FNR 1654-6628 1654-661X Food & Nutrition Research Vol. 54, June 2010, pp. 1–6 sici xxx PUBMED Abbreviation Ricardo Almon

*Ricardo Almon Family Medicine Research Centre School of Health and Medical Sciences Örebro UniversityBox 1613 Örebro S 701 16 Sweden +46 19 6025789 +46 19 6025797

Emma Patterson Torbjörn K. Nilsson Peter Engfeldt Michael Sjöström Family Medicine Research Centre, School of Health and Medical Sciences Örebro University

Örebro Sweden

Unit for Preventive Nutrition, Department of Biosciences and Nutrition Karolinska Institute

S-14157 Huddinge Sweden

School of Biological Sciences Dublin Institute of Technology

Dublin Ireland

Department of Laboratory Medicine, Clinical Chemistry Örebro University Hospital

Örebro Sweden

Food & Nutrition Research Original Article Examining of lactase gene polymorphism in children and adolescents Background : Lactase non-persistent (LNP) individuals may be lactose intolerant and therefore on a more restricted diet concerning milk and milk products compared to lactase persistent (LP) individuals. This may have an impact on body fat mass. Objective : This study examines if LP and LNP children and adolescents, defined by genotyping for the LCT-13910 C > T polymorphism, differ from each other with regard to milk and milk product intake, and measures of body fat mass. Design : Children (n=298, mean age 9.6 years) and adolescents (n=386, mean age 15.6 years), belonging to the Swedish part of the European Youth Heart Study, were genotyped for the LCT-13910 C > T polymorphism. Dietary intakes of reduced and full-fat dairy varieties were determined. Results : LNP (CC genotype) subjects consumed less milk, soured milk and yoghurt compared to LP (CT/TT genotype) subjects (p<0.001). Subsequent partitioning for age group attenuated this observation (p=0.002 for children and p=0.023 in adolescents). Six subjects were reported by parents to be ‘lactose intolerant’, none of whom were LNP. LNP children and adolescents consumed significantly less reduced fat milk and milk products than LP children and adolescents (p=0.009 for children and p = 0.001 for adolescents). Conclusions : We conclude that LP is linked to an overall higher milk and dairy intake, but is not linked to higher body fat mass in children and adolescents. milk consumption lactose intolerance LCT-13910 C > T polymorphism obesity nutrigenetics Lactase non-persistence (LNP) is an inherited condition defined as the inability to digest lactose due to a reduced production of lactase after weaning 1. A single nucleotide polymorphism (SNP) located 14 kb upstream of the lactase gene (LCT-13910 C > T) has been shown to be associated with lactase persistence (LP). The T allele appears to be dominant over the C allele that represents LNP 2 3 4 . LP (CT/TT genotype) is thus a dominant trait controlled by the lactase gene (LCT). LNP (CC genotype) is also known as adult-type hypolactasia (AtH) or primary hypolactasia 5. Homozygosity for the C allele contributes to the restricted consumption of fresh milk and some dairy products to approximately 12 g of lactose or two cups of milk daily 6. The perception of symptoms related to lactose maldigestion can nevertheless vary between single subjects with LNP and even LP. The underlying hypothesis of this study is that LP and LNP might influence the consumption of milk and milk products in children and adolescents, which in turn could be associated with differences in body fat mass between LP and LNP. Some studies suggest that dairy products may help prevent weight gain and promote weight loss in children and adolescents 7 8 9 10 . Other studies, in contrast, have not found an effect of milk and milk product intake on body fat mass 11 12 13 14 , or shown increased weight gain in children and adolescents 15. Also, a recent review suggests that the ‘dogma’ of promoting the intake of reduced fat diary should be readdressed by interventional studies. The authors conclude that reduced fat varieties of dairy products could constitute a significant contributory factor to hyperphagia and obesity observed in children and adolescents 16. Thus, the role of cow's milk consumption in relation to childhood and adolescent obesity is debated and remains controversial. The main purpose of this study is to examine if children and adolescents with and without the LCT-13910 C > T polymorphism differed in their intakes of milk and milk products, and thereby in measures of body fat mass. Full- and low-fat varieties of milk and milk products were studied. Blood samples were obtained from 686 subjects (300 children and 386 adolescents) belonging to the