Recently our group completed a genome-wide linkage study investigating Australian and Spanish families with inherited risk of colorectal cancer (CRC). A minor linkage peak from that study located on chromosome 1 correlates with the location of a known CRC risk-modifying gene, prostaglandin synthase (PTGS2). PTGS2 encodes the inducible prostaglandin synthase enzyme cyclooxygenase-2 (COX-2). Prostaglandins are implicated in the initiation of carcinogenesis and progression of tumours. Sequencing of PTGS2 in a small subset of affected individuals identified a high frequency of the minor C allele of single nucleotide polymorphism rs5275. We then genotyped the rs5275 polymorphism in 183 affected and 223 unaffected individuals from our CRC predisposed families. Tests for association in the presence of linkage were made using family-based association tests. The C allele was found to be significantly associated (P<0.01) with diagnosis of hereditary non-syndromic CRC (P=0.0094, dominant model) and an earlier age of diagnosis (P=0.0089, heterozygous-advantage model). Interestingly, by stratifying the age of diagnosis data, we observed a speculative gender-discordant effect. Relative to other groups, female CC carriers were diagnosed less when young, but by 60 years of age were the most at risk group. Conversely, CT carriers of both genders showed a consistently earlier diagnosis relative to TT carriers. Our results suggest potential differential age-and gender-dependent efficacies of chemopreventative COX-2 inhibitors in the context of non-syndromic colorectal cancer.
PTGS2; COX-2; rs5275; colorectal; cancer; hereditary
Genome-wide linkage studies have identified the 9q22 chromosomal region as linked with colorectal cancer (CRC) predisposition. A candidate gene in this region is transforming growth factor β receptor 1 (TGFBR1). Investigation of TGFBR1 has focused on the common genetic variant rs11466445, a short exonic deletion of nine base pairs which results in truncation of a stretch of nine alanine residues to six alanine residues in the gene product. While the six alanine (*6A) allele has been reported to be associated with increased risk of CRC in some population based study groups this association remains the subject of robust debate. To date, reports have been limited to population-based case–control association studies, or case–control studies of CRC families selecting one affected individual per family. No study has yet taken advantage of all the genetic information provided by multiplex CRC families.
We have tested for an association between rs11466445 and risk of CRC using several family-based statistical tests in a new study group comprising members of non-syndromic high risk CRC families sourced from three familial cancer centres, two in Australia and one in Spain.
We report a finding of a nominally significant result using the pedigree-based association test approach (PBAT; p = 0.028), while other family-based tests were non-significant, but with a p-value <; 0.10 in each instance. These other tests included the Generalised Disequilibrium Test (GDT; p = 0.085), parent of origin GDT Generalised Disequilibrium Test (GDT-PO; p = 0.081) and empirical Family-Based Association Test (FBAT; p = 0.096, additive model). Related-person case–control testing using the “More Powerful” Quasi-Likelihood Score Test did not provide any evidence for association (MQLS; p = 0.41).
After conservatively taking into account considerations for multiple hypothesis testing, we find little evidence for an association between the TGFBR1*6A allele and CRC risk in these families. The weak support for an increase in risk in CRC predisposed families is in agreement with recent meta-analyses of case–control studies, which estimate only a modest increase in sporadic CRC risk among 6*A allele carriers.
TGFBR1; 6*A; rs11466445; Colorectal; Cancer; Hereditary
With 14.234 diagnoses and over 4047 deaths reported in 2007, colorectal cancer (CRC) is the second most common cancer and second most common cause of cancer-related mortality in Australia. The direct treatment cost has recently been estimated to be around AU$1.2 billion for the year 2011, which corresponds to a four-fold increase, compared the cost reported in 2001. Excluding CRCs due to known rare genetic disorders, 20% to 25% of all CRCs occur in a familial aggregation setting due to genetic variants or shared environmental risk factors that are yet to be characterised. A targeted screening strategy addressed to this segment of the population is a potentially valuable tool for reducing the overall burden of CRC.
We developed a Markov model to assess the cost-effectiveness of three screening strategies offered to people at increased risk due to a strong family history of CRC. The model simulated the evolution of a cohort of 10,000 individuals from age 50 to 90 years. We compared screening with biennial iFOBT, five-yearly colonoscopy and ten-yearly colonoscopy versus the current strategy of the Australian National Bowel Cancer Screening Programme (i.e. base case).
