okamoto et.al. gpc5 - nature · okamoto et.al. gpc5 supplementary data common variation in gpc5 is...

OKAMOTO et.al. GPC5

Supplementary data

Common variation in GPC5 is associated with acquired

nephrotic syndrome

Koji Okamoto1,2, Katsushi Tokunaga2, Kent Doi1, Toshiro Fujita1, Hodaka Suzuki3,

Tetsuo Katoh3, Tsuyoshi Watanabe3, Nao Nishida2, Akihiko Mabuchi2, Atsushi

Takahashi4, Michiaki Kubo5, Shiro Maeda6, Yusuke Nakamura7 & Eisei Noiri1,8

1Department of Nephrology and Endocrinology, University Hospital, University of

Tokyo, Tokyo, Japan; 2Department of Human Genetics, Graduate School of

Medicine, University of Tokyo, Tokyo, Japan; 3Department of Internal Medicine

III, Fukushima Medical University School of Medicine, Fukushima, Japan; 4Laboratory for Statistical Analysis, Center for Genomic Medicine, RIKEN,

Yokohama, Japan; 5Laboratory for Genotyping Development, Center for

Genomic Medicine, RIKEN, Yokohama, Japan; 6Laboratory for Endocrinology

and Metabolism, Center for Genomic Medicine, RIKEN, Yokohama, Japan; 7Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical

Science, University of Tokyo, Tokyo, Japan; 8Science and Technology

Research Partnership for Sustainable Development

Correspondence should be addressed to E.N. ([email protected]).

Postal correspondence to E.N.: Eisei Noiri, MD, PhD,

Department of Nephrology and Endocrinology 107 Lab., The University of Tokyo

Hospital, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan

Nature Genetics: doi:10.1038/ng.792

OKAMOTO et.al. GPC5

SUPPLEMENTARY NOTE

1st panel and 2nd panel,

The subjects were recruited from the following medical institutes throughout

Japan; Fukujuji Hospital, Iizuka Hospital, Iwate Medical University School of

Medicine, Juntendo University, National Hospital Organization Osaka National

Hospital, Nihon University, Nippon Medical School, Osaka Medical Center for

Cancer and Cardiovascular Diseases, Shiga University of Medical Science, The

Cancer Institute Hospital of Japanese Foundation for Cancer Research,

Tokushukai Hospitals, and Tokyo Metropolitan Geriatric Hospital. Nephrotic

syndrome was diagnosed according to these criteria: The presence of heavy

proteinuria (>3.5 g/24 h), hypoalbuminemia (<3.0 g/dl) and peripheral edema.

Renal biopsy was performed in all NS patients. Control groups comprised 1,546

(control 1) and 1,548 individuals (control 2) registered as individuals with

diseases other than nephrotic syndrome, including bronchial asthma, myocardial

infarction, breast cancer, Basedow disease, cerebral infarction, cerebral

aneurism, osteoporosis, heart failure, unstable angina, pollinosis,

arteriosclerosis obliterans, emphysema, atopic dermatitis, stomach cancer, or

liver cirrhosis.

3rd panel

For the validation study (third panel), we obtained biopsy-proven NS case

samples from 201 cases at Fukushima Medical University, 142 cases at The

University Tokyo, and 88 cases from BioBank Japan as the previous criteria of

initial case 1 and case 2. We obtained control samples from 300

population-based control samples from the Pharma SNP consortium, and 3,071

disease control samples registered as individuals not having type 2 diabetes but

with diseases other than type 2 diabetes, having one of 13 other distinct

diseases, or healthy volunteers.


OKAMOTO et.al. GPC5

Supplementary Table 1. Study subjects for discovery, replication and extension

analysis

N Sex Age

at enrolment

(M-F) (years)

Case

1st panel 195 118 : 77 39.0±12.9

2nd panel 231 132 : 99 59.5±16.5

3rd panel 431 279 : 152 55.0±17.0

Combination 857 529 : 328 52.6

Control

1st panel 1,546 860 : 685 60.5±15.8

2nd panel 1,548 862 : 686 59.9±15.7

3rd panel 3,371 1,814 : 1,557 51.7±14.1

Combination 6,465 3,536 : 2,928 55.8

Case subjects include Minimal Change (n = 153), Focal segmental glomerulosclerosis

(n = 26), Membranous nephropathy (n = 117), Membranoproliferative glomerulonephritis

(n = 28), IgA nephropathy (n = 46), and Mesangial proliferative glomerulonephritis (n =

16), Crescentic glomerulonephritis (n = 5), Endocapillary proliferative glomerulonephritis

(n = 3), Diabetic nephropathy (n = 292), collagen disease related (n = 39), amyloidosis

(n = 7) and Drug induced (n = 2).Values are means ± s.d.


