Investigators > David Hughes > Research and Publications

The major goals of my research program are to make a contribution in delineating the inherited and somatic genetic factors together with nutritional factors and their molecular mechanisms that cause the initiation and progression of colorectal cancer (CRC). This is the principal disease on which my research is focused, but my work also includes similar projects in breast cancer (BC) and inflammatory bowel disease (IBD).

The separate project themes I wish to integrate in my genetic biomarker and genetic / nutritional epidemiology research program over the coming two years are described below.

1. Colorectal Cancer Genetic and Nutritional Epidemiology
2. Familial Colorectal Cancer Genetics
3. Gastrointestinal Disease Genetic and Nutritional Epidemiology
4. Colorectal Cancer Biomarkers for Detection, Prediction, and Prognosis
5. Infectious Agents and Colorectal Cancer
6. Metabolomics and Systems Biology

1. Colorectal Cancer Genetic and Nutritional Epidemiology
Studies are in progress and planned for the next few years investigating the effects of oxidative stress exposures, nutritional / dietary factors and potential chemopreventive agents such as Selenium (Se) and Vitamin D on the development and clinical outcome analyses of CRC using large cancer case control and prospective cohorts from several European collaborators (principally Dr Mazda Jenab, IARC, Lyon and Prof Pavel Vodicka, Prague, Czech Republic). I have also collected an Irish based case-control CRC and colorectal adenoma (CRA) series with around 400 cases and 500 controls. A new BC cohort from the Czech Republic (Dr Pavel Soucek) is now also available for study. These studies will be further integrated with analyses of nutritional biomarkers, genetic variation, gene expression, epigenetic markers, micro RNA profiling, and metabolomic profiling of these pathways.

Colorectal Cancer Risk: The Influence of Selenoprotein Gene Variants and Blood Selenium Status
The essential micronutrient selenium (Se) is incorporated as the amino acid selenocysteine in 25 functional selenoproteins implicated in cell protection from oxidative stress, redox control and inflammatory response. Previously, together with Professor John Hesketh at Newcastle University (England) we showed for the first time modification of CRC risk with functional variants of several selenoprotein pathway genes (Meplan et al, 2010). We are currently pursuing this work (via a grant I was awarded as PI by the Irish Health Research Board) in collaboration with Dr Mazda Jenab (IARC, Lyon, France) by examining variation in genes involved in Se metabolism using the large EPIC nested CRC cohort, comprising 1250 cases matched to an equal number of controls. We are also measuring the serum concentration of Se to examine the interaction between Se status and genetic variation.

However, this ongoing work restricts our analysis to a single Se status biomarker and only a few candidate variants in a limited number of selenoproteins (6/25) and it is not known which selenoproteins are critical in maintaining colonic health. We hypothesize that Se interactions due to a combination of dietary Se levels and variation in selenoprotein pathway genes plus other signalling pathways influenced by Se intake affect CRC development and survival outcomes.

We aim to examine this by a comprehensive assessment of 760 TagSNPs in the selenoprotein pathway, measurement of the major Se transport protein (SePP), and gene expression assays of key selenoproteins (current grant application to the Health Research Board, PI, D Hughes) using the EPIC, Irish and Czech CRC cohorts. The association between Se status and SePP levels with CRC risk will be assessed using conditional logistic regression, with multivariable adjustment for key confounding factors. Similar approaches will be used for assessment of CRC risk associated with genetic factors related to Se metabolism. Linear regression models will be employed to examine Se biomarker and miRNA associations. Gene-nutrient interactions will be assessed using interaction term approaches as well as pathway analysis approaches (multifactor dimensionality reduction (MDR), Bayesian and structural equation modeling). Sub-group analyses by sex and anatomical subsite of the colorectum will be conducted where appropriate.

