Analysis of variation in the sla drb1 allelic type and frequency among seven different pig breeds using a comprehensive high resolution genotyping

Chankyu Park Analysis of variation in the SLA-DRB1 allelic type and frequency among seven different pig breeds using a comprehensive high resolution genotyping Submitted by Le Minh

Supervisor: Prof Chankyu Park

Analysis of variation in the SLA-DRB1 allelic type and frequency among seven

different pig breeds using a

comprehensive high resolution

genotyping

Submitted by

Le Minh Thong

2010.02

Master’s Program in Department of Animal Biotechnology

Graduate school of Konkuk University

Seoul, Korea

석사학위 청구논문

지도교수 박 찬규

Analysis of variation in the SLA-DRB1 allelic type and frequency among seven

different pig breeds using a

comprehensive high resolution

genotyping

고해상도 SLA-DRB1 유전자형 분석

기술의 개발 및 품종 간 특성분석

2010 년 2 월 건국대학교 대학원 동물생명공학과

레민통

Supervisor: Prof Chankyu Park

Analysis of variation in the SLA-DRB1 allelic type and frequency among seven

different pig breeds using a

comprehensive high resolution

genotyping

Submitted by

Le Minh Thong

2010.02

A thesis submitted for the degree of Master of Science

Department of Animal Biotechnology

Graduate school of Konkuk University

Approved by Examination Committee:

Chairman:

Member:

Member:

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF TABLES

Abstract

1 Introduction 2

2 Materials and Methods 4

2.1 Animal 4

2.2 Isolation of genomic DNA and RNA 4

2.3 Primer designing and PCR amplification 4

2.4 Cloning and plasmid isolation 5

2.5 DNA sequencing 6

2.6 Alleles discrimination and population analysis 6

3 Results and discussion 7

3.1 Generation of intronic sequences to design a primer set for SLA-DRB1 exon2 genotyping 7

3.2 Designing of PCR primers for SLA-DRB1 specific amplification 11

3.3 Designing of a DRB1 genotyping protocol using direct sequencing 13

3.4 Evaluation of the accuracy of genotyping results 14

3.5 Allelic diversity of SLA-DRB1 16

References 22

초록 28

Appendix 29

LIST OF FIGURES

Figure 1: The amplification position of primers on exon 2 10

Figure 2: The PCR products amplified from exon 2 of DRB1 region 12

Figure 3: Effect of the specific primer of method 13

Figure 4: The structure of MHC class II and map of primers used in the method 14

Figure 5: The phylogenetic analysis of assigned alleles and new alleles C21-12 18

LIST OF TABLES

Table 1 PCR primers used for the amplification of SLA-DRB1 regions and

sequence analysis 8

Table 2 Comparison of allele types and frequencies of SLA-DRB1 alleles

among seven pig breeds by genomic sequence based typing 17

Table 3 Differences in SLA-DRB1 heterozygosity among seven pig breeds 19

Table 4 Summary of DQB1-DRB1 haplotypes detected in this study from KNP,

SNU minature and NIH miniature pigs 20

Abstract

As an effort to enable simple and comprehensive high resolution genotyping of swine leukocyte antigens (SLA) genes, a SLA-DRB1 genomic sequence based typing (GSBT) method was developed for genotype a hyperpolymorphic MHC class

II beta chain gene, DRB1 To obtain sequence information of intronic regions surrounding SLA-DRB1 exon 2 from diverse alleles, DNA fragments of introns 1 and 2 from 10 functional SLA-DRB1 alleles and 4 pseudogenes were cloned, and analyzed in sequence variations The results showed that the presence of extreme nucleotide variations including substitutions and deletions The information allowed design a unique strategy for comprehensive genotyping of SLA-DRB1 according to the criteria defined by Society for Animal Genetics Nomenclature Committee for Factors of the Swine Leukocyte Antigen System In this study, the efficiency, accuracy, and robustness of the method were demonstrated by comparing the results with various previous methods including cDNA SBT, PCR-SSP typing, allele segregation analysis, and heterozygous simulation typing Genotyping of 336 animals was performed with 15 DRB1 alleles identified including one new allele Ten DQB1-DRB1 haplotypes including 4 new haplotypes also were defined Population analysis using 7 different breeds showed difference in DRB1 allele frequency among pig breeds This is the first report on the development of high resolution comprehensive genotyping method of SLA-DRB1 developed by a systemic analysis of intronic polymorphisms for diverse SLA-DRB1 alleles

