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Genetic polymorphisms and susceptibility to lung disease

Journal of Negative Results in BioMedicine20065:5

https://doi.org/10.1186/1477-5751-5-5

Received: 03 March 2006

Accepted: 11 April 2006

Published: 11 April 2006

Abstract

Susceptibility to infection by bacterium such as Bacillus anthracis has a genetic basis in mice and may also have a genetic basis in humans. In the limited human cases of inhalation anthrax, studies suggest that not all individuals exposed to anthrax spores were infected, but rather, individuals with underlying lung disease, particularly asthma, sarcoidosis and tuberculosis, might be more susceptible. In this study, we determined if polymorphisms in genes important in innate immunity are associated with increased susceptibility to infectious and non-infectious lung diseases, particularly tuberculosis and sarcoidosis, respectively, and therefore might be a risk factor for inhalation anthrax. Examination of 45 non-synonymous polymorphisms in ten genes: p47phox (NCF1), p67phox (NCF2), p40phox (NCF4), p22phox (CYBA), gp91phox (CYBB), DUOX1, DUOX2, TLR2, TLR9 and alpha 1-antitrypsin (AAT) in a cohort of 95 lung disease individuals and 95 control individuals did not show an association of these polymorphisms with increased susceptibility to lung disease.

Introduction

Since October 2001, when Bacillus anthracis was released in the United States as an act of bioterrorism, there has been a greater interest in determining if there are risk factors for inhalation anthrax infection. Exposure to Bacillus anthracis spores does not cause infection in all exposed individuals [1]. Epidemiologic studies of individuals infected by inhalation anthrax have suggested that a weakened immune system might increase susceptibility to infection by Bacillus anthracis [2]. Some of the infected individuals had a history of chronic pulmonary disease, including asthma, sarcoidosis, and tuberculosis [24]. Studies in mice have demonstrated a genetic basis for anthrax sensitivity [5, 6]. For example, macrophages from C3H mice are 100,000 times more sensitive to the Bacillus anthracis toxin than macrophages from A/J mice [6]. The current study examines whether there are genetic polymorphisms in humans associated with increased susceptibility to lung disease. Identification of genes associated with an increased risk of lung disease might identify individuals who might also be of increased susceptibility to inhalation anthrax infection.

The N AD(P)H ox idases (NOX) are a family of enzymes that are essential in host defense against microbial infection, as reviewed by Quinn and Gauss [7]. The central enzyme of the NAD(P)H oxidase is a flavin and heme-containing protein, the most well known being the phagocytic gp91phox (CYBB, NOX2) protein. gp91phox, and a number of related proteins including DUOX1 and DUOX2, are transmembrane proteins which transport electrons and generate reactive oxygen species (ROS) at the expense of NADH or NADPH. The activity of the oxidases are highly regulated by accessory proteins, including p22phox (CYBA), p47phox (NOXO1, NCF1), p67phox (NOXA2, NCF2), and p40phox (NCF4). Chronic Granulomatous Disease (CGD), associated with severe, recurrent, and chronic non-specific bacterial and fungal infections, is most commonly caused by mutations in p47phox, gp91phox, p67phox, and p22phox that severely compromise the respiratory burst activity of neutrophils.

