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ABSTRACT

Epidemiologic and mechanistic evidence suggests that folate is involved in colorectal neoplasia. Some polymorphic genes involved in folate metabolism-methylenetetrahydrofolate reductase
(MTHFR C677T and A1298C ), methionine synthase ( MTR A2756G ), methionine synthase reductase ( MTRR A66G) , cystathionine ß-synthase ( CBS exon 8, 68-base-pair insertion), and thymidylate synthase ( TS enhancer region and 3' untranslated region)-have been investigated in colorectal neoplasia. For MTHFR C677T and A1298C, the variant allele is associated with reduced enzyme activity in vitro. For the other polymorphisms, functional data are limited and/or inconsistent. Genotype frequencies for all of the polymorphisms show marked ethnic and geographic variation. In most studies, MTHFR C677T (10 studies, >4,000 cases) and 1298CC (four studies, >1,500 cases) are associated with moderately reduced colorectal cancer risk. In four of five genotype-diet interaction studies, C677T subjects who had higher folate levels (or a "high-methyl diet") had the lowest cancer risk. In two studies, 677TT homozygote subjects with the highest alcohol intake had the highest cancer risk. Findings from six studies of MTHFR C677T and adenomatous polyps are inconsistent. There have been only one or two studies of the other polymorphisms; replication is needed. Overall, the roles of folate-pathway genes, folate, and related dietary factors in colorectal neoplasia are complex. Research priorities are suggested.

Key Words: CBS; colorectal neoplasms; epidemiology; folic acid; MTHFR; MTR; MTRR; TS

Abbreviations: CBS, cystathionine ß-synthase; CI, confidence interval; MSI, microsatellite instability; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; OR, odds ratio; rpt, repeat; TS, thymidylate synthase.

Evidence is accumulating for a role of folate in the etiology of colorectal carcinomas and adenomas (1). Many of the genes involved in folate metabolism are polymorphic (2). This paper reviews five polymorphic genes-methylenetetrahydrofolate reductase ( MTHFR ), methionine synthase ( MTR ), methionine synthase reductase ( MTRR ), cystathionine ß-synthase ( CBS ), and thymidylate synthase ( TS )-and their associations with colorectal neoplasia.

GENES

5,10-MTHFR plays a central role in folate metabolism (Figure 1), irreversibly converting 5,10-methylenetetrahydrofolate to 5-methylenetetrahydrofolate, the primary circulating form of folate. The substrate is vital for DNA synthesis. The product provides methyl groups for synthesis of methionine, a decreased pool of which may affect DNA methylation. The gene encoding 5,10-MTHFR, MTHFR, is located at 1p36.3 (3).

MTR, which is essential for maintaining adequate intracellular folate pools, catalyzes the remethylation of homocysteine to methionine, required for production of S -adenosylmethionine, the universal methyl group donor. Vitamin B 12 is a cofactor in this methylation process. The MTR gene is on 1q43 (4). MTR is maintained in its active form by MTRR (5), the gene for which, MTRR, is located at 5p15.3-p15.2. CBS catalyzes the conversion of homocysteine to cystathionine; vitamin B 6 is required in this reaction. The CBS gene is at 21q22.3. TS catalyzes the conversion of deoxyuridine monophosphate to thymidine monophosphate, requiring 5-10-methylenetetrahydrofolate as a methyl donor. The TS gene is located at 18p11.32.

Folate status could potentially be perturbed by polymorphisms in these genes. Two mechanisms have been proposed by which folate deficiency could affect malignancy: 1) by causing DNA hypomethylation and proto-oncogene activation and/or 2) by inducing uracil misincorporation during DNA synthesis, leading to catastrophic DNA repair, DNA strand breakage, and chromosome damage (6). Human evidence in support of these mechanisms is limited (6-7).

GENE VARIANTS

This section describes polymorphisms in the genes and their functional effects. With the exception of MTHFR , relatively few studies have investigated relations between the polymorphisms and blood levels of folate and related biomarkers in nondiseased persons. In subjects with medical conditions, it is possible that the condition or its treatment, rather than the underlying genotype, influences biomarker levels. Many studies have been small, with limited statistical power. A potential difficulty in interpretation is that any observed difference in biomarker levels by genotype may not be due to the polymorphism under study but to the presence of another polymorphism. Equally, a failure to observe differences in biomarkers by genotype could be due to the presence of another polymorphism with opposing functional effects. So far, there has been little investigation of the effects of combinations of polymorphisms. With regard to MTHFR C677T, only red cell folate measured by microbiologic assay is reliable; results of the radioimmune assay are biased (8). There is differential detection by the assays of various intracellular folates, the distribution of which is related to MTHFR genotype (9). Whether red cell folate results measured by radioimmunoassay are biased for other polymorphisms in the folate-pathway genes is not known.

