Characterization and specification of microsatellite markers in the HLA-DRB1 gene region: A revision to major histocompatibility complex database Maryam Shaykholeslam Esfahani, Sadeq Vallian ⇑ Division of Genetics, Department of Biology, Faculty of Science, University of Isfahan, Isfahan, Iran article info Article history: Received 6 November 2012 Accepted 10 April 2013 Available online xxxx abstract Association between HLA-DRB1 and a large number of diseases such as multiple sclerosis, type I diabetes and rheumatoid arthritis has been demonstrated. In the present study, we attempted to identify and characterize potential microsatellites in the HLA-DRB1 gene region to find specific markers for genotyp- ing and linkage analysis of this gene. The microsatellites including M2_3_22, M2_2_36, D6S2878, D6S2805, D6S2879 and D6S2880 were selected from microsatellite resource in the Major Histocompat- ibility Complex database (dbMHC). In silico analysis showed that only M2_3_22 was specific for HLA- DRB1. Moreover, our findings revealed some more accurate characteristics of the other investigated microsatellites. M2_3_22 existed as a single copy in all the MHC haplotype sequences and was located next to HLA-DRB1. Therefore, a new set of primers compatible with all the last published MHC haplotype sequences were designed and used to amplify M2_3_22 in 164 DNA samples obtained from unrelated Ira- nian individuals. M2_3_22 was successfully amplified in all DNA samples and three different alleles were identified. This locus was found in the Hardy–Weinberg equilibrium (P > 0.05) in the studied population. Together, the findings suggested that M2_3_22 could be introduced as a specific locus in the HLA-DRB1 gene region for linkage analysis and disease association studies. Ó 2013 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. 1. Introduction The human major histocompatibility complex (MHC) is the most variable and gene-dense region of the human genome, con- taining about 240 genes and pseudo-genes over a 3.6 Mb segment on chromosome 6 [1–3]. The MHC region has been divided into three non-overlapping segments called human leukocyte antigen (HLA) class I, II and III [4,5] (Fig. 1). The HLA class II genes exhibit an extensive degree of genetic polymorphism and encode cell sur- face molecules that present exogenous antigens to the CD4 + T cells [6,7]. Allelic variants of class II genes are associated with a large number of diseases such as rheumatoid arthritis [8], insulin- dependent diabetes mellitus (IDDM) [9,10], and multiple sclerosis (MS) [11,12]. The HLA class II region principally sub-divides into four sub- regions; DP, DO, DQ, and DR [6]. Five different DR haplotype groups exist in the human genome including DR1, DR8, DR51, DR52 and DR53 [13]. DR51, DR52, and DR53 have been sequenced. All DR haplotype groups have one of the highly polymorphic alleles of HLA-DRB1 locus [14]. Moreover, eight different MHC haplotype se- quences have been provided by the MHC haplotype project in 2008. Of the MHC haplotype sequences, PGF, COX, QBL, DBB, MANN, and SSTO contain the complete sequence of HLA-DRB1. PGF contained the DRB1 and DRB5 functional genes and exhibited the DR51 antigenic specificity [3]. COX and QBL contained the DRB1 and DRB3 functional genes and exhibited the DR52 antigenic specificity [15]. SSTO, DBB and MANN contained the DRB1 and DRB4 functional genes and exhibited the DR53 specificity [3]. The association of HLA-DRB1 or HLA-DQB gene with autoim- mune diseases such as MS and IDDM has been a consistent finding across nearly all investigated populations [10,11,16]. However, the complex etiology of MHC-associated diseases, along with the high degree of polymorphism, extensive linkage disequilibrium (LD), and incomplete knowledge of the allelic variation of the genes and regions flanking the classical HLA loci, has complicated the ex- act identification of disease susceptible variations [3,4]. Common methods for investigation of HLA loci including se- quence specific primer-PCR (SSP-PCR), sequence specific oligonu- cleotide probe hybridization (SSOPH) and direct sequencing are usually expensive and time consuming [7,17,18]. However, short tandem repeat (STR) markers, in view of their advantages, provide useful genetic markers in the HLA-related studies. Microsatellites 0198-8859/$36.00 - see front matter Ó 2013 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. http://dx.doi.org/10.1016/j.humimm.2013.04.032 ⇑ Corresponding author. Fax: +98 3117932456. E-mail addresses: persicarose@yahoo.com (M. Shaykholeslam Esfahani), svallian@biol.ui.ac.ir (S. Vallian). Human Immunology xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect www.ashi-hla.org journal homepage: www.elsevier.com/locate/humimm Please cite this article in press as: Shaykholeslam Esfahani M, Vallian S. Characterization and specification of microsatellite markers in the HLA-DRB1 gene region: A revision to major histocompatibility complex database. Hum Immunol (2013), http://dx.doi.org/10.1016/j.humimm.2013.04.032