Exome-Sequencing Identifies Novel Rheumatoid Arthritis-Susceptible Variants in the BTNL2

Exome-sequencing identifies novel rheumatoid arthritis-susceptible variants in the BTNL2

SupplementaryInformation

Supplementary Figure 1 Flowchart of this study.

Supplementary Figure 2 Amino acid changes caused by the 3 BTNL2 SNPs in humans andsome other mammalian species.

Supplementary Figure 3 LD plot of the region from NOTCH4 to DRB1 displayed in r2 using the Haploview program. LD blocks were created by the GAB algorithm. The region was divided into 3 LD blocks except for rs2076530 in the exon 5 of BTML2 and DRB1*04:05.

Supplementary Table 1 PCR primers used in this study

Supplementary Table 2 Filtering of SNV detected by exome sequencing

Supplementary Table 3 Top 30 Genes in the difference of average number of SNV

Supplementary Table 4 The difference of average number of SNV in the RA-susceptible genes

Supplementary Table 5 Comparison of the number of BTNL2 SNVs in the patients and controls with respect to HLA-DRB1 genotypes

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Supplementary Figure 1 Flowchart of this study.

Supplementary Figure 2 Amino acid changes caused by the 3 BTNL2 SNPs in humans andsome other mammalian species.

Supplementary Figure 3 LD plot of the region from NOTCH4 to DRB1 displayed in r2 using the Haploview program. LD blocks were created by the GAB algorithm. The region was divided into 3 LD blocks except for rs2076530 in the exon 5 of BTML2 and DRB1*04:05.

Supplementary Table 1 PCR primers used in this study

Target genePrimer name SequenceSNP ID

exonin amplicon

BTNL2

exon 6BNe6F5’-TCCAAACCTGAAGGAACATAAGGA-3’

BNe6R5’-ATGCTCCAGCCAGCCTCATC-3’

rs28362675

rs41521946

rs28362677

rs28362678

exon 5BNe5F5’-GACCTTCCCAGCCCATCCTC-3’

BNe5R5’-TCAGAGAAATTGTCCAGGAACTAGCA-3’

rs2076530

rs41441651

rs41417449

rs34423804

rs41355746

exon 3BNe3F5’-CTCCTGAACTCCGGCCACCT-3’

BNe3R5’-CGAGGTGCTATGGTGCAGTCC-3’

rs28362680

rs2076523

rs78587369

NOTCH4

intron 4/NCF5’-CAGCTTCGGGACTTCTGTTCAGC-3’

exon 4NCR5’-GACAGGGTCATGGATCAGCTGTG-3’

rs2071284

rs2071283

rs2071282

rs2071281

rs415929

Supplementary Table 2 Filtering of SNV detected by exome sequencing

Filtering StepCaseDisease control

Rheumatoid arthritisnon-autoimmune disease

(19 cases)(44 controls)

Total number of SNVs366,9481,036,369

Number of variants57,22597,452

Exclusion of synonymous SNVs31,53761,692

Exclusion of segmental duplications26,80655,135

Average supporting read ≥ 820,27535,997

Average SNP quality ≥ 10018,66034,313

Supplementary Table 3 Top 30 Genes in the difference of average number of SNV

GeneCytogeneticDifference inNumber of SNVb

locationaverage number of SNVa

BTNL26p21.326.3 16

CDC2717q21.326.0 14

PRRC2A6p21.333.7 9

FAM104BXp11.213.4 9

CTBP210q26.133.0 13

OR12D26p22.2-p21.312.9 7

ZNF8319q13.32.8 13

MUC216p21.322.5 19

VARS26p21.332.0 9

CCHCR16p21.331.9 14

UBD6p22.11.8 6

C6orf106p21.31.7 10

LCN159q34.31.7 3

NOTCH46p21.321.510

OR10C16p22.11.4 7

C6orf476p21.31.3 2

PRSS27q351.3 7

HSPA1L6p21.331.3 2

OR2J36p22.11.3 4

KIAA168319p13.11.3 10

MOG6p22.11.2 2

CDSN6p21.331.1 2

WDR466p21.31.1 2

EFCAB4B12p13.321.1 5

MCCD16p21.331.1 2

CDC42BPA1q42.131.0 7

MYPN10q21.31.0 6

KDM4DL11q211.0 2

PRG311q12.11.0 3

KIAA126717q21.311.0 2

a The difference in the average number of SNVs in each gene between 19 cases and 44 controls.

b The number of SNV in cases.