Görlach et al were the first to identify the presence of at least one pseudogene copy of the p47phox (NCF1) gene on chromosome 7q11.23 [8]. By construction of a detailed physical map of this region Hockenhull et al determined that there were one normal wildtype copy and two pseudogene copies of NCF1 per chromosome [9]. Heyworth et al elegantly demonstrated that in some individuals, one of the pseudogene copies of NCF1, possibly by recombination or gene conversion, has reverted to the normal wildtype GTGT sequence (i.e. pseudowildtype) [10]. Thus, individuals with this low frequency polymorphism of NCF1, have 2 "wildtype" copies and one pseudogene copy per chromosome [10]. Therefore, individuals (with 2 chromosomes) can have a NCF1 pseudogene: wt copy ratio of either 2:1, 1:1 or 1:2. Although two groups have examined the association of the minor 1:1 and 1:2 alleles with inflammatory bowel disease, the conclusions were in conflict primarily due to differences in allele frequencies of the control population and sample size [11, 12]. Other polymorphisms in p47phox, p67phox and gp91phox, have not been shown to be associated with human disease other than CGD. Recently p47phox has been shown by positional cloning to regulate the severity of arthritis in rats [13]. The H72Y polymorphisms in p22phox (CYBA), associated with reduced respiratory burst in isolated human neutrophils [14], but has yet to be shown to be clearly associated with a disease phenotype [1517]. DUOX1 and DUOX2, which are expressed in lung epithelium, regulates H2O2 [1820] and acid [21] production in the airway but have not been shown to be associated with lung disease. Mutations in DUOX2 have been shown to be associated with mild hypothyroidism [2224].

TLR2 is the receptor for peptidoglycans, lipoteichoic acid, lipoarabinomannan, mycolylarabinogalactan, and zymosan. Anthrax infection is thought to be partially mediated through the TLR2 pathway since TLR2 deficient mice are resistant to infection by the Sterne strain of Bacillus anthracis and HEK293 cells expressing TLR2, but not TLR4, are able to signal in response to exposure to heat-inactivated Bacillus anthracis [25]. Inactivation and killing of the tuberculosis mycobacterium is also mediated through TLR2 since macrophages from Tlr2-deficient mice or human macrophages blocked by anti-TLR2 antibodies failed to kill the bacteria [26]. Tlr9 and Tlr2 double knockout mice display a more pronounced susceptibility to infection by tuberculosis than single gene knockout mice [27]. The TLR2 polymorphism R753Q [28] and the R677W polymorphism in humans [2931] have been shown to be associated with increase risk for tuberculosis infection. The R753Q polymorphism was not associated with a generalized increased risk of infection, e.g. individuals with R753Q were less responsive to infection by Borrelia burgdorferi, which causes Lyme Disease [32] and R753Q was not associated with increased susceptibility to Staphylococcus aureus infection [33].

Alpha-1-anti-trypsin (AAT) deficiency has been associated with increased susceptibility to lung disease, particularly emphysema [34, 35]. Although more than 70 variants have been described, only a few are associated with reduced AAT protein expression and/or reduced activity [35]. Several studies have suggested that simple heterozygosity for mutant alleles of AAT may predispose individuals to chronic obstructive lung disease [3537]. The Z allele (E366K), which occurs at an allele frequency of 0.01–0.02 in people of European origin, is the most common allele associated with an increased risk of environmentally induced emphysema [34, 3840]. Homozygous individuals of the AAT S allele (E288V) are not at risk for emphysema but compound heterozygotes of the Z and S allele or a null allele are of increased risk [39, 41]. Carriers of the AAT S and Z alleles are over-represented in individuals with lung cancer [42]

In this study, we attempted to determine whether normal nonsynonymous genetic variations identified by the Genbank SNP database or previously described in the literature to be present in the normal population in the genes for p47phox (NCF1), p67phox (NCF2), p40phox (NCF4), gp91phox (CYBB), p22phox (CYBA), DUOX1, DUOX2, TLR2, TLR9 and alpha-1 anti-trypsin (AAT) are associated with an increased susceptibility to tuberculosis, sarcoidosis, recurrent pneumonia, and atypical mycobacterial infection.

