AFRL-SA-WP-SR-2018-0002
Retrospectively Estimating
Prevalence of Peanut Allergy
Genetic Markers in an Air
Force Population
Technical Support
Katherine Kohnen
Summer Hughes, MFS
John Trombley
Amy Walters
Shana Huntsberger
Technical Director
Richard R. Chapleau, Ph.D.
January 2018
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Air Force Research Laboratory
711 th Human Performance Wing
U.S. Air Force School of Aerospace Medicine
Aeromedical Research Department
Applied Technology and Genomics Division
2510 Fifth St., Bldg. 840
Wright-Patterson AFB, OH 45433-7913
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25 Jan 2018
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June 2017 - August 2017
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Retrospectively Estimating Prevalence of Peanut Allergy Genetic Markers in an Air
Force Population
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6. AUTHOR(S)
Katherine Kohnen, Summer Hughes, John Trombley, Amy Walters, Shana Huntsberger,
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Richard Chapleau
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7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)
USAF School of Aerospace Medicine
Aeromedical Research Department
Applied Technology & Genomics Division
2510 Fifth St., Bldg. 840
Wright-Patterson AFB, OH 45433-7913
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AFRL-SA-WP-SR-2018-0002
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14. ABSTRACT
Food allergies are complex, multifactorial traits that pose great risk to health and operational success. Latent food allergies could
manifest catastrophic outcomes during missions, resulting in potential failure and loss of life. Understanding an individual’s risk of
developing severe allergies is one of many components that can make up a high-quality surveillance and public health/preventive
medicine program. We therefore assessed whether two specific genetic markers identified as related to peanut allergies in a civilian
population of children were over- or under-represented in a U.S. Air Force population. Our results showed that for a single
polymorphism, there was a significant difference between the U.S. Air Force population and open-source literature genomes.
Although not strictly a validation of externally identified markers, our pilot study suggests that genetic markers derived from civilian
populations may not always correlate with military cohorts, and internally validating genetic tools prior to developing screens is of the
utmost importance for providing Trusted Care, Anywhere.
15. SUBJECT TERMS
Food allergies, peanuts, genetic markers
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OF ABSTRACT
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Richard Chapleau, PhD
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TABLE OF CONTENTS
Section Page
LIST OF FIGURES.ii
LIST OF TABLES.ii
1.0 SUMMARY.1
2.0 INTRODUCTION.1
3.0 METHODS.2
3.1 Sample Collection.2
3.2 SNP Genotyping.2
4.0 RESULTS.3
4.1 Genotyping a Collection of USAF Nasal Wash Samples.3
4.2 Comparing Risk Groups Between a USAF Population and the U.S. and Global
Populations.3
4.3 Allele Frequencies for Polymorphisms Associated with Peanut Allergy Risk.4
4.4 Combined Risk Group Distributions for USAF Population.4
5.0 DISCUSSION.4
6.0 CONCLUSION.5
7.0 REFERENCES.6
LIST OF ABBREVIATIONS AND ACRONYMS.7
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LIST OF FIGURES
Page
Figure 1. Genotype frequencies for USAF School of Aerospace Medicine nasal wash
archive samples.3
LIST OF TABLES
Page
Table 1. Frequency of Overall Risk Group Combinations.4
ii
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1.0 SUMMARY
Food allergies are complex, multifactorial traits that pose great risk to health and
operational success. Latent food allergies could manifest catastrophic outcomes during missions,
resulting in potential failure and loss of life. Understanding an individual’s risk of developing
severe allergies is one of many components that can make up a high-quality surveillance and
public health/preventive medicine program. We therefore assessed whether two specific genetic
markers identified as related to peanut allergies in a civilian population of children were over- or
under-represented in a U.S. Air Force population. Our results showed that for a single
polymorphism, there was a significant difference between the U.S. Air Force population and
open-source literature genomes. Although not strictly a validation of externally identified
markers, our pilot study suggests that genetic markers derived from civilian populations may not
always correlate with military cohorts, and internally validating genetic tools prior to developing
screens is of the utmost importance for providing Trusted Care, Anywhere.
