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Clinical Investigations in Critical Care |

Polymorphism in the Surfactant Protein-B Gene, Gender, and the Risk of Direct Pulmonary Injury and ARDS* FREE TO VIEW

Michelle Ng Gong; Zhou Wei; Li-Lian Xu; David P. Miller; B. Taylor Thompson; David C. Christiani
Author and Funding Information

*From the Pulmonary and Critical Care Unit (Drs. Gong, Thompson, and Christiani), Department of Medicine, Massachusetts General Hospital, Harvard Medical School; and the Environmental Health Department (Drs. Wei, Xu, and Miller), Harvard School of Public Health, Boston, MA.

Correspondence to: David C. Christiani, MD, FCCP, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115; e-mail: dchris@hohp.harvard.edu



Chest. 2004;125(1):203-211. doi:10.1378/chest.125.1.203
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Study objective: Major risk factors for ARDS have been identified. However, only a minority of patients with such risks develops ARDS. It is likely that, given the same type and degree of insult, there are heritable determinants of susceptibility to ARDS. To investigate the possibility of variable genetic susceptibility to ARDS, we examined the association between ARDS and a polymorphism in intron 4 of the surfactant protein-B (SP-B) gene.

Design: Nested case-control study conducted from September 1999 to March 2001.

Setting: Four adult medical and surgical ICUs at a tertiary academic center.

Patients: One hundred eighty-nine patients meeting study criteria for a defined risk factor for ARDS were enrolled and prospectively followed.

Measurements and results: Seventy-two patients (38%) developed ARDS. After stratification by gender and adjustment for potential confounders, there was a significantly increased odds for women with the variant SP-B gene to develop ARDS compared to women homozygous for the wild-type allele (odds ratio [OR], 4.5; 95% confidence interval [CI], 1.1 to 18.8; p = 0.03). Women with the variant SP-B polymorphism also had significantly increased odds of having a direct pulmonary injury such as aspiration or pneumonia as a risk factor for ARDS as opposed to an indirect pulmonary risk for ARDS (OR, 4.6; 95% CI, 1.1 to 19.9; p = 0.04). No such association with ARDS or direct pulmonary injury was found for men.

Conclusion: The variant polymorphism of the SP-B gene is associated with ARDS and with direct pulmonary injury in women, but not in men. Further study is needed to confirm the association between the variant SP-B gene, and gender, ARDS, and direct pulmonary injury.

Figures in this Article

The ARDS is a major cause of morbidity and mortality throughout the world. About 150,000 cases per year are reported in the United States alone,1 with a reported mortality rate of 40 to 60%.2 The American-European Consensus Committee on ARDS defines ARDS as an acute syndrome of lung inflammation and increased permeability that is associated with severe hypoxia and the presence of bilateral infiltrates on chest radiographs with no evidence of left heart failure.1 Major risk factors for the development of ARDS have been described and include sepsis, trauma, pneumonia, burns, multiple transfusions, cardiopulmonary bypass, and pancreatitis.34 Other factors such as older age,4 chronic alcoholism,5 tobacco abuse,6 absence of diabetes,7 and greater severity of illness4 also have been found to contribute to the risk of developing ARDS. Despite the common occurrence of these risk factors, only a minority of patients with the above conditions develops ARDS. Our current understanding about why some patients develop ARDS while others do not is incomplete. It is likely that, given the same type and degree of insult, there are individual differences in susceptibility to developing ARDS.

Individual differences in susceptibility are a subject of active molecular epidemiologic investigation for various common diseases such as lung cancer,8 coronary artery disease,9 traumatic brain injury,10 and osteoporosis.11 Other studies have suggested possible genetic susceptibility to developing cerebral malaria for individuals homozygous for the tumor necrosis factor (TNF)-α promoter polymorphism TNF2,12 septic shock in individuals with the TNF2 allele,13 or those who are homozygous for the polymorphism TNFB2 on the TNF-β gene.14

There have been some preliminary investigations into the genetic susceptibility of acute lung injury. Homozygosity for the deletion polymorphism in the angiotensin-converting enzyme gene, which is associated with higher angiotensin-converting enzyme levels and activity, was found in an increased frequency among patients with ARDS compared to the following three control groups in the United Kingdom: patients with non-ARDS respiratory failure; patients undergoing coronary artery bypass surgery; and healthy men.1516 In addition, the C allele of the –174GC polymorphism in the interleukin-6 gene, which has been associated with lower interleukin-6 plasma concentration, was found in a lower frequency in nonsurvivors with and without ARDS.,15 However, it is not clear whether the control subjects were at risk for lung injury initially.