Materials and methods

Study participants

Anonymized blood samples from control individuals of European, non-Hispanic origin (n = 95) were obtained from Kaiser Permanente [43] or from The Scripps Research Institute GCRC blood drawing program. From a group of 31,247 participants in a Kaiser Permanente study of European, non-Hispanic origin [43], all individuals that had a documented medical history with hospitalization for lung diseases: atypical mycobacterial infection (n = 1), repeated episodes of pneumonia (n = 5), sarcoidosis (n = 46), and tuberculosis (n = 43), were selected and will be referred to as the lung disease group (n = 95). The participants in the Kaiser Permanente study were members of Kaiser Permanente attending a Health Appraisal Clinic and were not selected for underlying acute or chronic disease. All human samples were obtained with written consent. Approvals for the protocols involving the use of human individuals were obtained from the institutional review boards of The Scripps Research Institute and Kaiser Permanente.

p47phox/NCF1 pseudogene: wildtype ratio

Amplification of the region of p47phox exon 2 with the wildtype GTGT sequence and the pseudogene delGT sequence were amplified using primers p47phox/NCF1 Ex2F GCTTCCTCCAGTGGGTAGTGGGATC and p47phox/NCF 161R GGAACTCGTAGATCTCGGTGAAGC and 32P-labeled p47phox/NCF1 Ex2F primer under standard PCR conditions for 25 cycles. The 32P-labeled amplified DNA products were separated on a 10% acrylamide/urea/TBE sequencing gel. Autoradiography was used to visualize the wildtype and pseudogene amplified products, which differ by 2 nucleotides in length.

Genotyping of single nucleotide polymorphisms (SNPs) by allele specific oligomer hybridization (ASOH)

For the genes of this study, non-synonymous SNPs identified in Genbank's SNP database and/or non-synonynous SNPs associated with lung disease were investigated. Amplification of DNA regions encompassing the SNPs were amplified using the primers listed in Table 1. ASOH was performed using standard hybridization conditions [44] using 32P radiolabeled probes and washing temperatures described in Table 1. Genotyping was determined following visualization of the hybridized probe by autoradiography.
Table 1

Primer List. List of primers used for DNA amplification and ASOH.

Primer name

Sequence

Temp °C

p47 Ex2F

GCTTCCTCCAGTGGGTAGTGGGATC

60

p47 161R

GGAACTCGTAGATCTCGGTGAAGC

 

Primer name

Sequence

Temp °C

p40 Ex2F

GTGCTGAGAGACGAATGTTGG

60

p40 Ex2R

GGGCAAGGTTCAGAGGTCAG

 

p40 Ex5F

GACGGGACATCTAGGCTGG

60

p40 Ex5R

GGCTCTGGCCATGTGGAAG

 

p40 Ex8F

TCTGAGGCGTGGCTCTGCTG

60

p40 Ex8R

GCTCATCTGGGAGCCACTGG

 

p40 Ex10F

ATGACACGGGCTTGTATCAGG

60

p40 Ex10R

GAGCTGAAGGTTTTTGCTGGTG

 

p40 86T

TGCTGACATCGAGGAGA

53

p40 86C

TGCTGACACCGAGGAGA

53

p40 353G

CCTGCTCAGCCTGCCGG

62

p40 353A

CCTGCTCAACCTGCCGG

61

p40 815C

ACGACCACCGCCCCTCA

58

p40 815T

ACGACCACTGCCCCTCA

56

p40 911C

GGACGTAGCGCTCATGG

57

p40 911A

GGACGTAGAGCTCATGG

55

Primer name

Sequence

Temp °C

p67 Ex3F

CTGGGCACCACAGGGAGCTA

58

p67 Ex3R

CACCAAGCCCGCAACACTGA

 

p67 Ex6F

GGGCTTCTATGTGGTTATCTCAA

60

p67 Ex6R

CCACAAGGAGGCTACCCTCTTCT

 

p67 Ex9F

GAGCCCAGGCAGGCTCAGTGTCAT

60

p67 Ex10R

GCCATCTCAAGGCGGGCTCAAGA

 

p67 Ex11F

GTGTTTCCCCACATCCAC

60

p67 Ex11R

AAGGCAGGGAGAGGAACT

 

p67 Ex13F

CAAGGGTTGGGCTAAAGGAC

60

p67 Ex14R

GTGTTCTCACACCACAGAGTCAG

 

