media.nature.com · Web [email protected]; [email protected] This file includes:...

37
Online Materials for Mutation in ST6GALNAC5 identified in family with coronary artery disease Kolsoum InanlooRahatloo 1,2α , Amir Farhang Zand Parsa 3 , Klaus Huse 2 , Paniz Rasooli 1 , Saeid Davaran 4 , Matthias Platzer 2 , Marcel Kramer 5 , Jian-Bing Fan 6 , Casey Turk 6 , Sasan Amini 6 , Frank Steemers 6 , Kevin Gunderson 6 , Mostafa Ronaghi 6 , Elahe Elahi 1,7* correspondence to: [email protected] ; [email protected] This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology & Radiology, Stanford School of Medicine, Stanford, CA 94305-5454 1 1 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 3 4

Transcript of media.nature.com · Web [email protected]; [email protected] This file includes:...

Page 1: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Online Materials for

Mutation in ST6GALNAC5 identified in family with coronary artery disease

Kolsoum InanlooRahatloo1,2α, Amir Farhang Zand Parsa3, Klaus Huse2, Paniz Rasooli1, Saeid Davaran4,

Matthias Platzer2, Marcel Kramer5, Jian-Bing Fan6, Casey Turk6, Sasan Amini6, Frank Steemers6, Kevin

Gunderson6, Mostafa Ronaghi6, Elahe Elahi1,7*

correspondence to: [email protected] ; [email protected]

This file includes:

Materials and Methods

Figures S1 to S7

Tables S1 to S10α, Present address: Dept. of Cardiology & Radiology, Stanford School of Medicine, Stanford, CA 94305-

5454

1

12

1

2

3

4

5

6

7

89

1011

12

13

1415

1617

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

Page 2: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Methods in detail

Genome-wide linkage analysis

Genome-wide SNP genotyping was carried out on DNA samples of eight individuals of the CAD-105

pedigree using HumanCytoSNP-12v1-0_D BeadChips and the iScan reader (Illumina; www.illumina.com)

(GEO accession no.: GSE42137). The individuals included six CAD affected and two CAD unaffected

individuals (Fig. 1). SNPs that had not been genotyped in one or more individual were removed from the

analysis with appropriate options in the GenomeStudio_Genotyping_Module_V1.0) (Illumina). MERLIN

was used to remove SNPs that exhibited Mendelian error and subsequently to attain parametric and

nonparametric logarithm of odds (LOD) scores under two sets of criteria: disease allele frequency of

0.001, penetrance of 90%, and 10% phenocopies; disease allele frequency of 0.0001, penetrance of 99%,

and 1% phenocopies1.

Genome wide exome sequencing

CAD affected individuals III-1 and III-2 were selected for exome sequencing. Genomic DNA was

isolated from blood samples by standard methods. DNA libraries were enriched using the TruSeq® Exome

Enrichment kit (Illumina, San Diego, CA, USA) and subsequently sequenced on an Illumina HiSeq® 2000

system (Illumina). The Truseq Exome assay targets 62 Mb of protein coding and regulatory untranslated

regions of the genome. Base calling was performed by the Illumina pipeline with default parameters. Over

eight gigabases of high quality sequences for each subject were generated. Sequence reads were mapped to

the human reference genome UCSC NCBI37/hg19 using ELANDv2 software (Illumina). Variant detection

was performed with CASAVA software (version 1.8.1; Illumina), and candidate variants were filtered to

have a CASAVA quality threshold of 10. CASAVA filtered out duplicate reads and reads without matched

pairs. In addition to CASAVA, variants were analyzed using Elnis Genomics (http://www.enlis.com) and

NextBio (http://www.nextbio.com/b/nextbio.nb) analysis softwares, again with reference to human genome

reference sequence NCBI37/hg19. Absence of the variants in 60 whole-exome sequence data available

within the Enlis Genomics data set (http://www.enlis.com/sample_genomes.html) and 15 other exome

sequences sequenced along with the CAD-105 patients but derived from healthy Iranians or Iranians

affected with unrelated disorders was also verified. The variants were finally systematically filtered to

identify those that were positioned within the linked loci, that affected splicing or amino acid changes, and

that were present in both patients. Development protocols and features of the TruSeq® Exome Enrichment

kit are described for the first time below.

TruSeq exome content

2

56

34

35

36

37

38

39

404142434445464748

49

505152535455565758596061626364656667

78

Page 3: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

The TruSeq Exome enrichment kit includes 340,427 probes, each constructed against the human

genome NCBI37/hg19 reference genome. The probe set was designed to enrich 201,121 exons spanning

20,794 genes targeting a total complexity of around 62Mb. For exons larger than 150 bases, the probes are

uniformly spaced roughly every 150 bases. Each 95-mer probe targets libraries of 300-400 bp (insert size

180-280bp) enriching 265-465 bases centered symmetrically on the midpoint of the probe. This means that,

in addition to comprehensive coverage of the major exon data bases, the kit also provides broad coverage

of non-coding DNA in exon flanking regions including promoters and UTRs. Databases covered by the kit

are CCDS coding Exons (31.3 Mb, hg19, 97.2% covered), RefSeq (33.2Mb, hg19, 96.4% covered), RefSeq

(regGene) exons plus (67.8 Mb, hg19, 88.3% covered), Encode/Gencode coding exons (25.6 Mb, hg19,

93.2% covered), and predicted microRNA targets (9.0 Mb, hg19, 77.6% covered).

TruSeq exome enrichment

Protocols, workflows, sequencing library preparation and pooling, and details regarding the TruSeq

Exome enrichment assay can be found in the “TruSeq Exome guide” and “TruSeq Exome enrichment kit

data sheet”: http://support.illumina.com/sequencing/sequencing_kits/truseq_exome_enrichment_kit.ilmn.

http://www.illumina.com/documents//products/datasheets/datasheet_truseq_exome_enrichment_kit.pdf

The method consists of a 2.5-3 day workflow. An indexing solution is supplied for each DNA sample,

which makes multi-sample pooling of up to twelve samples feasible in a single enrichment reaction. This is

a key feature of the technology that enables an automation friendly workflow and allows processing of

many samples simultaneously with minimum hands-on time. An overview of the enrichment scheme is

shown in Figure S2. In summary, the enrichment workflow steps are: (A) preparation of indexed libraries,

quantitation, and pooling of indexed libraries (see Preparation of sequencing libraries below), (B)

denaturing of libraries, (C) solution-phase hybridization of biotinylated oligonucleotide probes; (D) affinity

pull-down of targeted regions using magnetic streptavidin beads and high stringency washing, (E) elution

of captured regions of interest , repeat of pull-down step starting at (C) , and (F) PCR amplification using

universal primers (P5 and P7 in Figure S2) of the final eluted targeted libraries (not shown).

