1. 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

2. 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.

3. 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)

4. 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?

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

6. 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)

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

8. Project framework

9. 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

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

11. 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

12. 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 rs genotype: no associations
Maternal TCN2 rs 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

13. 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.

14. 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 rs 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
( )
0.64
0.02 rs 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

15. 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 rs GG
784
103.9 (16.6) CG
1978
104.1 (16.0)
1208
104.6 (17.0)
3970
0.35
(-0.38, 1.08) rs 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

16. 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 rs
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 rs
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

17. 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 rs 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 rs 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

18. 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)

19. 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.

20. 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

21. 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 rs GG 53
284 (197, 423) CG
139
277 (193, 417) 91
287 (207, 393)
283
1.00
( )
0.96 rs 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