1 Introduction

The major histocompatibility complex (MHC) of pigs, also called as swine leukocyte antigens (SLA), is encoded by one of the most polymorphic region of pig genome which consist of a group of genes designated as SLA class I and class II system (Kelley et al, 2005; Rothschild et al, 1998) SLA proteins function mainly in presenting self and non-self peptides on the surface of cells to T lymphocytes, and therefore they play a vital role in the development and control of the swine immune system (Lechler et al, 1994, Federica et al, 2009; Maiko et al, 2009) Numerous studies demonstrated the repeated association of SLA complex to immune responsiveness, disease resistance and susceptibility, as well as reproductive performance and production chacracteristics (Renard et al, 1989, Kristensen et al, 1995; Gautschi et al, 1990) Morever, not only have pigs served as an important nutritional source but also a valuable asset to the field of biomedical science (Vodicka et al, 2005) and possible organ or tissue donors for xenotransplantation (Dehoux et al, 2007; Lunney et al, 2007)

In order to study the influence of SLA polymorphisms on animal health and other traits, a robust typing method is needed to precisely characterize the alleles of major SLA genes SLA typing has been attempted using a several methods including serology (Ivanoska et al, 1991), mix lymphocyte culture (Rothschild et al, 1984), restriction fragment length polymorphism (RFLP) analysis (Chardon et al, 1985), PCR-RFLP (Ando et al, 2003; Lee et al 2005), analysis of SLA linked microsatellite (Tanaka et al, 2005; Nunez et al, 2004), sequence-specific oligonucleotide typing

products (Middleton et al, 1999; Ho et al, 2005, Erin et al, 2009) The International Society for Animal Genetics Nomenclature Committee for Factors of the Swine Leukocyte Antigen System recently established the principles of a systematic nomenclature system for SLA class I and class II genes and alleles by means of DNA sequencing (Ho et al, 2009), which requires the development of robust and accurate sequence-based typing (SBT) methods for characterization of SLA polymorphisms However, the existing methods including cDNA SBT methods still have several disadvantages such as needs for RNA isolation and DNA cloning which are not suitable for large scale studies and relatively labor extensive, difficulty in excluding the pseudogenes from related functional genes (Vage et al, 1994; Gustafsson et al, 1990), and unequal amplification of specific alleles in heterozygotes due to the hypervariability of SLA genes This study described the development of a comprehensive and simple genomic sequence based typing (GSBT) method which allows the identification of SLA-DRB1 alleles directly using combination of genomic DNA PCR and direct sequencing

In this study, as a trial for developing a high efficiency genotyping system for SLA, 336 pigs from seven breeds were successfully genotyped using SLA-DRB1 GSBT showed the efficiency, accuracy as well as robustness of the method This method can be widely used to overcome obstacles existing in field of SLA genotyping and contribute to establish the complete list of SLA polymorphism

2 Materials and Methods

2.1 Animals

Experiments were conducted using 336 pigs, consisted of 7 breeds, 114 SNU

miniature pigs derived from Chicago medical university, 105 Korean native pigs (KNP), 73 NIH miniature pigs, 11 Berkshire, 12 Duroc, 10 Landrace, and 11 Yorkshire KNP, SNU and NIH breeds had pedigree information This study also used 22 additional pigs consisted of 14 SNU, 8 KNP and 2 Yorkshire for the comparison of cDNA and genomic DNA typing results

2.2 Isolation of genomic DNA and RNA

Genomic DNA was isolated from blood or tissue samples as described by Miller

et al (1988) The isolation of total RNA was performed using the RNeasyTM Mini Kit according to manufacturer’s instructions (Qiagen, Germany)

