Mirzaie V; Ebrahimi H; Seyedi F; Tohidfar M; Nematollahi-Mahani NS

Research Article

Austin J Neurol Disord Epilepsy. 2019; 6(1): 1043.

HLA Association with Multiple Sclerosis in South- Eastern of Iran

Mirzaie V¹, Ebrahimi H², Seyedi F³, Tohidfar M⁴ and Nematollahi-Mahani NS⁵*

¹Department of Anatomy, Kerman University of Medical Sciences, Iran

²Neurology Research Center, Kerman University of Medical Sciences, Iran

³Department of Anatomy, Jiroft University of Medical Sciences, Iran

⁴Department of Plant Biotechnology, Shahid Beheshti University GC, Iran

⁵Kerman Neuroscience Research Center (KNRC), Institute of Neuropharmacology, and Afzal Research Institute, Iran

*Corresponding author: Seyed Noureddin Nematollahi-Mahani, Department of Anatomy, Afzalipour School of Medicine, Kerman University of Medical Sciences, Kerman, Iran

Received: March 25, 2019; Accepted: April 24, 2019; Published: May 01, 2019

Abstract

Introduction: Investigation of the HLA_DRB1 alleles frequency between Multiple Sclerosis (MS) patients in Kerman city, south-eastern of Iran, the association of rs3135388, tagging gene polymorphism of HLA_DRB1*15, with multiple sclerosis and comparing HLA_DRB1*11, the most frequent allele in this population, with HLA_DRB1*15, the most related allele to MS disease.

Methods: DNA samples were extracted and amplified by PCR with sequence-specific primers (PCR-SSP) for the HLA-DRB genes, using HLA SSP DRB Typing Kit. Based on HLA typing results, Samples having HLA_DRB1*15 were amplified with (PCR-SSP); rs3135388 primers, tagging gene for HLA_ DRB1 *15 polymorphism, then PCR product was digested by specific restriction enzyme for rs3135388, Cla I,

Results: Our findings indicated that HLA_DRB1*11 was the most frequent allele followed by HLA_DRB1*15. HLA_DRB1*15 allele was more frequent in females compared to the males, while there was no significant correlation between HLA_DRB1*11 and gender. Also, HLA_DRB1*15 was more related to disease severity based on Extended Disability Status Scale (EDSS) results. The rs3135388 T allele was more frequent in the MS group compared to the controls.

Conclusion: HLA_DRB1*15 is most probably related to MS disease with T allele as the most common polymorphism among the MS patients.

Keywords: Multiple Sclerosis; HLA_DRB1 Alleles; HLA Typing; rs3135388; Cla I Restriction Enzyme; Kerman-Iran Population

Abbreviations

MS: Multiple Sclerosis; RRMS: Relapsing-remitting Multiple Sclerosis; PPMS: Primary-progressive Multiple Sclerosis; SPMS: Secondary Progressive Multiple Sclerosis; EDSS: Expanded Disability Status Scale; NA: Not Applicable

Introduction

Multiple Sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system which causes progressive and relapsing neurological handicaps [1]. The disease is the most common human demyelinating illness [2,3] which afflicts twice as many women as men [4]. It is the first reason of non-traumatic neurological disabilities in young people [5]. The onset of MS typically is in young adults, thus it is associated with long term physical effects. It’s prevalence is 57.3 in 100000 populations in Kerman city, southern-east of Iran [6]. MS is a multifactorial disorder whose pathogenesis seems to involve interactions between genetic and environmental factors, which vary in different populations, ethnic groups and geographic locations [7,8]. Among the potential genetic factors, the Human Leukocyte Antigen (HLA) class II, the human form of MHC molecules on the 6p21 chromosome, has been identified as the main region associated with MS [9,10].

A main feature of HLAs is allelic polymorphism. Such polymorphism apparently enhances the probability of increasing an immune response by a subset of individuals among a population [11]. Various investigations have been done on HLA class II genes in different ethnic groups. Between these alleles, the HLA-DRB1*15 was found more frequent in most of patients in Caucasia [5], Greece [12], Latin American [13], Western Australia [7], Brazil [9], Moroccan [5], Portugal [14], Europe [15,16,17,18,19,20,21], Arabs [22] and African Americans [23].

So at present study we aimed to investigate the frequency and genetic polymorphisms of HLA class II alleles in Kerman province, southern-east of Iran, which has been postulated to suffer from high prevalence of MS, and to compare the findings with other populations worldwide.

