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HLA-DRB1 class II alleles in Indian asthmatic population: A candidate gene-environment interaction
Meta Gene 14 (2017) 49–52
Contents lists available at ScienceDirect
Meta Gene journal homepage: www.elsevier.com/locate/mgene
HLA-DRB1 class II alleles in Indian asthmatic population: A candidate geneenvironment interaction
MARK
Tejas Shah1, Siddhesh Solanke1, Kalpana Joshi⁎ Department of Biotechnology, Sinhgad College of Engineering, Savitribai Phule Pune University, Vadgaon Budruk, Pune 411041, Maharashtra, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Asthma Human Leukocyte Antigen Class II Immunoglobulin E HLA-DRB1 Human major histocompatibility complex (MHC)
Introduction: Asthma is a complex disease with interplay of genes and environment. Genome wide association studies (GWAS) have shown the systematic association of Human leukocyte antigen (HLA) genes with asthma. The objective of present study was to determine the frequency of HLA DRB1 alleles in adult Indian asthmatic and control groups. Material and methods: Polymerase Chain Reaction- sequence-specific typing (PCR-SSP) method was used for HLA genotyping in 98 healthy participants and 72 asthmatics. Results: The results revealed that DRB1*03 (OR = 2.63, 95% CI = 1.32–5.25), DRB1*04 (OR = 4.52, 95% CI = 1.06–22.02) and DRB1*07 (OR = 3.79, 95% CI = 1.03–15.10) were positively associated with risk of asthma while DRB1*15 (OR = 0.509, 95% CI = 0.257–1.01) were negatively associated with risk of asthma in Indian population. Association of low (> 150 IU/ml) and high (< 150 IU/ml) total serum immunoglobulin E, tIgE levels and HLA-DRB1 alleles in asthmatics showed no significant association. Conclusion: Our results suggest that HLA-DRB1*03, DRB1*04 and DRB1*07 are involved with susceptibility of asthma, while DRB1*15 has protective effect in adult Indian asthmatics.
1. Introduction Asthma is a complex disease with both genetic and environmental risk factors (Bijanzadeh et al., 2017). Asthma is an important public health concern due to increasing morbidity and mortality rates and thereby, prevention and control of asthma have great implication. Genetic studies have gained more focus with growing evidence from GWAS and genome wide linkage studies. For instance, FOXO3a gene rs13217795 polymorphism was associated with hyperactivity of T cells, neutrophils, and mast cells in asthmatics (Barkund et al., 2015). The human leukocyte antigen (HLA) super locus is a genomic region in the chromosomal position 6p21 that encodes the 6 classical transplantation HLA genes and at least 132 protein coding genes which play vital role in immune regulation and basic molecular and cellular metabolism (Shiina et al., 2009; Chavan et al., 2017). Especially, human major histocompatibility complex (MHC) class II genes on chromosome 6 are accountable for the most of familial clustering in asthma and are extremely polymorphic and immensely influence allergen-specific IgE response (Kontakioti et al., 2014). DR loci in HLA region is associated with asthma. Recent study in Indian paediatric population showed association of HLA-DRB1*03 with
⁎
1
Corresponding author. E-mail address: [email protected] (K. Joshi). Authors contributed equally.
http://dx.doi.org/10.1016/j.mgene.2017.08.001 Received 13 June 2017; Received in revised form 28 July 2017; Accepted 3 August 2017 Available online 04 August 2017 2214-5400/ © 2017 Published by Elsevier B.V.
childhood asthma (Lama et al., 2014). Cho et al. explored association of DRB1*04 and DRB1*07 in adult Korean asthma patients (Cho et al., 2000). The results from recent meta-analysis showed DRB1*03 was positively associated with asthma. HLA-DRB1*03, DRB1*04, DRB1*09 and DRB1*15 were associated with paediatric asthma while frequency of DRB1*07 and DRB1*14 were significantly associated in Asian asthmatic population. Previous finding in Caucasians suggest that the DRB1*03 appeared to be independent of ancestral haplotype mediated linkage disequilibrium between MHC class I and II alleles, thereby strengthening the case for DRB1*03 as an independent locus for asthma susceptibility (Hanchard et al., 2010).Though above studies support association of HLA-DRB1 alleles and asthma, other study dispute it (Dzurilla and Vrlik, 2013; Rihs and Barbalho-Kro, 1997). On basis of the results from previous population oriented studies, our aim was to study HLA class II, DRB1 allele frequency in Indian adult asthmatics and healthy control to determine the role of various alleles in protection or susceptibility of asthma. Further we also assessed the association of serum IgE levels and HLA DRB1 alleles in asthmatic population.
Meta Gene 14 (2017) 49–52
T. Shah et al.
reaction sequence-specific primer (PCR-SSP) techniques using Micro SSP DNA Typing Trays DRB (One Lambda Inc. Canoga Park, California, USA) according to manufacturer's instruction. The PCR was performed by using the Eppendorf Mastercycler® nexus (Hamburg, Germany). Amplified DNA fragments were detected using 2% agarose gel electrophoresis at 120 V for 15 mins, stained with Ethidium bromide in UV transillumination system. One Lambda HLA Fusion software version 3.0 was used to detect specific HLA-DRB1 alleles.
