Association study of ABCA1 polymorphisms in singapore populations 4

Sequence variations in the UTR and coding regions were numbered with reference to the ABCA1 cDNA Genbank accession NM_005502; in addition, coding SNPs cSNPs were also identified by their

4 Materials and Methods

4.1 General Methods

4.1.1 Nomenclature for ABCA1 SNPs

Locations of sequence variations in the ABCA1 proximal promoter were described relative to the transcriptional start site (TSS) mapped by Santamarina-Fojo et al (2000) Sequence variations in the UTR and coding regions were numbered with reference to the ABCA1 cDNA Genbank accession NM_005502; in addition, coding SNPs (cSNPs) were also identified by their amino acid positions Intronic SNPs were described in relation to their distances from the exon-intron splice junctions, with ‘+’ and ‘-‘ designating positions relative to the 5’ or 3’ splice junctions respectively, whichever was nearest to the SNP (den Dunnen and Antonarakis, 2001) To illustrate, IVS1+92T>C implies a T>C variant within intron 1, 92 bp downstream from the end of exon 1 Insertion/deletion polymorphisms were abbreviated as indels

4.1.2 Genomic DNA Extraction

Genomic DNA was isolated from whole blood using a slightly modified method of Parzer and Mannhalter (1991) Five ml of blood were transferred to a sterile 15 ml centrifuge tube, incubated in 10 ml of lysis buffer (0.32 M sucrose, 5 mM MgCl2, 0.01 M Tris-HCl,

1% Triton X-100, pH 8.0) for 15 min on ice, and then centrifuged at 2000 xg for 10 min at

room temperature The supernatant was discarded The pellet was resuspended in washing buffer (10 mM NaCl, 10 mM EDTA, pH 8.0) and spun as before The pellet,

completed in approximately 10-15 min, the tubes were placed on ice To precipitate DNA, ice-cold absolute ethanol was added and the tube gently inverted a few times The DNA, which might be visible as strands depending on yield, was transferred to 1.5 ml microfuge tubes and dried overnight on the bench The DNA pellet was resuspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and stored at 4oC

4.1.3 Polymerase Chain Reaction (PCR)

Generally, PCR was carried out in 20 ul reaction volumes comprising of 50-200 ng genomic DNA, 0.2 mM of each dNTP (MBI Fermentas), 0.4% dimethylsulfoxide (Sigma-Aldrich), 20 pmol of each primer (Operon), 1.5 mM MgCl2, 0.2 U Taq polymerase

(DynaZymeII, Finnzymes Oy) and 1x manufacturer’s buffer To accommodate for the high

GC content in the ABCA1 gene promoter, the PCR recipe was modified: 2x PCRx

enhancer (Invitrogen) in place of dimethylsulfoxide, and 2.5 U native Taq polymerase

from Invitrogen in place of DynaZymeII Thermal cycling was performed on a GeneAmp

9700 machine (Applied Biosystems, ABI) using a Touchdown protocol: initial denaturation

at 95oC for 3-5 min followed by 34 cycles of denaturation at 95oC for 30 sec, annealing at

63oC for 30 sec with temperature decrement of 0.5oC per cycle until 56oC and 56oC for the remaining 20 cycles, and extension at 72oC for 30 sec; and a final extension at 72oC for 5 min Touchdown PCR was chosen because it eliminates the need for optimization of annealing temperature as well as reduces spurious priming of templates (Don et al., 1991) PCR primers were obtained from literature or designed using Oligo 6 (Molecular Biology Insights Inc)

4.1.4 Preparation of Native Polyacrylamide Gels

The mini polyacrylamide vertical gel systems, miniVE or SE240 from Amersham Biosciences were used Each pair of glass plates (glass and notched silica plates for SE240) was rinsed in tap water and dried Prior to use, the plates were wiped with 70%

ethanol, spacers placed between the plates and clamped in the gel casting stand Sixteen

ml of gel mixture was sufficient for casting two mini gels The gel mixture consisted of an appropriate volume of acrylamide stock solution (prepared from 30% or 50% stocks for regular and single-strand conformation polymorphism (SSCP) gels respectively), glycerol (SSCP gels only) and stock 5x TBE buffer added to a final 1x Prior to casting of gels,

