Ethnic variation in methylation of birth weight and length Presenter: Zahra Sohani Supervisor: Dr. Sonia Anand

OVERVIEW 1.Brief background 2.Research question 3.The approach 4.The approach – revised 5.Brief findings 6.Putting it all in context with South Asians

BACKGROUND Rates of type 2 diabetes mellitus differ between South Asians and Europeans; South Asians are at a 2-5 fold greater risk Numerous studies have pointed to differences in body composition As adults, South Asians have greater visceral adiposity and develop metabolic complications at lower BMI and younger age In adolescence, South Asians have greater levels of impaired fasting glucose, impaired glucose tolerance, and insulin resistance At birth, South Asians are smaller, but comparably adipose to Europeans

The relationship between birth weight and type 2 diabetes is well characterized A systematic review synthesized data from 6,090 diabetes patients in 31 populations and reported a 25% reduction in odds of T2DM per kilogram of weight gained (OR: 0.75, 95% CI ) Another review classifying newborns into low birth weight (LBW) (<2.5 kg) compared with a birth weight of ≥ 2.5 kg found LBW to have a 32% increase in odds of T2DM (OR: 1.32, 95% CI )

As differences in body composition between these two groups are present at birth, the variation is likely a result of either altered genetic predisposition or the in-utero environment Since both genetic and environmental factors can independently and together alter methylation of genes, having downstream effects on the expression of genes, it has become a potentially important field of study to explain this ethnic variation

WHAT IS DNA METHYLATION? A methyl group to a nucleotide, commonly at the 5’ carbon of cytosine in CpG dinucleotides Methylation can transcriptionally regulate genes and miRNAs, control alternative promoter usage, and alternative splicing

THE QUESTION Are there differences in methylation of genes involved in birth weight and length among South Asians and white Caucasian newborns from the START and CHILD cohorts?

THE APPROACH STEP 1: Identify genes associated with birth weight and length in the literature

Table 1 – SNPs associated with birth weight and length at a genome wide threshold from the literature GeneSNPTraitstartposendpos LCORLrs724577brith length PTCH1rs473902birth length GPR126rs birth length HMGA2rs birth length DCST2rs905938birth length SF3B4rs birth length PTPDC1rs birth length HHIPrs birth length ADAMTSL3rs birth length ZBTB38rs724016birth length HMGA1rs birth length IGF1Rrs birth length GDF5rs143384birth length DTLrs birth length JAZF1rs birth length ACBD4rs birth length ANKRD13Brs birth length PMLrs birth length CCNL1rs900400birth weight CENPMrs birth weight ADCY5rs birth weight HMGA2rs birth weight CDKAL1rs birth weight CALCRrs birth weight ACTBL2rs birth weight LCORLrs724577birth weight ADRB1rs birth weight SLC2A4rs5415birth weight TCF7L2rs birth weight HHEX-IDErs birth weight

THE APPROACH STEP 2: Which methylation probe sites to investigate? Within 100 kilo base pairs of the SNP

12 birth weight SNPs on 12 genes 332 probe sites in total from all 12 genes 222 probe sites within 100 kilo base pairs of the SNP 18 birth length SNPs on 18 genes 422 probe sites in total from all 18 genes 295 probe sites within 100 kilo base pairs of the SNP

THE APPROACH STEP 3: Is there variation in level of methylation by ethnicity? Equation 1: CpG probe site = β 0 + β 1 *ethnicity + ɛ Equation 2: For probe sites showing ethnic variation based on above: Birth weight = β 0 + β 1 *CpG probe site + covariates + ɛ in South Asians and Europeans, separately

92% of the birth weight probe sites were statistically different between South Asians and European newborns

92% of the birth length probe sites were statistically different between South Asians and European newborns

THE APPROACH What could be causing this large-scale variation by ethnicity? (1) Variation in cell type composition Definitionbeta-coefficientp-value South Asian Mean European Mean Lymphocytes: LYMPHS Absolute: Monocytes: MONOS Absolute: Neutrophils: NEUTS Absolute: Nucleated Red Blood Cells: NRBC Absolute: Eosinophils: EOS Absolute: Basophils: BASOS Absolute:

(2) Is this difference restricted to birth weight genes or is this a global phenomenon? Meta-analysis of 1000 random sites from the epigenome in South Asians and Europeans to estimate global methylation levels in both groups Comparisonn_SAn_EUME Summary estimate (95% CI)Heterogeneity ME beta only CHILD mixed SA vs. CHILD EU ( , )X2 0, p= CHILD mixed and pure SA vs. CHILD EU ( , )X2: 0, p= CHILD pure SA vs. CHILD EU ( , )X2: 0, p= START SA vs. CHILD EU (0.0135, )X2 = , p=