2.0 INTRODUCTION
The Department of Defense considers peanut allergy an unwaiverable medical condition
[1], partially because much of the food provided to military personnel may be prepared and
manufactured in a few facilities. Additionally, downrange operations often require logistics
footprints that would be cost-prohibitive for accommodating food allergies in a small proportion
of active duty (AD) service members. For example, food allergies affect 2-10% of U.S. children
in the general population [2], and although such allergies are increasing in prevalence, they
account for a negligible fraction of potential AD warfighters. The scientific community currently
has little consensus about how and why food allergies develop, and there has been interest in
studying the possible genetic markers causing an increased risk of developing food allergies in
individuals.
A person can develop a food allergy at any age, with peaks in childhood and early
adulthood [3], and having knowledge about who is at risk of developing a food allergy may be
valuable to the U.S. Air Force (USAF), as Airmen potentially can develop allergies while they
are AD. Mild allergic reactions may include nasal drip, itchy skin and eyes, digestive problems,
and even respiratory distress [4]. Anaphylaxis is a rare, systemic reaction to an allergen that may
cause death in some severe cases and may be very difficult to treat in the field. In fiscal year
2009, the Department of Defense launched a research program funding four laboratories to
investigate the genetics of food allergies. A study from that program identified two single¬
nucleotide polymorphisms (SNPs) as being associated with the development of peanut allergies
[5]. An individual homozygous at either SNP for the risk allele resulted in a threefold higher risk
of developing a peanut allergy, while a heterozygous (one risk allele) individual had an increased
risk of 1.7-fold.
The overall goal of this pilot study was to determine if there are any differences between
allele and genotype frequencies in these two SNPs between a USAF population and the U.S. and
global populations. Here, we extracted DNA from a USAF nasal wash repository and genotyped
by polymerase chain reaction (PCR) to compare allele frequencies available from the 1000
Genomes Project [6]. Our results suggested a statistically significant increase in risk allele
frequency for one of the two SNPs and an overall increase in risk genotype frequency in the
USAF samples. Further research efforts are necessary to confirm these results and determine if
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the genetic predispositions are consistent across a current AD population and if they are
associated with childhood or adult-onset allergies. Ultimately, we hope that by identifying an
increased risk of developing food allergies, we may provide medical personnel with improved
surveillance on elevated risk Airmen for annual screening to prevent surprise allergic reactions
while on duty. In essence, our results in this pilot study may serve as support for additional
resources to investigate the genetic associations of these SNPs to peanut allergy risk and develop
screening tools.
3.0 METHODS
The Air Force Research Laboratory Institutional Review Board reviewed the research
protocol and classified the research as non-human subjects research (Protocol Number:
FWR20170097N).
3.1 Sample Collection
A de-identified nasal wash archive located at the USAF School of Aerospace Medicine
contains more than 2100 samples diagnosed with coronavirus collected from military medical
treatment facilities between 2013 and 2014. Of these, we extracted DNA from 465 samples using
the Maxwell 16 Cell DNA Purification Kit on a Maxwell 16 DNA extractor (Promega, Madison,
WI). Extractions were quantified for concentration of DNA and A260/A280 ratio using the
NanoDrop 1000 Spectrophotometer (ThermoFisher Scientific, Waltham, MA) with 2 pL of
sample. A quality control filter eliminated samples with a DNA concentration less than 1 ng/pL
or an A260/A280 ratio outside 0.8-3. After quality filtering, 247 samples remained available for
further analyses, putting the extraction efficiency at 53%.
3.2 SNP Genotyping
We used the BioMek FX P (Beckman Coulter Life Sciences, Indianapolis, IN) automated
liquid handler to prepare samples in 96-well plates for genotyping by PCR. Each plate consisted
of 46 samples assayed for both SNP assays (rs9275596 and rs7192) and four no template
controls. We performed PCR genotyping using the 7500 Fast Real-Time thermocycler (Applied
Biosystems, Foster City, CA) according to the manufacturer’s instructions for TaqMan-based
assays. We repeated samples once with insufficient amplification in either one of the two assays.
Automated genotype calling was supplemented with manual annotation, when necessary, using
the 7500 Fast software. Of the 247 samples, 204 had successful amplification in both SNPs after
the reruns, making the final PCR success rate 83%.
