Hereditary nonpolyposis colorectal cancer (HNPCC) is a syndrome of familial malignancies, presenting most frequently as colorectal or endometrial cancer (1). Carriers of mutations in mismatch repair (MMR) genes, especially in hMLH1 and hMSH2, are predisposed to HNPCC-related cancers (2). Among MMR mutation carriers, considerable interindividual variation exists in colorectal cancer age of onset. Polymorphisms of the metabolic genes NAT2 (3), NAT1, GSTM1, and GSTT1 (4) have been examined for their modifying effect in HNPCC populations and were associated with an earlier age of onset.

GSTM1 is a polymorphic metabolic gene that detoxifies electrophilic compounds such as heterocyclic amines. It is homozygously deleted (null genotype) in over 50% of some populations, including Caucasians, Chinese, and Indians (5). The null allele has a high frequency of about 0.7 (6). Absence of the gene may contribute to earlier onset of cancer in MMR mutation carriers (4). In this study, we examined GSTM1 genotype for association with an earlier colorectal cancer age of onset in a population of MMR mutation carriers from HNPCC families.

Study Subjects

We studied 104 confirmed MMR gene mutation carriers from 59 families in the institutional Hereditary Colon Cancer Registry. Study participant demographics are reported in Table 1. Each study subject contributed 10 ml of blood and DNA was extracted as described previously (7).

Table 1.

Study demographics

All study subjects
Caucasian subjects
GSTM1 nullGSTM1 gene carrierTotalGSTM1 nullGSTM1 gene carrierTotal
Cancer status       
    Colorectal cancer patients 20 (19.2%) 32 (30.8%) 52 (50.0%) 18 (20.7%) 25 (28.7%) 43 (49.4%) 
    Unaffected MMR mutation carriers 22 (21.2%) 30 (28.8%) 52 (50.0%) 20 (23.0%) 24 (27.6%) 44 (50.6%) 
Gender       
    Male 23 (22.1%) 24 (23.1%) 47 (45.2%) 22 (25.3%) 20 (23.0%) 42 (48.3%) 
    Female 19 (18.3%) 38 (36.5%) 57 (54.8%) 16 (18.4%) 29 (33.3%) 45 (51.7%) 
MMR gene mutated       
    hMLH1 14 (13.5%) 24 (23.1%) 38 (36.5%) 14 (16.1%) 22 (25.3%) 36 (41.4%) 
    hMSH2 28 (26.9%) 38 (36.5%) 66 (63.5%) 24 (27.6%) 27 (31.0%) 51 (58.6%) 
MMR gene mutation type       
    Truncation/Deletion 34 (32.7%) 8 (7.7%) 75 (72.1%) 30 (34.5%) 31 (35.6%) 61 (70.1%) 
    Missense 41 (39.4%) 21 (20.2%) 29 (27.9%) 8 (9.2%) 18 (20.7%) 26 (29.9%) 
All subjects 42 (40.4%) 62 (59.6%) 104 (100%) 38 (43.7%) 49 (56.3%) 87 (100%) 
Age of onseta       
    Time at risk 1798 2755 4553 1619 2233 3852 
    Incidence rate 0.0111 0.0116 0.0114 0.0111 0.0112 0.0112 
    Range 22–84 21–89 21–89 22–84 21–89 21–89 
    25% cancer-free 39 40 39 39 42 39 
    50% cancer-free 50 50 50 53 51 51 
    75% cancer-free 58 70 63 58 70 70 
Ethnic/Racial groups       
    Caucasians 38 (36.5%) 49 (47.1%) 87 (83.6%)    
    African American 4 (3.9%) 5 (4.8%) 9 (8.7%)    
    Hispanic 8 (7.7%) 8 (7.7%)    
All study subjects
Caucasian subjects
GSTM1 nullGSTM1 gene carrierTotalGSTM1 nullGSTM1 gene carrierTotal
Cancer status       
    Colorectal cancer patients 20 (19.2%) 32 (30.8%) 52 (50.0%) 18 (20.7%) 25 (28.7%) 43 (49.4%) 
    Unaffected MMR mutation carriers 22 (21.2%) 30 (28.8%) 52 (50.0%) 20 (23.0%) 24 (27.6%) 44 (50.6%) 
Gender       
    Male 23 (22.1%) 24 (23.1%) 47 (45.2%) 22 (25.3%) 20 (23.0%) 42 (48.3%) 
    Female 19 (18.3%) 38 (36.5%) 57 (54.8%) 16 (18.4%) 29 (33.3%) 45 (51.7%) 
MMR gene mutated       
    hMLH1 14 (13.5%) 24 (23.1%) 38 (36.5%) 14 (16.1%) 22 (25.3%) 36 (41.4%) 
    hMSH2 28 (26.9%) 38 (36.5%) 66 (63.5%) 24 (27.6%) 27 (31.0%) 51 (58.6%) 
MMR gene mutation type       
    Truncation/Deletion 34 (32.7%) 8 (7.7%) 75 (72.1%) 30 (34.5%) 31 (35.6%) 61 (70.1%) 
    Missense 41 (39.4%) 21 (20.2%) 29 (27.9%) 8 (9.2%) 18 (20.7%) 26 (29.9%) 
All subjects 42 (40.4%) 62 (59.6%) 104 (100%) 38 (43.7%) 49 (56.3%) 87 (100%) 
Age of onseta       
    Time at risk 1798 2755 4553 1619 2233 3852 
    Incidence rate 0.0111 0.0116 0.0114 0.0111 0.0112 0.0112 
    Range 22–84 21–89 21–89 22–84 21–89 21–89 
    25% cancer-free 39 40 39 39 42 39 
    50% cancer-free 50 50 50 53 51 51 
    75% cancer-free 58 70 63 58 70 70 
Ethnic/Racial groups       
    Caucasians 38 (36.5%) 49 (47.1%) 87 (83.6%)    
    African American 4 (3.9%) 5 (4.8%) 9 (8.7%)    
    Hispanic 8 (7.7%) 8 (7.7%)    
a