p67 542G

TGTGGGCAGGCTGTTTC

55

p67 542A

TGTGGGCAAGCTGTTTC

53

p67 836C

CTGGGCCACGGTCATGT

57

p67 836T

CTGGGCCATGGTCATGT

55

p67 983G

CCCTGGAAGACCCCAGC

47

p67 983A

CCCTGGAAAACCCCAGC

47

p67 1105G

CTCAGCCCGGGCTCCCC

50

p67 1105A

CTCAGCCCAGGCTCCCC

50

p67 1167C

GCTGGAACACACTAAGCTG

54

p67 1167A

GCTGGAACAAACTAAGCTG

54

p67 1183C

CCAGCTATCGGCCTCGG

57

p67 1183T

CCAGCTATTGGCCTCGG

57

Primer name

Sequence

Temp °C

p22 Ex 2F

GACCCTGTCACTGTGCTGTG

61

p22 Ex 2R

GAGGCAAACAGCTCACTGTG

 

p22 Ex 3F

CTGAGCTGGGCTGTTCCTT

63

p22 Ex 3R

CCACCCAACCCTGTGAGC

 

p22 Ex 4F

CAGCAAAGGAGTCCCGAGT

60

p22 Ex 4R

GGAAAAACACTGAGGTAAGT

 

p22 Ex 5F

AAGGCTGAGAACACCCAGG

60

p22 Ex 5R

GCTCAGCCTACAGAGCCG

 

p22 Ex 6F

GACCCAGGTCCTGGCTGTG

60+DMSO

p22 Ex 6R

AGGCTCACGCGCTCCCGG

 

p22 85A

TCGTGGCCACAGCTGGG

59

p22 85G

TCGTGGCCGCAGCTGGG

59

p22 113T

GTGGTACTTTGGTGCCT

52

p22 113C

GTGGTACTCTGGTGCCT

52

p22 179A

GAAGAGGAAGAAGGGCT

51

p22 179C

GAAGAGGACGAAGGGCT

53

p22 214C

GACAGAAGCACATGACC

53

p22 214T

GACAGAAGTACATGACC

51

p22 403G

CGCCCATCGAGCCCAAG

59

p22 403A

CGCCCATCAAGCCCAAG

56

p22 521C

GCTGCGGCGGCGGCG

62

p22 521T

GCTGCGGTGGCGGCG

60

Primer name

Sequence

Temp °C

gp91phox Ex 9F

CTAAAGCAGAGATCTAAGTGG

61

gp91phox Ex 9R

ACGGTGACCACAGAAATAGCTACCT

 

gp91phox Ex 11F

GTTTCTAGGCATTCTGAGCATCAAG

61

gp91phox Ex 11R

GTTCGTAAGCCCTGTACACTATG

 

gp91phox Ex 12F

GTGCCTTGGTTAGAATAGCTTGTG

61

gp91phox Ex 12R

GTTGAAGATATCTGGAATCTTCTGTTG

 

gp91phox 907C

TGGTCACTCACCCTTTC

50

gp91phox 907A

TGGTCACTAACCCTTTC

48

gp91phox 1414G

ACAATGCCGGCTTCCTC

55

gp91phox 1414A

ACAATGCCAGCTTCCTC

53

gp91phox 1499A

GGAGAAAGATGTGATC

48

gp91phox 1499G

GGAGAAAGGTGTGATC

50

Primer name

Sequence

Temp °C

DUOX1 27F

AGAGAGATCTCCTCTCAAGG

58

DUOX1 27R

GGTCACCGGAAGAGCTGAG

 

DUOX1 28F

GGGACCTTGGAAGCTCCAG

58

DUOX1 28R

GGACGTCGAGAAGTGAAGAG

 

DUOX1 3532T

GGTCTGAGTTCCCCCAG

58

DUOX1 3532C

GGTCTGAGCTCCCCCAG

60

DUOX1 3647G

GCCGCCGCCGCAGTTTCC

66

DUOX1 3647A

GCCGCCGCCACAGTTTCC

63

Primer name

Sequence

Temp °C

DUOX2 Ex5F

ATGTTCTTTCCGACGTGGTGAG

63

DUOX2 Ex6R

GCGCCGCCCACATGAGCAG

 

