A feasibility study of perioperative vitamin D supplementation in patients undergoing colorectal cancer resection.

Background: Vitamin D supplementation improves colorectal cancer (CRC) survival outcomes in randomized trials. The aim of this study was to test the feasibility, safety and efficacy of vitamin D supplementation in the pre- and perioperative period in patients undergoing CRC surgery. Methods: Patients were given 3200IU oral cholecalciferol (D3) per day perioperatively. Serial serum 25-hydroxyvitamin (25OHD) was measured by liquid chromatography tandem mass spectrometry and compared to untreated CRC controls. 25OHD and C-reactive protein (CRP) levels were compared using adjusted generalized linear mixed-effects models. Results: A total of 122 patients underwent serial perioperative sampling, including 41 patients given high-dose perioperative supplementation. Supplementation was well-tolerated with no adverse or serious adverse events related to supplementation reported. Pre-operative supplementation increased 25OHD levels on the day of surgery (103.9 vs. 42.5 nmol/l, P = 8.2E-12). Supplementation increased 25OHD levels at all post-operative timepoints (P < 0.001) and attenuated the post-operative drop in 25OHD (46 vs. 24% drop, P = 3.0E-4). Rate of vitamin D peri-operative insufficiency was significantly less in those on supplementation (e.g., day 3-5, 14 vs. 84%, P = 1.41E-08), with multivariate modeling across all timepoints indicating a ∼59 nmol/l higher 25OHD compared to control patients (P = 3.7E-21). Post-operative CRP was lower in patients taking supplementation (e.g., day 3-5 timepoint; 129 vs. 81 mg/l, P = 0.04). Conclusion: High dose pre-operative vitamin D supplementation is associated with higher perioperative 25OHD levels, lower rates of vitamin D insufficiency and reduced early post-operative CRP. Alongside published evidence for a beneficial effect of vitamin D on CRC survival outcomes, these novel findings provide strong rationale for early initiation of vitamin D supplementation after a diagnosis of CRC.

Oral vitamin D supplementation induces transcriptomic changes in rectal mucosa that are linked to anti-tumour effects.

BACKGROUND: The risk for several common cancers is influenced by the transcriptomic landscape of the respective tissue-of-origin. Vitamin D influences in vitro gene expression and cancer cell growth. We sought to determine whether oral vitamin D induces beneficial gene expression effects in human rectal epithelium and identify biomarkers of response. METHODS: Blood and rectal mucosa was sampled from 191 human subjects and mucosa gene expression (HT12) correlated with plasma vitamin D (25-OHD) to identify differentially expressed genes. Fifty subjects were then administered 3200IU/day oral vitamin D3 and matched blood/mucosa resampled after 12 weeks. Transcriptomic changes (HT12/RNAseq) after supplementation were tested against the prioritised genes for gene-set and GO-process enrichment. To identify blood biomarkers of mucosal response, we derived receiver-operator curves and C-statistic (AUC) and tested biomarker reproducibility in an independent Supplementation Trial (BEST-D). RESULTS: Six hundred twenty-nine genes were associated with 25-OHD level (P < 0.01), highlighting 453 GO-term processes (FDR<0.05). In the whole intervention cohort, vitamin D supplementation enriched the prioritised mucosal gene-set (upregulated gene-set P < 1.0E-07; downregulated gene-set P < 2.6E-05) and corresponding GO terms (P = 2.90E-02), highlighting gene expression patterns consistent with anti-tumour effects. However, only 9 individual participants (18%) showed a significant response (NM gene-set enrichment P < 0.001) to supplementation. Expression changes in HIPK2 and PPP1CC expression served as blood biomarkers of mucosal transcriptomic response (AUC=0.84 [95%CI 0.66-1.00]) and replicated in BEST-D trial subjects (HIPK2 AUC=0.83 [95%CI 0.77-0.89]; PPP1CC AUC=0.91 [95%CI 0.86-0.95]). CONCLUSIONS: Higher plasma 25-OHD correlates with rectal mucosa gene expression patterns consistent with anti-tumour effects, and this beneficial signature is induced by short-term vitamin D supplementation. Heterogenous gene expression responses to vitamin D may limit the ability of randomised trials to identify beneficial effects of supplementation on CRC risk. However, in the current study blood expression changes in HIPK2 and PPP1CC identify those participants with significant anti-tumour transcriptomic responses to supplementation in the rectum. These data provide compelling rationale for a trial of vitamin D and CRC prevention using easily assayed blood gene expression signatures as intermediate biomarkers of response.

