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Relation between Plasma Homocysteine Concentration, the 844ins68 Variant of the Cystathionine β-Synthase Gene, and Pyridoxal-5′-Phosphate Concentration
Molecular Genetics and Metabolism 67, 352–356 (1999) Article ID mgme.1999.2874, available online at http://www.idealibrary.com on
BRIEF COMMUNICATION Relation between Plasma Homocysteine Concentration, the 844ins68 Variant of the Cystathionine b-Synthase Gene, and Pyridoxal-5*-Phosphate Concentration eases have received considerable attention. Several case control (1,2) and prospective studies (3–5) have demonstrated that moderately elevated levels of tHcy measured after fasting are associated with increased risk of occlusive vascular diseases. In addition to fasting levels, tHcy can be measured 2– 6 h after a dose of methionine. Boushey et al. (6) showed, in a meta-analysis of 27 studies, that both fasting and post-methionine load (PML) hyperhomocysteinemia are risk factors for coronary artery disease (CAD). Recently, results from our laboratory (7) as well as from both the European Concerted Action Project (2) and the NHLBI Family Heart Study (8) showed that PML and fasting hyperhomocysteinemia are independent of each other in the majority of individuals, and that without methionine loading, approximately 27– 40% of the cases of elevated plasma tHcy could be missed. Moderately elevated levels of tHcy, whether measured during fasting or after a methionine load, can be caused by either genetic defects or nutritional deficiencies. Genetic defects can occur in either the remethylation or the transsulfuration pathways. Deficiencies of vitamin B 12 and folate, important cofactors in the remethylation pathway, and deficiency of pyridoxal-59-phosphate, the cofactor for the enzyme cystathionine b-synthase (CBS, EC 4.2.1.22), can also cause elevated levels of plasma tHcy (9,10). In the remethylation pathway, individuals who are homozygous for the “thermolabile” variant (C 677T) of the methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20) gene are thought to be predisposed to develop elevated plasma tHcy (11,12). However, the effect of this mutation is modulated by serum folate status (10). Thus, individuals who are
A moderately elevated plasma total homocysteine (tHcy), whether measured during fasting or postmethionine load (PML), is recognized as a risk factor for coronary artery diseases (CAD). Cystathionine b-synthase (CBS), a key enzyme in the transsulfuration pathway, is important for the metabolism of homocysteine. In recent years, a relatively prevalent mutation, the 844ins68 (68-bp insertion), was found to be carried by about 12% of the general population. In the current investigation, we studied 741 individuals with respect to the effect of the 68-bp insertion of the CBS gene on fasting and PML tHcy, and also determined the level of pyridoxal-5*-phosphate (vitamin B 6), a cofactor of the CBS enzyme. Our results showed that the mean fasting and PML increase in tHcy levels were lower in individuals carrying the 844ins68 variant compared to those without the insertion; although only the difference in PML increase in tHcy reached statistical significance (P 5 0.02). When these individuals were divided into two groups based on vitamin B 6 concentration, the PML increase in tHcy was significantly lower in individuals heterozygous for the insertion compared to those without the insertion only in the group of individuals whose vitamin B 6 concentrations were below the sample median (38.0 nmol/L). We speculate that the 68-bp insertion is associated with somewhat higher levels of CBS enzyme activity, and that the effect of this becomes more pronounced in the presence of relatively low concentrations of pyridoxal-5*-phosphate, a cofactor of the CBS enzyme. © 1999 Academic Press Key Words: homocysteine; methionine loading; cystathionine b-synthase; pyridoxal-5*-phosphate (vitamin B 6).
