DOI: 10.7860/JCDR/2013/4509.

2752
Original Article

The HLA Class II Associations with

Cardiology Section
Rheumatic Heart Disease in South Indian
Patients: A Preliminary Study
Divya Bajoria, Thangam Menon

ABSTRACT Methods: A total of 23 patients who were diagnosed with

Introduction: Rheumatic heart disease (RHD) occurs in 30-45% RHD and 6 control samples were included in this study. A low
of the patients with rheumatic fever (RF) and it leads to chronic resolution HLA Class II DR/DQ typing was performed on the
valvular lesions. The human leukocyte antigen (HLA) might blood samples by the PCR-SSP method.
confer a susceptibility to RHD. The aim of the present study Results and Conclusion: The DRB3*01:01:02:01 allele showed
was to determine the prevalent HLA class II DR/DQ allelic types a positive association with RHD, whereas the DQB1 loci alleles
which were associated with rheumatic heart disease (RHD) in a did not show any significant association.
small group of south Indian patients and to compare them with
those in the control subjects.

Key Words: HLA Class II DR/DQ typing, Rheumatic Heart Disease, South India

Introduction based on the clinical and the echocardiographic findings. The

Rheumatic fever (RF) and Rheumatic Heart Disease (RHD) are mean age of the patients was 29.6 ± 12.25 years (mean age ±
important public health problems which often lead to chronic S.D), and they included 16 males and 7 females. Six age and sex
valvular lesions [1]. The incidence per 1000 ranges from 0.6 to matched control samples from normal individuals in south India,
11 in the reports from different parts of India [2]. It occurs as a with no history of RHD or any autoimmune disease, were also
sequel to the throat infections which are caused by Streptococcus included in our study. The present study was conducted during
pyogenes, it manifests as polyarthritis, carditis, chorea, erythema the years 2010-2011. An ethical clearance was obtained from the
marginatum, and/or as subcutaneous nodules and it progresses to institutional ethical committee. Of the 23 patients, 13 (56.52%)
cause damage to the valve tissue of the heart, leading to congestive had mitral valve disease; 3(13.04%) had aortic valve disease; and
heart failure, stroke, endocarditis, and death [3]. 7(30.04%) had multi valvular disease.

Inspite of the knowledge of the inciting agent, the pathogenesis A low resolution HLA Class II DR/DQ typing was performed by
of RHD is not completely understood and only 2-3% of the the PCR-SSP (sequence specific priming) method. The assay
patients develop RF after occurrences of untreated streptococcal was performed by using the MICRO SSP™ HLA DNA TYPING
pharyngitis. Several studies have documented a high familial TRAYS in accordance with the protocol which was provided by the
incidence of RF, thus suggesting the involvement of host genetic manufacturers (One Lambda Diagnostics, USA). Two ml of blood
factors in the susceptibility to RF, with a consequential progression was collected in EDTA from each individual, and the genomic
to RHD [4, 5]. DNA was isolated from whole blood by using the DNAeasy Blood
As critical components in the antigen processing, the human and Tissue Kit (mini spin column-QIAGEN, Germany). Briefly, the
leukocyte antigen (HLA) molecules are attractive candidate procedure was as follows: The Micro SSP™ D mix, the primer set
antigens that might confer a susceptibility to RHD. Bryant et al., trays, and the DNA samples were thawed to room temperature. 2 µl
in his review, has provided an overall perspective of the several of Taq Polymerase (5units/µl) was added to the Micro SSP™ D-mix
HLA alleles which are associated with RF and RHD across various and the mixture was vortexed. 9 µl of this was added to the negative
regions of the world. A number of studies have suggested that the control reaction tube on the primer set tray. The DNA sample (39 µl)
HLA class II molecules appear to have a closer association with an was added to the Micro SSP™ D-mix tube and it was vortexed for
increased risk of RF or RHD than the class I molecules, although 5 seconds and centrifuged. 10 μl of the sample-reaction mixture
no single HLA haplotype or combination exists, that is consistently from the MicroSSP™ D-mix tube was pipetted into each reaction
associated with a susceptibility to RHD [6]. tube of the Micro SSP™ primer set tray, with the exception of the
negative control reaction tube. The reaction tubes with the tray
The aim of the present study was to determine the prevalent HLA
were sealed and the Micro SSP™ primer set tray was placed in
class II DR/DQ allelic types which were associated with Rheumatic
the PCR thermocycler. After the PCR process, the amplified DNA
Heart Disease (RHD) in a small group of south Indian patients and
fragments were separated by agarose gel electrophoresis and they
to compare them with those in the control subjects.
were visualized by using a gel documentation system (Bio-Rad,
Materials and Methods USA). The interpretation of the PCR-SSP results was based on the
We determined the HLA class II DR/DQ allelic types in 23 south presence or absence of a specific amplified DNA fragment. The
Indian patients who were diagnosed with rheumatic heart disease, pattern of the positive wells was matched with the information on

