METHODS Study subjects. Genotyping quality control. Statistical analysis. SNP selection

Transcription

1 METHODS Study subjects Among the African Caribbean families recruited in Barbados, asthma was defined as both a reported history of asthma and a history of physician-diagnosed asthma (past/current), plus a history of wheezing without an upper respiratory infection (URI) for 2 out of 4 hallmark symptoms (wheezing with a URI, cough without a URI, shortness of breath, and tightness in the chest). E1,E2 Among the African American families recruited through the Collaborative Study on the Genetics of Asthma (CSGA), E3,E4 asthma was defined by using criteria including at least 2 symptoms consistent with asthma, <3 pack-years of cigarette exposure, and a physician s diagnosis of asthma, in addition to either (1) bronchial hyperresponsiveness defined by a fall in baseline FEV 1 20% at 25 mg/ml methacholine or (2) in subjects whose FEV 1 was reduced, reversibility defined as a 15% increase in baseline FEV 1 after an inhaled bronchodilator (albuterol). Spirometry was performed according to American Thoracic Society guidelines E5 by using a KOKO (Pulmonary Data Services, Inc, Louisville, Colo) pneumotach connected to a laptop computer. African American patients and controls participating in the ALI and sepsis study were defined in accordance with the American College of Chest Physicians E6 and Society of Critical Care Medicine Consensus statements, E7 and subjects were recruited after local Institutional Review Board approval at Johns Hopkins University and the University of Maryland in Baltimore, Md, Emory University in Atlanta, Ga, and the Medical College of Wisconsin in Milwaukee, Wis. SNP selection In the initial study, we assessed the full length of the gene encoding MYLK (GenBank Accession number: U48959; kb, 32 exons) by direct sequencing of PCR amplicons in all exons, exonintron boundaries (including 100 bases of intronic sequence on either side), 59 and 39-untranslated regions (UTRs) and 2 kb upstream of 59 UTR, and identified 51 SNPs (10 exonic, 31 intronic, 9 59 UTR, and 1 SNP located in the noncoding region of exon 1), 4 of which were novel nonsynonymous coding SNPs (csnps) using individual DNA samples from 36 subjects equally divided into European Americans and African Americans as previously described. E1 Subsequently, a panel of 25 MYLK SNPs, including 4 novel SNPs previously identified by sequencing, was selected to genotype in an African American population selected for either severe sepsis or sepsis-associated ALI on the basis of (1) sufficient MAF (10%) in a population of African descent, (2) inter-snp distance (>0.5 kb), and (3) ready availability using the ABI TaqMan SNP Genotyping Assay platform. In the current study, we used both Clayton s htsnp E8 (available at: and Stram s TagSNPs E9,E10 (available at: ;stram/tagsnps.html) methods for the selection of haplotype tagging SNPs with genotypes for the 25 SNPs from 61 African American healthy controls participating in the ALI study described. E11 We selected these methods because, although both algorithms essentially rely on LD, they have slightly different approaches of acquiring this information. Briefly, htsnp relies on 2 measurements (proportion of diversity explained and r 2 ), which represent the ability of any SNP subset to maintain the overall SNP diversity of the entire region in question. TagSNP was written to circumvent problems that arise when there are multiple rare haplotypes in the haplotype estimation step, by calculating a haplotype dosage for the full set of SNPs, and then essentially performing a regression analysis on the selected subsets