Data used in this report were obtained from infants enrolled in a longitudinal cohort to study the impact of environmental exposures on asthma development and morbidity. Between September 1996 and December 1998, women delivering babies in six hospitals were invited to participate if they had at least one other child at home younger than 12 years of age with physician-diagnosed asthma. Because of the asthmatic sibling, these infants were considered at risk for this disease. A full description of the methods is provided elsewhere 222324. Our Human Investigations Committee as well as institutional review boards at each participating hospital reviewed and approved the study.

Of the initial 1,002 infants enrolled, data were available on otitis media during the first year of life for 889. Between the third and fifth year of participation, a second visit was made to the home at which time an effort was made to collect a blood sample from the cohort subjects. The analyses in this report include a convenience sample of 355 infants for whom whole blood and complete quarterly otitis media data were available.

Trained research assistants described the study to the infant's mother, obtained written informed consent, and administered standardized questionnaires to study participants during home interviews within 4 months of the infant's birth. We collected household demographic data such as maternal race and ethnicity, education, and number of children. We also collected detailed information regarding infant care such as use of daycare, any breastfeeding during the first year of life, and whether the infant was exposed to environmental tobacco smoke (ETS) in the home. Mothers were asked to report on their history of allergies or physician-diagnosed asthma. Infant respiratory symptoms were collected during quarterly telephone interviews at 6, 9 and 12 months of age. During these telephone interviews, we asked the mother to report her infant's respiratory symptoms and doctor or clinic visits (month and year of visit, reason for visit, and mother's report of the diagnosis). When the infant reached one year of age, each mother completed an extensive phone questionnaire covering her infant's health status during the previous year.

Otitis media was ascertained during each quarterly survey by mother's report of an episode of clinician-diagnosed otitis media with the month and year of diagnosis. Additional physician diagnoses of otitis media occurring in the same month or next consecutive month were counted as a single episode.

DNA from whole blood samples was extracted using the QIAamp DNA blood minikit (Qiagen) according to the manufacturer's instructions. Genotyping for the single nucleotide polymorphisms (SNPs) at aa19, aa50, aa62, aa133, and aa219 in SP-A1 and aa9, aa91, aa223 in SP-A2 were done using a sequence specific primer-PCR methodology described by Pantelidis et al.25. This method involves high stringency PCR amplification using allele-specific primers designed with the final 3' end nucleotide complementary to the nucleotide variants within a particular SNP locus. In our hands, this method did not give reproducible results for the CT polymorphism at aa140 within SP-A2. To detect polymorphisms in SP-A2 at aa140, we used PCR based cRFLP as described by DiAngelo et al. 26.

Unadjusted associations between otitis media episodes and selected study characteristics, SNPs, and individual SP-A1 or -A2 alleles were initially evaluated by χ² tests. Observed SNP frequencies were tested for Hardy-Weinberg equilibrium by χ² analysis. Allele haplotypes for SP-A1 and for SP-A2 were examined for linkage disequilibrium and haplotype frequencies were estimated using PROC HAPLOTYPE in the SAS/Genetics module of SAS version 9.1 (Cary, NC). Logistic regression was used to model the association between SP-A1 and SP-A2 haplotypes and otitis media. Note that specific haplotypes can only be inferred since each individual has two chromosomes, and the methods of genotyping provide only the two bases at each site without a chromosomal assignment. For example, homozygous SNPs result in one and only one haplotype assignment. In the presence of heterozygous SNPs, the allele haplotype could be one of several, each occurring with a probability based on observed SNP frequencies. Therefore, we made an effort to estimate the effect of uncertainty in haplotype assignment on model variability. We used the set of potential haplotypes (PROC HAPLOTYPE) and a regression calibration technique similar to that described by Carroll et al. 27 to create 100 separate data sets by randomly assigning a haplotype for a particular subject based on the probability of each of the possible haplotypes given for that subject. Next, 100 separate logistic regression models with coefficient and variance estimates for the i^th model of b_i and σi2 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaaiiGacqWFdpWCdaqhaaWcbaGaemyAaKgabaGaeGOmaidaaaaa@30F0@, respectively, were fit for each haplotype of interest. The estimate of the association (β^ MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaaiiGacuWFYoGygaqcaaaa@2E64@) between otitis media and each particular haplotype was calculated as the mean of the 100 logistic regressions. The variability of the estimate was calculated as

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.

The majority of the 355 infants in our study experienced otitis media during their first year. Thirty-eight percent of the infants experienced only one episode of otitis media, 24% experienced two, and 10% had three or more episodes during their first year of life (Table 1). Male infants were more likely to experience otitis media during their first year of life: 78% of males experienced otitis media compared to 67% of female infants (P = 0.02). Black infants tended to have less otitis media than white or Hispanic infants during their first year of life. For example, 58% of black infants experienced otitis media compared to 77% of white infants and 73% of Hispanic infants (P = 0.06). We did not find significant associations between maternal education, maternal allergies, maternal asthma, daycare before six months of age, breastfeeding, exposure to ETS in the home, or season of birth and otitis media during the first year of life.

