Supplementary material

MnSOD and CAT polymorphisms modulate the effect of the Mediterranean diet on breast cancer risk among Greek-Cypriot women

European Journal of Nutrition

Maria G. Kakkoura, Christiana A. Demetriou, Maria A. Loizidou, Giorgos Loucaides, Ioanna Neophytou, Simon Malas, Kyriacos Kyriacou, Andreas Hadjisavvas

M. G. Kakkoura, C. A. Demetriou, M. A. Loizidou, I. Neophytou, K. Kyriacou, A. Hadjisavvas Department of EM/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

M. G. Kakkoura, K. Kyriacou, A. Hadjisavvas The Cyprus School of Molecular Medicine, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

C. A. Demetriou Neurology Clinic D, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

G. Loucaides The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus

S. Malas Department of Oncology, Limassol General Hospital, Limassol, Cyprus

Correspondence to: Kyriacos Kyriacou and Andreas Hadjisavvas, The Cyprus School of Molecular Medicine and the Department of EM/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, 6 International Airport Avenue, 2370 Nicosia, Cyprus, Emails: [email protected] , [email protected] Supplementary Table 1: Genotype and minor allele frequencies for the MnSOD and CAT single nucleotide polymorphisms (SNPs) in the MASTOS study

Gene/SNP Cases Controls MnSOD p.Val16Ala (rs4880) Val/Val 342 343 Val/Ala 512 552 Ala/Ala 212 253 Total 1066 1148 MAFa 0.44 0.46 Hardy-Weinberg (p-value)b 0.28 CAT -262C>T (rs1001179) C/C 609 674 C/T 369 400 T/T 79 71 Total 1057 1145 MAFa 0.25 0.24 Hardy-Weinberg (p-value)b 0.25 aMAF – Minor Allele Frequency. bp-value from chi-square test performed for Hardy-Weinberg equilibrium (HWE) evaluation. Supplementary Table 2: Interactions between MnSOD, CAT single nucleotide polymorphisms (SNPs) and principal component analysis (PCA)- derived Mediterranean dietary patterna and their effect on breast cancer risk in the MASTOS study PCA-derived Mediterranean dietary pattern intake quartilesb: vegetables, fruit, legumes, fish Quartile 1 Quartile 2 Quartile 3 Quartile 4 Gene/SNP Adjustedc Adjustedc OR (95% Cases/Control Adjustedc OR Adjustedc OR Cases/Controls OR (95% Cases/Controls Cases/Controls CI) s (95% CI) (95% CI) CI) MnSOD p.Val16Ala (rs4880) Val/Val 102/75 1.00 78/77 0.74 (0.47-1.15) 82/89 0.64 (0.41-0.99) 79/102 0.54 (0.35-0.83) 0.87 (0.59- Val/Ala 147/125 144/131 0.77 (0.52-1.13) 120/150 0.55 (0.37-0.82) 100/140 0.51 (0.34-0.77) 1.29) 0.70 (0.43- Ala/Ala 53/52 54/66 0.56 (0.35-0.91) 59/53 0.79 (0.48-1.29) 46/81 0.39 (0.24-0.63) 1.16) p-interactiond 0.43 CAT -262C>T (rs1001179) C/C 171/148 1.00 160/165 0.80 (0.58-1.10) 148/161 0.73 (0.53-1.01) 129/195 0.54 (0.39-0.74) 1.01 (0.70- C/T 106/86 101/94 0.84 (0.58-1.22) 80/116 0.57 (0.39-0.82) 81/102 0.64 (0.44-0.93) 1.45) 1.17 (0.61- T/T 26/18 16/13 1.00 (0.45-2.18) 23/17 1.16 (0.58-2.31) 14/23 0.49 (0.24-1.01) 2.25) p-interactiond 0.58 aPCA-derived dietary pattern with high loadings of vegetables, fruit, legumes and fish, thus closely resembling the Mediterranean diet. bQuartiles of adherence to PCA-derived Mediterranean dietary pattern, with subjects in quartile 1, showing the lowest consumption of vegetables, fruit, legumes and fish and thus, lowest adherence to this dietary pattern and with subjects in quartile 4, showing the highest consumption of the same four food groups and therefore, highest adherence to this dietary pattern. cAdjusted for menopausal status, age and for the other PCA-derived dietary components (Patterns 1, 2 and 3) [11]. dp for interaction between quartiles of adherence to the PCA-derived Mediterranean dietary pattern and genotypes of each SNP, derived from a likelihood ratio test comparing regression models with and without gene-dietary pattern interactions. The odds ratios (ORs) [95% confidence interval (CI)], which correspond to the statistically significant p-values are presented in bold.

