Respiratory symptoms among infants at risk for asthma: association with surfactant protein A haplotypes

1471-2350-8-15 1471-2350 Research article Respiratory symptoms among infants at risk for asthma: association with surfactant protein A haplotypes Pettigrew M Melinda melinda.pettigrew@yale.edu Gent F Janneane janneane.gent@yale.edu Zhu Yong yong.zhu@yale.edu Triche W Elizabeth elizabeth.triche@yale.edu Belanger D Kathleen kathleen.belanger@yale.edu Holford R Theodore theodore.holford@yale.edu Bracken B Michael michael.bracken@yale.edu Leaderer P Brian brian.leaderer@yale.edu

Yale Center for Perinatal, Pediatric and Environmental Epidemiology, Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT, USA

BMC Medical Genetics 1471-2350 2007 8 1 15 http://www.biomedcentral.com/1471-2350/8/15 17407567 10.1186/1471-2350-8-15 24 10 2006 02 4 2007 02 4 2007 2007 Pettigrew et al; licensee 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.

Abstract

Background

We examined the association between single nucleotide polymorphisms (SNPs) in loci encoding surfactant protein A (SFTPA) and risk of wheeze and persistent cough during the first year of life among a cohort of infants at risk for developing asthma.

Methods

Between September 1996 and December 1998, mothers of newborn infants were invited to participate if they had an older child with clinician-diagnosed asthma. Each mother was given a standardized questionnaire within 4 months of her infant's birth. Infant respiratory symptoms were collected during quarterly telephone interviews at 6, 9 and 12 months of age. Due to the association of SFTPA polymorphisms and race/ethnicity, analyses were restricted to 221 white infants for whom whole blood and respiratory data were available. Ordered logistic regression models were used to examine the association between respiratory symptom frequency and SFTPA haplotypes.

Results

The 6A allele haplotype of SFTPA1, with an estimated frequency of 6% among our study infants, was associated with an increased risk of persistent cough (OR 3.69, 95% CI 1.71, 7.98) and wheeze (OR 4.72, 95% CI 2.20, 10.11). The 6A/1A haplotype of SFTPA, found among approximately 5% of the infants, was associated with an increased risk of persistent cough (OR 3.20, 95% CI 1.39, 7.36) and wheeze (OR 3.25, 95% CI 1.43, 7.37).

Conclusion

Polymorphisms within SFTPA loci may be associated with wheeze and persistent cough in white infants at risk for asthma. These associations require replication and exploration in other ethnic/racial groups.

Background

Wheeze and persistent cough in infants are serious respiratory symptoms that can be triggered by respiratory infections and/or a variety of environmental exposures 123. Surfactant protein A (SFTPA) is an abundant, multifunctional protein that is secreted by airway epithelial cells and functions as part of the innate immune response. SFTPA may be critical in protecting the lungs from infectious agents and environmental exposures early in life before the acquisition of specific immunity. SFTPA neutralizes respiratory viruses such as influenza and respiratory syncytial virus (RSV) 45. SFTPA also enhances the uptake of bacteria and viruses by phagocytes 678. In addition to its role in protecting the lungs from microorganisms, SFTPA has other important immunomodulatory functions including binding aeroallergens 9.

SFTPA's role in protection of the lungs has led to exploration of potential links between SFTPA and diseases of the respiratory tract in infants and young children. Polymorphisms within SFTPA1 and SFTPA2, two functional, highly homologous SFTPA genes 101112 have been linked to respiratory distress syndrome in infants 131415, severe RSV bronchiolitis 16, and otitis media 1718. In the present study, we used a candidate gene approach to investigate whether polymorphisms within the SFTPA1 and SFTPA2 genes were associated with wheeze and persistent cough during the first year of life among a prospective birth cohort at risk for developing asthma.

Methods

Cohort

Between September 1996 and December 1998, we invited women delivering babies in six hospitals in Connecticut and Massachusetts to participate in a longitudinal study of asthma development if they had at least one other child at home under 12 years of age with physician-diagnosed asthma. Infants enrolled in the cohort were considered at risk for asthma due to their asthmatic siblings. We provide a full description of the methods elsewhere 219.

