Journal of the Neurological Sciences 352 (2015) 37–40
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
HLA-DRB1 does not have a role in clinical response to interferon-beta among Iranian multiple sclerosis patients Sara Samadzadeh a,b, Elnaz Tabibian b, Tayebeh Sabokbar c,d, Abbas Shakoori d, Shahram Rahimi Dehgolan b, Saeed Azad Armaki a, Bahram Aslanbeigi a, Roya Abolfazli a,b,⁎ a
Shefa Neurosciences Research Center, Tehran, Islamic Republic of Iran Tehran University of Medical Sciences, Neurology Department, Tehran, Islamic Republic of Iran Neurology & Neurosciences Research Center, Qom University of Medical Sciences, Qom, Islamic Republic of Iran d Tehran University of Medical Sciences, Department of Medical Genetics, Cancer Institute, Tehran, Islamic Republic of Iran b c
a r t i c l e
i n f o
Article history: Received 4 October 2014 Received in revised form 9 February 2015 Accepted 2 March 2015 Available online 9 March 2015 Keywords: Multiple sclerosis HLA-DR beta-Chains Interferon-beta Frequencies in responders and non-responders Iran
a b s t r a c t Background & objectives: The role of human leukocyte antigen (HLA) in clinical response to immunotherapy is not completely known. In this study we evaluated the relationship between HLA-DRB1 genotype, which has been proved to be more common in Iranian MS patients, and clinical response to interferon-beta (IFNβ), which is the most common immunotherapy for relapsing–remitting MS. Design and setting: In this study 68 Iranian patients with confirmed diagnosis of RRMS who had been referred to and admitted in Neurology Department of Amiralam and Khatam Hospitals in Tehran were selected. Patients were followed prospectively for 2 years since initiation of therapy and clinical data, including EDSS scores were recorded every 3 months. MRI was performed at the time of diagnosis and each year. Methods: HLA-DRB1 typing was performed by polymerase chain reaction (PCR) for all patients and data was analyzed by STATA 12th edition. Results: There were 47 (69.1%) responders and 21 (30.9%) non-responders. These two groups were demographically and clinically comparable. Fisher's exact test did not show any difference between HLA-DRB1 allele frequencies in responders and non-responders. Conclusions: Our findings confirmed the lack of association between HLA-DRB1 and clinical response to IFNβ among MS patients as previous studies had done. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The etiology of MS is still unclear but according to current data, the disease develops in genetically susceptible individuals due to the complex interaction of a number of genes. However, MS development may require additional triggers, such as hormonal variables and environmental factors including infectious agents and emotional stress [1–3]. Interferon-beta (IFNβ) is the most common therapy for relapsing– remitting multiple sclerosis (RRMS) and also has a beneficial effect on disease activity. In fact it reduces the attack rate and probably suppresses the sustained progression of disability [4,5]. But near to thirty percent of patients do not completely respond to this immunotherapy. This group should be considered as non-responders. There are many criteria for defining and scoring response to IFNβ in RRMS patients based on their different characteristics including the number of relapses ⁎ Corresponding author at: Shefa Neurosciences Research Center, Tehran, Islamic Republic of Iran. Tel.: +98 9121070603; fax: +98 21 66704805. E-mail address: [email protected] (R. Abolfazli).
http://dx.doi.org/10.1016/j.jns.2015.03.004 0022-510X/© 2015 Elsevier B.V. All rights reserved.
per year, increase in Expanded Disability Status Scale (EDSS) or conversion to secondary progressive multiple sclerosis (SPMS). These criteria are changed and updated annually but no consensus has been reached yet [6–8]. Despite the recent improvements in our knowledge about genetic role in MS etiology, the role of human leukocyte antigen (HLA) in clinical response to immunotherapy is not completely known. Now it is well understood that alleles of HLA class II on chromosome 6p21 have a great role on genetic susceptibility to MS, particularly HLA-DR and DQ alleles (DRB1*1501, DRB5*0101, DQA1*0102, and DQB1*0602) [1–3,9–11] and among the studied and most probable alleles to be modifiers of clinical response to IFNβ are HLA-DRB1, DQA1, DQB1 and also HLA-DR2 haplotype; but there are not enough strong studies to support this important role for these genes till now [4,12–15]. Since we have some recent findings to support a role for HLA-DRB1 as the most important gene in susceptibility to MS in Caucasian and Iranian patients [11,16,17]. Therefore this study was designed to evaluate the association between HLA-DRB1 genotype and clinical response to IFNβ among Iranian MS patients.
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S. Samadzadeh et al. / Journal of the Neurological Sciences 352 (2015) 37–40
Table 1 Clinical summary of Iranian MS patients. Clinical information
All MS patients
Female only
Male only
Total no. of individuals with MS (n, %) Mean age in years (S.D.) Mean age of onset in years (S.D.) Mean disease duration in years (S.D.)