Table 1 Characteristic of study participants.
n Age (years) Duration of Asthma FEV (% predicted)a FVC (% predicted)b
Healthy participants
Asthmatics
98 41.5 ± 7.2 NA 92.1 ± 4.3 69.8 ± 2.7
72 42.3 ± 9.3 6.3 ± 5.7 62.4 ± 8.1 96.2 ± 4.8
Values are expressed as mean standard deviation (SD). a Forced vital capacity. b Forced expiratory volume in 1 sec.
2.3. IgE assay Serum concentrations of total IgE was quantified using singleplex kit on magnetic bead platform, Bio-Plex Pro™ Human IgE Isotyping according to manufacturer's instruction (Bio-Rad laboratories, Inc. Hercules, CA, USA). For association of HLA-DRB1 alleles and IgE, available data of total serum IgE levels of forty sir asthmatics from our previous study was used for this purpose (Joshi et al., 2017).
2. Material and methods 2.1. Study population Informed consent was obtained from the healthy and asthmatics participants before recruitment. The study design and protocol were approved by the Institutional Ethics Committee (IAEC) of Tilak Ayurved Mahavidyalaya and Seth Tarachand Ramnath Charitable Ayurvedic Hospital, Pune, India (RSTH/RES/IEC/429/2011). The trial protocol was registered and is available on Clinical Trial Registry of India: CTRI (CTRI/2014/12/005322). A total of 98 healthy participants and 72 asthmatics were recruited. Venous blood was collected from subjects to extract DNA and plasma. Healthy participants were non-smokers and had no history of asthma, allergy, or other diseases. Demographic and clinical characteristics of recruited participants are summarized in Table 1. Bronchial asthma was diagnosed by a chest physician with the help of diagnostic criteria as per ICD-9CM classifications (American Medical Association, 2005). Spirometry was performed in accordance with 2005 ATS/ERS norms to measure FEV1 and FVC (Joshi et al., 2017).
2.4. Statistical analysis The frequencies of HLA-DRB1 alleles within groups were determined by direct counting. Statistical analysis of distribution of allele frequencies between healthy control and asthmatics was performed by GraphPad PRISM software v5.0 (San Diego, CA, USA). Categorical data were analysed using Fisher's exact test and the goodness of fit: χ2 test. Odds ratio and 95% confidence intervals were also calculated. p < 0.05 was considered significant.
3. Result 3.1. Association of Asthma and HLA-DRB1 alleles
2.2. DNA isolation and HLA genotyping
Thirteen class II HLA-DRB1 alleles were tested in relation to incidences in asthma using genomic DNA for each allele (Table 2). Of these 13 alleles, HLA-DRB1*03 was 50% in the asthmatics, compared with 27.55% in healthy controls and were most significantly associated with an increased risk of asthma (OR: 2.63, 95% CI: 1.32–5.26, p < 0.01) followed by DRB1*04 (12.5% vs 3.06%, OR: 4.524, 95% CI: 1.065–22.025, p < 0.05) and DRB1*07 (13.88% vs 4.08%, OR: 3.790, 95% CI: 1.03–15.10, p < 0.05). In contrast, the HLA-DRB1*15 was significantly decreased in asthmatics as compared to healthy controls (47.95% vs 31.94%, OR: 0.509, 95% CI: 0.257–1.007, p < 0.05). The other 9 HLA-DRB1 alleles of interest were not identified as significantly associated with asthma incidence.
Venous blood was collected from healthy and asthmatic participants in an EDTA coated BD vacutainers (Becton, Dickinson Company Franklin Lakes, NJ, USA). Genomic DNA from peripheral blood mononuclear cells was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instruction. The purity and yield (ng) of isolated DNA was determined using NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, USA). The DNA samples used for genotyping had a purity ratio (260/ 280) between 1.7 and 2. Low resolution HLA typing was performed by polymerase chain Table 2 Allelic and genotype frequencies of HLA-DRB1 gene in healthy and asthmatic participants. Alleles
DRB1*01 DRB1*03 DRB1*04 DRB1*07 DRB1*08 DRB1*09 DRB1*10 DRB1*11 DRB1*12 DRB1*13 DRB1*14 DRB1*15 DRB1*16
Healthy controls (n = 98)
Asthmatics (n = 72)
AF
GF (%)
AF
GF (%)
9 27 3 4 12 2 14 11 7 14 15 47 5
9.18 27.55 3.06 4.08 12.24 2.04 14.28 11.22 7.14 14.28 15.30 47.95 5.10
5 36 9 10 5 4 12 11 3 6 14 23 2
6.94 50 12.5 13.88 6.94 5.55 16.66 15.27 4.16 8.33 19.44 31.94 2.77
AF: allelic frequency GF: genotype frequency, OR: odds ratio, CI: confidence interval, p < 0.05: significant.