OKAMOTO et.al. GPC5

Supplementary Table 2. Replication study of significant SNPs in both 1st and 2nd

panel

SNP ID Chr Nearest

Gene Control Case P value

rs1537378

G > A 9

CDKN2B

CDKN2A

1st 985/368/41 120/65/8 7.6 x 10-8

2nd 1,051/369/49 131/87/6

3rd 2,254/995/114 291/118/16 0.70

rs727915

G > A 15

SLCO3A1

ST8SIA2

1st 1,194/293/19 127/61/5 7.3 x 10-7

2nd 1,196/306/17 163/65/3

3rd 2,699/669/47 348/76/7 0.52

rs16974063

T > C 17 MAP2K2

1st 1,119/390/37 156/38/1 7.3 x 10-7

2nd 1,126/378/43 188/38/3

3rd 2,496/802/64 303/111/7 0.42

rs1537378

C > T 15

SLCO3A1

ST8SIA2

1st 798/619/129 80/88/27 1.2 x 10-5

2nd 784/636/123 94/102/28

3rd 1,733/1,335/298 221/172/35 0.80

rs11086243

C > T 20

SUF2

PREX1

1st 941/522/83 136/52/7 1.2 x 10-4

2nd 929/533/86 159/66/5

3rd 2,025/1,177/163 285/124/15 5.6 x 10-3

rs1479517

C > T 15

SLC03A1

ST8SIA2

1st 1,072/432/42 118/70/7 1.3 x 10-4

2nd 1,091/418/39 143/76/12

3rd 2,339/936/89 318/96/16 0.21

rs16946160

G > A 13 GPC5

1st 1,063/409/50 115/63/14 2.0 x 10-4

2nd 1,071/411/35 156/55/17

3rd 2,317/930/119 267/121/34 3.2 x 10-4

rs11742884

C > T 5

ADRB2

HTR4

1st 517/730/266 82/83/28 4.3 x 10-4

2nd 503/713/283 95/106/29

3rd 1,240/1,606/520 139/213/75 0.072

rs1940163

C > A 11

MAML2

MTMR2

1st 678/625/180 64/108/21 5.8 x 10-4

2nd 653/652/165 83/109/36

3rd 1,475/1,467/417 169/203/55 0.16

rs1340021

C > G 8 DDEF1

1st 657/695/194 73/86/36 1.8 x 10-3

2nd 691/669/188 81/115/32

3rd 1,383/1,569/409 184/198/45 0.30

Risk allele is denoted in boldface. P values were calculated with allele frequency.


OKAMOTO et.al. GPC5

Supplementary Table 3. The association study of rs16946160 in each pathological

diagnosis

rs16946160 Case Control P value OR (95%CI)

Minimal change 92/50/10

4,451 /

1,750 /

204

4.6 x 10-3 1.47 (1.12-1.92)

Focal segmental glomerulosclerosis; 15/7/1 0.62 1.21 (0.58-2.50)

Membranous nephropathy 72/33/12 2.4 x 10-3 1.59 (1.18-2.13)

Membranoproliferative glomerulonephritis 16/5/5 0.021 2.00 (1.10-3.70)

IgA nephropathy 33/8/3 0.81 0.94 (0.53-1.67)

Diabetic nephropathy 167/79/21 4.7 x 10-4 1.45 (1.18-1.79)

P value and OR were calculated with allele frequency.

Cases who have possibility of overlapping multiple nephropathies were excluded.


OKAMOTO et.al. GPC5

Supplementary Table 4. Primer sequences & genotype assay ID

Genotyping Annotation Primer sequence

rs727915 Inter Genes TaqMan(R) SNP genotyping assay ID: C_930406_10


rs11086243 Inter Genes TaqMan(R) SNP genotyping assay: C_31521161_10



rs16946160 GPC5 intron 2 TaqMan(R) SNP genotyping assay ID: C_33435996_10

rs16974063 MAP2K2 intron TaqMan(R) SNP genotyping assay ID: C_34548663_10


rs1340021 DDEF1 intron TaqMan(R) SNP genotyping assay ID: C_8340534_10


Promoter-Exon 1 GPC5 promoter 5'-CCTCTGCCTGATGCTTTTAACT-3'

5'-CCCTAAGCTGAGACACCAGAGT-3'

Exon 1 GPC5 exon 1 5'-ACTCTGGTGTCTCAGCTTAGGG-3'