Pulmonary surfactant is synthesized primarily by type II alveolar cells and lowers the surface tension by interfering with the intermolecular attractive forces of the aqueous layer on the alveolar surface, thereby allowing for normal expansion of the lung.17 Also, surfactant may have immunologic properties, including enhancement bacterial phagocytosis and chemotaxis of alveolar macrophages.1819 In ARDS, pulmonary surfactant does not function normally. For example, the lipid-protein complex obtained from ARDS patients via BAL does not appear to be surface-active, and the specific lipid composition of surfactant is altered in patients with ARDS.20 In addition, some plasma proteins such as albumin, which are characteristically elevated in the alveolar space of ARDS patients, inhibit the surface properties of the surfactant.21

Surfactant protein-B (SP-B) is one of the hydrophobic proteins crucial to the surface-lowering properties of surfactant.22 It is encoded on a relatively small gene of about 9,500 base pain (bp) on the short arm of chromosome 2.23 A polymorphism containing a variable number of tandem repeats has been localized to intron 4 of the SP-B gene in a study of infants with respiratory distress syndrome.24 About 90% of whites had an “invariant” or wild-type, 2.5-kb fragment, but some subjects had either a smaller or larger variant of this band due to a deletion or insertion polymorphism at intron 4 of this gene.25 Genomic DNA analysis revealed the frequency of insertion/deletion variants of this polymorphism to be 29.3% among infants with respiratory distress syndrome in contrast to 16.8% among control infants (p < 0.05).25 Polymorphisms in the SP-B gene were found to be associated with ARDS in two previous studies by the same group.27 In one study,24 the frequency of the insertion/deletion variants in intron 4 was 46.6% among 15 ARDS patients in contrast to 4.3% among control subjects (p < 0.05). However, this study was limited by the use of healthy blood donors as control subjects and by the lack of control of clinical factors known to contribute to ARDS.15 In the other study on the −1580C/T missense mutation in exon 4 of the SP-B gene,16 allele frequency in the control group deviated from that predicted by the Hardy-Weinberg equilibrium. It has been suggested28 that deviations from the Hardy-Weinberg equilibrium among control subjects in molecular epidemiology studies should prompt a repeat analysis and investigation for potential complications such as genotyping error and population stratification.

Therefore, we performed a hospital-based nested case-control study to investigate the possible association between the variant SP-B polymorphism and the development of ARDS in at-risk individuals.

Study Population

We conducted a nested case control study in the neurologic, cardiac, medical, and surgical ICUs of the Massachusetts General Hospital (Boston, MA). Every weekday, the research coordinator screens all patients admitted to the ICU in the preceding 24 h for ARDS risk factors, as defined in Table 1 . For patients admitted to the ICU with sepsis or severe sepsis, clinical data on the suspected source of infection was collected. Patients were considered to have pneumonia as a source of sepsis if they had two or more of the following factors: (1) new infiltrates seen on a chest radiograph; (2) temperature of > 38.3°C or < 36.0°C, or a WBC count of > 12,000 cells/μL or < 4,000 cells/μL, or > 10% bandemia; or (3) positive results of microbiological tests confirming pulmonary infection. On Mondays, all patients admitted to the ICU from the preceding weekend were screened. The exclusion criteria included the following: age < 18 years; absolute neutrophil count of < 500 cells/μL, unless secondary to sepsis; treatment with immunosuppressive agents or immunoenhancing agents such as granulocyte colony-stimulating factor in the preceding 21 days; a directive to withhold intubation; and a history of HIV infection, organ or bone marrow transplantation, or chronic lung disease, which may mimic ARDS radiologically or clinically. After the first year of recruitment (ie, in November 2000), immunosuppression therapy secondary to treatment with corticosteroids was removed as an exclusion criteria due to the exclusion of larger numbers of eligible patients.

Any ICU patient admitted with one or more of the required risk factors for ARDS and no exclusion criteria were approached for inclusion in the study. After consent was granted, patients were enrolled into the cohort and were followed prospectively during their ICU stay for the development of ARDS. The definition of ARDS is similar to that of the American European Consensus Committee,1 as follows: (1) intubated on positive ventilation; (2) Pao2/fraction of inspired oxygen ratio of ≤ 200 mm Hg; (3) bilateral infiltrates seen on chest radiographs not fully explained by masses, effusions, or collapse; and (4) pulmonary arterial occlusion pressure ≤ 18 mm Hg or no clinical evidence of left atrial hypertension. Subjects who fulfilled the criteria for ARDS during their ICU stay were selected as cases while at-risk patients who did not meet the criteria for ARDS at anytime during their ICU hospitalization were selected as control subjects. The Human Subjects Committees of the Massachusetts General Hospital and Harvard School of Public Health approved the study, and informed written consent was obtained from all subjects or their appropriate surrogates.

Alcohol abuse refers to any history of alcoholism, delirium tremors, alcohol-induced seizures, alcoholic hepatitis, or alcoholic cirrhosis. The term tobacco use includes all past and current cigarette smokers. The term corticosteroid use is defined as the use of ≥ 300 mg prednisone in the 21 days prior to admission to the ICU or ≥ 15 mg prednisone per day in the 7 days prior to admission to the ICU.

DNA Extraction and Genotyping

A 10-mL sample of whole blood was collected in ethylenediaminetetraacetic acid tubes from all subjects at the time of enrollment. DNA was extracted (PureGene kits; Gentra Systems, Inc; Minneapolis, MN) per the manufacturer’s instruction. This genomic DNA served as the template for the polymerase chain reaction (PCR) amplification of the SP-B gene, as previously described by Floros et al.24 The following oligonucleotides were used as PCR primers: 5′-CTGGTCATCGACTACTTCCA-3′; and 5′-TGTGTGTGAGAGTGAGGGTGTAAG-3′. Patients with 606 bp bands were considered to have a wild-type band. Those patients with any bands smaller or larger than 606 bp were considered to have a variant allele (Fig 1 ).

