Transcript Research Template - UMKC School of Medicine

Identification of Three Novel SNPs Associated With ARDS by Exome-Seq
K. Shortt1,2, S. Chaudhary2, D. Grigoryev1,2, D.P. Heruth2, L.Venkitachalam1, L.Q. Zhang2, and S.Q. Ye1,2. Department of Biomedical and Health
Informatics1 and Department of Pediatrics2, Children's Mercy Hospitals and Clinics, University of Missouri, Kansas City, MO.
Introduction
 Acute respiratory distress syndrome (ARDS) is
characterized by impaired gas exchange with a systemic
release of inflammatory mediators, hypoxemia, multiple
organ failure and high mortality rates (30-50%).
 The etiology and pathogenesis of ARDS are still not
fully understood and there are few effective therapeutic
options.
 It
is
increasingly
recognized
that
genetic
predisposition
significantly
contributes
to
the
pathogenesis of ARDS. Our objective of this study was
to identify novel genetic markers for susceptibility,
severity and mortality associated with ARDS.
Methods
Data
collection
Study
sample
• SNP Data of 96 patients were obtained using
exome-sequencing (Illumina HiScanSQ)
Table 1. Distribution of Race/Ethnicity
Table 3. Overall Associations of 3 SNPs
ARDS Patients
ARDS Total
Caucasian
African American
Other ancestry
ARDS with Sepsis total
Caucasian
African American
Other ancestry
ARDS with Pneumonia total
Caucasian
African American
Other ancestry
Secondary Controls from the 1000
Genomes Project
1000 genomes Total Control
1000 genomes European Ancestry Control
1000 genomes African Ancestry Control
rs3848719
rs9605146
rs78142040
Susceptibility(cases vs. controls)
Chi-squared p-value
5.47E-13
2.44E-116
4.38E-104
Odds Ratio (95% CI)1 2.30(1.83-2.89) 17.66(13.04-23.90) 153.54(56.03-420.77)
Severity(ventilator-free days/28 days)
p-value
NS
NS
NS
Odds Ratio(95% CI)2
NS
NS
NS
Severity(APACHE II score)
p-value
0.032
NS
0.061
Odds Ratio(95% CI)2 0.55(0.31-0.96)
NS
NS
Outcome(60-day mortality)
p-value
NS*
NS
0.017
Odds Ratio(95% CI)2
NS
NS
2.04(1.13-3.68)
NS: Not significant; * Significantly associated in genotyped Caucasian, pneumonia,
and Caucasian pneumonia subgroups;
1:Odds ratio for alternate allele (allelic test); 2:additive genotypic model
Participants (N)
213
145
43
25
107
71
20
16
106
74
23
9
Participants(N)
1092
379
246
 The outcomes of interest in ARDS cases are
described in Table 2.
• Selected SNPs were validated in additional
117 patients using the TaqMan Assay
Table 2. Outcomes among ARDS cases
• Cases: 213 ARDS patients in total
• Controls: 1092 participants from the 1000
Genomes Project
APACHE II Ventilator-free 60-day
ARDS cases
Score
days/28 days mortality
mean ±SD
mean ±SD
(%)
213(Total)
101.6± 31.9
12.3± 10.3
30.2
96(by Exome-Seq) 97.2±30.4
12.6±10.2
29.4
117(by TaqMan)
105.1±32.7
12.0±10.5
31
• Test association of SNPs with ARDS using
SNP & Variation Suite 7 software package
(Golden Helix, Bozeman, MT) for:
Data analysis • Susceptibility: Presence of disease
• Severity: APACHE II score, Ventilator-free
days/28 days
• Outcome: Mortality at 60 days
Results
 The distribution of race/ethnicity in the study sample is
given in Table 1.
• Among ARDS cases, 68% are Caucasian, 20% are
African American, and 12% are other races.
• Among controls, 35% are of European Ancestry, 23%
are of African ancestry, and 43% are of other
ancestries.
 These three novel SNPs that are associated with
susceptibility
(rs78142040,
rs9605146,
and
rs3848719), severity (rs3848719) and outcome
(rs78142040) of ARDS(Table 3).
