Replication of an association between 17q21 SNPs and asthma in a French-Canadian familial collection

Anne-Marie Madore, Karine Tremblay, Thomas J. Hudson, Catherine Laprise

ONLINE SUPPLEMENT


METHODS

Samples: The sample comprises 253 probands for a total of 1,275 subjects. This sample and all information concerning recruitment and clinical evaluation of the subjects were described in a recent report (Begin et al. 2007). Briefly, asthma phenotype was characterized by the investigators for all participants using a respiratory health questionnaire and function tests following American Thoracic Society standards (American Thoracic Society, 1987). Participants were defined as having asthma if (1) they had a reported history of asthma (validated by a physician), or (2) they showed asthma-related symptoms and a positive PC20 (provocative concentration causing a 20% drop in FEV1 [PC20] < 8 mg/ml (American Thoracic Society, 1987)) at the time of recruitment. Subjects with a PC20 greater than 8mg/ml; without history of physician-diagnosed asthma, and without both symptoms of asthma, and a PC20 greater than 8 mg/ml, were considered unaffected. See Table E1 for the clinical characteristics of the subjects.

Genotyping: Blood samples were drawn from all participants and DNA was extracted from blood leukocytes using Genomic-tip 100/G kit (Qiagen, Inc., Valencia, CA) according to manufacturer’s instructions. Genotyping of the rs11557467, rs2290400, and rs7216389 SNPs was performed with TaqManâ SNP Genotyping Assays (Applied Biosystems, Foster City, CA) using the Rotor-Gene 3000 (Corbett Research, Sydney, Australia) with 10 ng of genomic DNA, 2.15 μl of 2X TaqMan® Genotyping Master Mix and 0.11 μl of 20X TaqMan® SNP Genotyping Assay (C_9272244_10 , C_15884536_10 and C_29062108_10 respectively (Applied Biosystems, Foster City, CA)) for a final volume of 4.3 μl with the following cycling conditions: 95°C 10 min, 40 cycles of 92°C 15 sec and 58°C 1 min. The Rotor-Gene software (version 6.0) was used to determine genotypes using the scatter graph analysis option.

Genotyping for the seven remaining SNPs (rs2305479, rs4378650, rs6503525, rs7219923, rs8069176, rs8076131, rs9303281) was done using the Sequenom® matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass array spectrometer (Sequenom Inc., San Diego, CA, USA). Primers were designed using the Sequenom SNP Assay Design software version 3.0 for iPLEX reactions. The protocol and reaction conditions are in accordance with the manufacturer (Oeth P et al. 2005). The genotypes were viewed and analyzed using the MassARRAY Typer software version 3.4 (Sequenom).


REFERENCES

American Thoracic Society (1987). Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am Rev Respir Dis 136(1): 225-44.

Begin P, Tremblay K, Daley D, Lemire M, Claveau S, Salesse C, Kacel S, Montpetit A, Becker A, Chan-Yeung M, Kozyrskyj AL, Hudson TJ and Laprise C (2007). Association of urokinase-type plasminogen activator with asthma and atopy. Am J Respir Crit Care Med 175(11): 1109-16.

Oeth P, Beaulieu M, Park C, Kosman D, del Mistro G, van den Boom D and Jurinke C. (2005). iPLEX assay: Increased plexing efficiency and flexibility for MassArray system through single base primer extension with mass-modified terminators. SEQUENOM Application Note.


LEGENDS TO FIGURE

Figure E1: Pairwise linkage disequilibrium pattern of the nine 17q21 locus single nucleotide polymorphisms (SNPs).

The location of each tested SNP along the chromosome is indicated on top. The number in each diamond indicates the magnitude of linkage disequilibrium (D’) between respective pairs of SNPs (e.g., the pairwise magnitude of linkage disequilibrium for variants rs9303281 and rs7219923 is 0.99). Diamonds without numbers represent perfect linkage disequilibrium (D’=1.0).


Table E1

Clinical characteristics of the French-Canadian familial collection subjects

Families
Probands / Affected relatives / Unaffected relatives
n = 253 / n = 379 / n = 643
Male : Female ratio / 1 : 1.12 / 1 : 1.27 / 1 : 1.10
Mean age, yr (range) / 17 (2-50) / 39 (2-83) / 46 (3-93)
Median age, yr / 15 / 40 / 46
Smoking status, n (%)
Never / 212 (85) / 192 (51) / 272 (43)
Ex-smoker / 12 (5) / 112 (30) / 234 (37)
Smoker / 25 (10) / 71 (19) / 128 (20)
FEV1 *, % predicted (SD) / 92.7 (16.0) / 88.9 (22.4) / 99.1 (17.2)
PC20†, mg/ml (SD) / 2.50 (3.63) / 3.35 (4.43) / 21.36 (3.215)

Abbreviation used: FEV1 = Forced expiratory volume in one second, PC20 = Provocative concentration of methacholine that induces a 20% fall in FEV1, SD = Standard deviation.

*Geometric mean calculated for 218 probands, 325 affected relatives and 440 unaffected relatives

†Geometric mean of the log-transformed PC20 calculated for 193 probands, 285 affected relatives and 418 unaffected relatives


Figure E1