In recent years, plasma total homocysteine (tHcy) level and its association with cardiovascular dis352 1096-7192/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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homozygous for the C 677T mutation are most susceptible to elevated tHcy when their serum folate levels are relatively low. In the transsulfuration pathway, the presence of mutations in the CBS gene in the heterozygous carrier state was thought to be the major cause of mild PML hyperhomocysteinemia (13,14). However, recent studies have shown that heterozygous carriers of deleterious CBS mutations are present in less than 1% of the general population (7,15,16) and that while in heterozygous carriers defective CBS gene mutations play a role in causing hyperhomocysteinemia, they account for less than 5% of the patients with hyperhomocysteinemia (7,16). While most mutations of the CBS gene are rare, we have recently reported that a 68-bp insertion (844ins68) in the coding region of exon 8 is relatively prevalent, being present in the heterozygous state in approximately 12% of the general population (17). These results have also been confirmed in other laboratories (18 –20). In the current investigation, we studied the relatively prevalent 844ins68 variant of the CBS gene in a large number of individuals with respect to its effect on plasma tHcy levels, both during fasting and after a methionine load. In addition, we examined the influence of pyridoxal-59-phosphate, a cofactor of the CBS enzyme, on the relation between plasma tHcy levels and presence of the 844ins68 variant. METHODS
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The individuals studied were of mixed ancestry common to the upper midwestern region of the United States and belonged to no particular ethnic group. This study was approved by the Human Studies Committee of the University of Minnesota Institutional Review Board, and all subjects gave informed consent. Fasting and PML tHcy and Pyridoxal-59-Phosphate Assays Fasting blood samples were drawn and separated within one-half hour for the measurements of fasting plasma tHcy and pyridoxal-59-phosphate (vitamin B 6). Plasma vitamin B 6 was measured by a radioenzymatic assay (American Laboratory Products Company, Ltd. Windham, NH). Methionine (100 mg/kg body weight) was administered orally and a second blood sample was collected 4 h after loading for the determination of PML tHcy. Plasma tHcy for both fasting and PML samples was measured by HPLC with fluorometric detection (21). Because the PML tHcy level can be confounded by an elevated fasting tHcy level, we used the PML increase in tHcy, calculated as the difference between the fasting and the 4-h PML tHcy concentrations. Moderate hyperhomocysteinemia is defined as an elevated tHcy level based on the 90th percentile of the control population (fasting tHcy: males, $12 mmol/L, and females, $11 mmol/L; PML increase in tHcy: males, $30 mmol/L, and females, $31 mmol/L).
Study Population
Analysis of 844ins68 Variant of CBS Gene
We studied 743 individuals (518 males ranging in age from 24 to 76 years, mean age, 48.5 years; and 225 females ranging in age from 21 to 77 years, mean age 47.6 years). These individuals included 453 patients with premature CAD (documented by angiographically proven atherosclerosis, and/or one or more episodes of myocardial infarction or coronary artery bypass surgery before the age of 55 years), 81 patients undergoing evaluation for deep vein thrombosis, and 209 apparently healthy controls. Plasma fasting and 4 h PML tHcy concentrations and plasma pyridoxal-59-phosphate were routinely determined on all individuals. DNA was obtained from all individuals for mutation studies. Two individuals were found to be heterozygous for the T 833C mutation of the CBS gene and were therefore excluded from the study. No individuals carried the G 919A transition of the CBS gene.
Genomic DNA was extracted from peripheral leukocytes isolated from acid-citrate-dextrose-anticoagulated blood using a commercially available DNA isolation kit (Puregene, Gentra Systems, Minneapolis, MN). The 844ins68 variant was detected in conjunction with the T 833C and G 919A mutations of the CBS gene using digestion with the restriction enzymes BsrI and AluI, respectively (17). All digestion products were electrophoresed on a 12% polyacrylamide gel and silver-stained. Individuals carrying the 844ins68 variant were identified by the presence of digestion products of the 252-bp fragment in addition to the normal 184-bp fragment. Statistical Analysis Plasma tHcy and vitamin B 6 values had a skewed distribution; therefore, these variables were natural
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TABLE 1 Relation between Plasma tHcy, 844ins68 Variant of the CBS Gene, and Vitamin B 6 Status Genotype
a b
Plasma tHcy (n 5 733)
1/1 (n 5 633)
1/2 (n 5 100)
Fasting tHcy, mmol/L (95% CI) B 6 ,38.0 nmol/L B 6 $38.0 nmol/L PML Increase tHcy, mmol/L (95% CI) B 6 ,38.0 nmol/L B 6 $38.0 nmol/L
9.48 (9.25–9.73) 9.52 (9.15–9.90) 9.45 (9.16–9.75) 22.03 (21.43–22.64 23.06 (22.21–23.93) 21.05 (20.28–21.85)
9.16 (8.60–9.75) 9.32 (8.47–10.26) 8.99 (8.30–9.75) 20.20 (18.90–21.60) a 20.62 (18.76–22.65) b 19.85 (18.07–21.81)
1/2 significantly different from 1/1, P 5 0.02. 1/2 significantly different from 1/1, P 5 0.03.
