Supplement 1
Targeted enrichment of known ataxia genes (“Ataxia panel”)
Sequencing started with fragmentation of DNA by digestion with multiple restriction enzymes. A
set of gene-specific HaloPlex selector probes (Agilent, Santa Clara, CA, USA) was hybridized
with the fragmented DNA, inducing target fragments to form circular DNA molecules that
contained method-specific sequencing motifs as well as a biotin label. Probe/target DNA
molecules were captured and purified using magnetic streptavidin beads, and targets were then
ligated to adjacent oligonucleotides that were complementarily bound with selector probes.
Selector probe oligonucleotides contained sample barcodes for precise tracking afterwards. A
PCR amplification step provided an enriched library ready for sequencing. Finally, the enriched
library was checked for quality and quantity prior to sequencing on an Illumina MiSeq
sequencing device using a 2 x 150 bp paired end approach (MiSeq sequencing kit V2). Reads
were mapped against the hg19 standard reference genome to detect single-nucleotide
polymorphisms, single-nucleotide variants, short deletions, and insertions (SAMtools, IGV).
Variants were annotated by comparing them with openly accessible data from different
databases (the dbSNP, 1000 Genomes project, and exome sequencing project of 6500
controls) using ANNOVAR genetic variation analysis software.
Supplement 2
List of the 122 ataxia genes targeted by the custom-built high-coverage HaloPlex panel
KCNC3
BTD
WFS1
FGF14
SETX
ATCAY
ATP2B2
ATP2B3
CEP290
VPS13A
OPA1
POLH
POLG
KIF5C
MTPAP
EEF2
PDYN
PNPLA6
C10ORF2
NPC1
NPC2
CC2D2A
GPR56
CSTB
RELN
NEU1
VAMP1
GCLC
TMEM216
MRE11A
KCNA1
RRM2B
CACNB4
PDSS1
PDSS2
VRK1
TTBK2
GALC
NPHP1
GCDH
TSEN54
TRPC3
RARS2
EPM2A
SACS
RPGRIP1L
DLAT
CRAT
AFG3L2
ABCB7
ATM
SYNE1
GBE1
PRICKLE1
SYT14
SPTBN2
CACNA1A
ARL13B
ATP7B
CSH1
CSH2
KIAA0226
HEXA
HEXB
TTPA
PAX6
ALG6
KCNJ10
GLB1
PEX7
SLC1A3
ALAS2
FXN
PEX2
ABHD12
TSEN2
ADCK3
DNAJC19
ANO10
APTX
PRKCG
DARS2
PMM2
GRM1
TMEM67
XPA
XPC
CYP27A1
CA8
DDB2
SIL1
PLA2G6
CLN5
SLC19A3
NHLRC1
HPRT1
PLEKHG4
ERCC5
INPP5E
PIK3R5
ERCC3
ERCC4
PDHX
PEX10
TSEN34
ERCC2
GBA
AMACR
AHI1
POLR3A
POLR3B
EIF2B1
PHYH
ITPR1
MTTP
GBA2
SLC17A5
L2HGDH
TDP1
ARSA
CP
VLDLR
Supplement 3
NPC1 and NPC2 variants VUS 3 identified in the EVS
NPC1
Position
(start)
Position
(end)
Exon/
intron
Exon/
intron
no.
