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The Dıagnosıs of Human Leukocyte Antıgen Class I and Class II Allel ın Eastern Anatolıa Regıon

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INTRODUCTION: The aim of our study was to investigate the frequency of HLA class I and class II alleles in Eastern Anatolia. METHODS: The study included 1050 unrelated bone marrow donors from the archives. HLA class I and II polymorphisms were identified using polymerase chain reactions (PCR) using sequence-specific primers (SSP) and sequence-specific oligonucleotides (SSO). Ethical approval was obtained. RESULTS: The most common alleles were HLA-A*02 (21.1%), HLA-A*24 (18.4%), and HLA-A*03 (12.3%) in the HLA-A locus; HLA-B*35 (20%), HLA-B*51 (18.3%), and HLA-B*44 (6.3%) in the HLA-B locus; and HLA-DRB1*11 (22.3%), HLA-DRB1*04 (16.2%), and HLA-DRB1*15 (12.4%) in the HLA-DR locus. DISCUSSION AND CONCLUSION: : Our results are consistent with the HLA antigens identified in previous studies. We believe that our study will contribute to the determination of HLA diversity in our region.. Consolidating the data obtained in this study and other national data will facilitate the selection of bone marrow donors
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Van p D er g 26(2): 162-166, 2019
DOI: 10.5505/vtd.2019.42042
*Sorumlu Yazar: Dr Öğr Üyesi Eda Balkan, Ataturk Unıversıty Med ıcal B iology Erzurum /Turkey
E-m ail:ed a.diyarbakir@hotm ail.com, Phone: 0 (53 3) 540 28 26
Geliş Tarihi : 03.04.2018, Kabul Tarihi: 23.10.2018
KLİNİK ÇALIŞMA / CLINICAL RESEARCH
The Diagnosis of Human Leukocyte Antigen Class I
and Class Ii Allel In Eastern Anatolia Region
Doğuanadolu Bölgesinde İnsan Lökosit Antijennıf I ve Sınıf II Alel Dağılımı
Eda Balkan*, Ezgi Yaşar, Hasan Doğan
Atatürk Üniversitesi p Faltesi Tibbi Biyoloji Anabilim Dalı Erzurum Türkiye
Introduction
The MHC (major histocompatibility complex),
which is found in all vertebrates, is a diverse
group of genes encoding molecules with and
without immune-related functions. The human
MHC is called the human leukocyte antigen
(HLA) genes. HLAs are glycoproteins expressed
by the human MHC region. In humans, the MHC
is located on the short arm of chromosome 6.
HLA antigens are divided into three groups
according to their structural and functional
properties: Class I (HLA-A, -B, -C), Class II
(HLA-DR, -DQ, -DP), and Class III (properdin
factor B [FB], tumor necrosis factor [TNF], C4A,
C4B, and C2). The physiological function of HLA
molecules is to present peptides derived from
protein antigens to antigen-specific T lymphocytes
(1).
HLAs have considerable clinical importance. HLA
molecules are the main cause of acute rejection
after transplantation. Graft survival is associated
with HLA compatibility between the recipient and
donor.
In addition, it has been determined that some
diseases have a higher prevalence among
individuals with certain HLA alleles. These include
the common conditions ankylosing spondylitis
(B27), celiac disease (DR3), and Behçet's disease.
There are several hypotheses regarding the role of
HLAs in autoimmune disease, such as antigenic
mimicry between pathogens and HLA, and
antigenic hypo-and hyperresponsiveness regulated
ABSTRACT
Objective: The aim of our study was to investigate the
frequency of HLA class I and class II alleles in Eastern
Anatolia.
Materials and Methods: The study included 1050
unrelated bone marrow donors from the archives. HLA
class I and II polymorphisms were identified using
polymerase chain reactions (PCR) using sequence -
specific primers (SSP) and sequence-specific
oligonucleotides (SSO). Ethical approval was obtained.
Results: The most common alleles were HLA-A*02
(21.1%), HLA-A*24 (18.4%), and HLA-A*03 (12.3%) in
the HLA-A locus; HLA-B*35 (20%), HLA-B*51 (18.3%),
and HLA-B*44 (6.3%) in the HLA-B locus; and HLA-
DRB1*11 (22.3%), HLA-DRB1*04 (16.2%), and HLA-
DRB1*15 (12.4%) in the HLA-DR locus.