Swedish part of the European Youth Heart Study (EYHS). EYHS is a cross-sectional school-based study of risk factors for future cardiovascular disease among children (9–10 years old) and adolescents (15–16 years old). The mean ages in the Swedish sample for children and adolescents were 9.6 years and 15.6 years, respectively. Sampling procedures and participation rates have been described previously 17. Height, weight, hip and waist circumferences were measured by internationally accepted standardized procedures 18. Body mass index (BMI) was calculated as weight/height² (kg/m²). Body fat percentage was calculated by using Slaughter's equation based on skinfold thickness measurements 19. These measurements have been shown to correlate highly with dual-energy X-ray absorptiometry-measured body fat percentages in children of similar age groups 20. These measurements were taken using a Harpenden caliper at the biceps, triceps, subscapular, suprailiac and triceps surae sites on the left side of the body. All measurements were taken twice and then the mean thickness was calculated. If the first two measurements differed by more than 2 mm, a third measurement was taken and the two closest measurements were averaged. The consumption of milk and milk products was assessed by an interviewer-mediated 24-hour recall. In the nine-year olds, a qualitative food record completed the day before the interview with the help of parents served as a checklist for the data obtained during the recall. A food atlas was used to estimate portion sizes. Dietary data were processed by StorMats (version 4.02, Rudans Lättdata, Sweden) and analysed using the Swedish National Food database (version 99.1). The milk products of interest were milk, soured milk (a traditional milk product, ‘filmjölk’, in Swedish), yoghurt and cheese, both reduced- and full-fat versions. As part of a broad-ranging questionnaire, parents of the participants were asked if their child had a chronic illness or adhered to a special diet. For the genetic analysis genomic DNA was isolated from whole blood samples from the individuals with the QIAamp DNA Blood Mini Kit spin procedure. The DNA fragment spanning the -13910-C/T polymorphic site was amplified using a biotinylated forward-primer (5′-GGGCTGGCAATACAGATAAGATA-3′) and an unbiotinylated reverse-primer (5′-AGCAGGGCTCAAAGAACAATCTA-3′). The applied sequencing primer was: 5′-CTTTGAGGCCAGGG-3′. Sequencing was performed using a PSQ96 SNP reagent Kit and a PSQ 96MA system (Pyrosequencing AB) PSQ?96MA 2.0.1 software. The procedure has been previously described in detail 21 22. Statistical analyses were performed with the Statistical Package for Social Sciences software (SPSS, version 13.0 for Windows; SPSS Inc, Chicago, IL). The data are presented as means and SD in Tables 1 and 3. Quantitative effects on the selected anthropometric and food intake variables were tested in two- and three-way ANOVA/ANCOVA models with age group (children or adolescents), sex (girls or boys) and LCT-13910 C > T genotype CT/TT vs. CC (LNP vs. LP) as fixed factors. Adjustments for daily energy intake were performed using the residual method when body fat percentage was dependent variable in ANCOVA models. Children Adolescents Girls (n=138) Boys (n=162) Girls (n=196) Boys (n=190) Age (years) 9.6±0.4 9.6±0.3 15.6±0.4 15.6±0.4 Height (cm) 138±6.3 139±5.7 165±6.4 176±7.7 Weight (kg) 33.5±6.0 33.2±5.6 56.9±7.8 64.1±10.7 BMI (kg/m²) 17.3±2.1 17.1±2.2 20.9±2.4 20.6±2.6 Waist circumference (cm) 60.1±5.4 60.5±5.4 69.2±5.7 73.5±7.0 Body fat (%) 18.5±5.3 15.8±5.8 22.6±4.1 13.0±5.6 Energy intake (kJ/day) 8,481±1,920 8,891±2,368 8,753±2,665 12,372±3,686 Milk, soured milk and yoghurt intake (g/day) 562±315 683±340 501±380 801±521 Full-fat milk, soured milk and yoghurt intake (g/day) 218±286 253±292 134±233 246±381 Reduced fat milk, soured milk and yoghurt intake (g/day) 352±295 423±392 367±343 579±477 Milk intake (g/day) 489±291 566±367 410±345 696±477 Full-fat milk (g/day) 149±230 162±249 67±185 151±325 Reduced fat milk (g/day) 339±292 404±368 342±334 544±466 Soured milk intake (g/day) 48±118 78±169 52±126 80±192 Full-fat soured milk (g/day) 42±112 62±157 41±119 58±172 Reduced fat soured milk (g/day) 6.5±44 16±71 11±50 22±106 Yoghurt intake (g/day) 33±101 32±101 39±109 51±138 Full-fat yoghurt (g/day) 27±95 29±97 25±82 37±120 Reduced fat yoghurt (g/day) 5.9±40 3.1±29 14±77 13±70 Cheese intake (g/day) 18±33 23±38 30±39 47±63 Cream intake (g/day) 4.4±16 2.2±9 5.6±22 6.1±30 Ice cream intake (g/day) 21±39 19±44 13±35 21±52 LCT-genotype [n (%)]  CC 19 (13.8) 18 (11.1) 28 (14.3) 29 (15.3)  CT 55 (40.0) 65 (40.1) 77 (39.3) 76 (40.0)  TT 63 (46.2) 78 (48.8) 91 (46.4) 85 (44.7) Parents and 15-year olds gave specific written informed consent to participation in the present genetic study. The study was approved by the Research Ethics Committees of Örebro County Council and Huddinge University Hospital. The baseline characteristics of both girls and boys are shown in Table 1. Analysis of the LCT-13910 C > T genotype showed that the CT and TT genotypes, associated with LP, were found in 261 (87.0%) children and 329 (85.2%) adolescent subjects. The genotype LCT-13910 CC that is associated with LNP was found in 37 children (13.0%) and 57 adolescents (14.8%). Table 2 shows an analysis of the overall effect of the LCT-13910 C > T polymorphism on the studied variables using the levels TT/CT vs. CC in a three-way ANCOVA/ANOVA model. Taken together, milk, soured milk and yoghurt intakes, specifically intakes of reduced fat versions, were significantly lower in individuals with LNP (p < 0.001). Analysed separately, milk intake (g/d) and specifically reduced fat milk (g/d) was lower (p=0.001 and p<0.001, respectively) in LNP children and adolescents. Body fat percentage (adjusted and unadjusted for energy intake) as well as daily energy intake were not related to the LCT-13910 C > T genotype. Table 2 presents the distribution of milk and milk product intake-related variables, which were significantly related to the genotype in Table 2, stratified for age group and sex, and according to LCT-13910 C > T genotypes. Effect Genotype Sex Age group P value^a (P) (P) (P) ^aThree-way ANOVA, LCT-13910 C > T (CC vs. CT/TT), sex and age group as factors. ^bThree-way ANCOVA, LCT-13910 C > T (CC vs. CT/TT), sex and age group as factors; adjusted for daily energy intake (kJ/d). Body fat^b 0.906 <0.001 0.075 Energy 0.407 <0.001 <0.001 Milk, soured milk and yoghurt <0.001 0.223 0.258 Full-fat milk, soured milk and yoghurt 0.588 0.754 0.061 Reduced fat milk, soured milk and yoghurt <0.001 0.140 0.739 Milk 0.001 0.224 0.313 Full-fat milk 0.246 0.581 0.091 Reduced fat milk <0.001 0.116 0.867 Soured milk 0.320 0.438 0.941 Full-fat soured milk 0.495 0.759 0.586 Reduced fat soured milk 0.427 0.291 0.225 Yoghurt 0.502 0.392 0.486 Full-fat yoghurt 0.749 0.989 0.597 Reduced fat yoghurt 0.461 0.101 0.661 Cheese 0.255 0.467 0.294 Cream 0.901 0.780 0.889 Ice cream 0.680 0.919 0.469 As shown in Table 3, after subsequent partitioning of milk and milk product intake-related variables, the LCT-13910 CC genotype was associated with statistically significantly lower intake of total milk, soured milk and yoghurt (p=0.002 for children and p=0.023 for adolescents). Milk intake was significantly lower in LNP children (p=0.005) and in LNP adolescents (p=0.043) compared to LP children and adolescents. Analysed by fat content, the CC genotype was associated with significantly lower intake of total reduced fat milk, soured milk and yoghurt, in both children and adolescents (p=0.009 and p=0.019, respectively). Reduced fat milk intake was also significantly lower in LNP children and adolescents (p=0.010 and p = 0.001, respectively). No statistically significant interactions of the LCT-13910 C > T polymorphism with age and sex for the selected variables were found in either the two-way ANCOVA/ANOVA or three-way ANCOVA/ANOVA models. In response to a question about the presence of chronic conditions in, or adherence to special diets by, their children, parents reported lactose intolerance in just six cases (two children and four adolescents). None of these cases demonstrated genetically determined LNP. Genotype Age Sex Milk, soured milk and yoghurt Reduced fat milk, soured milk and yoghurt Milk intake (g/day) Reduced fat milk (g/day) ^aLCT-13910 C > T polymorphism. ^bTwo-way ANOVA: LCT-13910 C > T (CC vs. CT/TT) and sex as factor. CC Children Girls 471±299 269±235 412±276 203±292 Boys 427±192 355±316 352±183 269±235 Adolescents Girls 381±328 202±265 313±294 190±260 Boys 380±636 387±436 573±576 365±428 CT Children Girls 577±330 355±316 477±304 339±315 Boys 689±435 442±421 597±412 427±402 Adolescents Girls 891±516 390±629 424±734 368±390 Boys 891±557 629±510 734±502 581±497 TT Children Girls 598±306 390±291 524±287 381±290 Boys 717±380 438±397 582±344 412±361 Adolescents Girls 525±343 428±306 428±306 368±290 Boys 817±434 703±411 703±411 572±440 Effect LCT^a Children 0.002^b 0.009^b 0.005^b 0.010^b (P) Adolescents 0.023^b 0.019^b 0.043^b 0.001^b We conducted the current study to investigate the possible differences in dairy product intake and in body fat mass among LP and LNP children and adolescents. There was a statistically significant