Under the NBCSP scenario, 6,491 persons developed CRC with an average screening lifetime cost of AU$3,441 per person. In comparison, screening with biennial iFOBT, colonoscopy every ten years, and colonoscopy every five years reduced CRC incidence by 27%, 35% and 60%, and mortality by 15%, 26% and 46% respectively. All three screening strategies had a cost under AU$50,000 per life year gained, which is regarded as the upper limit of acceptable cost-effectiveness in the Australian health system. At AU$12,405 per life year gained and an average lifetime expectancy of 16.084 years, five-yearly colonoscopy screening was the most cost-effective strategy.
The model demonstrates that intensive CRC screening strategies targeting people at increased risk would be cost-effective in the Australian context. Our findings provide evidence that substantial health benefits can be generated from risk-based CRC screening at a relatively modest incremental cost.
Colorectal cancer; Family history; Screening; Cost-effectiveness
The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests.
Combined epigenomic methods – activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment – were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP).
Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas.
This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.
Colorectal cancer; DNA methylation; Biomarker
Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide and places a major economic burden on the global health care system. The time frame for development from premalignant to malignant disease typically spans 10-15 years, and this latent period provides an ideal opportunity for early detection and intervention to improve patient outcomes. Currently, early diagnosis of CRC is hampered by a lack of suitable non-invasive biomarkers that are clinically or economically acceptable for population-based screening. New blood-based protein biomarkers for early detection of CRC are therefore urgently required. The success of clinical biomarker discovery and validation studies is critically dependent on understanding and adjusting for potential experimental, analytical, and biological factors that can interfere with the robust interpretation of results. In this review we outline some important considerations for research groups undertaking biomarker research with exemplars from our studies. Implementation of experimental strategies to minimise the potential effects of these problems will facilitate the identification of panels of biomarkers with the sensitivity and specificity required for the development of successful tests for the early detection and surveillance of CRC.
Biomarker; Bias; Colorectal cancer; Diagnostic; Discovery; Validation
In strict carnivorous domestic cats, a metabolic competition arises between the need to
use amino acids for gluconeogenesis and for protein synthesis both in health and disease.
The present study investigated the amino acid-sparing potential of propionic acid in cats
using dietary propionylated starch (HAMSP) supplementation. A total of thirty cats were
fed a homemade diet, supplemented with either HAMSP, acetylated starch (HAMSA) or celite
(Control) for three adaptation weeks. Propionylated starch was hypothesised to provide
propionic acid as an alternative gluconeogenic substrate to amino acids, whereas acetic
acid from HAMSA would not provide any gluconeogenic benefit. Post-adaptation, a 5-d total
faecal collection was carried out to calculate apparent protein digestibility
coefficients. Fresh faecal and blood samples were collected to analyse fermentation
endproducts and metabolites. The apparent protein digestibility coefficients did not
differ between supplements (P = 0·372) and were not affected by the
protein intake level (P = 0·808). Faecal propionic acid concentrations
were higher in HAMSP than in HAMSA (P = 0·018) and Control
(P = 0·003) groups, whereas concentrations of ammonia
(P = 0·007) were higher in HAMSA than in HAMSP cats. Tendencies for or
higher propionylcarnitine concentrations were observed in HAMSP compared with HAMSA
(P = 0·090) and Control (P = 0·037) groups, and for
tiglyl- + 3-methylcrotonylcarnitine concentrations in HAMSP as compared with Control
(P = 0·028) cats. Methylmalonylcarnitine concentrations did not differ
between groups (P = 0·740), but were negatively correlated with the
protein intake level (r –0·459, P = 0·016). These
results suggest that HAMSP cats showed more saccharolytic fermentation patterns than those
supplemented with HAMSA, as well as signs of sparing of valine in cats with a sufficient
Acylated starch; Domestic cats; Fermentation; Gluconeogenesis; Propionic acid; DS, degree of substitution; HAMSA, acetylated high-amylose maize starch; HAMSP, propionylated high-amylose maize starch
Australia has one of the highest incidences of colorectal cancer (CRC) in the world. In 2006, the federal government introduced a screening programme consisting of a one-off faecal occult blood test offered to people turning 50, 55 or 65 years. We conducted a population-based study to estimate CRC screening practices existing outside the current programme.
1887 unaffected subjects categorised “at or slightly above average risk” of CRC were selected from the Australasian Colorectal Cancer Family Registry. We calculated proportions of participants that reported appropriate, under- and over-screening according to national guidelines. We performed a logistic regression analysis to evaluate associations between over-screening and a set of socio-demographic factors.
