Supplementary materials

Loss of podocalyxin causes a novel syndromic type of congenital nephrotic syndrome

Hee Gyung Kang1,2,7 , Moses Lee3,7, Kyoung Boon Lee4, Michael Hughes5, Bo Sang Kwon1,

Sangmoon Lee3, Kelly M. McNagny5, Yo Han Ahn1,2, Jeong Min Ko1,2, Il-Soo Ha1, 6, Murim

Choi1,3,6 and Hae Il Cheong1,2,6

Departments of 1Pediatrics, 3Biomedical Sciences, and 4Pathology, Seoul National University

College of Medicine, Seoul, Republic of Korea; 2Research Coordination Center for Rare

Diseases, Seoul National University Hospital, Seoul, Republic of Korea; 5The Biomedical

Research Centre, The University of British Colombia, Vancouver, Canada; 6Kidney Research

Institute, Medical Research Center, Seoul National University College of Medicine, Seoul,

Republic of Korea

7These authors contributed equally to this work.

Correspondance: Professor HI Cheong, Department of Pediatrics, Seoul National University

College of Medicine, 101 Daehang-no, Jongno-Gu, Seoul 03080, Republic of Korea. Tel:

82-2-2072-2810. Fax: 82-2-743-3455. E-mail: cheonghi@snu.ac.kr

or Professor M Choi, Department of Biomedical Sciences, Seoul National University College

of Medicine, 103 Daehang-no, Jongno-Gu, Seoul 03080, Republic of Korea. Tel: 82-2-740-

8912. Fax: 82-2-3673-2167. E-mail: murimchoi@snu.ac.kr

Supplementary Table 1. Whole exome sequencing statistics. Variant called and filtered as

previously descrived1.

Sample Proband Mother Father

Sex Male Female Male

Read length (bp) 100 100 100

Number of reads (M) 67.7 76.2 50.1

Mean coverage depth (x) 79.8 76.2 60.8

% of reads mapped on genome 99.44% 99.45% 99.50%

% of reads mapped on target 71.33% 70.60% 73.83%

% of targeted bases covered at least 4x 99.79% 99.66% 99.55%

% of targeted bases covered at least 8x 99.34% 99.30% 98.74%

% of targeted bases covered at least 20x 95.77% 96.68% 92.08%

Mean error rate 0.17% 0.17% 0.16%

Supplementary Table 2. List of notable variants discovered from the proband. Comp. het.: Compound heterozygotes.

Type Gene

Chromosome:

position

(hg19)

Nucleotide

substitution

Zygosit

y

Impact on

proteinAmino acid change

Amino acid

position/protein length

# of species different from

human/# of species with

ortholog

ExAC

frequency

Coverage depth

(alternative allele/total)

Proband Mother Father

De novo FUT1* 19:49253542 c.997T>C Het. Missense p.Ser333Pro 333/365 0/68 0

39/87

(44.8%)

0/129 0/72

Comp.

het.CPEB2†

4:15005328 c.1032_1046del Het.In-frame

deletionp.Pro345_His349del 345/1034 N.A.

0.053

(East Asian)

26/66

(44.8%)

51/51

(100%)

0/40

4:15005224 c.922_923insC Het.Frameshift

insertionp.Ala310ArgfsTer17 310/1034 N.A. 0

25/54

(46.2%)

0/8444/90

(48.9%)

Comp.

het. PODXL‡

7:131194125 c.1023G>A Het. Nonsense p.Trp341Ter 341/558 N.A. 031/62

(50%)

40/72

(55.6%)0/43

7:131241116 c.3G>T Het. Start lost p.0 1/558 N.A. 035/85

(41.2%)0/82

24/57

(42.1%)

*RefSeq ID : NG_007510.2, NP_000139.1,NM_000148.3.

†RefSeq ID : NP_001170853.1, NM_001177382.1.

‡RefSeq ID: NP_001018121.1, NM_001018111.2.

Supplementary Table 3. PCR primers used in this study.

Primer name Nucleotide sequences 5’-3’Length of

product (bp)

PODXL-M1I-F CAG GGC TCC GTG TGT GAC 415

PODXL-M1I-R AGC GGC AGG GAG GAA GAG 415

PODXL-W341X-F GGG AGG AGG GAA TGG GTA AG 210

PODXL-W341X-R CCA CAG CAG CAT CAA CTA CC 210

FUT1-S333P-F ACA CCA CAA GCT TCT CCA GA 294

FUT1-S333P-R GCA ACC ACA CCA TTA TGA CCA T 294

Supplementary Table 4. List of loss-of-function variants in Exome Aggregated Consortium (ExAC).