The enrichment method is unique in the sense that standard biotin-oligonucleotides are used, and that

non-stringent high concentration capture probe annealing is combined with two rounds of affinity

purification using Tm-normalizing stringency washes. The method is enabled by the high quality, relatively

uniform representation, and low preparation cost of the biotin-oligonucleotides, making this method

economically attractive for fixed sets with large sample volumes2.

Implementation of two rounds of biotin-oligonucleotide capture and release resulted in an increased

enrichment specificity (>80%) and overall assay robustness compared to a single round (40-50%). Two key

reagents, Cot-1 DNA (KREAcot DNA, Kreatech) and sequencing primer blockers (SBS3 and SBS12) in

step C (Figure S2) have a significant effect on the enrichment specificity. For example, 100, 50, 10, 5, 1,

and 0 ng/uL Cot-1 DNA yields 82.5%, 70.7%, 33.2%, 20.3%, 6.3%, and 2.1% enrichment specificity,

respectively. In the second enrichment step, the effect of cross-hybridizing repeat elements is effectively

blocked by Cot-1 DNA since fewer non-targeted libraries are present after the first enrichment step.

3

910

68

69707172737475767778

79

80818283

84858687888990919293

9495969798

99100101102103104

1112

Page 4: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Typical enrichment specificities are >80% and are relatively uniform across library insert sizes ranging

from 150-500 bp. The protocol results in a relatively uniform read count distribution across the targeted

regions. For example, at 0.2X of the mean coverage, >90% of the targeted bases are covered. This is

accomplished by a relatively non-stringent overnight hybridization capture at sub-nM concentrations of

capture probes to drive hybridization capture, and a highly-optimized Tm-normalizing stringency wash to

reduce off- target enrichment.

Preparation of sequencing libraries

Sequencing libraries were prepared using the TruSeq® DNA sample preparation kit v2 (

http://www.illumina.com/products/truseq_dna_sample_prep_kit_v2.ilmn)

using 1 ug of gDNA input. We also validated The Nextera® Exome enrichment kit with a few selected

samples using only 50ng of gDNA input

(http://www.illumina.com/documents/products/datasheets/datasheet_nextera_exome_enrichment.pdf).

Preparation of biotinylated capture probes

The synthesis and purification of the biotinylated oligonucleotides for the Exome Capture Target Oligo

(CTO) pool is described elsewhere2.

Effect of probe-target DNA mismatches on enrichment efficiency

We designed a set of oligonucleotide probes that enabled evaluation of the efficiency of target DNA

enrichment with various types and degrees of mismatches with respect to the probes. Capture probes were

designed to span a range of variant lesions consisting of deletions, insertions, consecutive substitutions, or

staggered substitutions relative to the hg19 reference genome. Each “variant” category was covered by 100

capture probes chosen from the TruSeq® Exome capture probe set with the following selection rules: (1)

random selection from regions with an average number of reads of a typical exome enrichment experiment,

(2) performing consistently well, (3) and positioned within regions covered by only a single probe. The

types of variant probes included probes with 0, 1, 2, 3, 4, 5, 7, 9, 12, 15 bp alterations where the alterations

consist of insertions, deletions, and consecutive- and staggered substitutions. For insertions, an insert of

specified length was inserted into the middle of the probe using a randomly generated sequence. Truncation

was performed symmetrically so probe length was maintained (e.g. for 2 base insertions: ATAT-

>ATGGAT->TGGA). For deletions, a deletion in the middle of the probe of specified length was

introduced with flanking sequence added symmetrically to maintain probe length (e.g. GGATATGG-

>NGGATGGN). For substitutions, homo-mismatched were used as a worst case scenario. Half of the

designs had consecutive mismatches (substitutions), and the other half had uniformly spaced mismatches

(staggered substitutions) (e.g. ATGATGAC->ATGTAGAC or ATGTTCAC). We also accessed the effect

of probe tiling in cases where more than one probe covers a given genomic region. This was accomplished

by designing a second set of probes such that three probes, rather than one probe, annealed to each target

region. This trio of probes included the original probe and two flanking probes of equal length. The latter

set is useful in determining how flanking probes can help recover the targeted region in cases of inefficient

affinity enrichment with a single central probe.

4

1314

105

106107108109110111

112

113

114

115116117

118

119120

121

122123124125126127128129130131132133134135136137138139140141

1516

Page 5: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Variant capture probe preparation

Capture probes were prepared using PCR amplification of a 90K pool of “in situ” array synthesized

oligonucleotides from CustomArray (Bothall, WA). Common amplification primers (Primer 1:

AGTCCGCGCAATCAG, and Primer 2: TGCAAGGATCACTCG) were included in the probe sequence

and flanked the 80 base capture probe sequence for a final array probe length of 110 bases. The PCR

reaction was performed with 1X Titanium Taq buffer (Clontech, Mountain view, CA), 1uM Biotin-Primer1

(Integrated DNA Technologies (IDT), Coralville, Iowa), 1uM Primer 2 (IDT), 200 uM dNTPs (Roche,

Indianapolis, IN), 1ul Titanium Taq (Clontech, Mountain view, CA), 0.1ng template 90K oligonucleotide

pool (CustomArray, 110 bp), and H2O to 100 uL. PCR cycling conditions: 95oC (5 min.), 95oC (30 s). 55oC

(30 s), 72oC (60 s), cycle 30 times, 72oC (5 min.), 10oC (forever). Sera-Mag Magnetic Streptavidin (100 uL;

MPB, Thermo Scientific, IN) were pre-washed with hybridization buffer (HB:1 M NaCl, 0.5 M phosphate

buffer, 0.1% Tween-20). The biotinylated PCR products (8 uL, 60 uM) were incubated in hybridization

buffer with the pre-washed MPB beads for 30 min at RT. The beads were subsequently washed with first

with 1X HB, then with 0.2X HB, NaOH (0.1N), and finally with 0.2X HB. The biotin-oligonucleotides

were eluted from MPB using water (100 uL) and heat (950C for 10 min) and obtained at a final

concentration of ~2 uM3, 4.