50
χ2
OR (95% CI)
p value
0.059 8.031 4.289 4.065 0.774 0.651 0.044 0.299 0.235 0.901 0.252 3.759 0.132
0.738[0.204–2.559] 2.630 [1.321–5.255] 4.524 [1.065–22.025] 3.790 [1.030–15.104] 0.535 [0.155–1.745] 2.82 [0.428–22.925] 1.200 [0.479–2.996] 1.426 [0.533–3.816] 0.56 [0.111–2.548] 0.545 [0.176–1.628] 1.336 [0.557–3.199] 0.509 [0.257–1.007] 0.531 [0.069–3.217]
0.779 < 0.01 < 0.05 < 0.05 0.308 0.403 0.673 0.492 0.521 0.336 0.538 < 0.05 0.700
Meta Gene 14 (2017) 49–52
T. Shah et al.
Kolarain, and the Mongoloid races. Participants in present study are residents of Western region of India which comprises of population from Australoid-Europoid origin (Ghodke et al., 2007; Karve, 1968). Hence it might be possible that both asthmatic populations: Asian as well as white share common protective allele i.e. HLA-DRB1*15. It is therefore possible that in different ethnic population different HLA antigens have role in predisposing and preventing disease. HLA class II antigens play an important role in displaying antigen for CD34+ T lymphocytes and hence regulate the specificity of the immune response. HLA-DR alleles have been involved in activating an allergen specific IgE response (Knapp et al., 2012). Analysis of HLA alleles in association with raised serum IgE showed that none of the HLA alleles was found to be associated with IgE in asthmatic groups. Previous study in Poland population exhibited significantly higher total serum IgE levels in asthmatics patients with HLA-DRB1*01, while a study in southern Chinese population showed no association between asthma and IgE levels (Woszczek et al., 2002; Wang et al., 1999). In this study, we did not found any association between asthmatics and IgE levels; probably due to relatively smaller number of subjects in IgE analysis in asthmatic group.
Table 3 HLA-DRB1 alleles and serum immunoglobulin-E (IgE) response. Alleles
IgE > 150 UI/ml n = 29 (%)
IgE < 150 UI/ml n = 17 (%)
DRB1*01 DRB1*03 DRB1*04 DRB1*07 DRB1*08 DRB1*09 DRB1*10 DRB1*11 DRB1*12 DRB1*13 DRB1*14 DRB1*15
1 (3.44) 3 (10.34) 2 (6.88) 4 (13.79) 2 (6.88) 2 (6.88) 3 (10.34) 2 (6.88) 2 (6.88) 3 (10.34) 2 (6.88) 3 (10.34)
2 (11.76) 1 (5.88) 2 (11.76) 3 (17.64) – – – 1 (5.88) 2 (11.76) 1 (5.88) 3 (17.64) 2 (11.76)
3.2. Association of HLA-DRB1 alleles and IgE levels in asthmatics Further analysis of the frequencies of HLA alleles in association with IgE in two asthmatic groups: IgE > 150 IU/ml (n = 29) and IgE < 150 IU/ml (n = 17) showed higher frequencies of DRB1*03 (10.34% vs 5.88%), DRB1*11 (6.88% vs 5.88%), DRB1*13 (10.34% vs 5.88%), and the lower frequencies of DRB1*01 (3.44% vs 11.76%), DRB1*04 (6.88% vs 11.76%), DRB1*07 (13.79% vs 17.64%), DRB1*12 (6.88% vs 11.76%), DRB1*14 (6.88% vs 17.64%) and DRB1*15 (10.34% vs 11.76%) respectively (Table 3). None of these showed the significance among the two asthmatic groups.
5. Conclusion The dissimilarity in allelic association with asthma in diverse population may be dependent on the ethnicity and environmental exposure. The present preliminary finding showed positive association of DRB1*03, DRB1*04 and DRB1*07 and a negative association with DRB1*15 allele in Indian asthmatics. A further study in large population based cohort of asthma patients is necessary to support the present findings.
4. Discussion
Conflict of interest
Asthma is a complex heterogeneous respiratory disease associated with genetic and environmental factor. Human major histocompatibility complex (MHC) is thought to play a vital role in regulation of inflammation on T helper cells. In last two decades many studies aimed on association between HLA-DRB1 alleles and asthma (Hanchard et al., 2010; Knutsen et al., 2010; Martyn et al., 2016; Smit et al., 2014; Muro et al., 2013). The result indicated that HLA-DRB1 alleles may lead to susceptibility or be protective in asthma patients. In this study we observed that the frequency of HLA-DRB1*03, DRB1*04 and DRB1*07 showed positive association with Indian asthmatics and results are in line with previous findings (Yao et al., 2016). A study conducted in Korean asthmatic adults, showed a significantly higher frequencies of HLA-DRB1*07 and HLA-DRB1*04 and results are in agreement with our findings (Cho et al., 2000). A study on Indian paediatric population showed higher frequency of HLA-DRB1*03 alleles (Lama et al., 2014). A recent meta-analysis on association of HLA-DRB1 gene polymorphism and risk of asthma indicated that individual carrying HLA-DRB1*03 have a 51% risk of asthma as compared to those who don't carry (Yao et al., 2016). In our study we found that HLA-DRB1*03 was significantly associated with risk of asthma and genotype frequency was found to be 50%. It has been reported that HLA-DRB1*03 allele play an important physiological role in determination of Th2 mediated inflammation (Rajagopalan