A pilot study of Vitamin D Receptor TaqI and ApaI Gene Variants in adult asthma Katrina Hutchinson MD Senior Clinical Biochemist at Biomnis Ireland School of Chemistry, National University of Ireland, Galway 19/06/2017

Table of Content Introduction Objectives and Methods Results 1 Introduction 2 Objectives and Methods 3 Results 4 Conclusions and Future Studies 5 Funding and Research Collaboration

Introduction Both vitamin D deficiency and asthma are frequent in Ireland Ireland has 4th highest prevalence of asthma worldwide (470,000 people) Vitamin D receptor (VDR) gene single nucleotide polymorphisms (SNPs) have been associated with asthma risk and allergy susceptibility

VDR gene and VDR polymorphisms associated with Asthma As the common polymorphic sites in VDR, ApaI and TaqI sites have been shown to influence gene transcription and mRNA stability.

Objectives To determine the VDR gene variants TaqI in exon 9 (T/C) (rs731236) and ApaI(C/T) (rs7975232) in intron 8 in adult asthmatics, and also in healthy volunteers in Ireland (using TaqMan® Assays) To investigate the impact of those polymorphisms that differed significantly between patients and control in asthma susceptibility and also in relation to vitamin D(25OHD) status, IgE and other biochemical and immunological indices To examine the possibility of using these polymorphisms as potential biomarkers for asthma  

Demographics of the subjects who completed the study   Asthma (n = 14) Control (n = 56) p value Sex 7 male ( 50%) 34 male Age 36 (29, 43) 46 (43, 49) 0.008* BMI kg/m2 28 (25, 31) 25 (24, 26) 0.035* 25OHD nmol/L 52 (35, 70) 54 (47, 62) 0.48 FEV1 % predicted 94.8 (80.8, 100.8) 109 (103.5, 114.5) 0.001* IL-10 pg/mL 31 ( 14, 49) 125 ( 102, 148) 0.0001* WCC x109/L 6.2 (5.6, 6.8) 6.5 ( 6.2, 6.8) 0.5 Neutrophils x109/L 3.3 (2.8, 3.8) 3.6 (3.3, 3.9) 0.41 Eosinophils x109/L 0.2 ( 0.15, 0.25) 0.18 ( 0.15, 0.21) 0.46 hs-CRP mg/L 2.3 (0.6, 4.0) 4.3 (0, 9) 0.59 IgE IU/ml 192 (66, 318) 81 (40, 122) 0.027*   Results are expressed as average (95% confidence intervals)

Genotypic association analysis of VDR RFLPs (restriction fragment length polymorphisms) Taq1 (T>C) and Apa1(C>T) between asthmatics and control Enzyme analysis Patients (14) Controls (56) χ 2 P value TaqI Genotypes   TT 3 (21.4 %) 34 (60.7 %) CT 9 (64.3%) 22 (39.3 %) CC 2 (14.3%) 0 (0 %) 12.85 0.002* ApaI Genotypes    6 (43 %) 5 (9 %) 14.9 0.001* 7 (50 %) 19 (34 %) 1 (7 %) 32 (57 %) Data are given as numbers and percentages. *p<0.05 is considered significant.

Allelic association analysis of VDR SNPs Taq1 (T>C) and Apa1(C>T) between asthmatics and control Enzyme analysis Patients (14) Controls (56) OR( 95 % CI) P value TaqI Allelic association   T 15 (54 %) 90 (80.4 %) C 13 (46 %) 22 (19.6 %) 3.55(1.48-8.52) 0.006* ApaI Allelic association 19 (68 %) 29 (26 %) 6.04 (2.5 -14.85) <0.0001* 9 (32 %) 83 (74 %) Data are given as numbers and percentages. OR = odds ratio; 95% CI (in parentheses) * Denotes statistically significant  

Haplotype frequencies estimation Haplotypes frequencies for VDR TaqI and ApaI polymorphisms for asthmatics (cases) and Control Haplotype frequencies estimation TaqI (SNP1) ApaI (SNP2) Total Control (freq.) Healthy adults (n=56) Cases (freq.) adults asthma (n=14) T C 0.61 0.68 0.32 0.20 0.14 0.46 0.12 0.22 0.05 0.06

Haplotype association with response (n=70) adults Haplotypes association with response (asthma) for VDR TaqI and ApaI polymorphisms Haplotype association with response (n=70) adults TaqI (SNP1) ApaI (SNP2) Frequency OR (95% CI) P-value T C 0.6082 1.00 --- 0.201 9.38 (2.39 - 36.86) 0.0021* 0.1418 2.56 (0.69 - 9.51) 0.16 0.049 0.00 1 Global haplotype association p-value: 0.0005* n = total number studied. * Denotes statistically significant. The haplotype analysis performed using the programme from the website: https://www.snpstats.net/snpstats/start.htm

Average FEV1% levels in asthmatics and healthy control with TC genotype for TaqI (a) and ApaI (b) polymorphisms t-test: TC genotypes between asthmatics and control , p < 0.05*

Average IgE levels in asthmatic adults with TC+CC genotypes vs TT for ApaI polymorphism t-test: TC+CC vs TT in asthmatics , p < 0.012*

Conclusions and Further Studies Our preliminary data are promising, and they are in line with our study carried out on paediatric asthmatics Our data suggest that TaqI and ApaI polymorphisms are more common in asthmatics It is possible that the ApaI polymorphism is associated with the atopic asthma phenotype in adults Further research is warranted to confirm our findings on the importance of VDR gene SNPs in asthma, to investigate the mechanisms by which they may influence the development and course of the disease, and also the possibility of using them as biomarkers for asthma susceptibility

Funding and Research Collaboration Research grant from IRC Abbott Diagnostics Thermo Fisher Scientific National Children’s Hospital Foundation Irish Thoracic Society Asthma Society of Ireland Irish Lung Foundation Dr. John Faul, Dr. Liam Cormican and Dr. Conor Kerley, The Asthma Research Centre at James Connolly Hospital and UCD Dr. Yury Rochev , School of Chemistry , NUI Galway, Ireland Dr. Michael Louw and Prof. Con Feighery , Biomnis Ireland The funding bodies had no involvement in study design, data collection, analysis or interpretation.