Variant probe enrichment assays

Enrichment assays were tested with various wash temperatures (32, 42, and 52oC) of step D (Figure S2)

in order to evaluate the effect of wash stringency on specificity, uniformity, and coverage of the targeted

regions across the probe variant categories. Not unexpected, higher stringency wash temperatures generated

higher enrichment efficiencies and lowered the uniformity across all probes. In Figures S3-S5, the average

read count per category is shown for the single probe and three probe designs at, respectively, 32, 42, and

52oC. The current protocol requires a 42oC stringency wash temperature. At this stringency, targeted

libraries with up to 11 bp staggered substitutions, 15 bp consecutive substitutions and 15 bp indels

compared to the probes are efficiently enriched without much loss of coverage in the targeted regions. With

the longer 95-mer probes in the current Truseq® Exome kit, it should be possible to detect even larger

variations. Larger variations can still be detected without loss in coverage using the 3 probe design. This

strongly indicates that flaking probes can mitigate against losses of capture efficiency of the mismatched

probe centered in the middle.

Sanger sequencing

Genomic DNA fragments containing each of the 12 candidate exomic variations distributed in 11 genes

and considered to be candidate CAD causing variations based on results of linkage analysis and exome

sequence data were amplified by PCR and sequenced. Reference sequences used for design of primers were

as follows: VPS13D: NC_000001.10,NM_015378.2; CRYZ: NC_000001.10, NM_001130042;

ST6GALNAC5: NC_000001.10, NM_030965; LPHN2: NC_000001.10, NM_012302; TTN:

NC_000002.11, NM_001256850; HSPD1: NC_000002.11, NM_002156; IRS1: NC_000002.11,

NM_005544; GPR35: NC_000002.11, NM_001195381; IL7R: NC_000005.9, NM_002185; LILRA2:

5

1718

142

143

144145146147148149150151152153154155156157158

159

160161162163164165166167168169170171

172

173174175176177178

1920

Page 6: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

NC_000019.9, NM_001130917; NLRP2: NC_000019.9, NM_001174081. DNAs from the seven affected

and three unaffected members of pedigree CAD-105 were analyzed with respect to these variations. The

amplicon containing the p.Val99Met causing variation in ST6GALNAC5 was also sequenced in 800

ethnically matched control Iranians not affected with cardiac disorders, and the amplicon containing the

p.*337Qext*20 variant was also sequenced in 800 controls. Finally all exons and adjacent intronic

sequences of ST6GALNAC5 were amplified and sequenced in 100 of the control individuals and in 160

Iranian CAD patients unrelated to each other and to pedigree CAD-105. Amplified DNA fragments were

sequenced using ABI Big Dye terminator chemistry and an ABI 3730XL genetic analyzer instrument

(Applied Biosystems, Foster city, CA). Sequences were analyzed with the Sequencher 4.8 software (Gene

Codes Corporation, Ann Arbor, MI). ST6GALNAC5 sequences derived from whole genome sequencing

performed in the United States on 150 CAD affected individuals and 800 individuals diagnosed not to be

affected with CAD were kindly provided by Dr. Leslie G. Biesecker (Genetic Diseases Research Branch,

National Human Genome Research Institute, USA). In this study, CAD diagnosis was based on having had

experienced MI, a stent placed, received a bypass, or presented with more than 50% occlusion upon

computed tomography angiography (CTA) or cardiac catheterization. In addition to reference sequences

described above, effects of nucleotide sequence variations observed were analyzed using protein reference

sequences NP_056193.2 (VPS13D), NP_001123514.1 (CRYZ), NP_112227.1 (ST6GALNAC5),

NP_036434.1 (LPHN2), NP_001243779.1 (TTN), NP_002147.2 (HSPD1), NP_005535.1 (IRS1),

NP_001182310.1 (GPR35), NP_002176.2 (IL7), NP_001124389.1 (LILRA2), and NP_001167552.1

(NLRP2). The sequences of all primers used are presented in Table S5.

Creation of plasmids pcDNA3.3- ST6GALNAC5, pcDNA3.3- ST6GALNAC5- p.Val99Met, and

pcDNA3.3- ST6GALNAC5- p.*337Qext*20

COOH-terminal FLAG-tagged ST6GALNAC5 cDNA was PCR amplified from a human heart cDNA

panel (Clontech, Mountain View, CA, USA) using forward primer 5'-AAAATGAAGACCCTGATGCGC-

3' and reverse primer 5′-TTACTTATCATCATCATCCTTATAATCGAACACAGGTTTATTCTCAGGA-

3′. The amplicon was cloned into pcDNA3.3 (Invitrogen, Karlsruhe, Germany) using the Topo TA Cloning

system (Invitrogen), and pcDNA3.3- ST6GALNAC5 was created. The plasmid was transformed into One

Shot® TOP10 Chemically Competent E. coli cells (Invitrogen, Karlsruhe, Germany), and the sequence of

the insert in plasmids isolated from ampicillin resistant cells was confirmed by direct sequencing.

Subsequently, the c.G295A mutation that causes p.Val99Met was introduced using the QuickChange site-

directed mutagenesis kit (Agilent Technology, Karlsruhe, Germany) according to the manufacturer’s

instructions. PcDNA3.3- ST6GALNAC5- p.Val99Met was thus created. Primers used contained the

sequence 5′- GGGACTGTGCCCTGATGACCAGCTCAG-3′ (nucleotide causing the mutation is

underlined) and the reverse complement of this sequence. Briefly, these primers which are complementary

to opposite DNA strands of pcDNA3.3- ST6GALNAC5, are extended during a cycling reaction to create

mutated plasmids with staggered nicks. DNA strands in plasmids without nicks are removed by digestion

with Dpn1. Nicks in surviving plasmids are repaired in vivo after transfection into TOP10 E. coli cells.