et al., 2006). Our result suggest association of HLA-DRB1*03 and HLA-DRB1*04 in Indian asthmatics and results are in line with previous finding in Finland's allergic asthmatic population (Kauppinen et al., 2012). In this study, HLA-DRB1*15 was found to have protective factor for asthma. Previous studies have shown DRB1*15 has a protective effect for asthma (Jiawei and Gesheng, 2007; Mishra et al., 2014). A metaanalysis report showed DRB1*03 significantly increases the risk of asthma and DRB1*15 significantly decreases risk of asthma in whites, while DRB1*07 increases risk of asthma and DRB1*14 had protective role against asthma in Asians (Yao et al., 2016), our results coincide with previous findings though we did not find significance in DRB1*14 allele. Indian population is an inter-mixture of the Aryan, Dravidian,
The authors report that there is no conflict of interests. Acknowledgments Dr. Kalpana Joshi thanks the Office of PSA, Government of India (SA/ADV/Ayurveda/4/Asthma/2010), for financial support (Prn.SA/ ADV/Ayurveda/4/Asthma/2010). Technical assistance offered by Mr. Avinash and Miss. Gitanjali is appreciated. The authors thank Dr. Shilpa Rakh for her guidance in HLA typing and data analysis. Authors thanks Dr. Tanuja Nesari and her team of TAMV for subject recruitment. The authors would like to thank Professor M. S. Valiathan and Professor K. Satyamoorthy of MAHE, Manipal University, for their continuous guidance. References American Medical Association, 2005. International Classification of Diseases: Physician ICD-9-CM, Vol.1–2 of Revision 9. AMA Press, Chicago, Ill, USA. Barkund, S., Shah, T., Ambatkar, N., Gadgil, M., Joshi, K., 2015. FOXO3a gene polymorphism associated with asthma in Indian population. Molecular Biology International 2015. Bijanzadeh, M., Mahesh, P.A., Ramachandra, N.B., 2017. An understanding of the genetic basis of asthma. Indian J. Med. Res. 134, 149–161. Chavan, Y., Mitra, T., Pawar, S., Wadgaonkar, M., Bhagwat, A., Bangar, S., Purohit, P., et al., 2017. Rapid detection of HLA-B*5701 allele by in-house developed tetraprimer amplification refractory mutation system PCR. Meta Gene. 12, 150–153. Cho, S., Kim, Y., Jung, J., Son, J., Lee, M., Kim, Y., Min, K., 2000. Association of HLADRB1*07 and DRB1*04 to citrus red mite (Panonychus citri) and house dust mite sensitive asthma. Clin. Exp. Allergy 30, 1568–1575. Dzurilla, M., Vrlik, M., 2013. No association between bronchial asthma and HLA-DRB1, -DQB1 alleles in the Slovak population. Bratisl Lek List. 114, 93–95. Ghodke, Y., Joshi, K., Arya, Y., Radkar, A., Chiplunkar, A., Shintre, P., Patwardhan, B., 2007. Genetic polymorphism of CYP2C19 in Maharashtrian population. Eur. J. Epidemiol. 22, 907–915. Hanchard, N., Jacobson, R., Poland, G., Juhn, Y., 2010. An assessment of the association between childhood asthma and HLA-DRB1*03 using extended haplotype analysis. Tissue Antigens 9, 491–494. Jiawei, W., Gesheng, W., 2007. Immunogenetic analysis of leukocyte antigen DRB1 and
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asthma. Allergy and Asthma Proceedings. 31, 120–125. Mishra, M., Dudeja, P., Gupta, R., 2014. Association of HLA-class II and IgE serum levels in pediatric asthma. Iran J. Immunol. 11, 21–28. Muro, M., López, P., Rosa, M., Quiles, M., Salgado, G., Dolores, M., et al., 2013. HLA DRB1 and HLA – DQB1 genes on susceptibility to and protection from allergic bronchopulmonary aspergillosis in patients with cystic fibrosis. Microbiol. Immunol. 57, 193–197. Rajagopalan, G., Iijima, K., Singh, M., Kita, H., Patel, R., David, C., 2006. Intranasal exposure to bacterial superantigens induces airway inflammation in HLA class II transgenic mice. Infect. Immun. 74, 1284–1296. Rihs, H., Barbalho-Kro, T., 1997. No evidence for the influence of HLA class II in alleles in isocyanate-induced asthma. Am. J. Ind. Med. 527, 522–527. Shiina, T., Hosomichi, K., Inoko, H., Kulski, J.K., 2009. The HLA genomic loci map: expression, interaction, diversity and disease. J. Hum. Genet. 15–39. Smit, L., Strachan, D., Vermeulen, R., De Bakker, P., Demenais, F., Dumas, O., et al., 2014. Human leukocyte antigen class II variants and adult-onset asthma: does occupational allergen exposure play a role ? The European Respiratory Journal. 44, 1234–1242. Wang, C., Peng, Z., Li, J., Liu, Z., 1999. Comparative study of HLA-DRB1 allele in patients with chronic bronchitis and bronchial asthma. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 16, 371. Woszczek, G., Kowalski, M., Borowiec, M., 2002. Association of asthma and total IgE levels with human leucocyte antigen-DR in patients with grass allergy. Eur. Respir. J. 20, 79–85. Yao, Y., Zhu, L., Li, J., Jin, Y., He, L., 2016. Association of HLA-DRB1 gene polymorphism with risk of asthma: a meta-analysis. Med Sci Monit Basic Res. 22, 80–86.