5'-AGTCCAGAGACCCCCTCATT-3'

Exon 2 GPC5 exon 2 5'-TGAAAAATGTTGTGTGTTCCAG-3'

5'-TTGCAGAATTCAAGATCCACTT-3'

Exon 3 GPC5 exon 3 5'-CTTGGAAAGCTGAATTTGGTTGAGTAGGCTGAGAGTTA-3'

5'-GATGCATTCTGAGTATTCCAGGGAACTGTCAGTCACA-3'

Exon 4 GPC5 exon 4 5'-TGGGCCCTATGAAACATCAC-3'

5'-TTTCTTTTCTCTTTCTGGTTGTAAA-3'

Exon 5 GPC5 exon 5 5'-TTTTGATGGCCTTTATTGTGG-3'

5'-TTCAGAAAAAGAAAGCAAGGATG-3'

Exon 6 GPC5 exon 6 3'-ACAGCAATACACACTACTTGATGAAAAACATTATTGAAGA-5'

5'-TGGCACGATAAGCATTTTAGCATTTTTCCTGGAAAAACATT-3'

Exon 7 GPC5 exon 7 5'-GGTGTCTACACCAAAAGAGTGG-3'

5'-TTTCCAATTCCTCTTGCTTG-3'

Exon 8 GPC5 exon 8 5'-GAAATGCAAAGGTAGCCAATTGTACAACATCAGGCTTTA-3'

5'-CATAAAGTCCCTAAAACTCAACGTTTAAATGACACACTTTA-3'

rs494371 for

Haplotype GPC5 intron 2

5'-TGCACATTTTGTTGACCGACTGAGTGAACGAGTAAA-3'

5'-CCAACTCTACCTCCAAAAACTCATCCAACTCATCCTA-3'

rs659614 for

Haplotype GPC5 intron 2

5'-AGTAGTTGCTGTTATATAAGAACAATGCTAATATCTAACTATA-3'

5'-CTGGCCAAGAATATGATAGAGAATTCACAGAAAAAGAAGACTA-3'

Mouse genotype-micro RNA-1 5'-TGAGAGCATTGCATCCTGTCTAT-3'

5'-CGTGCAGTAGGGCCCTCCTGAAC-3'


OKAMOTO et.al. GPC5

Mouse genotype-micro RNA-2 5'-GGCCACTGACTGACAGGACCTGCTTCAGGTTTG-3'

5'-GAACCTCGTCCACGTCCACTAC-3'

Mouse genotype-micro RNA-3 5'-GGCCACTGACTGACTCACCTCAGGGCAGAAATT-3'

5'-GAACCTCGTCCACGTCCACTAC-3'


OKAMOTO et.al. GPC5

Supplementary Table 5. RT-PCR primer sequences

Target Gene Primer sequence

Human GPC5 5'-GGTCGTTGGAAGAACTCTCG-3'

5'-GCGGCCACAAATCCTATTTA-3'

Rat gpc5 5'-GCTCCTTGGAAGAGCTGTCG-3'

5'-AAGGCCGCAAATCTTATTCA-3'

Mouse gpc5 5'-CGGTCTTTGGAAGAGTTGTCA-3'

5'-ATGGCCACAAATCGTATTCA-3'

Human GPC5 exon1-4 5'-CTGCGAAGAAGTTCGGAAAC-3'

5'-GGCTTTGTGTGGGTGTTCTT-3'

Human & Rat & Mouse B-actin 5'-CGCACCACTGGCATTGTCAT-3'

5'-TTCTCCTTGATGTCACGCAC-3'

Rat FGF-receptor2 5'-GCCAGCAGTCCCGCATCATCA-3'

5'-GACGCAACRGAGAARGAYTTG-3'


OKAMOTO et.al. GPC5

Supplementary Table 6. siRNA sequences

Target Gene siRNA sequence

Rat Gpc5 5’- GCAGGCGCUUAAUCUGGGCAUUGAA -3’,

5’- UUCAAUGCCCAGAUUAAGCGCCUGC-3’.

Mouse Gpc5 5’- GCAGGCGCUUAAUCUGGGCAUUGAA -3’

5’- UUCAAUGCCCAGAUUAAGCGCCUGC -3'

Mouse scrambled5’- CCUACAUGACGACGAUGUACCGUGA-3’

5’-UCACGGUACAUCGUCGUCAUGUAGG-3’


OKAMOTO et.al. GPC5

Supplementary Figure 1, Summary of GWAS results in the 1st panel

(a) A Manhattan plot showing the -log10(P values) of SNPs from the association analysis

of 195 Japanese individuals with NS and 1,546 control individuals. (b) The result of

quantile-quantil plots for the Fisher’s exact test in the 1st stage.