We assigned risk groups for each sample based on genotype per SNP. Samples
homozygous for the risk allele were “high risk,” heterozygous samples were “medium risk,” and
homozygous samples for the normal allele were “low risk.” The allele and risk group (genotype)
frequencies were then calculated and compared to global and U.S. frequencies obtained from the
1000 Genomes Project online database [6]. The risk alleles for rs9275596 and rs7192 are C and
T, respectively [5].
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4.0 RESULTS
4.1 Genotyping a Collection of USAF Nasal Wash Samples
For the SNP rs9275596, the low risk group was most frequent in the USAF samples
followed by the medium risk group. The highest risk group, associated with a threefold increase
of risk of peanut allergy development, was least frequent (Figure 1A). As peanut allergies are
disqualifying for military service, we expected to see a low frequency of elevated risk groups. In
contrast, the medium and low risk groups for rs7192 were nearly identical in frequency, while
the highest risk group had a higher frequency than in rs9275596 (Figure IB).
A (rs7192)
Low- GG
42%
Medium
45%
B (rs9275596)
Figure 1. Genotype frequencies for USAF School of Aerospace Medicine nasal wash archive samples.
4.2 Comparing Risk Groups Between a USAF Population and the U.S. and Global
Populations
Our analysis of the 1000 Genomes Project’s online database found the global risk group
frequencies for rs7192 are 12.1%, 46.7%, and 36.6%, for high, medium, and low risk groups,
respectively (Figure 1A). For the U.S. population, we calculated risk group frequencies of
13.7%, 39.3%, and 47.0%, respectively. Neither comparison between our sample population and
the global or the U.S. population showed significant difference (P=0.39 and 0.23, respectively).
The P-values were calculated using chi-square.
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In contrast, for rs9275596 we found significant deviation within the USAF population
compared to the global and U.S. populations. The global population distributions are 6.7%,
33.1%, and 60.2%, for high, medium, and low risk, while the U.S. distributions are 7.7%, 32.8%,
and 59.58%, respectively. There were significant differences in the risk group frequencies across
both populations. Our population’s distributions of 8.9%, 43.6%, and 47.4% (Figure IB) were
dramatic deviations from the global population (P=0.001) and the U.S. population (FA).002).
4.3 Allele Frequencies for Polymorphisms Associated with Peanut Allergy Risk
Comparing risk allele frequencies between our USAF population and either the global or
U.S. population resulted in finding significant differences for rs7192 (35.3%, 34.0%, and 32.1%,
respectively; P=0J and 0.3, respectively). In contrast, the risk allele frequency difference for
rs9275596 for our population and the global population was significant (68.9% vs. 76.7%,
P=0.008), while the difference from the U.S. population was not significant (73.4%, P=0.1).
4.4 Combined Risk Group Distributions for USAF Population
We calculated an overall risk classification based upon the genotypes of each SNP to
identify the frequency of overall risk group combinations between the two SNPs for our
population sampling. The most frequent risk group observed was low-low (LL, 35.78%),
indicating homozygous for the non-risk allele for both SNPs. A combined 16% of the USAF
population has at least one genotype suggesting heightened risk of developing peanut allergies
(Table 1). Overall, the difference between the risk group frequencies and allele frequencies for
both SNPs together was significant (USAF vs. global: P=0.006, USAF vs. U.S.: P=0.03).
Table 1. Frequency of Overall Risk Group Combinations
Overall Risk Group
USAF(%)
Low-Low
35.78
Low-Medium
15.20
Low-High
2.45
Medium-Medium
32.84
Medium-High
7.84
High-High
5.88
5.0 DISCUSSION
Despite decades of active research, the process by which food allergies develop remains a
complex issue. Evidence suggests that genetic predisposition makes up some portion of risk [5];
however, it does not explain all facets of food allergies. In particular, why some adults develop
allergies to foods they once were able to consume without a reaction is a puzzle open to
interpretation. Using genetic indicators to help predict risk of developing an allergy may be
advisable for the USAF as a precision-based care and risk management strategy.
Our work highlighted a specific polymorphism in the histocompatibility complex,
hypervariable region as having a reduced risk allele frequency compared to civilian populations.