MTHFR
Several polymorphisms in the MTHFR gene have been reported, and two have been investigated in colorectal neoplasia: 1) C -->T at nucleotide 677, leading to an alanine to valine conversion in the protein (10); and 2) A -->C in exon 7, causing an alanine to glutamate protein change
(11,12). These polymorphisms are located 2.1 kb apart. The other polymorphisms-T1059C, T1317C, and G1793A (12-14 )-are not discussed further in this paper.

For C677T , compared with homozygotes for the common variant ( CC ), heterozygotes have 65 percent of their enzyme activity levels in vitro and those who are homozygous variant ( TT ), 30 percent (15). From the microbiologic assay, compared with CC homozygotes, heterozygotes have 10 percent lower and TT homozygotes 18 percent lower red cell folate levels (16). Persons with the TT variant also have lowered plasma folate and vitamin B 12 levels and raised homocysteine levels (17,18). In two studies, the association with homocysteine held only when folate status was low (19, 20); in another, it occurred only when riboflavin status was poor (21). Regarding MTHFR and DNA methylation, one small study found that DNA from subjects with the TT variant had a significantly higher methyl group acceptance capacity than DNA from subjects with the CC variant (22), but this finding was not confirmed in a larger study (23). In 292 subjects (66 percent of whom had coronary atherosclerosis) selected by MTHFR genotype (187 CC, 105 TT), DNA methylation status was affected by genotype among only those with lower plasma folate levels; subjects with the TT variant who had lower plasma folate concentrations had lower methylation levels than all other groups of subjects (24). A few studies have investigated MTHFR and uracil misincorporation, DNA strand breaks, or genetic instability in vivo and in vitro, with inconclusive results (23, 25-27).

For A1298C , enzyme activity in vitro is decreased in homozygotes variants ( CC ) and, to a lesser extent, in heterozygotes compared with those without the variant (11). Studies of A1298C and plasma folate and homocysteine are inconsistent (12, 28-31), which may be due to methodological reasons (e.g., non-population-based study, small sample size), or it may be that there is a relation that depends on the status of folate and/or related nutrients. Enzyme activity in vitro for compound heterozygotes (i.e., heterozygotes for C677T and for A1298C) is unclear (29).

MTR
The A-G polymorphism at position 2756 in the protein binding region of MTR replaces aspartic acid with glycine (32). Most studies suggest that plasma homocysteine level is lower in those with the rarer, G , than the more common, A , allele (18, 33-36). One study found significantly higher plasma folate levels in GG than in AA subjects (34), but this finding was not observed in another study (18). Evidence on red cell folate and on plasma vitamin B 12 and vitamin B 6 is very limited (18, 35, 37).

MTRR
The A66G polymorphism in the MTRR gene results in the substitution of isoleucine with methionine at codon 22 (5). In two studies, subjects homozygous for the common allele ( AA ) had elevated homocysteine levels compared with those who had other genotypes (38, 39); in a third study, genotype was not a significant predictor of homocysteine level (40). No associations were found between genotype and serum folate, vitamin B 6 , or vitamin B 12 in the single known study ( 38 ).

CBS
Many mutations and several polymorphisms in the CBS gene have been reported (41). To our knowledge, the only variant investigated in colorectal neoplasia is the 68-base-pair insertion in the exon 8 coding region. Four studies found lower plasma homocysteine levels in persons carrying the insertion than in those without, although the difference was significant in only one
(35, 36, 39, 42). One study suggested that the effect was modulated by plasma vitamin B 6 concentration (43); another suggested an interaction with MTHFR C677T (35). The one available study that we know of found no associations between genotype and red cell folate or plasma vitamin B 12 level (35).