Micro RNA Expression Analysis: Vitamin D, Insulin Signalling, and Selenium Pathways
Accumulating recent evidence also strongly implicates miRNA biology in CRC carcinogenesis. A current project grant application to the WCRF (PI = Mazda Jenab, IARC-EPIC, Lyon; co-applicants = Valerie Fedirko and David Hughes) will measure miRNA expression in plasma samples from the EPIC CRC cohort. We will integrate the levels of pre-diagnostic circulating miRNAs to examine their involvement in vitamin D and insulin signalling pathways and Se mediated CRC pathogenesis. These plasma analyses will use the flow cytometry Firefly Bioworks Inc platform (Cambridge, MA, USA) and represent a novel use of miRNA levels in relation to Se status and genetic background.

2. Familial Colorectal Cancer Genetics
I collaborate with Michael Farrell, clinical genetics counsellor, and Dr David Gallagher, medical oncologist, in the Mater Private hospital in Dublin, on the genotype and phenotype interactions in Lynch syndrome (LS) CRC families. LS is an inherited predisposition to a range of cancers, notably uterine cancer in females and colorectal cancer (CRC) in both sexes, caused by mutated mismatch repair (MMR) genes.

Genetic counselling of at-risk individuals is based on identification of the deleterious germline MMR gene mutation in an appropriate family member who has developed cancer. Approximately 25% of mismatch repair (MMR) variants are exonic nucleotide substitutions. Some result in the substitution of one amino acid for another in the protein sequence, so-called missense variants, while others are silent. The interpretation of the effect of missense and silent variants as deleterious or neutral is challenging. These variants are termed ‘variants of uncertain significance’ or VUS. Pre-symptomatic testing for clinical use is not recommended for relatives of individuals with these variants. These relatives, including non-carriers, are considered at high-risk as long as the contribution of the variant to disease causation cannot be determined.

We have recently published two studies in the journal Familial Cancer that use a multivariate approach to detail the likely deleterious effect of particular missense variants in these MMR genes (Farrell et al 2012; Farrell et al 2013). Our integrative strategy is required to adequately assess the clinical significance of a VUS, especially in the absence of segregation data from multiple families harbouring the same base pair substitution.

3. Gastrointestinal Disease Genetic and Nutritional Epidemiology
I am collaborating with Dr Maria O’Sullivan (Trinity Centre, St James’s Hospital, Dublin) on case-control association studies of genetic and environmental risk modifiers of gastrointestinal diseases. Our current and upcoming studies are examining variation in selenoprotein, inflammation, and Vitamin D gene pathways. We are also examining levels of 25(OH)D in the serum of our IBD patients and controls and the IBD component of the prospective EPIC cohort, and will seek funds to test the interaction with variation in the whole vitamin D metabolism gene pathway.

4. Colorectal Cancer Biomarkers for Detection, Prediction, and Prognosis
The program over the next few years will progress and expand ongoing investigations of potential genetic, epigenetic, protein and biochemical markers for early detection of advanced adenomas and CRCs, as well as markers of prognosis and therapy response in CRC.

Epigenetics -TFAP2E gene hypermethylation and CRC: In collaboration with Dr Marc Taenzer within the group of Prof. Matthias Ebert (Technical University, Munich and University Hospital Mannheim) we recently published in the New England Journal of Medicine that TFAP2E gene hypermethylation was associated with clinical non-responsiveness to chemotherapy in CRC (Ebert et al 2012). We wish to further test genetic variation in the TFAP2E and DKK pathways genes for association with disease risk, clinical outcome and epidemiological risk factors using EPIC, Czech, German and Irish CRC cohorts. I am also currently working on other methylation targets for CRC screening and prognosis with Dr Taenzer.

DNA Markers: A CRC detection biomarker system in commercial development is the Randox (a diagnostic company based in Northern Ireland) RanplexCRCTM mutation biochip. They have a stool DNA CRC screening assay for 28 mutations in 4 genes (APC, P53, KRAS, and BRAF) involved in CRC initiation and progression. They also have a version specifically to aid in the selection of patients for anti-EGFR therapy. This biochip rapidly detects (3 hours) 20 point mutations within the genes KRAS, BRAF and PIK3CA from tissue DNA (fresh frozen biopsies or formalin fixed tissue). I am collaborating with Randox to use these assays to type DNA from stool samples from Irish patients, and on adenoma and CRC tissue DNA. We see increased efficacy of this technique compared to Sanger sequencing and pyrosequencing (manuscript in progress and then future validation screening work in next 2 years).

miRNA Markers: In collaboration with Prof Martin Clynes (Dublin City University) we are profiling serum to define miRNAs (Taqman arrays) differently expressed between adenoma and cancer stages, and from healthy controls (manuscript in preparation and then future validation screening work in next 2 years).

