Genetic Polymorphisms of Peptidase Inhibitor 3 (Elafin) Are. Associated with Acute Respiratory Distress Syndrome

Transcription

1 Genetic Polymorphisms of Peptidase Inhibitor 3 (Elafin) Are Associated with Acute Respiratory Distress Syndrome Paula Tejera 1, Zhaoxi Wang 1, Rihong Zhai 1, Li Su 1, Chau-Chyun Sheu 1, Deanne M. Taylor 2, Feng Chen 1, Michelle N. Gong 3, B. Taylor Thompson 4, and David C. Christiani 1,4 1 Department of Environmental Health, 2 Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts; 3 Division of Pulmonary and Critical Care Medicine, Mount Sinai School of Medicine, New York, New York; and 4 Pulmonaary and Critical Care Unit, Department of Medicine, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts Online Data Supplement

2 TABLE E1. RISK FACTORS FOR ARDS REQUIRED FOR STUDY INCLUSION AND EXCLUSION CRITERIA CONSIDERED Risk factor Definition Sepsis Known or suspected source of systemic infection plus at least two of the following: a) temperature > 38 C or < 36 C; b) heart rate > 90 beats/min; c) respiratory rate > 20 breaths/min or PaCO 2 < 32 mmhg; d) WBC count > 12,000/mm 3, < 4000/mm 3, or > 10% bandemia. Septic shock Fulfill requirements for sepsis plus one of the following: a) SBP < 90 mmhg or a reduction of 40 mmhg from baseline for 30 mins, unresponsive to 500 ml of fluid resuscitation; b) need for vasopressors to maintain SBP 90 mmhg or within 40 mmhg of baseline. Pneumonia Fulfill two or more of the following: a) new airspace opacity on chest radiograph; b) temperature > 38.3 C or < 36.0 C, WBC > 12,000/mm 3 or < 4000/mm 3 or > 10% bandemia; c) positive microbiological culture. Aspiration Defined as witnessed or documented aspiration event or the retrieval of gastric contents from the oropharynx, endotracheal tube, or bronchial tree. Trauma Defined as multiple fractures and/or pulmonary contusions. Multiple fractures are defined as a fracture of two long bones, an unstable pelvic fracture, or one long bone and a pelvic fracture. Pulmonary contusion is defined as airspace opacity on chest radiograph within 8 hrs of admission to the emergency room and evidence of blunt trauma to the chest, for example, fractured ribs or ecchymosis overlying airspace opacity. Multiple transfusion Defined as receiving 8 units of packed red blood cells within 24 hrs. WBC, white blood cell; SBP, systolic blood pressure. Exclusion criteria 1. Age < Diffuse alveolar hemorrhage. 3. Chronic lung diseases other than COPD or asthma. 4. Directive to withhold intubation. 5. Immunosuppression not secondary to corticosteroid. 6. Treatment with granulocyte colony-stimulating factor.

3 TABLE E2. COMPARE CHARACTERISTICS BETWEEN ARDS CASES AND CONTROLS WITH PLASMA SAMPLES. Controls (n = 59) ARDS (n = 54) P Age-yr, mean ± SD 60 ± ± Male, n (%) 34 (57.6%) 30 (55.6%) Caucasian, n (%) 56 (94.9%) 48 (88.9%) APACHE III score, mean ± SD * 71.3 ± ± 19.9 <0.001 On ventilation at ICU admission, n (%) 39 (66.1%) 49 (90.7%) Risk factors, n (%) Sepsis 22 (37.3%) 14 (26.0%) Septic shock 26 (44.1 %) 33 (61.1%) Pneumonia 21 (35.6%) 40 (74.1%) <0.001 Aspiration 6 (10.2%) 7 (13%) Pulmonary injury 29 (49.1%) 44 (81.5%) <0.001 Multiple transfusion 7 (11.9%) 5 (9.3%) Trauma 5 (8.5%) 2 (3.7%) Comorbidities, n (%) Diabetes 14 (24.0%) 14 (26.0%) Liver failure/cirrhosis 3 (5.1%) 5 (9.3%) Corticosteroid treatment before ICU admission, n (%) 3 (5.1%) 9 (16.7%) History of alcohol abuse 7 (11.9%) 10 (18.5%) Definition of abbreviations: ARDS, acute respiratory distress syndrome; APACHE, Acute Physiology and Chronic Health Evaluation; * APACHE III physiology score was calculated with all components on the day of ICU admission; Pneumonia, aspiration, pulmonary contusions, or sepsis from lower pulmonary source were categorized as pulmonary injury. Sepsis from an extrapulmonary source, trauma without pulmonary contusions, and multiple transfusions were categorized as extrapulmonary injury. Patients with both pulmonary and extrapulmonary injuries were considered to have pulmonary injury; Patient received 300 mg of prednisone or its equivalent within 21 days or 15 mg prednisone a day or its equivalent prior to ICU admission.

