Table S1. Investigated hGALE variants’ known properties

hGALE variant / UDP-Gal % Activity / UDP-GalNAc % Activity / UDP-Gal Km / UDP-GalNAc Km / NAD+ Limitation / Ability to rescue growth in yeast model / Protease stability / Thermal stability / Aggregation / References
p.A25V / 42 / ND / ND / ND / ND / ND / No detectable change / ND / Not observed / [57]
p.N34S / 88.88 / ND / No detectable change / ND / Decreased activity at low NAD+ concentrations against UDP-Gal / Yes / Decreased / ND / ND / [22, 52]
p.R40C / 38 / ND / ND / ND / ND / ND / No detectable change / ND / Not observed / [57]
p.D69E / 26 / ND / ND / ND / ND / ND / No detectable change / ND / Not observed / [57]
p.S81R / 25 / ND / No detectable change / ND / ND / Yes / No detectable change / ND / ND / [10]
p.G90E / 0.13 / 0 / No detectable change / ND / ND / No / Decreased / ND / ND / [14, 52]
hGALE variant / UDP-Gal % Activity / UDP-GalNAc % Activity / UDP-Gal Km / UDP-GalNAc Km / NAD+ Limitation / Ability to rescue growth in yeast model / Protease stability / Thermal stability / Aggregation / References
p.V94M / 3.1 / 23.6 / Increased / Increased / ND / No / Slight increase / ND / ND / [14, 51, 52]
p.D103G / 78 / 70 / Increased / ND / No detectable change / Yes / Decreased / Decreased / ND / [14, 52]
p.T150M / 24.7 / ND / No change / ND / ND / Yes / No detectable change / ND / ND / [10]
p.K161N / 0.5 / 72.5 / Increased / ND / No response and highly decreased activity against UDP-Gal. Decreased response and slightly decreased activity against UDP-GalNAc / No / Decreased / Increased / ND / [23]
p.E165K / 0 / ND / ND / ND / ND / ND / Decreased / ND / Yes / [57]
p.R169W / 60 / ND / ND / ND / ND / ND / No detectable change / ND / Not observed / [57]
hGALE variant / UDP-Gal % Activity / UDP-GalNAc % Activity / UDP-Gal Km / UDP-GalNAc Km / NAD+ Limitation / Ability to rescue growth in yeast model / Protease stability / Thermal stability / Aggregation / References
p.D175N / 43.57 / 72.5 / No detectable change / ND / Decreased response and activity against both UDP-Gal and UDP-GalNAc. / Yes / Decreased / Decreased / ND / [23]
p.L183P / 30.56 / ND / No detectable change / ND / ND / No / Decreased / ND / ND / [22, 52]
p.R220W / 47 / 98 / No detectable change / Increased / No detectable change / Yes / ND / ND / ND / [12]
p.R239W / 0 / ND / ND / ND / ND / ND / No detectable change / ND / Not observed / [57]
p.K257R / 106 / 106 / No detectable change / No detectable change / No detectable change / Yes / No detectable change / ND / ND / [23]
p.P293L / 56.11 / ND / No detectable change / ND / ND / Yes / No detectable change / ND / ND / [10]
hGALE variant / UDP-Gal % Activity / UDP-GalNAc % Activity / UDP-Gal Km / UDP-GalNAc Km / NAD+ Limitation / Ability to rescue growth in yeast model / Protease stability / Thermal stability / Aggregation / References
p.G302D / 0 / ND / ND / ND / ND / ND / No detectable change / ND / Not observed / [57]
p.L313M / 69 / 117 / Reduced / ND / No detectable change / Yes / No detectable change / Decreased / ND / [14, 52]
p.G319E / 124 / 124 / Increased / No detectable change / No detectable change / Yes / No detectable change / ND / ND / [23]
p.R335H / 41.67 / ND / Increased / ND / ND / ND / No detectable change / ND / Not observed / [52, 57]

ND: Not determined.


Table S2. SNP effect 4.0 and I-Mutant 3.0 predictions of investigated hGALE variants