DQB1 in childhood asthma. Zhejiang Pract Med. 12, 240–241. Joshi, K., Nesari, T., Dedge, A., Dhumal, V., Shengule, S., Gadgil, M., et al., 2017. Dosha phenotype specific Ayurveda intervention ameliorates asthma symptoms through cytokine modulations: results of whole system clinical trial. J. Ethnopharmacol. 197, 110–117. Karve, I., 1968. ‘Maharashtra-Land & it's People’ Maharashtra State Gazetteers General Series. Bombay Directorate of Government Printing, Stationary & publications, Maharashtra State. Kauppinen, A., Peräsaari, J., Taivainen, A., Kinnunen, T., Saarelainen, S., RytkönenNissinen, M., et al., 2012. Association of HLA class II alleles with sensitization to cow dander Bos d 2, an important occupational allergen. Immunobiology 217, 8–12. Knapp, B., Fischer, G., Van Hemelen, D., Fae, I., Maillere, B., Ebner, C., et al., 2012. Association of HLA-DRB1 with the allergic response to the major mugwort pollen allergen: molecular background. BMC Immunol. 13, 1–11. Knutsen, A., Vijay, H., Kariuki, B., Santiago, L., Graff, R., Wofford, J., Shah, M., 2010. Association of IL-4 RA single nucleotide polymorphisms, HLA-DR and HLA-DQ in children with Alternaria -sensitive moderate-severe asthma. Clinical and Molecular Allergy. 8, 1–9. Kontakioti, E., Domvri, K., Papakosta, D., Daniilidis, M., 2014. HLA and asthma phenotypes/endotypes: a review. Hum. Immunol. 75, 930–939. Lama, M., Chatterjee, M., Chaudhuri, T.K., 2014. A study of the association of childhood asthma with HLA alleles in the population of Siliguri, West Bengal, India. Tissue Antigens. 15, 316–320. Martyn, M., Molis, W., Jacobson, R., Poland, G., Weaver, A., Juhn, Y., 2016. Human leukocyte antigen type and progression from onset of symptoms to development of
52
Contents lists available at ScienceDirect
Meta Gene journal homepage: www.elsevier.com/locate/mgene
HLA-DRB1 class II alleles in Indian asthmatic population: A candidate geneenvironment interaction
MARK
Tejas Shah1, Siddhesh Solanke1, Kalpana Joshi⁎ Department of Biotechnology, Sinhgad College of Engineering, Savitribai Phule Pune University, Vadgaon Budruk, Pune 411041, Maharashtra, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Asthma Human Leukocyte Antigen Class II Immunoglobulin E HLA-DRB1 Human major histocompatibility complex (MHC)
Introduction: Asthma is a complex disease with interplay of genes and environment. Genome wide association studies (GWAS) have shown the systematic association of Human leukocyte antigen (HLA) genes with asthma. The objective of present study was to determine the frequency of HLA DRB1 alleles in adult Indian asthmatic and control groups. Material and methods: Polymerase Chain Reaction- sequence-specific typing (PCR-SSP) method was used for HLA genotyping in 98 healthy participants and 72 asthmatics. Results: The results revealed that DRB1*03 (OR = 2.63, 95% CI = 1.32–5.25), DRB1*04 (OR = 4.52, 95% CI = 1.06–22.02) and DRB1*07 (OR = 3.79, 95% CI = 1.03–15.10) were positively associated with risk of asthma while DRB1*15 (OR = 0.509, 95% CI = 0.257–1.01) were negatively associated with risk of asthma in Indian population. Association of low (> 150 IU/ml) and high (< 150 IU/ml) total serum immunoglobulin E, tIgE levels and HLA-DRB1 alleles in asthmatics showed no significant association. Conclusion: Our results suggest that HLA-DRB1*03, DRB1*04 and DRB1*07 are involved with susceptibility of asthma, while DRB1*15 has protective effect in adult Indian asthmatics.
1. Introduction Asthma is a complex disease with both genetic and environmental risk factors (Bijanzadeh et al., 2017). Asthma is an important public health concern due to increasing morbidity and mortality rates and thereby, prevention and control of asthma have great implication. Genetic studies have gained more focus with growing evidence from GWAS and genome wide linkage studies. For instance, FOXO3a gene rs13217795 polymorphism was associated with hyperactivity of T cells, neutrophils, and mast cells in asthmatics (Barkund et al., 2015). The human leukocyte antigen (HLA) super locus is a genomic region in the chromosomal position 6p21 that encodes the 6 classical transplantation HLA genes and at least 132 protein coding genes which play vital role in immune regulation and basic molecular and cellular metabolism (Shiina et al., 2009; Chavan et al., 2017). Especially, human major histocompatibility complex (MHC) class II genes on chromosome 6 are accountable for the most of familial clustering in asthma and are extremely polymorphic and immensely influence allergen-specific IgE response (Kontakioti et al., 2014). DR loci in HLA region is associated with asthma. Recent study in Indian paediatric population showed association of HLA-DRB1*03 with
⁎
1
Corresponding author. E-mail address: [email protected] (K. Joshi). Authors contributed equally.