2.3 Primer design and PCR amplification

DNA sequence alignments were performed using ClustalW (http://www.ebi.ac.uk/Tools/clustalw2) PCR primers were designed using Primer 3 (http://frodo.wi.mit.edu/primer3) For genomic PCR, amplification reactions were performed in a 20 μl reaction containing 50 ng genomic DNA, 0.5 μM of each primer, 200 μM dNTPs, PCR buffer [10 mM Tris (pH 8.3), 50 mM KCL, 1.5 mM MgCl], and 0.5 U of SuperthermTM DNA polymerase (JMR Holdings, Kent, UK) using Thermocycler 3000 (Biometra, Germany) Cycling profiles for PCR were an

initial denaturation at 95°C for 5 min followed by 35 cycles of 1 min at 94°C, 1 min

at 63°C, and 1,5 min at 72°C and finished with 5 min final extension at 72°C Genomic PCR conditions for different primers were identical except for indicated annealing temperature and extension time (Table 1) For RNA samples, reverse transcription was performed in a 25 μl reaction using oligo-(dT)15 and SuperScript™ III reverse transcriptase (Invitrogen, USA) for 50 min at 50°C and inactivated for 15 min at 72°C Two micro liters of cDNA from the reverse transcription of total RNA was used for PCR using Pyrobest polymerase (TaKaRa, Japan) PCR products were checked by electrophoresis on a 1% agarose gel in 1 X TAE buffer The gel was stained with ethidium bromide and visualized under UV light

2.4 Cloning and plasmid isolation

PCR products were gel-purified using QIAquickTM Gel Extraction Kit (Qiagen, Germany) and ligated into pGEM-T Easy vector (Promega, USA) The ligation products were electroporated into DH10B cells (Invitrogen, USA) using MicroPulserTM (Biorad, USA) Transformed bacteria were plated onto agar containing 50 μg/ml ampicillin, 40 mg/ml X-gal solution and 100 mM IPTG Five positive colonies were picked from each ligation and additional colonies were analyzed, if necessary The plasmids were isolated using Plasmid SVTM Miniprep Kit (GeneAll Biotechnology, Korea)

2.5 DNA sequencing

For direct sequencing of PCR products, 3 μl of amplified products were incubated for 30 min at 37°C together with 4 U of exonuclease I (Fermentas, Canada), and 0.8 U of shrimp alkaline phosphatase (USB corporation, USA) in 1.5

X reaction buffer to degrade primers and dephosphorylate dNTPs which were not consumed in the reaction for 30 min at 37°C, and the reaction was stopped by 15 min incubation at 80°C (Ibrahim et al, 2001) Sequencing reactions were performed using the ABI PRISM BigDyeTM Terminator Cycle Sequencing Kit (Applied Biosystems, USA) with the sequencing primer (Table 1) The products were analyzed on an automated DNA analyzer (3730XL, Applied Biosystem, USA) T7 and SP6 universal primers (Table 1) were used to obtain bidirectional sequence information of the cloned inserts within the pGEM-T Easy vector The procedure for sequencing plasmids were the same in direct sequencing except for excluding the step for removing unincorporated primers and dNTPs

2.6 Allele discrimination and population analysis

Analyses for allele discrimination was performed using CLC workbench (CLC Bio, Denmark) and NCBI blast analysis with 270 bp of SLA-DRB1 exon 2 sequences Calculation of allele frequency, observed heterozygosity and expected heterozygosity were estimated using the POPGENE 1.31 software package (Yeh et

al 1999) Alignment and phylogenetic analyses were performed using CLC workbench (CLC Bio, Denmark)

3 Results and discussion

3.1 Generation of intronic sequences to design a primer set for SLA-DRB1 exon 2 genotyping

The SLA-DRB1 gene consisted of six exons spanning approximately 12 kb together with introns (Renard et al, 2006) and, therefore, it is not possible to amplify the entire gene by genomic PCR Since the minimum requirement proposed by ISAG nomenclature committee for Factors for the SLA-DRB1 was the complete coverage of exon 2, a set of primers which can be used for the analysis of complete exon 2 from all SLA-DRB1 alleles was developed