of SNPs and the full set, to maximize the squared correlation between the true haplotype dosage and the predicted dosage in the haplotype tagging set of SNPs. Because haplotype tagging methods are still under much debate and development, and in part because of our recent experience in selecting tagging SNPs for populations of African descent, E12 we chose both of these methods in combination, with the aim of obtaining a more convincing view of which SNPs we may want to discard. Only SNPs retained by both programs using an R 2 cutoff of 0.8 were retained as tagging SNPs, resulting in the removal of the 6 most redundant markers from the previous panel, for a final panel of 19 MYLK SNPs (Table E2). Genotyping quality control Approximately 8% of samples were repeated for quality control with an average error rate of 1.3%. Mendelian inconsistencies identified using Sib-Pair (1.00a17) with 3.5% discordant genotype pairs in the Barbados family dataset. Genotypes were made missing as appropriate for members of nuclear families involved, which rendered 177 genotypes (1.2 %) missing for further analyses. Statistical analysis Association testing using FBAT. For haplotype analyses in the 2 family-based asthma populations, an expectation-maximization algorithm, which maximizes the likelihood of phased haplotype frequencies based on all observed genotypes in the nuclear family, was used. Adjusting of confounding caused by multiple comparisons. Empiric P values were calculated through permutation analysis by using Monte Carlo methods under the null hypothesis of complete independence between haplotypes and the observed phenotypes. FBAT s sampling procedure stops when at least 100 Monte Carlo based test statistic values are generated that are less than the observed value or after 100,000 replicate samples are generated, whichever comes first. Haplotype association testing in the severe sepsis and ALI population For re-evaluation of the 4-SNP haplotype in the case-control designed severe sepsis/ali population, haplotype frequency was estimated via a maximum likelihood method using the expectationmaximization algorithm under the assumption of Hardy-Weinberg equilibrium implemented in SNPem. E11,E13 The omnibus likelihood ratio test P value from SNPem was used as the global P value. RNA preparation and gene expression in human PBMCs Total RNA was extracted from human PBMCs of 28 unrelated subjects from Barbados (15 patients with asthma and 13 controls) by using the Trizol Reagent (catalog no ; Invitrogen, Carlsbad, Calif). Subjects were also selected on the basis of their rs genotypes (GG, GC, CC); the CC genotype was rare (6.9% in the Barbados population), and only 2 subjects with asthma with the CC genotype were available for the expression study. The result was a group divided with 6 or 7 in each subgroup (6 GG asthmatic, 7 GG nonasthmatic, 7 GC and 2 CC asthmatic, 6 GC nonasthmatic). Additional purification was performed on RNAeasy columns (catalog no ; Qiagen, Valencia, Calif). The quality of total RNA samples was assessed by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif). RNA samples were labeled according to Illumina-recommended protocols. In brief, 0.5 mg total RNA from each sample was labeled by using the Illumina TotalPrep RNA Amplification Kit (catalog no. IL1791; Ambion, Austin, Tex) in a process of cdna synthesis and in vitro transcription similar to that of other chip manufacturers. Single-stranded RNA (crna) was generated and labeled by incorporating biotin-16-uridine-59-triphosphate (catalog no ; Roche Diagnostics GmbH, Mannheim, Germany). Biotin-labeled crna 0.85 ug was hybridized (16 hours) to Illumina s Sentrix

