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Djulejic et al. Allele Frequency of HLA-DQB1 Locus in Macedonian Population
Macedonian Journal of Medical Sciences. 2012 Mar 15; 5(1):67-71.
http://dx.doi.org/10.3889/MJMS.1857-5773.2012.0223
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Allele Frequency of HLA-DQB1 Locus in Macedonian Population
Eli Djulejic1, 2, Aleksandar Senev1, Meri Kirijas1, Slavica Hristomanova1, Aleksandar Petlichkovski 1, Dejan Trajkov1, Mirko Spiroski 1
1
Institute of Immunobiology and Human Genetics, Faculty of Medicine, Ss. Cyril and Methodius University in Skopje, Skopje, Republic of
Macedonia; 2Quintiles, Belgrade, Republic of Serbia
Abstract
Citation: Djulejic E, Senev A, Kirijas M,
Hristomanova S, Petlichkovski A, Trajkov D,
Spiroski M. Allele Frequency of HLA-DQB1 Locus
in Macedonian Population. Maced J Med Sci.
2012 Mar 15; 5(1):67-71. http://dx.doi.org/10.3889/
MJMS.1957-5773.2012.0223.
Key words: HLA DQB1; Reverse Line Strip
typing; alleles; NMDP Codes; Macedonian
population.
Correspondence: Prof. Dr. Mirko Spiroski.
Institute of Immunobiology and Human Genetics,
Faculty of Medicine, Ss. Cyril and Methodius
University in Skopje, 50 Divizija No 16, 1109
Skopje, PO Box 60, Republic of Macedonia.
URL: http://www.iibhg.ukim.edu.mk
E-mail: [email protected]
Received: 17-Dec-2011; Revised: 09-Jan-2012;
Accepted: 18-Feb-2012; Online first: 22-Feb-2012
Copyright: © 2012 Djulejic E. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Aim: The aim of this paper was to genotype HLA DQB1 locus in healthy unrelated Macedonian
population.
Material and Methods: Reverse Line Strip typing for HLA DQB1 locus was performed on a population
of 217 samples from healthy individuals. The results were obtained as alleles, and as NMDP Codes.
Alleles were genotyped and expressed as high resolution with more than four digits with slash between
ambiguous alleles. NMDP Codes were expressed as four digits for unambiguous results, and with
combination of numbers and characters for the certain ambiguous combination. We did not found any
genotypic ambiguities in HLA-DQB1.
Results: We have found 33 different alleles of HLA DQB1 in Macedonian population, 12 of which were
unambiguous with the frequency of 40.55%, and 21 were ambiguous with the frequency of 58.53%. The
biggest frequency (33.64%) was found for HLA DQB1*05 unambiguous groups, and for HLA DQB1*03
ambiguous groups (36.40%). The biggest allele frequency in Macedonian population for HLA DQB1 was
found for HLA DQB1*03NX (0301/01/01/02/09/13) with 27.88%, followed with HLA DQB1*0502 with
13.82%, and HLA DQB1*02MN (0201/02/03) with 10.37%.
Conclusion: Allele frequency of HLA DQB1 in Macedonian population is similar with that found in other
European populations and can be used for anthropology and disease association studies.
Competing Interests: The authors have declared
that no competing interests exist.
Introduction
Highly polymorphic human leukocyte antigen
(HLA) gene, a cluster of closely linked genes, is located
on the short arm of chromosome 6. It is located alongside
a remarkably large number of other genes that are either
known or predicted to have immunological functions,
such as genes for tumour necrosis factor-alpha, a key
mediator of inflammatory response to infection and for
various complement and heat shock proteins. Previous
studies have highlighted that HLADQB1 polymorphism
influence individual immune response and thus affect
the outcome of diseases. Many diseases, especially
autoimmune disorders are related to HLA class II gene
polymorphisms. The relationship between the HLA gene
polymorphisms and diseases shows racial, ethnic and
geographic differences.