Statistical Analysis

The proportions of patients with ARDS by risk factor were calculated by dividing the number of case patients with the risk by all subjects with the risk. The clinical risk factors are not exclusive, as some patients have multiple risk factors such as aspiration and trauma. However, patients with sepsis syndrome and septic shock were considered to have septic shock, given that all patients with septic shock also qualified for having sepsis syndrome since sepsis syndrome is a required part of the defining criteria for septic shock. Patients with sepsis or septic shock were further divided into those having pneumonia as a source of their infection and those having a nonpulmonary source.

Comparisons between dichotomous variables were performed with the Fisher exact test, and continuous variables such as age and acute physiology and chronic health evaluation (APACHE) III scores were compared using the Wilcoxon rank sum test. A p value ≤ 0.05 was considered to be statistically significant. Conformity to the Hardy-Weinberg equilibrium was determined with a χ2 test. A multivariate logistic regression model was built to determine the association between the variant SP-B polymorphism and ARDS. Any covariate found to be significantly associated with ARDS on univariate analysis and any previously described predictors for ARDS, such as age, race, diabetes, and alcohol-related diseases, was included in the model. Tobacco use could not be adjusted for in the model as data were unavailable for 78 subjects. The likelihood functions for different logistic regression models were compared after assuming a linear, quadratic, and step function for continuous variables such as age and APACHE III scores. There were no statistically significant differences between the different models, but a stepwise function makes the least assumptions inherently. Thus, age and APACHE III score without the age component were categorized into decades and into intervals of 25, respectively, in the final models. The effect modification between the variant SP-B polymorphism and the development of ARDS and direct pulmonary injury were determined by the addition of an interaction term to the model. All analyses were performed using the SAS statistical software package (SAS; SAS Institute; Cary, NC).

Cohort Characteristics

From October 1999 to April 2000 and from June to September 2000, 2,294 consecutive patients admitted to the ICU were screened. A total of 428 patients were admitted to the ICU with one or more of the required risk factors for ARDS. Of those, 188 patients were excluded and 46 patients either refused consent or died before consent could be obtained, resulting in 194 patients being enrolled into the cohort. In addition, nine patients were enrolled as part of the pilot study between January and March 1999. Of the 203 enrolled subjects, 7 had been previously enrolled (these subjects were not reanalyzed), 4 patients died or were transferred before DNA collection, and 3 patients were excluded from analyses secondary to the initiation of therapy with steroids after enrollment. This left 189 patients for analyses. A total of 72 patients (38%) developed ARDS during their ICU stay. They were selected as case patients, while the other 117 subjects served as control subjects. The characteristics of the cohort are summarized in Table 2 . Greater severity of illness, as indicated by APACHE III scores, was significantly associated with ARDS. There were no significant differences in age, gender, history of alcoholic diseases, diabetes, tobacco use, and the frequency of the variant SP-B gene between the case patients and the control subjects.

The occurrence of ARDS by clinical risk factor on admission to the ICU is described in Table 3 . The clinical risk factors are not exclusive as some patients have multiple risk factors such as aspiration and trauma.

The highest incidence of ARDS occurred in those patients admitted to the ICU with septic shock or aspiration, while the lowest incidence occurred in trauma patients. Among patients with sepsis, those with pneumonia as a source of infection had a significantly greater rate of progression to ARDS (45%) than did those with nonpulmonary sources of infection (17%; p = 0.05), and in patients with pneumonia and septic shock the rate of progression to ARDS was 54% compared with 25% in those with extrapulmonary sources of infection (p = 0.01). The occurrence of ARDS for conditions that result in direct pulmonary injury such as sepsis or septic shock from pneumonia and aspiration are significantly higher than conditions that result in indirect pulmonary injury such as extrapulmonary sepsis, trauma, or multiple transfusions. When the risk factors of sepsis or septic shock from pneumonia and aspiration are combined as direct pulmonary injury, the rate of ARDS is 50.0% compared to 27.3% (p = 0.002) in those patients with extrapulmonary sources of sepsis, trauma, or multiple transfusions. Subjects with more than one risk factor for ARDS were not significantly more likely to progress to ARDS (p = 0.8).

Gender, Variant SP-B Polymorphism, and the Risk of ARDS and Direct Pulmonary Injury

Thirty-seven subjects (20%) in the cohort had the SP-B-variant genotype with an allele frequency of 0.10. No patient was homozygous for the variant polymorphism. The genotype frequency of the control subjects did not depart significantly from that determined by the Hardy-Weinberg equilibrium (p > 0.25). There was no significant difference in race, gender, age, APACHE III scores, and history of alcohol abuse or diabetes between those with the variant SP-B genotype and those with the wild-type genotype.

The association between the variant SP-B genotype and the development of ARDS in the cohort after stratification by gender and adjustment for age, white race, alcohol abuse, history of diabetes, multiple risk factors, and APACHE III score is shown in Table 4 . The odds of developing ARDS was not significantly elevated in the cohort (odds ratio [OR], 2.1; 95% confidence interval [CI], 0.9 to 4.7; p = 0.08), but after stratification by gender significantly increased odds of developing ARDS were found in women with a variant SP-B allele (OR, 4.5; 95% CI, 1.1 to 18.8; p = 0.03) [Table 4] , but not in men. Despite the differences in the ORs for ARDS between men and women, our model lacked the power to detect a statistically significant effect modification by gender and variant SP-B genotype after the addition of an interaction term (p = 0.1).