Supplementary Table 4 The difference of average number of SNV in the RA-susceptible genes a

GeneCytogeneticaverage number of SNV

locationRAbControlc

C6orf106p21.31.7 0.0

PADI41p36.131.7 1.6

NOTCH46p21.31.5 0.0

AFF32q11.2-q121.2 1.3

LONRF22q11.21.1 1.5

PTPN221p13.21.1 1.0

ZNF77415q26.11.1 1.1

SH2B312q241.0 1.0

C59q34.10.9 0.9

ARHGEF33p14.30.7 0.3

C1QTNF622q13.10.7 0.7

CSF25q31.10.6 0.5

CTLA42q330.6 0.6

GSDMB17q120.6 0.8

TNFRSF141p36.320.6 0.6

MMEL11p360.4 0.5

AIRE21q22.30.3 0.4

PRKCQ10p150.3 0.4

TMEM172p150.3 0.3

ANKRD555q11.20.3 0.2

DSCAM21q22.20.3 0.4

ZPBP217q120.3 0.2

KIAA11094q270.2 0.4

ARAP111q13.40.2 0.3

IL6ST5q11.20.2 0.2

PRKCH14q23.10.2 0.3

PLD414q32.330.1 0.1

FOXF116q240.1 0.1

IL2RB22q13.10.1 0.3

PIP4K2C12q13.30.1 0.1

TNFAIP36q230.1 0.1

ANAPC44p15.20.0 0.0

ARID5B10q21.20.0 0.0

CD4020q12-q13.20.0 0.0

CD836p230.0 0.0

IL24q26-q270.0 0.0

IRF816q24.10.0 0.0

PDE2A11q13.40.0 0.1

PHF199q33.20.0 0.0

ANXA34q21.21-d-

APOM6p21.33--

B3GNT22p15--

BATF14q24.3--

BDP1P18q23--

BLK8p23-p22--

C21orf13021q22.2--

C4orf524p15.2--

C5orf305q21.1--

C9orf1449p13.2--

C9orf144B9p12--

CCL219p13--

CCR66q27--

CD2471q24.2--

CDK67q21-q22--

DKFZp667F071110p15.1--

ETS111q23.3--

FAM107A3p21.1--

FAM167A8p23-p22--

FLI111q24.1-q24.3--

GCH114q22.1-q22.2--

GEMIN8P115q23--

HCP5P126p21.3--

IKZF317q21--

IL214q26-q27--

IL2RA10p15-p14--

IRF57q32--

JDP214q24.3--

KIF31p36.12--

KIF5A12q13.13--

KRT18P392q33.2--

MICA6p21.33--

NFKBIE6p21.1--

NR6p21.32--

OLIG36q23.3--

P4HA25q31--

PFKL21q22.3--

POU3F11p34.1--

PRKCB16p11.2--

PRKCB116p11.2--

PTPN218p11.3-p11.2--

PXK3p14.3--

RBPJ4p15.2--

REL2p13-p12--

RNF1826p23--

RPLP115q22--

RPS12P32p16.1--

RPS14P107q32.3--

RPS15AP227q32.3--

RPS2P141p13--

RRAGC1p34--

RSBN11p13.2--

SALL318q23--

SPRED22p14--

STAT42q32.2-q32.3--

TNPO37q32.1--

TRAF19q33-q34--

TRHDE12q15-q21--

UBASH3A21q22.3--

a Genes listed in “A Catalog of Published Genome-Wide Association Studies”(

b 19 RA cases

c 44 disease control

dNo non-synonymous SNV was observed in exome sequencing of 19 RA followed by filtering in this study.“0.0” in the column of the average number of SNV indicates 0 < N < 0.1.

Supplementary Table 5 Comparison of the number of BTNL2 SNVs in the patients and controls with respect to HLA-DRB1 genotypes

GenotypeNumber of cases Average num. of BTNL2 SNV

RACont.RACont.

DRB1*04:05, 09:01361312.0 11.5

DRB1*04:05, 04:0524612.0 10.8

DRB1*04:05, 15:0222812.0 11.9

DRB1*09:01, 09:011872.0 0.0

DRB1*09:01, 15:02122011.7 12.0

DRB1*01:01, 04:0511711.2 11.7

DRB1*01:01, 09:011174.1 4.4

DRB1*08:03, 09:0110131.9 2.1

DRB1*01:01, 15:029611.8 11.8

DRB1*04:05, 08:0391211.8 11.1

DRB1*04:05, 04:0681110.8 10.2

DRB1*04:05, 13:028812.0 7.8

DRB1*08:03, 15:01662.0 2.0

DRB1*09:01, 15:01691.0 0.0

DRB1*08:03, 15:025612.0 11.8

The overall average number of BTNL2 SNV as follows: Case (n = 411), 7.8; Control (n = 420), 5.8.

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