log transformed and geometric means were used. Mean tHcy and vitamin B 6 levels and 95% confidence intervals (CI) were calculated after adjustment for age and gender. The F test was used to determine if there were differences in tHcy and vitamin B 6 concentrations between the various genotypes. The statistics were computed with SPSS for Windows (Release 7.5, SPSS, Chicago, IL). RESULTS We determined the fasting and PML increase in tHcy, 844ins68 genotype of the CBS gene, and vitamin B 6 concentration in 741 individuals. Of these, 633 (85.4%) did not carry the 844ins68 variant, 100 (13.5%) were heterozygous, and 8 (1.1%) were homozygous for the 68-bp insertion. Table 1 shows that the geometric mean fasting and PML increases in tHcy concentrations were both lower in individuals heterozygous for the 844ins68 variant compared to those without the insertion; however, only the difference in PML increase in tHcy concentrations reached statistical significance (P 5 0.02). To examine the influence of vitamin B 6 concentration on the relation between plasma tHcy levels and presence of the 844ins68 variant, we divided the group of individuals without the insertion and those heterozygous for the insertion into subgroups based on the median (, or $38.0 nmol/L) vitamin B 6 concentration. As shown in Table 1, the PML increase in tHcy was significantly lower (P 5 0.03) in individuals heterozygous for the insertion compared to those without the insertion only in the subgroup of individuals whose vitamin B 6 concentrations were below the sample median of 38.0 nmol/L.
DISCUSSION The 68-bp insertion (844ins68) was reported initially by Sebastio et al. (22) as a mutation in an Italian patient with classic homocystinuria due to CBS deficiency. The insertion in exon 8 of the CBS gene was presumed to introduce a premature termination codon resulting in a nonfunctional protein. Subsequently, we found the 68-bp insertion to be a highly prevalent mutation, being present in the heterozygous state in 12% of the general population (17). The inserted sequence introduces an alternate splice site. However, splicing seems to take place exclusively at the distal 39-splice site as only normal-size mRNA was found in experiments using reverse transcription-PCR (17). Since the presence of this mutation was not associated with hyperhomocysteinemia, we suggested that this mutation is likely to be a benign polymorphism. Sperandeo et al. (18), while confirming our finding that the 68-bp insertion is highly prevalent, suggested that the allele carrying the insertion yields a lower amount of mRNA, thus predisposing individuals carrying the mutation to elevated tHcy levels. The results of the current study showed conclusively that carriers of the 68-bp insertion have lower plasma tHcy levels compared to those who do not carry this polymorphism. The difference is most significant for PML increases in tHcy measured as the difference between the tHcy value 4 h after a methionine loading and the fasting tHcy level. These results suggest that the presence of the insertion is associated with higher CBS enzyme activities in vivo. The exact mechanism for this is not known. Previous studies have shown that intronic sequences are important for transcription initiation, transcription maturation, and export of RNA to the
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cytoplasm (23–25). In the case of the 844ins68 variant, the inserted 68 bp may contain sequences that promote these steps. Alternatively, the 68-bp insertion may be in linkage disequilibrium with a polymorphic site in the regulatory region which upregulates the transcription of the CBS gene. Regardless of the mechanism of how the 68-bp insertion affects CBS enzyme activity, the association of the 844ins68 variant with lower plasma tHcy levels, especially after a methionine load, adds to our understanding of the multifactorial regulation of homocysteine metabolism. The interplay between mild mutations and vitamin levels was first documented for the C 677T mutation of the MTHFR gene and folate concentration by Jacques et al. (10). These investigators showed that individuals who were homozygous for the C 