Proteomics: I am collaborating with Professor Martin Clynes and Dr Paul Dowling in Dublin City University (DCU) on whole proteomic approaches to define predictive and prognostic protein biomarkers for CRC.

5. Infectious Agents and Colorectal Cancer
An estimated 15% or more of the cancer burden worldwide is attributable to known infectious agents. I have also recently started working on infectious agent biomarkers and CRC carcinogenesis. Some of this work is in collaboration with Dr Massimo Tommasino, head of the Infections and Cancer Biology Group, IARC, Lyon.

Dr Lorna Flanagan, a postdoctoral researcher in our group at the RCSI, has examined a bacterial strain (Fusobacterium nucleatum, Fn) newly suspected of involvement in CRC. This study provided further new evidence that this common gut bacterium is involved in CRC (Flanagan et al 2014 European Journal of Clinical Microbiology and Infectious Diseases [epub March 6, see publications]. The manuscript describes the presence of the Fn in tissue and stool samples from patients with CRC and colorectal adenomas (CRA). It confirms in European patients previous observations from North America that Fn is found more abundantly in disease biopsy samples of CRC patients compared to the matched normal biopsies. The research further showed that Fn infection levels may be associated with both adenoma progression from early to advanced stages and the transition from adenoma to cancer. Potentially the most important new finding of the study was the association observed between Fn infection and CRC prognosis. Cancer patients with low bacterial levels had a significantly longer overall survival time than patients with moderate and high levels of the bacterium. Finally, the study also showed that stool screening for Fn is a potential biomarker indicating the presence of colorectal neoplasia, but that this needs tested in a large study.

I would also like to further this work in the next 2 years by incorporating data on infectious status and nutritional exposures to my cohort studies on CRC risk and outcomes, and host genetic / nutritional status susceptibility to infection.

6. Metabolomics and Systems Biology
In collaboration with Dr Mazda Jenab and Dr Augustin Scalbert (IARC) we are currently using quantitative tandem mass spectrometry with a targeted metabolomic metabolite kite (Biocrates, Germany) to try to locate metabolite biomarkers of the adenoma to CRC progression. We are examining serum samples and in the future wish to examine matched sets of disease+normal tissue pairs from patients with CRCs and CRAs.

I also collaborate with Dr Paul Dowling (DCU) in a further recent metabolomic profiling analysis of my CRC and adenoma samples using the diagnostics and services company, Metabolon, where liquid and gas chromatography is coupled to single-stage mass spectrometers. In the coming year we will validate the key leads from this work. We propose to compare the data from the different analyses between the Lyon and Dublin experiments.

Publications (listed by research area topic)

Colorectal cancer: screening, biomarkers, nutrition, epidemiology, infectious agents, familial genetics