Supplementary Table 3: Interactions between MnSOD, CAT single nucleotide polymorphisms (SNPs) and vegetables or fruit intake and their effect on breast cancer risk in the MASTOS study Vegetables intakea Group 1 Group 2 Group 3 Gene/SNP Adjustedc OR (95% Adjustedc OR (95% Adjustedc OR (95% Cases/Controls Cases/Controls Cases/Controls CI) CI) CI) MnSOD p.Val16Ala (rs4880) Val/Val 87/91 1.00 203/159 1.33 (0.92-1.92) 52/93 0.56 (0.35-0.88) Val/Ala 133/143 0.97 (0.65-1.42) 291/246 1.22 (0.86-1.74) 87/160 0.55 (0.37-0.83) Ala/Ala 59/55 0.99 (0.61-1.59) 104/116 0.91 (0.61-1.37) 49/81 0.64 (0.40-1.03) p-interactiond 0.44 CAT -262C>T (rs1001179) C/C 148/162 1.00 346/318 1.20 (0.91-1.59) 114/191 0.66 (0.47-0.92) C/T 105/102 1.08 (0.75-1.55) 201/178 1.24 (0.91-1.69) 63/119 0.57 (0.39-0.84) T/T 23/20 1.25 (0.65-2.41) 45/27 1.80 (1.05-3.09) 11/24 0.52 (0.24-1.11) p-interactiond 0.68 Fruit intakeb Group 1 Group 2 Gene/SNP Case s/Co Cases/Controls Adjustedc OR (95% CI) Adjustedc OR (95% CI) ntrol s MnSOD p.Val16Ala (rs4880) 147/ Val/Val 195/178 1.00 0.77 (0.56-1.05) 165 210/ Val/Ala 301/263 1.03 (0.78-1.35) 0.65 (0.49-0.86) 286 85/1 Ala/Ala 127/125 0.91 (0.66-1.27) 0.56 (0.39-0.79) 27 p-interactiond 0.54 CAT -262C>T (rs1001179) 237/ C/C 371/344 1.00 0.64 (0.50-0.80) 327 172/ C/T 197/196 0.90 (0.70-1.16) 0.74 (0.57-0.96) 203 26/4 T/T 53/29 1.63 (1.00-2.66) 0.56 (0.33-0.94) 2 p-interactiond 0.05

aVegetables intake is categorised in three groups: Group 1 = Quartile 1, Group 2 = Quartiles 2 and 3, Group 3 = Quartile 4. Subjects in Group 1 show the lowest consumption of vegetables and subjects in Group 3 show the highest consumption of vegetables. bFruit intake is categorised in two groups: Group 1 = below median, Group 2 = above median. Subjects in Group 1 show the lowest consumption of fruit and subjects in Group 2 show the highest consumption of fruit. cAdjusted for menopausal status, age and for the other PCA-derived dietary components (Patterns 1, 2 and 3) [11]. dp for interaction between groups of vegetables or fruit intake and genotypes of each SNP, derived from a likelihood ratio test comparing regression models with and without gene-food intake interactions. The odds ratios (ORs) [95% confidence interval (CI)] which correspond to the statistically significant p-values are presented in bold.

