1. 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

2. Table of Content Introduction Objectives and Methods Results1
Introduction 2
Objectives and Methods 3
Results 4
Conclusions and Future Studies 5
Funding and Research Collaboration

3. 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

4. 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.

5. Objectives
To determine the VDR gene variants TaqI in exon 9 (T/C) (rs731236) and ApaI(C/T) (rs ) 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

6. 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)

7. 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.

8. 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( )
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

9. 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

10. 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 ( )
0.0021*
0.1418
2.56 ( )
0.16
0.049
0.00 1
Global haplotype association p-value: *
n = total number studied. * Denotes statistically significant.
The haplotype analysis performed using the programme from the website:

11. 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*

12. 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*

13. 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

14. 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.