Maced J Med Sci. 2012 Mar 15; 5(1):67-71.
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The genes for the heteromeric major
histocompatibility complex class II proteins, the alpha
and beta subunits, are clustered in the 6p21.3 region.
Todd et al. (1987) presented a map of the class II loci.
They suggested that the structure of the DQ molecule, in
particular residue 57 of the beta-chain, specifies the
autoimmune response against insulin-producing islet
cells that leads to insulin-dependent diabetes mellitus
(IDDM; OMIM: 222100) [1]. Of the approximately 14
class II HLA genes within the HLA-D region, the DQ3.2beta gene accounts for the well-documented association
of HLA-DR4 with insulin-dependent diabetes mellitus
and is the single allele most highly correlated with this
disease. Kwok et al. (1989) found that amino acid 45 was
critical for generating serologic epitopes characterizing
the DQ3.2-beta gene and its nondiabetic allele, DQ3.1beta [2]. Todd et al. (1990) found that in Japanese IDDM
was more strongly associated with HLA-DQ than with
HLA-DR (OMIM: 142857); that the A3 allele at the DQA1
(OMIM: 146880) locus was most strongly associated
with disease; that the DQw8 allele of the DQB1 locus,
which is associated with susceptibility to type I diabetes
in Caucasians and blacks, was not increased in frequency
in Japanese patients; and that asp57-encoding DQB1
alleles, which are associated with reduced susceptibility
to type I diabetes in Caucasians, was present in all
except 1 of 49 Japanese patients and in all of 31 controls,
in at least heterozygous state. Forty percent of patients
were homozygous for asp57-encoding DQB1 alleles
versus 35% of controls [3].
In addition to insulin-dependent diabetes
mellitus, an increased frequency of specific alleles at the
DQB1 locus has been claimed for narcolepsy (OMIM:
161400), pemphigus vulgaris (169610), and ocular
cicatricial pemphigoid (OCP; OMIM: 164185). Mignot et
al. (1997) found that narcolepsy is tightly associated with
the HLA-DQB1*0602 allele, especially when the
manifestations include typical cataplexy [4].
The molecule encoded by DQA1*0102/
DQB1*0602, termed DQ0602, confers strong
susceptibility to narcolepsy but dominant protection
against type I diabetes. To elucidate the molecular
features underlying these contrasting genetic properties,
Siebold et al. (2004) determined the crystal structure of
the DQ0602 molecule at 1.8-angstrom resolution [5].
Structural comparisons to homologous DQ molecules
with differential disease associations highlighted a
previously unrecognized interplay between the volume
of the P6 pocket and the specificity of the P9 pocket,
which implies that presentation of the expanded peptide
68
repertoire is critical for dominant protection against type
I diabetes. In narcolepsy, the volume of the P4 pocket
appears central to the susceptibility, suggesting that the
presentation of a specific peptide population plays a
major role [5].
Cicatricial pemphigoid (CP) is a chronic
autoimmune blistering disease affecting multiple mucous
membranes derived from stratified squamous epithelium
and occasionally the skin. CP has a wide spectrum of
disease manifestations. Patients with oral pemphigoid
(OP) have a benign self-limited disease in which
pathologic changes are restricted to the oral mucosa.
On the other hand, patients with ocular cicatricial
pemphigoid, a chronic condition marked by relapses
and remissions, have ocular involvement and also
perhaps involvement of other mucous membranes. All
clinical types are characterized by the presence of
similar anti-basement zone autoantibody. The factors
that determine the development of one form of CP or the
other are not known. Yunis et al. (1994) studied class II
alleles by DNA testing in 22 Caucasian patients with OP
and their families (19 families) [6]. The results were
compared to those obtained from 17 OCP patients and
their family controls and those of 42 control Caucasian
families studied for bone marrow transplantation. The
results indicated that HLA-DQB1*0301 is a marker of
susceptibility for both oral and ocular forms of CP. The
analysis of the amino acid sequence of the DQB1 alleles
present in both OP and OCP suggested that amino acid
residues at position 57 and positions 71-77 may also be
markers [6].