 Profile of the 3 potential SNP markers are
described in Table 4
• rs78142040 (C>T) is suspected to occur within a
histone mark in intron 6 of the Arylsulfatase D
gene
• rs9605146 (G>A) causes a coding change from
proline to leucine with a deleterious effect in the
XK, Kell blood group complex subunit-related
family, member 3 gene.
• rs3848719 (G>A) is a synonymous SNP in the
5th exon of the Zinc-Finger/Leucine-Zipper CoTransducer NIF1 gene.
Table 4. Profile of the 3 SNPs
rs3848719
rs9605146
rs78142040
Location
20:44596545 22:17265194
X:2832771
Gene ID
ZNF335
XKR3
ARSD
Call Rate (Cases + Controls)
0.981
0.998
0.995
Call Rate (Cases)
0.883
0.986
0.972
HWE P-value (Cases)
2.86E-04
4.22E-01
1.18E-03
HWE P-value (Controls)
6.69E-10
7.80E-19
1.81E-239
HWE P-value (Cases + Controls)
6.14E-15
2.06E-30
7.47E-02
Reference: Alternate Allele
G>A
G>A
C>T
Alt. Allele Freq. (Cases)
39.4%
38.6%
22.0%
Alt. Allele Freq. (Controls)
22.0%
3.4%
0.2%
Number AA(%) (Cases)
41(21.8%)
34(16.2%)
2(1.0%)
Number AA(%) (Controls)
88(8.1%)
11(1.0%)
2(0.2%)
Number Ar(%) (Cases)
66(35.1%)
94(44.8%)
87(42.0%)
Number Ar(%) (Controls)
305(27.9%)
53(4.9%)
0(0.0%)
Number rr(%) (Cases)
81(45.1%)
82(39.1%)
118(57.0%)
Number rr(%) (Controls)
699(64.0%)
1028(94.1%)
1090(99.8%)
SNP classification
Coding
Coding
Coding
Coding classification
Synonymous Nonsynonymous
Intronic
Reference amino acid
S
P
NK
Alternate amino acid
S
L
NK
Provean prediction (cutoff=-25)1
Neutral
Deleterious
NK
Sift prediction (cutoff=0.05)1
Tolerated
Tolerated
NK
Pph2 prediction2
NK
benign
NK
NK: Not known, HWE: Hardy-Weinberg equilibrium, AA: alternate genotype or
homozygous minor genotype, Ar: heterozygous genotype, rr: reference genotype or
homozygous major genotype
1:http://provean.jcvi.org/genome_submit.php, 2:http://genetics.bwh.harvard.edu/pph2/
Summary
 In our study cohort, 49,834 SNPs were
common across the ARDS subgroups (all
ARDS, Caucasian ARDS, African American
ARDS, all sepsis, and all pneumonia) and the
1000 Genomes Project.
• Of these, the significance level for
association with susceptibility was p<0.01
for 156 SNPs, and p<2.95x10-7 for 102
SNPs
• 53 of the 102 SNPs were located in coding
regions - 24 caused nonsynonymous
changes in amino acid coding; 10 of these
changes had a negative predicted
functional effect.
• Of
the
27
SNPs
analyzed
(24
nonsynonymous , 2 synonymous, 1 splice
variant), the following SNPs emerged as
potential markers to be associated with:
• Susceptibility: rs78142040, rs9605146,
rs3848719
• Severity (APACHEII): rs3848719
• Outcome (60-day Mortality): rs7814204
Conclusion
 In our cohort of ARDS patients, we identified
three
novel
genetic
biomarkers
of
susceptibility, severity, and mortality to ARDS.
 Further studies are warranted to validate
these findings in larger populations and to
identify the underlying molecular mechanisms.
Contact: Katherine Shortt( [email protected])
IRB Approval: This study protocol was determined to be
IRB exempt.
Funded By: NHLBI/NIH Grant (HL080042 & HL080042S1, Ye, SQ), start-up fund of Children's Mercy Hospitals
and Clinics, UMKC (Ye, SQ), and a Sarah Morrison
Student Research Award of UMKC (Shortt, K).