From supplemental tables S2 and S3 (authors used hg19 alignment).

MONDO: MONDO:0005145 for SALS

Case#1: Smith 2014 SALS, G43V, M, 52 y.o. onset, Italy

CasePresentingHPOs: HP:0002145, HP:0007354

CaseHPOFreeText: spinal-onset ALS with upper and lower motor neuron signs

CasePreviousTesting: Only the FALS cases were pre-screened for other mutations

CaseMethod1: This case identified through case-control study

CaseGenotypingMethod: Exome sequencing, confirmed with Sanger sequencing.

Variant: NM_006000.3(TUBA4A):c.128G>T p.Gly43Val

CAID: CA2122536

gnomAD: 2-220116828-C-A, Total overall AF (non-neuro) = 0.00002402; TUBA4A_p.G43V_gnomAD

CaseAddInfo: This is the sole SALS case in this report of TUBA4A variants, while the others are FALS. This is also the only N-terminal variant, while the rest are C-terminal variants with more supporting functional evidence. This variant found in 1/13023 control samples.

From supplemental tables S2 and S3 (authors used hg19 alignment).

MONDO: MONDO:0005144 for FALS

Case#4: Smith 2014 FALS R320C, M, 64 y.o. onset, Italy

CasePresentingHPOs: HP:0007354

CaseHPOFreeText: spinal-onset ALS with upper and lower motor neuron signs

CasePreviousTesting: All FALS were screened for known causative mutations/repeat expansions in SOD1, C9orf72, TARDBP, FUS, PFN1, UBQLN2, OPTN, VCP and ANG and removed from cohort if positive for variant.

CaseMethod1: This case identified through case-control study

CaseGenotypingMethod: Exome sequencing, confirmed with Sanger sequencing.

Variant: NM_006000.3(TUBA4A):c.958C>T (p.Arg320Cys)

ClinVar: 180182

gnomAD: 2-220115463-G-A; Total overall AF (non-neuro) = 0.000009609; TUBA4A_p.Arg320Cys_gnomAD

CaseAddInfo: This variant found in 0/13023 control samples.

From supplemental tables S2 and S3 (authors used hg19 alignment).

MONDO: MONDO:0005144 for FALS

Case#6: Smith 2014 FALS A383T, M, 71 y.o. onset, Italy

CasePresentingHPOs: HP:0007354

CaseHPOFreeText: spinal-onset ALS with upper and lower motor neuron signs

CasePreviousTesting: All FALS were screened for known causative mutations/repeat expansions in SOD1, C9orf72, TARDBP, FUS, PFN1, UBQLN2, OPTN, VCP and ANG and removed from cohort if positive for variant.

CaseMethod1: This case identified through case-control study

CaseGenotypingMethod: Exome sequencing, confirmed with Sanger sequencing.

Variant: NM_006000.3(TUBA4A):c.1147G>A (p.Ala383Thr)

ClinVar: 180186

gnomAD: 2-220115274-C-T; Total overall AF (non-neuro) = 0.000009611; TUBA4A_p.Ala383Thr_gnomAD

CaseAddInfo: This variant found in 1/13023 control samples.

From supplemental tables S2 and S3 (authors used hg19 alignment).

MONDO: MONDO:0005144 for FALS

Case#7: Smith 2014 FALS K430N, F, 64 y.o. onset, Italy

CasePresentingHPOs: HP:0007354

CaseHPOFreeText: spinal-onset ALS with upper and lower motor neuron signs

CasePreviousTesting: All FALS were screened for known causative mutations/repeat expansions in SOD1, C9orf72, TARDBP, FUS, PFN1, UBQLN2, OPTN, VCP and ANG and removed from cohort if positive for variant.

CaseMethod1: This case identified through case-control study

CaseGenotypingMethod: Exome sequencing, confirmed with Sanger sequencing.

Variant: NM_006000.3(TUBA4A):c.1290G>C (p.Lys430Asn)

CAID: CA350721313

CaseAddInfo: This variant found in 0/13023 control samples. This variant did not co-segregate with an affected cousin of the sequenced proband.

From supplemental tables S2 and S3 (authors used hg19 alignment).

MONDO: MONDO:0005144 for FALS

Case#5: Smith 2014 FALS R320H, F, 41 y.o. onset, Italy

CasePresentingHPOs: HP:0007354

CaseHPOFreeText: spinal-onset ALS with upper and lower motor neuron signs

CasePreviousTesting: All FALS were screened for known causative mutations/repeat expansions in SOD1, C9orf72, TARDBP, FUS, PFN1, UBQLN2, OPTN, VCP and ANG and removed from cohort if positive for variant.

CaseMethod1: This case identified through case-control study

CaseGenotypingMethod: Exome sequencing, confirmed with Sanger sequencing.

Variant: NM_006000.3(TUBA4A):c.959G>A (p.Arg320His)

ClinVar: 180183

gnomAD: 2-220115462-C-T; Total overall AF (non-neuro) = 0.000 ; TUBA4A_p.Arg320His_gnomAD

CaseAddInfo: This variant found in 0/13023 control samples.