The differences in baseline characteristics between the male and female subjects in the cohort were examined (Table 5 ). More men than women had a risk for multiple transfusions (p = 0.03). Otherwise, there were no significant gender differences in baseline characteristics, risk factors for ARDS, direct vs indirect pulmonary injury as a predisposing condition for ARDS, and the frequency of the variant SP-B polymorphism.

Since SP-B is a locally active molecule in the lung, it is possible that the variant SP-B gene may be a factor not only in ARDS but also in the development of the intermediary steps to ARDS, specifically, primary lung injury such as aspiration or pneumonia that is severe enough to warrant an admission to the ICU. Therefore, we examined the odds of developing a direct lung injury such as sepsis from pneumonia or aspiration in those patients with a variant SP-B allele (Table 4) . Although there was no association between female gender and the development of direct pulmonary injury (crude OR, 1.1; 95% CI, 0.6 to 2.0 [p = 0.8]; adjusted OR, 1.1; 95% CI, 0.6 to 2.1 [p = 0.7]), there was a strong association between the presence of the variant SP-B allele and the admission of women to the ICU with a direct pulmonary injury such as pneumonia or aspiration compared to women admitted to the ICU with other injuries (OR, 4.6; 95% CI, 1.0 to 19.9; p = 0.4) [Table 4] . There was no such statistically significant association between the presence of the variant SP-B allele and direct pulmonary injury for men. Our model lacked the power to detect a statistically significant effect modification by gender and presence of the variant SP-B polymorphism on direct pulmonary injury after the inclusion of an interaction term (p = 0.1).

The limitations of this study should be mentioned. As this was a preliminary report, the study is small. Thus, confirmatory studies on a larger group of patients will need to be performed. The critical presentation of many of the patients and the inability to interview them for clinical history limits the ability to obtain data for certain parameters such as tobacco history, which were missing for 78 patients. To our knowledge, the variant SP-B genotype has not been associated previously with tobacco abuse. There was no significant difference in tobacco use between men and women in the study (p = 0.2). Thus, the missing tobacco information is unlikely to explain the significant association seen between the variant SP-B polymorphism and ARDS or direct pulmonary injury in women. In addition, we did not have the power to determine whether the variant SP-B genotype influenced mortality in ARDS patients. We chose to focus on one polymorphism, but it is highly unlikely that a single gene can explain variable individual susceptibility to the complex syndrome designated as ARDS. In addition, we were not able to measure bronchoalveolar concentrations of SP-B in our cohort for phenotypic correlation to genotype. Thus, the functional consequence of the variant SP-B polymorphism remains unclear.

Nevertheless, this cohort of critically ill patients with clearly defined risk factors for ARDS revealed significant and novel findings in the risk factors for the development of ARDS. Despite the limited study size, we found a significant association between the presence of the variant SP-B gene and increased susceptibility to developing ARDS in at-risk women, but not in at-risk men. Given the small sample size, the CIs are wide but still statistically significant. The variant polymorphism in intron 4 of the SP-B gene is known to vary between whites and African Americans with a higher frequency of the variant found in African Americans and Nigerians.26 We lack the power to confirm such population stratification in the frequency of this polymorphism. However, it is unlikely that the relationship found is secondary to racial differences between case patients and control subjects, as our cohort was predominantly white and race was adjusted for in the analysis. It is also unlikely that the association between the variant SP-B polymorphism and ARDS or direct pulmonary injury in women is falsely inflated because of a difference in prevalence in ARDS between men and women. Previous studies,35 have not indicated any gender differences in the development of ARDS except perhaps in female trauma or burn patients. Indeed, we also did not find any significant gender difference in the development of ARDS (Table 2) .

Max et al26 detected a similar genetic association with the variant SP-B polymorphism in 15 patients with ARDS compared to 23 healthy control subjects. The ARDS patients were defined according to the American-European Consensus Committee criteria and included 7 men and 8 women, with 9 of the 15 case patients developing ARDS as a result of pneumonia or aspiration. Max et al,26 reported significant increased odds for patients with ARDS to have the variant SP-B allele compared to control subjects. However, the data were not stratified by gender, and important clinical factors related to the variant SP-B polymorphism and ARDS, such as race, age, alcohol history, diabetes, or severity of illness, were not controlled for in the analysis. In addition, their control subjects consisted of healthy individuals with no preceding clinical condition that placed them at risk for ARDS. As the variant SP-B allele may play a role in the intermediate conditions leading to ARDS, the choice of using at-risk control subjects in our cohort, while more clinically relevant, may bias the results toward the null and would not explain the positive association found.

Although this cohort was not designed originally to assess pneumonia and aspiration as an outcome, the study design generated a group of critically ill patients with homogenous, defined problems that are intermediary steps in the development of ARDS. Hence, these intermediate steps can be evaluated separately. A similar approach was utilized in genetic association studies29 examining bronchial hyperresponsiveness as an intermediate step in the development of asthma.