hGALE variant / Predictions (TANGO, WALTZ, LIMBO and FoldX) / FoldX
kcal/mol
1EK6 / FoldX
kcal/mol
1HZJ / Average FoldX
kcal/mol / I-Mutant 3.0
kcal/mol
1EK6 / I-Mutant 3.0
kcal/mol
1HZJ / Average I-Mutant 3.0
kcal/mol
Mild
p.G319E / Increases the ability of binding to chaperones Reduced stability / -3.19 / -2.36 / -2.78 ± 0.59 / -0.18 (3) / -0.11 (5) / -0.15 ± 0.05
p.K257R / 0.26 / -0.71 / -0.23 ± 0.69 / -0.31 (4) / -0.44 (5) / -0.38 ± 0.09
p.N34S / 0.16 / -0.91 / -0.38 ± 0.76 / -0.18 (5) / -0.23 (5) / -0.21 ± 0.04
p.D103G / Slightly reduced stability / -0.79 / -0.86 / -0.83 ± 0.05 / -1.14 (7) / -1.18 (7) / -1.16 ± 0.03
p.L313M / -0.02 / -0.10 / -0.06 ± 0.06 / -0.65 (3) / -0.64 (3) / -0.65 ± 0.01
p.R169W / -0.31 / 0.59 / 0.14 ± 0.64 / -0.28 (6) / -0.42 (7) / -0.35 ± 0.10
p.P293L / Decreases the ability of binding to chaperones Reduced stability / -2.39 / -2.37 / -2.38 ± 0.01 / -0.49 (6) / -0.58 (6) / -0.54 ± 0.06
Intermediate
p.R220W / Reduced stability / -1.34 / -0.27 / -0.81 ± 0.8 / -0.45 (4) / -0.39 (4) / -0.42 ± 0.04
p.D175N / Slightly reduced stability / -0.53 / -0.43 / -0.48 ± 0.07 / -0.50 (0) / -0.53 (2) / -0.52 ± 0.02
p.A25V / -0.27 / -0.04 / -0.16 ± 0.16 / 0.39 (2) / 0.42 (3) / 0.41 ± 0.02
p.R335H / Decreases the ability of binding to chaperones / -0.42 / -0.25 / -0.34 ± 0.12 / -0.88 (6) / -0.83 (5) / -0.86 ± 0.04
p.R40C / -0.57 / 0.17 / -0.2 ± 0.52 / -0.21 (2) / -0.19 (2) / -0.20 ± 0.01
p.L183P / Severely reduced stability / -6.58 / -6.51 / -6.55 ± 0.05 / -2.12 (7) / -2.29 (7) / -2.21 ± 0.12
p.D69E / Reduced stability / -2.32 / -2.72 / -2.52 ± 0.28 / 0.21 (3) / -0.07 (2) / 0.07 ± 0.2
p.S81R / Decreases propensity to aggregate Increased stability / 1.46 / 1.45 / 1.46 ± 0.01 / 0.18 (2) / 0.27 (4) / 0.23 ± 0.06
p.T150M / Slightly increased stability / 0.53 / 0.64 / 0.59 ± 0.08 / 0.19 (4) / 0.14 (3) / 0.17 ± 0.04
Severe
p.V94M / Slightly increased stability / 0.66 / 0.59 / 0.63 ± 0.05 / -0.59 (7) / -0.53 (7) / -0.56 ± 0.04
p.K161N / -0.11 / 0.65 / 0.27 ± 0.54 / -1.36 (7) / -1.36 (7) / -1.36 ± 0.00
p.G90E / Reduced stability / -2.7 / -4.10 / -3.4 ± 0.99 / -0.57 (3) / -0.59 (2) / -0.58 ± 0.01
p.E165K / Decreases the ability of binding to chaperones Reduced stability / -3.90 / -3.28 / -3.59 ± 0.44 / -1.46 (8) / -1.62 (9) / -1.54 ± 0.11
p.R239W / Decreases the ability of binding to chaperones Slightly reduced stability / -0.82 / -1.34 / -1.08 ± 0.38 / -0.16 (1) / -0.16 (1) / -0.16 ± 0.00
p.G302D / Reduced stability / -3.18 / -3.03 / -3.11 ± 0.11 / -0.68 (6) / -0.70 (6) / -0.69 ± 0.01


Table S3. Known patient’ genotypes, clinical phenotypes and individual mutation rankings

Genotype / Clinical Phenotype / Activity Groups (UDP-Gal) / Reference
N34S/L183P / Mild/Peripheral / M/I / [16]
R40C/R169W / Mild/Peripheral / I/M / [79]
A25V/R40C / Mild/Peripheral / I/I / [79]
D69E/E165K / Mild/Peripheral / I/S / [79]
D69E/G302D / Mild/Peripheral / I/S / [79]
G90E/unknown / Mild/Peripheral / S/? / [18]
D103G/D103G / Mild/Peripheral / M/M / [18]
A25V/R169W / Mild/Peripheral / I/M / [79]
D175N/unknown / Mild/Peripheral / I/? / [9]
R239W/R335H / Mild/Peripheral / S/I / [79]
K257R/WT / Mild/Peripheral / M/M / [18]
G302D/R335H / Mild/Peripheral / S/I / [79]
L313M/WT / Mild/Peripheral / M/M / [18]
G319E/unknown / Mild/Peripheral / M/? / [18]
K161N/unknown / Mild/Peripheral / S/? / [9]
R220W/R220W / Mild/Peripheral / I/I / [12]
S81R/G319E / Intermediate / I/M / [9]
T150M/T150M / Intermediate / I/I / [9]
P293L/unknown / Intermediate / M/? / [9]
V94M/V94M / Severe/Generalised / S/S / [8]