From supplemental tables S2 and S3 (authors used hg19 alignment).

MONDO: MONDO:0005144 for FALS

Case#2: Smith 2014 FALS, T145P, M, 69 y.o. onset, Italy

CasePresentingHPOs: HP:0007354

CaseHPOFreeText: spinal-onset ALS with upper and lower motor neuron signs

CasePreviousTesting: All FALS were screened for known causative mutations/repeat expansions in SOD1, C9orf72, TARDBP, FUS, PFN1, UBQLN2, OPTN, VCP and ANG and removed from cohort if positive for variant.

CaseMethod1: This case identified through case-control study

CaseGenotypingMethod: Exome sequencing, confirmed with Sanger sequencing.

Variant: NM_006000.3(TUBA4A):c.433A>C (p.Thr145Pro)

ClinVar: 180187

CaseAddInfo: This variant found in 0/13023 control samples.

From supplemental tables S2 and S3 (authors used hg19 alignment).

MONDO: MONDO:0005144 for FALS

Case#3: Smith 2014 FALS R215C, F, 78 y.o. onset, Italy

CasePresentingHPOs: HP:0002145, HP:0007354

CaseHPOFreeText: spinal-onset ALS with upper and lower motor neuron signs

CasePreviousTesting: All FALS were screened for known causative mutations/repeat expansions in SOD1, C9orf72, TARDBP, FUS, PFN1, UBQLN2, OPTN, VCP and ANG and removed from cohort if positive for variant.

CaseMethod1: This case identified through case-control study

CaseGenotypingMethod: Exome sequencing, confirmed with Sanger sequencing.

Variant: NM_006000.3(TUBA4A):c.643C>T (p.Arg215Cys)

ClinVar: 180185

gnomAD: 2-220115778-G-A; total overall AF (non-neuro) = 0.000009609; TUBA4A_pArg215Cys_gnomAD

CaseAddInfo: This variant found in 0/13023 control samples.

From supplemental tables S2 and S3 (authors used hg19 alignment).

MONDO: MONDO:0005144 for FALS

Case#8: Smith 2014 FALS W407X, F, 66 y.o. onset, Italy

CasePresentingHPOs: HP:0007354

CaseHPOFreeText: spinal-onset ALS with upper and lower motor neuron signs

CasePreviousTesting: All FALS were screened for known causative mutations/repeat expansions in SOD1, C9orf72, TARDBP, FUS, PFN1, UBQLN2, OPTN, VCP and ANG and removed from cohort if positive for variant.

CaseMethod1: This case identified through case-control study

CaseGenotypingMethod: Exome sequencing, confirmed with Sanger sequencing.

Variant: NM_006000.3(TUBA4A):c.1220G>A (p.Trp407Ter)

ClinVar: 180184

CaseAddInfo: This variant found in 0/13023 control samples.

Case#: Li 2018 fALS H393Pfs*19, Chinese

MonDO: MONDO:0005144 for fALS

CasePresentingHPOs: HP:0007354

CaseGenotypingMethod: Sanger sequencing. TUBA4A sequencing was performed using Sanger. No further details provided.

Variant: NM_006000.3(TUBA4A):c.1177dup (p.His393ProfsTer19)

CAID: CA2018007640

CaseAddInfo: This variant failed to cosegregate with affected family members. This proband also carried the SOD1 p.E133V variant (chr21:33040827A>T, c.401A>T).

Case#: Li 2018 sALS A383T, Chinese, 65 y.o. onset

MonDO: MONDO:0005145 for sALS

CasePresentingHPOs: HP:0007354

CaseGenotypingMethod: Sanger sequencing. TUBA4A sequencing was performed using Sanger. No further details provided.

Variant: NM_006000.3(TUBA4A):c.1147G>A (p.Ala383Thr)

ClinVar: 180186

gnomAD: 2-220115274-C-T; total overall AF (non-neuro) = 0.000009611; TUBA4A_A383T_gnomAD

Case#: Li 2018 sALS I243T, Chinese, 51 y.o. onset

MonDO: MONDO:0005145 for sALS

CasePresentingHPOs: HP:0007354

CaseGenotypingMethod: Sanger sequencing. TUBA4A sequencing was performed using Sanger. No further details provided.

Variant: NM_006000.3(TUBA4A):c.701T>C (p.Ile234Thr)

CAID: CA350726345

Case#: Li 2018 sALS I42T, Chinese

MonDO:* MONDO:0005145 for sALS

CasePresentingHPOs: HP:0007354

CaseGenotypingMethod: Sanger sequencing. TUBA4A sequencing was performed using Sanger. No further details provided.