Supplementary Table 4: Interactions between MnSOD, CAT single nucleotide polymorphisms (SNPs) and legumes or fish intake and their effect on breast cancer risk in the MASTOS study Legumes intakea Q ua Gene/SN Quartile 1 rti Quartile 3 Quartile 4 P le 2 Cases/Control Cases/Co Adjustedc OR (95% Adjustedc OR Cases/Contr Adjustedc OR (95% Adjustedc OR (95% CI) Cases/Controls s ntrols CI) (95% CI) ols CI) MnSOD p.Val16Ala (rs4880) Val/Val 106/102 1.00 110/107 0.93 (0.63-1.38) 48/43 0.99 (0.59-1.64) 78/91 0.74 (0.49-1.13) Val/Ala 164/181 0.87 (0.61-1.24) 145/153 0.87 (0.61-1.26) 84/83 0.84 (0.55-1.28) 118/131 0.79 (0.54-1.15) Ala/Ala 61/81 0.71 (0.46-1.11) 62/65 0.82 (0.52-1.29) 32/42 0.63 (0.37-1.10) 57/64 0.77 (0.49-1.23) p-interactiond 0.91 CAT -262C>T (rs1001179) C/C 187/220 1.00 162/178 0.97 (0.72-1.31) 97/97 0.99 (0.69-1.41) 162/175 0.98 (0.73-1.32) C/T 116/114 1.13 (0.81-1.58) 130/130 1.10 (0.80-1.52) 52/58 0.92 (0.60-1.43) 71/97 0.71 (0.49-1.03) T/T 27/27 1.01 (0.56-1.81) 21/16 1.42 (0.71-2.86) 13/14 1.01 (0.46-2.25) 18/14 1.50 (0.71-3.19) p-interactiond 0.46 Gene/SN Fish intakeb P Q ua Quartile 1 rti Quartile 3 Quartile 4 le 2 Cases/Control Cases/Co Adjustedc OR (95% Adjustedc OR Cases/Contr Adjustedc OR (95% Adjustedc OR (95% CI) Cases/Controls s ntrols CI) (95% CI) ols CI) MnSOD p.Val16Ala (rs4880) Val/Val 86/72 1.00 82/83 0.86 (0.55-1.34) 103/86 1.06 (0.68-1.65) 71/102 0.63 (0.40-0.98) Val/Ala 135/140 0.82 (0.55-1.23) 148/138 0.93 (0.62-1.38) 118/127 0.79 (0.52-1.20) 110/144 0.71 (0.47-1.08) Ala/Ala 61/61 0.79 (0.49-1.28) 47/60 0.67 (0.40-1.11) 54/59 0.80 (0.48-1.31) 50/72 0.63 (0.38-1.03) p-interactiond 0.71 CAT -262C>T (rs1001179) C/C 157/157 1.00 167/163 1.04 (0.76-1.43) 159/152 1.08 (0.78-1.49) 125/199 0.66 (0.47-0.92) C/T 108/93 1.09 (0.76-1.57) 87/105 0.83 (0.57-1.20) 93/105 0.88 (0.61-1.27) 81/96 0.93 (0.63-1.37) T/T 18/23 0.75 (0.39-1.47) 25/12 2.10 (1.00-4.43) 18/13 1.41 (0.65-3.07) 18/23 0.85 (0.43-1.66) p-interactiond 0.08

aQuartiles of legumes intake, with subjects in quartile 1 showing the lowest consumption of legumes and subjects in quartile 4, showing the highest consumption of legumes; Q1 = Quartile 1, Q2 = Quartile 2, Q3 = Quartile 3, Q4 = Quartile 4. bQuartiles of fish intake, with subjects in quartile 1 showing the lowest consumption of fish and subjects in quartile 4, showing the highest consumption of fish; Q1 = Quartile 1, Q2 = Quartile 2, Q3 = Quartile 3, Q4 = Quartile 4. cAdjusted for menopausal status, age and for the other PCA-derived dietary components (Patterns 1, 2 and 3) [11]. dp for interaction between quartiles of legumes or fish intake and genotypes of each SNP, derived from a likelihood ratio test comparing regression models with and without gene-food intake interactions. The odds ratios (ORs) [95% confidence interval (CI)] which correspond to the statistically significant p-values are also presented in bold. Supplementary Table 5: Quantities of antioxidant micronutrients in a serving of 100g of the foods loaded in the principal component analysis (PCA)- derived dietary patterns 1a, 2b, 3c and 4d in the MASTOS study