Conclusion: Our results are consistent with the HLA
antigens identified in previous studies. We believe that
our study will contribute to the determination of HLA
diversity in our region. Consolidating the data obtained
in this study and other national data will facilitate the
selection of bone marrow donors.
Key Words: HLA; Class I; Class II
ÖZET
Amaç: Çalışmamızın amacı Doğu Anadolu bölgesindeki
HLA sınıf I ve sınıf II alelerinin sıklığının araştırılmasıdır.
Gereç ve Yöntemler: Çalışmaya aralarında herhangi bir
akrabalık ilişkisi olmayan 1050 kemik iliği vericisi alındı.
HLA nıf I ve II polimorfizmleri, polimeraz zincir
reaksiyonu (PCR) metodu ile sekansa öz primerler
(SSP) ve sekansa spesifik oligonükleotidler (SSO)
kullanılarak belirlendi. Etik kurul onayı alınmıştır.
Bulgular: En yaygın aleller, HLA -A lokusunda, HLA-
A*02 (21,1%), HLA-A*24 (18,4%), HLA-A*03 (12,3%),
HLA-B lokusunda HLA-B*35 (20%), HLA-B*51 (18,3%),
HLA-B*44 (6,3), HLA-DR lokusunda HLA-DRB1*11
(22,3%), HLA-DRB1*04 (16,2%), HLA-DRB1*15
(12,4%) olarak belirlendi.
Son: Çaşma sonuçlarımız, daha önce yapılan
çalışmalarda tespit edilen HLA antijenleri ile uyumludur.
Çalışmamız bölgemizdeki HLA çeşitliliğinin
belirlenmesinde kat sağlayacağı şünmekteyiz.
Çalışmamızda elde ettiğimiz veriler ve der ulusal
verilerin toplanılması kemik iliği bağışçılarının seçiminde
yardımolacaktır
Anahtar Kelimeler: HLA; sınıf I; sınıf II
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Van Tıp Derg Cilt:26, Sayı:2, Nisan/2019
163
Table 1. Distribution of human leukocyte antigen (HLA)-A allele frequencies
HLA-A alleles
Allele Frequency (%)
A*02
21.1%
A*24
18.4%
A*03
12.3%
A*11
11.3%
A*01
9.2%
A*26
6%
A*68
4.8%
A*32
3.5%
A*29
3.5%
A*23
3.1%
A*30
2.9%
A*31
1.5%
A*31
1%
A*28
0.5%
A*66
0.4%
A*25
0.3%
A*69
0.1%
by class II genes (2).
There is a broad range of HLA molecules in the
population. The frequencies of the HLA alleles
show considerable variation between different
populations. HLA diversity results in significant
immunological differences among individuals. A
total of 17,695 HLA alleles have been reported to
date, 12,893 in the class I region and 4,802 in the
class II region, and this number continues to
increase (3).
In the present study, we aimed to determine the
frequency of HLA alleles in healthy individuals
tested by the Tissue Typing Laboratory of a
Health Research and Application Center Hospital
between 2007 and 2017, and to assess the results
of HLA genotyping (4-6).
Materials and Methods
A total of 1,050 unrelated volunteer bone marrow
donors who applied for HLA tissue typing were
included. Genomic DNA was isolated from whole
blood using EZ-1 isolation kit (QIAGEN GmbH,
Hilden, Germany). HLA Class I and II (HLA-A, -
B, -DRB1) tissue typing was performed on all
samples using low-resolution polymerase chain
reaction using sequence-specific primers (PCR-
SSP) (One lambda, SSP AB, Stockholm, Sweden)
and Luminex-based sequence-specific
oligonucleotide primed PCR (PCR-SSO)
(Lifecodes, Immucor, Norcross, GA, USA).
PCR-SSO amplification was performed in a
Labcycler (SensoQuest GmbH, Germany).
Following the oligonucleotide probes and
conjugated streptavidin on hybridized fluorescent
beads step, the results were analyzed using
MatchIT Software (version 3.27). PCR-SSP
amplification was performed in a PE 9700 thermal
cycler and the resulting PCR products were loaded
in an agarose gel, visualized in ultraviolet (UV)
light. Positives were documented and analyzed
using the Start Scored program.