100 ul of 10% ammonium persulphate solution and 20 ul of TEMED were added to initiate polymerization The gel mixture was poured between the clamped plates using a serological pipette, combs put in place and gels left to set at room temperature After the gels had set, the combs were removed and the wells were rinsed thrice with distilled water before use

4.1.5 Gel Visualization by Ethidium Bromide Staining

Ethidium bromide was added to molten agarose at a final concentration of 0.2 ug/ml before pouring into the gel tray Polyacrylamide gels were stained post-electrophoresis in water containing 10 ug/ml of ethidium bromide for 5 min Stained gels were viewed under ultraviolet (UV) illumination and images captured on Polaroid T57 black and white film

4.1.6 Gel Visualization by Silver Staining

Polyacrylamide gels were removed carefully from the glass plates and soaked in 10% ethanol for 5 min (fixation) in plastic containers, followed by freshly prepared 1% nitric acid (oxidization of non-DNA containing areas) for 3 min, and then transferred to a transparent glass dish containing 0.1% silver nitrate for 20 min with shaking Excess silver ions were rinsed away in deionized water, followed by a final rinse in the developing

dark, the solution was replaced; this was necessary to reduce background staining When sufficient intensity of the DNA bands had been achieved (often within ~10 min), the developing solution was discarded and 10% acetic acid added to quench the reaction To maintain a permanent record of the stained gels, they were shrunken first in 80% methanol to ~70% of original size, placed between cellophane sheets, clamped in place with a Perspex drying frame and binder clips, and air-dried for two days Although the gels were shrunken to ~70% of original size, it was necessary since high percentage polyacrylamide gels tended to crack upon drying Despite the smaller gel size upon preservation, there was no loss in resolution of bands Indeed, shrunken gels gave sharper bands (Mohamed et al., 1989)

4.1.7 Big Dye Terminator Sequencing

PCR products were purified directly or following agarose gel electrophoresis using the QIAQuick kit (Qiagen) Purified PCR templates were quantitated using UV absorbance at

260 nm before they were sequenced using fluorescent dye terminator chemistry Each 20

ul sequencing reaction comprised of 2 ul BigDye Ready Terminator mix (version 2 or 3, ABI), 3 ul sequencing buffer (400 mM Tris-HCl, pH 8.0, 10 mM MgCl2), 3.2 pmol primer and 3-10 ng purified PCR template Thermal cycling was performed on a GeneAmp9700 machine (ABI) with 25 cycles of 96oC for 10 sec, 50oC for 5 sec and a final extension of

60oC for 4 min in 9600 emulation mode Sequencing products were precipitated using isopropanol (BigDye Terminator Ready Mix version 2) or sodium acetate-ethanol (BigDye Terminator Ready Mix version 3) Precipitated sequencing products were then resuspended in 10 ul of HiDi formamide (ABI), transferred into 96-well optical microplates and ran on the ABI3100 Genetic Analyzer with the appropriate instrument settings The DNA traces were inspected visually for SNPs

4.2.2 Resequencing of ABCA1 Proximal Promoter

A segment of the ABCA1 proximal promoter spanning 594 bp downstream and 297 bp upstream of the transcriptional start site was amplified using Touchdown PCR, purified and sequenced using the amplification as well as internal primers (Figure 4.1)

4.2.3 DHPLC Screening of ABCA1 Exons

An outline of the DHPLC procedure is provided in Figure 4.2 For DHPLC assay development, primers were designed to amplify most of the 50 ABCA1 exons individually using Oligo 6 (Table 4.1) Amplicon sizes ranged between 200 and 800 bp Oven temperature for the optimal separation of heteroduplex molecules in each amplicon was selected using DHPLCMelt (http://insertion.stanford.edu/melt.html) or WAVEMAKER™ (Transgenomic) Ideally, amplicons with one predominant melting domain are preferred since it means that heteroduplex detection could be achieved at a single optimal melting temperature (Topt) In some cases, this criterion could be simply fulfilled by redesigning

Transgenomic WAVE DHPLC system comprised of a 96-well autosampler mounted on a Peltier cooling rack, pumps, buffer degasser, 20 ul sample loop, DNASep (Transgenomic) separation column housed in a temperature-controlled oven, online variable wavelength