There were socioeconomic differences between the group from the study cohort who chose to participate in the blood draw (355 out of 889 [40%]) and those who did not (n = 554): whole blood samples were obtained from more white infants (64% vs. 57%) and fewer Hispanic infants (18% vs. 26%) (P = 0.04), more of the mothers in the blood draw group had college degrees (39% vs. 31%, P = 0.03) and chose to breastfeed their infants (68% vs. 60%, P = 0.02). The rates of otitis media between the two blood draw groups did not differ: 28% compared to 26% for otitis media in the first 6 months; 73% vs. 71% for any otitis media in the first year; and 10% vs. 9% for 3 or more episodes of otitis media in the first year for the blood draw participants vs. non-participants, respectively.

Table 2 contains unadjusted associations between each of the nine candidate SNPs and otitis media for all 355 infants in our sample. Each of the individual SNPs was in Hardy-Weinberg equilibrium in the full sample of 335 subjects and within each racial/ethnic category (P > 0.05). Polymorphisms in SP-A1 at codon 19 were associated with otitis media during the first year of life. Infants with an alanine (CT) at codon 19 were also more likely to have a greater number of otitis media episodes during their first year of life. The percentage of infants with an alanine at codon 19 and 0, 1, and 2 or more episodes of otitis media during their first year were 10%, 49%, and 41% respectively. In contrast, the percentage of infants with valine (homozygote TT) at codon 19 and 0, 1, and 2 or more episodes of otitis media during their first year were 31%, 36%, and 33% respectively (P = 0.01).

We did not find associations between polymorphisms within any of the SNPs and gender, maternal education, maternal allergies, maternal asthma, daycare before six months, exposure to ETS, or season of birth. Codons 19, 62, and 133 in SP-A1, and 223 in SP-A2 were associated with race/ethnicity (Figure 1). Given the association of otitis media with both race/ethnicity and specific polymorphisms within our study population, we restricted our haplotype analyses to the ethnic group with the largest number of subjects (226 white infants).

The unadjusted relationship between polymorphisms in each of the SNPs and any episode of otitis media was examined in logistic regression models in the white infants. White infants having an alanine (CT) at SP-A1 codon 19 were more than 3.8 times more likely (95% CI 1.3, 11.2) than those with a valine (TT) to have otitis media during their first year of life.

We designated the SP-A1 and SP-A2 alleles by 6Aⁿ and 1A^m respectively as has been previously described 2126. The associated nomenclature as well as the nucleotide and amino acid changes in the major SP-A1 and SP-A2 alleles are given in Table 3. The five SNPs in SP-A1 were in linkage disequilibrium. The most common SP-A1 allele haplotypes among the white infants in our study population were 6A², 6A³, 6A⁴, and 6A in decreasing order of frequency. The four SNPs in SP-A2 were also in linkage disequilibrium (P < 0.0001). 1A⁰ was the most common SP-A2 allele among the white infants in our study followed by 1A¹, 1A², 1A, 1A⁵, 1A⁶, and 1A³. All others combined made up 4% of the SP-A2 alleles.

We examined associations between any otitis media during the first year of life and specific allele haplotypes in SP-A1 and SP-A2 (Table 3). The 6A⁴ allele of SP-A1 was protective for otitis media in white infants (OR 0.34; 95% CI 0.15, 0.79). The 1A⁵ allele in SP-A2 was underrepresented among white infants with otitis media (OR 0.38; 95% CI 0.14, 1.05). White infants with the 1A allele in SP-A2 were over 3 times more likely to have otitis media during the first year of life (OR 3.03; 95% CI 0.88, 10.39), but with wide confidence intervals that included unity.

SP-A1 and SP-A2 alleles are in close proximity and are known to be in strong linkage disequilibrium 2829. Within our population of white infants, the SP-A1 and SP-A2 allele haplotypes were also in linkage disequilibrium (P < 0.0001). Since the alleles at these two loci cosegregate as one unit, we examined the distribution of SP-A1 and SP-A2 alleles together. Unadjusted relationships between the SP-A haplotypes and any episode of otitis media are shown in Table 4. We did not adjust for other known otitis media risk factors (e.g. daycare, breastfeeding) because these were not associated with polymorphisms in SP-A. Consistent with our allele analysis, white infants with the 6A⁴/1A⁵ haplotype experienced decreased risk for otitis media during their first year of life (OR 0.23; 95% CI 0.07,0.73).