302 Journal of Clinical and Diagnostic Research. 2013 February, Vol-7(2): 302-304
www.jcdr.net Divya Bajoria and Thangam Menon, HLA Class II DR/DQ Typing of RHD Patients

the Micro SSP™ worksheet to obtain the HLA typing of the sample II DRB1*07-DQB1*0401-2 and DRB1*07-DQB1*0302 could be the
DNA. risk alleles and that the HLA class II DRB1*06 and DQB1*0602-8
could be the protective alleles among the RHD patients in Latvia.
The statistical analysis was performed by using the Fisher’s exact
The DRB1*07 allele was also found to be associated with RHD
test to determine the degree of association.
in Pakistan [9]. Guedez et al., [10] also found the DRB1*0701
-DQA1*0201 haplotype to be significantly associated with RHD. On
Results the contrary, in our study, we found that DRB1*07:01:01:01 was
The frequency of DRB3*01:01:02:01 was 39.13% in the RHD
present in both the RHD patients and the controls and therefore, it
patients, whereas it was not present in the controls. The difference
could not be linked with RHD in the south Indian patients.
in the prevalence of the DRB3*01:01:02:01 allele among the RHD
patients and the controls was statistically significant (p= 0.01). Although several studies have been done in different parts of the
DRB1*09:01:02, on the other hand, occurred at a frequency world, studies on the HLA association with RHD is lacking in India,
of 16.6% in the controls but was not found in any of the RHD especially in the south Indian population. This was a pilot study
patients [Table/Fig-1]. The DQB1 alleles did not show a significant on the HLA Class II types which were seen in the RHD patients
association in the RHD patients as compared to that in the controls in south India. Previous studies which had been done in north
[Table/Fig-2]. India were based on serological assays and the HLA alleles which
were associated with RHD were reported to be DR3, DR4 and
RHD patients Controls DQ3 [11-13].
Allele Allele
frequency frequency In the present study, DRB3*01:01:02:01 was seen with an allele
DRB allele AF (%) AF (%) p value frequency of 39.13% in the patients and it was seen in none of the
DRB3*01:01:02:01 18/46 39.13 0/12 0 0.011 controls. This difference was unlikely to be incidental and it was
DRB4*01:01:01:01 16/46 34.78 6/12 50 0.505 certainly worth investigating in future studies. We also found that
the alleles of the DQB1 loci were not significantly associated with
DRB1*07:01:01:01 10/46 21.73 3/12 25 1
RHD.
DRB1*15:01:01:01 6/46 13.04 4/12 33.33 0.191
DRB1*04:01:01 6/46 13.04 1/12 8.33 1 The HLA types which were seen in the RHD patients in south India
DRB1*03:01:01:01 5/46 10.86 0/12 - 0.354
were different from the HLA types which had been reported in other
countries, and hence there appeared to be ethnic differences in the
DRB1*10:01:01 5/46 10.86 1/12 8.33 1
distribution of the HLA alleles. In addition, the earlier studies which
DRB5*01:01:01 6/46 13.04 4/12 33.33 0.190
were done on HLA were based on serological assays and thus, a
DRB1*13:01:01 7/46 15.21 0/12 - 0.354 comparison of the results of our study with earlier reports was not
DRB1*14:01:01 2/46 4.3 0/12 - 1 possible. The molecular techniques for HLA typing are superior to
DRB1*14:04 2/46 4.3 0/12 - 1 the serological assays and they are also less cumbersome. The
DRB1*14:02 1/46 2.17 0/12 - 1 limitation of this study was the small number of patients in the
DRB1*08:09 1/46 2.17 0/12 - 1 control group.
DRB1*08:01:01 0/46 - 1/12 8.33 0.207
DRB1*09:01:02 0/46 - 2/12 16.66 0.0399
Conclusion
The present study was a pilot study; however, the preliminary
DRB1*11:01:01 1/46 2.17 0/12 - 1
results are interesting and there is a need to do a larger case-
[Table/Fig-1]: Frequency of distribution of DRB locus alleles in RHD control study in India to confirm whether DRB3*01:01:02:01 is a
patients and controls
risk allele for RHD.