TS
The TS enhancer region contains a series of 28-base-pair tandem repeats. Two repeats (2 rpt) or three repeats (3 rpt) are most common, with 3 rpt occurring most frequently. More repeats have been observed but are rare (44, 45). In vitro, compared with the double repeat, the triple repeat has been associated with 2.6-fold greater thymidylate synthase expression (46). Among 497 Singapore Chinese, plasma folate levels were significantly lower, and homocysteine levels nonsignificantly higher, in 3 rpt/3 rpt subjects than in those with other genotypes (47). When MTHFR and TS were considered together, plasma folate levels were highest (15.3 nM) in 677CC or 677CT and not 3 rpt/3 rpt subjects, intermediate (13.8 nM) in 677CC or 677CT and 3 rpt/3 rpt subjects, and lowest (11.6 nM) in 677TT subjects (irrespective of TS genotype).

The 3' untranslated region contains a 6-base-pair deletion at base pair 1494, the functional consequences of which are not known (48). The two polymorphisms appear to be in linkage disequilibrium (48).

Refer to the Appendix for Internet sites pertaining to the genes discussed in this review.

POPULATION FREQUENCIES

This section includes information on studies reporting genotype frequencies in persons without cancer or other diseases. Using appropriate Medical Subject Headings (MeSH) and text words, we searched MEDLINE, EMBASE, and PubMed databases for papers published from 1990 to December 2002. Further relevant articles were identified by hand-searching reference lists in published papers. MTHFR frequencies are from the Human Genome Epidemiology (HuGE) reviews by Botto and Yang (49) and by Robien and Ulrich (50). The A1298C data reported by Robien and Ulrich are augmented with results from less-studied geographic areas and ethnic groups. For the studies tabulated here, Hardy-Weinberg equilibrium of the genotype frequencies was assessed by using the Pearson X ² test.

MTHFR
There is considerable ethnic and geographic variation in the frequency of the C677T variant (49). The TT prevalence ranged from around 1 percent in Black populations in the United States, sub-Saharan Africa, and South America to more than 20 percent in US Hispanics, Colombians, and Amerindians in Brazil. TT genotype frequency in White populations in Europe, North America, and Australia was 8-20 percent. In Europe, there appears to be a trend of increasing frequency of the variant from north to south. Twelve percent of Japanese were TT homozygotes.

For A1298C , the CC prevalence in North American studies, which included mainly White subjects, was 7-12 percent (50). In four Hispanic series ( n < 90), the frequency was 4-5 percent (51-54). In two African-American series, 2 and 4 percent were CC subjects. In Europe, the prevalence of CCranged from 4 to 12 percent in most studies. In two northeast Scotland series of subjects randomly selected from general practitioner registers, the frequencies were 15 percent (95 percent confidence interval (CI): 11.8, 19.2) and 18 percent (95 percent CI: 9.5, 30.4) (55, 56). In Chinese, Japanese, and Hawaiian populations, 1-4 percent were CC (50, 54) subjects. In the single studies in Brazil, Morocco, South Africa, and Turkey and among Israeli Jews, the frequencies were 6 percent (95 percent CI: 2.8, 9.6), 3 percent (95 percent CI not available), 4 percent (95 percent CI: 1.4, 9.9), 6 percent (95 percent CI: 1.7, 14.8), and 13 percent (95 percent CI: 9.7, 16.5), respectively (31, 33, 57-59).

In some series, but not all, a few persons with three or four variant alleles (i.e., 677TT / 1298AC , CT / CC, TT / CC) have been reported (35, 60-64).

MTR
In Japanese, Chinese, and Korean populations, the frequency of the GG genotype was 2-3 percent (18, 32- 37, 54, 65-82; Web table 1). (This information is described in the first of four supplementary tables; each is referred to as "Web table" in the text and is posted on the Web site of the Human Genome Epidemiology Network (http://www.cdc.gov/genomics/hugenet/default.htm) as well as on the Journal 's Web site (http://aje.oupjournals.org/).) In most European series, approximately 3 percent of the subjects had the GG genotype. Frequencies from all but two North American studies were 1-5 percent. The frequency was 10-11 percent in these two series-one of White children and their mothers in Canada and the other of White persons in Hawaii. In the single African-American population, 6 percent (95 percent CI: 4.3, 8.7) of the subjects had the GG genotype. In three studies, the genotype frequencies were not in Hardy-Weinberg equilibrium (73-75).

