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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.
Nature Genetics: doi:10.1038/ng.792
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.
Nature Genetics: doi:10.1038/ng.792
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.
Nature Genetics: doi:10.1038/ng.792
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.
Nature Genetics: doi:10.1038/ng.792
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
rs1551990 Inter Genes TaqMan(R) SNP genotyping assay ID: C_1903155_10
rs11086243 Inter Genes TaqMan(R) SNP genotyping assay: C_31521161_10
rs1479517 Inter Genes TaqMan(R) SNP genotyping assay ID: C_8718108_10
rs1940163 Inter Genes TaqMan(R) SNP genotyping assay ID: C_11473612_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
rs1537378 Inter Genes TaqMan(R) SNP genotyping assay ID: C_83169_10
rs1340021 DDEF1 intron TaqMan(R) SNP genotyping assay ID: C_8340534_10
rs11742884 Inter Genes TaqMan(R) SNP genotyping assay ID: C_31987068_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'
Nature Genetics: doi:10.1038/ng.792
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'
Nature Genetics: doi:10.1038/ng.792
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'
Nature Genetics: doi:10.1038/ng.792
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’
Nature Genetics: doi:10.1038/ng.792
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.
Nature Genetics: doi:10.1038/ng.792
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.
Nature Genetics: doi:10.1038/ng.792
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.
Nature Genetics: doi:10.1038/ng.792
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.
Nature Genetics: doi:10.1038/ng.792
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).
Nature Genetics: doi:10.1038/ng.792
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).
Nature Genetics: doi:10.1038/ng.792
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.
Nature Genetics: doi:10.1038/ng.792
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.).
Nature Genetics: doi:10.1038/ng.792
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.).
Nature Genetics: doi:10.1038/ng.792
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.
Nature Genetics: doi:10.1038/ng.792
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).
Nature Genetics: doi:10.1038/ng.792