The aim of the study was to analyze the allele
frequency of HLA-DQB1 locus in Macedonian population.
Material and Methods
Population samples
Two hundred and seventeen unrelated random
healthy Macedonian volunteers of Macedonian origin
and nationality residents of different regions of the
Republic of Macedonia were included in this study. Their
ages ranged between 20 and 59 years. Peripheral blood
was drawn after signing of the informed consent. Genomic
DNA was extracted from the peripheral blood leukocytes
using the standard phenol/chloroform procedure
previously described [7], and stored in the anthropology
project field of our DNA Bank (hDNAMKD) until
processing [8].
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Djulejic et al. Allele Frequency of HLA-DQB1 Locus in Macedonian Population
PCR amplification
Table 1: The distribution of unambiguous and ambiguous results
of HLA-DQB1 typing by RLS method in Macedonian population.
PCR amplification was performed with
biotinylated primers of HLA-DQB1 genes. The PCR
cycling conditions were: 96°C, 60 sec; 96°C, 15sec;
58°C, 45sec; 72°C, 15s; for 35 cycles, extension at 72°C
for 5 min. and hold program for 15°C indefinitely.
HLA-DQB1 genotyping
HLA-DNA typing of HLA-DQB1 genes was
performed by using the reverse hybridization method
(Dynal RELI SSO HLA-DQB1 Typing Kit, UK). The
labeled PCR products were hybridized in a single reaction
to an array of SSO probes immobilized on nylon
membrane with Bee Robotics, UK. Different probes for
HLA-DQB1 typing were used with corresponding motifs.
The presence of biotinylated PCR product bound to a
specific probe was detected using streptavidinhorseradish peroxidase (HRP) and a chromogenic,
soluble substrate to produce a blue “line” at the position
of the positive probe. Genotyping software (LIRAS for
LIPA HLA v.6.00) can interpret the probe reactivity
pattern as a genotype and indicates potential ambiguities
[9].
Statistical analysis
The statistical analysis of the observed allele
frequencies was performed by using the Arlequin
Software version 2.000 [10].
Results
The results were obtained as alleles, and as
NMDP Codes. Alleles were genotyped and expressed
as high resolution with more than four digits with slash
between ambiguous alleles. NMDP Codes were
expressed as four digits for unambiguous results, and
with combination of numbers and characters for the
certain ambiguous combination. We did not found any
genotypic ambiguities in HLA-DQB1.
We found 33 different alleles of HLA-DQB1 in
Macedonian population, 12 of which were unambiguous
with the frequency of 40.55% and 21 were ambiguous
with the frequency of 58.53%. The biggest frequency
(33.64%) was found for 05 unambiguous groups, and for
03 ambiguous groups (36.40%). The biggest allele
frequency in Macedonian population for HLA-DQB1 was
found for 03NX (030101/0102/09/13) with 27.88%,
followed with 0502 with 13.82%, and 02MN (0201/02/
03) with 10.37% (Table 1).
The most frequent allele in Macedonian
population is HLA-DQB1*03 with frequencies between
0.346 and 0.433 followed by HLA-DQB1*05 with
frequencies between 0.276 and 0.345. HLA-DQB1*04
allele is present in lowest frequencies in Macedonian
population from 0 to 0.016 (Table 2).
Table 2: HLA-DQB1 allele frequencies in several Macedonian
populations [11].
Discussion
In this paper we presented HLA-DQB1 allele
frequencies in the Macedonian population. We found
that HLA-DQB1*03 was the most frequent allele (0.377),
followed by HLA-DQB1*05 (0.345), HLA-DQB1*06
(0.152), HLA-DQB1*02 (0.108), and the lowest frequency
Maced J Med Sci. 2012 Mar 15; 5(1):67-71.