However, we still found that approximately 16% of the sampled population had at least one
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“high risk” genotype, indicating at least a threefold increase in chance of peanut allergy
development. It is important to remember that the higher risk genotypes do not guarantee peanut
allergies, but merely suggest and increased risk of allergy development. While having a peanut
allergy is disqualifying and an unwaiverable medical condition, being able to survey for risk of
peanut allergy development could allow an Airman to be more aware of reactions to food
sources and possibly self-limit intake of trigger foods, strategies that may reduce the chance of
severe allergic reaction [7].
Our original hypothesis was that the risk alleles and high-risk genotypes should be
present less frequently in USAF populations. However, the data suggest that for one
polymorphism (rs9275596), our population has an increased frequency of elevated risk and there
is no decrease in risk group frequency for the other polymorphism (rs7192). As the nasal washes
used in our study came from military medical treatment facilities that treat both military
personnel and their families, a portion of our population is likely to have been non-AD. The
number of diagnosed peanut allergies should be limited to the family members treated at the
facility, which may account for a portion of the genetic risk alleles. However, as discussed
above, merely having an elevated risk genetic profile does not guarantee an individual will
develop an allergy. We also expect, therefore, that a systematic investigation of diagnoses vs.
genotype of only AD Airmen would still find a portion of the population with risk genotypes,
albeit we expect a lower value than for the civilian population. We suggest thoroughly
examining this assertion in a formal manner before any effort begins to develop administrative,
policy, or medical decisions.
6.0 CONCLUSION
Food allergies are complex, multifactorial traits that pose great risk to health and
operational success. Latent food allergies could manifest catastrophic outcomes during missions,
resulting in potential failure and loss of life. Understanding an individual’s risk of developing
severe allergies is one of many components that can make up a high-quality surveillance and
public health/preventive medicine program. We therefore assessed whether two specific genetic
markers identified as related to peanut allergies in a civilian population of children were over- or
under-represented in a USAF population. Our results showed that for a single polymorphism,
there was a significant difference between the USAF population and open-source literature
genomes. Although not strictly a validation of externally identified markers, our pilot study
suggests that genetic markers derived from civilian populations may not always correlate with
military cohorts, and internally validating genetic tools prior to developing screens is of the
utmost importance for providing Trusted Care, Anywhere.
5
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7.0 REFERENCES
1. Department of Defense. Medical standards for appointment, enlistment, or induction in the
military services. Washington (DC): Department of Defense; 2011. Department of Defense
Instruction 6130.03. [Accessed 17 Oct 2017]. Available from http://www.esd.whs.mil/
Portals/54/Documents/DD/issuances/dodi/613003p.pdf .
2. Sicherer SH, Sampson HA. Food allergy: epidemiology, pathogenesis, diagnosis, and
treatment. J Allergy Clin Immunol. 2014; 133(2):291-307.
3. Kamdar TA, Peterson S, Lau CH, Saltoun CA, Gupta RS, Bryce PJ. Prevalence and
characteristics of adult-onset food allergy. J Allergy Clin Immunol Pract. 2015; 3(1): 114-
115.
4. Mayo Clinic Staff. Peanut allergy, (n.d.). [Accessed 17 Oct 2017]. Available from
https://www.mavoclinic.org/diseases-conditions/peanut-allergv/basics/symptoms/con-
20027898 .
5. Hong X, Hao K, Ladd-Acosta C, Hansen KD, Tsai HJ, et al. Genome-wide association study
identifies peanut allergy-specific loci and evidence of epigenetic mediation in US children.
Nat Commun. 2015; 6:6304.
6. 1000 Genomes Project Consortium, Auton A, Brooks LD, Durbin RM, Garrison EP, et al. A
global reference for human genetic variation. Nature. 2015; 526(7571):68-74.
7. Al-Muhsen S, Clarke AE, Kagan RS. Peanut allergy: an overview. CMAJ. 2003;
168(10): 1279-1285.
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LIST OF ABBREVIATIONS AND ACRONYMS
AD
PCR
SNP
USAF
active duty
polymerase chain reaction
single nucleotide polymorphism
U.S. Air Force
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