Of 532 participants at average risk of CRC, eligible for screening, 4 (0.75%) reported appropriate screening, 479 (90%) reported never having been screened, 18 (3 %) reported some but less than appropriate screening and 31 (6%) reported over-screening. Of 412 participants aged 50 years or over, slightly above average risk of CRC, 1 participant (0.25%) reported appropriate screening, 316 (77%) reported no screening and 11 (3%) reported some but less than appropriate screening. Among participants under age 50 years, 2% of those at average risk, and 10% of those slightly above average risk reported over screening. Middle-aged people, those with a family history of CRC and those with a university degree were more likely to be over-screened.
Overall, the level of CRC screening participation was low and the vast majority of screening tests undertaken were inappropriate in terms of timing, modality or frequency.
The current models for colorectal cancer (CRC) are essentially linear in nature with a sequential progression from adenoma through to carcinoma. However, these views of CRC development do not explain the full body of published knowledge and tend to discount environmental influences. This paper proposes that CRC is a cellular response to prolonged exposure to cytotoxic agents (e.g., free ammonia) as key events within a sustained high-risk colonic luminal environment. This environment is low in substrate for the colonocytes (short chain fatty acids, SCFA) and consequently of higher pH with higher levels of free ammonia and decreased mucosal oxygen supply as a result of lower visceral blood flow. All of these lead to greater and prolonged exposure of the colonic epithelium to a cytotoxic agent with diminished aerobic energy availability. Normal colonocytes faced with this unfavourable environment can transform into CRC cells for survival through epigenetic reprogramming to express genes which increase mobility to allow migration and proliferation. Recent data with high protein diets confirm that genetic damage can be increased, consistent with greater CRC risk. However, this damage can be reversed by increasing SCFA supply by feeding fermentable fibre as resistant starch or arabinoxylan. High protein, low carbohydrate diets have been shown to alter the colonic environment with lower butyrate levels and apparently greater mucosal exposure to ammonia, consistent with our hypothesis. Evidence is drawn from in vivo and in vitro genomic and biochemical studies to frame experiments to test this proposition.
ammonia; colorectal cancer; dietary protein; resistant starch; short chain fatty acid
Our objective was to determine screening practices of unaffected people in the general population at moderately increased and potentially high risk of colorectal cancer (CRC) due to their family history of the disease. 1627 participants in the Australasian Colorectal Cancer Family Registry (ACCFR) study were classified into two CRC risk categories according to the strength of their family history of the disease. We calculated the proportion of participants that adhered to national CRC screening guidelines by age-group and for each familial risk category. We performed a multinomial logistic regression analysis to evaluate the associations between screening and socio-demographic factors. Of the 1236 participants at moderately increased risk of CRC, 70 (6%) reported having undergone guideline-defined “appropriate” screening, 251 (20%) reported some, but less than appropriate screening and 915 (74%) reported never having had any CRC screening test. Of the 392 participants at potentially high risk of CRC, 3 (1%) reported appropriate screening, 140 (36%) reported some, but less than appropriate screening and 249 (64%) reported never having had any CRC screening test. On average, those of middle-age, higher education and who had resided in Australia longer were more likely to have had screening for CRC. The uptake of recommended screening by unaffected people at the highest familial risk of developing CRC is extremely low. Guidelines for CRC screening are not being implemented in the population. More research is needed to identify the reasons so as to enable development of strategies to improve participation in screening.
colorectal cancer; family history; screening; guidelines
The acute response to genotoxic carcinogens in rats is an important model for researching cancer initiation events. In this report we define the normal rat colonic epithelium by describing transcriptional events along the anterior-posterior axis and then investigate the acute effects of azoxymethane (AOM) on gene expression, with a particular emphasis on pathways associated with the maintenance of genomic integrity in the proximal and distal compartments using whole genome expression microarrays.
There are large transcriptional changes that occur in epithelial gene expression along the anterior-posterior axis of the normal healthy rat colon. AOM administration superimposes substantial changes on these basal gene expression patterns in both the distal and proximal rat colonic epithelium. In particular, the pathways associated with cell cycle and DNA damage and repair processes appear to be disrupted in favour of apoptosis.
The healthy rats’ colon exhibits extensive gene expression changes between its proximal and distal ends. The most common changes are associated with metabolism, but more subtle expression changes in genes involved in genomic homeostasis are also evident. These latter changes presumably protect and maintain a healthy colonic epithelium against incidental dietary and environmental insults. AOM induces substantial changes in gene expression, resulting in an early switch in the cell cycle process, involving p53 signalling, towards cell cycle arrest leading to the more effective process of apoptosis to counteract this genotoxic insult.