UV detector and a Windows NT computer running the WAVEMAKER™ software for analysis protocols and data collection Buffers were: buffer A, 0.1 M triethylammonium acetate (TEAA; Transgenomic) pH 7.0; and buffer B, 0.1 M TEAA and 25% HPLC-grade acetonitrile (Fisher Scientific) Eluted products were monitored online by UV absorbance

at 260 nm Sample injection volumes were 3-5 ul Starting and ending gradient conditions were determined using WAVEMAKER™ with necessary adjustment when the peaks eluted too close to the injection or wash peaks A standard linear gradient of 2% buffer B and a flow rate of 0.9% buffer B per ml were used throughout Prior to heteroduplex detection, a subset of PCR samples was analyzed by DHPLC in non-denaturing mode at

50oC or on agarose gels to ensure specificity of PCR The actual optimal temperature (Topt) for heteroduplex analysis was determined semi-empirically by serial injections at temperatures starting 2oC below the predicted Topt (given by DHPLCMelt or WAVEMAKER™) to 2oC above in 1oC increments The temperature that resulted in first major time shift of DNA peak was selected for variant detection The actual DHPLC screening temperatures used are provided in Table 4.1 Representative samples showing heteroduplex peaks were re-amplified and sequenced with the amplification primers Samples that yielded homoduplex peak profiles served as reference

4.2.4 In silico SNP Discovery from ABCA1 ESTs

The SNPFINDER (Buetow et al., 1999; http://lpgws.nci.nih.gov:82/perl/snp/snp_cgi.pl) program was used to mine candidate SNPs from overlapping regions of ABCA1-specific, non-redundant ESTs in the UniGene cluster Hs.211562 To maximize the number of sequences for multiple alignment, no reverse strand confirmation was specified High

confidence putative SNPs in which mismatches were seen across multiple, independent sequences and were flanked by high quality bases, were validated by sequencing

4.2.5 Inferring Functional Significance of ABCA1 SNPs

The functional importance of ABCA1 variants was inferred by various methods Putative transcription factor binding sites in the proximal were identified by searching against the TRANSFAC database (Kel et al., 2003) using MATCH (http://www.gene-regulation.com/cgi-bin/pub/programs/match/bin/match.cgi) Coding SNPs causing amino acid replacements were classified according to BLOSUM62 (Henikoff and Henikoff, 1992) and Grantham’s scores (Grantham, 1974) The BLOSUM62 scoring matrix is derived from the observed amino acid substitutions in a large set of approximately 2000 conserved amino acid blocks from 500 protein families Positive and negative BLOSUM62 scores denote frequent and rare amino acid substitutions respectively

Grantham’s D values are judged on three physiochemical properties of amino acids

(polarity, volume and composition) and are correlated to the evolutionary exchangeability

of amino acid residues Sequences from ABCA1 orthologues from diverse species were aligned using ClustalW

-515 400U

-417 +297L

+280-Figure 4.1 Resequencing of the ABCA1 gene proximal promoter A 891 bp fragment

was amplified with the primers PrU and +280-+297L and sequenced using nested as

well as the PCR primers Positions of SNPs identified in this study are indicated and

are numbered with respect to the transcriptional start site (TSS) identified by

Santamina-Fojo et al (2001)

TSS +1

ABCA1 genomic sequence AF275948 Exon and flanking intron sequence

Experimental Topt determination using

1oC increment starting 2oC below predicted melting temperature(s) to 2oC above

Figure 4.2 Outline of exon-centric SNP discovery in the ABCA1 gene using

DHPLC

Table 4.1 PCR primers and DHPLC analysis temperatures for SNP screening

in ABCA1 exons

Region Forward primer

Reverse primer

Amplicon size (bp)

Exon size (bp)

T opt

Exon 1 5’-CCGGCTCCACGTGCTTTC-3’

5’-CCTCCGGTCCCATCTGAGTTGC-3’

514 221 60oC Exon 1A 5’-GGGTTTGGCTCTGGGTACTGC-3’

5’-GCTGCTCTCCAGGAATCAGTTCG-3’

510 136 56oC Exon 2 5’-GGCTTTGCAGCAATAACTGATG-3’

5’-AGCCACATGCCATATCCAGAC-3’

382 66 60oC Exon 3 5’-GGGAAGACACAGAGGTATATGG-3’

5’-AGTCTCCTGAGTTCATGCCTTA-3’

470 94 54,

59oC Exon 4 5’-GAGCCTCAAAATCGCTTCA-3’

5’-TACTCGAGGTGATGGATACCC-3’

