Genome Wide Association Study of Sepsis in Extremely
Premature Infants
Lakshmi Srinivasan, MBBS, MTR
1
, Grier Page, PhD
2
, Haresh Kirpalani, BM, MSc
1
, Jeffrey
C. Murray, MD
3
, Abhik Das, PhD
4
, Rosemary D. Higgins, MD
5
, Waldemar A. Carlo, MD
6
,
Edward F. Bell, MD
3
, Ronald N. Goldberg, MD
7
, Kurt Schibler, MD
8
, Beena G. Sood, MD,
MS
9
, David K. Stevenson, MD
10
, Barbara J. Stoll, MD
11
, Krisa P. Van Meurs, MD
10
, Karen J.
Johnson, RN
3
, Joshua Levy, MS
2
, Scott A. McDonald, BS
2
, Kristin M. Zaterka-Baxter, RN,
BSN
2
, Kathleen A. Kennedy, MD, MPH
12
, Pablo J. Sánchez, MD
13
, Shahnaz Duara, MD
14
,
Michele C. Walsh, MD, MS
15
, Seetha Shankaran, MD
16
, James L. Wynn, MD
17
, and C.
Michael Cotten, MD, MHS
7
for the Eunice Kennedy Shriver National Institute of Child
Health and Human Development Neonatal Research Network
1
Department of Pediatrics, The Children’s Hospital of Philadelphia and The University of
Pennsylvania, Philadelphia, PA
2
Social, Statistical and Environmental Sciences Unit, RTI International, Research Triangle Park,
NC
3
University of Iowa, Department of Pediatrics, Iowa City, IA
4
Social, Statistical and Environmental Sciences Unit, RTI International, Rockville, MD
5
Eunice Kennedy Shriver National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, MD
6
Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL
7
Department of Pediatrics, Duke University, Durham, NC
8
Department of Pediatrics, University of Cincinnati, Cincinnati, OH
9
Department of Pediatrics, Wayne State University, Detroit, MI
10
Department of Pediatrics, Division of Neonatal and Developmental Medicine, Stanford
University School of Medicine and Lucile Packard Children’s Hospital, Palo Alto, CA
11
Emory University School of Medicine, Department of Pediatrics, Children’s Healthcare of
Atlanta, Atlanta, GA
12
Department of Pediatrics, University of Texas Medical School at Houston, Houston, TX
13
Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX
Contact information for the corresponding author (and reprint request author): Lakshmi Srinivasan, MBBS MTR, Clinical Associate in
Pediatrics, The Children’s Hospital of Philadelphia, Room 2NW17, 34
th
and Civic Center Boulevard, Philadelphia, PA 19104,
srinivasanl@email.chop.edu, Phone: 215-590-1653, Fax: 215-590-3051.
CONFLICTS OF INTEREST STATEMENT:
The authors do not have any conflicts of interest to report.
HHS Public Access
Author manuscript
Arch Dis Child Fetal Neonatal Ed
. Author manuscript; available in PMC 2017 September
01.
Published in final edited form as:
Arch Dis Child Fetal Neonatal Ed
. 2017 September ; 102(5): F439–F445. doi:10.1136/
archdischild-2016-311545.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
14
University of Miami Miller School of Medicine, Miami, FL
15
Department of Pediatrics, Rainbow Babies & Children’s Hospital, Case Western Reserve
University, Cleveland, OH
16
Department of Pediatrics, Wayne State University, Detroit, MI
17
Department of Pediatrics, University of Florida, Gainesville, FL
Abstract
Objective—To identify genetic variants associated with sepsis (early and late-onset) using a
genome wide association (GWA) analysis in a cohort of extremely premature infants.
Study Design—Previously generated GWA data from the Neonatal Research Network’s
anonymized genomic database biorepository of extremely premature infants were used for this
study. Sepsis was defined as culture-positive early-onset or late-onset sepsis or culture-proven
meningitis. Genomic and whole genome amplified DNA was genotyped for 1.2 million single
nucleotide polymorphisms (SNPs); 91% of SNPs were successfully genotyped. We imputed 7.2
million additional SNPs. P values and false discovery rates were calculated from multivariate
logistic regression analysis adjusting for gender, gestational age and ancestry. Target statistical
value was p<10
−5
. Secondary analyses assessed associations of SNPs with pathogen type. Pathway
analyses were also run on primary and secondary end points.
Results—Data from 757 extremely premature infants were included: 351 infants with sepsis and
406 infants without sepsis. No SNPs reached genome-wide significance levels (5×10
−8
); two SNPs
in proximity to FOXC2 and FOXL1 genes achieved target levels of significance. In secondary
analyses, SNPs for ELMO1, IRAK2 (Gram positive sepsis), RALA, IMMP2L (Gram negative
sepsis) and PIEZO2 (fungal sepsis) met target significance levels. Pathways associated with sepsis
and Gram negative sepsis included gap junctions, fibroblast growth factor receptors, regulators of
cell division and Interleukin-1 associated receptor kinase 2 (p values<0.001 and FDR<20%).