Of the 1,002 infants originally enrolled, respiratory symptom data were available for the first year of life for 889. Between the third and fifth year of participation, we made a second visit to the home to collect a blood sample from our cohort subjects. The current analyses are based on a convenience sample of 355 for whom whole blood and complete respiratory symptom data were available. Nucleotide changes at amino acid (aa) 19, aa 62, and aa 133 in SFTPA1, and aa 223 in SFTPA2 were significantly associated with ethnicity. To prevent identification of invalid associations due to population stratification, we restricted our haplotype analyses to the ethnic group with the largest number of subjects (221 white infants). The Yale Human Investigations Committee and institutional review boards at each participating hospital reviewed and approved the study.

Data collection

A research assistant visited the home within 4 months of the infant's birth in order to describe the study to the infant's mother, obtain written informed consent, and administer a standardized questionnaire. We collected household demographic data including maternal race and ethnicity, education and number of children; detailed information regarding infant care (e.g. breastfeeding and daycare use); and maternal health status (e.g. self-reported history of allergies or physician-diagnosed asthma). Infant respiratory symptoms were collected during quarterly telephone interviews at 6, 9 and 12 months of age at which time each mother reported on her infant's respiratory symptoms (number of symptom-days per month of wheeze or persistent cough) and doctor or clinic visits (month and year of visit, reason for visit, and diagnosis). Around the time of the infant's first birthday, the mother completed an additional phone questionnaire covering her infant's health status during the previous year.

Genotyping of SFTPA genes

DNA was extracted from whole blood using the QIAamp DNA blood minikit (Qiagen) according to the manufacturer's instructions. We used sequence specific primer-PCR methodology to genotype the SNPs at aa 19, aa 50, aa 62, aa 133, and aa 219 in SFTPA1 and aa 9, aa 91, aa 223 in SFTPA2 20. We used PCR based cRFLP as described by DiAngelo et al. 21 to detect polymorphisms in SFTPA2 at aa 140.

Data analysis

Outcomes of interest were respiratory symptom frequency in the first year of life categorized as none or as 1 to 7, 8 to 14, 15 to 21, 22 to 28, or more than 28 days of persistent cough or wheeze. Between 1 and 6 months of symptom information were missing for 18 infants. Data from these infants were included in analyses based on their symptom rates. All other infants had 12 months of complete symptom data. Unadjusted associations between respiratory symptoms and selected study characteristics, SNPs, and individual SFTPA1 or SFTPA2 alleles were evaluated with tests for linear trend (Cochran-Armitage or Somers' D statistic). For statistical tests involving SNPs, frequencies for the minor homozygous allele and heterozygous alleles were combined and compared to the dominant homozygous group. Observed SNP frequencies were tested for Hardy-Weinberg equilibrium by χ²analyses. We examined pairwise linkage disequilibrium with r²measures (Fig. 1) using PROC ALLELE of the SAS/Genetics module of SAS version 9.1 22. Allele haplotypes for SFTPA1 and for SFTPA2 were assigned and examined for linkage disequilibrium using PROC HAPLOTYPE 22. We examined the association between haplotype and respiratory symptom frequency (as categorical variables) using ordered logistic regression. Separate analyses were conducted for each symptom and each haplotype compared to all other haplotypes. We estimated the effect of uncertainty in haplotype assignment using a regression calibration technique 1823 and the SFTPA1 and SFTPA2 haplotype probabilities obtained from PROC HAPLOTYPE. As described previously 18, this technique involved first creating 100 separate haplotype data sets by randomly assigning subject haplotypes based on each subject's haplotype probabilities. Next, 100 separate ordered logistic regression models with coefficient and variance estimates (β_iand σ_i²for the i^thmodel) were fit for each haplotype of interest. The estimate of the association between the respiratory symptom and the particular haplotype of interest ( β^ MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaaiiGacuWFYoGygaqcaaaa@2E64@) was calculated as the mean of the 100 regression models. The variability of the estimate was calculated as