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of HLA-DQB1*04 (0.016). Our results, obtained in the
Macedonian laboratory with reverse line strip assay, are
very similar with other published results obtained in
different laboratories of European countries: Finland
[12], Netherlands [13], and Spain [14].
HLA-DQB1 locus was not included in the genetic
structure separation of European populations. But for
Europe, the results obtained for the 3 loci (HLA-A, HLAB
and HLA-DRB1) indicate a major genetic structure
separating West-Central (Austrians and Czech, plus
Polish, French and Spanish, at locus DRB1) and North
(Irish, Finns) from Southeast (Macedonians, Greeks,
Albanians and Croatians, plus Italians, Greek Pomaki,
Slovenians, Romanians and Macedonians, at locus
DRB1) Europeans along the first principal axis of the
PCoordA (Figures 1c, 2c and 3c), while the second axis
separates either the Irish on one side and the Finns on
the other side (loci A and B in Figures 1c and 2c), or the
Sardinians (locus DRB1 in Figure 3c). The sample from
Quebec has been included in this analysis because of
the recent European descent of Quebecers. All loci
indicate that this population is closer to West than to
Southeast Europeans [15]. We expect that HLA-DQB1
allele frequencies in European population follow the
similar genetic structure separation.
Several diseases were found to be associated
with HLA-DQB1 alleles. Type IA diabetes is an
autoimmune disease with genetic and environmental
factors contributing to its etiology. Twin studies, family
studies, and animal models have helped to elucidate the
genetics of autoimmune diabetes. Most of the genetic
susceptibility is accounted for by human leukocyte antigen
(HLA) alleles. The most-common susceptibility
haplotypes are DQA1*0301-DQB1*0302 and
DQA1*0501-DQB1*0201. Less-common haplotypes
such as DQA1*0401-DQBl*0402 and DQA1*0l0lDQB1*0501 are associated with high risk for diabetes;
however, large study populations are needed to analyze
their effect. The DQA1*0102-DQB1*0602 haplotype is
associated with diabetes resistance [16]. Narcolepsy is
classically associated with HLA DQB1*0602, the most
specific genetic marker for narcolepsy across all ethnic
groups [17].
In summary we can say that frequency of HLADQB1 alleles in Macedonian population are very similar
with other European populations and can be used for
anthropological comparisons and association studies
with different diseases.
Acknowledgement
The authors thank Elena Cvetkovska for isolation
of genomic DNA and taking care of the Macedonian
Human DNA Bank (www.hdnamkd.org.mk), as well as
Dr. Ana Strezova which was involved in the routine HLADNA genotyping. This work was supported in part from
the ICGEB collaborative research program (CRP/
MAC03-01), Trieste, Italy.
References
1. Todd JA, Bell JI, McDevitt HO. HLA-DQ beta gene
contributes to susceptibility and resistance to insulin-dependent
diabetes mellitus. Nature. 1987;329(6140):599-604.
2. Kwok WW, Lotshaw C, Milner EC, Knitter-Jack N, Nepom
GT. Mutational analysis of the HLA-DQ3.2 insulin-dependent
diabetes mellitus susceptibility gene. Proc Natl Acad Sci U S
A. 1989;86(3):1027-30.
3. Todd JA, Fukui Y, Kitagawa T, Sasazuki T. The A3 allele of
the HLA-DQA1 locus is associated with susceptibility to type 1
diabetes in Japanese. Proc Natl Acad Sci U S A.
1990;87(3):1094-8.
4. Mignot E, Hayduk R, Black J, Grumet FC, Guilleminault C.
HLA DQB1*0602 is associated with cataplexy in 509 narcoleptic
patients. Sleep. 1997;20(11):1012-20.