DUOX2 Ex17F

GCCTGCTCAGACTCACAGAG

62

DUOX2 Ex17R

ACTCCTTAGGGATCTTGAGCAG

 

DUOX2 Ex24F

GATGCCTGCCAGATCCCCAG

62

DUOX2 Ex25R

TGGCCGCCGTGCCTCGTG

 

DUOX2 413T

TGGAGACCTCGTGTTCG

54

DUOX2 413C

TGGAGACCCCGTGTTCG

56

DUOX2 429A

CCGAACAGCGCGGGGAC

60

DUOX2 429C

CCGACCAGCGCGGGGAC

63

DUOX2 597-8GG

GCTTCTCGGGGGGACAG

58

DUOX2 597-8GA

GCTTCTCGAGGGGACAG

56

DUOX2 597-8CG

GCTTCTCCGGGGGACAG

58

DUOX2 597-8CA

GCTTCTCCAGGGGACAG

56

DUOX2 2048G

TGTGCTCCGTGTGGTCC

56

DUOX2 2048A

TGTGCTCCATGTGGTCC

54

DUOX2 3026G

CACTCCCCGGCTGTACA

56

DUOX2 3026A

CACTCCCCAGCTGTACA

52

DUOX2 3200T

CTTTGCCTTGCCACCCT

53

DUOX2 3200C

CTTTGCCTCGCCACCCT

55

Primer name

Sequence

Temp °C

TLR2 450F

ATTGCAAATCCTGAGAGTGG

58

TLR2 688R

GCAGTTCCAAACATTCCACG

 

TLR2 1141F

GCCTGTGAGGATGCCTGG

60

TLR2 1827R

GCACAGGACCCCCGTGAG

 

TLR2 1782F

GTGCTGTGCTCTGTTCCTG

60

TLR2 2392R

TCCCAACTAGACAAAGACTGG

 

TLR2 170T

GAAAAGATTTTGCTGGAC

53

TLR2 170Tdel

GAAAAGATTTGCTGGAC

53

TLR2 1892C

GGAAGCCCAGGAAAGCT

55

TLR2 1892A

GGAAGCACAGGAAAGCT

53

TLR2 2258G

CAAGCTGCGGAAGATAA

50

TLR2 2258A

CAAGCTGCAGAAGATAA

48

Primer name

Sequence

Temp °C

TLR9 Ex2F

GTGGGTGGAGGTAGAGCTG

60

TLR9 365R

ACAGCCAAGAAGGTGCTGG

 

TLR9 2501F

TGCTGCATCACCTCTGTGG

54

TLR9 2794R

TGCGGCTGCCATAGACCG

 

TLR9 13C

GTTTCTGCCGCAGCGCC

60

TLR9 13T

GTTTCTGCTGCAGCGCC

58

TLR9 237T

CACCTCCATGATTCTGA

52

TLR9 237G

CACCTCCAGGATTCTGA

54

TLR9 296C

GAACTGCCCGCCGGTTG

58

TLR9 296T

GAACTGCCTGCCGGTTG

60

TLR9 2588G

AAGTGGGCGAGATGAGG

57

TLR9 2588A

AAGTGGGCAAGATGAGG

55

TLR9 2644G

CGCAGAGCGCAGTGGCA

60

TLR9 2644A

CGCAGAGCACAGTGGCA

58

Primer name

Sequence

Temp °C

AAT Ex2F

TGTCGGCAAGTACTTGGCACAG

60

AAT Ex2R

CATAATGCATTGCCAAGGAGAG

 

AAT Ex3F

CAGATGATGAAGCTGAGCCTCG

65

AAT Ex3R

AGCCCTCTGGCCAGTCCTGATG

 