Low plasma vitamin D is associated with adverse colorectal cancer survival after surgical resection, independent of systemic inflammatory response.

OBJECTIVE: We assessed the effect of surgical resection of colorectal cancer (CRC) on perioperative plasma vitamin D (25OHD) and C-reactive protein (CRP) level. We investigated the relationship between circulating vitamin D level and CRC survival. DESIGN: We sequentially sampled 92 patients undergoing CRC resection, and measured plasma 25OHD and CRP. For survival analyses, we assayed 25OHD and CRP in two temporally distinct CRC patient cohorts (n=2006, n=2100) and investigated the association between survival outcome, circulating vitamin D and systemic inflammatory response. RESULTS: Serial sampling revealed a postoperative fall (mean 17.3 nmol/L; p=3.6e-9) in plasma 25OHD (nadir days 1-2). CRP peaked 3-5 days postoperatively (143.1 mg/L; p=1.4e-12), yet the postoperative fall in 25OHD was independent of CRP. In cohort analyses, 25OHD was lower in the 12 months following operation (mean=48.8 nmol/L) than preoperatively (54.8 nmol/L; p=1.2e-5) recovering after 24 months (52.2 nmol/L; p=0.002). Survival analysis in American Joint Committee on Cancer stages I-III demonstrated associations between 25OHD tertile and CRC mortality (HR=0.69; 95% CI 0.46 to 0.91) and all-cause mortality (HR=0.68; 95% CI 0.50 to 0.85), and was independent of CRP. We observed interaction effects between plasma 25OHD and rs11568820 genotype (functional VDR polymorphism) with a strong protective effect of higher 25OHD only in patients with GG genotype (HR=0.51; 95% CI 0.21 to 0.81). We developed an online tool for predicted survival (https://apps.igmm.ed.ac.uk/mortalityCalculator/) that incorporates 25OHD with clinically useful predictive performance (area under the curve 0.77). CONCLUSIONS: CRC surgery induces a fall in circulating 25OHD. Plasma 25OHD level is a prognostic biomarker with low 25OHD associated with poorer survival, particularly in those with rs11568820 GG genotype. A randomised trial of vitamin D supplementation after CRC surgery has compelling rationale.

The impact of vitamin D pathway genetic variation and circulating 25-hydroxyvitamin D on cancer outcome: systematic review and meta-analysis.

BACKGROUND: Vitamin D has been linked with improved cancer outcome. This systematic review and meta-analysis investigates the relationship between cancer outcomes and both vitamin D-related genetic variation and circulating 25-hydroxyvitamin D (25OHD) concentration. METHODS: A systematic review and meta-analysis of papers until November 2016 on PubMed, EMBASE and Web of Science pertaining to association between circulating vitamin D level, functionally relevant vitamin D receptor genetic variants and variants within vitamin D pathway genes and cancer survival or disease progression was performed. RESULTS: A total of 44 165 cases from 64 studies were included in meta-analyses. Higher 25OHD was associated with better overall survival (hazard ratio (HR=0.74, 95% CI: 0.66-0.82) and progression-free survival (HR=0.84, 95% CI: 0.77-0.91). The rs1544410 (BsmI) variant was associated with overall survival (HR=1.40, 95% CI: 1.05-1.75) and rs7975232 (ApaI) with progression-free survival (HR=1.29, 95% CI: 1.02-1.56). The rs2228570 (FokI) variant was associated with overall survival in lung cancer patients (HR=1.29, 95% CI: 1.0-1.57), with a suggestive association across all cancers (HR=1.26, 95% CI: 0.96-1.56). CONCLUSIONS: Higher 25OHD concentration is associated with better cancer outcome, and the observed association of functional variants in vitamin D pathway genes with outcome supports a causal link. This analysis provides powerful background rationale to instigate clinical trials to investigate the potential beneficial effect of vitamin D in the context of stratification by genotype.