http://dx.doi.org/10.1016/j.mgene.2017.08.001 Received 13 June 2017; Received in revised form 28 July 2017; Accepted 3 August 2017 Available online 04 August 2017 2214-5400/ © 2017 Published by Elsevier B.V.
childhood asthma (Lama et al., 2014). Cho et al. explored association of DRB1*04 and DRB1*07 in adult Korean asthma patients (Cho et al., 2000). The results from recent meta-analysis showed DRB1*03 was positively associated with asthma. HLA-DRB1*03, DRB1*04, DRB1*09 and DRB1*15 were associated with paediatric asthma while frequency of DRB1*07 and DRB1*14 were significantly associated in Asian asthmatic population. Previous finding in Caucasians suggest that the DRB1*03 appeared to be independent of ancestral haplotype mediated linkage disequilibrium between MHC class I and II alleles, thereby strengthening the case for DRB1*03 as an independent locus for asthma susceptibility (Hanchard et al., 2010).Though above studies support association of HLA-DRB1 alleles and asthma, other study dispute it (Dzurilla and Vrlik, 2013; Rihs and Barbalho-Kro, 1997). On basis of the results from previous population oriented studies, our aim was to study HLA class II, DRB1 allele frequency in Indian adult asthmatics and healthy control to determine the role of various alleles in protection or susceptibility of asthma. Further we also assessed the association of serum IgE levels and HLA DRB1 alleles in asthmatic population.
Meta Gene 14 (2017) 49–52
T. Shah et al.
reaction sequence-specific primer (PCR-SSP) techniques using Micro SSP DNA Typing Trays DRB (One Lambda Inc. Canoga Park, California, USA) according to manufacturer's instruction. The PCR was performed by using the Eppendorf Mastercycler® nexus (Hamburg, Germany). Amplified DNA fragments were detected using 2% agarose gel electrophoresis at 120 V for 15 mins, stained with Ethidium bromide in UV transillumination system. One Lambda HLA Fusion software version 3.0 was used to detect specific HLA-DRB1 alleles.
Table 1 Characteristic of study participants.
n Age (years) Duration of Asthma FEV (% predicted)a FVC (% predicted)b
Healthy participants
Asthmatics
98 41.5 ± 7.2 NA 92.1 ± 4.3 69.8 ± 2.7
72 42.3 ± 9.3 6.3 ± 5.7 62.4 ± 8.1 96.2 ± 4.8
Values are expressed as mean standard deviation (SD). a Forced vital capacity. b Forced expiratory volume in 1 sec.
2.3. IgE assay Serum concentrations of total IgE was quantified using singleplex kit on magnetic bead platform, Bio-Plex Pro™ Human IgE Isotyping according to manufacturer's instruction (Bio-Rad laboratories, Inc. Hercules, CA, USA). For association of HLA-DRB1 alleles and IgE, available data of total serum IgE levels of forty sir asthmatics from our previous study was used for this purpose (Joshi et al., 2017).
2. Material and methods 2.1. Study population Informed consent was obtained from the healthy and asthmatics participants before recruitment. The study design and protocol were approved by the Institutional Ethics Committee (IAEC) of Tilak Ayurved Mahavidyalaya and Seth Tarachand Ramnath Charitable Ayurvedic Hospital, Pune, India (RSTH/RES/IEC/429/2011). The trial protocol was registered and is available on Clinical Trial Registry of India: CTRI (CTRI/2014/12/005322). A total of 98 healthy participants and 72 asthmatics were recruited. Venous blood was collected from subjects to extract DNA and plasma. Healthy participants were non-smokers and had no history of asthma, allergy, or other diseases. Demographic and clinical characteristics of recruited participants are summarized in Table 1. Bronchial asthma was diagnosed by a chest physician with the help of diagnostic criteria as per ICD-9CM classifications (American Medical Association, 2005). Spirometry was performed in accordance with 2005 ATS/ERS norms to measure FEV1 and FVC (Joshi et al., 2017).
2.4. Statistical analysis The frequencies of HLA-DRB1 alleles within groups were determined by direct counting. Statistical analysis of distribution of allele frequencies between healthy control and asthmatics was performed by GraphPad PRISM software v5.0 (San Diego, CA, USA). Categorical data were analysed using Fisher's exact test and the goodness of fit: χ2 test. Odds ratio and 95% confidence intervals were also calculated. p < 0.05 was considered significant.
3. Result 3.1. Association of Asthma and HLA-DRB1 alleles
2.2. DNA isolation and HLA genotyping
Thirteen class II HLA-DRB1 alleles were tested in relation to incidences in asthma using genomic DNA for each allele (Table 2). Of these 13 alleles, HLA-DRB1*03 was 50% in the asthmatics, compared with 27.55% in healthy controls and were most significantly associated with an increased risk of asthma (OR: 2.63, 95% CI: 1.32–5.26, p < 0.01) followed by DRB1*04 (12.5% vs 3.06%, OR: 4.524, 95% CI: 1.065–22.025, p < 0.05) and DRB1*07 (13.88% vs 4.08%, OR: 3.790, 95% CI: 1.03–15.10, p < 0.05). In contrast, the HLA-DRB1*15 was significantly decreased in asthmatics as compared to healthy controls (47.95% vs 31.94%, OR: 0.509, 95% CI: 0.257–1.007, p < 0.05). The other 9 HLA-DRB1 alleles of interest were not identified as significantly associated with asthma incidence.