677T mutation were predisposed to elevated plasma tHcy levels when their plasma folate level was below the sample median. Likewise, when we divided the group of individuals without the insertion and those heterozygous for the insertion into subgroups based on the median vitamin B 6 concentration, we found that the 68-bp insertion, when present, is associated with a significantly lowered PML increase in tHcy levels only in the subgroup of individuals whose vitamin B 6 concentrations were below the sample median of 38.0 nmol/L. We speculate that the 68-bp insertion is associated with a small increase in CBS enzyme activity. The effect of this increase may be masked when the concentration of vitamin B 6, a cofactor of the CBS enzyme, is present at relatively high levels. In conclusion, the present study documents that the 68-bp insertion (844ins68), a common variant in the CBS gene, influences plasma tHcy, and this influence is modulated by plasma vitamin B 6 levels. The finding of yet another example of the interplay between micronutrient and prevalent polymorphism in influencing plasma tHcy levels demonstrates the intricacies of the regulation of plasma tHcy levels by both genetic and acquired factors.
concentrations and extracranial carotid artery stenosis. N Engl J Med 332:286 –291, 1995. 2.
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Stampfer MJ, Malinow MR, Willet WC, Newcomer LM, Upson B, Ullmann D, Tishler PV. A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. J Am Med Assoc 268:877– 881, 1992.
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Arnesen E, Refsum H, Bonaa KH, Ueland PM, Forde OH, Nordrehaug JE. Serum total homocysteine and coronary heart disease. Int J Epidemiol 24:704 –709, 1995.
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Perry II, Refsum H, Morris RW, Ebrahim SB, Ueland PM, Shaper AG. Prospective study of serum homocysteine concentration and risk of stroke in middle-aged British men. Lancet 346:1395–1398, 1995.
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Boushey CJ, Beresford SAA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: Probable benefits of increasing folic acid intakes. J Am Med Assoc 274:1049 –1057, 1995.
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Tsai MY, Welge BC, Hanson NQ, Bignell MK, Vessey J, Schwichtenberg K, Yang F, Bullemer FE, Rasmusen R, Graham KJ. Genetic causes of mild hyperhomocysteinemia in patients with premature occlusive coronary artery diseases. Atherosclerosis 143:163–170, 1999.
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Bostom AG, Selhub J, Jacques PF, Nadeau MR, Williams RR, Ellison RC. Methionine intolerance with normal fasting total plasma homocysteine: Initial results from the NHLBI Family Heart Study. Irish J Med Sci 164s:3, 1995.
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Selhub J, Jacques PF, Wilson PWF, Rush D, Rosenberg IH. Vitamin status and intake as primary determinants of homocysteinemia in the elderly. J Am Med Assoc 270:2693– 2698, 1993.
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Jacques PF, Bostom AG, Williams RR, Ellison RC, Eckfeldt JH, Rosenberg IH, Selhub J, Rozen R. Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 93:7–9, 1996.
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Kang SS, Wong P, Susmano A, Sora J, Norusis M, Ruggie N. Thermolabile methylenetetrahydrofolate reductase: An inherited risk factor for coronary artery disease. Am J Hum Genet 48:536 –545, 1991.
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Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJH, den Heijer M, Kluijtmans LAJ, van den Heuvel LP, Rozen R. A candidate genetic risk factor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nature Genet 10:111–113, 1995.
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Boers GHJ, Smals AGH, Trijbels FJM, Fowler B, Bakkeren JAJM, Schoonderwaldt HC, Kleijer W, Kloppenborg PW. Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease. N Engl J Med 313: 709 –715, 1985.
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ACKNOWLEDGMENT This study was supported in part by a grant from the American Heart Association, Minnesota Affiliate.