406 142 58oC Exon 5 5’-GGAGCCAGGCTGAGTTCTATG-3’

5’-GCAACCAGCTCCCCTAGTTTC-3’

406 119 61oC Exon 6 5’-CCTGATATGGCGATGCTC-3’

5’-CTGGATGGTTTGGCAATTCCT-3’

59oC Exon 7 5’-TGCCTTGTGCTTTGATACATTC-3’

5’-CCCAAAAGTCTGAAAGAACACTA-3’

538 177 57oC Exon 8 5’-AATGAACAAAATGCAAACTTAC-3’

5’-ACCAATTAGCTCTGAGACATCTA-3’

485 93 53,

58oC Exon 9 5’-GTAATGCTGATGCTGCTCGTC-3’

5’-GTGGTGCCCTGTGACTTTAGC-3’

347 117 60oC Exon 12 5’-GTGCTCCTCAAGATTTAGTTGG-3’

5’-CCTGCCTGAACCTTATTGTAAC-3’

333 198 59oC Exon 13 5’-TGGCTGCCAACGTTCTCATGAA-3’

5’-CCCAAGACCAGGCTGGTGTG-3’

281 206 60oC Exon 14 5’-GGCTTGGTAAGGGTAGTAGGGT-3’

5’-CCCCATCTGGCACAGTATAAACT-3’

57oC Exon 15 5’-TCTACATTGCATTGCTCGTGAA-3’

5’-TTCTCCTCCCTTAGCCCGTGTT-3’

474 223 61oC Exon 16 5’-CGCCTCTGCATTGTCTCTAACA-3’

5’-CCAGAGGTACTCACAGCGAAGA-3’

354 222 61oC Exon 17 5’-GGTAGCCCACCACTCCCCTAAAG-3’

5’-ATCAGCTGCCTGTCCTTGGACTA-3’

525 205 60oC Exon 18 5’-ATGATGCTGAGCTTGGCTCATAC-3’

5’-CAGCAGCACTAGGTTAAAGAAAG-3’

200 114 58oC Exon 19 5’-TGCTCCTTTATCATCGTATTCCC-3’

5’-ACATGTTCACATTCGGCAACTCC-3’

517 172 61oC Exon 20 5’-CCCATAGGCCCACTTGTAGTTA-3’

5’-GCTGAGGCAGGAGAATCGCTTGA-3’

61oC Exon 21 5’-TGGGCCAGGGGACACTGTATTCT-3’

5’-GCCAGGGACAAGTTTCTGTTACC-3’

339 143 62oC Exon 22 5’-GGTGCGGTTGGTAACAGA-3’

5’-AGCCTAGCCATGAGATACAGC-3’

333 138 61oC Exon 23 5’-CCAGTGCTTACCCCTGCTAA-3’

5’-AACAGAGCAGGGAGATGGTG-3’

446 221 59oC Exon 24 5’-GAGGCTGTTCACACGGCACAC-3

5’-AGGCACCCCAGCAAGCATTAG-3’

457 73 62oC Exon 25 5’-CCAGCTGTCCACCCATTCTACAC-3’

5’-TTCCTGCTATCTCCCACTACCA-3’

636 203 59oC

Table 4.1 Continued from previous page PCR primers and DHPLC analysis

temperatures for SNP screening in ABCA1 exons

Region Forward primer

Reverse primer

Amplicon Size (bp)

Exon size (bp)

5’-CTCCGGCATCCATATCTTGAC-3’

310 149 60oC Exon 29 5’-TTGGTGCAGCACAGTCATAGG-3’

5’-TGGTCTGCTCGAATCTTACCC-3’

327 125 61oC Exon 30 5’-CCCTGATGCCGAATACAG-3’

5’-TGCACGGTGTTAGAAGACTCTCC-3’

496 99 55,

60oC Exon 31 5’-CCATCTCTGGTAATCCTACTCTTG-3’

5’-GATATCTCACTCATTCCTGCTTCC-3’

318 190 61oC Exon 32 5’-TCAGGTTTCCGGTCACACTG-3’

5’-CCCACTGTTTCAGTCTGTTATTTG-3’

289 95 58oC Exon 33 5’-TTGCATCATTAGGAATAGGCTC-3’

5’-CCAAGGCTTTCTTCAATCCAAG-3’