AAT Ex5F

GAGCAAGGCCTATGTGACAGG

60

AAT Ex5R

AGCTCAACCCTTCTTTAATGTCAT

 

AAT 374G

ACTCCTCCGTACCCTCA

56

AAT 374A

ACTCCTCCATACCCTCA

54

AAT 863A

GCACCTGGAAAATGAAC

50

AAT 863T

GCACCTGGTAAATGAAC

50

AAT 1096G

CCATCGACGAGAAAGGG

56

AAT 1096A

CCATCGACAAGAAAGGG

54

Statistics

The Fisher's Exact test was performed with GraphPad InStat using the raw data entered into a 2 × 2 contingency table. Power calculations were performed to give the probability of finding the differences between the gene frequencies as statistically significant, given the sample size.

Results

We examined 95 individuals of European, non-Hispanic origin with documented medical history with hospitalization for lung disease (46 with sarcoidosis, 43 with tuberculosis, five with recurrent pneumonia, and one with atypical mycobacterial infection) and 95 control individuals of European, non-Hispanic origin for differences in allele frequencies in genes involved in innate immunity.

P47phox/(NCF1)

Examination of the pseudogene: wt copy ratio of control versus lung disease individuals demonstrated no statistically significant difference in the frequencies of the pseudogene: wt ratios in the lung disease group as compared to the control group (Table 2).
Table 2

Pseudogene versus gene ratio. p47phox/NCF1 pseudogene: wt gene ratio in lung disease and control individuals. The data are presented as number of individuals with the indicated pseudogene:wt ratio and the number within parentheses indicates the calculated frequency.

p47phox/NCF1 (Pseudogene: wt)

control (n = 59)

Lung Disease (n = 64)

2:1

46 (0.78)

51 (0.80)

1:1

13 (0.22)

12 (0.19)

1:2

0 (0)

1 (0.02)

p67phox (NCF2), p40phox (NCF4), p22phox (CYBA), gp91phox (CYBB), DUOX1, DUOX2

SNPs in the p67phox (NCF2), p40phox (NCF4), p22phox (CYBA) and gp91phox (CYBB), DUOX1 and DUOX2 genes were examined. Some SNPs did not occur at a high enough frequency to be detected in our samples. None of the allele frequencies differed significantly between the lung disease and the control groups (Table 3).
Table 3

Summary of SNP Analyses. SNP analyses of candidate genes in lung disease versus control groups. Numbering of SNPs start from the ATG initiator methionine of the cDNA. Data are presented as number of alleles identified divided by total number of alleles examined. Numbers within parentheses are the calculated allele frequencies. Power calculations were performed using number of subjects.

p67phox (NCF2)

dbSNP rs#

SNP

amino acid

Control

Lung Disease

Power to detect 2× increase

Power to detect 1.5× increase

Exon 6

rs2274064

542 A/G

K181R

79/186 (0.43)

91/190 (0.48)

0.98

0.96

Exon 9

rs13306581

836 C/T

T279M

0

0

  

Exon 11

 

983 G/A

R 328K

0

0

  

Exon 13

 

1105 G/A

G369R

0

0

  

Exon 13

rs17849502

1167 C/A

H389Q

12/190 (0.06)

10/188 (0.05)

0.22

 

Exon 14

rs13306575

1183 C/T

R395W

0

0

  

p22phox (CYBA)

dbSNP rs#

SNP

amino acid

Control

Lung Disease

Power to detect 2× increase

Power to detect 1.5× increase

Exon 2

 

85 A/G

T29A

0

0

  

Exon 2

 

113 T/C

F38S

0

0

  

Exon 3

 

179 A/C

K60S

4/190 (0.02)

0

0.06

 

Exon 4

rs4673

214 C/T

H72Y

61/180 (0.34)

60/190 (0.37)

0.99

0.61

Exon 6

 

403 G/A

E135K

0

0

  

Exon 6

rs17845095

521 C/T

A174V

93/176 (0.41)