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BRIEF COMMUNICATION Fowler B, Graham I. Hyperhomocysteinemia: An independent risk factor for vascular disease. N Engl J Med 324: 1149 –1155, 1991. Kluijtmans LA, van den Heuvel LPWJ, Boers GHJ, Frosst P, Stevens E, van Oost BA, den Heijer M, Trijbels FJ, Rozen R, Blom HJ. Molecular genetic analysis in mild hyperhomocysteinemia: A common mutation in the methylenetetrahydrofolate reductase gene is a genetic risk factor for cardiovascular disease. Am J Hum Genet 58:35– 41, 1996. Folsom AR, Nieto FJ, McGovern PG, Tsai MY, Malinow MR, Eckfeldt JH, Hess DL, Davis CE. Prospective study of coronary heart disease incidence in relation to fasting total homocysteine, related genetic polymorphisms, and B vitamins: The Atherosclerosis Risk in Communities (ARIC) study. Circulation 98:204 –210, 1998. Tsai MY, Bignell M, Schwichtenberg K, Hanson NQ. High prevalence of a mutation in the cystathionine b-synthase gene. Am J Hum Genet 59:1262–1267, 1996. 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 b-synthase mRNA. Am J Hum Genet 59:1391–1393, 1996. Kluijtmans LAJ, Boers GHJ, Trijbels FJM, van LithZanders HMA, van den Heuvel LPWJ, Blom HJ. A common 844ins68 insertion variant in the cystathionine b-synthase gene. Biochem Mol Med 62:23–25, 1997. Ramsbottom D, Scott JM, Molloy A, Weir DG, Kirke PN, Mills JL, Gallagher PM, Whitehead AS. Are common mutations of cystathionine b-synthase involved in the aetiology of neural tube defects? Clin Genet 51:39 – 42, 1997. Ubbink JB, Hayward VWJ, Bissbort S. Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum. J Chromatogr 565:441– 446, 1991.
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Sebastio G, Sperandeo MP, Panico M, de Franchis R, Kraus JP, Andria G. The molecular basis of homocystinuria due to cystathionine b-synthase deficiency in Italian families, and report of four novel mutations. Am J Hum Genet 56:1324 – 1333, 1995.
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Michael Y. Tsai 1 Feng Yang Michelle Bignell ¨ Omer Aras Naomi Q. Hanson Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, Minnesota 55455-0392 Received April 23, 1999
1 To whom correspondence should be addressed at 420 Delaware St. SE, Box 609, Mayo, Minneapolis, MN 55455-0392. Fax: (612) 625-6994. E-mail: [email protected].
BRIEF COMMUNICATION Relation between Plasma Homocysteine Concentration, the 844ins68 Variant of the Cystathionine b-Synthase Gene, and Pyridoxal-5*-Phosphate Concentration eases have received considerable attention. Several case control (1,2) and prospective studies (3–5) have demonstrated that moderately elevated levels of tHcy measured after fasting are associated with increased risk of occlusive vascular diseases. In addition to fasting levels, tHcy can be measured 2– 6 h after a dose of methionine. Boushey et al. (6) showed, in a meta-analysis of 27 studies, that both fasting and post-methionine load (PML) hyperhomocysteinemia are risk factors for coronary artery disease (CAD). Recently, results from our laboratory (7) as well as from both the European Concerted Action Project (2) and the NHLBI Family Heart Study (8) showed that PML and fasting hyperhomocysteinemia are independent of each other in the majority of individuals, and that without methionine loading, approximately 27– 40% of the cases of elevated plasma tHcy could be missed. Moderately elevated levels of tHcy, whether measured during fasting or after a methionine load, can be caused by either genetic defects or nutritional deficiencies. Genetic defects can occur in either the remethylation or the transsulfuration pathways. Deficiencies of vitamin B 12 and folate, important cofactors in the remethylation pathway, and deficiency of pyridoxal-59-phosphate, the cofactor for the enzyme cystathionine b-synthase (CBS, EC 4.2.1.22), can also cause elevated levels of plasma tHcy (9,10). In the remethylation pathway, individuals who are homozygous for the “thermolabile” variant (C 677T) of the methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20) gene are thought to be predisposed to develop elevated plasma tHcy (11,12). However, the effect of this mutation is modulated by serum folate status (10). Thus, individuals who are