68 31.9(±7.2) 20.1(±5) 6.8(±5)
53(77.9) 53.5(±6.6) 25.1(±5.2) 6.4(±4.1)
15(22.1) 33.3(±9.3) 24.9(±4.6) 8.4(±7.3)
Familial autoimmune disease (n, %) Yes No
6 62
6(100) 47(68.1)
0 15(31.9)
16 52 1.9(±0.8) 1.7(±0.7)
13(81.3) 40(76.9) 1.9(±0.8) 1.6(±0.6)
3(18.7) 12(23.1) 1.8(±0.8) 1.8(±0.8)
11 25 32
9(81.8) 21(84) 23(71.9)
2(18.2) 4(16) 9(28.1)
MS family history (n, %) Yes No EDSS at entry (mean, S.D.) Relapse no. 2 years before starting INFβ (mean, S.D.) Types of INFβ (n, %) INFβ 1b SC INFβ 1b IM INFβ 1a SC No. = numbers/S.D. = standard deviation. EDSS: Expanded Disability Status Scale. IM: Intramuscular. SC: Subcutaneous.
considered as non-responder when there is at least 1 point increase in his/her EDSS confirmed for 6-months of follow-up or when at least 1 relapse occurs during the follow-up period which must be two years. 2.2. Genomic DNA extraction Genomic DNA was obtained from peripheral blood by standard techniques. Samples comprising 5 ml of peripheral blood were collected in a vacuum with anti-coagulant EDTA and centrifuged at 2500 rpm for 15 min to produce a buffy coat. Genomic DNA was extracted from cells in the buffy coat by using the “Gene extract” extraction method. The test requires DNA with a ratio (A260/A280) of N1.6 and the DNA was used preferentially at a concentration of 75 ng (± 25 ng) per reaction. 2.3. Performing the SSP typing test 24 pair primers were combined into 16 primer mixes and used to amplify the DRB1 alleles. Each primer-pair combination was tested against positive and negative control DNA. The PCR accomplished with these primers produce well-defined DNA fragments of different lengths. The amplified DNA fragments will be detected in an agarose gel by dyeing the double stranded DNA. 2.4. HLA genotyping
2. Materials and methods 2.1. Study subjects In this study 68 Iranian patients with confirmed diagnosis of RRMS who had been referred to and admitted in Neurology Department of Amiralam and Khatam Hospital in Tehran were selected. The study protocol was approved by the Research Ethics Committee of Tehran University of Medical Sciences. The enrolment was performed from “December 2010 until May 2011” and patients were followed for two years. Subjects were invited to participate and were informed in detail about the research. Voluntary consent was obtained from all the individuals included in the study. Demographic and clinical data for each patient were gathered from his/her hospital file and include the patient's age, sex, age at disease onset, disease duration, EDSS at the beginning of study, family history of MS, family history of auto-immune diseases and type of immunotherapy. Inclusion criteria are as follows: 1. the age at MS onset ranged from 18 to 56 years old, 2. adult males and females, 3. diagnosis of MS as defined by the revised (2005) McDonald diagnostic criteria for Multiple Sclerosis (MS), and 4. signed informed consent prior to initiation of any study-mandated procedures. Exclusion criteria are included pregnancy, inability to give informed consent, dementia, cancer or other chronic internal and neurological diseases and recreational drug abuse. The different treatment groups (IFB 1a IM once daily, IFB 1a SC TIW, IFB 1b SC EOD) reflect the gradual availability of the three different IFNB therapies in IRAN. Patients were followed prospectively for 2 years since initiation of therapy and clinical data, including EDSS scores were recorded every 3 months. The primary end point was the suppression of relapses during follow-up and no increase in the EDSS score confirmed by two consecutive visits. Patients were instructed to inform doctors if new symptoms appeared and they were examined within a period of less than 3 days. MRI was performed at the time of diagnosis and also each year, but was not used to monitor the treatment. At the end of the following period each patient was to be classified as responder or non-responder. Since there are not any common and international criteria to define the response to IFNβ, in this study we used Rio et al. (2006) that are stringent criteria in which a patient is
The HLA-DRB1 genetic polymorphism was determined by DNA amplification with polymerase chain reaction and hybridized by specific sequence oligonucleotide primers (PCR-SSO) using R.O.S.E. HLA SSPTyping for HLA genotyping (R.O.S.E. GenTec Ltd. United Kingdom). Reading and interpretation of the results were undertaken according to the manufacturer's instructions. Typifying resolution ranged from low to intermediate. Result interpretation is easy by using provided worksheets in the special soft ware. 2.5. Statistical analysis: finally all data was entered in STATA 12th edition software Differences in the HLA-DRB1 subtype frequencies between two groups of responders and non-responders were compared using Fisher's exact test separately for all alleles and p-value b 0.05 was considered to be statistically significant. 3. Results Total number of individuals was 68 Iranian MS patients, 53 (77.9%) female and 15 (22.1%) male with mean aged in years (SD) 31.9(±7.2), mean aged of onset in years (S.D.) 20.1(±5), mean disease duration in years (S.D.) 6.8(±5), and familial autoimmune disease was only (8%) female. Also EDSS at entry (mean, S.D.) was 1.9(±0.8) and relapse no. 2 years before starting INFβ (mean, S.D.) was 1.7(±0.7).
