ANALYSIS OF FUNCTIONAL VARIATION IN HUMAN
METABOLIC GENES

The interplay between the environment and our genes ultimately determines the state of our health. The focus of this laboratory's research is to identify and understand how alterations in DNA influence the formation of disease states. Much of the laboratory's effort is spent on the development of yeast-based functional assays to determine whether a particular alteration affects the function of the underlying protein. Our current work focuses on the genes controlling the metabolism of two different metabolic pathways: 1) the homocysteine metabolic pathway and 2) the phase I /phase II detoxification pathway.

Homocysteine is a thiol-containing amino acid that can have two fates -- condensation with serine to form cystathionine or methylation to form methionine (Figure 1). Over the past two decades, a link between alterations in the metabolism of homocysteine and human disease has been firmly established. High levels of total plasma homocysteine (tHcy) are a recognized risk factor in the development of vascular disease and birth defects. Oral administration of folic acid can lower tHcy. The major goal of this laboratory is to identify mutations in the genes involved in homocysteine metabolism and determine the relationship between these changes and specific human diseases.

C FACTORS INFLUENCING PLASMA HOMOCYSTEINE LEVELS. WANG, KRUGER, in collaboration with MALINOW^a

We have been examining DNA polymorphisms in three genes in the tHcy metabolic pathway to determine whether these alterations are associated with increased risk of coronary heart disease, increased basal tHcy, or increased "responsiveness" to lowering of tHcy by folic acid. Our population consists of 140 coronary heart disease patients and 100 controls recruited from Providence St. Vincent hospital in Portland, Oregon. We used PCR/ Restriction fragment length polymorphism (RFLP) technology to type several polymorphisms in genes in the homocysteine metabolic pathway. We have found that one common alteration in the methylene-tetrahydrofolate reductase (MTHFR) gene, c677T, and two alterations in the cystathionine beta-synthase (CBS) gene, c699t and c1080t, are significantly over-represented in heart patients relative to controls. We have also found that these three alterations are also associated with tHcy responsiveness to folate. These findings suggest particular alleles of the MTHFR and CBS genes may influence the risk of homocysteine related heart disease. In addition, it may be possible to predict a priori people who will respond to folate.

We have developed a yeast functional assay for the human MTHFR gene in the yeast Saccharomyces cerevisiae. The basis for this assay is that wild type human MTHFR can functionally substitute for its yeast counterpart, the MET11 gene. Based on this observation, we developed a yeast functional assay to detect and characterize mutant alleles of human MTHFR. This assay can distinguish mutant alleles with MTHFR activities ranging from undetectable to 50% of wild type, including the common C677T mutation. Yeast growth rates correlate well with in vitro enzyme activity. This yeast assay should be useful in the characterization of previously unknown MTHFR mutations, and structure/function studies on the MTHFR protein.

Our laboratory has previously developed a yeast functional assay for the human CBS gene. In this assay, expression of human CBS cDNA rescues the cysteine requirement of a yeast strain deleted for the endogenous CBS enzyme. In the course of expressing and characterizing various human mutant CBS proteins in this yeast strain, we have identified a mutation that can partially suppress a number of different disease causing CBS alleles. This suppressor mutation when expressed in cis can partially suppress the yeast growth defect of at least eight different CBS mutations. This mutation creates a stop codon at amino acid 411 of the CBS gene, and thus truncates about one quarter of the protein. Our hypothesis is that this truncated region of the protein acts as a negative regulatory domain, and truncation of this region allows an increase in the residual activity of the mutant proteins. To confirm this hypothesis, we have expressed and purified various forms of CBS and measured enzyme activity in vitro. These studies have shown that truncated CBS protein is ten times more active than the full-length form. In addition, we have discovered that this inhibition by the Cterminus appears to be responsible for regulation by Sadenosylmethionine, an allosteric affector of CBS. These findings suggest that the Cterminus of CBS would be a good potential target for homocysteine lowering drugs.