6

2122

179

180181182183184185186187188189190191192193194195196197198199

200201

202203204205206207208209210211212213214215

2324

Page 7: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

PcDNA3.3- ST6GALNAC5- p.*337Qext*20 was created by performing overlap extension PCR using

forward primer 5′- AAAATGAAGACCCTGATGCGC-3′ and three reverse primers

5′- TGGGATTACAGTCTGGCATGCTCATTCCTTGGAACACAG-3′,

5′- GTGTCTCGGTGTCTGATGCAGTGAATACCTGGGATTACAG-3′,and

5′- TTACTTATCATCATCATCCTTATAATCGTGTCTCGGTGTCTGA-3′. The third reverse primer

contained the Flag sequence. Presence of the mutations was confirmed in isolated plasmids by sequencing.

ST6GALNAC5 expression in COS-7 cells

Transfection was performed using Lipofectamin 2000 (Invitrogen) according to the manufacturer’s

instructions. African green monkey kidney COS-7 cells (ATCC, Rockville, MD, USA) were seeded at a

density of 2 × 104/100 μl in a 12-well plate (for RT-PCR and Western blotting analyses) or onto poly-L-

lysine coated coverslips (for immunofluorescent microscopy) and cultured in Dulbecco’s MEM medium

GlutaMAX™ (Invitrogen) supplemented with 10% fetal calf serum and 1% antibiotic (Penicillin -

Streptomycin; Sigma, Hamburg, Germany). Growth was in an atmosphere with 5% CO2 and 95% humidity

at 37°C. Cells were transfected with pcDNA3.3- ST6GALNAC5, pcDNA3.3- ST6GALNAC5- p.Val99Met,

and pcDNA3.3- ST6GALNAC5- p.*337Qext*20 after 24 hours. ST6GALNAC5 sequences in plasmids

isolated from COS-7 cells were confirmed by sequencing. Expression analyses were performed 24 hours

post transfection.

For RT-PCR, RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA

synthesis was performed with Sprint RT Complete-Random Hexamer first-strand cDNA synthesis kit

(Clontech-Takara Bio Europe, Saint-Germain-en-Laye, France). Five micrograms of total RNA was used

for reverse transcription.

For Western blotting, PBS washed COS-7 cells were lysed in 1% NP-40 (v/v), 20 mM Tris-HCl, pH 7.6,

150 mM NaCl, 1:1000 protease inhibitor cocktail, and 1 mM EDTA5. Total protein content in supernatants

recovered after centrifugation was determined using the BCA assay (Bio-Rad, Munich, Germany). Aliquots

containing 20 microgram of protein were size-fractionated in NuPAGE 10% Bis-Tris gels (Invitrogen)

using a buffer that contained 50 mM MOPS, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA, pH 7.7. Proteins

were then transferred onto nitrocellulose membranes, and probed with mouse monoclonal M2-anti-FLAG

(1:1000; F3165, Sigma-Aldrich, Munich, Germany), rabbit anti-human sialylytransferase 7e (1:1000;

ab69855, abcam, Cambridge, MA, USA), or goat anti-human lamin B (1:6000; sc-6216, Santa Cruz

Biotechnology, Santa Cruz, CA, USA) primary antibody and appropriate (anti- mouse IG, 1:2500, W4021,

Promega, Mannheim, Germany; anti-rabbit IG,1: 4500, 65-6120, Invitrogen; and anti-goat IG, 1: 50000 ;

sc-2768, Santa Cruz Biotechnology) secondary anti-IgG antibody anti-coupled to horseradish peroxidase.

Lamin B served as internal control. Detection was performed using the enhanced chemiluminescence

(ECL) Western blotting detection system (Invitrogen). Exposure times were 5 to15 seconds.

Sialyltransferase enzyme assay

Sialyltransferase enzyme activity in protein extracts of untransfected COS-7cells and COS-7 cells

transfected with pcDNA3.3- ST6GALNAC5, pcDNA3.3- ST6GALNAC5- p.Val99Met, and pcDNA3.3-

7

2526

216

217218

219

220

221

222

223

224225226227228229230231232233

234235236237

238239240241242243

244245

246247248249250

251

252

2728

Page 8: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

ST6GALNAC5- p.*337Qext*20 was assayed using the Sialyltransferase Activity Kit (R&D Systems,

Wiesbaden-Nordenstadt, Germany), according to the manufacturer’s instructions. This kit utilizes the 5’-

nucleotidase CD73 as a coupling phosphatase to remove inorganic phosphate quantitatively from the

leaving nucleotide cytidine 5’-monophosphate that is generated during sialyltransferase reactions7. Assays

were performed on protein extracts isolated from cells 24 hours after transfection; three independent

transfections with each vector were performed and protein extractions and enzyme assays were done on

cells of each transfection experiment. Varying amounts of protein were incubated with 25 nmol of CMP-

Neu5Ac (C8271; Sigma), 1 mg of asialofetuin (A4781, Sigma), and 50 ng of Coupling Phosphatase 2 in 1X

Assay Buffer for 20 minutes at 37° C. Released inorganic phosphate was measured by spectrophotometry.

ST6GALNAC5 ELISA assay

ST6GALNAC5 protein concentrations in the extracts of untransfected COS-7cells and COS-7 cells

transfected with pcDNA3.3- ST6GALNAC5, pcDNA3.3- ST6GALNAC5- p.Val99Met, and pcDNA3.3-

ST6GALNAC5- p.*337Qext*20 that were used for sialyltransferase enzyme assays were measured using an

ST6GALNAC5 ELISA kit (antibodies-online Inc., Atlanta, GA, USA) according to the instructions of the

manufacturer. The assay employs a quantitative sandwich enzyme immunoassay technique. Briefly,

purified standard ST6GALNAC5 provided with the kit and protein extracts of cultured cells were added to

wells of microplates previously coated with antibody specific for ST6GALNAC5. After incubation to allow

binding of ST6GALNAC5 to the immobilized antibodies and removal of unbound substances, biotin-

conjugated antibody specific for ST6GALNAC5, avidin-conjugated horseradish peroxidase, and

horseradish peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) were sequentially added to the

wells. Appropriate incubations, removal of liquids from the wells, and washings were performed.