Table 2 Classification of responder and non-responder according to Rio 2006 criteria. (Ann Neurol 2006;59:344–52.) Criteria elements
All MS patients
Female only
Male only
EDSS at entry (mean, S.D.) EDSS at month 6 (mean, S.D.) EDSS at month 24 (mean, S.D.) Relapse no. at month 24 Relapse no. 2 years before starting INFβ (mean, S.D.) Increased EDSS ≥ 1 at month 6 Relapse rate ≥ 1 at month 24
1.9(±0.8) 2.5(±1.1) 3(±1.5) 1(±1.5) 1.7(±0.7)
1.9(±0.8) 2.4(±1.1) 2.9(±1.4) 0.8(±1.4) 1.6(±0.6)
1.8(±0.8) 2.8(±1.1) 3.6(±1.7) 1.6(±1.6) 1.8(±0.8)
21 21
13 13
8 8
No. = numbers/S.D. = standard deviation. EDSS: Expanded Disability Status Scale.
S. Samadzadeh et al. / Journal of the Neurological Sciences 352 (2015) 37–40
39
Table 3 Demographic and clinical characteristics of responders and non-responders. Characteristics
Responders
Total no. of individuals with MS (n, %) Mean age in years (S.D.) Mean age of onset in years (S.D.) Mean disease duration disease in years (S.D.) MS family history + (n, %+) Autoimmune family history + (n, %+) EDSS at entry (mean, S.D.) Relapse no. 2 years before starting INFβ (mean, S.D.) Type of IFNβ (n, %) IFNβ 1a IM IFNβ 1b SC sIFNβ 1a SC
Non-responders
Total
Female
Male
Total
Female
Male
47(69.1) 30.4(5.9) 25.4(5.0) 4.9(2.5) 12(25.5) 5(10.6) 1.9(0.5) 1.4(0.5)
40(85.1) 30.4(6.1) 25.4(5.1) 5(2.6) 10(83.3) 5(100) 1.9(0.4) 1.5(0.5)
7(14.9) 30.1(4.9) 25.6(5.2) 4.6(1.3) 2(16.7) 0(0) 1.8(0.6) 1.1(0.4)
21(30.9) 35.3(8.7) 24.2(5.1) 11.0(6.4) 4(19.0) 1(4.7) 2.0(1.2) 2.3(0.6)
13(61.9) 34.8(7.0) 24.2(5.6) 10.6(4.9) 3(75.0) 1(100) 2.0(1.4) 2.2(0.6)
8(38.1) 36.1(11.5) 24.4(4.4) 11.8(8.8) 1(25.0) 0(0) 1.9(1.0) 2.4(0.5)
24(51) 2(4.3) 21(44.7)
21(87.5) 2(100) 17(80.9)
3(12.5) 0(0) 4(19.1)
1(4.8) 9(42.8) 11(52.4)
0(0) 7(77.7) 6(54.5)
1(100) 2(22.3) 5(45.5)
EDSS: Expanded Disability Status Scale. IM: Intramuscular. SC: Subcutaneous.
Types of INFβ were INFβ 1b SC, INFβ 1b IM, and INFβ 1a SC (11, 25, 32 MS patients) (Table 1). Followed for two years and finally classified as responder or non-responder based on clinical Rio 2006 criteria; more than one relapse in follow-up period or a 1-point progression in Expanded Disability Status Scale (EDSS) (Table 2). There were 47 (69.1%) responders and 21 (30.9%) non-responders. These two groups were demographically and clinically comparable as demonstrated in Table 3. Fisher's exact test did not show any difference between HLA-DRB1 allele frequencies in responders and non-responders. Fisher's exact test was performed to show the difference between HLA-DRB1 allele frequencies and a summary of the absolute numbers and percentages obtained for each allele is shown in (Table 4). We can see that none of the results have relieved a significant value. In fact these findings do not show any difference between HLA-DRB1 allele frequencies in responders and non-responders. 4. Discussion As we mentioned before there is a strong data supporting association between HLA-DRB1 genotype and MS susceptibility in some populations [9,18] but we have only few studies addressing the role of HLA genes in clinical response to IFNβ and all of these studies have been against this association till now. Fusco et al. (2001) with a cohort of 39 MS patients did not find any association between HLA-DRB1*1501 allele and response to IFNβ [13]. In another study Villoslada et al. (2002) analyzed the distribution of HLA-DR2 haplotype in a cohort of 134 RRMS patients treated with IFNβ and finally found no difference between responder and non-responder groups [4]. In a more recent
Table 4 HLA-DRB1 allele frequencies in responders and non-responders. HLA-DRB1
Non-responders (N = 21) n (%)
Responders (N = 47) n (%)
p-value a
*01 *03 *04 *07 *08 *10 *11 *12 *13 *14 *15 *16
2 (4.76) 4 (9.52) 7 (16.67) 6 (14.29) 1 (2.38) 1 (2.38) 9 (21.43) 1 (2.38) 3 (7.14) 2 (4.76) 6 (14.29) 0 (0)
6 (6.38) 11 (11.70) 14 (14.89) 9 (9.57) 0 (0) 0 (0) 21 (22.34) 1 (1.06) 6 (6.38) 3 (3.19) 22 (23.40) 1 (1.06)