4 TABLE E3. PROTEINASE INHIBITOR 3 (PI3) GENOTYPE FREQUENCIES AND COMPARISONS BETWEEN ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) AND CONTROLS, AND SURVIVORS AND NONSURVIVORS ARDS Development ARDS 28-Day Mortality ARDS 60-Day Mortality Overall ARDS Controls Survivors Nonsurvivors Survivors Nonsurvivors (n = 1480) (n = 449) (n = 1031) P (n = 299) (n = 150) P (n = 270) (n = 179) P rs AA 508 (34.3%) 149 (33.2%) 359 (34.8%) 95 (31.8%) 54 (36.0%) 80 (29.6%) 69 (38.5%) TA 710 (48.0%) 215 (47.9%) 495 (48.0%) 147 (49.2%) 68 (45.3%) 138 (51.1%) 77 (43.0%) TT 262 (17.7%) 85 (18.9%) 177 (17.2%) 57 (19.2%) 28 (18.7%) 52 (19.3%) 33 (18.4%) rs AA 1027 (69.4%) 315 (70.2%) 712 (69.1%) 206 (70.5%) 103 (69.1%) 189 (70.0%) 126 (70.4%) AT 405 (27.4%) 123 (27.4%) 282 (27.3%) 79 (27.0%) 42 (28.2%) 75 (27.8%) 48 (26.8%) TT 48 (3.2%) 11 (2.4%) 37 (3.6%) 7 (2.4%) 4 (2.7%) 6 (2.2%) 5 (2.8%) rs * AA 984 (66.5%) 277 (61.7%) 707 (68.6%) 178 (59.5%) 99 (66.0%) 158 (58.5%) 119 (66.5%) AC 444 (30.0%) 152 (33.8%) 292 (28.3%) 107 (35.8%) 45 (30.0%) 99 (36.7%) 53 (29.6%) CC 52 (3.5%) 20 (4.4%) 32 (3.1%) 14 (4.7%) 6 (4.0%) 13 (4.8%) 7 (3.9%) * FDR P =

5 TABLE E4. BASELINE CHARACTERISTICS BETWEEN SURVIVORS AND NONSURVIVORS OF ARDS ARDS 28-day mortality ARDS 60-day mortality Nonsurvivors Survivors Nonsurvivors Survivors Characteristic (n = 150) (n = 299) P (n = 179) (n = 270) P Age-yr mean ± SD 68 ± ± 19 < ± ± 18 <0.001 Male 87 (58.0%) 182 (60.9%) (59.2%) 163 (60.4%) APACHE III score, mean ± SD * 90.6 ± ± 21.4 < ± ± 21.4 <0.001 On ventilation at ICU admission, n (5) 131 (87.3%) 261 (87.3%) (87.8%) 155 (86.6%) Risk factors, n (%) Sepsis 32 (21.3%) 85 (28.4%) (24.6%) 73 (27.0%) Septic shock 105 (70.0%) 162 (54.2%) (64.8%) 151 (55.9%) Pneumonia 108 (72.0%) 194 (64.9%) (70.9%) 175 (64.8%) Aspiration 17 (11.3%) 28 (9.4%) (11.2%) 25 (9.3%) Multiple Transfusion 14 (9.3%) 33 (11.0%) (11.2%) 27 (10.0%) Trauma 2 (1.3%) 30 (10.0%) < (1.1%) 30 (11.1%) <0.001 Pulmonary injury 108 (72.0%) 213 (71.2%) (70.9%) 194 (71.8%) Comorbidity, n (%) Diabetes 30 (20.0%) 48 (16.0%) (17.9%) 46 (17.0%) Liver cirrhosis/failure 16 (10.7%) 15 (5.0%) (11.7%) 10 (3.7%) Corticosteroid treatment before ICU admission n(%) 21 (14.0%) 26 (8,7%) (15.6%) 19 (7.0%) History of alcohol abuse 23 (15.3%) 41 (13.7%) (15.6%) 36 (13.3%) Definition of abbreviations: ARDS, acute respiratory distress syndrome; APACHE, Acute Physiology and Chronic Health Evaluation; SD, standard deviation. * APACHE III physiology score was calculated with all components on the day of ICU admission;