2 HumanRef-8 Expression BeadChips (catalog no ; Illumina, San Diego, Calif). The hybridized biotinylated crna was detected with streptavidin-cy3 and quantitated by using Illumina s BeadStation 500GX Genetic Analysis Systems scanner. Array data analysis Preliminary analysis of the scanned data was performed by using Illumina BeadStudio software. The basic Illumina data are returned from the scanner in the form of an.idat file that contains single intensity data values/gene after the computation of a trimmed mean average for each probe type represented by a variable number of bead probes/gene on the array. The BeadStudio software returns information on the number and SD of all the bead measurements per probe/ gene as well as a detection call based on a comparison between the measured intensity calculated for a single probe/gene and the intensities measured for a large number of negative control beads built-in to the BeadChip arrays (D 5 % above negative/100, 1 5 perfect, ie, the intensity value of a gene is greater than all the intensities for every negative control tested). The background filtering (any gene below D for 75% of samples was eliminated from further analysis) was performed. Then the data were log-transformed and normalized to a chip-wise mean by using the CLUSTER tool (available at: rana.stanford.edu/clustering). E14 Significance testing was performed by comparing expression levels among patients with asthma and healthy controls stratified by SNP rs G>C genotypes (GG or GC/CC) by using the unpaired t test. Statistical significance (P values) was defined at the 5% level for all analyses. RESULTS Study populations Among the 848 African Caribbean family members in Barbados and 368 African American family members, distributions of tige concentrations were similar to those described previously. E1,E15 Families from both populations were originally selected through an asthmatic proband. Total serum IgE was significantly higher (P < ) among patients with asthma compared with subjects without asthma in both populations (Table E1). Both populations were highly atopic, especially the Caribbean families (mean total serum IgE was 622 ng/ml and 378 ng/ml for the Caribbean and American populations, respectively). Association among SNPs Frequencies of the 19 MYLK SNPs in both populations are summarized in Table E2. All SNPs were in Hardy- Weinberg equilibrium among both sets of founders. Moderate to high LD (according to the definition of Gabrieletal E16 ) was observed for 8 out of 18 pairings of contiguous SNPs (D9 ranging from 0.88 to 1.0) in the African Caribbean population. Because of an average inter-snp distance of 11 kb, the resulting blocks were small (2 blocks observed in the 39 smmylk isoform, composed of markers rs and rs in block 1, and rs and ss in block 2, with D for both blocks), and overall patterns across the region revealed low LD (Fig E1). A similar pattern was observed in the African American founders except that there was relatively higher LD among markers in the border of the nmmylk and smmylk isoforms of the gene, resulting in a 7kb block composed of markers rs and rs Genetic association analyses in the African Caribbean population All individual SNP analyses between the 19 markers across the 3 MYLK regions and asthma and log[tige] were first performed under the robust genotype model (data not shown) with results giving the strongest signals suggesting the additive model. Analyses performed under dominant and recessive models (data not shown) supported the additive model results and did not identify any additional associations. In addition to the negative association among the Caribbean families between both asthma and log[tige] (P and P 5.05, respectively) and a promoter SNP (rs936170; Fig 2, A and B) of the smmylk isoform and the 3-SNP and 4-SNP haplotype associations in this region, a negative association for log[tige] (P 5.03) was also observed for a nonsynonymous SNP that creates a proline to histidine amino acid change in the second exon of the nmmylk isoform (rs , referred to as MYLK_007 in Gao et al E11 ; Table E3); this SNP had previously been shown to be part of a 3-SNP haplotype that was significantly associated with risk of ALI, but only among African American patients. E11 Additional associations (single-marker and haplotype) observed for markers in the nmmylk isoform included significant evidence for linkage and association in the 59 region of MYLK (nmmylk) between the 2 phenotypes (asthma and log[tige]) and 2-SNP, 3-SNP, and/or 4-SNP haplotypes (Table E4). The strongest associations were for a 4-SNP haplotype (rs rs ; GTAC) in the 59 region (region 1, Fig 1) for asthma and log[tige] (P and.001, respectively), and a 2-SNP haplotype beginning with the second SNP (rs rs ; CG) for log[tige] (P 5.013). In the third region, near the 39 end of MYLK (smmylk), a 4-SNP haplotype (rs ss ; CTCC) provided the strongest signal for asthma and log[tige] (P and P 5.01, respectively). An intronic SNP (rs ) at the 39 end of MYLK contributed significantly in determining the direction of this association, and similar to the other associations, this SNP had previously been implicated in a haplotype conferring risk of sepsis. E11 DISCUSSION Another limitation in our study design is the ability to directly test for association between causal variants because we did not use a large panel of SNPs that could fully represent variability in the MYLK gene. Therefore, we cannot be certain the causal variant is included in our panel of markers. Because the primary objective of this study was to compare associations previously observed for a sepsis/ali study to associations between the same set of SNPs and asthma, the SNPs used here was selected from our previous resequencing project of the 32 MYLK exons, exon-intron boundaries, and 59 and 39-UTRs of MYLK. At the time of this publication, there are 388 SNPs

