A linkage disequilibrium map of the human major histocompatibility complex in singapore chinese conserved extended haplotypes and ancestral blocks 2

B-lymphoblastoid cell lines BLCL were established from whole blood obtained from these subjects.. For the high-resolution SNP map Section 3.2, subjects consisted of 214, unrelated blood

CHAPTER 2:

METHODS AND MATERIALS

2.1 Subjects

All subjects in this study were Singaporean blood donors who described themselves

as being of Chinese ancestry (tracing back to their grandparents) and self-declared as having no personal history of autoimmune conditions Appropriate informed consent was obtained

For the first SNP map (Section 3.1), subjects consisted of 93 males and 105 females B-lymphoblastoid cell lines (BLCL) were established from whole blood obtained from these subjects For the high-resolution SNP map (Section 3.2), subjects consisted of 214, unrelated blood donors and 41 individuals from 12 nuclear families B-lymphoblastoid cell lines (BLCL) were established from whole blood obtained from these subjects Included in this second set of samples were DNA from selected archived B-lymphoblastoid cell-lines established previously from healthy, unrelated Singaporean Chinese blood donors The archived samples were genotyped at 3 classical HLA loci (HLA-A, -B, -DRB1) and screened to see if they carried homozygous HLA alleles at either 2 loci (HLA-A, -B) or 3 loci (HLA-A, -B, -DRB1)

A total of 29 HLA homozygous samples were eventually selected and genotyped in the high-resolution SNP map The gender distribution of this set of samples was 159 males and 125 females

2.2 Establishing B-Lymphoblastoid Cell Lines from Whole Blood Samples

To establish a renewable source of DNA, BLCLs were established from whole blood

of subjects; peripheral blood mononuclear cells were first isolated from anti-coagulant blood by density-gradient separation, followed by Epstein-Barr virus (EBV) transformation of B cells Briefly, heparinised blood was first layered over

Ficoll-Histopaque (Gibco, USA) in a tube, followed by centrifuging at 900xg for 15mins at room temperature The interface containing mononuclear cells was collected and rinsed twice with 10ml of RPMI medium To infect the cell pellets with EBV, the 2ml

of a cell culture medium (RPMI with 10% FCS and PIS) was first added followed by 0.5ml of supernatant from EBV infected B95-8 cell lines The cells were then incubated overnight at 37ºC Incubated cells were rinsed with 10ml of RPMI and re-suspended in the cell culture medium The cells were then split and re-incubated at 37ºC The density of the cells was monitored daily and culture medium added when necessary As the cell culture approaches confluency, it is transferred into 25ml and subsequently 75ml culture flasks With each transfer 20ml of fresh cell culture medium is added

A portion of the cultured cells are reserved for freezing These cells are first rinsed in RPMI and re-suspended in 1.5ml of ice-cold freezing medium (see Appendix) They were then kept at -70ºC for a minimum of 2 hours before being stored in liquid nitrogen Remaining cells kept for DNA extractions were first rinsed in 1ml of phosphate buffered saline and cell pellets stored at -20ºC for up to 5 days

2.3 DNA Extraction from Cell Pellets

Genomic DNA was extracted from cell pellets either by using Gentra’s Autopure LS system with Gentra’s Puregene DNA extraction kit (Gentra Systems, USA), or Chemicon’s Non-organic DNA extraction kit (Millipore, USA) Extracted DNA was re-suspended in 10mM Tris/1mM EDTA (TE buffer) and kept at -20ºC for short-term storage (less than 2 weeks) or -80ºC for longer-term storage Special care was taken to avoid repetitive freeze-thaw cycles that may degrade the DNA

For SNP genotyping, the most important criteria for success are DNA quality and accurate quantification DNA was checked for degradation using gel electrophoresis with 0.7% agarose gels, and ensuring that genomic DNA was not fragmented The minimum quantity of DNA needed for SNP genotyping was 80µl of DNA at a concentration of not less than 50ng/µl (minimum of 4mg of DNA per sample) DNA quantification was mostly performed by DNA staining with PicoGreen (Invitrogen, USA) an ultra-sensitive fluorescent nucleic acid stain for dsDNA Concentrations for several isolated samples were determined using a ND-1000 Nanodrop spectrophotometer (Thermo Fisher Scientific, USA)