6 Pneumonia, aspiration, pulmonary contusions, or sepsis from lower pulmonary source were categorized as pulmonary injury. Sepsis from an extrapulmonary source, trauma without pulmonary contusions, and multiple transfusions were categorized as extrapulmonary injury. Patients with both pulmonary and extrapulmonary injuries were considered to have pulmonary injury; Patient received 300 mg of prednisone or its equivalent within 21 days or 15 mg prednisone a day or its equivalent prior to ICU admission.

7 TABLE E5. COX PROPORTIONAL HAZARD ANALYSIS OF ASSOCIATION BETWEEN GENETIC VARIANTS OF PI3 AND 60-DAY SURVIVAL IN PATIENTS WITH ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) All ARDS (n = 449) Extrapulmonary injury (n = 123) Pulmonary injury (n = 326) Genotype HR * (95% CI) P HR * (95% CI) p HR * (95% CI) p rs ( ) ( ) ( ) rs ( ) ( ) ( ) rs ( ) ( ) ( ) Haplotype Global test Hap1 ATA Hap2 TTC 1.02 ( ) ( ) ( ) Hap3 TAA 0.93 ( ) ( ) ( ) Hap4 TTA 1.20 ( ) ( ) ( ) Definition of abbreviations: HR, Hazard ratio * Adjusted for age, gender, APACHE III score, risk factors for ARDS, comorbidities (diabetes, liver cirrhosis/failure) and alcohol abuse. HR were expressed per difference in the number of minor alleles (additive model).

8 Global test for the haplotypes by likehood-rario test (LTR), with 4 degrees of freedom.

9 TABLE E6. COMPARE CHARACTERISTICS BETWEEN ARDS CASES WITH PLASMA SAMPLES AND THOSE WITHOUT PLASMA (EXCLUDED IN THIS STUDY) With Plasma (n = 54) Without Plasma (n = 401) P Age-yr, mean ± SD 61 ± ± Male, n (%) 30 (55.6%) 244 (60.8%) Caucasian, n (%) 48 (88.9%) 401 (100%) <0.001 APACHE III score, mean ± SD * 86.6 ± ± On ventilation at ICU admission, n (%) 49 (90.7%) 349 (87.0%) Risk factors, n (%) Sepsis 14 (26.0%) 105 (26.2%) Septic shock 33 (61.1 %) 237 (59.1%) Pneumonia 40 (74.1%) 266 (66.3%) Aspiration 7 (13%) 41 (10.2%) Pulmonary injury 42 (77.8%) 284 (71.0%) Multiple transfusion 5 (9.3%) 42 (10.5%) Trauma 2 (3.7%) 30 (7.5%) Comorbidities, n (%) Diabetes 14 (26.0%) 68 (17.0%) Liver failure/cirrhosis 5 (9.3%) 26 (6.5%) Corticosteroid treatment before ICU admission, n (%) 9 (16.7%) 40 (10.0%) History of alcohol abuse 10 (18.5%) 54 (13.5%) Definition of abbreviations: ARDS, acute respiratory distress syndrome; APACHE, Acute Physiology and Chronic Health Evaluation; * APACHE III physiology score was calculated with all components on the day of ICU admission; Pneumonia, aspiration, pulmonary contusions, or sepsis from lower pulmonary source were categorized as pulmonary injury. Sepsis from an extrapulmonary source, trauma without pulmonary contusions, and multiple transfusions were categorized as extrapulmonary injury. Patients with both pulmonary and extrapulmonary injuries were considered to have pulmonary injury; Patient received 300 mg of prednisone or its equivalent within 21 days or 15 mg prednisone a day or its equivalent prior to ICU admission.