• Flanagan L, Schmid J, Neary P, Ebert MP, Soucek P, Kunicka T, Liska V, Bruha J, Dezeeuw N, Tommasino M, Jenab M, Prehn JH, Hughes DJ (2014) Fusobacterium nucleatum associates with stages of colorectal neoplasia development, colorectal cancer and disease outcome. European Journal of Clinical Microbiology & Infectious Diseases. March 6. [Epub ahead of print] PMID:24599709
• Kelley L, Swan N, Hughes DJ (2013). An analysis of the duplicate testing strategy of an Irish immunochemical FOBT colorectal cancer screening programme. Colorectal Disease, 15, e512–e521. PMID: 23746062
•Farrell MP, Hughes DJ, Drost M, Wallace AJ, Cummins RJ, Fletcher TA, Meany MA,Kay EW, de Wind N, Power DG, Andrews EJ, Green AJ, Gallagher DJ (2013). Multivariate analysis of MLH1 c.1664T>C (p.Leu555Pro) mismatch repair gene variant demonstrates its pathogenicity. Familial Cancer, 12, 741-747. PMID: 23712482
• Ebert MPA, Tänzer M, Balluff B, Burgermeister E, Kretzschmar AK, Hughes DJ, Tetzner R, Lofton-Day C, Rosenberg R, Reinacher-Schick A, Schulmann K, Hofheinz R, Röcken C, Keller G, Langer R, Stöhlmacher J, Schuster T, Ströbel P, Schmid R (2012). TFAP2E/DKK4 and Chemoresistance in Colorectal Cancer. New England Journal of Medicine, 366, 44-53. PMID:22216841
• Farrell MP, Hughes DJ, Berry IR, et al, Daly PA (2012) Clinical correlation and molecular evaluation confirm that the hMLH1 p.R182G (c.544a/g) mutation is pathogenic and causes lynch syndrome. Familial Cancer 11(3):509-518. PMID: 22773173
• Hughes DJ, Soucek P, Hlavatá I, Pardini B, Naccarati A, Vodickova L, Jenab M, and Vodicka P (2011) Variation in the Vitamin D receptor gene is not associated with Colorectal Cancer Risk in the Czech Republic. Journal of Gastrointestinal Cancer 42, 149-154. PMID: 20585998
• Hughes DJ, Hlavatá I, Soucek P, Pardini B, Naccarati A, Vodickova L, O’Morain C, and Vodicka P (2011) Ornithine Decarboxylase G316A Genotype and Colorectal Cancer Risk. Colorectal Disease 13, 860-864. PMID: 20456464
• Méplan C, Hughes DJ, Pardini B, Naccareti A, Soucek P, Vodickova L, Hlavata I, Vrana D, Vodicka P and Hesketh J (2010) Genetic Variants in Selenoprotein Genes Increase Risk of Colorectal Cancer. Carcinogenesis, Jun;31(6):1074-9. PMID: 20378690.

Gastrointestinal disease genetics
• Hughes DJ, McManus R, Neary P, O’Morain C, O’Sullivan M (2011) Common Variation in the Vitamin D Receptor Gene and Risk of Inflammatory Bowel Disease in an Irish Case-Control Study. Eur J Gastroenterol Hepatol 23, 807-812. PMID:21818054
• Murphy G, Thornton J, McManus R, Swan N, Ryan B, O’Morain CA, Hughes DJ, O’Sullivan M (2009) Association of gastric disease with polymorphisms in the inflammatory related genes IL-1B, IL-1RN, IL-10, TNF and TLR4. Eur J Gastroenterol Hepatol 21, 630–635. PMID: 19295440

Renal cancer genetics
• Morris MR, Hughes DJ, Tian YM, Ricketts CJ, Lau KW, Gentle D, Shuib S, Serrano-Fernandez P, Lubinski J, Wiesener MS, Pugh CW, Latif F, Ratcliffe PJ, Maher ER (2009) Mutation analysis of hypoxia-inducible factors HIF1A and HIF2A in renal cell carcinoma. Anticancer Res 29, 4337-43. PMID: 20032376