Table S4. Identified missense mutations of hGALE from the NCBI dbSNP database

Transcript / Mutation / No. of Independent Observations / rs ID / Submitter(s)
c.41T>C / p.Ile14Thr / 1 / rs143264410 / NHLBI Exome Sequencing Project
c.77G>A / p.Gly26Asp / 1 / rs150919841 / NHLBI Exome Sequencing Project
c.172C>T / p.Arg58Cys / 1 / rs142821993 / NHLBI Exome Sequencing Project
c.416C>G / p.Pro139Arg / 1 / rs141583863 / NHLBI Exome Sequencing Project
c.421T>C / p.Tyr141His / 3 / rs11546126 / Cancer Genome Anatomy Project (CGAP)
Illumina Inc
Korea Research Institute of Bioscience and Biotechnology
c.506G>A / p.Arg169Gln / 1 / rs140878602 / NHLBI Exome Sequencing Project
c.518A>C / p.Gln173Pro / 1 / rs149888063 / NHLBI Exome Sequencing Project
c.503C>T / p.Thr177Ile / 1 / rs151234596 / NHLBI Exome Sequencing Project
c.539C>T / p.Ala180Val / 2 / rs3204468 / UCLA-DOE Laboratory for Structural Biology and Molecular Medicine Sequenom, Inc.
c.551G>A / p.Arg184His / 1 / rs140515600 / NHLBI Exome Sequencing Project
c.611T>C / p.Ile204Thr / 1 / rs143924570 / NHLBI Exome Sequencing Project
c.643G>T / p.Val215Leu / 1 / rs3180382 / UCLA-DOE Laboratory for Structural Biology and Molecular Medicine
c.680G>C / p.Gly227Ala / 1 / rs3177811 / UCLA-DOE Laboratory for Structural Biology and Molecular Medicine
c.691G>T / p.Asp231Tyr / 1 / rs3179862 / UCLA-DOE Laboratory for Structural Biology and Molecular Medicine
c.704G>C / p.Gly235Ala / 2 / rs3180549 / UCLA-DOE Laboratory for Structural Biology and Molecular Medicine Sequenom, Inc.
c.712G>A / p.Val238Ile / 1 / rs112843559 / Stephan C. Schuster Lab Penn State Univeristy
c.728A>C / p.His243Pro / 1 / rs143015594 / NHLBI Exome Sequencing Project
c.733G>A / p.Val245Met / 1 / rs145762332 / NHLBI Exome Sequencing Project
c.736G>A / p.Asp246Asn / 1 / rs148015366 / NHLBI Exome Sequencing Project
c.755T>A / p.Ile252Thr / 1 / rs148834880 / NHLBI Exome Sequencing Project
c.808C>A / p.Asn268Lys / 1 / rs3179863 / UCLA-DOE Laboratory for Structural Biology and Molecular Medicine
c.842A>C / p.Tyr275Ser / 1 / rs139134986 / NHLBI Exome Sequencing Project
c.848C>T / p.Ala283Val / 2 / rs3179864 / UCLA-DOE Laboratory for Structural Biology and Molecular Medicine Sequenom, Inc.
c.904G>C / p.Gly302Arg / 1 / rs146052241 / NHLBI Exome Sequencing Project
c.979G>C / p.Asp327His / 1 / rs150326189 / NHLBI Exome Sequencing Project
c.991G>T / p.Glu331Ter / 4 / rs3209973 / UCLA-DOE Laboratory for Structural Biology and Molecular Medicine
Sequenom, Inc.
Illumina Inc.
Korea Research Institute of Bioscience and Biotechnology
c.1034G>A / p.Gly345Asp / 1 / rs141944470 / NHLBI Exome Sequencing Project


