V ITAMIN B12 STATUS IN PREGNANCY AND CHILD S IQ AT AGE 8: A M ENDELIAN RANDOMIZATION STUDY IN ALSPAC EUCCONET International Workshop Bristol, October 18-19,

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