1.00 1.00 0.79 0.42 0.31 0.31 0.91 0.52 1.00 0.66 0.22 1.00
study by Fernández et al. (2005) who aimed to investigate the influence of the HLA class II alleles − DRB1, − DQA1, and − DQB1 in 96 MS patients, no association was found between HLA class II alleles and response to treatment [14]. Finally Comabella et al. (2009) studied 149 RRMS patients classified as responder or non-responder based on the stringent clinical criteria but distribution of HLA class I (HLA-A, − B, and − C genes) and class II alleles (− DRB1, − DQA1, − DQB1 and HLA-DR2 haplotype) was similar between these two groups and the findings did not support a role for HLA class I and class II genes in response to IFNβ [15]. In our study a cohort of 68 RRMS patients were genotyped for HLA-DRB1 individually. For all the HLA-DRB1 genomic types, allelic frequencies were similar between responders and non-responders (Table 4). It should be mentioned that stringency level of a criterion to define response to IFNβ can potentially alter the significance of associations. Fernández et al. used the less rigorous criteria requiring one or more relapses or sustained progression of 0.5 or more points on EDSS after 1 year of IFNβ treatment. In the studies by Fusco et al. and Villoslada et al. intermediate criteria were used to define treatment failure and the criterion used to define response in our study and in Comabella et al. was suggested by Rio et al. (2006) which is the most stringent criteria used till now requiring presence of any relapses and sustained progression of 1 or more points on the EDSS after two years of treatment. 5. Conclusion Regardless of the criteria used, all these studies including ours confirm the lack of association between HLA class II alleles and clinical response to IFNβ and we can conclude that currently available data do not support a role for HLA class II genes as modifiers of response to immunotherapy. Surely more investigations are needed to extend this finding to all types of HLA alleles and also to the interactions of specific ones. Acknowledgments
a p-value of Fisher's exact test between frequency of alleles in responders and nonresponders.
All the authors thank Shefa Neuroscience Research Center for the sponsorship especially Dr. Hadi Kazemi, the chairman, and also they appreciate Pirhossein Koulivand, the chief of Khatam-ol-Anbia Hospital, for his cooperation and kindness and they finally thank the patients who participated in the study so patiently. References [1] Sospedra M, Martin R. Immunology of multiple sclerosis. Annu Rev Immunol 2005; 23:683–747. [2] Oksenberg JR, et al. Multiple sclerosis: genomic rewards. J Neuroimmunol 2001; 113(2):171–84.
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[3] Kaimen-Maciel DR, et al. HLA-DRB1* allele-associated genetic susceptibility and protection against multiple sclerosis in Brazilian patients. Mol Med Rep 2009;2(6): 993–8. [4] Villoslada P, et al. The HLA locus and multiple sclerosis in Spain. Role in disease susceptibility, clinical course and response to interferon-beta. J Neuroimmunol 2002;130(1–2):194–201. [5] Goodin DS, et al. Disease modifying therapies in multiple sclerosis: report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology and the MS Council for Clinical Practice Guidelines. Neurology 2002; 58(2):169–78. [6] Durelli L, et al. Does high-dose interferon beta-1b improve clinical response in more severely disabled multiple sclerosis patients? J Neurol Sci 2000;178(1):37–41. [7] Cohen JA, et al. Therapy of relapsing multiple sclerosis. Treatment approaches for nonresponders. J Neuroimmunol 1999;98(1):29–36. [8] Sormani MP, De Stefano N. Defining and scoring response to IFN-beta in multiple sclerosis. Nat Rev Neurol 2013;9(9):504–12. [9] Oksenberg JR, Barcellos LF. The complex genetic aetiology of multiple sclerosis. J Neurovirol 2000;6(Suppl. 2):S10–4.