Colorectal cancer; Azoxymethane; Rats; Gene expression
Up to 25% of colorectal cancer (CRC) may be caused by inherited genetic variants that have yet to be identified. Previous genome-wide linkage studies (GWLSs) have identified a new loci postulated to contain novel CRC risk genes amongst affected families carrying no identifiable mutations in any of the known susceptibility genes for familial CRC syndromes. To undertake a new GWLS, we recruited members from 54 non-syndromic families from Australia and Spain where at least two first-degree relatives were affected by CRC. We used single-nucleotide polymorphism arrays to genotype 98 concordant affected relative pairs that were informative for linkage analyses. We tested for genome-wide significance (GWS) for linkage to CRC using a quantile statistic method, and we found that GWS was achieved at the 5% level. Independently, using the PSEUDO gene-dropping algorithm, we also found that GWS for linkage to CRC was achieved (P=0.02). Merlin non-parametric linkage analysis revealed significant linkage to CRC for chromosomal region 10p15.3–p15.1 and suggestive linkage to CRC for regions on 14q and 9q. The 10p15.3–p15.1 has not been reported to be linked to hereditary CRC in previous linkage studies, but this region does harbour the Kruppel-like factor 6 (KLF6) gene that is known to be altered in common CRC. Further studies aimed at localising the responsible genes, and characterising their function will give insight into the factors responsible for susceptibility in such families, and perhaps shed further light on the mechanisms of CRC development.
colorectal cancer; linkage; 10p; 14q; 9q
Nutrigenetics and nutrigenomics hold much promise for providing better nutritional advice to the public generally, genetic subgroups and individuals. Because nutrigenetics and nutrigenomics require a deep understanding of nutrition, genetics and biochemistry and ever new ‘omic’ technologies, it is often difficult, even for educated professionals, to appreciate their relevance to the practice of preventive approaches for optimising health, delaying onset of disease and diminishing its severity. This review discusses (i) the basic concepts, technical terms and technology involved in nutrigenetics and nutrigenomics; (ii) how this emerging knowledge can be applied to optimise health, prevent and treat diseases; (iii) how to read, understand and interpret nutrigenetic and nutrigenomic research results, and (iv) how this knowledge may potentially transform nutrition and dietetic practice, and the implications of such a transformation. This is in effect an up-to-date overview of the various aspects of nutrigenetics and nutrigenomics relevant to health practitioners who are seeking a better understanding of this new frontier in nutrition research and its potential application to dietetic practice.
Dietetics; Nutrigenetics; Nutrigenomics; Nutrition Research; Personalised nutrition
Resistant starch (RS), fed as high amylose maize starch (HAMS) or butyrylated HAMS (HAMSB), opposes dietary protein-induced colonocyte DNA damage in rats. In this study, rats were fed Western-type diets moderate in fat (19%) and protein (20%) containing digestible starches [low amylose maize starch (LAMS) or low amylose whole wheat (LAW)] or RS [HAMS, HAMSB, or a whole high amylose wheat (HAW) generated by RNA interference] for 11 wk (n = 10/group). A control diet included 7% fat, 13% protein, and LAMS. Colonocyte DNA single-strand breaks (SSB) were significantly higher (by 70%) in rats fed the Western diet containing LAMS relative to controls. Dietary HAW, HAMS, and HAMSB opposed this effect while raising digesta levels of SCFA and lowering ammonia and phenol levels. SSB correlated inversely with total large bowel SCFA, including colonic butyrate concentration (R2 = 0.40; P = 0.009), and positively with colonic ammonia concentration (R2 = 0.40; P = 0.014). Analysis of gut microbiota populations using a phylogenetic microarray revealed profiles that fell into 3 distinct groups: control and LAMS; HAMS and HAMSB; and LAW and HAW. The expression of colonic genes associated with the maintenance of genomic integrity (notably Mdm2, Top1, Msh3, Ung, Rere, Cebpa, Gmnn, and Parg) was altered and varied with RS source. HAW is as effective as HAMS and HAMSB in opposing diet-induced colonic DNA damage in rats, but their effects on the large bowel microbiota and colonocyte gene expression differ, possibly due to the presence of other fiber components in HAW.