88/190 (0.46)

0.99

0.79

p40phox (NCF4)

dbSNP rs#

SNP

amino acid

Control

Lung Disease

Power to detect 2× increase

Power to detect 1.5× increase

Exon 2

rs13057803

86 T/C

I29T

0

0

  

Exon 5

rs9610595

353 G/A

S118N

0

0

  

Exon 8

 

815 G/A

P272L

30/190 (0.16)

29/190 (0.15)

0.68

0.22

Exon 10

rs5995361

911 C/A

A304E

0

0

  

gp91phox (CYBB)

dbSNP rs#

SNP

amino acid

Control

Lung Disease

Power to detect 2× increase

Power to detect 1.5× increase

Exon 9

rs28935182

907 C/A

H303N

0

0

  

Exon 11

rs13306300

1414 G/A

G472S

0

0

  

Exon 12

rs28935181

1499 A/G

D500G

0

0

  

Duox 1

dbSNP rs#

SNP

amino acid

Control

Lung Disease

Power to detect 2× increase

Power to detect 1.5× increase

Exon 27

rs2458236

3532 T/C

F1178L

64/184 (0.35)

56/154 (0.36)

0.99

0.63

Exon 28

rs2292466

3647 G/A

R1216H

0

0

  

Duox 2

dbSNP rs#

SNP

amino acid

Control

Lung Disease

Power to detect 2× increase

Power to detect 1.5× increase

Exon 5

rs2001616

413 C/T

P138L

26/188 (0.14)

22/190 (0.12)

0.59

 

Exon 5

rs7166994

429 C/A

D143E

0

0

  

Exon 6

rs2467827

598 G/A

G200R

1/188 (0.01)

1/190 (0.01)

0.05

 

Exon 17

rs8028305

2048 G/A

R683H

0

0

  

Exon 24

rs2277611

3026 G/A

A1009Q

0

0

  

Exon 25

rs269868

3200 T/C

L1067S

22/186 (0.12)

15/190 (0.08)

0.5

 

TLR2

dbSNP rs#

SNP

amino acid

Control

Lung Disease

Power to detect 2× increase

Power to detect 1.5× increase

Exon 2

rs3840097

510Tdel

F170Lfs

0

0

  

Exon 2

rs5743699

1232C/T

T411I

nd

0

  

Exon 2

rs5743702

1667T/C

I556T

nd

0

  

Exon 2

rs5743703

1736G/A

R579H

nd

0

  

Exon 2

rs5743704

1892C/A

P631H

9/184 (0.05)

8/188 (0.04)

0.18

 

Exon 2

 

2029C/T

R677W

nd

0

  

Exon 2

rs5743706

2143T/A

Y715N

nd

0

  

Exon 2

rs5743707

2145T/G

Y715Stop

nd

0

  

Exon 2

rs5743708

2258G/A

R753Q

2/182 (0.01)

4/188 (0.02)

0.05

 

Exon 2

 

2304G/T

E768D

nd

0

  

TLR9

dbSNP rs#

SNP

amino acid

Control

Lung Disease

Power to detect 2× increase

Power to detect 1.5× increase

Exon 2

rs5743842

13 C/T

R5C

2/190 (0.01)

0

0.05

 

Exon 2

rs5743843

237T/G

H79Q

0

0

  

Exon 2

rs5743844

296 C/T

P99L

0

0

  

Exon 2

rs5743845

2588 G/A

R863Q

6/170 (0.04)

0/186 (0*)

0.14

 

Exon 2

rs5743746

2644 G/A

A882T

0

0

  

AAT (SERPINA1)

dbSNP rs#

SNP

amino acid

Control

Lung Disease

Power to detect 2× increase

Power to detect 1.5× increase

Exon 2

rs709932

374G/A

R125H

38/178 (0.21)

29/182 (0.16)

0.85

0.31

Exon 3

rs17580

863A/T

E288V

5/190 (0.03)

