UMOD Protein Structure and Mutation Analysis in UAKD

(1)

Legend for Supplemental Figure 1. Schematic representation of the UMOD molecule showing the locations of the studied UAKD mutations. Italic numbers denote amino acid sequence positions for boundaries between different functional domains. White circles denote mutations with low cell surface expression; black circles denote mutations with high cell surface expression; grey circles denote mutations with no cell surface expression data; branched structures denote N-linked glycosylation sites.

H₂N: N-terminus; SP: signal peptide; EGF: epidermal growth factor-like domain; Cys-rich:

cysteine-rich domain; D8C: domain of eight cysteines; ZP: zona pellucida domain; TM:

transmembrane domain; COOH: C-terminus.

(2)

Supplement Table 1. UMOD protein sequence and correlated functional domains

Functional Domain

UMOD Amino Acid Sequence Range

Number of Individuals with Mutations and ESRD

status (Families)

Signal Peptide 0-24 0

Epidermal Growth Factor 1 (EGF1)

31-64 5 (4)

64-107 20 (4)

107-149 14 (5)

Cysteine-Rich 1 (Cys-Rich 1) 149-199 40 (7)

Domain of Eight Cysteines (D8C)

199-287 73 (24)

Cysteine-Rich 2 (Cys-Rich 2) 287-334 39 (10)

Zona Pellucida (ZP) 334-585 9 (4)

Transmembrane 614-640 0

(3)

UMOD functional domains with their amino acid sequence positions. The number of individuals and families with mutations in each domain is also included in this table. There were no mutations found in the signal peptide and transmembrane regions.

(4)

Supplemental Table 2. UMOD Mutation and cellular surface expression Mutation Exon Functional

Domain

Cell Line Uromodulin Cell Surface Expression

(% of Wild Type)

Reference

p.Cys126Arg 4 EGF3 HeLa, 9.42 – Low (12)

AtT-20 43.1 - Low (8)

p.Cys223Arg 4 D8C HeLa 16.8 – Low (12)

p.Cys150Ser 4 Cys-Rich1 HEK293 30.7 – Low (10)

p.Val273Phe 4 D8C AtT-20 40.7 - Low (8)

p.Cys77Tyr 4 EGF2 LLC-PK1 48.0 – Low (15)

p.Asp196Asn 4 Cys-Rich1 HeLa 45.0 – Low (12)

p.Asp59Ala 4 EGF1 HEK293 47.0 - Low (10)

p.Cys315Arg 5 Cys-Rich2 HEK293 52.2 - High (10)

p.Cys148Trp 4 EGF3 HEK293 48.3 – High (9)

HEK293 53.0 – High (10)

p.Arg204Gly 4 D8C HEK293 56.9 - High (10)

p.Gly488Arg 8 ZP HeLa 58.90 - High (12)

p.Met229Arg 4 D8C AtT-20 64.9 - High (8)

HEK293 71.1 - High (10)

p.Cys32Tyr 4 EGF1 AtT-20 68.2 - High (8)

p.Cys317Tyr 5 Cys-Rich2 AtT-20 70.9 - High (8)

HEK293 74.0 - High (10)

p.Thr225Lys 4 D8C HEK293 84.7 - High (10)

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UMOD mutations with their exons, functional domain, and cell surface expression. Also included is the cell line used in each reference. Mutations which had a cell surface expression

<50% were considered as low and mutations which had a cell surface expression >50% were considered as high. If there was a difference in percent expression between two published reports, the average between them was used as the marker for expression.

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Supplemental Table 3 A. Amino acid affected by UMOD mutation vs. time to ESRD

Factor Coefficient

Hazard Ratio

Standard Error

Chi square

Degrees of Freedom

P-value

Frailty Model – Amino acid affected vs ESRD (Baseline Cysteine)

Non-Polar -1.04 0.35 0.87 1.42 1 0.23

Polar -0.21 0.81 0.59 0.12 1 0.73

Frailty 45.83 19.8 < 0.01

Cox-Proportional Hazards Test of the effect of amino acid type on time to ESRD. The amino acid affected is not statistically associated with time to ESRD

(7)

Supplemental Table 3 B. Amino Acid Affected and time to ESRD

The median manifestation age (in years) of ESRD manifestation was based on the Kaplan Meier estimates. NA, not available

UMOD mutation

Age (years) at ESRD manifestation

Estimate of Median

95% Confidence Interval

Lower Bound Upper Bound

Cysteine 57 52 62

Polar 58 49 67

Non-Polar 60 56 NA

(8)

Supplemental Figure 1