3 reported for the MYLK gene in the West African Yoruban International HapMap Project population, which most closely matches our 2 populations of African descent, of which 53 SNPs are tagging SNPs (R 2 > 0.80; International HapMap Consortium, HapMap data Rel 21a/phaseII Jan07, on NCBI B35 assembly, dbsnp b125). There are 4 coding, nonsynonymous MYLK SNPs, and 7 known 59 UTR and three 39 UTR SNPs. On the basis of our previous sequencing in African Americans, E11 we identified 10 SNPs in 59 UTR and 8 SNPs in coding region of MYLK with 4 of them conferring an amino acid change, and 3 out of the 4 nonsynonymous SNPs were included in these studies. The sliding window haplotype analysis presented here does provide a more comprehensive search for evidence of association than could be attained by analysis of individual SNPs, but we acknowledge the limitations of our approach. Intronic SNPs gave our strongest statistical evidence of linkage in the presence of disequilibrium, but our SNPs are limited to the first 100 base pairs on either side of each exon. Further effort is clearly warranted to identify the likely causal variants associated with atopic asthma. REFERENCES E1. Barnes KC, Neely JD, Duffy DL, Freidhoff LR, Breazeale DR, Schou C, et al. Linkage of asthma and total serum IgE concentration to markers on chromosome 12q: evidence from Afro-Caribbean and Caucasian populations. Genomics 1996;37: E2. Barnes K, Hizawa N, Neely J, Duffy D, Freidhoff L, Ehrlich E, et al. Linkage analysis of atopy to chromosome 12q markers in an Afro- Caribbean population [abstract]. J Allergy Clin Immunol 1997;99:S476. E3. CSGA. The Collaborative Study on the Genetics of Asthma: a genomewide search for asthma susceptibility loci in ethnically diverse populations. Nat Genet 1997;15: E4. Xu J, Meyers DA, Ober C, Blumenthal MN, Mellen B, Barnes KC, et al. Genomewide screen and identification of gene-gene interactions for asthma-susceptibility loci in three U.S. populations: collaborative study on the genetics of asthma. Am J Hum Genet 2001;68: E5. Standardization of spirometry, 1994 update. American Thoracic Society. Am J Respir Crit Care Med 1995;152: E6. Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K, Hudson L, et al. Report of the American-European Consensus conference on acute respiratory distress syndrome: definitions, mechanisms, relevant outcomes, and clinical trial coordination. Consensus Committee. J Crit Care 1994;9: E7. American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med 1992;20: E8. Clayton D. Choosing a set of haplotype tagging SNPs from a larger set of diallelic loci. Available at: software/stata/htsnp/htsnp.pdf. Accessed April 9, E9. Stram DO, Haiman CA, Hirschhorn JN, Altshuler D, Kolonel LN, Henderson BE, et al. Choosing haplotype-tagging SNPS based on unphased genotype data using a preliminary sample of unrelated subjects with an example from the Multiethnic Cohort Study. Hum Hered 2003;55: E10. Stram DO. Tag SNP selection for association studies. Genet Epidemiol 2004;27: E11. Gao L, Grant A, Halder I, Brower R, Sevransky J, Maloney JP, et al. Novel polymorphisms in the myosin light chain kinase gene confer risk for acute lung injury. Am J Respir Cell Mol Biol 2006;34: E12. Chi PB, Duggal P, Kao WH, Mathias RA, Grant AV, Stockton ML, et al. Comparison of SNP tagging methods using empirical data: association study of 713 SNPs on chromosome 12q q24.21 for asthma and total serum IgE in an African Caribbean population. Genet Epidemiol 2006;30: E13. Fallin D, Cohen A, Essioux L, Chumakov I, Blumenfeld M, Cohen D, et al. Genetic analysis of case/control data using estimated haplotype frequencies: application to APOE locus variation and Alzheimer s disease. Genome Res 2001;11: E14. Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A 1998;95: E15. Zambelli-Weiner A, Ehrlich A, Stockton ML, Grant AV, Zhang S, Levett PN, et al. Evaluation of the CD14/-260 polymorphism and house dust endotoxin exposure in the Barbados asthma genetics study. J Allergy Clin Immunol 2005;115: E16. Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, et al. The structure of haplotype blocks in the human genome. Science 2002;296:

4 TABLE E1. Demographic characteristics of the 2 asthma populations* African Caribbean African American Characteristics Founders Total Founders Total No. of subjects Percent male sex (%) 49.7% 49.1% 44.2% 48.5% Age (y) Percent asthmatic (%) 10.4% 41.6% 37.7% 70.4% Geometric mean IgE concentration (95% CI) ( ) ( ) (276.3, 453.5) (318.9, 448.2) P value (patients with asthma vs.001 < subjects without asthma) *Plus-minus values are means 6 SDs. Serum total IgE concentrations were expressed as geometric mean in ng/ml, adjusted for age and sex, centered around the predicted mean for 20-year-old men.

5 TABLE E2. Chromosome 3 location, MAF, and type of MYLK SNPs genotyped in 342 African Caribbean founders from Barbados and 165 African American founders from Baltimore Washington, DC* SNP Chromosome position (build 125) Inter-SNP distance (bp) Type Nucleotide/amino acid change Minor allele frequency (N) Caribbean founders American founders rs Intron G/C (288) (154) rs ,777 C.62 C/A, Pro>His (282) (146) rs Intron G/A (271) (151) rs Intron G/T (288) (156) rs ,871 Intron A/G (287) (150) rs ,892 C.439 T/C, Pro>Ser (207) (159) rs C.782 T/C, Val>Ala (287) 0.43 (158) rs Intron C/A (249) (150) rs Intron C/T (281) (154) rs ,511 Promoter, 23269à G/C (284) (152) rs Intron G/A (289) (153) ss IVS C/T (292) (156) rs Intron G/C (289) (156) rs Intron T/C (284) (153) rs ,928 Intron G/A (267) (142) rs ,108 Intron C/T (287) (151) rs ,532 C.4842 T/C (269) (153) rs ,203 Intron C/T (278) (158) ss IVS31172 C/G (281) (155) *SNPs were named relative to the representative mrna (Genbank accession U48959) of full-length MYLK (32 exons) in reference genomic sequence NT_ Since submission of the original report, E11 information on MYLK SNPs identified by sequencing was uploaded into National Center for Biotechnology Information dbsnp ( Marker rs was previously referred to as hcv , rs was MYLK_007, rs was hcv , rs was MYLK_002, rs was MYLK_003, ss was MYLK_025, and ss was MYLK_037. àcounted from the transcriptional initiation site of the smmylk form of the gene.

6 TABLE E3. FBAT in 2 independent population of African descent (Barbados and CSGA)* Marker Barbados CSGA Asthma log[tige] Asthma log[tige] rs G (0.144) (0.230) ( ) ( ) (45) (61) (22) (22) C (0.144) (0.230) ( ) ( ) (45) (61) (22) (22) rs C (0.095) (0.037) ( ) ( ) (86) (115) (40) (45) A (0.095) (0.037) ( ) ( ) (86) (115) (40) (45) rs G (0.529) (0.195) ( ) ( ) (30) (39) (19) (23) A (0.529) (0.195) ( ) ( ) (30) (39) (19) (23) rs G (0.789) (0.842) ( ) ( ) (72) (104) (44) (48) T (0.789) (0.842) ( ) ( ) (72) (104) (44) (48) rs A (0.344) (0.534) ( ) ( ) (32) (38) (14) (18) G (0.344) (0.534) ( ) ( ) (32) (38) (14) (18) rs T (0.771) (0.301) ( ) ( ) (65) (79) (43) (48) C (0.771) (0.301) ( ) ( ) (65) (79) (43) (48) rs T (0.899) (0.947) ( ) ( ) (94) (128) (48) (56) C (0.899) (0.947) ( ) ( ) (94) (128) (48) (56) rs C (0.808) (0.567) ( ) ( ) (47) (72) (29) (31) A (0.808) (0.567) ( ) ( ) (47) (72) (29) (31) rs C (0.829) (0.506) (1.000) ( ) (97) (131) (44) (53) T (0.829) (0.506) (1.000) ( ) (97) (131) (44) (53) rs G (0.0097) (0.052) ( ) ( ) (83) (119) (45) (54) C (0.0097) (0.052) ( ) ( ) (83) (119) (45) (54) rs G (0.528) (0.489) ( ) ( ) (52) (69) (22) (28) A (0.528) (0.489) ( ) ( ) (52) (69) (22) (28) ss C (0.974) (0.284) ( ) ( ) (73) (105) (46) (51) T (0.974) (0.284) ( ) ( ) (73) (105) (46) (51)