Animal studies show that increasing large bowel butyrate concentration through ingestion of butyrylated or resistant starches opposes carcinogen-induced tumorigenesis, which is consistent with population data linking greater fiber consumption with lowered colorectal cancer (CRC) risk. Butyrate has been shown to regulate the apoptotic response to DNA damage. This study examined the impact of increasing large bowel butyrate concentration by dietary butyrylated starch on the colonic epithelium of rats treated with the genotoxic carcinogen azoxymethane (AOM). Four groups of 10 male rats were fed AIN-93G based-diets containing either low amylose maize starch (LAMS), LAMS with 3% tributyrin, 10% high amylose maize starch (HAMS) or 10% butyrylated HAMS (HAMSB). HAMS and HAMSB starches were cooked by heating in water. After 4 weeks, rats were injected once with AOM and killed 6 h later. Rates of apoptosis and proliferation were measured in colonic epithelium. Short-chain fatty acid concentrations in large bowel digesta and hepatic portal venous plasma were higher in HAMSB than all other groups. Apoptotic rates in the distal colon were increased by HAMSB and correlated with luminal butyrate concentrations but cellular proliferation rates were unaffected by diet. The increase in apoptosis was most marked in the base and proliferative zone of the crypt. Regulation of luminal butyrate using HAMSB increases the rates of apoptotic deletion of DNA-damaged colonocytes. We propose this pro-apoptotic function of butyrate plays a major role reducing tumour formation in the AOM-treated rat and that these data support a potential protective role of butyrate in CRC.
It was hypothesised that colorectal cancer (CRC) could be diagnosed in biopsies by measuring the combined expression of a small set of well known genes. Genes were chosen based on their role in either the breakdown of the extracellular matrix or with changes in cellular metabolism both of which are associated with CRC progression
Gene expression data derived from quantitative real-time PCR for the solute transporter carriers (SLCs) and the invasion-mediating matrix metalloproteinases (MMPs) were examined using a Linear Descriminant Analysis (LDA). The combination of MMP-7 and SLC5A8 was found to be the most predictive of CRC.
A combinatorial analysis technique is an effective method for both furthering our understanding on the molecular basis of some aspects of CRC, as well as for leveraging well defined cancer-related gene sets to identify cancer. In this instance, the combination of MMP-7 and SLC5A8 were optimal for identifying CRC.
Hammerhead ribozymes are RNA-based molecules which bind and cleave other RNAs specifically. As such they have potential as laboratory reagents, diagnostics and therapeutics. Despite having been extensively studied for 15 years or so, their wide application is hampered by their instability in biological media, and by the poor translation of cleavage studies on short substrates to long RNA molecules. This work describes a systematic study aimed at addressing these two issues.
A series of hammerhead ribozyme derivatives, varying in their hybridising arm length and size of helix II, were tested in vitro for cleavage of RNA derived from the carbamoyl phosphate synthetase II gene of Plasmodium falciparum. Against a 550-nt transcript the most efficient (t1/2 = 26 seconds) was a miniribozyme with helix II reduced to a single G-C base pair and with twelve nucleotides in each hybridising arm. Miniribozymes of this general design were targeted to three further sites, and they demonstrated exceptional cleavage activity. A series of chemically modified derivatives was prepared and examined for cleavage activity and stability in human serum. One derivative showed a 103-fold increase in serum stability and a doubling in cleavage efficiency compared to the unmodified miniribozyme. A second was almost 104-fold more stable and only 7-fold less active than the unmodified parent.
Hammerhead ribozyme derivatives in which helix II is reduced to a single G-C base pair cleave long RNA substrates very efficiently in vitro. Using commonly available phosphoramidites and reagents, two patterns of nucleotide substitution in this derivative were identified which conferred both good cleavage activity against long RNA targets and good stability in human serum.
Accurately identifying accessible sites in RNA is a critical
prerequisite for optimising the cleavage efficiency of hammerhead
ribozymes and other small nucleozymes. Here we describe a simple
RNase H-based procedure to rapidly identify hammerhead ribozyme-accessible
sites in gene length RNAs. Twelve semi-randomised RNA–DNA–RNA
chimeric oligonucleotide probes, known as ‘gapmers’,
were used to direct RNase H cleavage of transcripts with the specificity
expected for hammerhead ribozymes, i.e. after NUH sites (where H
is A, C or U). Cleavage sites were identified simply by the mobility
of RNase H cleavage products relative to RNA markers in denaturing
polyacrylamide gels. Sites were identified in transcripts encoding
human interleukin-2 and platelet-derived growth factor. Thirteen
minimised hammerhead ribozymes, miniribozymes (Mrz), were synthesised
and in vitro cleavage efficiency (37°C, pH
7.6 and 1 mM MgCl2) at each site was analysed. Of the
13 Mrz, five were highly effective, demonstrating good initial rate
constants and extents of cleavage. The speed and accuracy of this
method commends its use in screening for hammerhead-accessible sites.