A moderately elevated plasma total homocysteine (tHcy), whether measured during fasting or postmethionine load (PML), is recognized as a risk factor for coronary artery diseases (CAD). Cystathionine b-synthase (CBS), a key enzyme in the transsulfuration pathway, is important for the metabolism of homocysteine. In recent years, a relatively prevalent mutation, the 844ins68 (68-bp insertion), was found to be carried by about 12% of the general population. In the current investigation, we studied 741 individuals with respect to the effect of the 68-bp insertion of the CBS gene on fasting and PML tHcy, and also determined the level of pyridoxal-5*-phosphate (vitamin B 6), a cofactor of the CBS enzyme. Our results showed that the mean fasting and PML increase in tHcy levels were lower in individuals carrying the 844ins68 variant compared to those without the insertion; although only the difference in PML increase in tHcy reached statistical significance (P 5 0.02). When these individuals were divided into two groups based on vitamin B 6 concentration, the PML increase in tHcy was significantly lower in individuals heterozygous for the insertion compared to those without the insertion only in the group of individuals whose vitamin B 6 concentrations were below the sample median (38.0 nmol/L). We speculate that the 68-bp insertion is associated with somewhat higher levels of CBS enzyme activity, and that the effect of this becomes more pronounced in the presence of relatively low concentrations of pyridoxal-5*-phosphate, a cofactor of the CBS enzyme. © 1999 Academic Press Key Words: homocysteine; methionine loading; cystathionine b-synthase; pyridoxal-5*-phosphate (vitamin B 6).
In recent years, plasma total homocysteine (tHcy) level and its association with cardiovascular dis352 1096-7192/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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homozygous for the C 677T mutation are most susceptible to elevated tHcy when their serum folate levels are relatively low. In the transsulfuration pathway, the presence of mutations in the CBS gene in the heterozygous carrier state was thought to be the major cause of mild PML hyperhomocysteinemia (13,14). However, recent studies have shown that heterozygous carriers of deleterious CBS mutations are present in less than 1% of the general population (7,15,16) and that while in heterozygous carriers defective CBS gene mutations play a role in causing hyperhomocysteinemia, they account for less than 5% of the patients with hyperhomocysteinemia (7,16). While most mutations of the CBS gene are rare, we have recently reported that a 68-bp insertion (844ins68) in the coding region of exon 8 is relatively prevalent, being present in the heterozygous state in approximately 12% of the general population (17). These results have also been confirmed in other laboratories (18 –20). In the current investigation, we studied the relatively prevalent 844ins68 variant of the CBS gene in a large number of individuals with respect to its effect on plasma tHcy levels, both during fasting and after a methionine load. In addition, we examined the influence of pyridoxal-59-phosphate, a cofactor of the CBS enzyme, on the relation between plasma tHcy levels and presence of the 844ins68 variant. METHODS
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The individuals studied were of mixed ancestry common to the upper midwestern region of the United States and belonged to no particular ethnic group. This study was approved by the Human Studies Committee of the University of Minnesota Institutional Review Board, and all subjects gave informed consent. Fasting and PML tHcy and Pyridoxal-59-Phosphate Assays Fasting blood samples were drawn and separated within one-half hour for the measurements of fasting plasma tHcy and pyridoxal-59-phosphate (vitamin B 6). Plasma vitamin B 6 was measured by a radioenzymatic assay (American Laboratory Products Company, Ltd. Windham, NH). Methionine (100 mg/kg body weight) was administered orally and a second blood sample was collected 4 h after loading for the determination of PML tHcy. Plasma tHcy for both fasting and PML samples was measured by HPLC with fluorometric detection (21). Because the PML tHcy level can be confounded by an elevated fasting tHcy level, we used the PML increase in tHcy, calculated as the difference between the fasting and the 4-h PML tHcy concentrations. Moderate hyperhomocysteinemia is defined as an elevated tHcy level based on the 90th percentile of the control population (fasting tHcy: males, $12 mmol/L, and females, $11 mmol/L; PML increase in tHcy: males, $30 mmol/L, and females, $31 mmol/L).