The age of onset is the age at first diagnosis for cancer patients and the age at blood draw for unaffected MMR mutation carriers. The age of onset values were determined from Kaplan-Meier estimates, with the median age of onset defined as the age at which 50% of the population is cancer-free.

Genotyping

PCR and acrylamide gel electrophoresis analyses were used to determine the GSTM1 genotype of the participants. Primers specific to GSTM1, as described by Xu et al. (8), were used to amplify the gene by PCR, using 100 ng of genomic DNA in a 30-μl reaction mixture containing a 1× dilution of GeneAmp10× PCR buffer (Applied Biosystems, Foster City, CA); 2.8 mm MgCl2 (Applied Biosystems); 0.25 mm each dNTP (Invitrogen Corporation, Carlsbad, CA); 0.5 units AmpliTaq Gold (Applied Biosystems); 0.1 μl [32P]DCTP (3000 Ci/mmol; PerkinElmer Life Sciences, Inc., Boston, MA), and 30 pmol of each GSTM1 primer (Applied Biosystems). The PCR reactions were performed for 10 min at 94°C followed by 25 cycles of 94°C for 30 s, 57°C for 20 s, and 72°C for 45 s, and a final extension step at 72°C for 5 min. DNA sequencing analysis confirmed that the primers produced the GSTM1 sequence. Primers for β-globin (10 pmol each) and IFN (30 pmol each), as described by Chen et al. (9), were included in the PCR to generate positive controls.

The PCR product was mixed with an equal volume of loading buffer containing 95% formamide, 20 mm EDTA, 0.05% xylene cyanol, and 0.05% bromphenol blue. The prepared samples were electrophoresed on a non-denaturing 5% acrylamide gel (10% glycerol, 10% Tris-borate-EDTA) in 1× Tris-borate-EDTA running buffer. The gels were vacuum-dried and autoradiographed. The positive controls confirmed success of amplification by producing a band above (β-globin) and below (IFN) the GSTM1 band location in each lane. Absence of the GSTM1 band identified the participant as homozygous for the null deletion. Presence of the band identified the participant as a GSTM1 gene carrier, although homozygous carriers could not be distinguished from heterozygous carriers. Gene frequencies are reported in Table 1.