7 TABLE E3. Continued Barbados Marker Asthma log[tige] Asthma log[tige] rs G (0.829) (0.560) ( ) ( ) (55) (74) (28) (32) C (0.829) (0.560) ( ) ( ) (55) (74) (28) (32) rs T (0.331) (0.668) ( ) ( ) (92) (123) (39) (50) C (0.331) (0.668) ( ) ( ) (92) (123) (39) (50) rs G (0.716) (0.679) ( ) ( ) (61) (78) (33) (40) A (0.716) (0.679) ( ) ( ) (61) (78) (33) (40) rs C (0.806) (0.941) ( ) ( ) (56) (83) (26) (33) T (0.806) (0.941) ( ) ( ) (56) (83) (26) (33) rs T (0.848) (0.289) ( ) ( ) (77) (115) (42) (52) C (0.848) (0.289) ( ) ( ) (77) (115) (42) (52) rs C (0.512) (0.605) ( ) ( ) (77) (105) (52) (60) T (0.512) (0.605) ( ) ( ) (77) (105) (52) (60) ss C (0.425) (0.451) ( ) ( ) (58) (77) (34) (40) G (0.425) (0.451) ( ) ( ) (58) (77) (34) (40) *z Scores (P value) and allele frequencies (number of families) were tested for the 19 MYLK SNPs and asthma-associated phenotypes under an additive model (significant P values in boldface). CSGA

8 TABLE E4. Association testing results for 2-SNP to 4-SNP haplotype windowing of 19 MYLK SNPs in 3 populations of African descent for asthma, log[total IgE], severe sepsis, and ALI *Global P value generated by the Omnibus overall haplotype profile testing. Frequencies in combined case groups versus controls. àx 2 values generated by permutation in SNPem.

9 FIG E1. LD estimation for 19 variants in MYLK spanning 217 kb on chromosome 3. Pairwise LD estimated using D9 values in 342 African Caribbean founders in Barbados (right panel) and 165 African American founders (left panel) is represented as red squares for strong LD (LD 2, D9 51), pink squares for weak LD (LD 2, D9 < 1), blue squares for nonsignificant LD (LD < 2, D9 51), and white squares for little or no LD (LD < 2, D9 < 1).

10 FIG E2. Distributions of 2log 10 (P value) for single MYLK SNP or haplotypes from SNP windowing across the MYLK gene in the African American families. Results of 2-SNP to 4-SNP haplotype windowing for asthma (A) and log[total serum IgE concentrations] (log[tige]; B) are displayed. The y axis indicates the value of 2log 10 (P); the x axis indicates the relative position for each SNP marker locus at the MYLK gene region on human chromosome 3 in the 59 to 39 direction. The vertical line in black represents the position of each SNP marker on x axis and its height on y axis, indicating the value of 2log 10 (P). The horizontal lines in red represent the 2-SNP windowing, and the lines in blue represent the 3-SNP windowing.