Materials and Methods

Patients

Kerman university of Medical Sciences ethics committee approved the study. The subjects provided a written consent before the initiation of the study. The study included 50 unrelated MS patients (33 women and 17 men) introduced by the neurological clinic of Shafa hospital at Kerman University of Medical Sciences, Kerman-Iran. Diagnosis was based on the revised Mc-Donald criteria, which were registered at the Iranian MS society, Kerman branch. Ten healthy individuals from the same community entered the study as controls. Through medical evaluation, it was ensured that the control individuals were free of acute or chronic internal and neurological diseases

Sample collections

Peripheral blood samples (10 ml) from MS patients and controls were collected in EDTA. Each subject was informed about the study and provided a written informed consent for the genetic analysis.

DNA extraction and HLA typing

Genomic DNA was obtained by manual techniques.

1000 μl RBC lysis buffer was added to 500 μl of peripheral blood and centrifuged at 7000 rpm for 2 minutes. The step was repeated 2-3 times and the supernatants were discarded each time. 400 μl nuclear lysis buffer, 100 μl Nacl solution and 600 μl chloroform were added and centrifuged at 7000 rpm for 2 minutes.

800 μl cold absolute ethanol was then added to 400 μl of supernatant and centrifuged at 12000 rpm for 1 minute. The supernatants were finally discarded and 50 μl elution buffer was added to the tube.

The test requires DNA with a ratio (A260/A280) of >1.6 and at least a concentration of 75 ng per reaction.

DNA was amplified by PCR with Sequence-Specific Primers (PCR-SSP) for the HLA-DRB genes, using MorganTM HLA SSP DRB Typing Kit. PCR products were visualized under UV illuminator after electrophoretic separation on 2% agarose gel containing ethidium bromide. Genotypes were deduced from the amplification patterns using the worksheet provided in kit in the special software.

Restriction enzyme digestion

Based on HLA typing results, Samples having HLA_DRB1*15 were amplified with Polymerase Chain Reaction using Sequence- Specific Primers (PCR-SSP); rs3135388 primers, tagging gene for HLA_DRB1*15 polymorphism. The data showing primer, PCR and digestion information are presented elsewhere [24].

PCR product was digested by Cla I, specific restriction enzyme for rs3135388 provided from Thermo fisher scientific company, after electrophoresis the results were compared in order to examine the genotype differences between the samples.

Statistical analysis

HLA-DRB1 allele results were set up using the Excel software, and allelic frequency was obtained using SPSS version 16.1.

Results

Demographic and clinical data

To determine the association of HLA-DRB1 and MS disease, a total of 50 MS patients and 10 controls were analyzed. The original city of birth of the patients and control group were recorded. They all were from the same ethnicity and regional location; Kerman province, southeastern Iran.

Among 50 patients with MS and 10 controls there were 35 females and 15 males in MS and 7 females and 3 males in the control group.

Mean age was 33.78 years in MS and 28.8 years in the control group. Mean age at onset was 27.9 years, mean disease duration was 7.4 years. Other clinical features are recorded in Table 1.

Table 1: Demographic and clinical characteristics of MS patients and controls.




  
    Clinical information
    MS patients
    controls
  
  
    Total number of individuals
    50
    10
  
  
    Gender
    male
    16
    3
  
  
    female
    34
    7
  
  
    Mean age (y)
    33.78
    28.8
  
  
    Mean age at onset (y) (S.D.)
    27.9 ± (8.8)
    NA
  
  
    Mean disease duration(y)(S.D)
    7.4 (4.8)
    NA
  
  
    Type of initial symptoms, n (%)
    
    NA
  
  
    Optic neuritis
    18 (36%)
    NA
  
  
    Long motor tracts
    33 (66%)
    NA
  
  
    Long sensory tracts
    10 (20%)
    NA
  
  
    Brainstem-Cerebellum
    17 (34%)
    NA
  
  
    Disease course, n (%)
    
    NA
  
  
    RRMS
    28 (56%)
    NA
  
  
    PPMS
    3 (6%)
    NA
  
  
    SPMS
    14 (28%)
    NA
  
  
    NEW CASE
    5 (10%)
    NA
  
  
    EDSS, n (%)
    
