EUCCONET International Workshop Bristol, October 18-19, 2011 Vitamin B12 status in pregnancy and child’s IQ at age 8: A Mendelian randomization study in ALSPAC Carolina Bonilla School of Social and Community Medicine EUCCONET International Workshop Bristol, October 18-19, 2011

Vitamin B12 facts Only synthesised by microorganisms Main sources: fish, shellfish, eggs, meat, dairy products Recommended Daily Amount: 2-3 ug/day Dietary deficiency rare (vegans at risk) B12 deficiency: <150 pmol/l Main functions: red blood cell formation, DNA synthesis, maintenance of healthy nervous system Transport: 80% bound to transcobalamin I (HC) 20% bound to transcobalamin II (holoTC). holoTC delivers B12 to cells. Stored in the liver Vitamin B12 plays a role in the metabolism of the fatty acids needed to produce myelin the sheath around the neuronal axon. Thus deficiency can result in vitamin B12 neuropathy with its associated degeneration of nerve fibres and irreversible brain damage.

indicators of B12 deficiency Birth defects Spontaneous abortion Pre-eclampsia Prematurity Low birth weight Cardiovascular disease Cognitive deficit Dementia modified from Nexo and Hoffmann-Lucke (2011)

Vitamin B12 status during pregnancy and cognition in children Lower cognition tests scores among offspring of mothers with deficient intake of B12 (Mexico; del Rio Garcia et al., 2009) Children of mothers with low B12 levels performed worse in sustained-attention and working memory tests (India; Bhate et al., 2008) No association of maternal B12 levels with cognitive performance in children. Although verbal ability scores were higher in children of mothers with low B12 (India; Veena et al., 2010) Problem: residual confounding?

Mendelian Randomization Mendelian Randomization uses genetic variants to make causal inferences about (modifiable) environmental risk factors for disease related outcomes

Mendelian Randomization Assumptions genotype associated with exposure of interest genotype not associated with confounders no direct effect of the genotype on outcome, only through exposure Advantages not affected by confounding not affected by reverse causation Bochud and Roussod (2010)

Instrumental variables rs492602 (A68A) FUT2 GWAS Hazra et al. (2008) Tanaka et al. (2009) rs1801198 (P259R) rs9606756 (I23V) TCN2 candidate gene studies

Project framework

ALSPAC (Avon Longitudinal Study of Parents and Children) Population-based prospective study conducted in Bristol, England, to evaluate factors that affect health and development of children ~ 14,000 pregnant women enrolled between April 1991 and December 1992 Information on mother and child collected at regular intervals and ongoing DNA samples available for mothers and children (~10000 each, ~7000 duos) IQ scores available for 6259 children aged 8 y.o. Maternal dietary intake: FFQ Cord blood vitamin B12 levels available for 331 children

Main outcome: full scale IQ at age 8 years: WISC-III (Wechsler, Golombok and Rust, 1992), age-adjusted mean (SD): 104.4 (16.4) Exposures: maternal dietary intake during pregnancy (ug/day) cord blood vitamin B12 levels (pmol/l)

Potential confounders/mediators Mother Child Age Education Social class Parity Any infection during pregnancy Ever smoked Alcohol consumption (before and during pregnancy) Folate supplementation in pregnancy Sex Age Gestation Birth weight Breastfeeding duration

Association with covariables IQ: associated with 12/14 covariables (p<0.01), except for gestation and sex Maternal dietary intake: associated with 10/14 covariables (p<0.01), except for having an infection in pregnancy, smoking, gestation and sex Maternal FUT2 genotype: associated with having an infection in pregnancy (p=0.03) Maternal TCN2 rs1801198 genotype: no associations Maternal TCN2 rs9606756 genotype: associated with parity (p=0.01) Offspring FUT2 genotype: associated with parity (p=0.01), alcohol intake before (p=0.02) and during pregnancy (p=0.03) Offspring TCN2 genotypes: no associations

per doubling of maternal vitamin B12 intake Observational Study N = 5004 mean difference in child IQ per doubling of maternal vitamin B12 intake 95% CI p-value Model 1 2.03 1.30, 2.76 < 0.001 Model 2 0.74 0.04, 1.44 0.04 Model 3 0.70 0.002, 1.39 0.05 Model 1: Adjusted for offspring sex and age at time of IQ assessment, and maternal energy intake. Model 2: Model 1 + maternal education, social class, age at delivery, parity, any infection in pregnancy, ever smoked, alcohol consumption before and during pregnancy, folate supplementation. Model 3: Model 2 + gestational length and birth weight.