MTRR
The lowest reported prevalence of GG homozygotes was 8-10 percent in Japanese in Hawaii and in Hawaiians (5, 14, 38-40, 54, 83-85; Web table 2). Among 558 subjects in Northern Ireland, 12 percent (95 percent CI: 9.1, 14.6) were GG homozygotes, but this series was not in Hardy-Weinberg equilibrium. In most of the remaining series, the frequency was 19-29 percent. Among 97 African Americans and 96 Hispanics, the frequencies were 42 percent (95 percent CI: 32.3, 52.7) and 50 percent (95 percent CI: 39.6, 60.4), respectively.

CBS
Homozygosity for the 68-base-pair insertion is rare in all populations (35, 36, 39, 42, 54, 65, 70, 71, 77, 82, 86-98; Web table 3). The highest reported frequency was 3 percent among Blacks from Brazil and Africa. In four other series, the homozygote prevalence also reached 3 percent, but the genotype frequencies were not in Hardy-Weinberg equilibrium (42, 70, 71, 96). In Europe, Australia, and most US populations, the frequency of heterozygotes was 8-19 percent, with most around 13-15 percent. Two Japanese series found no heterozygotes. Heterozygosity occurred in 5 percent (95 percent CI: 1.6, 11.3) of the single Chinese series.

TS
In three studies in the United Kingdom, and in three of mainly White populations in the United States, 19-23 percent of subjects were 2 rpt/2 rpt (44-47, 99-102; Web table 4). The prevalence was 14-20 percent in two African and one African-American series and 17 percent among volunteers born in four southwest Asian countries living in Scotland. Two to 4 percent of two Chinese populations were homozygous variant. In all studies, genotype frequencies were in Hardy-Weinberg equilibrium.

In a single study of US Whites, 10 percent (95 percent CI: 7.7, 12.5) were homozygotes for the 3' untranslated region deletion (102).

Combinations of genotypes
Most studies reporting frequencies of combinations of genotypes are small (33, 35, 70, 80, 94, 103). In the largest, of almost 1,300 males in the United Kingdom, 8 percent carried the CBS68-base-pair insertion and the MTHFR T allele; 5 percent of subjects had the CBS 68-base-pair insertion and the MTR G allele; and 20 percent carried both the MTR G and MTHFR T alleles (35).

Comments on studies of population frequencies
Few of the studies reviewed here were population based; many relied on convenience samples. Selection and participation biases may therefore explain some of the apparent variations in genotype prevalence. In a few studies, genotype frequencies were not in Hardy-Weinberg equilibrium. Although lack of Hardy-Weinberg equilibrium might indicate that the series were subject to selection or participation biases, there are other reasons why Hardy-Weinberg equilibrium might not hold, including migration or genotyping error (104). Many of the studies are relatively small, so the estimates of genotype frequency lack precision.

In many studies, the ethnic makeup of the participants is not described. Most well characterized are White populations in the United States and western Europe. Other populations, geographic areas, and ethnic groups, particularly in Africa, Asia (other than Japan), and South America, have been less studied. The generalizability from, for example, one "Black African" population to another may be limited since it is not always straightforward to establish ethnicity (105).

DISEASE

An estimated 945,000 new cases of colorectal cancer were diagnosed worldwide in 2000, and 492,000 persons died from the disease (106). Two thirds of incident cases occur in developed countries, where it is the third most common cancer in males and second most common in females (107). There are substantial international variations in incidence (108). Sixty to 70 percent of colorectal cancers arise in the colon (108).

Although most evidence is indirect, the majority of colorectal carcinomas are believed to develop from adenomatous polyps (109). Hyperplastic polyps may be precursors of some right-sided colon cancers (110). Investigation of the first occurrence, and the recurrence, of polyps may reveal factors important in early stages of the neoplastic process.

Fewer than 10 percent of incident colorectal tumors are due to hereditary nonpolyposis colorectal cancer and familial adenomatous polyposis (111). When these syndromes are excluded, there is still familial aggregation of cancers and adenomas (112-114), which is unlikely to be entirely accounted for by familial clustering of environmental factors (115). This information points to the potential importance of genetic susceptibility factors, and the interaction of these with each other and with environmental factors, in the disease causation.

The studies of Japanese migrants to the United States in the 1960s revealed the overwhelming importance of environmental factors in colorectal cancer etiology (116). Established risk factors for the disease are shown in Table 1 (109, 117-125).