177 33 58oC Exon 34 5’-ACCTTGTACACACTCGCACTG-3’

5’-CGTTTAACCTGCCAACTACTC-3’

270 105 58oC Exon 35 5’-ACTTGGTTCTCAGGAGCC-3’

5’-CCCCTGCCAACTTTACC-3’

337 75 55oC Exon 36 5’-GTGTTTTGCCTTGGTATGTGAC-3’

5’-GCTGTTCCCCTACAATGAG-3’

390 170 59oC Exon 37 5’-GTCCATGTCCTCACTGATTG-3’

5’-TTCCTGATGATAGCCAGAGC-3’

344 178 60oC Exons 38-39 5’-GCATTGTATATTTGATTTAGGGGTG-3’

5’-AATTAAACACTGTCCTCTGGCT-3’

60oC Exon 40 5’-CTTCACTCCCATATTTCAGAACTTG-3’

5’-ATCTCCATTAAAGCATCCTACAGC-3’

59oC Exon 41 5’-TCTGACCGGATCTCTGCATTGTG-3’

5’-CAGGTGCTCCACGGGTTCTAAG-3’

194 130 60 oC Exon 42 5’-ATCCTGGAGACTGTGGCAAGTAG-3’

5’-GCCCTTTTATTAAGCAAGTCAGC-3’

321 121 57 oC Exon 43 5’-TGGCTTAGGGTTATTGACAAGT-3’

5’-TCTGAAATCATTCTCTTGACATACA-3’

369 63 56oC Exon 44 5’-AGAATTGAAGGTTTGAGGTAGTTAC-3’

5’-TTTATGTGCTTCTTTACCCTTTACT-3’

342 107 55oC Exon 45 5’-TTTCAAGTAGCAGCAGATGTATTGG-3’

5’- CAGCCTGAAGTCAATGCGTGTG-3’

364 142 55oC Exon 46 5’-TGTATGTGTAGGACAGCATGATAA-3’

5’-GGGAAGACAAGCCAATCATACAAC-3’

311 135 59oC Exon 47 5’-AAATGAATTTGAAAGTTACTCTAAT-3’

5’-GATCGCATATTCTACTTGGA-3’

333 104 57oC Exon 48 5’-ACTGAACAGCATCATCCCTATATCC-3’

5’-GTTAAGTAACTTGCCCAAGAGTCAC-3’

448 93 56oC Exon 49 5’-GGGTTCCCAGGGTTCAGTAT-3’

5’-GATCAGGAATTCAAGCACCAA-3’

436 245 59oC Exon 50 5’-TAATTCTGTATGCTCCTACTTGACC-3’

5’-CATTGCATTGCATTGAATAGTATCAG-3’

57oC

4.3 Genetic Association Study

4.3.1 Cases and Controls

Cases were unrelated patients who had been admitted to the Cardio-Thoracic Surgery Unit of the Singapore General Hospital for coronary bypass graft surgery between 1989 and 2000 Eligibility criterion included at least 50% stenosis in one or more major coronary arteries as confirmed by coronary angiography A detailed family history for CAD, hypertension, diabetes and smoking, along with clinical data such as height, weight, blood pressure and smoking habits were documented Further inclusion criteria for patients in the association study were absence of valve disease, cardiomyopathy, hypertension or diabetes The mean age of the male cases analyzed in this study was 58.17 ± 9.14 years and ranged between 32 and 97 years

Unrelated controls were obtained from two sources The first set of controls was randomly selected when they were undergoing mandatory pre-employment and annual medical checkups at the Singapore Anti-Tuberculosis Association Chest and Heart Clinic between 1989 and 1995 Physical examination and laboratory tests such as blood hemoglobin estimation, urine analysis for albumin and sugar, chest X-ray and resting electrocardiogram were carried out The second set of controls were randomly called during a national rhinitis survey study in 2000 to attend a rhinologic examination in the Ear, Neck and Throat outpatient clinic of the National University Hospital in Singapore Medical histories were documented by questionnaire Inclusion criteria for controls were

an absence of personal or familial history of CAD, hypertension and diabetes The mean age of the first collection of male controls analyzed in the study was 45.71 ± 12.98 years and ranged between 10 and 84 years while the mean age of the male controls from the second collection was 34.36 ± 14.25 years and ranged between 7 and 74 years Controls from the two collections were combined and analyzed together