The common problem in genotyping of genes is the difficulty of obtaining comprehensive and consistent amplification of a specific locus because of the presence of extreme intra-locus polymorphisms and inter-locus similarity including pseudogenes This is more serious in the case of SLA-DRB1 typing where there are

at least 4 pseudogenes, SLA-DRB2, -DRB3, -DRB4 and -DRB5, adjacent to one functional DRB1 If sequence information of introns 1 and 2 for all SLA-DRB1 alleles and pseudogenes is available, a strategy for effectively discriminate alleles of SLA-DRB1 may be possible to design

Since very limited sequence information for intronic sequences of any SLA genes is currently available in the public database, a primer set was designed to amplify intronic regions surrounding SLA-DRB1 exon 2 from diverse DRB1 alleles The sequence information (accession no AY303991) of a BAC containing SLA-DRB1 was obtained from NCBI and 8 pairs of primers were designed to clone SLA-

DRB1 exon 2 flanking regions (primer data not shown) Among the primers, SLADRBi1F3 and SLADRBi2R9 (Table 1), which amplify an amplicon consisted

of 441 bp of intron 1, 270 bp of exon 2, and 980 bp of intron 2, allowed to clone target regions to design exon 2 genotyping primers Using the primer set, 14 different sequences with a size of 1691 bp from 10 SLA-DRB1 alleles and 4 pseudogenes were obtained through the repeated amplification, cloning and sequence analysis (data not shown) Although it is possible to obtain amplicons containing the complete DRB1 exon 2 from using SLADRBi1F3 and SLADRBi2R9, the primers were not able to used into practical genotyping since they also amplified pseudogenes in addition to functional DRB1 at the same time

Table 1 PCR primers used for the amplification of SLA-DRB1

regions and sequence analysis

1

Ando et al, 2005

The 610~618 bp of the sequences consisted of 295 bp of intron 1, 56 bp of intron

2, and 270 bp of exon 2 from 10 DRB1 alleles and 4 pseudogenes alleles were

Target region or

Annealing Tm(°C)

Product size(bp)

shown in Fig 1 The analysis results of these sequences showed a number of SNPs and deletion polymorphisms within the introns in addition to sequence variation from exon 2 The presence of such diverse polymorphism within the introns was the main cause of hindering the development of efficient genotyping method for SLA-DRB1 The Genbank accession numbers for the 14 sequences from SLA-DRB1*0404, Wu01, 0402, 0201, 0301, 1001, 0101, 1301, 0901, 0701, SLA-DRB2*kn02, kn03 and kn04, kn01 are GU263811 ~ GU263824

Fig 1 The amplification position of primers on exon 2

Analysis of nucleotide polymorphisms of the SLA-DRB1 exon 2 and flanking region including partial sequences from introns 1 and 2 for 10 expressed alleles and 4 pseudogenes Allele names are indicated

on the left Identical nucleotides are shown as dots The gray boxes indicate the nucleotide deletions exon/intron boundaries are shown by the vertical lines and short symmetrical arrows through the alignment Thin arrows show the locations of a sequencing primer, while the bold arrows denote the PCR primers

3.2 Designing of PCR primers for SLA-DRB1 specific amplification

The criteria in developing new SLA-DRB1 genotyping were 1) to use genomic DNA for PCR templates, 2) to eliminate pseudogene amplifications, 3) to permit the analysis of the entire sequence of SLA-DRB1 exon 2 after sequencing, 4) to comprehensively genotype all the alleles, and 5) to avoid complexity in the experimental procedure The successful primer design for locus specific and non-biased allele amplification and sequencing was essential to achieve the goals

To design primers to comprehensively amplify the complete exon 2 sequence of SLA-DRB1 without being influenced by the highly polymorphic allelesm of MHC genes, the systematic analysis of intronic sequences of different alleles around the exon 2 were performed through the comparison of all available 14 sequences (Fig 1) Candidate regions for DRB1 specific amplification was identified from introns 1 and