Study Population
Analysis of 844ins68 Variant of CBS Gene
We studied 743 individuals (518 males ranging in age from 24 to 76 years, mean age, 48.5 years; and 225 females ranging in age from 21 to 77 years, mean age 47.6 years). These individuals included 453 patients with premature CAD (documented by angiographically proven atherosclerosis, and/or one or more episodes of myocardial infarction or coronary artery bypass surgery before the age of 55 years), 81 patients undergoing evaluation for deep vein thrombosis, and 209 apparently healthy controls. Plasma fasting and 4 h PML tHcy concentrations and plasma pyridoxal-59-phosphate were routinely determined on all individuals. DNA was obtained from all individuals for mutation studies. Two individuals were found to be heterozygous for the T 833C mutation of the CBS gene and were therefore excluded from the study. No individuals carried the G 919A transition of the CBS gene.
Genomic DNA was extracted from peripheral leukocytes isolated from acid-citrate-dextrose-anticoagulated blood using a commercially available DNA isolation kit (Puregene, Gentra Systems, Minneapolis, MN). The 844ins68 variant was detected in conjunction with the T 833C and G 919A mutations of the CBS gene using digestion with the restriction enzymes BsrI and AluI, respectively (17). All digestion products were electrophoresed on a 12% polyacrylamide gel and silver-stained. Individuals carrying the 844ins68 variant were identified by the presence of digestion products of the 252-bp fragment in addition to the normal 184-bp fragment. Statistical Analysis Plasma tHcy and vitamin B 6 values had a skewed distribution; therefore, these variables were natural
354
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TABLE 1 Relation between Plasma tHcy, 844ins68 Variant of the CBS Gene, and Vitamin B 6 Status Genotype
a b
Plasma tHcy (n 5 733)
1/1 (n 5 633)
1/2 (n 5 100)
Fasting tHcy, mmol/L (95% CI) B 6 ,38.0 nmol/L B 6 $38.0 nmol/L PML Increase tHcy, mmol/L (95% CI) B 6 ,38.0 nmol/L B 6 $38.0 nmol/L
9.48 (9.25–9.73) 9.52 (9.15–9.90) 9.45 (9.16–9.75) 22.03 (21.43–22.64 23.06 (22.21–23.93) 21.05 (20.28–21.85)
9.16 (8.60–9.75) 9.32 (8.47–10.26) 8.99 (8.30–9.75) 20.20 (18.90–21.60) a 20.62 (18.76–22.65) b 19.85 (18.07–21.81)
1/2 significantly different from 1/1, P 5 0.02. 1/2 significantly different from 1/1, P 5 0.03.
log transformed and geometric means were used. Mean tHcy and vitamin B 6 levels and 95% confidence intervals (CI) were calculated after adjustment for age and gender. The F test was used to determine if there were differences in tHcy and vitamin B 6 concentrations between the various genotypes. The statistics were computed with SPSS for Windows (Release 7.5, SPSS, Chicago, IL). RESULTS We determined the fasting and PML increase in tHcy, 844ins68 genotype of the CBS gene, and vitamin B 6 concentration in 741 individuals. Of these, 633 (85.4%) did not carry the 844ins68 variant, 100 (13.5%) were heterozygous, and 8 (1.1%) were homozygous for the 68-bp insertion. Table 1 shows that the geometric mean fasting and PML increases in tHcy concentrations were both lower in individuals heterozygous for the 844ins68 variant compared to those without the insertion; however, only the difference in PML increase in tHcy concentrations reached statistical significance (P 5 0.02). To examine the influence of vitamin B 6 concentration on the relation between plasma tHcy levels and presence of the 844ins68 variant, we divided the group of individuals without the insertion and those heterozygous for the insertion into subgroups based on the median (, or $38.0 nmol/L) vitamin B 6 concentration. As shown in Table 1, the PML increase in tHcy was significantly lower (P 5 0.03) in individuals heterozygous for the insertion compared to those without the insertion only in the subgroup of individuals whose vitamin B 6 concentrations were below the sample median of 38.0 nmol/L.