Briefly, the PicoGreen DNA quantification protocol first involved constructing a single-replicate 8-point DNA standard curve, ranging from 0 to 75ng/µl, from Lamda DNA (Invitrogen, USA) DNA working standards were created by serial dilution of Lamda DNA with TE buffer to the appropriate concentrations, and pipetted into 16 wells of a spectrofluorometer plate Equal amounts of working concentration PicoGreen was added to each DNA standard and incubated at room temperature for 5 mins, protected from light to prevent photodegradation of PicoGreen Spectrofluorometric analysis was then performed on a Tecan Genios fluorescence reader (Tecan, Switzerland) and analysed using the Megallan v4.0 software (Tecan, Switzerland) Fluorescence readings from the DNA standards were used to construct a standard curve DNA samples were subsequently stained with PicoGreen using the same protocol and concentrations determined by comparing fluorescence readings against the standard curve

2.4 SNP Selection and Genotyping

The 1152 SNPs that make up the first generation map (Section 3.1) were selected from release 121 of dbSNP (Smigielski et al 2000) Using a “picket fence” approach, SNPs were selected to achieve a desired coverage of 1 SNP per 100kb across the chromosome 6p, with a higher resolution of 1 SNP per 10kb in the segment between positions 30.0Mb to 37.0Mb of the chromosome arm that contains the MHC In order

to avoid SNPs that may not be informative in an Oriental population, SNPs that were annotated as “validated” in dbSNP, as well as those reported to have a minor allele frequency of at least 5% in an East Asian population were preferentially chosen

For the high-resolution SNP map (Section 3.2), SNPs from Illumina’s MHC Panel Set (Illumina, USA) were used This set comprised of 2360 SNPs that were previously genotyped successfully and established to be informative (Miretti et al 2005) It was also designed to provide a 1 SNP per 2kb resolution from positions 28.97Mb to 33.88Mb, covering known gene loci as well as intergenic regions across the MHC

SNP genotyping was carried out with the Illumina GoldenGate Assay on a BeadArray Platform (Illumina, USA) Briefly, the GoldenGate assay is a highly multiplexed PCR-based genotyping assay that uses a combination of allele-specific oligonucleotides and a locus-specific oligonucleotide to determine the genotype of each targeted SNP in a sample Genomic DNA is first immobilised to a solid support and sets of oligonucleotides targeted to specific SNPs are annealed to the DNA Unbound oligonucleotides are next removed in a series of stringency washes Following this, allele specific extension ligation is used to extend correctly matched allele-specific oligonucleotides to the locus-specific oligonucleotides A subsequent

PCR reaction using fluorescently labelled primers, with a specific dye for each allele, amplifies the appropriate product using universal PCR sites on the oligonucleotides The PCR products are then hybridised onto a 1152-spot microarray that captures locus specific signatures, and by analysing the fluorescent signals on the microarray the genotype of each assayed SNP in a sample can be accurately determined

Genotype results were next filtered using a set of quality controls:

i SNP genotypes were checked that they did not deviate from the

Hardy-Weinberg equilibrium using a Fisher’s exact test at a significance level

of 0.001

ii SNPs with a minor allele frequency of less than 5% were discarded Additionally, for the high-resolution SNP map with family data:

iii SNPs that had genotypes disconcordant with pedigree structure in

more than one family were discarded

Illumina provides a list of flanking sequences that were used for designing the SNP assays To ensure that the location of the SNPs map to the genome as annotated, the flanking sequences were mapped back to the human genome assembly using BLAST (Altschul et al 1990)

2.5 HLA Sequence-Based Typing

The HLA genotype at the 4 classical HLA loci HLA-A, -B, -C and -DRB1 was determined for each sample by sequence-based typing For the HLA-A, -B and -C genes, this involved PCR amplification of exons 2 and 3 using specific primers, followed by direct DNA sequencing of the PCR products in opposite directions For

HLA-DRB1, exon 2 was amplified using a cocktail of allele-specific primers at the 5´ end and a loci-specific primer containing a M13 priming sequence at the 3´ end The sequences of the PCR primers are shown in Table 2.1

PCR reactions for HLA-A,-B and -C were performed in total reaction volumes of 50µl comprising of: 5µl of 2mM dNTPs, 5µl of 10xPCR buffer, 5µl of 5µM 5´ primer, 5µl of 5µM 3´ primer, 100ng of genomic DNA and 0.2µl of High Fidelity Taq DNA polymerase (Lifecodes Corporation, USA) The following thermal cycling profile was used:

98ºC for 2mins,

[94ºC for 30secs, 65ºC for 1min, 72ºC for 2mins] – 32 cycles,

72ºC for 10mins

PCR reactions for HLA-DRB1 was performed in total reaction volumes of 30µl comprising of: 3µl of 2mM dNTPs, 3µl of 10xPCR buffer, 0.3µl of 20µM of each

Loci Name Sequence (5´to 3´)

A1 Tgg CCC CTg gTA CCC gT A2 gAA ACS gCC TCT gYg ggg AgA AgC AA B1 ggg TCC CAg TTC TAA AgT CCC CAC B2 CCA TCC CCg gCg ACC TAT

C1 AgC gAg gKg CCC gCC Cgg CgA C2 ggA gAT ggg gAA ggC TCC CCA CT DRB1-10 CAg gAA ACA gCT ATg ACC TgA AgA CCA CgT TTC TTg gAg gAg g

DRB1-04 Agg AAA CAg CTT ATg ACC TgA gAC gCA CgT TTC TTg gAg CAg gTT AAA C DRB1-01 CAg gAA ACA gCT ATg ACC TgA gAC gCA CgT TTC TTg Tgg SAg CTT AAg TT DRB1-09 CAg gAA ACA gCT ATg ACC TgA CCA gCA CgT TTC TTg AAg CAg gAT AAg TT DRB1-52.1 CAg gAA ACA gCT ATg ACC CCC ACA gCA CgT TTC TTg gAg TAC YCT A

DRB1-07 CAg gAA ACA gCT ATg ACC TgA gAC TCA CgT TTC CTg Tgg CAg ggT AAR TAT A DRB1-15 CAg gAA ACA gCT ATg ACC TgA gAC TCA CgT TTC CTg Tgg CAg CCT AAg A DRB1-M13 TgT AAA ACg ACg gCC AgT gCT YAC CTC gCC KCT gCA C

DRB1

Table 2.1: List of Oligonucleotide Primers for HLA-A,-B,-C and -DRB1 Amplification

A

B

C

primer, 200ng of genomic DNA and 0.2µl of High Fidelity Taq DNA polymerase (Roche Diagnostics, Germany) The following thermal cycling profile was used: 95ºC for 5mins,

[95ºC for 30secs, 62ºC for 10secs, 72ºC for 30secs] – 30 cycles,

72ºC for 10mins

All PCR reactions were carried out on a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, USA) PCR products were purified using the QIAquick gel extraction kit (Qiagen, Germany) following the manufacturer’s instructions The purified PCR templates were sequenced with the corresponding primers listed in Table 2.2 to derive HLA genotype of each sample

2.6 PCR Amplification for Sequencing Recombination Hotspots

For re-sequencing of recombination hotspots, PCR primers were designed such that overlapping PCR fragments tile across the hotspots interval Primers were designed from repeat-masked human genome sequence with the help of the Primer3 application (Rozen and Skaletsky 2000) Table 2.3 lists the pairs PCR primers used for each hotspot and includes the annealing temperatures that were specific to the thermal cycling profiles for each PCR reaction

Table 2.2: List of Oligonucleotide Primers Used for HLA Sequencing

PCR reactions were performed in total reaction volumes of 30µl comprising of: 3µl of 2mM dNTPs, 3µl of 10xPCR buffer with 1.5mM MgCl2, 1.5µl of 5µM of the forward primer, 1.5µl of 5µM of the reverse primer, 1.5µl of 100% DMSO (Sigma-Aldrich, USA), 100ng of genomic DNA and 0.2µl of High Fidelity Taq DNA polymerase (Roche Diagnostics, Germany) The following thermal cycling profile was used:

94ºC for 2mins,

[94ºC for 15secs, TA for 30secs, 72ºC for 2mins] – 30 cycles,

72ºC for 7mins

(Where TA refers to the primer annealing temperature specific to each pair of primers and listed in Table 2.2)

Hotspot Primer Name Sequence (5´to 3´) Chromosome

Location 1

Fragment Size (bp)

Annealing Temp (ºC)