About 50% of human tumor cells exhibit a methionine dependent growth phenotype. This phenotype is characterized by the tumor cells requirement for methio-nine and its inability to utilize methionine's immediate metabolic precursor, homocysteine, to support growth. Our laboratory is interested in understanding the metabolic basis for this phenotype. Since homocysteine is converted to methionine by the action of methionine synthase (MS), we examined the status of the MS gene in a dozen previously characterized tumor cell lines. In earlier work (Li et al., Hum. Mol. Genet. 5:1851, 1996), our laboratory cloned and characterized the human MS encoding cDNA. We used this information to characterize the MS cDNA from methionine-dependent tumor cells. In all cases the MS cDNA and protein appears to be normal, suggesting that defects in MS are not the cause of methionine dependent growth.

Recently we have looked for mutations in another pathway involved in methionine biosynthesis, the so-called "methionine salvage pathway." Our data indicates that there is a direct correlation between methionine dependent growth and defects in 5'deoxy-5'-methylthioadenosine phosphorylase (MTAP), a critical enzyme in this pathway. This finding suggests that the methionine salvage pathway may be playing a more important role in the production of methionine then previously thought.

A new direction in the laboratory concerns the identification of DNA alterations that influence one's susceptibility to carcinogenesis by aflatoxin. Aflatoxin is a potent carcinogen produced by the common mold Aspergillus. Animal models clearly indicate that aflatoxin is a potent inducer of liver cancer (hepatocellular carcinoma). The presence of aflatoxin in a country's food supply strongly correlates with high levels of liver cancer in the population. Thus, aflatoxin is suspected of being a important cause of liver cancer world-wide.

Aflatoxin must first be metabolically activated to become carcinogenic. This activation occurs via hydroxylation by the P450 family of cytochrome oxidases. In humans, the P450 family consists of over fifty genes, many of which contain DNA variants that are different in individuals throughout the population. The current interest in this laboratory is to identify which P450 proteins are capable of metabolizing aflatoxin, and determine how variation in these genes affects this ability. Our ultimate goal is to identify genotypes of aflatoxin "sensitive" versus "insensitive" individuals.

We are doing this work by modeling the human P450 genes in yeast. Yeast lack an endogenous P450 capable of activating aflatoxin and therefore are insensitive to its effect. However, one can express a human P450 allowing for aflatoxin activation. In yeast the consequence of this activation is an increase in the rate of mitotic recombination and translocation (Sengstag et al., Cancer Res. 56:5457, 1996), which can be monitored by growth in a suitably marked strain. We plan to use this system to determine the relative activities of the human P450 proteins in aflatoxin activation.

CHEN, P., PODDAR, R., TIPA, E.V., DIBELLO, P.M., MORAVEC, C.D., ROBINSON, K., GREEN, R., KRUGER, W.D., GARROW, T.A., JACOBSEN, D.W. Homocysteine metabolism in cardiovascular cells and tissues: implications for hyperhomocysteinemia and cardiovascular disease. Adv. Enzyme Regul. (in press).

LOBO, A., NASO, A., ARHEART, K., KRUGER, W.D., BAOU-GHAZALA, T., ALSOUS, A., NEHLAWI, M., GUPTA, A., MOUSTAPHA, A., VAN LENTE, F., JACOBSEN, D.W., ROBINSON, K. Reduction of homocysteine levels in coronary disease by low-dose folic acid combined with vitamins B6 and B12: A placebo-controlled study. Am. J. Cardiol. 83:821-825, 1999.

MALINOW, M.R., NUELL, P.B., HESS, D.L., ANDERSON, P.H., KRUGER, W.D., PHILLIPSON, B.E., GLUCKMAN, R.A., BLOCK, P.C., UPSON, B.M. Reduction of plasma homocyst(e)ine by breakfast cereal fortified with folic acid in patients with coronary heart disease. N. Eng. J. Med. 338:1009-1015, 1998.

SHAN, X., KRUGER, W.D. correction of disease-causing CBS mutations in yeast. Nature Genet. 19:91-94, 1998.

TAOKA, S., OHJA, S., SHAN X., KRUGER, W.D., BANERJEE, R. Evidence for heme-mediated redox regulation of human cystathionine b-synthase activity. J. Biol. Chem. 273(30):25179-25184, 1998.

^§   Fox Chase researcher

^a   M.R. Malinow: Oregon Regional Primate Research Center, Beaverton, OR 97006

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998