[10] Noseworthy JH, et al. Multiple sclerosis. N Engl J Med 2000;343(13):938–52. [11] Abolfazli R, et al. Relationship between HLA-DRB1* 11/15 genotype and susceptibility to multiple sclerosis in Iran. J Neurol Sci 2014;345(1–2):92–6. [12] Byun E, et al. Genome-wide pharmacogenomic analysis of the response to interferon beta therapy in multiple sclerosis. Arch Neurol 2008;65(3):337–44. [13] Fusco C, et al. HLA-DRB1*1501 and response to copolymer-1 therapy in relapsing– remitting multiple sclerosis. Neurology 2001;57(11):1976–9. [14] Fernandez O, et al. HLA class II and response to interferon-beta in multiple sclerosis. Acta Neurol Scand 2005;112(6):391–4. [15] Comabella M, et al. HLA class I and II alleles and response to treatment with interferon-beta in relapsing–remitting multiple sclerosis. J Neuroimmunol 2009; 210(1–2):116–9. [16] Shahbazi M, et al. High frequency of the IL-2–330T/HLA-DRB1*1501 haplotype in patients with multiple sclerosis. Clin Immunol 2010;137(1):134–8. [17] Chao MJ, et al. HLA class I alleles tag HLA-DRB1*1501 haplotypes for differential risk in multiple sclerosis susceptibility. Proc Natl Acad Sci U S A 2008;105(35):13069–74. [18] Weinstock-Guttman B, et al. Pharmacogenetics of MXA SNPs in interferon-beta treated multiple sclerosis patients. J Neuroimmunol 2007;182(1–2):236–9.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
HLA-DRB1 does not have a role in clinical response to interferon-beta among Iranian multiple sclerosis patients Sara Samadzadeh a,b, Elnaz Tabibian b, Tayebeh Sabokbar c,d, Abbas Shakoori d, Shahram Rahimi Dehgolan b, Saeed Azad Armaki a, Bahram Aslanbeigi a, Roya Abolfazli a,b,⁎ a
Shefa Neurosciences Research Center, Tehran, Islamic Republic of Iran Tehran University of Medical Sciences, Neurology Department, Tehran, Islamic Republic of Iran Neurology & Neurosciences Research Center, Qom University of Medical Sciences, Qom, Islamic Republic of Iran d Tehran University of Medical Sciences, Department of Medical Genetics, Cancer Institute, Tehran, Islamic Republic of Iran b c
a r t i c l e
i n f o
Article history: Received 4 October 2014 Received in revised form 9 February 2015 Accepted 2 March 2015 Available online 9 March 2015 Keywords: Multiple sclerosis HLA-DR beta-Chains Interferon-beta Frequencies in responders and non-responders Iran
a b s t r a c t Background & objectives: The role of human leukocyte antigen (HLA) in clinical response to immunotherapy is not completely known. In this study we evaluated the relationship between HLA-DRB1 genotype, which has been proved to be more common in Iranian MS patients, and clinical response to interferon-beta (IFNβ), which is the most common immunotherapy for relapsing–remitting MS. Design and setting: In this study 68 Iranian patients with confirmed diagnosis of RRMS who had been referred to and admitted in Neurology Department of Amiralam and Khatam Hospitals in Tehran were selected. Patients were followed prospectively for 2 years since initiation of therapy and clinical data, including EDSS scores were recorded every 3 months. MRI was performed at the time of diagnosis and each year. Methods: HLA-DRB1 typing was performed by polymerase chain reaction (PCR) for all patients and data was analyzed by STATA 12th edition. Results: There were 47 (69.1%) responders and 21 (30.9%) non-responders. These two groups were demographically and clinically comparable. Fisher's exact test did not show any difference between HLA-DRB1 allele frequencies in responders and non-responders. Conclusions: Our findings confirmed the lack of association between HLA-DRB1 and clinical response to IFNβ among MS patients as previous studies had done. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The etiology of MS is still unclear but according to current data, the disease develops in genetically susceptible individuals due to the complex interaction of a number of genes. However, MS development may require additional triggers, such as hormonal variables and environmental factors including infectious agents and emotional stress [1–3]. Interferon-beta (IFNβ) is the most common therapy for relapsing– remitting multiple sclerosis (RRMS) and also has a beneficial effect on disease activity. In fact it reduces the attack rate and probably suppresses the sustained progression of disability [4,5]. But near to thirty percent of patients do not completely respond to this immunotherapy. This group should be considered as non-responders. There are many criteria for defining and scoring response to IFNβ in RRMS patients based on their different characteristics including the number of relapses ⁎ Corresponding author at: Shefa Neurosciences Research Center, Tehran, Islamic Republic of Iran. Tel.: +98 9121070603; fax: +98 21 66704805. E-mail address: [email protected] (R. Abolfazli).
http://dx.doi.org/10.1016/j.jns.2015.03.004 0022-510X/© 2015 Elsevier B.V. All rights reserved.
per year, increase in Expanded Disability Status Scale (EDSS) or conversion to secondary progressive multiple sclerosis (SPMS). These criteria are changed and updated annually but no consensus has been reached yet [6–8]. Despite the recent improvements in our knowledge about genetic role in MS etiology, the role of human leukocyte antigen (HLA) in clinical response to immunotherapy is not completely known. Now it is well understood that alleles of HLA class II on chromosome 6p21 have a great role on genetic susceptibility to MS, particularly HLA-DR and DQ alleles (DRB1*1501, DRB5*0101, DQA1*0102, and DQB1*0602) [1–3,9–11] and among the studied and most probable alleles to be modifiers of clinical response to IFNβ are HLA-DRB1, DQA1, DQB1 and also HLA-DR2 haplotype; but there are not enough strong studies to support this important role for these genes till now [4,12–15]. Since we have some recent findings to support a role for HLA-DRB1 as the most important gene in susceptibility to MS in Caucasian and Iranian patients [11,16,17]. Therefore this study was designed to evaluate the association between HLA-DRB1 genotype and clinical response to IFNβ among Iranian MS patients.
38
S. Samadzadeh et al. / Journal of the Neurological Sciences 352 (2015) 37–40
Table 1 Clinical summary of Iranian MS patients. Clinical information
All MS patients
Female only
Male only
Total no. of individuals with MS (n, %) Mean age in years (S.D.) Mean age of onset in years (S.D.) Mean disease duration in years (S.D.)