Table S5. SNP effect 4.0 and I-Mutant 3.0 predictions of reported hGALE variants from the NCBI dbSNP database

hGALE variant / Predictions (TANGO, WALTZ, LIMBO and FoldX) / FoldX
kcal/mol
1EK6 / FoldX
kcal/mol
1HZJ / Average FoldX
kcal/mol / I-Mutant 3.0
kcal/mol
1EK6 / I-Mutant 3.0
kcal/mol
1HZJ / Average I-Mutant 3.0
kcal/mol
p.I14T / Reduced stability / -2.64 / -2.00 / -2.32 ± 0.45 / -1.42 (5) / -1.44 (6) / -1.43 ± 0.01
p.G26D / Reduced stability / -3.95 / -4.19 / -4.07 ± 0.17 / -0.80 (3) / -0.71 (3) / -0.76 ± 0.06
p.R58C / Reduced stability / -1.98 / -2.33 / -2.16 ± 0.25 / -0.80 (5) / -0.74 (5) / -0.77 ± 0.04
p.P139R / Reduced stability / -3.51 / -3.17 / -3.34 ± 0.24 / -0.66 (6) / -0.69 (6) / -0.68 ± 0.02
p.Y141H / Reduced stability / -1.62 / -2.40 / -2.01 ± 0.55 / -0.55 (1) / -0.64 (2) / -0.60 ± 0.06
p.R169Q / -0.44 / -0.39 / -0.42 ± 0.04 / -0.92 (9) / -1.10 (9) / -1.01 ± 0.12
p.Q173P / Reduced stability / -3.56 / -3.71 / -3.64 ± 0.11 / -0.02 (4) / -0.11 (4) / -0.07 ± 0.06
p.T177I / Increased aggregation tendency Reduced stability / -1.63 / -1.80 / -1.72 ± 0.12 / -0.15 (0) / -0.14 (2) / -0.15 ± 0.01
p.A180V(1EK6)
p.V180A(1HZJ) / Increased aggregation tendency (1EK6)
Decreased aggregation tendency (1HZJ)
Reduced stability / -1.16 / -1.08 / -1.12 ± 0.06 / -0.58 (1) / -2.19 (10) / -1.39 ± 1.14
p.R184H / Increased aggregation tendency Increased amyloid propensity Severely reduced stability / -7.03 / -5.21 / -6.12 ± 1.29 / -1.37 (7) / -1.31 (6) / -1.34 ± 0.04
p.I204T / -0.39 / -0.41 / -0.4 ± 0.01 / -0.47 (6) / -0.72 (8) / -0.60 ± 0.18
p.V215L / Decreased amyloid propensity Reduced stability / -2.07 / -4.27 / -3.17 ± 1.56 / -0.61 (3) / -0.67 (4) / -0.64 ± 0.04
p.G227A / Reduced stability / -3.5 / -3.68 / -3.59 ± 0.13 / -1.22 (6) / -1.23 (6) / -1.23 ± 0.01
p.D231Y / -0.29 / -0.06 / -0.18 ± 0.16 / 0.25 (7) / 0.33 (7) / 0.29 ± 0.06
p.G235A / Severely reduced stability / -5.01 / -4.72 / -4.87 ± 0.21 / -1.08 (5) / -1.08 (5) / -1.08 ± 0.00
p.V238I / 0.47 / 0.37 / 0.42 ± 0.07 / -0.24 (3) / -0.24 (3) / -0.24 ± 0.00
p.H243P / -1.09 / -0.22 / -0.66 ± 0.62 / -0.43 (0) / -0.43 (1) / -0.43 ± 0.00
p.V245M / Increased stability / 1.42 / 0.80 / 1.11 ± 0.44 / -0.16 (2) / -0.14 (2) / -0.15 ± 0.01
p.D246N / Increased aggregation tendency Reduced stability / -1.37 / -1.12 / -1.25 ± 0.18 / 0.33 (5) / 0.24 (4) / 0.29 ± 0.06
p.I252T / Reduced stability / -1.71 / -1.30 / -1.51 ± 0.29 / -1.73 (8) / -1.84 (7) / -1.79 ± 0.08
p.N268K / Severely reduced stability / -5.38 / -6.71 / -6.05 ± 0.94 / -0.46 (0) / -0.53 (0) / -0.50 ± 0.05
p.Y275S / Reduced stability / -2.77 / -2.44 / -2.61 ± 0.23 / -1.10 (6) / -1.17 (7) / -1.14 ± 0.05
p.A283V / Decreased ability to bind chaperones Slightly reduced stability / -0.02 / -0.84 / -0.43 ± 0.58 / -0.35 (5) / -0.27 (5) / -0.31 ± 0.02
p.G302R / Reduced stability / -2.78 / -2.37 / -2.58 ± 0.29 / -0.34 (5) / -0.37 (5) / -0.36 ± 0.02
p.D327H / Reduced stability / -1.21 / -1.75 / -1.48 ± 0.38 / -0.09 (0) / 0.08 (2) / -0.01 ± 0.12
p.G345D / Reduced stability / -2.34 / -1.51 / -1.93 ± 0.59 / -0.62 (4) / -0.63 (4) / -0.95 ± 0.01

14