THE APPROACH - REVISED 1.Linear regression analysis of CpG probe sites on birth weight/length separately in CHILD and START birth weight = β 0 + β 1 *CpG probe site + covariates + ɛ 2.Meta-analysis of b-coefficients for probe sites from both cohorts Heterogeneity from meta-analysis to estimate ethnic heterogeneity

THE FINDINGS Table 2 – CpG probe sites showing ethnic variation in association with birth weight GeneCpG siteSouth Asian β-coefficient p-value European Caucasians β-coefficient p-value Summary estimate p-value heterogeneity TCF7L2cg x x x10 -5 CALCRcg x x x10 -5 HMGA2cg x x x10 -4 HMGA2 cg x x x10 -4 TCF7L2cg x x x10 -4 CDKAL1cg x x x10 -3

Table 3 – Association between methylation level and single nucleotide polymorphisms for birth weight GeneSNPcpg% methylationβ-coefficient [SE]P-valueSummary estimateHeterogeneity p-value TCF7L2rs cg GGGAGAAA START [0.0046]9.21 x x10 -1 CHILD [0.0039]3.23 x10 -1 cg GGGAGAAA START [0.0066]7.73 x x10 -2 CHILD [0.0066]5.92 x10 -3 CALCRrs cg GGGAGAAA START [0.0032]3.29 x x10 -1 CHILD [0.0038]2.36 x10 -1 HMGA2rs cg GGGAGAAA START [0.0020]4.97 x x10 -1 CHILD [0.0028]2.61 x10 -1 cg GGGAGAAA START [0.0052]3.25 x x10 -1 CHILD [0.0062]3.79 x10 -3 CDKAL1rs cg CCCACAAA START [0.0076]5.72 x x10 -1 CHILD [0.0070]2.62 x10 -1

Table 4 – Variance explained Variables in the modelCHILDSTART HMGA2 cg gestational age rs _G sex cg gestational age rs _G sex TCF7L2 cg gestational age rs _A sex cg gestational age rs _A sex CDKAL1 cg gestational age rs _A sex CALCR cg gestational age rs _A sex

IN CONTEXT WITH LITERATURE ON SOUTH ASIANS Overwhelming majority of studies in European populations Some candidate gene studies exploring birth weight in South Asians Busch et al found that among South Asians, those homozygous for PON2 A148/A148 had significantly lower birth weight (n=290) Is it possible that these genes are less important in governing birth weight in South Asians?

GeneLead SNPProxy SNPAlleleSouth AsianEuropean Caucasians Beta (SE)p-valueBeta (SE)p-value rs rs A (0.044)6.99E (0.049)3.91E-01 rs rs A ( )9.93E (0.044)7.27E-01 rs rs G ( )8.58E (0.042)8.55E-01 TCF7L2rs rs A ( )2.66E (0.044)2.27E-02 rs rs740746G ( )9.37E (0.048)5.30E-01 HMGA2rs rs G ( )7.66E (0.044)1.52E-01 rs rs G ( )3.79E (0.041)3.26E-01 rs A ( )7.52E (0.048)1.99E-01 CDKAL1rs rs A ( )3.98E (0.047)6.46E-01

NEXT STEPS 1.Epigenome-wide search for cpgs associated with birth weight in South Asians 2.This analysis is conducted in only a subset of START / CHILD – use more data when available

Illumina Infinium HumanMethylation450 assay. (a) Infinium I assay. Each individual CpG is interrogated using two bead types: methylated (M) and unmethylated (U). The probe design assumes that all CpGs underlying the probe body have the same methylation status as the target CpG. Both bead types will incorporate the same labeled nucleotide for the same target CpG, thereby producing the same color fluorescence. The nucleotide that is added is determined by the base downstream of the 'C' of the target CpG. The proportion of methylation, β, can be calculated by comparing the intensities from the two different probes in the same color: β= M/(U + M). (b) Infinium II assay. Each target CpG is interrogated using a single bead type. A probe may have up to three underlying CpG sites, with a degenerate R base corresponding to the 'C' of each CpG. Methylation state is detected by single base extension at the position of the 'C' of the target CpG, which always results in the addition of a labeled 'G' or 'A' nucleotide, complementary to either the 'methylated' C or 'unmethylated' T, respectively. Each locus is detected in two colors, and methylation status is determined by comparing the two colors from the one position: β = Green (M)/(Red (U) + Green (M)).