The contributions of adjusted ambient ultraviolet B radiation at place of residence and other determinants to serum 25-hydroxyvitamin D concentrations.

BACKGROUND: Solar ultraviolet B (UVB) radiation is the major source of vitamin D (vitD) for humans. OBJECTIVES: To describe ambient UVB radiation at wavelengths that induce vitD synthesis (vitD-UVB) in Scotland, and to examine the relationship to serum 25-hydroxyvitamin D (25OHD). METHODS: We estimated the average vitD-UVB dose for each day of the year and for each postcode area in Scotland, using the Tropospheric Emission Monitoring Internet Service database. Cumulative and weighted vitD-UVB (CW-vitD-UVB) exposure at place of residence was calculated for each participant. Plasma 25OHD was assayed in 1964 healthy participants. RESULTS: Significant seasonal and geographical variation in vitD-UVB was observed. Ambient vitD-UVB exposure at place of residence was significantly associated with plasma 25OHD (P < 0·01). An average increase in 25OHD of 1 ng mL(-1) was observed for every 1000 mJ cm(-2) higher CW-vitD-UVB dose or for every 2·5 µg of daily supplement taken. Adequate 25OHD concentration (> 16 ng mL(-1)) was observed in the majority when CW-vitD-UVB dose was > 6000 mJ cm(-2), a level of ambient radiation achieved only in summer months in Scotland. When predicting vitD deficiency, dramatic improvement in the area under the curve was observed (from 0·55 to 0·70) after CW-vitD-UVB dose was added to the model, in addition to a range of other covariates. CONCLUSIONS: Ambient vitD-UVB can be a useful predictor of vitD status. Geotemporally mapped measurements of vitD-UVB can be used as a proxy for vitD status or as a covariate in epidemiological research, particularly if 25OHD is unavailable.

A simple method to overcome the inhibitory effect of heparin on DNA amplification.

BACKGROUND: Genetic material from large patient cohorts is increasingly central to translational genetic research. However, patient blood samples are a finite resource and their supply and storage are often dictated by clinical and not research protocols. Our experience supports difficulty in amplifying DNA from blood stored in herparin; a scenario that other researchers may have or will encounter. This technical note describes a number of simple steps that enable successful PCR amplification. METHODS: DNA was extracted using the Illustra Nucleon Genomic DNA Extraction Kit. PCR amplification was attempted using a number of commercially available PCR mastermixes. RESULTS: PCR DNA amplification failed using ReddyMix™ PCR Master Mix, Thermo-Start® (Thermo Scientific Inc. US) and ZymoTaq™ (Zymo research, US) PCR mastermixes, as demonstrated absence of products on gel electrophoresis. However, using the Invitrogen™ (Thermo Scientific Inc., US) Platinum® Taq DNA Polymerase, PCR products were identified on a 1% agarose gel for all samples. PCR products were cleaned with ExoSAP-IT® (Affymetrix Inc., US) and a sequencing reaction undertaken using a standard Big Dye protocol. Subsequent genotyping was successful for all samples for alleles at the CDH1 locus. CONCLUSION: From our experience a standard phenol/chloroform purification and using the Invitrogen™ Platinum® Taq has enabled the amplification of whole blood samples taken into lithium heparin and stored frozen for up to a month. This simple method may enable investigators to utilise blood taken in lithium heparin for DNA extraction and amplification.

A large-scale meta-analysis to refine colorectal cancer risk estimates associated with MUTYH variants.

BACKGROUND: defective DNA repair has a causal role in hereditary colorectal cancer (CRC). Defects in the base excision repair gene MUTYH are responsible for MUTYH-associated polyposis and CRC predisposition as an autosomal recessive trait. Numerous reports have suggested MUTYH mono-allelic variants to be low penetrance risk alleles. We report a large collaborative meta-analysis to assess and refine CRC risk estimates associated with bi-allelic and mono-allelic MUTYH variants and investigate age and sex influence on risk. METHODS: MUTYH genotype data were included from 20 565 cases and 15 524 controls. Three logistic regression models were tested: a crude model; adjusted for age and sex; adjusted for age, sex and study. RESULTS: all three models produced very similar results. MUTYH bi-allelic carriers demonstrated a 28-fold increase in risk (95% confidence interval (CI): 6.95-115). Significant bi-allelic effects were also observed for G396D and Y179C/G396D compound heterozygotes and a marginal mono-allelic effect for variant Y179C (odds ratio (OR)=1.34; 95% CI: 1.00-1.80). A pooled meta-analysis of all published and unpublished datasets submitted showed bi-allelic effects for MUTYH, G396D and Y179C (OR=10.8, 95% CI: 5.02-23.2; OR=6.47, 95% CI: 2.33-18.0; OR=3.35, 95% CI: 1.14-9.89) and marginal mono-allelic effect for variants MUTYH (OR=1.16, 95% CI: 1.00-1.34) and Y179C alone (OR=1.34, 95% CI: 1.01-1.77). CONCLUSIONS: overall, this large study refines estimates of disease risk associated with mono-allelic and bi-allelic MUTYH carriers.

