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VITAMIN B12 STATUS IN PREGNANCY AND
CHILD’S IQ AT AGE 8: A MENDELIAN RANDOMIZATION STUDY IN ALSPAC
EUCCONET International Workshop Bristol, October 18-19, 2011
Carolina BonillaSchool of Social and Community Medicine
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
indicators of B12 deficiency
modified from Nexo and Hoffmann-Lucke (2011)
Birth defects Spontaneous abortion Pre-eclampsia Prematurity Low birth weight Cardiovascular disease Cognitive deficit Dementia
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)FUT2GWASHazra et al. (2008)Tanaka et al. (2009)
rs1801198 (P259R)rs9606756 (I23V)TCN2candidate 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
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
Mother Child
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
OBSERVATIONAL STUDY
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.
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 < 0.001Model 2 0.74 0.04, 1.44 0.04Model 3 0.70 0.002, 1.39 0.05
MATERNAL SNPS VS DIETARY INTAKE AND CORD BLOOD B12
SNP genotype N
median (IQR) of maternal vitamin
B12 dietary intake
(μg/day)
Nmedian (IQR) of vitamin B12 cord blood (pmol/L)
FUT2rs492602
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 1639 4.3 (3.0, 6.2) 50 294 (214, 534)
ratio of geometric
means per C allele
(95% CI)
64631.00
(0.98, 1.02)240
1.08(0.97, 1.19)
p-value 0.98 0.15
TCN2rs180119
8
GG 1279 4.1 (3.0, 5.9) 41 276 (173, 369)CG 3210 4.3 (3.1, 6.2) 125 279 (196, 479)CC 1994 4.1 (3.1, 6.1) 68 318 (226, 436)
ratio of geometric
means per C allele
(95% CI)
64831.00
(0.99, 1.02)234
1.13(1.02-1.25)
p-value 0.64 0.02
TCN2rs960675
6
AA 5476 4.3 (3.2, 6.2) 193 288 (205, 397)AG 1646 4.3 (3.1, 6.1) 58 298 (206, 484)GG 121 4.2 (3.0, 6.2) 2 238 (82, 394)
ratio of geometric
means per G allele
(95% CI)
72430.99
(0.97, 1.01)253
1.02(0.89, 1.18)
p-value 0.28 0.73
MATERNAL SNPS VS OFFSPRING IQ
SNP genotype NIQ
mean (SD)
FUT2rs492602
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)
39610.86
(0.14, 1.58)
p-value 0.02
TCN2rs1801198
GG 784 103.9 (16.6)CG 1978 104.1 (16.0)CC 1208 104.6 (17.0)
mean difference in child IQ per C allele (95% CI)
39700.35
(-0.38, 1.08)
p-value 0.35
TCN2rs9606756
AA 3673 104.5 (16.0)AG 1038 104.7 (17.0)GG 75 108.3 (14.5)
mean difference in child IQ per G allele (95% CI)
47860.60
(-0.39, 1.58)
p-value 0.24
MATERNAL SNPS VS OFFSPRING IQ - ADJUSTED
SNP unadjustedadjusted 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)
1.04(-0.02, 2.09)
p-value 0.08 0.05 0.05
TCN2 rs1801198
mean difference in child IQ per C allele
(95% CI)
0.52(-0.42, 1.45)
0.36(-0.72, 1.45)
0.34(-0.74, 1.43)
p-value 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)
p-value 0.33 0.18 0.19
MATERNAL SNPS VS OFFSPRING IQ BY RDA
SNP genotype N < RDA N ≥ RDA
p-value for
interaction
mean IQ (SD)mean IQ
(SD)
FUT2rs49260
2
TT 120 98.9 (17.2) 852 104.2 (16.7) 0.12TC 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)
32640.94
(0.15, 1.72)
p-value 0.30 0.02
TCN2rs18011
98
GG 106 102.1 (17.2) 640 104.4 (16.5) 0.52CG 258 100.3 (15.9) 1631 105.0 (15.9)CC 160 101.0 (17.2) 994 105.5 (16.8)
mean difference in child IQ per C allele
(95% CI)524
-0.44(-2.46, 1.57)
32650.56
(-0.24, 1.37)
p-value 0.67 0.17
TCN2rs96067
56
AA 471 101.5 (16.5) 3057 105.2 (15.9) 0.69AG 132 101.1 (16.9) 873 105.5 (17.0)GG 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)
39880.60
(-0.47, 1.68)
p-value 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 OxfordSarah Lewis David SmithDebbie Lawlor Helga RefsumAndy Ness
Yoav Ben-Shlomo AustraliaDavid Gunnell Marie-Jo BrionGeorge Davey Smith Craig PennellAmy Taylor Raine team and
participantsPauline EmmettNic TimpsonBeate St. PourcainKate NorthstonePhil Lobb & Sue RingALSPAC team and participants
OFFSPRING SNPS VS CORD BLOOD VITAMIN B12
SNP genotype Nmedian (IQR) of
vitamin B12 cord blood (pmol/L)
FUT2rs492602
TT 64 224 (168, 318)TC 143 290 (207, 399)CC 79 368 (239, 503)
ratio of geometric
means per C allele
(95% CI)
2861.24
(1.13, 1.35)
p-value 1.79x10-6
TCN2rs1801198
GG 53 284 (197, 423)CG 139 277 (193, 417)CC 91 287 (207, 393)
ratio of geometric
means per C allele
(95% CI)
2831.00
(0.92-1.01)
p-value 0.96
TCN2rs9606756
AA 232 301 (204, 420)AG 59 273 (196, 394)GG 6 223 (108, 227)
ratio of geometric
means per G allele
(95% CI)
2530.88
(0.78, 1.01)
p-value 0.06