2 on the basis of the degree of sequence conservation among DRB1 alleles, elimination possibility of pseudogens, and distances from exon 2 A primer set, SLADRBi1F14 and SLADRBi2R17 (Table 1) at nucleotide positions 175 – 193 and

583 - 621, amplified a 447 bp DNA fragment while satisfying the required conditions (Fig 2) Because of the presence of a few nucleotide variations within

the primer regions, among different alleles we included degenerated bases in the primer sequences

Fig 2 The PCR products amplified from exon 2 of DRB1 region

The results of PCR amplification using the SLA-DRB1 specific primers for 10 different alleles A 447

bp of the SLA-DRB1 specific band (marked by an arrow) were consistently amplified from all different alleles The nonspecific bands appeared together with expected PCR products did not affect the quality

of sequencing results since the sequencing primer were different from PCR primers Allele names were indicated on the top

In detail, reverse primer was designed on the region containing the highest level

of differences between alleles of DRB1 and four pseudogenes at 4 nucleotide positions, 584, 587, 590 and 594 However, the difference was only 12 nucleotides long with 100% GC content Moreover, there was a region showing 1-3 nucleotides deletions at the adjacent downstream region To overcome this, the annealing control primer (ACP) strategy (Hwang et al, 2003) was applied Therefore, the 39 bp long SLADRBi2R17 primer was designedby adding nontemplate based additional sequences resulting in a tripartite structure with a polydeoxyinosine linker between the 3′ end target core sequence (12 nucleotides) and the 5′ end nontarget universal sequence (21 nucleotides) In addition, to meet the unbiased amplification of all

DRB1 alleles, the core region of the primer was degenerated at two positions (Table 1) This ACP primer showed dramatic improvement of annealing specificity

The forward primer, SLADRBi1F14, was designed to eliminate a pseudogene, DRB2-kn01, which still was co-amplified and generated noise peaks in sequencing results SLADRBi1F14 contained three degenerated nucleotide positions and one specific nucleotide difference at the 3’ end of the primer from pseudogene DRB2-kn01, resulting in complete elimination of the pseudogene (Fig 3) The results of SLA-DRB1 specific amplification using genomic DNA from animals having 10 different alleles were shown in Fig 2

Fig 3 Effect of the specific primer of method

The effect of SLA-DRB1 genotyping primer selection to specifically exclude pseudogene amplification The chromatogram on the top is the direct sequencing result of PCR amplicons produced by a less perfect genotyping primer set In addition to the peaks corresponding to correct SLA-DRB1 signals from heterozygous sample (0403/1001), signals from a pseudogene (DRB2-kn01) were also appeared

in the sequencing chromatogram, resulting in triple peaks (indicated by rectangles) or a number of ambiguous base callings The chromatogram at the bottom shows the removal of pseudogene signals

by redesigning the genotyping primer set, resulting in preferable genotyping results The same DNA and sequencing primer were used

3.3 Designing of a DRB1 genotyping protocol using direct sequencing

The most accurate and efficient genotyping of SLA-DRB1 can be achieved by comprehensive amplification of DRB1 alleles using a set of common primers and

subsequent sequence analysis by directly sequencing the products However, the presence of nucleotide deletions at the intronic regions near to the beginning and end

of exon 2 prevented from obtaining readable results from the direct sequencing of many heterozygote animals (Fig 1)

There was one nucleotide deletion at the nucleotide position 254 in DRB1*1001 provided unreadable results in many genotyping results due to the abundant presence of the allele in pigs when a regular type of sequencing primer was used A 24 bp universal sequencing primer was designed within the most conserved region within the intron 1 starting from the nucleotide position 237 - 260

SLA-in Fig 1, which covers the deletion poSLA-int The general strategy and the positions of all primers used in this study were described in Fig.4

Fig 4 The structure of MHC class II and map of primers used in the method 3.4 Evaluation of the accuracy of genotyping results

To assess the accuracy and reliability of genotyping results from our GSBT method, several test experiments were carried out Firstly, allele segregation