Association studies on 11 published colorectal cancer risk loci.

BACKGROUND: Recently, several genome-wide association studies (GWAS) have independently found numerous loci at which common single-nucleotide polymorphisms (SNPs) modestly influence the risk of developing colorectal cancer. The aim of this study was to test 11 loci, reported to be associated with an increased or decreased risk of colorectal cancer: 8q23.3 (rs16892766), 8q24.21 (rs6983267), 9p24 (rs719725), 10p14 (rs10795668), 11q23.1 (rs3802842), 14q22.2 (rs4444235), 15q13.3 (rs4779584), 16q22.1 (rs9929218), 18q21.1 (rs4939827), 19q13.1 (rs10411210) and 20p12.3 (rs961253), in a Swedish-based cohort. METHODS: The cohort was composed of 1786 cases and 1749 controls that were genotyped and analysed statistically. Genotype-phenotype analysis, for all 11 SNPs and sex, age of onset, family history of CRC and tumour location, was performed. RESULTS: Of eleven loci, 5 showed statistically significant odds ratios similar to previously published findings: 8q23.3, 8q24.21, 10p14, 15q13.3 and 18q21.1. The remaining loci 11q23.1, 16q22.1, 19q13.1 and 20p12.3 showed weak trends but somehow similar to what was previously published. The loci 9p24 and 14q22.2 could not be confirmed. We show a higher number of risk alleles in affected individuals compared to controls. Four statistically significant genotype-phenotype associations were found; the G allele of rs6983267 was associated to older age, the G allele of rs1075668 was associated with a younger age and sporadic cases, and the T allele of rs10411210 was associated with younger age. CONCLUSIONS: Our study, using a Swedish population, supports most genetic variants published in GWAS. More studies are needed to validate the genotype-phenotype correlations.

Common genetic variants at the MC4R locus are associated with obesity, but not with dietary energy intake or colorectal cancer in the Scottish population.

BACKGROUND: Common single-nucleotide polymorphism (SNP) variants around the melanocortin 4 receptor (MC4R) gene have recently been associated with obesity risk and insulin resistance. Obesity is a known risk factor for colorectal cancer (CRC) and we hypothesized that there might be a common inherited genetic component. METHODS AND RESULTS: Four of the variants reported earlier were genotyped and tested for association with body mass index (BMI), waist circumference (WC), dietary energy intake (DEI) and CRC. Using a case-control genetic association study, we replicated the association with BMI (P=0.0001, additive genetic effect=0.37 kg/m(2)) and WC (P=0.005, additive genetic effect=0.70 cm) using over 3800 individuals. However, there was no association between these variants and CRC risk. Rare (highly penetrant) variants within the MC4R gene have been shown to influence eating behaviour and hyperphagia. We hypothesized that the newly identified common variants might also influence hyperphagia. Using DEI data recorded from a validated food frequency questionnaire, we found no significant genetic association between MC4R SNPs and DEI. CONCLUSIONS: As the MC4R locus explains only 0.28% of the BMI and 0.14% of the WC phenotypic variance in the Scottish population, most of the genetic contribution to obesity remains to be identified.

Germline mutation prevalence in the base excision repair gene, MYH, in patients with endometrial cancer.