10 TABLE E7. COMPARE CHARACTERISTICS BETWEEN CONTROLS SELECTED IN THIS STUDY AND THOSE EXCLUDED With Plasma (n = 59) Without Plasma (n = 975) P Age-yr, mean ± SD 60 ± ± Male, n (%) 34 (57.6%) 595 (61.0%) Caucasian, n (%) 56 (94.9%) 975 (100%) <0.001 APACHE III score, mean ± SD * 71.3 ± ± On ventilation at ICU admission, n (%) 39 (66.1%) 638 (65.4%) Risk factors, n (%) Sepsis 22 (37.3%) 363 (37.2%) Septic shock 26 (44.1 %) 422 (43.3%) Pneumonia 21 (35.6%) 423 (43.4%) Aspiration 6 (10.2%) 81 (8.31%) Pulmonary injury 26 (44.1%) 481 (49.3%) Multiple transfusion 7 (11.9%) 111 (11.4%) Trauma 5 (8.5%) 76 (7.8%) Comorbidities, n (%) Diabetes 14 (23.7%) 270 (27.7%) Liver failure/cirrhosis 3 (5.1%) 37 (3.8%) Corticosteroid treatment before ICU admission, n (%) 3 (5.1%) 90 (9.2%) History of alcohol abuse 7 (11.9%) 95 (9.7%) Definition of abbreviations: ARDS, acute respiratory distress syndrome; APACHE, Acute Physiology and Chronic Health Evaluation; * APACHE III physiology score was calculated with all components on the day of ICU admission; Pneumonia, aspiration, pulmonary contusions, or sepsis from lower pulmonary source were categorized as pulmonary injury. Sepsis from an extrapulmonary source, trauma without pulmonary contusions, and multiple transfusions were categorized as extrapulmonary injury. Patients with both pulmonary and extrapulmonary injuries were considered to have pulmonary injury; Patient received 300 mg of prednisone or its equivalent within 21 days or 15 mg prednisone a day or its equivalent prior to ICU admission.

11 ONLINE SUPPLEMENT FIGURE LEGENDS Figure E1. Flow diagram of study design and patient selection for case-control study. Figure E2. Comparison of elafin (PI3) propeptide sequence among different species. Elafin peptide sequences from different species were retrieved from National Center for Biotechnology Information (NCBI) database using BLAST search engine. Sequence alignment was conducted on Jalview software (39). Arrow indicates the position of non-synonymous polymorphism (T34P, rs ). Black underlines indicate the positions of 5 transglutaminase substrate motifs. Accession numbers in alignment are H. sapiens NP_ ; P. troglodytes AB ; G.g.gorilla AB ; P. abelii AB ; M. mulatta NP_ ; P. anubis AB ; C. guereza AB052934; C. aethiops AB ; A. nancymae AB ; S. boliviensis ABO ; C. jacchus AB ; O. garnetti AB ; L. catta AB ; E. caballus XP_ ; C. familiaris XP_ Figure E3. Haploview LD (D ) of SNPs of PI3 gene constructed using genetic data derived from HapMap. The D for any two SNPs is presented in the box representing their intersection. The black boxes represent the strongest LD, the gray ones represent lower linkage disequilibrium values. Asterisks indicate tagging SNPs

12 predicted by the HapMap CEU sample. Figure E4. Flow diagram of study design and patient selection for plasma PI3 analysis.

13 Admitted to ICU with required risk for ARDS and did not meet exclusion criteria (n = 2642) No consent because of refusal (n = 318) or lack of surrogate (n = 467) or death prior to consent obtained (n = 136) Excluded from the study because of previous history of ARDS or previous enrollment as controls (n = 70) Enrolled into prospective cohort and followed for ARDS development (n = 1651) Non Caucasian (n = 153) Enrolled into the present study and subject to genotyping (n = 1498) Excluded from analysis due to missing genotype data (n = 9) or clinical data (n = 9) Did not develop ARDS (n = 1031) Developed ARDS (n = 449) Survivors (n = 299) Non-survivors (n = 150) Figure E1

14 Figure E2 1

15 * * * Figure E3 1

16 Figure E4