4/190 (0.02)

0.1

 

Exon 4

rs28929474

1096G/A

E366K

4/192 (0.02)

2/190 (0.01)

0.07

 

TLR2, TLR9, AAT

TLR2, TLR9, and AAT genes were examined. Again, many SNPs did not occur at high enough frequency to be observed. Most of the allele frequencies did not differ between the lung disease and control groups. The TLR2 polymorphism R753Q, associated with tuberculosis, was not shown to be different between the control or lung disease group. The TLR2 R677W polymorphism, also associated with tuberculosis, was not observed in either group. The R863Q SNP in TLR9 was absent from the lung disease group indicating that this polymorphism was not associated with increased lung disease. The AAT S (Glu288Val) and Z (E366K) alleles, associated with chronic obstructive lung disease, were examined and there was no difference in allele frequencies between the control and lung disease groups (Table 3).

Discussion

Since only a subset of individuals exposed to Bacillus anthracis spores develop pulmonary disease, the most life-threatening form of anthrax infection, it would be important to identify factors that lead to susceptibility to this type of infection. This might make it possible to identify those individuals who are at greatest risk and to provide them with the most aggressive treatment at the outset of infection. The ability to thus triage individuals in the case of a bioterrorism attack would be valuable. Moreover, understanding genetic susceptibility could lead to better management of individuals with pulmonary anthrax infection.

The genetic influences on resistance to infection are very strong. Indeed, genetic influences on resistance to infection appear to be greater than genetic influences on cancer or cardiovascular disease [45]. In the past few decades a considerable number of polymorphisms have been shown to cause infectious disease susceptibility in mice [6] and in humans [28, 31, 46]. Because infections caused by Bacillus anthracis are rare it was impossible to examine candidate polymorphisms in patients who actually developed pulmonary anthrax. Instead, it was necessary to use surrogate infections such as unusual mycobacterial infections, recurrent pneumonia, and tuberculosis or examine lung diseases such as sarcoidosis, which has been reported in cases of inhalation anthrax, for this study. The "lung disease group" in this study represented all the individuals with documented hospitalization for lung disease from a collection of 31,247 individuals of European, non-Hispanic origin unselected for any particular acute or chronic health problem. Candidate genes were chosen on the basis of their role in immunity against chronic infection as well as their participation in the innate immune response. This is a reasonable approach, since defects in the immune system generally increases susceptibility not to a single organism, but rather to multiple organisms that share some features in the pathogenesis of the disease that they produce.

Our analyses of genes of the NAD(P)H oxidase, p47 (NCF1), p67phox (NCF2), p40phox (NCF4), p22phox (CYBA), and gp91phox (CYBB), as well as other genes involved in innate immunity such as DUOX1 and 2, TLR2, TLR9 and AAT demonstrated that there were no differences between the control and lung disease group comprised of primarily sarcoidosis and tuberculosis individuals. There may, of course, be many other polymorphisms that affect susceptibility to Bacillus anthracis. Although the genes that we chose seemed to be reasonable candidates; there are many additional genes encoding products that could be important in effecting the course of anthrax in humans. For example, it has been suggested that susceptibility to Bacillus anthracis might involve myD88 [25]. Furthermore, susceptibility to infection by tuberculosis may be altered by variations in the vitamin D receptor gene [47]. Similarly, sarcoidosis has been shown to be associated with particular alleles in BTNL2 [48, 49], IL18 [50], and IFNa [51], and SLC11A1 [52].

Declarations

Acknowledgements

This is manuscript number MEM18018. This work was supported by the CDC 5PO1 CI000095 and the Stein Endowment Fund. The authors would like to thank Dr. Jill Waalen for performing the power calculations and Drs. Ernest Beutler, Gary Bokoch, Bruce Beutler, Ulla Knaus, and Bruce Zuraw for their helpful discussions.

Authors’ Affiliations

(1)
Department of Molecular and Experimental Medicine, The Scripps Research Institute

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.