Chr Position RSID Ref AlternateProtein

ConsequenceTranscript Consequence Annotation Allele frequency

7 131189145 . C CATTCCACT p.Gly535ValfsTer14 c.1601_1602insAGTGGAAT Frameshift1/121406

(8.24E-06)

7 131189147 . G

GTTCATCTATAAAGTCCCTTACGTGGCTTTTTCTTGATCTCCCTCATCATCCAGC

AGCA

p.Leu534CysfsTer19

c.1599_1600insTGCTGCTGGATGATGAGGGAGATCAAGAAAAAGCCACGTAAGGGACTTT

ATAGATGAA

Stop gained1/121406

(8.24E-06)

7 131189268 . C T c.1480-1G>A Splice acceptor1/121314

(8.24E-06)

7 131190795rs14561

4497C T c.1312-1G>A Splice acceptor

1/100808

(9.92E-06)

7 131191386 . AT A p.Ser401LeufsTer28 c.1200delA Frameshift1/121400

(8.24E-06)

7 131193761 . CTG C p.Gln350AspfsTer4 c.1048_1049delCA Frameshift1/121332

(8.24E-06)

7 131193782 . CTT C p.Lys343ValfsTer2 c.1027_1028delAA Frameshift1/121312

(8.24E-06)

7 131193789 . T C c.1024-2A>G Splice acceptor1/121298

(8.24E-06)

7 131193997 . TG T p.His331IlefsTer14 c.991delC Frameshift4/22794

(1.76E-04)

7 131193997 . TGG T p.His331PhefsTer11 c.990_991delCC Frameshift4/22794

(1.76E-04)

7 131194093 . C A p.Glu320Ter c.958G>T Stop gained1/120000

(8.33E-06)

7 131194104 . C CT p.Gly316GlufsTer32 c.946_947insA Frameshift3/120940

(2.48E-05)

7 131195586rs13790

7090C T c.706+1G>A Splice donor

1/121176

(8.25E-06)

7 131196199 . GT G c.101-2delA Splice acceptor1/120002

(8.33E-06)

7 131241039 . GAC G p.Ser27AlafsTer16 c.78_79delGT Frameshift35/24220

(1.45E-03)

7 131241042 .GGCGA

G p.Ser25ArgfsTer141 c.73_76delTCGC Frameshift37/25628

(1.44E-03)

Supplementary Table 5. Comparison of phenotypes between the mouse mutant model and the human

patient.

Phenotype Human PODXLM1I/W341X Mouse model Reference

Anuric Renal Failure + 100% 1

Omphalocele at birth + ~25% 1

Generalized Edema + ~30% 1

Microcoria + No 1

Patent ductus arteriosus + N.A. 1

Pulmonary Hypertension + Suggestive 2

Neural symptoms + Suggestive 3, 4

Recurrent infections + N.A. -

Supplementary Figure 1. Genome-wide (A) copy number variation and (B) loss-of-

heterozygosity patterns of the patient genome. No notable structural variation was

detected.

Supplementary Figure 2. Sanger traces of FUT1 S333P de novo variant from the patient

and parents. CPEB2 sequences were CG-rich (GC ratio: 76.1%) and not amplifiable. Primer

sequences used for PCR amplifications are shown at Table S2.

Supplementary Figure 3. Selected isoform structures and expression profile for CPEB2.

(A) Among the CPEB2 isoforms, two variants (isoforms 7 and 8) extend long enough to

contain the compound heterozygous variants on the 310th and 345th amino acid residues. (B)

The expression profiles of the four isoforms across 53 human tissues. The compound

heterozygous variants containing isoforms 7 and 8 are either expressed exclusively in testis

(red; isoform 8) or barely detectable in all tissues (yellow; isoform 7), while isoform 3 (light

blue) displays a more dynamic pattern throughout multiple tissues. The expression data was

obtained from GTEx (http://www.gtexportal.org/).

Supplementary Figure 4. Western blot analysis of PODXL in HeLa cells.

Supplementary Figure 5. Immunohistochemistry of omphalocele from second control.

H&E (A), CD34 (B), and PODXL (C) staining (Original magnification x400).

Supplementary Figure 6. Coverage profile of LAMB2 by WES.

Supplementary Figure 7. Eye structure of Podxl-/- mice at postnatal day 1. (A-C) Podxl+/+

and (D-F) Podxl-/- gross (A, D), and H&E stained (B, C, E, F) eyes, displaying largely normal

eye and head structures in the mutant mice. Scale bars: 1 mm (B) and 200 µm (C). D: dorsal,

V: ventral, P: posterior, A: anterior.