Antioxidant micronutrients (quantities)e Food (1 Vitamin E (α- Lutein + serving of Vitamin Carotene- Carotene-α Cryptoxanthi Lycopene 20:5 n-3 (g) 22:6 n-3 (g) tocopherol) zeaxanthin 100g) C (mg) β (mcg) (mcg) n (mcg) (mcg) (mg) (mcg) PCA-derived dietary pattern 1 (meat, potatoes)a Meat Meat (not further 0.003 0.001 0 0 0 0 0 0 0 specified) Beef (not specified as to cut, 0.003 0.001 0 0 0 0 0 0 0 cooked, not specified as to fat eaten) Pork (not specified as to cut, 0 0 0 0.17 0 0 0 0 0 cooked, not specified as to fat eaten) Lamb (not specified as 0 0 0 0.14 0 0 0 0 0 to cut, cooked) Chicken (not 0.003 0.006 0 0.54 0 0 0 27 0 specified as to part and cooking method, not specified as to skin eaten) Rabbit (not specified as to domestic 0 0 0 0.44 0 0 0 0 0 or wild, cooked) Turkey (not further 0.004 0.004 0 0.06 0 0 0 0 0 specified) Sausage (not further 0.002 0.003 0 0.9 0 0 0 0 0 specified) Cold cut (not further 0 0 0 0.29 0 0 0 0 0 specified) Potatoes White potato (not further 0 0 7.1 0.27 20 0 0 9 0 specified) Supplementary Table 1 (Continued)

Antioxidant micronutrients (quantities)e Food (1 Vitamin E (α- Lutein + serving of Vitamin Carotene- Carotene-α Cryptoxanthi Lycopene 20:5 n-3 (g) 22:6 n-3 (g) tocopherol) zeaxanthin 100g) C (mg) β (mcg) (mcg) n (mcg) (mcg) (mg) (mcg) PCA-derived dietary pattern 2 (cereals, milk, dairy)b Cereals Corn flakes (not further 0 0 21 0.12 83 54 0 339 0 specified) Milk Milk, cow's, 0 0 0 0.07 7 0 0 0 0 fluid, whole (includes 3.5% or 3.8% fat milk, leche fresca) Milk, cow's, fluid, skim or nonfat, 0.5% or less 0 0 0 0.01 0 0 0 0 0 butterfat (includes fat free milk) Dairy Yogurt (not specified as to type of 0 0 0.6 0.02 2 0 0 0 0 milk or flavour) Cheese (not further 0.003 0.001 0 0.4 97 0 9 27 0 specified) PCA-derived dietary pattern 3 (cakes, sweets, nuts, crackers, pasta, rice)c Cakes Cake or cupcake (not 0 0 0.1 4.33 0 0 0 3 0 specified as to type)

Supplementary Table 1 (Continued)

Antioxidant micronutrients (quantities)e Food (1 Vitamin E (α- Lutein + serving of Vitamin Carotene- Carotene-α Cryptoxanthi Lycopene 20:5 n-3 (g) 22:6 n-3 (g) tocopherol) zeaxanthin 100g) C (mg) β (mcg) (mcg) n (mcg) (mcg) (mg) (mcg) PCA-derived dietary pattern 3 (cakes, sweets, nuts, crackers, pasta, rice)c (continued) Sweets Chocolate, sweet or 0 0 0 0.4 12 4 0 24 0 dark Cookie (not further 0 0 0 2.11 0 0 0 1 0 specified) Biscuit (baking powder or buttermilk type, not specified as 0 0 0 1.32 0 0 0 9 0 to made from mix, refrigerated dough, or home recipe) Bakery Pie, fried (not further 0 0 1.3 1.72 5 0 5 8 0 specified) Nuts Nuts (not further 0 0 0.4 10.94 3 0 0 21 0 specified) Crackers Crackers (not specified as 0 0 0 3.27 0 0 0 12 0 to sweet or nonsweet) Pasta/Rice Macaroni 0 0 0 0.06 0 0 0 7 0 (cooked, not specified as to fat added in cooking)

Supplementary Table 1 (Continued)