The study was conducted in accordance with the
principles of the Declaration of Helsinki and was
approved by the local ethics committee (Atatürk
University Faculty of Medicine Ethics Committee
no:21). Written informed consent forms were
obtained from each volunteer bone marrow
donor. The frequencies of HLA-A, -B, and -DR
alleles were analyzed using SPSS the study data
were analysed using SPS version 23.0 (IBM
Corp., Armonk, NY, USA) statistical software
package. Frequency distributions were used for
comparisons.
Results
Seventeen different HLA-A alleles were identified
in our group of 1,050 bone marrow donors. The
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Van Tıp Derg Cilt:26, Sayı:2, Nisan/2019
164
Table 2. Distribution of human leukocyte antigen (HLA)-B allele frequencies
HLA-B alleles
Allele Frequency (%)
B*35
20.0%
B*51
18.3%
B*44
6.3%
B*49
6.2%
B*52
5.1%
B*18
4.5%
B*38
4.3%
B*55
4.2%
B*07
4.2%
B*50
3.7%
B*08
3.6%
B*41
2.4%
B*40
2.3%
B*27
2.2%
B*15
2.2%
B*13
1.9%
B*57
1.5%
B*58
1.4%
B*14
1.4%
B*39
0.9%
B*53
0.6%
B*37
0.6%
B*45
0.4%
B*47
0.3%
B*56
0.2%
B*60
0.2%
Table 3. Distribution of human leukocyte antigen (HLA)-DR allele frequencies
HLA-DR alleles
Allele Frequency (%)
DRB1*11
22.3%
DRB1*04
16.2%
DRB1*15
12.4%
DRB1*13
11.9%
DRB1*03
9.0%
DRB1*07
6.9%
DRB1*14
6.5%
DRB1*01
5.2%
DRB1*16
3.1%
DRB1*08
2.7%
DRB1*10
2.1%
most common alleles in the HLA-A locus were
HLA-A*02 (21.1%), HLA-A*24 (18.4%), and
HLA-A*03 (12.3%) (Table 1). Twenty-six
different alleles were detected in the HLA-B locus.
The most common HLA-B alleles were HLA-
B*35 (20%), HLA-B*51 (18.3%), HLA-B*44
(6.3%) (Table 2). In the HLA-DR locus, the most
common alleles were HLA-DRB1*11 (22.3%),
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Van Tıp Derg Cilt:26, Sayı:2, Nisan/2019
165
HLA-DRB1*04 (16.2%), and HLA-DRB1*15
(12.4%) (Table 3).
Discussion
The most frequently detected HLAs in our study
were A*02, B*35, and DRB1*11. Previous studies
have identified HLA antigen types in different
populations and groups using a variety of
methods. Our findings are similar to the most
common HLA alleles found in other regional
studies conducted in Turkey.
Choukri et al. (7) reported the distribution of
HLA-A and -B alleles among 100 healthy,
unrelated individuals living in the Casablanca
region of Morocco as HLA-A*02 (21%), A*01
(11%), A*03 (10%), B*44 (11.4%), B*50 (9.9%),
B*05 (8.5%), and B*35 (6.5%).
In a study by Bardi et al., (8) the most common
alleles were HLA-A*02, 24, 01, HLA-B*35, 44, 51,
and DRB1*11, 13, 07 among Caucasians; HLA-
A*02, 03, 30, HLA-B*35, 15, 44, and DRB1*13,
11, 03 among Afro-Brazilians; and HLA-A*24, 02,
26, HLA-B*40, 51, 52, and DRB1*04, 15, 09
among Asians.
Chen et al. (9) reported the distribution of HLA-
A, -B, and -Cw as A*24 (27.2%), B*51 (16.8%),
Cw*04 (13.3%), and Cw*070201G1 (13.3%)
among 158 unrelated Tibetan Chinese individuals.
In studies conducted in Turkey, Patıroğlu et al.
(10) investigated the distribution of HLA antigens
in the Kayseri region and identified 185 HLA
Class I and 125 HLA Class II molecules using
microcytotoxicity assay. They determined the most
common alleles in that region to be HLA-A*02,
A*09, A*03, HLA-B*05, B*35, B*12, HLA-
Cw*04, HLA-DR*05, DR*02, and HLA-DQ*03.
Pala et al. (11) reported that HLA-A*02 (20.5%),
HLA-B*35 (22.9%) and HLA-DR*11 (17.6%)
were the most common HLAs in a Thracian
population.