Statistical Analysis

Age of onset of colorectal cancer was defined as the patient's age at diagnosis or, for the unaffected MMR mutation carriers, the age at which blood was drawn. We defined colorectal cancer age of onset as the outcome and GSTM1 genotype as the independent variable. We tested for an association between GSTM1 genotype and an earlier colorectal cancer age of onset by comparing Kaplan-Meier survival curves, evaluating the homogeneity of the curves with the log-rank test and Wilcoxon's test.

By Kaplan-Meier survival analysis, the age of onset for colorectal cancer in subjects who were GSTM1 gene carriers was not different from that in subjects who were null for GSTM1 (P = 0.88 by the log-rank test; P = 0.82 by Wilcoxon's test). The median age of onset, or the age at which 50% of the population is cancer-free, was 50 years for both carrier and null genotypes.

Because of possible heterogeneity in susceptibility to colorectal cancer among different racial and ethnic groups, we repeated the analysis in Caucasians. The other groups were not analyzed due to small sample sizes. Limiting analysis to Caucasians did not reveal a difference in colorectal cancer age of onset (P = 0.72 by the log-rank test; P = 0.94 by Wilcoxon's test). Median age of onset was 53 years in GSTM1 null subjects, compared to 51 years in GSTM1 gene carriers. Stratification by other factors also yielded no significant differences in age of onset. Other factors examined (by log-rank test) were MMR gene mutated (P = 0.94 for MLH1; P = 0.98 for MSH2), MMR gene mutation type (P = 0.64 for truncating/deleting mutation; P = 0.81 for missense mutation), and gender (P = 0.66 for males; P = 0.76 for females).

We found no significant association between the GSTM1 null genotype and earlier age of onset for colorectal cancer in our HNPCC population. In contrast, Moisio et al. (4) reported a 6-year earlier age of onset in HNPCC patients from the Finnish Cancer Registry who were GSTM1 null. There were several differences between the two studies that might account for the conflicting results.

Their study population was composed mostly of 150 MMR mutation carriers with a specific mutation (“Mutation 1”) in hMLH1. Of these 150 “Mutation 1” carriers, they found that 87 (58%) also carried the GSTM1 mutation and 44 (50.5%) of the 87 had colorectal cancer, with a median age of onset of 42 years. Conversely, nearly two thirds of our study population carried mutations in the hMSH2 gene and we included all mutations found in both hMLH1 and hMSH2 in our initial GSTM1 genotype analysis. For a more direct comparison, we also analyzed GSTM1 genotype in hMLH1 mutation carriers alone and still found no significance. However, as the hMLH1 mutation carriers composed only one third of our population, the sample size was much smaller in our study.

Also, the methods for calculating the median age at first diagnosis were different between the two studies. We used Kaplan-Meier survival analysis to determine the median age of onset. By this method, the median age of 50 years represents the age at which 50% of our population was cancer-free, thus includes the unaffected participants in the calculation. In their study, only colorectal cancer cases were used in the calculation. They reported median values of 43.5 years among patients who carried the GSTM1 gene and 37.5 years among GSTM1 null patients. For direct comparison, we calculated the median age at diagnosis by their methods. Again, we observed no difference between GSTM1 carriers and nulls, because the median age at diagnosis for both groups was 42 years.

Additionally, their study participants were Finnish while ours were largely American Caucasians with much more genetic heterogeneity. Although we repeated the analysis, limiting the population to Caucasians, we still found no difference between the GSTM1 genotypes. Thus, heterogeneity in susceptibility to HNPCC may be related to ethnic differences between our study populations as well as underlying germline MMR mutations.

In summary, while some metabolic and other genes might be responsible for the interindividual variation in cancer phenotype seen in HNPCC, GSTM1 genotype alone was not responsible for modifying the age of onset for colorectal cancer in this study. We know that there are other genetic factors that contribute to this variation, such as polymorphisms in cyclin D1 and N-acetyltransferase 2 (3, 7). Future studies that examine GSTM1 genotype combined with other genetic and/or environmental factors may reveal a modifier effect for GSTM1 in MMR gene mutation carriers.

Grant support: Grant CA70759 from the National Cancer Institute, by NIH Cancer Center Support Grant CA16672, and also by a cancer prevention fellowship from the National Cancer Institute, Grant R25 CA57730.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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