Venous blood was collected from healthy and asthmatic participants in an EDTA coated BD vacutainers (Becton, Dickinson Company Franklin Lakes, NJ, USA). Genomic DNA from peripheral blood mononuclear cells was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instruction. The purity and yield (ng) of isolated DNA was determined using NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, USA). The DNA samples used for genotyping had a purity ratio (260/ 280) between 1.7 and 2. Low resolution HLA typing was performed by polymerase chain Table 2 Allelic and genotype frequencies of HLA-DRB1 gene in healthy and asthmatic participants. Alleles
DRB1*01 DRB1*03 DRB1*04 DRB1*07 DRB1*08 DRB1*09 DRB1*10 DRB1*11 DRB1*12 DRB1*13 DRB1*14 DRB1*15 DRB1*16
Healthy controls (n = 98)
Asthmatics (n = 72)
AF
GF (%)
AF
GF (%)
9 27 3 4 12 2 14 11 7 14 15 47 5
9.18 27.55 3.06 4.08 12.24 2.04 14.28 11.22 7.14 14.28 15.30 47.95 5.10
5 36 9 10 5 4 12 11 3 6 14 23 2
6.94 50 12.5 13.88 6.94 5.55 16.66 15.27 4.16 8.33 19.44 31.94 2.77
AF: allelic frequency GF: genotype frequency, OR: odds ratio, CI: confidence interval, p < 0.05: significant.
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χ2
OR (95% CI)
p value
0.059 8.031 4.289 4.065 0.774 0.651 0.044 0.299 0.235 0.901 0.252 3.759 0.132
0.738[0.204–2.559] 2.630 [1.321–5.255] 4.524 [1.065–22.025] 3.790 [1.030–15.104] 0.535 [0.155–1.745] 2.82 [0.428–22.925] 1.200 [0.479–2.996] 1.426 [0.533–3.816] 0.56 [0.111–2.548] 0.545 [0.176–1.628] 1.336 [0.557–3.199] 0.509 [0.257–1.007] 0.531 [0.069–3.217]
0.779 < 0.01 < 0.05 < 0.05 0.308 0.403 0.673 0.492 0.521 0.336 0.538 < 0.05 0.700
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Kolarain, and the Mongoloid races. Participants in present study are residents of Western region of India which comprises of population from Australoid-Europoid origin (Ghodke et al., 2007; Karve, 1968). Hence it might be possible that both asthmatic populations: Asian as well as white share common protective allele i.e. HLA-DRB1*15. It is therefore possible that in different ethnic population different HLA antigens have role in predisposing and preventing disease. HLA class II antigens play an important role in displaying antigen for CD34+ T lymphocytes and hence regulate the specificity of the immune response. HLA-DR alleles have been involved in activating an allergen specific IgE response (Knapp et al., 2012). Analysis of HLA alleles in association with raised serum IgE showed that none of the HLA alleles was found to be associated with IgE in asthmatic groups. Previous study in Poland population exhibited significantly higher total serum IgE levels in asthmatics patients with HLA-DRB1*01, while a study in southern Chinese population showed no association between asthma and IgE levels (Woszczek et al., 2002; Wang et al., 1999). In this study, we did not found any association between asthmatics and IgE levels; probably due to relatively smaller number of subjects in IgE analysis in asthmatic group.
Table 3 HLA-DRB1 alleles and serum immunoglobulin-E (IgE) response. Alleles
IgE > 150 UI/ml n = 29 (%)
IgE < 150 UI/ml n = 17 (%)
DRB1*01 DRB1*03 DRB1*04 DRB1*07 DRB1*08 DRB1*09 DRB1*10 DRB1*11 DRB1*12 DRB1*13 DRB1*14 DRB1*15
1 (3.44) 3 (10.34) 2 (6.88) 4 (13.79) 2 (6.88) 2 (6.88) 3 (10.34) 2 (6.88) 2 (6.88) 3 (10.34) 2 (6.88) 3 (10.34)
2 (11.76) 1 (5.88) 2 (11.76) 3 (17.64) – – – 1 (5.88) 2 (11.76) 1 (5.88) 3 (17.64) 2 (11.76)
3.2. Association of HLA-DRB1 alleles and IgE levels in asthmatics Further analysis of the frequencies of HLA alleles in association with IgE in two asthmatic groups: IgE > 150 IU/ml (n = 29) and IgE < 150 IU/ml (n = 17) showed higher frequencies of DRB1*03 (10.34% vs 5.88%), DRB1*11 (6.88% vs 5.88%), DRB1*13 (10.34% vs 5.88%), and the lower frequencies of DRB1*01 (3.44% vs 11.76%), DRB1*04 (6.88% vs 11.76%), DRB1*07 (13.79% vs 17.64%), DRB1*12 (6.88% vs 11.76%), DRB1*14 (6.88% vs 17.64%) and DRB1*15 (10.34% vs 11.76%) respectively (Table 3). None of these showed the significance among the two asthmatic groups.