Although diet appears to be important in colorectal cancer (120), it has been difficult to identify the specific components involved. Observational epidemiologic evidence shows that a high vegetable intake is related to decreased risk (120), although recent work suggests that the relation is complex (124, 125). Vegetables, particularly green, leafy vegetables, are a major source of folate. The majority of prospective and case-control studies of serum folate, red cell folate, or reported dietary or total folate intake are compatible with inverse associations with colon cancer and adenomas (17, 54, 76, 125-146). There is no consistent association between rectal cancer and folate intake (126, 131, 133-135, 137, 138). One small trial of folic acid supplementation in persons from whom polyps had been removed observed a reduced recurrence rate in the supplemented group (147). Some studies are compatible with a positive association between alcohol intake, which adversely affects folate metabolism (148), and colorectal neoplasia (109). A "low-methyl diet," comprising high alcohol intake and low folate and methionine (and/or vitamins B 6 and B 12 ) intakes, has been associated with increased colon cancer risk (126, 130, 132, 140).

Internet sites providing data and information on colorectal neoplasia are contained in the Appendix.

ASSOCIATIONS

This section appraises studies of the polymorphisms and colorectal neoplasia risk. These studies were identified by using the search strategy described above with the addition of disease-specific Medical Subject Headings and text words.

MTHFR
C677T
To our knowledge, there have been 10 cancer studies: five in the United States, two in the United Kingdom, and one each in Australia, Mexico, and Korea (17, 54, 56, 98, 149-155 ;
Table 2). Two included only colon cancers (150, 154); the remainder included colon and rectal tumors. On the basis of the functional effects of the polymorphism, and the inverse association between folate status and disease, it might have been expected that the variant would be associated with increased disease risk. In contrast, seven studies were consistent with reduced risk in homozygous variant ( TT ) subjects compared with homozygotes for the common allele
(17, 54, 149-151, 153, 154). Observed relative risks ranged from 0.45 to 0.9, although most did not reach statistical significance. A significant trend of decreasing risk with increasing number of T alleles has been reported (54). As has been observed in several meta-analyses of gene-disease associations (156, 157), the strongest effects were found in the two earliest studies (17, 149). Both were nested within cohort studies of predominantly White male populations in the United States. These populations were likely to have relatively high average intakes of total folate as a consequence of comparatively frequent use of vitamin supplements (158).

INTERACTIONS

Gene-environment interactions
MTHFR C677T
The gene-environment interactions explored have concerned features of the "low-methyl" diet and genotype. Four of five studies suggest interactions between folate, methionine, or alcohol and C677T in relation to cancer. Chen et al. (149) reported that the inverse association with the TT genotype was greatest among persons in the highest tertiles of folate and methionine intake. The results of Ma et al. (17), who examined plasma folate, and Le Marchand et al. (54), who analyzed food and total folate intake, were compatible with this finding. Keku et al. (154), however, did not observe this pattern with regard to total folate intake.

Slattery et al. (150) categorized subjects as consuming low-, intermediate-, and high-methyl diets. The lowest odds ratio was for subjects with the TT genotype consuming a high-methyl diet (OR for high-methyl and TT vs. low-methyl and CC = 0.4, 95 percent CI: 0.1, 0.9), while the odds ratios for subjects consuming a low-methyl diet did not vary by genotype (150). Consistent with this finding, Ma et al. (17) observed an increased risk among the folate deficient (plasma folate <3.0 ng/ml) irrespective of genotype.

Two cancer studies found significant interactions between C677T and alcohol (17, 149). High intake abolished the reduced risk associated with the TT genotype to the extent that subjects with this TT genotype who consumed the largest quantities of alcohol were at the greatest risk of cancer (greater even than for those without the T allele who were in the highest alcohol group). Keku et al. (154) found no interaction with alcohol but did not consider quantity, only whether subjects had "ever" or "never" consumed alcohol.

High blood riboflavin levels may improve MTHFR activity in TT persons because the cofactor for MTHFR is a metabolite of riboflavin (171). Le Marchand et al. (54) observed the lowest relative risk for cancer among TT persons with the highest riboflavin intake. Genotype-folate-riboflavin combinations were not considered.

Little is published on gene-diet interactions and adenomas. In the two known studies, the stratum of highest risk comprised TT persons with the lowest red cell or plasma folate levels
(160) or the lowest intakes of folate, methionine, vitamin B 6 , and vitamin B 12 (159), but the gene-nutrient interactions were not statistically significant. With regard to alcohol and genotype, the pattern observed is similar to that for cancer (159, 160).