Breast cancer genetic epidemiology, familial genetics
• Hughes DJ (2008) Use of Association Studies to Define Genetic Modifiers of Breast Cancer Risk in BRCA1 and BRCA2 Mutation Carriers. Familial Cancer 7, 233-244. PMID: 18283561
• Sinilnikova OM, Antoniou AC, Simard J, Healey S, Léoné M, et al, Hughes DJ, et al, Easton D, Chenevix-Trench G, Stoppa-Lyonnet D, on behalf of CIMBA (2009) The TP53 Arg72Pro and MDM2 309G/T polymorphisms are not associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers. British J Cancer 101, 1456–1460. PMID: 19707196
• Antoniou AC, Sinilnikova OM, Simard J, Léoné M, Dumont M, Neuhausen SL, Struewing JP, Stoppa Lyonnet D, Barjhoux L, Hughes DJ, et al, Couch F, Easton DF and Chenevix-Trench G on behalf of CIMBA (2007). RAD51 135G/C modifies breast cancer risk among BRCA2 mutation carriers: Results from a combined analysis of 19 studies. Am J Hum Genet 81, 1186-1200. PMID: 17999359
• Cox A, Dunning AM, Garcia-Closas M, Balasubramanian S, Reed MW, Pooley KA, et al, Sangrajrang S, Hughes D, Odefrey F, et al, Pharoah PD, Easton DF; on behalf of the Breast Cancer Association Consortium (2007) A common coding variant in CASP8 is associated with breast cancer risk. Nature Genetics 39, 352-8. PMID: 17293864
• Couch FJ, Antoniou AC, Sinilnikova O, Vierkant RA, Pankratz VS, Fredericksen ZS, Stoppa-Lyonnet D, Coupier I, Hughes D, et al, Easton DF, Chenevix-Trench G on behalf of the Consortium of Investigators of Modifiers of BRCA1/2 (2007) AURKA F31I Polymorphism and Breast Cancer Risk in BRCA1 and BRCA2 Mutation Carriers: A CIMBA study. Cancer Epi Bio Prev 16, 1416-21. PMID: 17627006
• Sinilnikova OM, McKay JD, Tavtigian SV, Canzian F, DeSilva D, Biessy C, Monnier S, Dossus L, Boillot C, Gioia L, Hughes DJ, et al, Kaaks R (2007) Haplotype-Based Analysis of Common Variation in the Acetyl-CoA Carboxylase alpha Gene and Breast Cancer Risk: a Case-Control Study Nested within the European Prospective Investigation into Cancer and Nutrition. Cancer Epi Bio Prev 16, 409-15. PMID: 17372234
• Chenevix-Trench G, Milne RL, Antoniou AC, et al* (2007) An international initiative to identify genetic modifiers of cancer risk in BRCA1 and BRCA2 mutation carriers: the Consortium of Investigators of Modifiers of BRCA1 and BRCA2 (CIMBA). Breast Cancer Res 9, 104. (*CIMBA co-author)
• The Breast Cancer Association Consortium (BCAC) (2006) Commonly Studied Single-Nucleotide Polymorphisms and Breast Cancer: Results from the Breast Cancer Association Consortium. Journal of the National Cancer Institute 98, 1382-96. PMID: 17018785
• Hughes DJ, Sophie M. Ginolhac, Isabelle Coupier, Laure Barjhoux, Valérie Gaborieau, et al, Olga M. Sinilnikova (2005) Modification of breast cancer risk in BRCA1 and BRCA2 mutation carriers with polyglutamine repeat length in the AIB1 gene. Int J Cancer 117;230-233. PMID: 15900600
• Hughes D, Ginolhac SM, Coupier I, Corbex M, et al, Sinilnikova OM (2005) Common BRCA2 variants and modification of breast and ovarian cancer risk in BRCA1 mutation carriers. Cancer Epi Bio Prev 14;265-267. PMID: 15668505
• Coupier I, Cousin PY, Hughes D, Legoix-Né P, Trehin A, Sinilnikova O, Stoppa-Lyonnet D (2005) BAP1 and breast cancer risk. Familial Cancer 4 ;273-7. PMID: 16341802
• Sinilnikova OM, Ginolhac SM, Magnard C, Léoné M, Anczukow O, Hughes D, Moreau K, Thompson D, Gaborieau V, Hall J, Gérard JP, Bonadona V, Lasset C, Goldgar DE, Joulin V, Dalla Venezia N, Lenoir GM (2004) Acetyl-CoA Carboxylase α Gene and Breast Cancer Susceptibility. Carcinogenesis 25;2417-2424. PMID: 15333468

Circulating tumour cells
• Commission written solely by Hughes DJ for Rarecells company – Paterlini-Brechot P (2011) Organ-specific markers in circulating tumor cell screening: an early indicator of metastasis-capable malignancy. Invited Review. Future Oncology 7, 849-871. PMID:21732757

Genome biology

• The C. elegans Sequencing Consortium* (1998) Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282: 2012-8 PMID:9851916. *member

