Evidence of geneenvironment interaction Click to edit Master subtitle style for the RUNX2 gene and Environmental tobacco

smoke in controlling the risk of cleft lip with/without cleft palate

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The paper

Examined 49 markers in the Runt-related transcription factor 2 (RUNX2) gene and nonsyndromic cleft lip with/without cleft palate Chinese case-parent trios at GxE interactions and parent of origin

326

Looked

effects
5

SNPs showed significant evidence of linkage in association with CL/P evidence between markers in RUNXC2 and ETS (environmental tobacco smoke)
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Compelling

Background RUNX2
Gene

located on chromosome 6p21

Encode

for a TF critical for osteoblastic differentiation to be under oestrogen control for osteoblast- and osteoclasto-gensis of RUNX2 in humans lead to cleidocranial dysplasia (heritable)

Thought

Mutations

Missing clavicles dental abnormalities and craniofacial abnormalities, can include clefting

Animal

models suggests mutations and expression levels of RUNX2 maybe involved in the development of several craniofacial defects
4/16/12 genotypes for association with CL/P but

Has

Approach taken

Trios recruited in 3 sites mainly in China

None of the parents had the disorder

Collected a lot of data but only looked at ETS and multivitamin supplementation because the numbers for maternal cigarette smoking and alcohol consumption were so low (only 1%) High rate of Chinese men smoke = exposure An association analyses of case-parent trios from four populations also suggested RUNX2 may influence risk of CL/P through imprinting effects 69 selected SNPs were selected from designed cores provided by illumina, 20 monomorphic ones dismissed. No mendelian inconsistencies found in minor allele frequencies from the remainder polymorphic 49 and ranged from 5% to 47.6%
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Approach

Pairwise linkage disequilibrium (LD) was measured as r2 for all SNPs using the Haploview program, and was used to identify LD blocks To assess the significance of TDT (Transmission Disequilibrium Test) findings while considering multiple tests, the permutation test in PLINK was used to get corrected p values The family association test program (FBAT) was used for haplotype anaylsis Sliding window approach for haplotypes to systematically analyze all adjacent 2-SNP and 3-SNP combinations Plots of log10 (p value) for individual SNPs and haplotypes were generated using the R package snp.plotter Conditional logistic regression under additive models to estimate the relative risk (RR) for being a case with or without environmental exposures for each SNP 4/16/12 Tested for log-additive GxE interaction

Approach

Estimated haplotype-environment interaction where family-based association tests are evaluated while allowing for a potential GxE interaction in a 2 degree of freedom (df) test, followed by a separate 1 df test for GxE interaction alone The 2 df test examines the genetic effect of a haplotype while taking into account possible effects of GxE interaction, while the 1 df test investigates the effect of GxE interaction alone Parent-of-origin effects were also tested using several methods

transmission asymmetry test (TAT) used to check for potential parent-of-origin effects, A significant TAT could reflect an imprinting effect or a maternal genotype effect
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Replication study

In silico replication study using samples from European and Asian ancestry from a GWAS of nonsyndromic CL/P using a caseparent design by another study SNPs in RUNX2 were genotyped and analysed similarly

78

TDT analysis in PLINK, tested GxE interaction using conditional logistic regression models, PBAT to confirm findings seen in Chinese trios
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Results
9

independent SNPs and 8 blocks of linkage disequilibrium were identified

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Results

5 SNPs in the RUNX2 gene showed significant linkage in the presence of associations with nonsyndromic CL/P. One of these remained significant after in permutation tests.

These findings were replicated in 825 European case parent trios.

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Results
Single

SNP analysis using conditional logistic regression models and haplotype analysis using PBAT suggested the RUNX2 gene may also influence the risk of CL/P through interaction with ETS and multivitamin supplementation.

Independent samples from European populations showed consistent evidence of significant GxETS interaction at two SNPs.

One

SNP showed marginally significant maternal over-transmission after considering maternal genotypic effects
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Criticism

Average sample size

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Criticism
Very Did

poorly defined criteria for ETS

not specify what multivitamin supplementation was (Contained folic acid? Dose?) look at null effects of having genotype but no phenotype (are there any kids with the genotype but no phenotype?) diversity in sample population, could have incorporated other ethnicities looked at one gene for interaction with ETS/ multivitamin supplementation
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Didnt

Low

Only

Other environmental factors

General Alcohol

pollution / radiation exposure intake

Binge drinking?

Diet

and food hygiene

Other

physiological diseases/ disorder carried by the parents and precautions for planned pregnancies
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Stress Awareness

Other genes in the pathway

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Other genes in the pathway

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Other genes in the pathway

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Other genes
Other

studies have investigated common environmental factors (eg. smoking, alcohol, multivitamin/ folic acid supplementation, midication) with allele variants in other genes

TGFA, TGB3, MSX1, BCL3, RARA, MTHFR, CYP1A1, MAT1, MAT2, GSTT1, EPHX1
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GxE GWAS approach

Possible

given the new wave of sequencing approaches and computational advances caveats will be problematic

Many

Large sample size with systematic accuracy Easier with a disease/disorder with a singular, easily characterized phenotype Well defined parameters

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