Germline mutations in the base excision repair gene, MutY human homolog (MYH), have recently been associated with a recessively inherited multiple adenoma polyposis syndrome and colorectal cancer. The spectrum of extracolonic lesions is still being characterized, although preliminary reports suggest that bi-allelic mutation carriers may share some of the clinical features of other hereditary colon cancer syndromes. Of 225 endometrial cancer patients, we identified one individual as a compound heterozygote, carrying mutations Y165C and G382D of MYH, and five individuals with heterozygous defects (three G382D and two Y165C). The patient with the bi-allelic Y165C/G382D mutation also had a sebaceous carcinoma, a feature of Muir-Torre syndrome. Although several intronic polymorphisms were detected in the heterozygous carriers, no other pathogenic variants were identified. While not conclusive, this novel and interesting finding provides evidence that bi-allelic germline mutations in MYH may increase susceptibility to endometrial cancer.

Contribution of the NOD1/CARD4 insertion/deletion polymorphism +32656 to inflammatory bowel disease in Northern Europe.

BACKGROUND: NOD1/CARD4 and NOD2/CARD15 are both intracellular pattern-recognition receptors. The NOD1/CARD4 gene lies within a previously described inflammatory bowel disease (IBD) locus (7p14). An association has been suggested between the NOD1/CARD4+32656 deletion*1 variant of a complex deletion*1/insertion*2 polymorphism and IBD in 1 recent study in Europe. Our aim was to assess the influence of NOD1/CARD4+32656 on disease susceptibility and phenotype in the Scottish and Swedish IBD populations. METHODS: A total of 3,962 individuals (1,791 IBD patients, 522 parents, 1,649 healthy controls) from 2 independent populations (Scotland and Sweden) were genotyped for NOD1/CARD4+32656 A/C by TaqMan and direct sequencing. Case-control, Transmission Disequilibrium Testing (TDT) and detailed genotype-phenotype (Montreal) analyses were performed. The case-control analysis had 80% power to detect an effect size of odds ratio (OR) 1.21 for IBD. RESULTS: In case-control analyses in Scottish and Swedish patients, none of the genotypes studied in IBD, Crohn's disease (CD) or ulcerative colitis (UC), differed significantly from controls (deletion*1 allelic frequency 73.9%, 73.6%, 73.9%, and 73.6%, respectively: all P > 0.8). No epistatic interaction with NOD2/CARD15 was seen for CD susceptibility. TDT analysis in our Scottish early onset cohort was negative. CONCLUSIONS: This variant allele of NOD1/CARD4+32656 is not associated with a strong effect on susceptibility to IBD in children and adults in Northern Europe. A gene-wide haplotype-based approach may be preferable to analysis of individual variants to assess the contribution of the NOD1/CARD4 gene to IBD.

Association of MUTYH and colorectal cancer.

Mutations in the MUTYH gene have been reported to be associated with increased risk of developing colorectal cancer. In this study, we confirmed this association using original data on 928 colorectal cancer cases and 845 healthy controls from Scotland. We then conducted a meta-analysis from published data on the association between mutations at MUTYH and colorectal cancer risk. We show for the first time a small but significant mono-allelic effect with a genotype relative risk (GRR) of 1.27 (95% confidence interval (CI): 1.01-1.61), and confirm and give a more precise estimate of the strong bi-allelic effect with an estimated GRR of 117 (95% CI: 74-184). This study underscores the need for large sample sizes in order to identify small gene effects when the disease allele frequency is low.

Prevalence of family history of colorectal cancer in the general population.

BACKGROUND: Robust estimates of the prevalence of a family history of colorectal cancer in the general population are essential to inform planning of provision for colonoscopic surveillance and for clinical genetics services. However, there is a paucity of high-quality data. METHODS: Computerized record linkage was used to assess systematically the family history of 160 cancer-free community subjects and thereby provide prevalence data that are independent of participant recall. The data set comprised 2664 first- and second-degree relatives of study subjects, with 148 068 years at risk. RESULTS: Of people in the 30-70 years age range, 9.4 (95 per cent confidence interval (c.i.) 5.8 to 14.9) per cent had a first-degree relative affected by colorectal cancer, and 28.8 (95 per cent c.i. 22.3 to 36.2) per cent had an affected first- or second-degree relative. Between 0 and 3.1 per cent of study subjects merited colonic surveillance, depending on the stringency of the guidelines used. CONCLUSION: An appreciable proportion of the general population has a relative affected by colorectal cancer, sufficient to merit screening under certain criteria. In the absence of good-quality evidence supporting colonoscopic surveillance in groups at moderate risk, these data directly inform the planning of services for people with a family history of colorectal cancer. However, the clinical risk and financial implications of screening should be taken into account.