5. Siebold C, Hansen BE, Wyer JR, Harlos K, Esnouf RE,
Svejgaard A, Bell JI, Strominger JL, Jones EY, Fugger L. Crystal
structure of HLA-DQ0602 that protects against type 1 diabetes
and confers strong susceptibility to narcolepsy. Proc Natl Acad
Sci U S A. 2004;101(7):1999-2004.
6. Yunis JJ, Mobini N, Yunis EJ, Alper CA, Deulofeut R,
Rodriguez A, Foster CS, Marcus-Bagley D, Good RA, Ahmed
AR. Common major histocompatibility complex class II markers
in clinical variants of cicatricial pemphigoid. Proc Natl Acad
Sci U S A. 1994;91(16):7747-51.
7. Towner P. Purification of DNA. Essential molecular biology
(ed. Brown TA). Oxford University Press, Oxford 1995; 47-54.
8. Spiroski M, Arsov T, Petlichkovski A, Strezova A, Trajkov D,
Efinska-Mladenovska O, Zaharieva E. Case Study:
Macedonian Human DNA Bank (hDNAMKD) as a source for
public health Genetics. In: Georgieva L, Burazeri G (eds.).
Health determinants in the scope of new public health. Hans
Jacobs Company: Sofia 2005; 33-44.
9. Saiki RK, Walsh PS, Levenson CH, Erlich HA. Genetic
analysis of amplified DNA with immobilized sequence-specific
oligonucleotide probes. Proc Natl Acad Sci U S A.
1989;86(16):6230-4.
10. Schneider S, Roessli D, Excoffier L. Arlequin version 2.000:
a software for population genetics data analysis. Geneva
70
http://www.mjms.ukim.edu.mk
Unauthenticated
Download Date | 6/18/17 8:11 AM
Djulejic et al. Allele Frequency of HLA-DQB1 Locus in Macedonian Population
(Switzerland): Genetics and Biometry Laboratory, University
of Geneva; 2000.
and the sub-Saharan origin of the Greeks. Tissue Antigens.
2001;57(2):118-27.
11. Gonzalez-Galarza FF, Christmas S, Middleton D and Jones
AR. Allele frequency net: a database and online repository for
immune gene frequencies in worldwide populations. Nucleic
Acid Research. 2011; 39:D913-D919.
15. Nunes JM, Riccio ME, Buhler S, Di D, Currat M, Ries F,
Almada AJ, Benhamamouch S, Benitez O, Canossi A,
Fadhlaoui-Zid K, Fischer G, Kervaire B, Loiseau P, de Oliveira
DC, Papasteriades C, Piancatelli D, Rahal M, Richard L,
Romero M, Rousseau J, Spiroski M, Sulcebe G, Middleton D,
Tiercy JM, Sanchez-Mazas A. Analysis of the HLA population
data (AHPD) submitted to the 15th International
Histocompatibility/Immunogenetics Workshop by using the
Gene[rate] computer tools accommodating ambiguous data
(AHPD project report). Tissue Antigens. 2010;76(1):18-30.
12. Ilonen J, Kocova M, Lipponen K, Sukarova-Angelovska E,
Jovanovska A, Knip M. HLA-DR-DQ haplotypes and type 1
diabetes in Macedonia. Hum Immunol. 2009;70(6):461-3.
13. Hristova-Dimceva A, Verduijn W, Schipper RF, Schreuder
GM. HLA-DRB and -DQB1 polymorphism in the Macedonian
population. Tissue Antigens. 2000;55(1):53-6.
14. Arnaiz-Villena A, Dimitroski K, Pacho A, Moscoso J, GómezCasado E, Silvera-Redondo C, Varela P, Blagoevska M,
Zdravkovska V, Martínez-Laso J. HLA genes in Macedonians
16. Redondo MJ, Fain PR, Eisenbarth GS. Genetics of type
1A diabetes. Recent Prog Horm Res. 2001;56:69-89.
17. Sullivan SS. Narcolepsy in adolescents. Adolesc Med State
Art Rev. 2010 Dec;21(3):542-55, x-xi.
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