MTHFR A1298C
Keku et al. (154) observed a significant interaction ( p = 0.03) between total folate intake and A1298C genotype among White but not African-American subjects; fewer African-American subjects were involved in the study. Unlike the pattern for C677T , White 1298CC subjects who consumed less than 400 ng of folate per day had a greater reduced cancer risk than those whose folate intake was higher. No interactions were observed between A1298C and "ever" or "never" consuming alcohol.

Two further studies of A1298C reported no significant interactions with blood levels or intake of folate or related nutrients and colorectal neoplasia (28, 54). The results were not shown.

MTR
For cancer, Ma et al. (18) reported a significant interaction between MTR and alcohol intake (Table 5); persons with the GG genotype consuming more than one drink a day had an increased disease risk (OR for GG and X1 drink/day vs. AA and <1 drink/day = 2.64, 95 percent CI: 0.65, 10.82), while those consuming less than one drink a day had a reduced risk (OR = 0.27, 95 percent CI: 0.09, 0.81; p for interaction = 0.04). There was also a nonsignificant 50 percent risk reduction among GG subjects whose plasma folate levels were in the upper two tertiles compared with those with the same folate level and the AA /AG genotype; persons with the GG genotype in the lowest plasma folate tertile did not have a reduced risk ( P for interaction = 0.22).

MTRR and CBS
Le Marchand et al. (54) reported no significant interactions between MTRR or CBS and dietary folate, vitamin B 12 , vitamin B 6 , riboflavin, or methionine. Results were not shown.

TS
For adenomas, Ulrich et al. (102) found a statistically significant interaction between the tandem repeat polymorphism and folate intake. Among 3 rpt/3 rpt persons, higher folate intake (>440 ng/day) was associated with a 50 percent reduced risk compared with lower folate intake. However, among 2 rpt/2 rpt persons, higher folate intake was associated with a 50 percent increased risk ( P for interaction = 0.03). A similar pattern was observed for vitamin B 12 intake ( P for interaction = 0.08). No interactions were found with intakes of vitamin B 6 , methionine, or alcohol, nor were there interactions between the 3' untranslated region polymorphism and dietary variables.

Gene-gene interactions
Metabolism of any exposure is likely to depend on the balance between the relative activities of all of the enzymes active within the metabolic pathway (172). So far, we know of two studies that have considered joint effects of folate-pathway genes (54, 102; Table 5).

For cancer, Le Marchand et al. (54) observed that the MTHFR T allele had the greatest effect among subjects with the MTR G allele (OR for CT / TT and AG / GG vs. CC and AA = 0.7, 95 percent CI: 0.5, 1.0; p for interaction = 0.05). Considering MTHFR C677T and CBS, they reported that the group with both variants appeared to be at reduced risk; however, this result was based on small numbers, and the interaction was not significant. Meanwhile, MTRR did not interact with MTHFR C677T .

For adenomas, Ulrich et al. (102) investigated interactions between C677T , TS tandem repeat, and folate intake. The association of higher folate intake with reduced risk among 3 rpt/3 rpt subjects was not modified by MTHFR. The increased risk associated with lower folate intake in TT subjects appeared limited to 3 rpt homozygotes. These findings were not statistically significant.

Comments on studies of gene-disease associations and interactions
Some of the heterogeneity in the findings with regard to the genotype main effects is likely to be due to differences between the populations studied in average levels of intake of folate, alcohol, and related dietary factors. If there truly are interactions between genotype and folate, for example, they may be seen only in populations with high or low folate levels (depending on the direction of the interaction). Such an effect has recently been observed for MTHFR C677T and coronary heart disease (158).

Methodological factors are also important. Five cancer studies ( 17, 56, 149, 151, 152) and four adenoma studies (76, 152, 161, 162) each included fewer than 300 cases and thus had limited statistical power, particularly for subgroup and interaction analyses. The nonprospective studies are most susceptible to bias. Some were not population based. In some, it is not clear whether the controls came from the population that gave rise to the cases. In others, the case series were limited to subjects still alive to provide a DNA sample (prevalent cases), which would have resulted in bias if any of the genotypes were associated with survival (currently not known). Few studies provided information on participation rates, making it difficult to assess bias and generalizability. It is likely that a proportion of the controls in the cancer studies may have been harboring undiagnosed polyps. Depending on the relations between each polymorphism and adenomas, this may have introduced random error or bias. The presence of undetected polyps among controls would not be important if the genotype was etiologically relevant only after an adenoma had developed, as seems likely for MTHFR C677T . For the other genotypes, it is not clear at what stage in the adenoma-carcinoma sequence they may be relevant. Finally, the possibility cannot be discounted that the findings do not reflect an association between the specified polymorphisms and colorectal neoplasia but rather are a consequence of linkage disequilibrium.