Prognosis in DNA mismatch repair deficient colorectal cancer: are all MSI tumours equivalent?

Microsatellite instability (MSI) in colorectal tumours is the hallmark of defective DNA mismatch repair (MMR) and high level MSI can be detected in up to 15% of incident colorectal cancers. MSI in sporadic colorectal tumours is primarily due to epigenetic silencing of MLH1 while MSI is almost universal in tumours from HNPCC family members due to germline MMR gene mutation with loss or mutational inactivation of the second copy as a somatic event. There is evidence that tumour MSI is associated with a better outcome than the generality of large bowel malignancy. However, although MSI occurs in both sporadic colorectal cancer and in tumours arising in patients with germline MMR gene mutations, cancer survival should not be considered to be equivalent for these two groups with MSI tumours simply because both exhibit similarities in molecular phenotype. Here, we review the evidence on prognosis in patients with sporadic MSI tumours compared to those who have inherited a germline DNA MMR repair gene defect. In addition, we explore whether there are variables that afford opportunity to distinguish three groups on the basis of MSI status, namely: sporadic MSI tumours; MSI tumours in carriers of germline MMR gene defects; microsatellite stable (MSS) tumours. Differences in prognosis between these three groups is important because it underpins the rationale for surveillance and early identification of tumours in MMR gene carriers, as well as refining understanding of the influence of MSI on cancer progression. Furthermore, we discuss the effect of MSI on the effectiveness of chemotherapy regimens.

Mismatch repair genes hMLH1 and hMSH2 and colorectal cancer: a HuGE review.

Evidence to support a role for the mismatch repair genes human mutL homolog 1 (hMLH1) and human mutS homolog 2 (hMSH2) in the etiology of colorectal cancer has come from linkage analysis, segregation studies, and molecular biologic analysis. More recently, carriers of potentially pathogenic mutations in the hMLH1/hMSH2 genes have consistently been shown to be at a greatly increased risk of developing colorectal cancer compared with the general population. When considered together, the available evidence shows a strong, consistent, and biologically plausible association between mismatch repair gene mutations and colorectal cancer. The penetrance of mutations in hMLH1/hMSH2 is incomplete and is significantly higher in males (approximately 80%) than in females (approximately 40%). To date, evidence for gene-gene or gene-environment interactions is limited, although preliminary studies have revealed a number of avenues that merit exploration. Population screening for mutation carriers is not currently a feasible option, and mutation analysis remains restricted to either relatives of mutation carriers or colorectal cancer cases selected on the basis of phenotype.

Whole-gene APC deletions cause classical familial adenomatous polyposis, but not attenuated polyposis or "multiple" colorectal adenomas.

Familial adenomatous polyposis (FAP) is a dominantly inherited colorectal tumor predisposition that results from germ-line mutations in the APC gene (chromosome 5q21). FAP shows substantial phenotypic variability: classical polyposis patients develop more than 100 colorectal adenomas, whereas those with attenuated polyposis (AAPC) have fewer than 100 adenomas. A further group of individuals, so-called "multiple" adenoma patients, have a phenotype like AAPC, with 3-99 polyps throughout the colorectum, but mostly have no demonstrable germ-line APC mutation. Routine mutation detection techniques fail to detect a pathogenic APC germ-line mutation in approximately 30% of patients with classical polyposis and 90% of those with AAPC/multiple adenomas. We have developed a real-time quantitative multiplex PCR assay to detect APC exon 14 deletions. When this technique was applied to a set of 60 classical polyposis and 143 AAPC/multiple adenoma patients with no apparent APC germ-line mutation, deletions were found exclusively in individuals with classical polyposis (7 of 60, 12%). Fine-mapping of the region suggested that the majority (6 of 7) of these deletions encompassed the entire APC locus, confirming that haploinsufficiency can result in a classical polyposis phenotype. Screening for germ-line deletions in APC mutation-negative individuals with classical polyposis seems warranted.