Variant: NM_006000.3(TUBA4A):c.125T>C (p.Ile42Thr)

CAID: CA2122537

gnomAD: 2-220116831-A-G; total overall AF (non-neuro) = 0.0001787 --> exceeds VCEP limit of 0.00005 therefore score at 0; TUBA4A_pI42T_gnomAD

Supplementary Table 1 (7 cols x 6 rows)

Nucleotide position Type of variation rs ID 1000 Genome MAF % ESP MAF %<br /> Italian Controls (MAF%) SALS (MAF %) c.909A>G p.Val303Val rs141922502 0.18 0.477 37/3960 (0.47) 14/1106 (0.6) c.999C>T p.Ala333Ala rs147351423 0.32 0.19 20/3960 (0.25) 2/1106 (0.1) c.1257C>T p.Ser419Ser rs199590938 0.01 § - - 1/1106 (0.05) c.376-16C>T Intronic rs116486324 0.16 0.21 n.c. 1/1106 (0.05) c.8G>A 3’UTR rs142720001 0.02 0.023 4/3960 (0.05) 1/1106 (0.05) c.48G>A 3’UTR rs11556349 1.7 0.845 46/3960 (0.58) 4/1106 (0.18)

Case#: N6287, M, 59 y.o., Italian

CasePresentingHPOs: HP:0007354, HP:0100543, HP:0002483

CaseNotHPOs:

CaseGenotypingMethod: Sanger sequencing

Variant: NM_006000.3(TUBA4A):c1312G>A p.Asp438Asn

CAID: CA65993021

gnomAD: 2-220115109-C-T; Total overall AF (non-neuro) = 0.000009617; gnomAD_NM_006000.3(TUBA4A):c1312G>A p.Asp438Asn

CaseAddInfo: dbSNP ID: rs913033084. While most cases were found to be negative during pre-screening for known mutations, some cases were positive for mutations in SOD1 (n=4), C9orf72 (n=12), TARDBP (n=4), & FUS/TLS (n=1) genes. Whether this case tested positive for any of these is not specified.

Case#: N5214, F, 62 y.o., Italian

CasePresentingHPOs: HP:0007354, HP:0002015, HP:0001260, HP:0002380, HP:0003690, HP:0003202

CaseNotHPOs: HP:0100543

CaseGenotypingMethod: Sanger sequencing

Variant: NM_006000.3(TUBA4A):c.1045A>T p.Thr349Ser

CAID: CA350723588

CaseAddInfo: While most cases were found to be negative during pre-screening for known mutations, some cases were positive for mutations in SOD1 (n=4), C9orf72 (n=12), TARDBP (n=4), & FUS/TLS (n=1) genes. Whether this case tested positive for any of these is not specified.

Case#: P937, F, Italian

CasePresentingHPOs: HP:0007354

CaseGenotypingMethod: Sanger sequencing

Variant: c.19C>A p.Val7Ile

CaseAddInfo: This allele is not found in the reference NM_006000.2 or NM_006000.3 (CAID can not be created or retrieved because there is a G in this location instead of the C reported here).

CaseAddInfo: While most cases were found to be negative during pre-screening for known mutations, some cases were positive for mutations in SOD1 (n=4), C9orf72 (n=12), TARDBP (n=4), & FUS/TLS (n=1) genes. Whether this case tested positive for any of these is not specified.

Case#: B1103, M, 57 y.o., Italian

CasePresentingHPOs: HP:0007354, HP:0003202, HP:0007340

CaseNotHPOs: HP:0100543

CaseGenotypingMethod: Sanger sequencing

Variant: NM_006000.3(TUBA4A):c.226+4A>G IVS2+4A>G

CAID: CA2018007638

CaseAddInfo: While most cases were found to be negative during pre-screening for known mutations, some cases were positive for mutations in SOD1 (n=4), C9orf72 (n=12), TARDBP (n=4), & FUS/TLS (n=1) genes. Whether this case tested positive for any of these is not specified.

Case#: Perrone 2017, F, 47 y.o. onset, Belgium

CasePresentingHPOs: HP:0007354, HP:0009027, HP:0001260, HP:0007289, HP:0001324, HP:0003487

CasePreviousTesting: Multiplex exome NGS panel for major FTD, ALS, AD, and PD genes: GRN, MAPT, VCP, TARDBP, FUS, SOD1, TBK1, APP, PSEN1, PSEN2, PRNP, LRRK2, PARK2, and SNCA

CaseMethod1:

CaseMethod2:

CaseGenotypingMethod: Whole-genome sequencing, exome resequencing using a multiplex NGS panel for major FTD, ALS, AD, and PD genes, and multiplex PCR amplification of the 4 TUBA4A exons (if all patients from the "Belgian Neurology consortium" are also the "patients of Project MinE").

Variant: NM_006000.3(TUBA4A):c.1142C>T p.Thr381Met

CAID: CA2122379

gnomAD: 2-220115279-G-A, Total overall AF (non-neuro) = 0.00001922; TUBA4A_Thr381Met_gnomAD

CaseAddInfo: The reported variant c.1142G>A is not a valid allele for TUBA4A. That is the genomic coordinate of the reference, not the coding sequence (TUBA4A is on the antisense strand). The CAID was found using fuzzy query at the protein level.