68 31.9(±7.2) 20.1(±5) 6.8(±5)
53(77.9) 53.5(±6.6) 25.1(±5.2) 6.4(±4.1)
15(22.1) 33.3(±9.3) 24.9(±4.6) 8.4(±7.3)
Familial autoimmune disease (n, %) Yes No
6 62
6(100) 47(68.1)
0 15(31.9)
16 52 1.9(±0.8) 1.7(±0.7)
13(81.3) 40(76.9) 1.9(±0.8) 1.6(±0.6)
3(18.7) 12(23.1) 1.8(±0.8) 1.8(±0.8)
11 25 32
9(81.8) 21(84) 23(71.9)
2(18.2) 4(16) 9(28.1)
MS family history (n, %) Yes No EDSS at entry (mean, S.D.) Relapse no. 2 years before starting INFβ (mean, S.D.) Types of INFβ (n, %) INFβ 1b SC INFβ 1b IM INFβ 1a SC No. = numbers/S.D. = standard deviation. EDSS: Expanded Disability Status Scale. IM: Intramuscular. SC: Subcutaneous.
considered as non-responder when there is at least 1 point increase in his/her EDSS confirmed for 6-months of follow-up or when at least 1 relapse occurs during the follow-up period which must be two years. 2.2. Genomic DNA extraction Genomic DNA was obtained from peripheral blood by standard techniques. Samples comprising 5 ml of peripheral blood were collected in a vacuum with anti-coagulant EDTA and centrifuged at 2500 rpm for 15 min to produce a buffy coat. Genomic DNA was extracted from cells in the buffy coat by using the “Gene extract” extraction method. The test requires DNA with a ratio (A260/A280) of N1.6 and the DNA was used preferentially at a concentration of 75 ng (± 25 ng) per reaction. 2.3. Performing the SSP typing test 24 pair primers were combined into 16 primer mixes and used to amplify the DRB1 alleles. Each primer-pair combination was tested against positive and negative control DNA. The PCR accomplished with these primers produce well-defined DNA fragments of different lengths. The amplified DNA fragments will be detected in an agarose gel by dyeing the double stranded DNA. 2.4. HLA genotyping
2. Materials and methods 2.1. Study subjects In this study 68 Iranian patients with confirmed diagnosis of RRMS who had been referred to and admitted in Neurology Department of Amiralam and Khatam Hospital in Tehran were selected. The study protocol was approved by the Research Ethics Committee of Tehran University of Medical Sciences. The enrolment was performed from “December 2010 until May 2011” and patients were followed for two years. Subjects were invited to participate and were informed in detail about the research. Voluntary consent was obtained from all the individuals included in the study. Demographic and clinical data for each patient were gathered from his/her hospital file and include the patient's age, sex, age at disease onset, disease duration, EDSS at the beginning of study, family history of MS, family history of auto-immune diseases and type of immunotherapy. Inclusion criteria are as follows: 1. the age at MS onset ranged from 18 to 56 years old, 2. adult males and females, 3. diagnosis of MS as defined by the revised (2005) McDonald diagnostic criteria for Multiple Sclerosis (MS), and 4. signed informed consent prior to initiation of any study-mandated procedures. Exclusion criteria are included pregnancy, inability to give informed consent, dementia, cancer or other chronic internal and neurological diseases and recreational drug abuse. The different treatment groups (IFB 1a IM once daily, IFB 1a SC TIW, IFB 1b SC EOD) reflect the gradual availability of the three different IFNB therapies in IRAN. Patients were followed prospectively for 2 years since initiation of therapy and clinical data, including EDSS scores were recorded every 3 months. The primary end point was the suppression of relapses during follow-up and no increase in the EDSS score confirmed by two consecutive visits. Patients were instructed to inform doctors if new symptoms appeared and they were examined within a period of less than 3 days. MRI was performed at the time of diagnosis and also each year, but was not used to monitor the treatment. At the end of the following period each patient was to be classified as responder or non-responder. Since there are not any common and international criteria to define the response to IFNβ, in this study we used Rio et al. (2006) that are stringent criteria in which a patient is
The HLA-DRB1 genetic polymorphism was determined by DNA amplification with polymerase chain reaction and hybridized by specific sequence oligonucleotide primers (PCR-SSO) using R.O.S.E. HLA SSPTyping for HLA genotyping (R.O.S.E. GenTec Ltd. United Kingdom). Reading and interpretation of the results were undertaken according to the manufacturer's instructions. Typifying resolution ranged from low to intermediate. Result interpretation is easy by using provided worksheets in the special soft ware. 2.5. Statistical analysis: finally all data was entered in STATA 12th edition software Differences in the HLA-DRB1 subtype frequencies between two groups of responders and non-responders were compared using Fisher's exact test separately for all alleles and p-value b 0.05 was considered to be statistically significant. 3. Results Total number of individuals was 68 Iranian MS patients, 53 (77.9%) female and 15 (22.1%) male with mean aged in years (SD) 31.9(±7.2), mean aged of onset in years (S.D.) 20.1(±5), mean disease duration in years (S.D.) 6.8(±5), and familial autoimmune disease was only (8%) female. Also EDSS at entry (mean, S.D.) was 1.9(±0.8) and relapse no. 2 years before starting INFβ (mean, S.D.) was 1.7(±0.7).