    NA
  
  
    <3
    19 (38%)
    NA
  
  
    3=EDSS=6
    17 (34%)
    NA
  
  
    >6
    14 (28%)
    NA
  
  
    Symptoms at enrollment time, n (%)
    Optic neuritis
    21 (42%)
    NA
  
  
    Long motor tracts
    34 (68%)
    NA
  
  
    Long sensory tracts
    10 (20%)
    NA
  
  
    Brainstem-Cerebellum
    17 (34%)
    NA
  
  
     
      Family history, n (%)
    With MS
    4 (8%)
    NA
  
  
    Without MS
    46 (92%)
    
  
  
    RRMS,    Relapsing-remitting MS; PPMS, Primary-progressive MS; SPMS, Secondary    progressive MS; EDSS, Expanded Disability Status Scale; NA, not applicable.



Table 1: Demographic and clinical characteristics of MS patients and controls.

HLA typing parameters

Allele frequencies in MS and control group: As the results show in Table 2, HLA_DRB1*11 was significantly the most frequent allele in MS patients and after that alleles *15, *04 and *16 are more frequent among the others. Also, HLA_DRB1*08, *10 and *12 have the least frequency in MS group.

Table 2: Associations of HLA-DRB1 alleles with MS and carriage frequencies in the MS and control groups of subjects.




  
    HLA_DRB1
    Case carriage frequency% (n)
    Control carriage frequency% (n)
    Odds ratio
    p-value
  
   
  
    *01
    9 (7.5%)
    2 (1.6%)
    0.89
    0.05
  
  
    *03
    10 (8.3%)
    2 (1.6%)
    1
    0.05
  
  
    *04
    11 (9.1%)
    0
    0
    0.05
  
  
    *07
    8 (6.6%)
    2 (1.6%)
    0.78
    0.05
  
  
    *08
    1 (0.8%)
    0
    0
    0.05
  
  
    *10
    1 (0.8%)
    0
    0
    0.05
  
  
    *11
    23 (19.1%)
    1 (1.6%)
    5.67
    0.05
  
  
    *12
    1 (1.6%)
    0
    0
    0.05
  
  
    *13
    7 (5.8%)
    4 (3.3%)
    0.3
    0.05
  
  
    *14
    6 (5%)
    4 (3.3%)
    0.2
    0.05
  
  
    *15
    12 (10%)
    4 (3.3%)
    0.54
    0.05
  
  
    *16
    11 (9.1%)
    1 (1.6%)
    2.34
    0.05



Table 2: Associations of HLA-DRB1 alleles with MS and carriage frequencies in
the MS and control groups of subjects.

Genotype frequencies in MS and control group: The HLA_ DRB1*11/11 and 11/01 genotypes were the most frequent in MS group followed by HLA_DRB1*16/16, 11/07, 11/15 and 13/03. Among the control samples HLA_DRB1*13/14 had the highest frequency (Tables 3,4).

Table 3: Distribution of homozygote and heterozygote HLA_DRB1 alleles in MS group.




  
    HLA_DRB1 alleles in case group
    *01
    *03
    *04
    *07
    *08
    *10
    *11
    *12
    *13
    *14
    *15
    *16
  
  
    *01
    .
    .
    
    
    
    
    …
    
    
    
    
    .
  
  
    *03
    
    
    .
    
    
    
    
    
    ..
    
    .
    
  
  
    *04
    
    .
    ..
    .
    
    
    .
    .
    .
    
    
    
  
  
    *07
    
    
    
    .
    
    
    ..
    
    
    
    .
    
  
  
    *08
    
    
    
    
    
    
    
    
    
    .
    
    
  
  
    *10
    
    
    
    
    
    
    
    
    
    
    .
    
  
  
    *11
    .
    
    .
    .
    
    
    ….
    
    
    ..
    .
    .
  
  
    *12
    
    
    
    
    
    
    
    
    
    
    
    
  
  

    *13
    
    .
    
    
    
    
    
    
    
    ..
    
    
  
  
    *14
    
    
    
    
    
    
    
    
    
    
    .
    
  
  
    *15
    .
    .
    
    .
    
    
    ..
    
    
    
    .
    
  
  
    *16
    
    ..
    
    
    
    
    
    
    .
    
    
    …



Table 3: Distribution of homozygote and heterozygote HLA_DRB1 alleles in MS group.

Table 4: Distribution of homozygote and heterozygote HLA_DRB1 alleles in control group.