Our results indicate that women with a variant SP-B allele have a statistically significant fourfold increase in the odds of being admitted to an ICU with direct pulmonary injury such as pneumonia or aspiration as opposed to extrapulmonary sepsis, trauma, or multiple transfusions. Our results are consistent with those of a recent report16 that looked at a different polymorphism of the SPB gene, the −1580C/T missense mutation in exon 4, in ARDS patients. Although their study was flawed by a deviation from the Hardy-Weinberg equilibrium among the control subjects, the 1580C allele was found to be associated with ARDS when healthy control subjects and at-risk individuals were compared. This association was due entirely to the increased frequency of the allele in a group of 23 patients with what the authors termed “idiopathic ARDS.” This group consisted of 20 patients (87%) with the direct pulmonary injury of pneumonia leading to ARDS. No such association was found in a group of 29 patients with “exogetic ARDS,” which consisted of 25 patients (86%) with indirect pulmonary injury such as surgery or trauma leading to ARDS. This result suggests that this SP-B polymorphism may contribute either to the etiology of ARDS or to the pathogenesis of ARDS in those patients with pneumonia. Because of the limited sample size, we were unable to determine whether the variant SP-B polymorphism contributes further to the development of ARDS in those patients with direct pulmonary injury.

Our results indicate that gender may contribute to disease susceptibility in critically ill patients, even among patients who share the same genotype. It is becoming increasingly clear that there are gender differences in disease susceptibility, severity, and outcome in a large variety of diseases.30 Male gender has been found to be associated with increased risk for sepsis and increased mortality from sepsis in some studies,31 but not all.32 Although women with acute respiratory failure from all causes do not appear to have a higher mortality rate than men,33 women have been found to have a higher mortality rate than men in the setting of pneumonia.3435 In addition, women have been found to be more sensitive to other types of pulmonary injury than men. Female smokers experience a greater loss of FEV1 with a greater risk of hospitalization for COPD than do male smokers, even after controlling for pack-years of smoking.36 Women treated with mediastinal radiation and/or chemotherapy for Hodgkin disease have a greater risk of cardiac and/or pulmonary sequelae that cannot be attributed to differences in age, smoking habits, radiation dose, or chemotherapy.37

Our results indicate that gender alone does not increase the risk for ARDS or direct pulmonary injury. However, female gender in association with the presence of the variant SP-B genotype increases the odds of developing ARDS, pneumonia, or aspiration severe enough to require care in an ICU. As the variant SP-B polymorphism in intron 4 is not transcribed, it is likely that this polymorphism is closely linked to another locus on the SP-B gene that affects the phenotype of the SP-B gene. However, it is clear that SP-B is essential for the normal properties of the lung. Monoclonal antibodies against SP-B produce acute respiratory failure and histologic damage to the lungs of rabbits that are identical to ARDS.38 Full-term infants who are unable to produce SP-B due to a frameshift mutation in exon 4 of this gene die of neonatal respiratory disease, which is characterized histopathologically by alveolar proteinosis.39 Mice that are homozygous for a null mutation in the SP-B gene die, while heterozygous mice have a normal survival and no signs or symptoms under normal conditions except for decreased lung compliance.40 However, these heterozygous mice have an increased risk of pulmonary inflammation, hemorrhage, and edema similar to ARDS after exposure to a direct pulmonary insult such as hyperoxia.41 Although humans heterozygous for SP-B deficiency have normal lung function at baseline,42 it is not clear what the response of these individuals to age and environmental insults such as tobacco use or pneumonia would be. In addition, there is evidence that SP-B production can be compromised in patients with pneumonia and ARDS. Immunosuppressed mice with Pneumocystis carinii pneumonia developed an acquired SP-B deficiency compared to immunosuppressed, uninfected mice.43 ARDS patients have been found to have significantly decreased concentrations of SP-B in their BAL fluid compared to healthy control subjects and patients who are at risk for ARDS,44 and decreased SP-B levels correlated with increased surface activity in the ARDS patients.

It is, therefore, biologically possible that individuals with the variant SP-B genotype are susceptible to greater lung dysfunction after a pulmonary insult. Coupled with their increased sensitivity to lung injury, the presence of the variant SP-B allele could increase further the odds of women developing either ARDS or a direct pulmonary injury, such as pneumonia or aspiration, that is severe enough to warrant an admission to an ICU.

In summary, in our cohort of critically ill patients with the clearly defined ARDS risk factors of sepsis, severe sepsis, trauma, aspiration, and multiple transfusion, the variant polymorphism in intron 4 of the SP-B gene is associated with an increased likelihood of developing ARDS and of being admitted to an ICU with direct pulmonary injury in women, but not in men. A larger study is needed to confirm these results and to investigate further whether the variant SP-B polymorphism in intron 4 contributes to the development of ARDS in those patients with direct or indirect pulmonary risk factors.

Abbreviations: APACHE = acute physiology and chronic health evaluation; bp = base pair; CI = confidence interval; OR = odds ratio; PCR = polymerase chain reaction; SP-B = surfactant protein-B; TNF = tumor necrosis factor

This research was supported by research grants HL60710 and ES00002 from the National Institutes of Health. Dr. Gong was supported by grant K23 HL67197 from the National Heart, Lung, and Blood Institute, and Drs. Xu and Miller were supported by grant T32 ES07069 from the National Institute of Environmental Health Sciences.