Conclusions—No SNPs met genome-wide significance in this cohort of ELBW infants;
however, areas of potential association and pathways meriting further study were identified.
List of keywords
infection; ELBW; extreme prematurity; genetics
BACKGROUND
Sepsis is a potentially life threatening illness, affecting 10–20% of neonates worldwide, with
a higher incidence in preterm and very low birth weight infants.
12
Up to one-third of
extremely premature infants develop sepsis, which carries a high mortality and morbidity.
1–3
Susceptibility to neonatal sepsis is mediated by complex interactions between environmental
and maternal factors and the neonatal immune response, which may be modified by
differences in genetic composition or function.
3–5
Twin studies and ethnic variations suggest
that host genetic factors may contribute to susceptibility to sepsis.
4–8
Genetic studies of
Srinivasan et al.
Page 2
Arch Dis Child Fetal Neonatal Ed
. Author manuscript; available in PMC 2017 September 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
sepsis in newborns have previously used candidate gene approaches, with varied
findings.
9–14
While genome-wide association studies (GWAS) have been performed in adult sepsis, no
GWAS has reported genetic variation in risk of sepsis in extremely preterm infants.
15–17
An
agnostic GWAS approach rather than a study on known variant candidate genes has potential
to identify gene variants across many genes (1 million or more variants), including disease
associations with previously unknown genes. In addition, investigators do not possess
sufficient knowledge of the function of all genes to predict candidate genes well. The high
rate of sepsis in extremely low birth weight (ELBW) neonates establishes them as a unique
population for unbiased study of genetic associations with sepsis.
4
METHODS
Study population and data source
We performed a secondary analysis utilizing an anonymized DNA biorepository from a prior
study of cytokines and neurodevelopmental outcomes in ELBW infants conducted by the
Eunice Kennedy Shriver NICHD Neonatal Research Network (NRN).
1819
Enrolled infants
were <1000 grams birth weight and <72 hours old at enrollment; infants with major
congenital anomalies were excluded. The NRN’s anonymized DNA database and
biorepository was previously approved by all site institutional review boards (IRBs) for
candidate gene analyses and genome wide analyses of diseases associated with extreme
prematurity, including sepsis.
20
We received a waiver of IRB authorization for use of this
anonymized data set. In this multi-racial and multi-ethnic study, blood spot samples were
collected on more than 800 ELBW infants and analyzed using the Illumina Omni-1 Quad
array.
20
Primary Outcome
Our primary outcome was the identification of single nucleotide polymorphisms (SNPs)
significantly associated with the occurrence of all sepsis in extremely preterm infants.
Secondary Outcomes
Given that EOS and LOS have distinct etiologies and likely differences in mechanisms
underpinning their pathogenesis, we planned
a priori
to identify SNPs significantly
associated with late-onset sepsis (LOS) alone.
321
Due to the relatively low incidence of
early-onset sepsis (EOS), it was not feasible to examine SNPs associated with EOS alone.
We also identified SNPs associated with Gram positive, Gram negative, and fungal
pathogens. While infants who experienced multiple episodes of sepsis with different
bacterial pathogens were included only once in the primary analysis, they were included in
each pathogen category that they fit in secondary analyses. Finally, we also reviewed SNPs
tested in previous candidate gene association studies in neonatal sepsis.
Study definitions
Cases consisted of infants with one or more episodes of culture-proven sepsis (EOS or LOS)
or meningitis, treated with antibiotics for ≥ 5 days, or, who died before treatment was
Srinivasan et al.
Page 3
Arch Dis Child Fetal Neonatal Ed
. Author manuscript; available in PMC 2017 September 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
completed.
12
Controls were infants not classified as cases. Infants with positive blood or
CSF cultures were defined as having culture proven sepsis. EOS comprised infants with
culture proven sepsis in the first 72 hours of life, while LOS comprised cases beyond that
timeframe. All subjects included in the study were followed through the duration of their
hospital course for occurrence of sepsis.
Sample source and storage
Our study utilized DNA data obtained from samples originally collected as part of a
prospective multicenter cohort study of cytokines in premature infants (the Cytokine Study,
performed in 17 centers of the National Institute of Child Health and Human Development
Neonatal Research Network).
18
In the parent study, whole blood spots were obtained on
filter paper within 4 hours after birth and at serial timepoints (days 3, 7, 14, 21). These filter
paper blood spots were frozen as soon as possible. For our study and other secondary
genomic analyses performed utilizing this dataset, DNA was extracted from filter paper
blood spots for the earliest age available for each subject, amplified and analyzed using the
Illumina Omni-1 Quad array, as described below. Additional details regarding sample
methods are available in prior studies utilizing this sample set.