Maternal SNPs vs dietary intake and cord blood B12 genotype N median (IQR) of maternal vitamin B12 dietary intake (μg/day) median (IQR) of vitamin B12 cord blood (pmol/L) FUT2 rs492602 TT 1639 4.2 (3.0, 6.1) 62 291 (183, 397) TC 3185 4.2 (3.1, 6.1) 128 300 (196, 424) CC 4.3 (3.0, 6.2) 50 294 (214, 534) ratio of geometric means per C allele (95% CI) 6463 1.00 (0.98, 1.02) 240 1.08 (0.97, 1.19) p-value 0.98 0.15 TCN2 rs1801198 GG 1279 4.1 (3.0, 5.9) 41 276 (173, 369) CG 3210 4.3 (3.1, 6.2) 125 279 (196, 479) 1994 4.1 (3.1, 6.1) 68 318 (226, 436) 6483 (0.99, 1.02) 234 1.13 (1.02-1.25) 0.64 0.02 rs9606756 AA 5476 4.3 (3.2, 6.2) 193 288 (205, 397) AG 1646 4.3 (3.1, 6.1) 58 298 (206, 484) 121 4.2 (3.0, 6.2) 2 238 (82, 394) ratio of geometric means per G allele 7243 0.99 (0.97, 1.01) 253 1.02 (0.89, 1.18) 0.28 0.73

Maternal SNPs vs offspring IQ genotype N IQ mean (SD) FUT2 rs492602 TT 1009 103.3 (16.8) TC 1940 104.2 (16.1) CC 1012 105.0 (16.7) mean difference in child IQ per C allele (95% CI) 3961 0.86 (0.14, 1.58) p-value 0.02 TCN2 rs1801198 GG 784 103.9 (16.6) CG 1978 104.1 (16.0) 1208 104.6 (17.0) 3970 0.35 (-0.38, 1.08) rs9606756 AA 3673 104.5 (16.0) AG 1038 104.7 (17.0) 75 108.3 (14.5) mean difference in child IQ per G allele (95% CI) 4786 0.60 (-0.39, 1.58) 0.24

Maternal SNPs vs offspring IQ - adjusted unadjusted adjusted for child’s genotype adjusted for child’s genotype and AIMs FUT2 rs492602 mean difference in child IQ per C allele (95% CI) 0.77 (-0.14, 1.68) 1.04 (-0.02, 2.09) p-value 0.08 0.05 TCN2 rs1801198 0.52 (-0.42, 1.45) 0.36 (-0.72, 1.45) 0.34 (-0.74, 1.43) 0.28 0.51 0.53 TCN2 rs9606756 mean difference in child IQ per G allele (95% CI) 0.69 (-0.71, 2.09) 1.09 (-0.52, 2.71) 1.08 (-0.53, 2.69) 0.33 0.18 0.19

Maternal SNPs vs offspring IQ by RDA genotype N < RDA ≥ RDA p-value for interaction mean IQ (SD) FUT2 rs492602 TT 120 98.9 (17.2) 852 104.2 (16.7) 0.12 TC 259 101.5 (15.1) 1591 105.0 (16.1) CC 146 101.1 (17.9) 821 106.0 (16.3) mean difference in child IQ per C allele (95% CI) 525 1.05 (-0.93, 3.04) 3264 0.94 (0.15, 1.72) p-value 0.30 0.02 TCN2 rs1801198 GG 106 102.1 (17.2) 640 104.4 (16.5) 0.52 CG 258 100.3 (15.9) 1631 105.0 (15.9) 160 101.0 (17.2) 994 105.5 (16.8) 524 -0.44 (-2.46, 1.57) 3265 0.56 (-0.24, 1.37) 0.67 0.17 rs9606756 AA 471 101.5 (16.5) 3057 105.2 (15.9) 0.69 AG 132 101.1 (16.9) 873 105.5 (17.0) 12 108.3 (9.2) 58 108.3 (15.3) mean difference in child IQ per G allele (95% CI) 615 0.71 (-2.02, 3.45) 3988 0.60 (-0.47, 1.68) 0.61 0.27

Limitations Dietary intake measurements do not account for bioavailability No maternal B12 levels available Small group of children with measured B12 concentration SNPs explain a small proportion of trait variance (<5%) Replication in an Australian birth cohort did not confirm these results (awaiting meta-analysis)

Conclusions Observational association of maternal B12 dietary intake and offspring IQ attenuated markedly with adjustment for confounding factors There was some evidence of association between maternal genotypes and cord blood B12 levels (although power is low) Maternal FUT2 genotype was associated with offspring IQ No statistical evidence of association found between maternal TCN2 SNPs and offspring IQ B12 levels during pregnancy may not have an important causal effect on offspring’s cognitive ability However, larger Mendelian randomization studies are needed to further explore this issue.

Acknowledgements Bristol Oxford Sarah Lewis David Smith Debbie Lawlor Helga Refsum Andy Ness Yoav Ben-Shlomo Australia David Gunnell Marie-Jo Brion George Davey Smith Craig Pennell Amy Taylor Raine team and participants Pauline Emmett Nic Timpson Beate St. Pourcain Kate Northstone Phil Lobb & Sue Ring ALSPAC team and participants

Offspring SNPs vs cord blood vitamin b12 genotype N median (IQR) of vitamin B12 cord blood (pmol/L) FUT2 rs492602 TT 64 224 (168, 318) TC 143 290 (207, 399) CC 79 368 (239, 503) ratio of geometric means per C allele (95% CI) 286 1.24 (1.13, 1.35) p-value 1.79x10-6 TCN2 rs1801198 GG 53 284 (197, 423) CG 139 277 (193, 417) 91 287 (207, 393) 283 1.00 (0.92-1.01) 0.96 rs9606756 AA 232 301 (204, 420) AG 59 273 (196, 394) 6 223 (108, 227) ratio of geometric means per G allele 253 0.88 (0.78, 1.01) 0.06