Table Graphic Jump Location
Table 1. Clinical Risk Factors for ARDS Required on Admission to the ICU for Subjects in Cohort*
* 

SCCM = Society of Critical Care Medicine

Figure Jump LinkFigure 1. PCR results of the polymorphism in intron 4 of the SP-B gene (2% agarose gel containing the amplified SPB gene). Lane 1 is the DNA ladder, and lanes 2 to 13 are the results from six subjects in the cohort performed in duplicate. Subjects with a single 606-bp band (patients 2 to 4 in lanes 4 to 9) have the wild-type genotype. Subjects with one or more bands larger or smaller than 606 bp are considered to have the insertion variant (patient 1 in lanes 2 and 3, and patient 5 in lanes 10 and 11) or the deletion variant (patient 6 in lanes 12 and 13), respectively. Pt = patient.Grahic Jump Location
Table Graphic Jump Location
Table 2. Baseline Characteristics of the Study Population*
* 

Values given as No. (%) or median (range), unless otherwise indicated.

 

Comparing case patients with ARDS to control subjects.

 

Tobacco history was not available for 78 subjects.

Table Graphic Jump Location
Table 3. Occurrence of ARDS by Clinical Risk Factor*
* 

Values given as No. (%), unless otherwise indicated.

 

Number of ARDS patients with risk factor/total number of patients with risk factor × 100.

 

p = 0.05 (comparing proportion of ARDS in those with sepsis from a pneumonia source to those with extrapulmonary source).

§ 

p = 0.01 (comparing proportion of ARDS in those with septic shock from a pneumonia source to those with extrapulmonary source).

 

Pneumonia or aspiration.

 

Trauma, sepsis from an extrapulmonary source, or multiple transfusions.

Table Graphic Jump Location
Table 4. Logistic Regression Analysis for Variant SP-B Genotype and Development of ARDS and Direct Pulmonary Injury
* 

Adjusted for white race, age, history of alcohol abuse, diabetes, multiple (> 1) risk factors for ARDS, and APACHE III score. APACHE II score was recalculated without the age component prior to inclusion in the model.

 

OR for developing ARDS compared to at-risk control subjects.

 

OR for being admitted to the ICU with direct pulmonary injury such as pneumonia or aspiration compared to patients admitted to the ICU with indirect pulmonary injury such as extrapulmonary infections, trauma or multiple transfusions.

Table Graphic Jump Location
Table 5. Characteristics of Patients by Gender*
* 

Values given as No. (%) or median (range), unless otherwise indicated.

 

Tobacco history was unknown for 78 patients in the cohort.

 

Sepsis or septic shock from pneumonia or aspiration.

§ 

Trauma, sepsis, or septic shock from an extrapulmonary source or multiple transfusions.

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Holm, B, Notter, R, Finkelstein, J Surface property changes from interactions of albumin with natural lung surfactant and extracted lung lipids.Chem Phys Lipids1985;38,287-298. [CrossRef] [PubMed]
 
Hawgood, S, Clements, JA Pulmonary surfactant and its apoproteins.J Clin Invest1990;86,1-6. [CrossRef] [PubMed]
 
Nogee, LM Genetics of the hydrophobic surfactant proteins.Biochim Biophys Acta1998;1408,323-333. [CrossRef] [PubMed]
 
Floros, J, Veletza, SU, Kotikalapudi, P, et al Dinucleotide repeats in human surfactant protein-B gene and respiratory distress syndrome.Biochem J1995;305,583-590. [PubMed]
 
Veletza, SV, Rogan, PK, TenHave, T, et al Racial differences in allelic distribution at the human pulmonary surfactant protein B gene locus (SP-B).Exp Lung Res1996;22,489-494. [CrossRef] [PubMed]
 
Max, M, Pison, U, Floros, J Frequency of AP-B and SP-A1 gene polymorphism in the acute respiratory distress syndrome (ARDS).Appl Cardiopulm Pathophysiol1996;6,111-118
 
Lin, Z, Oearson, C, Chinchilli, V, et al Polymorphisms of the humanSP-A, SP-B, andSP-Dgenes: association ofSP-BThr131Ile with ARDS.Clin Genet2000;58,181-191. [PubMed]
 
Silverman, EK, Palmer, LJ Case-control association studies for the genetics of complex respiratory diseases.Am J Respir Cell Mol Biol2000;22,645-648. [PubMed]
 
Howard, TD, Whittaker, PA, Zaiman, AL, et al Identification and association of polymorphisms in the interleukin-13 gene with asthma and atopy in a Dutch population.Am J Respir Cell Mol Biol2001;25,377-384. [PubMed]
 
Committee on Understanding the Biology of Sex and Gender Differences. Sex affects health. Wizemann, TM Pardue, M-L eds.Exploring the biological contributions to human health: does sex matter?2001,117-172 National Academies Press. Washington, DC:
 
Schroder, J, Kahlke, V, Staubach, KH, et al Gender differences in human sepsis.Arch Surg1998;133,1200-1205. [CrossRef] [PubMed]
 
Eachempati, SR, Hydo, L, Barie, PS Gender-based differences in patients with sepsis.Arch Surg1999;134,935-940. [CrossRef] [PubMed]
 
Epstein, SK, Vuong, V Lack of influence of gender on outcomes of mechanically ventilated medical ICU patients.Chest1999;116,732-739. [CrossRef] [PubMed]
 