Variant
effect
Alleles
cDNA
change
Protein
change
All Allele
ESP
MAF (%) (EA/AA/All)
All Genotype ESP
ExAC_allele
_freq
Polyphen
prediction
Disease
relevance
classification
21140382
21140379
exon
6
inframe
na
c.688_693
delTCTGT
G
p.S230_V23
1del
A1=3/R=1
2517
0.0363/0.0/0.024
A1A1=0/A1R=3/RR
=6257
0.0000318
na
variants of
unclear
significance
21119967
21119964
intron
17
unknown,
noncoding
na
c.26056_26053del
IVS1846T>C
A1=1/R=1
2519
0.0121/0.0/0.008
A1A1=0/A1R=1/RR
=6259
n/a
na
variants of
unclear
significance
21153543
21153543
intron
1
unknown,
noncoding
T/G
c.58-5t>g
IVS2-5t>g
C=1/A=13
005
0.0116/0.0/0.0077
CC=0/CA=1/AA=65
02
0.00001579
na
variants of
unclear
significance
21152148
21152148
intron
2
unknown,
noncoding
A/C
c.181-4a>c
IVS3-4a>c
G=2/T=13
004
0.0233/0.0/0.0154
GG=0/GT=2/TT=65
01
0.00001606
na
variants of
unclear
significance
21152147
21152147
intron
2
unknown,
noncoding
A/G
c.1813a>g
IVS3-3a>g
C=1/T=13
005
0.0/0.0227/0.0077
CC=0/CT=1/TT=65
02
0.00000802
4
na
variants of
unclear
significance
21148904
21148904
exon
4
stop
mutation
C/T
c.346C>T
p.R116X
A=1/G=13
005
0.0/0.0227/0.0077
AA=0/AG=1/GG=6
502
0.00000791
2
na
pathogenic
21148840
21148840
exon
4
missense
C/T
c.410C>T
p.T137M
A=1/G=13
005
0.0116/0.0/0.0077
AA=0/AG=1/GG=6
502
0.0000158
possibly
damaging
variants of
unclear
significance
21148825
21148825
exon
4
frameshift
na
c.424_425i
nsGA
p.K142Rfs*8
0
A1=1/R=1
2519
0.0121/0.0/0.008
A1A1=0/A1R=1/RR
=6259
0.00000790
2
na
likely
pathogenic
21141474
21141474
exon
5
missense
C/T
c.481C>T
p.R161W
A=2/G=13
004
0.0233/0.0/0.0154
AA=0/AG=2/GG=6
501
0.00003159
probably
damaging
variants of
unclear
significance
21141324
21141324
exon
5
missense
G/T
c.631G>T
p.D211Y
A=1/C=13
0.0/0.0227/0.0077
AA=0/AC=1/CC=65
0.00000789
probably
variants of
unclear
005
02
3
damaging
significance
21140279
21140279
exon
6
missense
A/G
c.797A>G
p.D266G
C=1/T=13
005
0.0116/0.0/0.0077
CC=0/CT=1/TT=65
02
n/a
possibly
damaging
variants of
unclear
significance
21140203
21140203
exon
6
missense
G/T
c.873G>T
p.W291C
A=7/C=12
999
0.0116/0.1362/0.0538
AA=0/AC=7/CC=64
96
0.00009559
probably
damaging
variants of
unclear
significance
21137071
21137071
intron
7
unknown,
noncoding
T/A
c.955+10t>
a
IVS7+10t>a
T=1/A=13
005
0.0116/0.0/0.0077
TT=0/TA=1/AA=65
02
0.00000808
7
na
variants of
unclear
significance
21136325
21136325
exon
8
missense
T/C
c.1208T>C
p.F403S
G=1/A=13
005
0.0/0.0227/0.0077
GG=0/GA=1/AA=6
502
0.00002371
possibly
damaging
variants of
unclear
significance
21136322
21136322
exon
8
missense
G/A
c.1211G>
A
p.R404Q
T=1/C=13
005
0.0116/0.0/0.0077
TT=0/TC=1/CC=65
02
0.00003951
probably
damaging
pathogenic
21134957
21134957
intron
8
unknown,
noncoding
T/A
c.13279t>a
IVS9-9t>a
T=1/A=13
005
0.0/0.0227/0.0077
TT=0/TA=1/AA=65
02
0.00001772
na
variants of
unclear
significance
21134854
21134854
exon
9
missense
C/T
c.1421C>T
p.P474L
A=1/G=13
005
0.0/0.0227/0.0077
AA=0/AG=1/GG=6
502
0.00003158
possibly