Table 2 Classification of responder and non-responder according to Rio 2006 criteria. (Ann Neurol 2006;59:344–52.) Criteria elements
All MS patients
Female only
Male only
EDSS at entry (mean, S.D.) EDSS at month 6 (mean, S.D.) EDSS at month 24 (mean, S.D.) Relapse no. at month 24 Relapse no. 2 years before starting INFβ (mean, S.D.) Increased EDSS ≥ 1 at month 6 Relapse rate ≥ 1 at month 24
1.9(±0.8) 2.5(±1.1) 3(±1.5) 1(±1.5) 1.7(±0.7)
1.9(±0.8) 2.4(±1.1) 2.9(±1.4) 0.8(±1.4) 1.6(±0.6)
1.8(±0.8) 2.8(±1.1) 3.6(±1.7) 1.6(±1.6) 1.8(±0.8)
21 21
13 13
8 8
No. = numbers/S.D. = standard deviation. EDSS: Expanded Disability Status Scale.
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Table 3 Demographic and clinical characteristics of responders and non-responders. Characteristics
Responders
Total no. of individuals with MS (n, %) Mean age in years (S.D.) Mean age of onset in years (S.D.) Mean disease duration disease in years (S.D.) MS family history + (n, %+) Autoimmune family history + (n, %+) EDSS at entry (mean, S.D.) Relapse no. 2 years before starting INFβ (mean, S.D.) Type of IFNβ (n, %) IFNβ 1a IM IFNβ 1b SC sIFNβ 1a SC
Non-responders
Total
Female
Male
Total
Female
Male
47(69.1) 30.4(5.9) 25.4(5.0) 4.9(2.5) 12(25.5) 5(10.6) 1.9(0.5) 1.4(0.5)
40(85.1) 30.4(6.1) 25.4(5.1) 5(2.6) 10(83.3) 5(100) 1.9(0.4) 1.5(0.5)
7(14.9) 30.1(4.9) 25.6(5.2) 4.6(1.3) 2(16.7) 0(0) 1.8(0.6) 1.1(0.4)
21(30.9) 35.3(8.7) 24.2(5.1) 11.0(6.4) 4(19.0) 1(4.7) 2.0(1.2) 2.3(0.6)
13(61.9) 34.8(7.0) 24.2(5.6) 10.6(4.9) 3(75.0) 1(100) 2.0(1.4) 2.2(0.6)
8(38.1) 36.1(11.5) 24.4(4.4) 11.8(8.8) 1(25.0) 0(0) 1.9(1.0) 2.4(0.5)
24(51) 2(4.3) 21(44.7)
21(87.5) 2(100) 17(80.9)
3(12.5) 0(0) 4(19.1)
1(4.8) 9(42.8) 11(52.4)
0(0) 7(77.7) 6(54.5)
1(100) 2(22.3) 5(45.5)
EDSS: Expanded Disability Status Scale. IM: Intramuscular. SC: Subcutaneous.
Types of INFβ were INFβ 1b SC, INFβ 1b IM, and INFβ 1a SC (11, 25, 32 MS patients) (Table 1). Followed for two years and finally classified as responder or non-responder based on clinical Rio 2006 criteria; more than one relapse in follow-up period or a 1-point progression in Expanded Disability Status Scale (EDSS) (Table 2). There were 47 (69.1%) responders and 21 (30.9%) non-responders. These two groups were demographically and clinically comparable as demonstrated in Table 3. Fisher's exact test did not show any difference between HLA-DRB1 allele frequencies in responders and non-responders. Fisher's exact test was performed to show the difference between HLA-DRB1 allele frequencies and a summary of the absolute numbers and percentages obtained for each allele is shown in (Table 4). We can see that none of the results have relieved a significant value. In fact these findings do not show any difference between HLA-DRB1 allele frequencies in responders and non-responders. 4. Discussion As we mentioned before there is a strong data supporting association between HLA-DRB1 genotype and MS susceptibility in some populations [9,18] but we have only few studies addressing the role of HLA genes in clinical response to IFNβ and all of these studies have been against this association till now. Fusco et al. (2001) with a cohort of 39 MS patients did not find any association between HLA-DRB1*1501 allele and response to IFNβ [13]. In another study Villoslada et al. (2002) analyzed the distribution of HLA-DR2 haplotype in a cohort of 134 RRMS patients treated with IFNβ and finally found no difference between responder and non-responder groups [4]. In a more recent
Table 4 HLA-DRB1 allele frequencies in responders and non-responders. HLA-DRB1
Non-responders (N = 21) n (%)
Responders (N = 47) n (%)
p-value a
*01 *03 *04 *07 *08 *10 *11 *12 *13 *14 *15 *16
2 (4.76) 4 (9.52) 7 (16.67) 6 (14.29) 1 (2.38) 1 (2.38) 9 (21.43) 1 (2.38) 3 (7.14) 2 (4.76) 6 (14.29) 0 (0)
6 (6.38) 11 (11.70) 14 (14.89) 9 (9.57) 0 (0) 0 (0) 21 (22.34) 1 (1.06) 6 (6.38) 3 (3.19) 22 (23.40) 1 (1.06)
1.00 1.00 0.79 0.42 0.31 0.31 0.91 0.52 1.00 0.66 0.22 1.00
