit comprises a nucleic acid probe specific for the insertion and comprises at least two nucleic acid sequences capable of serving as nucleic acid primers for a fragment of nucleic acid surrounding the 844 nucleotide of the cystathionine β-synthase gene.

14. Kit according to any of claims 10-13 wherein said kit comprises a further nucleic acid sequence suitable as probe and/or primer specific for ascertaining the presence or absence of sequences characteristic for increased risk of arterosclerosis.

15. Kit according to any of claims 10-14 wherein said kit comprises a further nucleic acid sequence suitable as probe and/or primer specific for ascertaining the presence or absence of sequences characteristic for increased risk of a debilitating or fatal hereditary disease.