  
    HLA_DRB1 alleles in control group
    *01
    *03
    *04
    *07
    *08
    *10
    *11
    *12
    *13
    *14
    *15
    *16
  
  
    *01
    
    
    
    
    
    
    
    
    
    
    
    
  
  
    *03
    .
    
    
    .
    
    
    
    
    
    
    
    
  
  
    *04
    
    
    
    
    
    
    
    
    
    
    
    
  
  
    *07
    
    
    
    
    
    
    
    
    
    
    
    
  
  
    *08
    
    
    
    
    
    
    
    
    
    
    
    
  
  
    *10
    
    
    
    
    
    
    
    
    
    
    
    
  
  
    *11
    .
    
    
    
    
    
    
    
    
    
    
    
  
  
    *12
    
    
    
    
    
    
    
    
    
    
    
    
  
  
    *13
    
    
    
    
    
    
    
    
    
    …
    .
    
  
  
    *14
    
    
    
    
    
    
    
    
    
    
    
    
  
  
    *15
    
    
    
    .
    
    
    
    
    
    
    .
    
  
  
    *16
    
    
    
    
    
    
    
    
    
    .



Table 4: Distribution of homozygote and heterozygote HLA_DRB1 alleles in control group.

Genotype characteristics: According to other studies HLA_ DRB1*15 is the most related allele to multiple sclerosis. Our results also showed high frequency in this allele (Table 5). So we decided to study the allele frequencies of HLA-DRB1*1501 rs3135388 gene polymorphism, a tagging polymorphism for HLA_DRB1*15, in MS patients who had had this allele based on the HLA typing results. Among all, 11 samples in MS and 3 samples in control group had HLA_DRB1*15 allele.

Table 5: Genotype differences of rs3135388 Polymorphism.




  
    Polymorphism
    Genotype
    Genotype
    Genotype
    Allele
    Allele
  
  
    rs3135388
    TT (%)
    TC (%)
    CC (%)
    T
    C
  
  
    MS (N=11)
    4/11 (36.3%)
    1/11/2019 -9%
    6/11 (54%)
    9/22(40.9%)
    13/22(59%)
  
  
    Control (N=3)
    0/3
    2/3/2019 (66.6%)
    1/3/2019 (33.3%)
    2/6 (33.3%)
    4/6 (66.6%)



Table 5: Genotype differences of rs3135388 Polymorphism.

The homozygotes TT and heterozygotes TC were more frequent in MS patients and a higher frequency of the rs3135388 T allele was found in MS group compared to the controls.

Comparing HLA_DRB1*11 and *15 features: As the frequency of HLA_DRB1*11 is so high among MS patients in this study and also in Iranian population and HLA_DRB1*15 is reported to be severely associated to MS disease we compared some features of these two allele carriers.

According to Table 6 among 50 MS patients 19 of them carried HLA_DRB1*11 and 11 patients carried HLA_DRB1*15 allele in either homozygote or heterozygote forms.

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Citation: Mirzaie V, Ebrahimi H, Seyedi F, Tohidfar M and Nematollahi-Mahani NS. HLA Association with Multiple Sclerosis in South-Eastern of Iran. Austin J Neurol Disord Epilepsy. 2019; 6(1): 1043.

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Polymorphism	Genotype	Genotype	Genotype	Allele	Allele
rs3135388	TT (%)	TC (%)	CC (%)	T	C
MS (N=11)	4/11 (36.3%)	1/11/2019 -9%	6/11 (54%)	9/22(40.9%)	13/22(59%)
Control (N=3)	0/3	2/3/2019 (66.6%)	1/3/2019 (33.3%)	2/6 (33.3%)	4/6 (66.6%)

HLA_DRB1		*11	*15
gender		9 female (47.3%)	8 female (72.7%)
gender		10 male (52.6%)	3 male (27.2%)
Mean disease duration		6.9 y	6.6 y
Disease course, n (%)	RRMS	12 (63.1%)	4 (36.3%)
	PPMS	2 (10.5%)	2 (18.1%)
	SPMS	5 (26.3%)	4 (36.3%)
NEW CASE		0	1 (9%)
EDSS, n (%)	<3	9 (47.3%)	2 (18.1%)
	3=EDSS=6	7 (36.8%)	4 (36.3%)
	>6	3 (15.7%)	5 (45.4%)