difference between milk and milk product consumption in LP and LNP subjects. LNP was associated with a lower consumption of milk, soured milk and yoghurt. Interestingly, LP children and adolescents consumed statistically significantly more reduced fat milk than LNP subjects. Concerning the key question of our study, whether LP and LNP influence the consumption of milk and milk products, which in turn could be associated with differences in body fat mass, we found no differences between LP and LNP as regards body fat mass. A recent study, performed on the National Health and Nutrition Survey (NHANES) 1999–2004, showed that a dietary pattern characterised by greater milk intake was associated with increased BMI among preschool children 23. Sweden has traditionally one of the highest consumption rates of milk in the world 24 and we hypothesise that the potential physiologic effects of single food components, such as milk and milk products, may differ between countries due to their particular food and farming culture, agricultural inheritance and geographical as well as climatic singularities. The higher consumption of reduced fat milk, compared to full-fat milk, in all age and genotype groups might reflect a secular trend. Krachler et al. 25 reported in 2005 a switch from 3% of fat milk to 1.5% of fat milk among northern Swedes that occurred between 1986 and 1999. About 83% of the LNP individuals consumed some amount of milk. On average, they consumed 421 g (95% CI 399, 502) milk per day. This is compatible with many studies showing that LNP subjects can tolerate a certain amount of lactose intake per day 6 26 27 28 . Concerning the high prevalence of LNP (14%) compared to the traditional prevalence data of LNP in Sweden (2%), we have found a trend towards an increasing prevalence of LNP in Sweden. This increase seems to have taken place in the course of the second half of the twentieth century due to higher immigration rates to Sweden after the Second World War 29. The strengths to be considered in this study are that we use the concept of Mendelian randomisation (MR), by subdividing genotypes in LP and LNP, to readdress the question of whether nutrigenetically defined factors, like milk and milk product intake, affect body fat mass in children and adolescents. MR studies modifiable causes of disease in genetic epidemiology. A functional genetic variant, in our study LCT-13910 C > T polymorphism, acts as proxy for modifiable lifetime exposure patterns, i.e. milk consumption. The LCT-13910 C > T polymorphism is known to influence the exposure pattern, milk consumption 2 5 6 30 31 32 . In addition, we analysed both, full fat and reduced fat varieties as regards the key question of this work. Limitations of this study include the drawbacks inherent to 24-hour dietary recall, which may not fully reflect long-term food intake behaviour 33. Yet the genetically determined condition of LP or LNP, which we studied in this work, is not subject to changes during life. The results should be interpreted bearing in mind the limitations inherent in any cross-sectional study. Furthermore, results of genetic epidemiological studies obtained in one defined population should not be applied without reserve to other populations or ethnic groups. The age of onset of LNP shows a wide regional and ethnical variation 4. In persons of European descent, the onset of genetically determined LNP is rarely seen before five years of age 34 35 36 ; Finnish children who develop LNP have done so by the age of 10 4. Thus, it could be assumed that the majority of children and almost all adolescents with LCT-13910 CC genotype had developed manifest LNP at the time of inclusion in this study. However, the correlation between LNP and self-reported ‘lactose intolerance’ has been previously suggested to be poor 37 38 39 , which we confirm in this study: six cases parents reported ‘lactose intolerance’ in their children, in two children and four adolescents, and none of them were LNP according to genotyping data. In conclusion, LP is linked to an overall higher milk and dairy intake, but is not linked to higher measures of body fat mass in children and adolescents. This study was supported by grants from Örebro Läns Landstings Forskningskommitté and by Nyckelfonden, Örebro, Sweden. We thank the Stockholm County Council, Sweden, for creating and guaranteeing the study base. We are grateful to Associate Professor Olle Carlsson for statistical advice and Lewis Burgess, Ph.D., applied anthropologist in New York City for revising the manuscript Swallow DM. 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