Figure 1

Pairwise linkage disequilibrium measure (r²) for surfactant protein A SNPs (5 SFTPA1 SNPs: aa 19, aa 50, aa 62, aa 133, aa 219; 4 SFTPA2 SNPs: aa 9, aa 91 aa 140, aa 223) from 221 white infants at risk for developing asthma. Gray scale represents strength of association from white (r²= 0) to black (r²= 1). (CT and MA, 1998 – 2000)

var (β^ MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaaiiGacuWFYoGygaqcaaaa@2E64@) = mean (σi2 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaaiiGacqWFdpWCdaqhaaWcbaGaemyAaKgabaGaeGOmaidaaaaa@30F0@) + var (b_is)

where mean (σi2 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaaiiGacqWFdpWCdaqhaaWcbaGaemyAaKgabaGaeGOmaidaaaaa@30F0@) is defined as the mean of the 100 logistic regression variances and var (b_is) is defined as the variance of the 100 logistic regression b coefficients.

Results

Close to half of the 221 infants in our study experienced persistent cough (51%) or wheeze (46%) during their first year of life (Table 1). Thirty-two percent of the infants experienced from 1 to 28 days of persistent cough, 19% experienced more than 28 days of persistent cough during their first year; 36% experienced 1 to 28 days of wheeze and 10% experienced more than 28 days of wheeze during their first year of life. Male infants and infants of asthmatic mothers were more likely to experience persistent cough and wheeze during their first year of life (Table 1). We did not find significant associations with either persistent cough or wheeze and maternal allergies, age daycare attendance began, number of months of breastfeeding, or exposure to environmental tobacco smoke (ETS).

Table 1

Unadjusted associations between personal characteristics and respiratory symptoms during the first year of life for 221 white infants at risk for developing asthma. (CT and MA, 1998 – 2000)

Persistent cough (days/year) (%)

Wheeze (days/year) (%)

Characteristic

n (%)

1 – 7

8 – 14

15 – 21

22 – 28

> 28

p-value ^a

1 – 7

8 – 14

15 – 21

22 – 28

> 28

p-value ^a

White infants N (%)

221

109 (49.3)

30 (13.6)

19 (8.6)

12 (5.4)

10 (4.5)

41 (18.6)

119 (54.3)

49 (22.4)

17 (7.8)

7 (3.2)

6 (2.7)

21 (9.6)

Gender

male

115 (52.0)

42.6

14.8

11.3

7.0

17.4

0.12

44.2

27.4

8.0

3.5

4.4

12.4

0.005

female

106 (48.0)

56.6

14.3

5.7

3.8

1.9

19.8

58.0

17.0

7.6

2.8

0.9

6.6

Maternal allergies

81 (36.7)

44.4

17.3

8.6

3.7

2.5

23.5

0.20

52.5

21.2

10.0

3.8

2.5

10.0

0.35

Yes

140 (63.4)

52.1

11.4

8.6

6.4

5.7

15.7

55.4

23.0

6.5

2.9

9.4

Maternal asthma

153 (69.2)

52.3

15.0

9.2

5.2

2.0

16.3

0.02

57.2

23.7

7.9

2.6

0.7

7.9

0.01

Yes

68 (30.7)

42.6

10.3

7.4

5.9

10.3

23.5

47.8

19.4

7.5

4.5

7.5

13.4

Daycare (starting age in months)

1 – 5

43 (19.6)

48.8

14.0

9.3

2.3

7.0

18.6

0.26

55.8

14.0

11.6

2.3

4.6

11.6

0.45

6 – 12

21 (9.6)

57.1

9.5

19.0

4.8

0.0

9.5

55.0

30.0

10.0

0.0

0.5

None in first year

155 (70.8)

49.0

14.2

6.4

4.5

19.4

53.9

24.0

6.5

3.9

2.6

9.1

Breast feeding (duration in months)

None

49 (22.3)

53.1

14.3

6.1

4.1

0.0

22.4

0.25

49.0

18.4

10.2

2.0

6.1

14.3

0.15

1 – 5

57 (25.9)

49.1

19.3

3.5

7.0

5.3

15.8

57.9

19.3

3.5

1.8

14.0

6 – 12

114 (51.8)