study by Fernández et al. (2005) who aimed to investigate the influence of the HLA class II alleles − DRB1, − DQA1, and − DQB1 in 96 MS patients, no association was found between HLA class II alleles and response to treatment [14]. Finally Comabella et al. (2009) studied 149 RRMS patients classified as responder or non-responder based on the stringent clinical criteria but distribution of HLA class I (HLA-A, − B, and − C genes) and class II alleles (− DRB1, − DQA1, − DQB1 and HLA-DR2 haplotype) was similar between these two groups and the findings did not support a role for HLA class I and class II genes in response to IFNβ [15]. In our study a cohort of 68 RRMS patients were genotyped for HLA-DRB1 individually. For all the HLA-DRB1 genomic types, allelic frequencies were similar between responders and non-responders (Table 4). It should be mentioned that stringency level of a criterion to define response to IFNβ can potentially alter the significance of associations. Fernández et al. used the less rigorous criteria requiring one or more relapses or sustained progression of 0.5 or more points on EDSS after 1 year of IFNβ treatment. In the studies by Fusco et al. and Villoslada et al. intermediate criteria were used to define treatment failure and the criterion used to define response in our study and in Comabella et al. was suggested by Rio et al. (2006) which is the most stringent criteria used till now requiring presence of any relapses and sustained progression of 1 or more points on the EDSS after two years of treatment. 5. Conclusion Regardless of the criteria used, all these studies including ours confirm the lack of association between HLA class II alleles and clinical response to IFNβ and we can conclude that currently available data do not support a role for HLA class II genes as modifiers of response to immunotherapy. Surely more investigations are needed to extend this finding to all types of HLA alleles and also to the interactions of specific ones. Acknowledgments
a p-value of Fisher's exact test between frequency of alleles in responders and nonresponders.
All the authors thank Shefa Neuroscience Research Center for the sponsorship especially Dr. Hadi Kazemi, the chairman, and also they appreciate Pirhossein Koulivand, the chief of Khatam-ol-Anbia Hospital, for his cooperation and kindness and they finally thank the patients who participated in the study so patiently. References [1] Sospedra M, Martin R. Immunology of multiple sclerosis. Annu Rev Immunol 2005; 23:683–747. [2] Oksenberg JR, et al. Multiple sclerosis: genomic rewards. J Neuroimmunol 2001; 113(2):171–84.
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[3] Kaimen-Maciel DR, et al. HLA-DRB1* allele-associated genetic susceptibility and protection against multiple sclerosis in Brazilian patients. Mol Med Rep 2009;2(6): 993–8. [4] Villoslada P, et al. The HLA locus and multiple sclerosis in Spain. Role in disease susceptibility, clinical course and response to interferon-beta. J Neuroimmunol 2002;130(1–2):194–201. [5] Goodin DS, et al. Disease modifying therapies in multiple sclerosis: report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology and the MS Council for Clinical Practice Guidelines. Neurology 2002; 58(2):169–78. [6] Durelli L, et al. Does high-dose interferon beta-1b improve clinical response in more severely disabled multiple sclerosis patients? J Neurol Sci 2000;178(1):37–41. [7] Cohen JA, et al. Therapy of relapsing multiple sclerosis. Treatment approaches for nonresponders. J Neuroimmunol 1999;98(1):29–36. [8] Sormani MP, De Stefano N. Defining and scoring response to IFN-beta in multiple sclerosis. Nat Rev Neurol 2013;9(9):504–12. [9] Oksenberg JR, Barcellos LF. The complex genetic aetiology of multiple sclerosis. J Neurovirol 2000;6(Suppl. 2):S10–4.
[10] Noseworthy JH, et al. Multiple sclerosis. N Engl J Med 2000;343(13):938–52. [11] Abolfazli R, et al. Relationship between HLA-DRB1* 11/15 genotype and susceptibility to multiple sclerosis in Iran. J Neurol Sci 2014;345(1–2):92–6. [12] Byun E, et al. Genome-wide pharmacogenomic analysis of the response to interferon beta therapy in multiple sclerosis. Arch Neurol 2008;65(3):337–44. [13] Fusco C, et al. HLA-DRB1*1501 and response to copolymer-1 therapy in relapsing– remitting multiple sclerosis. Neurology 2001;57(11):1976–9. [14] Fernandez O, et al. HLA class II and response to interferon-beta in multiple sclerosis. Acta Neurol Scand 2005;112(6):391–4. [15] Comabella M, et al. HLA class I and II alleles and response to treatment with interferon-beta in relapsing–remitting multiple sclerosis. J Neuroimmunol 2009; 210(1–2):116–9. [16] Shahbazi M, et al. High frequency of the IL-2–330T/HLA-DRB1*1501 haplotype in patients with multiple sclerosis. Clin Immunol 2010;137(1):134–8. [17] Chao MJ, et al. HLA class I alleles tag HLA-DRB1*1501 haplotypes for differential risk in multiple sclerosis susceptibility. Proc Natl Acad Sci U S A 2008;105(35):13069–74. [18] Weinstock-Guttman B, et al. Pharmacogenetics of MXA SNPs in interferon-beta treated multiple sclerosis patients. J Neuroimmunol 2007;182(1–2):236–9.