Antioxidant micronutrients (quantities)e Food (1 Vitamin E (α- Lutein + serving of Vitamin Carotene- Carotene-α Cryptoxanthi Lycopene 20:5 n-3 (g) 22:6 n-3 (g) tocopherol) zeaxanthin 100g) C (mg) β (mcg) (mcg) n (mcg) (mcg) (mg) (mcg) PCA-derived dietary pattern 3 (cakes, sweets, nuts, crackers, pasta, rice)c (continued) Rice (white, cooked, fat added in cooking, made with oil, not specified as to fat added 0 0 0 0.38 0 0 0 0 0 in cooking, not specified as to type of fat added in cooking, basmati or jasmine rice) Couscous, plain (cooked, not 0 0 0 0.13 0 0 0 25 0 specified as to fat added in cooking) Cereal 0 0 0 0.07 0 0 0 0 0 (cooked, instant, not specified as to grain) PCA-derived Mediterranean dietary pattern 4 (vegetables, fruit, legumes, fish)d Vegetables Vegetables (not specified as to type, 0 0 3.1 0.9 2034 938 0 618 0 cooked, not specified as to fat added in cooking) Lettuce, salad with assorted vegetables (including 0 0 9.1 0.5 2783 255 2 2139 266 tomatoes and/or carrots, no dressing)

Supplementary Table 1 (Continued)

Antioxidant micronutrients (quantities)e 2 Food (1 2: 22:5 6 Vitamin E (α- Lutein + serving of Vitamin C Carotene- Carotene- Cryptoxanthin Lycopene 20:5 n-3 (g) n-3 n tocopherol) zeaxanthin 100g) (mg) β (mcg) α (mcg) - β (mcg) (mcg) (g) -3 (mg) (mcg) (g ) PCA-derived Mediterranean dietary pattern 4 (vegetables, fruit, legumes, fish)d (continued) Vegetables (continued) Mixed vegetables (corn, lima beans, peas, green beans, and carrots, 0 0 0 3.1 0.91 2034 938 0 617 0 cooked, not specified as to form, not specified as to fat added in cooking) Fruit Fruit (not specified as 0 0 0 19.3 0.14 293 11 21 39 118 to type) Legumes Beans and tomatoes (not specified as to fat added in cooking, 0 0 0 2.6 1.28 84 0 1 7 4517 including beans, all kinds, beans with tomato sauce) Rice, white, with lentils (not 0 0 0 1.3 0.62 12 0 2 6 0 specified as to fat added in cooking) Fish Fish (not 0.078 0.031 0. 0.4 0.81 0 0 0 0 0 specified as 1 to type, cooked, not 7 specified as 2 to cooking method) Fish (not 0. specified as 1 0.028 0.004 0 0.33 0 0 0 0 0 to type, 9 canned) 6 aPCA-derived dietary pattern with high loadings of meat and potatoes [11]. bPCA-derived dietary pattern with high loadings of cereals, milk and dairy products [11]. cPCA-derived dietary pattern with high loadings of cakes, sweets, nuts, crackers, pasta, and rice [11]. dPCA-derived dietary pattern with high loadings of fish, fruit, legumes and vegetables, thus closely resembling the Mediterranean diet [11]. eQuantities for each of the antioxidant micronutrient of interest [20:5, 22:5, 22:6 n-3 polyunsaturated fatty acids, Vitamin C, Vitamin E, Carotene α and β, Cryptoxanthin- β, Lutein + zeaxanthin, Lycopene] were obtained from the online USDA FNDDS site "What's in the Foods You Eat? Search Tool", which calculates quantities of nutrients in grams (g), micrograms (mcg) or milligrams (mg) for several measures (e.g grams) of various servings for the foods in the database. More general foods [e.g. vegetables (not specified as to type, cooked, not specified as to fat added in cooking)] and foods which correspond more to the dietary habits of the Cypriot population were chosen from the database in order to better represent each food group loaded in the PCA-derived dietary patterns, since the 32-item food frequency questionnaire of the study did not include detailed food items.

FNDDS U. USDA Food and Nutrient Database for Dietary Studies: What’s In The Foods You Eat Search Tool 2011-2012 [https://reedir.arsnet.usda.gov/codesearchwebapp/%28h1zdt555qfcnfm55pjp44h45%29/codesearch.aspx]. 2011-2012. Accessed 12 January 2015.