Napolitano, LM, Greco, ME, Rodriguez, A, et al Gender differences in adverse outcomes after blunt trauma.J Trauma2001;50,274-280. [CrossRef] [PubMed]
 
Crabtree, TD, Pelletier, SJ, Gleason, TG, et al Gender-dependent differences in outcome after treatment of infection in hospitalized patients.JAMA1999;282,2143-2148. [CrossRef] [PubMed]
 
Prescott, E, Bjerg, AM, Andersen, PK, et al Gender differences in smoking effects on lung function and risk of hospitalization for COPD: results from a Danish longitudinal population study.Eur Respir J1997;10,822-827. [PubMed]
 
Lund, MB, Kongerud, J, Nome, O, et al Cardiopulmonary sequelae after treatment for Hodgkin’s disease: increased risk in females?Ann Oncol1996;7,257-264. [CrossRef] [PubMed]
 
Grossman, G, Suzuki, Y, Robertson, B, et al Pathophysiology of neonatal lung injury induced by monoclonal antibody to surfactant protein B.J Appl Physiol1997;82,2003-2010. [PubMed]
 
Nogee, L, Garnier, G, Dietz, H, et al A mutation in the surfactant protein B gene responsible for fatal neonatal respiratory disease in multiple kindreds.J Clin Invest1994;93,1860-1863. [CrossRef] [PubMed]
 
Clark, JC, Weaver, TE, Iwamoto, HS, et al Decreased lung compliance and air trapping in heterozygous deficient mice.Am J Respir Cell Mol Biol1997;16,46-52. [PubMed]
 
Tokieda, K, Iwamoto, HS, Bachurski, C, et al Surfactant protein-B-deficient mice are susceptible to hyperoxic lung injury.Am J Respir Cell Mol Biol1999;21,463-472. [PubMed]
 
Yusen, RD, Cohen, AH, Hamvas, A Normal lung function in subjects heterozygous for surfactant protein-B deficiency.Am J Respir Crit Care Med1999;159,411-414. [PubMed]
 
Beers, MF, Atochina, EN, Preston, AM, et al Inhibition of lung surfactant protein B expression duringPneumocystis cariniipneumonia in Mice.J Lab Clin Med1999;133,406-407. [CrossRef] [PubMed]
 
Gregory, TJ, Longmore, WJ, Moxley, MA, et al Surfactant chemical composition and biophysical activity in acute respiratory distress syndrome.J Clin Invest1991;88,1976-1981. [CrossRef] [PubMed]
 
Bone, R, Sibbald, W, Sprung, C The ACCP-SCCM consensus conference on sepsis and organ failure.Chest1992;101,1481-1483. [CrossRef] [PubMed]
 

Figures

Figure Jump LinkFigure 1. PCR results of the polymorphism in intron 4 of the SP-B gene (2% agarose gel containing the amplified SPB gene). Lane 1 is the DNA ladder, and lanes 2 to 13 are the results from six subjects in the cohort performed in duplicate. Subjects with a single 606-bp band (patients 2 to 4 in lanes 4 to 9) have the wild-type genotype. Subjects with one or more bands larger or smaller than 606 bp are considered to have the insertion variant (patient 1 in lanes 2 and 3, and patient 5 in lanes 10 and 11) or the deletion variant (patient 6 in lanes 12 and 13), respectively. Pt = patient.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1. Clinical Risk Factors for ARDS Required on Admission to the ICU for Subjects in Cohort*
* 

SCCM = Society of Critical Care Medicine

Table Graphic Jump Location
Table 2. Baseline Characteristics of the Study Population*
* 

Values given as No. (%) or median (range), unless otherwise indicated.

 

Comparing case patients with ARDS to control subjects.

 

Tobacco history was not available for 78 subjects.

Table Graphic Jump Location
Table 3. Occurrence of ARDS by Clinical Risk Factor*
* 

Values given as No. (%), unless otherwise indicated.

 

Number of ARDS patients with risk factor/total number of patients with risk factor × 100.

 

p = 0.05 (comparing proportion of ARDS in those with sepsis from a pneumonia source to those with extrapulmonary source).

§ 

p = 0.01 (comparing proportion of ARDS in those with septic shock from a pneumonia source to those with extrapulmonary source).

 

Pneumonia or aspiration.

 

Trauma, sepsis from an extrapulmonary source, or multiple transfusions.

Table Graphic Jump Location
Table 4. Logistic Regression Analysis for Variant SP-B Genotype and Development of ARDS and Direct Pulmonary Injury
* 

Adjusted for white race, age, history of alcohol abuse, diabetes, multiple (> 1) risk factors for ARDS, and APACHE III score. APACHE II score was recalculated without the age component prior to inclusion in the model.

 

OR for developing ARDS compared to at-risk control subjects.

 

OR for being admitted to the ICU with direct pulmonary injury such as pneumonia or aspiration compared to patients admitted to the ICU with indirect pulmonary injury such as extrapulmonary infections, trauma or multiple transfusions.

Table Graphic Jump Location
Table 5. Characteristics of Patients by Gender*
* 

Values given as No. (%) or median (range), unless otherwise indicated.

 

Tobacco history was unknown for 78 patients in the cohort.

 

Sepsis or septic shock from pneumonia or aspiration.

§ 

Trauma, sepsis, or septic shock from an extrapulmonary source or multiple transfusions.