OKAMOTO et.al. GPC5

Supplementary Figure 2, Summary of replication study in the 2nd panel

(a) A Manhattan plot showing the -log10(P values) of SNPs from the association analysis

of 231 Japanese individuals with NS and 1,548 control individuals. (b) The result of

quantile-quantile plots for the Fisher’s exact test in the 2nd stage.


OKAMOTO et.al. GPC5

Supplementary Figure 3. Variation screening and association study of GPC5

LD block of in nearby genes. Structure of GPC5 and variations used for association

study. Black blocks represent coding regions and white blocks are the untranslated

regions. 3 SNPs (rs7322083, rs3759452, and rs553717) were found as variations with

direct sequencing for promoter region and all exons in 201 cases and 300 controls. P

value was calculated with allele frequency.


OKAMOTO et.al. GPC5

Supplementary Figure 4. Estimated haplotype frequencies in promoter and intron

2 in case and control samples

Structure of GPC5 and SNPs used for haplotype analysis. Black blocks represent

coding regions and white blocks are the untranslated regions. Five Tag SNPs

(rs7322083 and rs3759452 for promoter; rs494371, rs659614 and rs16946160 for intron

2) were selected for haplotype estimation. Haplotypes with frequencies of less than 1%

were excluded. The permutation P value was calculated by Haploview ver. 4.1 with

10,000 permutations.


OKAMOTO et.al. GPC5

Supplementary Figure 5, Urinary GPC5

(a) Western blot images of urinary GPC5. Each samples were standardized with urinary

creatinine. (b) Representative western blot image of each genotype. Each samples

were standardized with urinary creatinine (10 G/G, 6 G/A and 4 A/A).


OKAMOTO et.al. GPC5

Supplementary Figure 6, Production of podocyte specific knockdown mice

(a) Schematic drawing of transgenic vector for generating podocin induced three kinds

of micro RNA against Gpc5. The construct of the vector contains podocin promoter and

primary micro RNA sequences, which directs the excision of the engineered three kinds

of micro RNA against mice Gpc5. (b) Representative image of renal cortical western

blot. (c) Representative images of GPC5 (green) expression in mice glomerulus counter

stained by Nephrin (blue).


OKAMOTO et.al. GPC5

Supplementary Figure 7, Characterization data of rat-cultured glomerular

epithelial cells (GEC)

Differential interference contrast microscopic image and immunocytochemical staining.

Several markers of podocytes were expressed by this cell line. 516, GSA3,

synaptopodin has been detected. Thy1.1 as Mesangial cell marker has not been

detected in this cell line.


OKAMOTO et.al. GPC5

Supplementary Figure 8, In vivo knockdown efficacy

(a) Quantitative real-time PCR data showed expression levels of Gpc5 relative to that of

β actin on 3rd day after hydrodynamic siRNA transfection (n = 3, Error bars are s.e.m.).

(b) Representative image of renal cortical western blot and quantification. Protein levels

of GPC5 of renal cortex were quantified on 5th day after hydrodynamic siRNA

transfection (n = 3, Error bars are s.e.m.).


OKAMOTO et.al. GPC5

Supplementary Figure 9, siRNA against Gpc5 ameliorates PAN-FGF2 induced

pathological injury

(a) Periodic acid-Schiff stain of renal cortex at day 28. Focal segmental sclerosis

and cast formation by Tamm-Horsfall protein were found in both groups, though

pathological changes were more severe in scrambled-siRNA group. (b)

Sclerosis score of glomeruli in nephrotic model at day 28. The sclerosis score of

Gpc5-siRNA group mice were significantly lower than those of scrambled-siRNA

group (n = 6, Error bars are s.e.m.).


OKAMOTO et.al. GPC5

Supplementary Figure 10, PCA analysis in the 1st panel and 2nd panel

Samples in the 1st panel (a) and in the 2nd panel (b) in HapMap database are analyzed

by a program of smartpca1, and plotted for the first (X axis) and the 2nd (Y axis)

principal components. Japanese samples are plotted in a single cluster.


OKAMOTO et.al. GPC5

SUPPLEMENTARY REFERENCES 1. Price, A.L. et al. Principal components analysis corrects for stratification in

genome-wide association studies. Nat Genet 38, 904-9 (2006).


okamoto et.al. gpc5 - nature · okamoto et.al. gpc5 supplementary data common variation in gpc5 is...

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