1.1F TCT CAT gTC ATC TgC TTC TTg g 29,790,748 1.1R CTC ACA Tgg CTA TTg gAA AAg g 29,792,689 1.2F gAC CAg ACA ACA Agg AAC TgA g 29,792,666 1.2R TTg gTT gAA TAA CTg ggg TCA g 29,796,136 1.3F CCT gAC CCC AgT TAT TCA ACC 29,796,134 1.3R TTC TgC CTC CCA TTC CAT AC 29,798,757 2.1F gCT ggC Tgg ATg TAg TTg Ag 31,676,300 2.1R TgA TTg gAg Tgg ACA Agg Tg 31,678,495 2.2F TCA CCT TgT CCA CTC CAA TC 31,678,494 2.2R gCC TTC CTT CAT TCT CTC ACC 31,680,416 2.3F gAT ggg TAg TgT ggT TTC TgC 31,679,635 2.3R CCT TTg AgC CTg ggA gTg g 31,681,306 3.1F TCT Tgg gTA CAT gCT AAC TTC C 33,128,468 3.1R ggA CAg ggC ACA TTC TTA gg 33,131,576 3.2F CTg AAT gAA CCC TgC TCT gg 33,130,471 3.2R TgA ATA ACA AAT ggg CAA ACC 33,132,863 3.3F CCT AAg AAT gTg CCC TgT CC 33,131,576 3.3R gAg CAA Agg gCT gAA gAT TAT g 33,134,373 4.1F ACT CTg CCT TTC CTC ATC AAA C 32,907,046 4.1R TCC TCA TCA ACA CCA CTT TCT g 32,909,388 4.2F TCC CAA ggA gCC ACA gAT Ag 32,908,868 4.2R TCA CAA TAg CAg Cgg AgA Ag 32,910,930 4.3F gCC TCC TTT CCC TAT gCT g 32,910,481 4.3R ggA CgT TgT gAg TTg gAg gT 32,913,463 4.4F CCA AAg gAg AAg Agg CAC AT 32,913,295 4.4R Tgg gAg gTg gTT TCA ATC Tg 32,915,552

Table 2.3: List of Oligonucleotide Primers Used to Amplify the Hotspots Intervals for Re-Sequencing

62 62 HLA-F Telo

NCR3 - AIF1

2,623 2,195 1,941

1,922 1,671

64 64 62

62 3,470

64

2,392 2,797

2,062 3,108

1 Chromosome Location - Coordinates are based on the reference human genome sequence assembly (NCBI36.1)

64

DPA1-Telo

TAP2

64 62 62

2,982

All PCR reactions were carried out on a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, USA) PCR products were purified using the QIAquick gel extraction kit (Qiagen, Germany) following the manufacturer’s instructions The purified PCR templates were sequenced with the corresponding primers listed in Table 2.4 below

Table 2.4: List of Oligonucleotide Primers Used for Hotspot Re-Sequencing

A Hotspot Telomeric to HLA-F

Primer

1.1F TCT CAT gTC ATC TgC TTC TTg g 29,790,748 1.s1 TCT gAg TAA AAA gTg CCT ggT g 29,791,266 1.s2 CAC CAg gCA CTT TTT ACT CAg A 29,791,284 1.s3 CCT ATT gTg TTT TCC ATT CC 29,791,689 1.s4 gAA TgC CCA CCC AgT AgC 29,791,829 1.s5 CCT CAA TAC CCA Agg CTC Tg 29,792,260 1.s6 CAg AgC CTT ggg TAT TgA gg 29,792,279 1.2F gAC CAg ACA ACA Agg AAC TgA g 29,792,666 1.s7 gCC TTT TCC AAT AgC CAT gT 29,792,687 1.1R CTC ACA Tgg CTA TTg gAA AAg g 29,792,689 1.s8 TAC Agg CAT gAg CCA CCA 29,793,275 1.s9 Tgg Tgg CTC ATg CCT gTA 29,793,297 1.s10 TgA ATg ACC AAg gTT ACA Cg 29,796,114 1.3F CCT gAC CCC AgT TAT TCA ACC 29,796,134 1.2R TTg gTT gAA TAA CTg ggg TCA g 29,796,136 1.s11 CCC TAA ACA gAg AAC CCT CCA 29,796,539 1.s12 ggC TgC AAg TAA TCC TCC Tg 29,796,665 1.s13 Tgg ATA ACA gAg ggA gAC CA 29,796,880 1.s14 gTg ACA gAg Tgg Tgg ggA CT 29,797,355 1.s15 CAg TCA CAA TgC CCC TCA C 29,797,704 1.s16 TCA ggg CTA Tgg AAT gAA gg 29,797,838 1.s17 gTT AgC CAg gAT ggT CTC g 29,798,083 1.s18 AAA CTg gTC TCT gTC CTA TTT CA 29,798,392 1.3R TTC TgC CTC CCA TTC CAT AC 29,798,757