In a study by Erikoğlu et al. (12) including 362
subjects (57% male, 43% female), the most common
HLAs were HLA-A*02 (48%), HLA*B35 (33%), and
HLA-DR*B11 (48%).
In another study, Kayhan et al. (13) reported that
the most frequent alleles in the HLA-A, -B, and -
DRB1 groups were HLA-A*02, 24, 11; HLA-
B*35, 51, 44; and HLA-DRB1*11, 04, 13. They
also reported the most common haplotypes as
HLA-A*02-B*51-DRB1*11, HLA-A*11-B*35-
DRB1*11, and A*24-B*35-DRB1*11.
Söyöz et al. (6) determined that the highest
frequency alleles among 450 bone marrow donors
were HLA-A*02, HLA-B*35, and HLA-DRB1*11.
The results of our study corroborate the findings
of other studies conducted in Turkey. We believe
our study makes an important contribution to
HLA allele frequency data in the Eastern Anatolia
region of Turkey. Our next study will investigate
HLA haplotype frequency in a larger Turkish
population.
Conflict of Interest: None
Source of Funding: None
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... Ayrıca çalışmamızda ki A*02, A*24, A*03 sıralamasının, A lokus antijenleri için daha önce toplumumuzla ilgili yapılan diğer çalışmalarla uyumlu olduğu görülmektedir [16][17][18][19][20]. Sonuçlarımız ülkemizde yapılan diğer çalışmalarda tanımlanan HLA antijenleri ile karşılaştırıldığında bazı farklılıklar göstermesine karşın büyük ölçüde uyumluydu. ...
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We have studied the distribution of HLA-A and -B alleles and haplotypes by sequence-specific primer amplification in a sample of 100 unrelated healthy individuals belonging to both Berber and Arabic-speaking groups from the region of Casablanca in Morocco. Among the 17 HLA-A and 23 HLA-B alleles observed, the most frequent were HLA-A2 (21%), -A1 (11%), -A3 (10%), -B44 (11.4%), -B50 (9.9%), -B5(8.5%) and -B35 (6.5%). Six two-locus haplotypes were observed with a frequency above 5%: A2-B50 (9.6%), A23-B44 (7.4%), A2-B15 (6.4%), A68-B39 (5.3%), A1-B51 (5.3%) and A68-B44 (4.3%). Our data confirm that, on the basis of genetic distances, the majority of present-day North Africans from Morocco are closely related to Berbers and also to Iberians. They cluster apart from Middle-Eastern Mediterranean populations, and show greater genetic distances to Eastern and other Mediterranean populations. This study will serve as a reference for further anthropological studies, as well as studies of HLA and disease associations.
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Tibetans live in Qinghai-Tibet Plateau rising about 4000 m a.s.l. in south-west China. Archaeological evidences suggested that there have been humans living in Tibet at least 5000 years ago. However, Tibetan earlier history remains elusive. In the present study, allelic distribution of human leucocyte antigen (HLA)-A, -B and -Cw in 158 unrelated Tibetan Chinese was investigated using sequencing-based typing methods, and a total of 25 HLA-A, 45 HLA-B and 20 HLA-Cw alleles were identified. A*24G1 (27.2%), B*51G1 (16.8%), Cw*04G1 (13.3%) and Cw*070201G1 (13.3%) are the most common HLA-A, -B and -Cw alleles. The most frequently detected haplotypes were A*24G1-B*51G1-Cw*140201 (3.6%), A*24G1-B*51G1 (6.8%), A*02G1-Cw*070201G1 (6.5%) and B*51G1-Cw*140201 (5.0%). Chi-squared test suggested that all three loci fitted the Hardy-Weinberg expectations. No evidence for a departure from selective neutrality at the HLA-A and -B loci was observed. However, significant departure of the observed homozygosity from the expected values was found for HLA-Cw. Though the contemporary Tibetans inhabit the south-west China, Nei's genetic distance measure based on frequencies of HLA-A, -B and -Cw indicated that Tibetans were closer to northern Han Chinese, Mongolian Chinese, Koreans and Japanese rather than to southern Han Chinese. The corresponding dendrogram constructed according to the neighbour-joining method supported that Tibetans separated from southern Han and located in North-East Asian cluster which included northern Han Chinese and Mongolian Chinese. These data were in good agreement with language classification and with a recent hypothesis that Tibetan might originate from northern China along Yellow river.