References

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Holm, B, Notter, R, Finkelstein, J Surface property changes from interactions of albumin with natural lung surfactant and extracted lung lipids.Chem Phys Lipids1985;38,287-298. [CrossRef] [PubMed]
 
Hawgood, S, Clements, JA Pulmonary surfactant and its apoproteins.J Clin Invest1990;86,1-6. [CrossRef] [PubMed]
 
Nogee, LM Genetics of the hydrophobic surfactant proteins.Biochim Biophys Acta1998;1408,323-333. [CrossRef] [PubMed]
 
Floros, J, Veletza, SU, Kotikalapudi, P, et al Dinucleotide repeats in human surfactant protein-B gene and respiratory distress syndrome.Biochem J1995;305,583-590. [PubMed]
 
Veletza, SV, Rogan, PK, TenHave, T, et al Racial differences in allelic distribution at the human pulmonary surfactant protein B gene locus (SP-B).Exp Lung Res1996;22,489-494. [CrossRef] [PubMed]
 
Max, M, Pison, U, Floros, J Frequency of AP-B and SP-A1 gene polymorphism in the acute respiratory distress syndrome (ARDS).Appl Cardiopulm Pathophysiol1996;6,111-118
 
Lin, Z, Oearson, C, Chinchilli, V, et al Polymorphisms of the humanSP-A, SP-B, andSP-Dgenes: association ofSP-BThr131Ile with ARDS.Clin Genet2000;58,181-191. [PubMed]
 
Silverman, EK, Palmer, LJ Case-control association studies for the genetics of complex respiratory diseases.Am J Respir Cell Mol Biol2000;22,645-648. [PubMed]
 
Howard, TD, Whittaker, PA, Zaiman, AL, et al Identification and association of polymorphisms in the interleukin-13 gene with asthma and atopy in a Dutch population.Am J Respir Cell Mol Biol2001;25,377-384. [PubMed]
 
Committee on Understanding the Biology of Sex and Gender Differences. Sex affects health. Wizemann, TM Pardue, M-L eds.Exploring the biological contributions to human health: does sex matter?2001,117-172 National Academies Press. Washington, DC:
 
Schroder, J, Kahlke, V, Staubach, KH, et al Gender differences in human sepsis.Arch Surg1998;133,1200-1205. [CrossRef] [PubMed]
 
Eachempati, SR, Hydo, L, Barie, PS Gender-based differences in patients with sepsis.Arch Surg1999;134,935-940. [CrossRef] [PubMed]
 
Epstein, SK, Vuong, V Lack of influence of gender on outcomes of mechanically ventilated medical ICU patients.Chest1999;116,732-739. [CrossRef] [PubMed]
 
Napolitano, LM, Greco, ME, Rodriguez, A, et al Gender differences in adverse outcomes after blunt trauma.J Trauma2001;50,274-280. [CrossRef] [PubMed]
 
Crabtree, TD, Pelletier, SJ, Gleason, TG, et al Gender-dependent differences in outcome after treatment of infection in hospitalized patients.JAMA1999;282,2143-2148. [CrossRef] [PubMed]
 
Prescott, E, Bjerg, AM, Andersen, PK, et al Gender differences in smoking effects on lung function and risk of hospitalization for COPD: results from a Danish longitudinal population study.Eur Respir J1997;10,822-827. [PubMed]
 
Lund, MB, Kongerud, J, Nome, O, et al Cardiopulmonary sequelae after treatment for Hodgkin’s disease: increased risk in females?Ann Oncol1996;7,257-264. [CrossRef] [PubMed]
 
Grossman, G, Suzuki, Y, Robertson, B, et al Pathophysiology of neonatal lung injury induced by monoclonal antibody to surfactant protein B.J Appl Physiol1997;82,2003-2010. [PubMed]
 
Nogee, L, Garnier, G, Dietz, H, et al A mutation in the surfactant protein B gene responsible for fatal neonatal respiratory disease in multiple kindreds.J Clin Invest1994;93,1860-1863. [CrossRef] [PubMed]
 
Clark, JC, Weaver, TE, Iwamoto, HS, et al Decreased lung compliance and air trapping in heterozygous deficient mice.Am J Respir Cell Mol Biol1997;16,46-52. [PubMed]
 
Tokieda, K, Iwamoto, HS, Bachurski, C, et al Surfactant protein-B-deficient mice are susceptible to hyperoxic lung injury.Am J Respir Cell Mol Biol1999;21,463-472. [PubMed]
 
Yusen, RD, Cohen, AH, Hamvas, A Normal lung function in subjects heterozygous for surfactant protein-B deficiency.Am J Respir Crit Care Med1999;159,411-414. [PubMed]
 
Beers, MF, Atochina, EN, Preston, AM, et al Inhibition of lung surfactant protein B expression duringPneumocystis cariniipneumonia in Mice.J Lab Clin Med1999;133,406-407. [CrossRef] [PubMed]
 
Gregory, TJ, Longmore, WJ, Moxley, MA, et al Surfactant chemical composition and biophysical activity in acute respiratory distress syndrome.J Clin Invest1991;88,1976-1981. [CrossRef] [PubMed]
 
Bone, R, Sibbald, W, Sprung, C The ACCP-SCCM consensus conference on sepsis and organ failure.Chest1992;101,1481-1483. [CrossRef] [PubMed]
 
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