1820
Genomic analysis
Genomic and whole genome amplified DNA were genotyped on the Human-OMNI1-
Quad_v1-0_B BeadChip platform (Illumina, San Diego, California). Initially, 1.2 million
SNPs were tested, of which over 900,000 (91%) were successfully genotyped. We employed
standard quality control thresholds, and included samples and SNPs with > 97% call rate,
while excluding minor allele frequencies < 1%. Additionally, 7.2 million SNPs were
imputed against the HapMap 3 cosmopolitan reference panel with Impute2.
20
All SNP-gene
assignments were made based on the UCSC gene tracks within the GRCh37/hg19 genome
build.
2223
Genotypes, and a subset of subject information inclusive of key characteristic and
outcome phenotype data have been stored, with no identifying links, in the National Human
Genome Research Institute Database of Genotypes and Phenotypes (dbGaP Study
Accession: phs000353.v1.p1).
1920
.
Data Analysis
Following standard quality checks, single and multiple logistic regression models were
developed for the primary and secondary analyses. Covariates included in the multiple
regression model included gestational age, small for gestational age (SGA) status, ancestry,
surgery, C-section, and the first four eigenvalues for ancestry (described below). In previous
analyses of this dataset, these covariates were determined to be the most important
predictors of sepsis.
8
To minimize false discoveries, we established a conservative
significance level of 10
−5
as the ‘target’ for reporting, while a p value of 5×10
−8
is regarded
as a “genome-wide” association. The latter value is widely accepted to be the threshold in
genome wide association studies that would adequately adjust for the multiple comparisons
involved, and is estimated to correspond to a p value of 0.05 in a classical epidemiological
study testing a single hypothesis.
22–25
Srinivasan et al.
Page 4
Arch Dis Child Fetal Neonatal Ed
. Author manuscript; available in PMC 2017 September 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
We used this two-tiered approach in order to screen for genes of interest, which may be
missed if only the more stringent threshold were applied. When SNPs met threshold
significance, we did not limit reporting based on FDR thresholds, in order to better identify
any genes of putative biologic importance that should be examined closely in future
validation studies.
25
P values were calculated per SNP for a one degree of freedom additive
model. For the secondary analysis of previously identified candidate SNPs, we reported p
values <0.05 as trends of interest.
Correction for population stratification
Spurious associations may be obtained, or true associations overlooked, if allele frequencies
differ between subpopulations and are represented unequally between cases and controls due
to systematic ancestry differences.
26–28
The first four eigenvalues derived from linkage
disequilibrium pruned for genotyped SNPs were included as covariables in primary and
secondary analyses, to adjust for individual ancestry (eigenvalues generated using GWAS-
Tools).
20
Exploratory pathway analysis
We assigned genes to biological pathways using the Reactome database
(www.reactome.org), using the adaptive rank truncated product (ARTP) method (pathway
tools package ARTP).
29–31
SNPs were notated to genes based on their location within 50 kb
of the gene models defined in Refseq, and p-values assigned based on the results of the
GWA analysis. A summary of the association between each gene and sepsis was generated,
based on the multiple-testing adjusted P-value associated with the most significant SNP
within that gene. The ARTP method was used to combine gene-level P-values into a test
statistic for the pathway association with sepsis.
31
Given the exploratory nature of this
analysis, we designated pathways with p values <0.005 as the cut-off for reporting, and
reported false discovery rates (FDR) of less than 20% to facilitate assessment of the strength
of the evidence.
25
RESULTS
Data from 751 ELBW infants passed quality control checks and were included in the
analysis. This cohort included 345 infants with sepsis (Supplementary Table 1). Most infants
with sepsis had experienced LOS (320 infants, 393 episodes). Ninety-nine infants
experienced more than one episode of sepsis. Gram positive pathogens were present in more
than three-fourths of the LOS events (252 episodes), while Gram negative and fungal sepsis
occurred with lower frequency (72 and 69 episodes respectively, Supplementary Table 1).
Infants could be positive for more than one organism per sepsis event. Of the infants with
sepsis, 42 were diagnosed with culture proven meningitis. Eighty-one infants in the overall
cohort developed necrotizing enterocolitis (NEC), including 40 cases of surgical NEC. NEC
events occurred more frequently in infants with sepsis compared to uninfected infants
(14.1% versus 8.7%, p = 0.02, Chi square test); however the dataset lacked information on
timing of NEC in relation to sepsis event.
Srinivasan et al.
Page 5
Arch Dis Child Fetal Neonatal Ed
. Author manuscript; available in PMC 2017 September 01.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript