THE WHOLE HOST

Effect-measure modification by host genetics of the

relationship between the oral microbial community and
early childhood caries: a case-cohort study

Freida Blostein
04/23/2019
 WHAT IS ECC?

Early childhood caries (ECC) is caries occurring in the primary dentition of children aged
under 6 years1.

In 2011, 22.7% of US children aged 2-5 had caries in their primary teeth, with even higher
prevalence in poor, socially disadvantaged, and minority groups2.

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 WHAT CAUSES ECC?

ECC etiology involves a process of

accelerated tooth demineralization caused by
certain oral microbiota producing acids from
dietary sugars3.

Yet associations between candidate

microorganisms and caries development vary
in different studies.

This may in part be due to other host and

environmental characteristics which confound
or modify the microbe-sugar etiology.

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 WHAT CAUSES ECC?

One proposed host characteristic is tooth enamel

integrity.

Defects in the enamel can result from host genetics,

prenatal exposures and childhood illness.

Genome wide association studies (GWAS) have

identified associations between caries and variants in
genes related to enamel formation4.

One such variant is rs7738851, located in NEDD9.

Introduction Research Question & Hypotheses Study Design Analysis

Analysis Plan Strengths &Discussion
Results Limitations
 RESEARCH QUESTION:

Do host genetic factors modify the association of the oral microbiome with
ECC?

SPECIFICALLY: Does the association between taxonomic composition of early life

oral microbiome and ECC vary by allele of rs7738851?

Introduction Research Question & Hypotheses Study Design Analysis

Analysis Plan Strengths &Discussion
Results Limitations
 HYPOTHESES

1) We hypothesize there will be a positive association between early life oral

microbial communities dominated by acidogenic taxa, such as Veillonella and
mutans streptococci, and ECC.

2) We hypothesize there will be evidence of effect modification by rs7738851

allele A on the additive scale (super-additive effect modification).

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 DAG

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 COHRA2 6

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 COHORT

Prenatal 2 month First tooth 1 year 2 year 3 year 4 year 5 year

visit visit visit visit visit visit visit visit

Demographic questionnaire
Salivary & plaque samples Dietary questionnaire One time host genotyping
Dental exam Tooth hygiene questionnaire

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 CASE-COHORT RATIONALE

X 7 sample points X 1000 samples = $$$$

Prenatal 2 month First tooth 1 year 2 year 3 year 4 year 5 year

visit visit visit visit visit visit visit visit

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 CASE-COHORT SAMPLING

COHORT

SUB-COHORT

CASES

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 CASE-COHORT SAMPLING

COHORT

SUB-COHORT
0.20*1000=200 total

CASES

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 CASE-COHORT SAMPLING

COHORT

SUB-COHORT
0.20*1000=200 total

CASES
0.20*1000
=200 total

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 CASE-COHORT SAMPLING

COHORT

SUB-COHORT
0.20*1000=200 total

200*0.2
CASES =
0.20*1000 40
=200 total

=160 outside sub-

cohort

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 CASE-COHORT SAMPLING

COHORT 200+160=
360 individuals in
analysis sample

200 cases
SUB-COHORT 160 controls
0.20*1000=200 total

200*0.2
CASES =
0.20*1000 40
=200 total

=160 outside sub-

cohort

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 ANALYSIS PLAN

A weighted Cox proportional hazard model with Barlow weighting method to

account for the study design will be used to test for effect modification.

Exposure variable: Microbial composition will be assessed using 16S rRNA amplicon
sequencing of salivary samples from all available visits. Samples will be clustered using
Dirichlet multinomial mixture modeling, a Bayesian algorithm which groups samples
together based on inferred abundance of taxa7.

Time variant covariates: Reported weekly frequency of sugary drink and food intake
and frequency of tooth brushing.

Time invariant covariates: Child’s gender, baseline household income, and allele
variant of rs7738851.

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 WEIGHTING SCHEME

COHORT
𝑤𝑐𝑜𝑛𝑡𝑟𝑜𝑙
1
=
SUB-COHORT
𝑝𝑚 ∗
𝑤𝑐𝑎𝑠𝑒 = 1 1
CASES = =5
0.2

# 𝑐𝑜𝑛𝑡𝑟𝑜𝑙𝑠 𝑖𝑛 𝑠𝑢𝑏𝑐𝑜ℎ𝑜𝑟𝑡 160

∗ 𝑝𝑚 = = = 0.2
𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑟𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙𝑠 800

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 ANALYSIS PLAN

Include an interaction term between categorical exposure variable and dichotomous

variable for A allele of rs7738851.
5
Equation 1: 𝜆 𝑡: 𝐺, 𝐸, 𝐶 = 𝜆0 𝑡 𝑒 𝛽1𝐺+𝛽2𝐸2+𝛽3𝐸3+𝛽4𝐸2∗𝐺+𝛽5𝐸3∗𝐺+∑𝑘=1 𝛾𝑘 𝐶𝑘

Where:

G: binary allele variant

E: Hypothetical dummy variables for categorical microbiome CST (3 levels depicted,

more possible)

C: Sugary drink frequency, sugary food frequency, tooth brushing frequency, child
gender, & baseline household income.

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 MAIN ANALYSIS

5
𝜆 𝑡: 𝐺, 𝐸, 𝐶 = 𝜆0 𝑡 𝑒 𝛽1𝐺+𝛽2𝐸2+𝛽3𝐸3+𝛽4𝐸2∗𝐺+𝛽5𝐸3∗𝐺+∑𝑘=1 𝛾𝑘 𝐶𝑘

Use contrast statements to asses HRs for each combination of genotype and CST
compared to referent of non-risk alleles and low-risk CST to assess effect-measure
modification on multiplicative scale

Calculate HRs of CSTs within strata of genotype.

Calculate RERI to asses effect-measure modification on additive scale

෣ 𝐻𝑅:𝐶𝑆𝑇2 = 𝑒 𝛽෢1+𝛽෡2+𝛽෡4 − 𝑒 𝛽෡1 − 𝑒 𝛽෡2 + 1

Equation 2: 𝑅𝐸𝑅𝐼

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 SENSITIVITY ANALYSIS

Treatment of missing data (main analysis)

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 SENSITIVITY ANALYSIS

Treatment of missing data (main analysis)

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 SENSITIVITY ANALYSIS

Treatment of missing data (sensitivity analysis)

Multiple imputation using entire cohort

SUB-COHORT Substantive Model (Equation 1):

𝜆 𝑡: 𝐺, 𝐸, 𝐶

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 RESULTS

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 RESULTS

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 RESULTS

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 RESULTS

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 RESULTS

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 DISCUSSION

Main effects unsurprising:

Many studies have found associations between acidogenic bacterial communities and ECC.

Although we expect to find only a small effect size of risk allele on ECC, this is also unsurprising:
Enamel quality is likely a polygenic.

Our measure captures only a small amount of variation in genetic determinants of enamel quality.

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 PUBLIC HEALTH IMPORTANCE OF
EFFECT MODIFICATION

Effect-measure modification on additive scale, such as our expected result, is more relevant
public health measure and is useful in identifying target groups for intervention.

Children with poor enamel quality already recommended to undergo increased screening and
preventive efforts against dental decay.

But enamel defects can only be identified once teeth erupt. Identify at risk genetic groups can
allow for earlier prevention and screening – even prenatally?

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 LIMITATIONS IN INTERPRETATION

Biological interaction is plausible

Grooves in hypoplastic enamel  better bacterial adherence
Hypocalcified enamel  more susceptible to acid

BUT

Effect-measure modification ≠ evidence for biological interaction

Additionally….

Can’t draw inference on sufficient cause interaction

Assumptions not met

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 LIMITATIONS

Small sample size  underpowered

Limited generalizability

Ascertainment of the exposure variable

- Collapsing high-dimensional data on microbial community to a single,

categorical variable may result in lower resolution of exposure

- 16S rRNA amplicon sequencing is also inherently limited in that low

abundance organisms may fail to amplify

- If such organisms differ between cases and controls, differential exposure

misclassification may result

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 STRENGTHS

Longitudinal assessment of exposure and outcome information: temporal order of

exposure and outcome ensured

Case-cohort design allows for multiple time point sampling

- Allows for control of time variant confounders.

High retention rate in cohort

Introduction Research Question & Hypotheses Study Design Analysis Results Discussion
 REFERENCES
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• Image References
• Slide 1, Slide 5, Slide 6: Tooth by Ryan Farishian from the Noun Project
• Slide 2, Slide 3: Sad Tooth by Akshar Pathak from the Noun Project
• Slide 3, Slide 6: teeth by HeadsOfBirds from the Noun Project
• Slide 3, Slide 5, Slide 6, Slide 10: Bacteria by mungang kim from the Noun Project
• Slide 4: 1. Pitts NB, Zero DT, Marsh PD, et al. Dental caries. Nat Rev Dis Prim.
2017;3(May):17030. doi:10.1038/nrdp.2017.30.
• Slide 6, Slide 8, Slide 10: dna helix by Olena Panasovska from the Noun Project
• Slide 8: COHRA image: COHRA project
• Slide 8: Map of the Appalachian region of the United States, from the Web site of the
Appalachian Regional Commission, at http://www.arc.gov/images/regionmap.gif PD-
USGov (Public Domain Usage)
• Slide 10. S;ode 9: Test Tube by ProSymbols from the Noun Project
• Slide 10, Slide 9: Survey by Jean-Philippe Cabaroc from the Noun Project
• Slide 10, Slide 9: nursing mother by Sofía Franco from the Noun Project
• Slide 10, Slide 9: Child by Adrien Coquet from the Noun Project
• Slide 11, Slide 10: soda by lipi from the Noun Project
• Slide 10, Slide 9: By OCHA Visual, US In the OCHA Humanitarian Icons Collection
(Public Domain Usage)
• Slide 10, Slide 9: Child by Ludovic Riffault from the Noun Project
• Slide 16: Happy Tooth by Akshar Pathak from the Noun Project
THANKS!