5. Conclusion The dissimilarity in allelic association with asthma in diverse population may be dependent on the ethnicity and environmental exposure. The present preliminary finding showed positive association of DRB1*03, DRB1*04 and DRB1*07 and a negative association with DRB1*15 allele in Indian asthmatics. A further study in large population based cohort of asthma patients is necessary to support the present findings.
4. Discussion
Conflict of interest
Asthma is a complex heterogeneous respiratory disease associated with genetic and environmental factor. Human major histocompatibility complex (MHC) is thought to play a vital role in regulation of inflammation on T helper cells. In last two decades many studies aimed on association between HLA-DRB1 alleles and asthma (Hanchard et al., 2010; Knutsen et al., 2010; Martyn et al., 2016; Smit et al., 2014; Muro et al., 2013). The result indicated that HLA-DRB1 alleles may lead to susceptibility or be protective in asthma patients. In this study we observed that the frequency of HLA-DRB1*03, DRB1*04 and DRB1*07 showed positive association with Indian asthmatics and results are in line with previous findings (Yao et al., 2016). A study conducted in Korean asthmatic adults, showed a significantly higher frequencies of HLA-DRB1*07 and HLA-DRB1*04 and results are in agreement with our findings (Cho et al., 2000). A study on Indian paediatric population showed higher frequency of HLA-DRB1*03 alleles (Lama et al., 2014). A recent meta-analysis on association of HLA-DRB1 gene polymorphism and risk of asthma indicated that individual carrying HLA-DRB1*03 have a 51% risk of asthma as compared to those who don't carry (Yao et al., 2016). In our study we found that HLA-DRB1*03 was significantly associated with risk of asthma and genotype frequency was found to be 50%. It has been reported that HLA-DRB1*03 allele play an important physiological role in determination of Th2 mediated inflammation (Rajagopalan et al., 2006). Our result suggest association of HLA-DRB1*03 and HLA-DRB1*04 in Indian asthmatics and results are in line with previous finding in Finland's allergic asthmatic population (Kauppinen et al., 2012). In this study, HLA-DRB1*15 was found to have protective factor for asthma. Previous studies have shown DRB1*15 has a protective effect for asthma (Jiawei and Gesheng, 2007; Mishra et al., 2014). A metaanalysis report showed DRB1*03 significantly increases the risk of asthma and DRB1*15 significantly decreases risk of asthma in whites, while DRB1*07 increases risk of asthma and DRB1*14 had protective role against asthma in Asians (Yao et al., 2016), our results coincide with previous findings though we did not find significance in DRB1*14 allele. Indian population is an inter-mixture of the Aryan, Dravidian,
The authors report that there is no conflict of interests. Acknowledgments Dr. Kalpana Joshi thanks the Office of PSA, Government of India (SA/ADV/Ayurveda/4/Asthma/2010), for financial support (Prn.SA/ ADV/Ayurveda/4/Asthma/2010). Technical assistance offered by Mr. Avinash and Miss. Gitanjali is appreciated. The authors thank Dr. Shilpa Rakh for her guidance in HLA typing and data analysis. Authors thanks Dr. Tanuja Nesari and her team of TAMV for subject recruitment. The authors would like to thank Professor M. S. Valiathan and Professor K. Satyamoorthy of MAHE, Manipal University, for their continuous guidance. References American Medical Association, 2005. International Classification of Diseases: Physician ICD-9-CM, Vol.1–2 of Revision 9. AMA Press, Chicago, Ill, USA. Barkund, S., Shah, T., Ambatkar, N., Gadgil, M., Joshi, K., 2015. FOXO3a gene polymorphism associated with asthma in Indian population. Molecular Biology International 2015. Bijanzadeh, M., Mahesh, P.A., Ramachandra, N.B., 2017. An understanding of the genetic basis of asthma. Indian J. Med. Res. 134, 149–161. Chavan, Y., Mitra, T., Pawar, S., Wadgaonkar, M., Bhagwat, A., Bangar, S., Purohit, P., et al., 2017. Rapid detection of HLA-B*5701 allele by in-house developed tetraprimer amplification refractory mutation system PCR. Meta Gene. 12, 150–153. Cho, S., Kim, Y., Jung, J., Son, J., Lee, M., Kim, Y., Min, K., 2000. Association of HLADRB1*07 and DRB1*04 to citrus red mite (Panonychus citri) and house dust mite sensitive asthma. Clin. Exp. Allergy 30, 1568–1575. Dzurilla, M., Vrlik, M., 2013. No association between bronchial asthma and HLA-DRB1, -DQB1 alleles in the Slovak population. Bratisl Lek List. 114, 93–95. Ghodke, Y., Joshi, K., Arya, Y., Radkar, A., Chiplunkar, A., Shintre, P., Patwardhan, B., 2007. Genetic polymorphism of CYP2C19 in Maharashtrian population. Eur. J. Epidemiol. 22, 907–915. Hanchard, N., Jacobson, R., Poland, G., Juhn, Y., 2010. An assessment of the association between childhood asthma and HLA-DRB1*03 using extended haplotype analysis. Tissue Antigens 9, 491–494. Jiawei, W., Gesheng, W., 2007. Immunogenetic analysis of leukocyte antigen DRB1 and
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