Ultimately, a stop solution was added to prevent further enzymatic activity and optical densities were

measured at 450 nm. Amounts of ST6GALNAC5 protein per nanogram total protein in the extracts was

calculated based on a standard curve derived from readings of the standard ST6GALNAC5. All samples

were assayed in triplicate.

1. Abecasis, G.R., Cherny, S.S., Cookson, W.O., Cardon, L.R. (2002). Merlin rapid analysis of dense

genetic maps using sparse gene flow trees. Nat. Genet. 30, 97–101.

2. York, K.T., et al (2011). Highly parallel oligonucleotide purification and functionalization using

reversible chemistry. NAR. doi:10.1093/nar/gkr910.

3. Maurer, K., et al. (2006). Electrochemically generated acid and its containment to 100 micron reaction

areas for the production of DNA microarrays. PlosOne. 1, e34. doi:10.1371/journal.pone.0000034.

4. Holmberg, A., Blomstergren, A., Nord, O., Lukacs, M., Lundeberg, J., Uhlén, M. (2005). The biotin-

streptavidin interaction can be reversibly broken using water at elevated temperatures. Electrophoresis. 26,

501-510.

8

2930

253

254255256257258259260261262

263

264265266267268269270271272273274275276277278

279

280281

282283

284285

286287

288289

3132

Page 9: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

5.  Hosoya KI, Kim KJ, Lee VHL. (1996). Age-dependent expression of P-glycoprotein gp170 in Caco-2

cell monolayers. Pharm Res (NY). 13, 885–890.

6. Wacker I, Kaether C, Krömer A, et al. Microtubule-dependent transport of secretory vesicles visualized

in real time with a GFP-tagged secretory protein. J Cell Sci 1997;110(pt 13):1453-63.

7. Wu ZL, Ethen CM, Prather B, Machacek M, Jiang W. Universal Phosphatase-coupled

Glycosyltransferase Assay. Glycobology 2010; DOI: 10.1093/glycob/cwq187.

9

3334

290

291292

293294

295296

3536

Page 10: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Figure S1

Figure S1- LOD plots of chromosomal regions showing best linkage to CAD status in pedigree CAD-105. A, Plot of chromosome 1. B, Plot of chromosome 19. The

LOD plots are under assumption of autosomal dominant model of inheritance. Two close peaks

on chromosome 1 and one peak on chromosome 19 are evident.

10

3738

297

298

299

300

301

302303304

3940

Page 11: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Figure S2

Figure S2- The Truseq® Exome enrichment workflow. Indexed libraries can be

prepared with the TruSeq DNA sample preparation kit v2 or with the Nextera® sample preparation

solution (as shown).

11

4142

305

306307308309

4344

Page 12: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Figure S3

Figure S3- Average read count of the targeted regions using the one probe design (blue) vs. the three probe design (green) across the categories: deletions, insertions, consecutive- and staggered substitutions at the 320C wash temperature.

12

4546

310

311

312

313

314

315

316

4748

Page 13: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Figure S4

Figure S4- Average read count of the targeted regions using the one probe design (blue) vs. the three probe design (green) across the categories: deletions, insertions, consecutive- and staggered substitutions at the 420C wash temperature.

13

4950

317

318319

320

321

322

5152

Page 14: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Figure S5

Figure S5- Average read count of the targeted regions using the one probe design (blue) vs. the three probe design (green) across the categories: insertions, consecutive- and staggered substitutions categories at the 520C wash temperature.

14

5354

323

324325

326

327

328

5556

Page 15: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Figure S6

Figure S6-DNA sequence chromatograms. A, Chromatograms verifying presence of

12 exome sequence-based candidate CAD causing mutations in affected individual III-1. B,

Chromatogram showing stop-loss mutation in CAD patient.

15

5758

329

330331

332333334335

5960

Page 16: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Figure S7

Figure S7- Confirmation of expression of ST6GALNAC5 in COS-7 transfected cells.  A,

Confirmation by RT-PCR on RNA extracted from COS-7 culture transfected with pcDNA3.3-

ST6GALNAC5 (Wt), culture transfected with pcDNA3.3- ST6GALNAC5- p.Val99Met (Mut1), and

culture transfected with pcDNA3.3- ST6GALNAC5- p.*337Qext*20 (Mut2). NT: RT-PCR on RNA

extracted from non-transfected cells. B-D, Confirmation by Western blotting on protein extracted

from COS-7 culture transfected with pcDNA3.3- ST6GALNAC5 (Wt), culture transfected with

pcDNA3.3- ST6GALNAC5- p.Val99Met (Mut1) and culture transfected with pcDNA3.3-

ST6GALNAC5- p.*337Qext*20 (Mut2). The primary antibodies used were either against

FLAG (B), sialyltransferase 7e (C), or lamin B (D). A less intensely stained band in

addition to the major protein band was consistently observed when antibodies against

FLAG or sialytransferase 7e were used. This may represent a modified form of the

encoded protein. Lamin B served as internal loading control. NT: Western blot on protein

extracted from non-transfected cells.

16

6162

336

337

338

339

340341342343344345

346

347

348

349

350

351

6364

Page 17: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Table S1- Phenotypic and genotypic data on families with mutations in ST6GALNAC5 A: CAD-105 pedigree with p.V99M mutation

ID CAD status¥ Sex Age at Present ST6GALNAC

5 LDL Triglycerides HDL Fasting Systolic

BPDiastolic

PB BMI

diagnosis age genotype (mg/dl)δ (mg/dl)δ (mg/dl)δ blood

glucose (mm Hg)β (mm Hg)β (kg/m2)