Mutation frequency in coding and non-coding repeat sequences in mismatch repair deficient cells derived from normal human tissue.

Repetitive tracts within the coding regions of TGFBR2 and BAX are frequently mutated in mismatch repair deficient tumours and are implicated in tumour progression. However, there has been little study of the balance between selection pressure and inherent instability at sequences within these genes. To determine whether TGFBR2 and BAX are inherently prone to mutations in the presence of MMR defects, we studied MMR deficient cells derived from B-lymphocytes. By analysing cells derived from normal tissue we aimed to minimize the effects of selection pressures that bias the apparent frequency of mutation. We definitively show that certain sequences, usually repaired by MMR, are inherently unstable. Using a small pool PCR technique we confirmed these cells exhibit microsatellite instability. Additionally, we demonstrate that MMR deficiency results in an excess of mutations, specifically at the poly(A)(10) tract compared to other regions of the TGFBR2 gene (P<0.001). Conversely, an excess of mutations does not appear to arise at the poly(G)(8) tract of the BAX gene. These studies provide insight into the mechanism by which TGFBR2 and BAX genes become mutated during tumorigenesis. These findings invoke the notion of "unmasking" specific hypermutable sequences in particular genes adding further complexity to the concept of the mutator phenotype.

Hypermutability at a poly(A/T) tract in the human germline.

Poly(A/T) tracts are abundant simple sequence repeats (SSRs) within the human genome. They constitute part of the coding sequence of a variety of genes, encoding polylysine stretches that are important for protein function. Assessment of poly(A/T) tract stability is also used to identify microsatellite unstable colorectal cancers, which are characteristic of tumours defective in DNA mismatch repair. Despite their importance, little is known about the stability of poly(A/T) SSRs in the human germline. We have determined the stability of a paradigm poly(A/T) tract, BAT-40, by study of population allele frequencies, mutation frequency in families and mutation frequency in sperm DNA. We show that the locus is polymorphic, with a level of heterozygosity of 59.7%. Germline mutation was observed in 13 of 187 germline transmissions (7.0%) in 10 families suggesting BAT-40 is unstable in the germline. Further evidence for germline instability at BAT-40 was provided by small pool PCR analysis of matched blood and sperm DNA templates, revealing a significantly elevated frequency of mutation in the germline (P < 0.001). These findings provide insight into poly(A/T) tract stability in the germline. They also have relevance to the study of gene expression and to determination of microsatellite instability in tumours.

Early-onset colorectal cancer with stable microsatellite DNA and near-diploid chromosomes.

Colorectal cancer has been described in terms of genetic instability selectively affecting either microsatellite sequences (MIN) or chromosome number and structure (CIN). A subgroup with apparently stable, near-diploid chromosomes and stable microsatellites (MACS) also exists. These distinctions are important, partly because of their value in highlighting different pathways of carcinogenesis, and partly because of their direct relevance to prognosis. Study of early-onset cancer has often proved a fruitful resource for the identification of the nature and function of cancer susceptibility genes. In a study of colorectal cancer with stable microsatellite DNA, we describe 22 early-onset tumours (mean age=33), compared with 16 late-onset tumours (mean age=68). Both groups contained carcinomas with the MACS phenotype, characterized by near diploid DNA content, as defined by flow cytometry, and minimal chromosome arm deletion or amplification (six or less events per genome), determined by comparative genomic hybridization (CGH). Minimal chromosome imbalance correlated strongly with diploid DNA content (P<0.001). The proportion of MACS cancers was significantly greater in early-onset as compared to late-onset tumours (64 vs 13%, P=0.005). Of the chromosome arm imbalances commonly observed in late-onset tumours, only 18q- was observed more than twice amongst the 14 early-onset MACS tumours. Seventy-nine per cent of these MACS tumours were located in the distal colon, and 69% were at advanced clinico-pathological stages (with lymph node or distant metastasis). A positive family history of colorectal or other cancers was elicited in seven patients in the MACS early-onset group, and one additional patient in this group had a metachronous ovarian cancer. The results suggest that MACS cancer may have a genetic basis different from either MIN or CIN, and further studies of these cancers may lead to discovery of new mechanisms of colorectal carcinogenesis and cancer susceptibility.