LABORATORY TESTING

MTHFR C677T and A1298C are detected by means of DNA amplification using polymerase chain reaction followed by restriction fragment length polymorphism analysis; HinfI for C677T and MboII (12) for A1298C (10, 11) are used. The MTR and MTRR polymorphisms and the 3' untranslated region variant in TS are also detected by restriction fragment length polymorphism, with digestion with Maell for MTR, with NdeI or AflIII for MTRR , and with Dral for TS (4, 5, 48, 54). The TS tandem repeat and CBS insertion are detected by DNA amplification and visualization on agarose gels (46, 97).

Most studies did not report the success rate in extracting DNA from samples, the proportion of eligible subjects for whom genotyping failed, or the degree of genotyping reproducibility, all of which are important indicators of the analytical validity of genotyping (173).

Laboratories are increasingly using high-throughput genotyping methods, an area of considerable development and innovation. Although quality control and analytical validity in this context are important (173), published data are currently lacking.

POPULATION TESTING

Companies in the United States and the United Kingdom are offering consumer tests for genotypic or phenotypic markers of polymorphisms influencing nutrient metabolism, including MTHFR (174, 175). However, the scientific evidence currently is not strong enough to advocate population testing for any polymorphisms reviewed here.

Testing for these polymorphisms might be valuable in cancer patients. 5-Fluorouracil, commonly used in colorectal cancer chemotherapy, is a thymidylate synthase inhibitor and can cause severe folate depletion. Knowledge of patient genotype could be used to tailor chemotherapy regimes to 1) minimize toxicity and side effects, thus improving quality of life, and/or 2) increase the effectiveness of treatment and ultimately lengthen survival. So far, evidence in this area is limited to the TS tandem repeat and MTHFR C677T . Among 51 stage III colon cancer patients treated with 5-fluorouracil and leucovorin (folinic acid), presence of the MTHFR T allele had little effect on probability of death or length of survival in those who had died, except in 12 patients with rectosigmoid colon cancer (176). In a study of 365 nonadjuvant-treated patients, the TT genotype was associated with improved survival, but this result did not persist after adjustment for disease stage (98).

For TS, some (177-179) but not all (180, 181) studies of colorectal cancer patients concluded that higher TS tumor expression levels were related to shorter survival. Consistent with this finding, one genotype study suggested that carrying the 3 rpt allele increased risk of death (179). Four studies of genotype and response to 5-fluorouracil (182- 185) suggested that 2 rpt/2 rpt patients may be more responsive to therapy but subject to greater toxicity (186). Most of the studies (of genotype or phenotype) have been small, included selected patient groups, and made limited adjustment for potentially important factors.

CONCLUSIONS AND RESEARCH PRIORITIES

The observed association of the MTHFR homozygous variant genotypes with reduced carcinoma risk was the opposite of what might have been expected a priori. This finding has led investigators to reconsider the folate metabolism pathway, putting a greater emphasis on the functions of folate and MTHFR in DNA synthesis. The evidence is compatible with interactions between MTHFR genotype and folate, alcohol, and/or related nutrients in relation to colorectal cancer. Evidence on polymorphisms other than MTHFR C677T is extremely limited. The associations observed between MTR, CBS, MTRR, and TS genotypes and colorectal neoplasia are tentative at best and require replication. The few studies of combinations of polymorphisms suggest the possibility of gene-gene interactions; again, further investigation is needed to confirm initial findings. Altogether, the evidence suggests that the roles of folate-metabolizing genes, folate, and related dietary factors in colorectal neoplasia are complex. Methodologies are currently lacking for specification of hypotheses, clarification of functional effects, and statistical analysis relating to such complex gene-environment pathways. This area of research must be a priority if advancements in understanding of disease etiology are to be achieved. Table 6 lists other areas for further research.

Table 6  Research priorities.

NOTES

Correspondence to Linda Sharp, Epidemiology Group, Department of Medicine and Therapeutics, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD, Scotland (e-mail: L.Sharp@abdn.ac.uk )

SUPPLEMENTARY TABLES

• WebTable 1
• WebTable 2
• WebTable 3
• WebTable 4
  

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