(yrs) (yrs) (mg/dl)δ

II-1 +: CABG M 55 65 Mut/Wt 180 142 42 105 120 80 23

II-2 - F 75 Wt/Wt 163 92 41 99 160 90 26

II-3 - M 72 Wt/Wt 145 82 42 75 140 90 23

II-4 - M 78 Wt/Wt 154 85 40 110 130 90 25

II-5 +: MI M 55 dead

II-6 +: MI F 68 dead

II-12 +: MI F 52 dead

II-13 +: MI F 50 dead

II-21 +: MI M 61 dead

III-1 +: MI/CABG

F 38 40 Mut/Wt 190 156 42 76 110 90 25

III-2 +: CABG M 43 47 Mut/Wt 183 107 47 78 120 90 26

III-3 +: MI/CABG

M 47 51 Mut/Mut 192 80 41 81 120 80 25

III-4 +: CABG M 50 62 Mut/Wt 174 151 40 92 120 90 25

III-5 +: CABG M 43 55 Mut/Wt 185 199 44 107 110 80 24

III-6 +: Angina

F 50 54 Mut/Wt 186 126 41 71 120 90 26

III-7 +: MI M 42 dead

III-9 M 35 Mut/Mut 83 202 49 148 25

17

6566

6768

Page 18: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

-

III-10 - M 47 Mut/Mut 173 143 44 98 120 80 24

III-11 - F 36 Mut/Mut 209 158 41 110 130 90 26

III-21 - F 47 Wt/Wt 262 198 31 79 130 80 25

III-25 +: MI M 50 dead

IV-3 - M 28 Mut/Wt 122 218 43 95 100 60 24

IV-4 - M 25 Wt/Wt 127 68 36 73 110 70 25

IV-6 - F 20 Mut/Wt 200 59 66 88 100 70 21

IV-7 - M 30 Mut/Wt 279 117 43 92 118 70 25

IV-8 - M 21 Mut/Wt 52 74 50 91 100 60 22

IV-9 - M 19 Mut/Wt 175 77 63 87 120 90 24

IV-10 - F 25 Mut/Wt 85 72 69 87 110 90 25

P-valuesϒ 0.017 0.087 0.348 0.569 0.01 0.199 0.905

B: Family with p.*337Qext*20 mutation

ID CAD status¥ Sex Age at Present ST6GALNAC

5 LDL Triglycerides HDL Fasting Systolic

BPDiastolic

PB BMI

diagnosis age genotype (mg/dl)δ (mg/dl)δ (mg/dl)δ blood

glucose (mm Hg)β (mm Hg)β (kg/m2)

(yrs) (yrs) (mg/dl)δ

II-1 - F 62 Wt/Wt 173 91 42 190 170 90 23

II-2 - M 61 Wt/Wt 138 106 41 145 160 90 24

II-3 +: MI/CABG

M 51 53 Mut/Wt 181 132 39 203 160 90 24

II-4 +: CABG M 54 58 Mut/Wt 125 110 40 189 150 90 25

18

6970

7172

Page 19: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Tables A & B: ¥: +, CAD affected and -, CAD unaffected; δMeasured after 12 hour fast; β average of four measurements taken at five minute intervals in the lying position using a mercury sphygmomanometer; ϒ derived using Mann-Whitney test which is appropriate for small sample sizes. P-values were calculated using available data on individual III-7 and individuals listed above him in the table. The remaining data which pertain to individuals who are relatively young and who may later experience CAD were not used in the calculations; CABG, coronary artery bypass graft; MI, myocardial infarction; M, male; F, female; Mut, mutated allele; Wt, wild type allele; LDL, low- density lipoprotein cholesterol; HDL, high-density lipoprotein cholesterol; BP, blood pressure; BMI, body mass index.

C: Averages of data of subgroups of CAD-105 individuals grouped on basis of CAD status or mutation status¥

CAD (7)*

Without CAD (>70 yrs)(3)*

With mut

(16)*

With mut - het (12)*

With mut - homo (4)*

Without mut (5)*

LDL (mg/dl)184.3 (±5.6) 154.0 (±7.3) 166.8

(±54.1)167.6

(±55.8)164.3

(±48.6)170.2

(±47.4)

Triglycerides (mg/dl)

137.3 (±35.2

)86.3 (±4.2) 130.1

(±48.4)124.8

(±48.7)145.8

(±43.7)105.0

(±47.1)

HDL (mg/dl)

42.4 (±2.2) 41.0 (±0.8) 47.8

(±9.2)49.2

(±10.1) 43.8 (±3.3) 38.0 (±4.0)

Glucose (mg/dl)

87.1 (±13.3

)94.7 (±14.6) 94.1

(±17.5)89.1

(±10.2)109.2

(±24.6)87.2

(±14.7)

Systolic BP (mm Hg)

117.1 (±4.5) 143.3 (±12.5) 114.5

(±8.8)β112.3 (±8.1)

123.3 (±4.7)δ

134.0 (±16.2)

Diastolic BP (mm Hg)

85.7 (±4.9) 90.0 (±0.0)

80.7 (±10.6)

β 80 (±11.5) 83.3 (±4.7)δ

84.0 (±8.0)

BMI ((kg/m2)

24.9 (±1.0) 24.7 (±1.2) 24.4

(±1.4) 24.2 (±1.5) 25.0 (±0.7)24.8

(±1.0)Table C: ¥Averages based on data of part A of this table. *Number in parenthesis indicates number of individuals in the group for whom data is available; β, data on 15 individuals available; δ, data on 3 individuals available. With mut = with mutation p.V99M; Without mut = without mutation p.V99M; het, heterozygous for the mutation; homo, homozygous for the mutation.

19

7374

7576

Page 20: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Table S2- Genomic regions showing maximum linkage to CAD status in pedigree CAD-105*

Chromosome ChromosomalSNPs

bordering Nucleotide positions Size of linkedLOD

scoreβ No. annotated

bandlinked

regionsbordering linked

region (bp) region (cM) protein coding

genesψ

1 1p31.3rs11208724

& 66179203-68588299 3.49 2.2 21

rs1430751

1 1p22.3-rs7522428

& 75178360-79595720 3.94 2.2 47

1p31.1 rs6690294

19 19q13.33rs6509789

& 54042830-55879872 8.96 2.2 136

rs1126757 * Six CAD affected and two CAD unaffected individuals were included in the analysis; β analysis with MERLIN under autosomal dominant model. ψ based on human reference genome UCSC NCBI37/hg19.

20

7778

352

7980

Page 21: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Table S3- Summary of exome sequencing data

ID Target covered Total no. No. novel No. novel variants No. novel variants

at 10X (%)* variants δ variantsα affecting splicing affecting amino acid changesψ

CD-105-III-1 92 149274 7402 8 576

CD-105-III-2 92 167597 8282 10 581* target size was 62 Mb; δ variants detected with CASAVA (V1.8.1), Illumina- reference sequence UCSC NCB137/hg19); α variants that were absent in the NCBI dbSNP v130 and 1000 genomes databases and in the control exome sequences; ψ 283 of these were common to both patients.