47.4

10.5

12.3

5.3

6.1

18.4

54.5

25.9

8.9

3.6

1.8

5.4

ETS

197 (89.6)

48.7

12.2

9.6

5.6

4.6

19.3

0.19

55.4

20.5

8.7

3.1

9.2

0.38

Yes

23 (10.4)

52.2

26.1

0.0

4.4

13.0

43.5

39.1

0.0

4.4

0.0

13.0

^ap-values from test for trend: Cochran-Armitage exact test (1-sided) for gender, maternal allergies, maternal asthma, Environmental Tobacco Smoke (ETS); Somer's D statistic (1-sided) for age when starting daycare and duration of breastfeeding. Significant p-values (p < 0.05) in bold-face type.

Table 2 contains unadjusted associations between each of the nine candidate SNPs and persistent cough or wheeze. Individual SNPs were in Hardy-Weinberg equilibrium (p > 0.05 for the χ²test). Having a C nucleotide at aa 19 in SFTPA1 was associated with greater frequency of wheeze. The G nucleotide at aa 133 was associated with both wheeze and persistent cough during the first year of life. We did not find any significant associations between each of the nine SNPs and personal characteristics listed in Table 1.

Table 2

Unadjusted associations between SNPs from surfactant protein A alleles (SFTPA1,SFTPA2) and respiratory symptoms^aduring the first year of life for white infants at risk for developing asthma. (CT and MA, 1998 – 2000)

Persistent cough (days/year) (%)

Wheeze (days/year) (%)

Characteristic

n (%)

1 – 7

8 – 14

15 – 21

22 – 28

> 28

p-value ^b

1 – 7

8 – 14

15 – 21

22 – 28

> 28

p-value ^b

White infants N (%)

221

109 (49.3)

30 (13.6)

19 (8.6)

12 (5.4)

10 (4.5)

41 (18.6)

119 (54.3)

49 (22.4)

17 (7.8)

7 (3.2)

6 (2.7)

21 (9.6)

SFTPA1 ^c

aa 19 (rs1059047)

1 (0.4)

42 (19.0)

46.5

7.0

11.6

7.0

2.3

25.6

0.15

44.2

18.6

11.6

4.6

7.0

14.0

0.02

178 (80.5)

49.7

14.9

8.0

5.1

17.1

56.6

23.1

6.9

2.9

1.7

8.7

aa 50 (rs1136450)

48 (21.4)

103 (46.0)

51.7

10.9

8.2

6.1

3.4

19.7

0.50

53.4

22.6

8.9

2.7

3.4

8.9

0.51

73 (32.6)

45.2

17.8

9.6

4.1

6.8

16.4

55.6

22.2

5.6

4.2

1.4

11.1

aa 62 (rs1136451)

5 (2.2)

61 (27.5)

45.3

7.8

14.1

6.2

3.1

23.4

0.11

46.9

21.9

10.9

3.1

6.2

10.9

0.07

156 (70.3)

51.3

15.6

6.5

4.6

5.2

16.9

57.9

21.7

6.6

3.3

1.3

9.2

aa 133 (rs1059057)

1 (0.4)

38 (16.9)

36.8

7.9

13.2

7.9

2.6

31.6

0.02

39.5

18.4

10.5

5.3

10.5

15.8

0.003

186 (82.7)

51.9

14.8

7.6

4.9

15.8

57.5

23.2

7.2

2.8

1.1

8.3

aa 219 (rs4253527)

1 (0.4)

34 (15.1)

52.9

11.8

20.6

2.9

0.0

11.8

0.12

55.9

29.4

11.8

0.0

2.9

0.06

190 (84.4)

48.7

13.9

6.4

5.9

5.4

19.8

54.0

21.1

7.0

3.9

3.2

10.8

SFTPA2 ^c

aa 9 (rs1059046)

42 (18.7)

109 (48.4)

52.4

11.6

9.5

4.1

3.4

19.0

0.26

55.1

21.1

8.8

2.0

3.4

9.5

0.50

74 (32.9)