RHD patients Controls References

Allele Allele p [1] Guilherme L, Kohler KF, Kalil J. Rheumatic heart disease: mediation by
frequency frequency value complex immune events. Adv Clin Chem. 2011; 53:31-50.
DQB allele AF (%) AF (%) [2] Kumar RK, Paul M, Francis PT. RHD in India: are we ready to shift
DQB1*05:01:01 11/46 23.91 3/12 25 1 from secondary prophylaxis to vaccinating high-risk children? Current
Science. 2009; 97:397-404.
DQB1*06:01:01 12/46 26.08 2/12 16.66 0.710 [3] Cunningham MW. Pathogenesis of group A streptococcal infections
DQB1*02:01:01 9/46 19.56 3/12 25 0.698 and their sequelae. Adv Exp Med Biol. 2008; 609:29-42.
[4] Di Sciascio G, Taranta A. Rheumatic fever in children. Am Heart J.
DQB1*03:03:02 5/46 10.86 2/12 16.66 0.626 1980; 99(5): 635–58.
DQB1*03:02:01 4/46 8.6 1/12 8.33 1 [5] Pickles WN. Rheumatic family. Lancet. 1943;2:241.
[6] Bryant PA, Robins-Browne R, Carapetis JR, Curtis N. Some of the
DQB1*03:01:01 3/46 6.5 0/12 - 0.597
People, Some of the Time: Susceptibility to Acute Rheumatic Fever.
DQB1*04:01:01 1/46 2.17 1/12 8.33 0.374 Circulation. 2009 Feb 10; 119(5):742-53.
DQB1*05:03:01 1/46 2.17 0/12 - 1 [7] El-Hagrassy N, El-Chennawi F, Zaki Mel-S, Fawzy H, Zaki A, Joseph
N. HLA class I and class II HLA DRB profiles in Egyptian children with
[Table/Fig-2]: Frequency of distribution of DQB1 locus alleles in RHD rheumatic valvular disease. Pediatr Cardiol. 2010 Jul;31(5):650-56.
patients and controls [8] Stanevicha V, Eglite J, Sochnevs A, Gardovska D, Zavadska D,
Shantere R. HLA class II associations with rheumatic heart disease
among clinically homogeneous patients in children in Latvia. Arthritis
Discussion Res Ther. 2003;(5):R340-46.
In a study which was done by EL-Hagrassy et al., [7] on Egyptian [9] Rehman S, Akhtar N, Ahmad W, Ayub Q, Mehdi SQ, Mohyuddin A.
children, DR*04-02 and DR *10-0101 were found to be common in Human leukocyte antigen (HLA) class II association with rheumatic
heart disease in Pakistan. J Heart Valve Dis. 2007 May;16(3):300-04.
the RHD patients. Stanevicha et al., [8] reported that the HLA class

Journal of Clinical and Diagnostic Research. 2013 February, Vol-7(2): 302-304 303
 Divya Bajoria and Thangam Menon, HLA Class II DR/DQ Typing of RHD Patients www.jcdr.net

[10] Guedez Y, Kotby A, El-Demellawy M, Galal A, Thomson G, Zaher [12] Reddy KS, Narula J, Bhatia R, Shailendri K, Koicha M, Taneja
S, et al. HLA Class II associations with rheumatic heart disease are V, Jhingan B, Pothineni RB, Malaviya AN, Mehra NK et al. Immunologic
more evident and consistent among clinically homogeneous patients. and immunogenetic studies in rheumatic fever and rheumatic heart
Circulation. 1999 Jun 1; 99(21):2784–90. disease. Indian J Pediatr. 1990 Sep-Oct;57(5):693–700.
[11] Bhat MS, Wani BA, Koul PA, Bisati SD, Khan MA, Shah SU. HLA [13] Wani BA. Study of HLA-A, B, C, DR, DQ profile of patients with
antigen pattern of Kashmiri patients with rheumatic heart disease. established rheumatic heart disease in Kashmir. Indian Heart J. 1997
Indian J Med Res. 1997 Jun;105: 271–74. Mar-Apr; 49(2):152–54.

AUTHOR(S): NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING
1. Divya Bajoria AUTHOR:
2. Thangam Menon Dr. Thangam Menon,
Professor & Head, Department of Microbiology,
PARTICULARS OF CONTRIBUTORS:
Dr. A.L. Mudaliar Post Graduate Institute of Basic Medical
1. PhD Research Scholar,
Sciences, University of Madras,
Department of Microbiology,
Taramani, Chennai – 600113, India.
Dr.A.L.Mudaliar Post Graduate Institute of Basic Medical
Phone: 91-044-24547100
Sciences, University of Madras, Taramani,
Fax: 91-44-24540709
Chennai–600113, India.
E-mail: [email protected]
2. Professor & Head,
Department of Microbiology, Financial OR OTHER COMPETING INTERESTS:
Dr. A.L. Mudaliar Post Graduate Institute of Basic Medical None.
Sciences, University of Madras, Taramani, Date of Submission: May 09, 2012
Date of Peer Review: Jun 24, 2012
Chennai–600113, India. Date of Acceptance: Nov 24, 2012
Date of Publishing: Feb 01, 2013

304 Journal of Clinical and Diagnostic Research. 2013 February, Vol-7(2): 302-304