21

8182

353

8384

Page 22: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Table S4- Candidate CAD causing mutations in pedigree CAD-105 based on linkage analysis and exome sequence data analysis

ChromosomeChromosomal

position Variation Gene Effect on Bioinformatics Segregation

with

of variation (bp)* protein sequenceprediction of

effectCAD status

in

on protein functionψ

CAD-105 pedigree

1 12336823 G>A VPS13D p.D1060N Tolerated -

1** 75172042 C>T CRYZp.E310K, p.E173K,

p.E276Kβ Tolerated -1** 77509922 G>A ST6GALNAC5 p.V99M Damaging +

1** 82437577 T>C LPHN2 p.I982T Damaging -2 179634421 T>G TTN p.T2963P, p.T2917Pβ Tolerated -2 198358148 T>C HSPD1 p.I257V Tolerated -2 227660612 G>C IRS1 p.P948R Tolerated -2 241569440 A>T GPR35 p.Y55F Tolerated -5 35871197 G>A IL7R p.R140Q Tolerated -5 59284478 G>T PDE4D p.L37I Tolerated -

19 14200072 T>C SAMD1 p.R247G Tolerated -19 15734118 A>T CYP4F8 p.N284Y Tolerated -

19** 55087463 G>C LILRA2 p.R381P Tolerated -19** 55087468 G>A LILRA2 p.G383S Tolerated -

19** 55493047 C>G NLRP2 p.P154A, p.P131Aβ Tolerated -*human reference genome UCSC NCBI37/hg19; ** variations within peak linked loci, β in different isoforms of encoded protein; ψ both SIFT and PolyPhen predictions

22

8586

354

355

356

357

8788

Page 23: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Table S5- Primer sequences used for amplification and sequencing of ST6GALNAC5 exons and amplicons containing candidate CAD causing variations

Gene/exon no. Forward Reverse

VPS13D/19* 5'-CTGGTGGATACCATGCAGACA-3' 5'-TGGGCACTCTGAACTCACAAA-3'

CRYZ/9* 5'-CACATAAGAAGCTCATGGAATCG-3' 5'-TTTTCTTAGGAGGAATTTCAGCA-3'

ST6GALNAC5/1 5'-GTGGGTACACTGGCTCGGTTA-3' 5'-GGAGCGGGTAGAAAGTTGTCC-3'

ST6GALNAC5/2 5'-TCTCCCCCACTAGAGTGACCA-3' 5'-AATGCGCTGGAGAGATCAGAG-3'

ST6GALNAC5/3 5'-CTGACATGATGGGGAGGAGAG-3' 5'-CCCACAACAAAAGCCTGTAGC-3'

ST6GALNAC5/4 5'-CAAATGGAGGAGAGAGGGAGA-3' 5'-GAATGAGAACTTGGGACATGC-3'

ST6GALNAC5/5aα 5'-CCTCAAAACCTCTCCACTTCC-3' 5'-ACTGGCCCAGATTGCACTAAA-3'

ST6GALNAC5/5bα 5'-ATGATGGTTGGAAATGGCCTA-3' 5'-GCCAGGTCATGTCTGTAAAACAA-3'

ST6GALNAC5/5cα 5'-CAAATTTGAAGGCACCAGCA-3' 5'-TGATGACACAGCTGGGAGAGA-3'

LPHN2/15* 5'-TAAGGATTCAGTGGTGGAGTGGT-3' 5'-TGGCCTATATGTAAGCATGTTTTT-3'

TTN/37* 5'-ATATCGAGGTGCCTGAGACCA-3' 5'-TTGGTTCTCATCTGGCACTTGT-3'

HSPD1/7* 5'-TGAAGGCATGAAGTTTGATCG-3' 5'-CCAAACACTGCACCACCAGTA-3'

IRS1/1* 5'-TCCACAGCTCACCTTCTGTCA-3' 5'-CTGTTCGCATGTCAGCATAGC-3'

GPR35/6* 5'-CCTGCTCACTCTCTGCTGACC-3' 5'-CGGCGTGTCTGAGGTGTCT-3'

IL7R/4* 5'-TGGAGGTAAAGTGCCTGAATTT-3' 5'-TGATCAGGGATGGATCGAACT-3'

LILRA2/6* 5'-CCCTCACCCATCCTTCTTCTC-3' 5'-GGTCCCTGCCTATTTCCACTC-3'

NLRP2/5* 5'-GCCGGAAAACACATTTGTAGC-3' 5'-CAGGCAGATCACCTGACATTG-3'*Amplified fragments contain position of candidate disease causing variation; αAll exons of ST6GALNAC5 amplified, long exon 5 encoding 3'UTR amplified in multiple reactions.

23

8990

358

9192

Page 24: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Table S6- Conservation of p.99Val in human sialyltransferase 7e at corresponding positions in paralogous and orthologous proteins.Paralogous p.V99 Sequence ID.*sialyltransferases    