DISCUSSION The 68-bp insertion (844ins68) was reported initially by Sebastio et al. (22) as a mutation in an Italian patient with classic homocystinuria due to CBS deficiency. The insertion in exon 8 of the CBS gene was presumed to introduce a premature termination codon resulting in a nonfunctional protein. Subsequently, we found the 68-bp insertion to be a highly prevalent mutation, being present in the heterozygous state in 12% of the general population (17). The inserted sequence introduces an alternate splice site. However, splicing seems to take place exclusively at the distal 39-splice site as only normal-size mRNA was found in experiments using reverse transcription-PCR (17). Since the presence of this mutation was not associated with hyperhomocysteinemia, we suggested that this mutation is likely to be a benign polymorphism. Sperandeo et al. (18), while confirming our finding that the 68-bp insertion is highly prevalent, suggested that the allele carrying the insertion yields a lower amount of mRNA, thus predisposing individuals carrying the mutation to elevated tHcy levels. The results of the current study showed conclusively that carriers of the 68-bp insertion have lower plasma tHcy levels compared to those who do not carry this polymorphism. The difference is most significant for PML increases in tHcy measured as the difference between the tHcy value 4 h after a methionine loading and the fasting tHcy level. These results suggest that the presence of the insertion is associated with higher CBS enzyme activities in vivo. The exact mechanism for this is not known. Previous studies have shown that intronic sequences are important for transcription initiation, transcription maturation, and export of RNA to the
BRIEF COMMUNICATION
cytoplasm (23–25). In the case of the 844ins68 variant, the inserted 68 bp may contain sequences that promote these steps. Alternatively, the 68-bp insertion may be in linkage disequilibrium with a polymorphic site in the regulatory region which upregulates the transcription of the CBS gene. Regardless of the mechanism of how the 68-bp insertion affects CBS enzyme activity, the association of the 844ins68 variant with lower plasma tHcy levels, especially after a methionine load, adds to our understanding of the multifactorial regulation of homocysteine metabolism. The interplay between mild mutations and vitamin levels was first documented for the C 677T mutation of the MTHFR gene and folate concentration by Jacques et al. (10). These investigators showed that individuals who were homozygous for the C 677T mutation were predisposed to elevated plasma tHcy levels when their plasma folate level was below the sample median. Likewise, when we divided the group of individuals without the insertion and those heterozygous for the insertion into subgroups based on the median vitamin B 6 concentration, we found that the 68-bp insertion, when present, is associated with a significantly lowered PML increase in tHcy levels only in the subgroup of individuals whose vitamin B 6 concentrations were below the sample median of 38.0 nmol/L. We speculate that the 68-bp insertion is associated with a small increase in CBS enzyme activity. The effect of this increase may be masked when the concentration of vitamin B 6, a cofactor of the CBS enzyme, is present at relatively high levels. In conclusion, the present study documents that the 68-bp insertion (844ins68), a common variant in the CBS gene, influences plasma tHcy, and this influence is modulated by plasma vitamin B 6 levels. The finding of yet another example of the interplay between micronutrient and prevalent polymorphism in influencing plasma tHcy levels demonstrates the intricacies of the regulation of plasma tHcy levels by both genetic and acquired factors.
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ACKNOWLEDGMENT This study was supported in part by a grant from the American Heart Association, Minnesota Affiliate.
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Michael Y. Tsai 1 Feng Yang Michelle Bignell ¨ Omer Aras Naomi Q. Hanson Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, Minnesota 55455-0392 Received April 23, 1999
1 To whom correspondence should be addressed at 420 Delaware St. SE, Box 609, Mayo, Minneapolis, MN 55455-0392. Fax: (612) 625-6994. E-mail: [email protected].