Cystic fibrosis genetics
• Hughes D, Dörk T, Sthurmann M, Graham C (2001) Mutation and haplotype analysis of the CFTR gene in atypically mild cystic fibrosis patients from Northern Ireland. J Med Genet 38: 136-139. PMID: 11288718
• Estivill X, Bancells C, Ramos C, Biomed Cystic Fibrosis Genetic Analysis Consortium* (1997) Geographic distribution and regional origin of 272 cystic fibrosis mutations in European populations. Hum Mutation 10: 135–54. PMID: 9259197. *I was a contributing member
• Hughes D, Hill A, Macek M Jr, Redmond A, Nevin N, Graham C (1996) Mutation Characterization of the CFTR Gene in 206 Northern Irish CF Families: 30 Mutations, Two of which are Novel, Account for 94% of CF Chromosomes. Human Mutation 8: 340-347. PMID: 8956039
• Hughes D, Wallace A, Taylor J, Tassabehji M, McMahon R, Hill A, Nevin N, Graham C (1996) Fluorescent Multiplex Microsatellites used to Define Haplotypes Associated with 75 CFTR Mutations from the UK on 437 CF Chromosomes. Human Mutation 8: 229-235. PMID: 8889582
• Hughes D, Hill A, Redmond A, Nevin N, Graham C (1995) Fluorescent Multiplex Microsatellites used to identify Haplotype Associations with 15 CFTR Mutations in 124 Northern Irish CF Families. Human Genetics 95: 462-464. PMID: 7535745
• The Cystic Fibrosis Genetic Analysis Consortium* (1994) Population variation of common cystic fibrosis mutations. Human Mutation 4: 167-177. PMID:7530552. *contributor / member

Immune function genetics
• Williams F, Hughes D, Middleton D (1994) A new HLA-DRB1*11 allele (DRB1*1109) differing at codons 32, 34 and 37. Tissue Antigens 44: 63-64. PMID: 7974472
• Middleton D, Hughes D, Trainor F, Graham C, Savage D (1994) An HLA-DRB*04 first domain sequence (DRB1*0416) which differs from HLA-DRB1*0401 at codon 59. Tissue Antigens 43: 44-46. PMID: 8023318
• Middleton D, Hughes D, Williams F, Graham C, Martin J, Savage D (1993) A new DRB1 allele DRB1*1107 a combination of DRB1*11 and DRB1*03. Tissue Antigens 42:160-3. PMID: 8284792

Neurological disease genetics
• Mullan M, Bennett C, Figueredo C, Hughes D, et al. (1995) Clinical features of early onset, familial Alzheimer’s disease linked to chromosome 14. American Journal of Medical Genetics 60: 44-52. PMID:7485234
• Hughes D, Brown J, Hardy J, Chartier-Harlin MC (1992) A polymorphic dinucleotide repeat in intron 2 of the human cystatin-C gene. Human Molecular Genetics 1: 143. PMID:1301159
• Fidani L, Rooke K, Chartier-Harlin MC, Hughes D, Tanzi R, Mullan M, Roques P, Hardy J, Goate A (1992) Screening for mutations in the open reading frame and promoter of the beta -amyloid precursor protein gene in familial Alzheimer’s disease: Identification of a further family with APP717 Val/Ile. Human Molecular Genetics 1: 165-168. PMID:1303172
• Chartier-Harlin MC, Crawford F, Houlden H, Warren A, Hughes D, Fidani L, Goate A, Rossor M, Roques P, Hardy J, Mullan M (1991) Mutations at codon 717 of the amyloid precursor protein gene cause Alzheimer’s disease. Nature 353: 844-846. PMID:1944558
• Crawford F, Hardy J, Mullan M, Goate A, Hughes D, Fidani L, Roques P, Rossor M, Chartier-Harlin MC (1991) Sequencing of codon 16 and 17 of the B-amyloid precursor protein gene in 14 families with early onset Alzheimer’s disease fails to reveal mutations in the B-amyloid sequence. Neuroscience Letters 133: 1-2. PMID:1791986