hST6GALNAC5 CRDCALVTSSGHLLHSR gi|21759442|ref|NP_112227.1

hST6GALNAC1 CITCAVVGNGGILNNSH gi|18490673|ref|NP_060884.1

hST6GALNAC2 CIRCAVVGNGGILNGSR gi|26996844|ref|NP_006447.2

hST6GALNAC3 CDLCAIVSNSGQMVGQ gi|48428651|ref|NP_001153483.1

hST6GALNAC4 CRSCAVVSSSGQMLGSG gi|21759443|ref|NP_778204.1

hST6GALNAC6 CHQCVIVSSSSHLLGTKL gi|48146753|emb|CAG33599.1

hST3GAL1 CRRCAVVGNSGNLRESS gi|1705559|ref|NP_003024.1

hST3GAL2 CRRCAVVGNSGNLRGSG gi|21759433|ref|NP_008858.1

hST3GAL3 RCRRCIIVGNGGVLANKS gi|284055255|ref|NP_777623.2

hST3GAL4 CRRCVVVGNGHRLRNSS gi|14714972|ref|gb|AAH10645.1

hST3GAL5 CRRCVVIGSGGILHGLEL gi|109633044|ref|NP_003887.3

hST3GAL6 CKKCVVVGNGGVLKNKT gi|403225034|ref|NP_001258075.1

hST8SIA1 LKKCAVVGNGGILKKSG gi|28279804|gb|AAH46158.1

hST8SIA2 FGTCAIVGNSGVLLNSG gi|64654294|gb|AAH96205.1

hST8SIA3 YNICAVVGNSGILTGSQC gi|110815855|ref|NP_056963.2

hST8SIA4 -KTCAVVGNSGILLDSEC gi|2494834|ref|NP_005659.1

hST8SIA5 KKCAVVGNGGILKNSR gi|80478739|gb|AAI08911.1

Orthologous proteins  

Species

Human KMHCRDCALVTSSGHLLH gi|21759442|ref|NP_112227.1

Chimpanzee KMHCRDCALVTSSGHLLH gi|397472622|ref|XP_003807839.1

Cow KMHCRDCALVTSSGQLLR gi|300796298|ref|NP_001179733.1

Mouse KMHCKDCALVTSSGHLLR gi|148540137|ref|NP_036158.3

Chicken KMHCKSCALVTSSGHLLG gi|46425420|emb|CAG26706.1

Xenopus KMHCKTCALVTSSGHLLG gi|288562694|ref|NP_001165748.1

Zebrafish LKTHCRSCALVTSSGHMT gi|112182837|emb|CAL18608.1

*Sequence ID number from NCBI (http://www.ncbi.nlm.nih.gov/)

24

9394

359

360

9596

Page 25: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

25

9798

361

99100

Page 26: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Table S7- ST6GALNAC5 sequence variations* observed in 160 CAD patients and 100 control individuals from Iran

In controls& patients

Only incontrols

Only in patients

No. controls

with variation

No. patiens

with variation

Allele frequency

inENSEMBL

**

Predictedeffect***

rs number

C>T/5' NC 2 3 0.03 Benign rs76305167

c.141G>A/p.Q47Q 2 3 0.167 Benign rs62637703

c.381C>A/p.P127P 3 8 0.02 Benign rs35763299

c.711A>G/p.T237T 3 5 0.004 Benign rs143357043

c.357T>C/p.C119C 1 0.003 Benign rs147610766

c.492G>C/p.Q164H 1 0.003 Benign rs189362082

c.536G>A/p.R179Q 2 <0.001 Benign rs200875685

c.759G>A/p.D257N 1 Benign Novel

p.*337Qext*20 2 Damaging Novel* All observed in heterozygous state; **http://www.ensembl.org; ***SIFT and PolyPhen predictions

26

101102

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

103104

Page 27: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Table S8- ST6GALNAC5 sequence variations* observed in 150 CAD patients and 800 control individuals from the USA

In controls In patients No. individuals Allele Predicted rs number

with variation frequency in effect***

ENSEMBL** c.474C>A/p.D158E 1 Benign Novel

c.607A>G/p.M203V 1 0.001 Benign rs151200060

c.616C>T/p.R206C 1 Damaging Novel

c.492G>C/p.Q164H 1 < 0.01 Benign rs189362082

c.301A>G/p.N101D 7 Damaging Novel

c.19C>G/p.H7D 1 0 Damaging rs200073725

c.1000C>A/p.P334T 1 Benign Novel

* All observed in heterozygous state; **http://www.ensembl.org; ***SIFT and PolyPhen predictions

27

105106

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

107108

Page 28: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Table S9- Statistical analysis on numbers of control and CAD affected individuals with rare sequence variations in ST6GALNAC5 that cause amino acid changes Rare ST6GALNAC5 variations that cause amino acid change

Iranian cohort US cohort¥ Combined

Iranian & US cohorts

No.controls with the variations/total no. controls 0/100 4/800 4/900

No. with the variations/total no. patients 6/160 2/150 8/310P value* 0.085 0.242 0.003βOdds Ratio -δ 2.69 5.93

(95% CI: 0.49-14.8) (95%CI: 1.77-19.85)βRare ST6GALNAC5 variations that cause amino acid change predicted to be damagingβ

Iranian cohort US cohort¥ Combined

No.controls with the variations/total no. controls 0/100 1/800 1/900

No. with the variations/total no. patients 2/160 1/150 3/310

P value* 0.525 0.291 0.054Odds Ratio (OR) -δ 5.36 8.79

(95%CI: o.33-86.21) (95%CI: 0.91-84.77)¥ Individuals with the p.N101D variation not included in the analysis because it appears to be a common polymorphism in the the population studied; *, Fisher exact test; δ, OR can not be calculated because of the zero value; β, the P value and the OR decrease, respectively, to 0.022 and 4.42 (95% CI: 1.24-15.77) if the p.R179Q mutation, which was not tested for enzyme activity and which was observed in of two Iranian patients, is not included in the analysis. Figures consistent with ST6GALNAC5 having a role in CAD are shown in bold.

28

109110

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

111112

Page 29: media.nature.com · Web viewelahe.elahi@gmail.com; elaheelahi@ut.ac.ir This file includes: Materials and Methods Figures S1 to S7 Tables S1 to S10 α, Present address: Dept. of Cardiology

Table S10- ST6GALNAC5 protein concentrations in extracts of untransfected COS-7 cells and cells transfected with vectors expressing wild type and mutatedST6GALNAC5 proteins as measured by an ELISA assay

Source of extractsβpg ST6GALNAC5/ ng total

proteinUntransfected cells 4.3 ± 0.1Cells expressing wild type protein (transfection 1) 16.4 ± 2.4Cells expressing mutated protein: p.Val99Met (transfection 1) 18.0 ± 0.42Cells expressing mutated protein: p.Val99Met (transfection 2) 16.8 ± 3.7Cells expressing mutated protein: p.Val99Met (transfection 3) 18.3 ± 1.7Cells expressing wild type protein (transfection 2) 19.5 ± 0.23Cells expressing mutated protein: p.*337Qext*20 (transfection 1) 18.10 ± 0.07Cells expressing mutated protein: p.*337Qext*20 (transfection 2) 17.6 ± 0.99Cells expressing mutated protein: p.*337Qext*20 (transfection 3) 18.2 ± 0.28 β: Each extract was assayed in triplicate.

29

113114

458

115116