43.2

17.6

6.8

8.1

6.7

17.6

52.8

25.0

5.6

1.4

9.7

aa 91 (rs17886395)

3 (1.4)

57 (25.7)

50.8

8.5

10.2

5.1

3.4

22.0

0.33

54.2

18.6

6.8

3.4

8.5

0.23

162 (73.0)

49.1

15.1

8.2

5.7

5.0

17.0

54.8

23.6

8.3

3.2

0.6

9.6

aa 140 (rs1965707)

13 (5.8)

87 (38.8)

53.1

15.3

8.2

2.0

19.4

0.15

53.1

24.5

10.2

3.1

2.0

7.1

0.24

124 (55.4)

45.9

12.3

9.0

8.2

6.6

18.0

55.0

20.8

5.8

3.3

11.7

aa 223 (rs1965708)

7 (3.1)

71 (31.6)

54.0

11.8

9.2

2.7

4.0

18.4

0.27

55.3

23.7

9.2

4.0

1.3

6.6

0.18

147 (65.3)

46.9

14.5

8.3

6.9

4.8

18.6

53.8

21.7

7.0

2.8

3.5

11.2

^aN = 221 infants had data for persistent cough and N = 219 for wheeze during the first year of life. ^bp-values from test for trend: Cochran-Armitage exact test (1-sided). Minor homozygotes and heterozygous alleles were combined and compared to the dominant homozygous group. Significant p-values (p < 0.05) in bold-face type. ^cSNPs were in Hardy-Weinberg equilibrium. rs numbers are given in parentheses next to each amino acid.

By convention, SFTPA1 and SFTPA2 allele haplotypes are denoted by 6Aⁿand 1A^mrespectively 1721. The estimated frequency of each allele haplotype is given in Table 3. The five SNPs in SFTPA1 were in linkage disequilibrium as were the four SNPs in SFTPA2 (p < 0.0001, χ²test of allelic associations).

Table 3

Unadjusted associations between surfactant protein A (SFTPA1,SFTPA2) haplotypes and persistent cough or wheeze in the first year of life.^aUnadjusted odds ratios (OR) and 95% confidence intervals (CI) are from ordered logistic regression models predicting symptom frequency. ^b(CT and MA, 1998 – 2000)

Allele, haplotype ^c

Nucleotide/amino acid ^d

Estimated Frequency Distribution^e% (95% CI)

Persistent Cough OR (95% CI)

Wheeze OR (95% CI)

SFTPA1

aa 19

aa 50

aa 62

aa 133

aa 219

C/Ala

C/Leu

C/Arg

6.1% (4.0 – 8.3)

3.69 (1.71, 7.98)

4.72 (2.20, 10.11)

6A ²

T/Val

G/Val

C/Arg

54.0% (49.3 – 58.7)

1.18 (0.63, 2.19)

1.13 (0.60, 2.14)

6A ³

T/Val

C/Leu

C/Arg

25.6% (21.6 – 29.6)

0.81 (0.49, 1.36)

0.83 (0.49, 1.40)

6A ⁴

T/Val

C/Leu

T/Trp

6.2% (4.1 – 8.4)

0.94 (0.44, 2.03)

0.70 (0.31, 1.60)

all others

SFTPA2

aa 9

aa 91

aa 140

aa 223

C/Thr

C/Pro

C/Gln

8.4% (6.0 – 10.8)

1.53 (0.75, 3.14)

1.30 (0.63, 2.71)

1A ⁰

A/Asn

G/Ala

C/Gln

54.2% (49.5 – 58.8)

1.13 (0.60, 2.13)

1.14 (0.59, 2.19)

1A ¹

C/Thr

G/Ala

A/Lys

13.7% (10.7 – 16.7)

0.82 (0.45, 1.48)

0.87 (0.47, 1.58)

1A ²

C/Thr

G/Ala

C/Gln

10.2% (7.6 – 12.7)

0.95 (0.49, 1.84)

0.83 (0.42, 1.64)

1A ³

A/Asn

G/Ala

A/Lys

1.8% (0.7 – 2.9)

0.45 (0.08, 2.48)

0.55 (0.10, 3.06)

1A ⁵