damaging
likely
pathogenic
21134723
21134723
exon
9
missense
C/T
c.1552C>T
p.R518W
A=2/G=13
004
0.0233/0.0/0.0154
AA=0/AG=2/GG=6
501
0.00006573
probably
damaging
likely
pathogenic
21131617
21131617
exon
10
missense
C/T
c.1628C>T
p.P543L
A=1/G=13
005
0.0116/0.0/0.0077
AA=0/AG=1/GG=6
502
n/a
probably
damaging
likely
pathogenic
21125090
21125090
exon
12
frameshift
na
c.1780_17
81insT
p.Y594Lfs*1
3
A1=1/R=1
2519
0.0121/0.0/0.008
A1A1=0/A1R=1/RR
=6259
n/a
na
likely
pathogenic
21124448
21124448
exon
13
missense
G/A
c.1990G>
A
p.V664M
T=1/C=13
005
0.0116/0.0/0.0077
TT=0/TC=1/CC=65
02
0.00000791
2
probably
damaging
likely
pathogenic
21124355
21124355
exon
13
missense
C/G
c.2083C>
G
p.L695V
C=1/G=13
005
0.0116/0.0/0.0077
CC=0/CG=1/GG=6
502
0.00000791
3
probably
damaging
likely
pathogenic
21123523
21123523
exon
14
missense
G/A
c.2141G>
A
p.R714H
T=2/C=13
004
0.0116/0.0227/0.0154
TT=0/TC=2/CC=65
01
0.0001671
probably
damaging
variants of
unclear
significance
21120491
21120491
exon
17
missense
T/C
c.2525T>C
p.P842S
G=1/A=13
0.0/0.0227/0.0077
GG=0/GA=1/AA=6
0.00000811
probably
variants of
unclear
005
502
6
damaging
significance
21120465
21120465
exon
17
missense
G/A
c.2551G>
A
p.A851T
T=2/C=13
004
0.0/0.0454/0.0154
TT=0/TC=2/CC=65
01
0.00004803
probably
damaging
variants of
unclear
significance
21119949
21119949
exon
18
missense
A/T
c.2621A>T
p.D874V
A=1/T=13
005
0.0116/0.0/0.0077
AA=0/AT=1/TT=65
02
0.00003173
benign
likely
pathogenic
21119438
21119438
intron
18
unknown,
noncoding
C/T
c.27964c>t
IVS19-4c>t
A=1/G=13
005
0.0/0.0227/0.0077
AA=0/AG=1/GG=6
502
0.00000912
7
na
variants of
unclear
significance
21119430
21119430
exon
19
stop
mutation
C/T
c.2800C>T
p.R934X
A=1/G=13
005
0.0116/0.0/0.0077
AA=0/AG=1/GG=6
502
0.00000893
na
pathogenic
21119411
21119411
exon
19
missense
C/T
c.2819C>T
p.S940L
A=1/G=13
005
0.0/0.0227/0.0077
AA=0/AG=1/GG=6
502
0.00003414
probably
damaging
pathogenic
21119357
21119357
exon
19
missense
G/A
c.2873G>
A
p.R958Q
T=1/C=13
005
0.0116/0.0/0.0077
TT=0/TC=1/CC=65
02
0.00002438
probably
damaging
pathogenic
21119321
21119321
exon
19
frameshift
na
c.2908_29
09insTT
p.S970fs*14
A1=1/R=1
2519
0.0/0.0234/0.008
A1A1=0/A1R=1/RR
=6259
n/a
na
likely
pathogenic
21119309
21119309
intron
19
unknown,
noncoding
C/T
c.2911+10
c>t
IVS19+10c>
t
A=1/G=13
005
0.0/0.0227/0.0077
AA=0/AG=1/GG=6
502
0.00003325
na
variants of
unclear
significance
21118640
21118640
intron
19
unknown,
noncoding
G/A
c.29125g>a
IVS20-5g>a
T=1/C=13
005
0.0116/0.0/0.0077
TT=0/TC=1/CC=65
02
0.00006366
na
variants of
unclear
significance
21118573
21118573
exon
20
missense
G/T
c.2974G>T
p.G992W
A=1/C=13
005
0.0116/0.0/0.0077
AA=0/AC=1/CC=65
02
0.00002369
probably
damaging
pathogenic
21118528
21118528
exon
20
missense
C/G
c.3019C>
G
p.P1007A
C=2/G=13
004
0.0233/0.0/0.0154
CC=0/CG=2/GG=6
501
0.0001184
probably
damaging
pathogenic
21116835
21116835
exon
21
missense
A/G
c.3047A>
G
p.H1016R
C=1/T=13
005
0.0/0.0227/0.0077
CC=0/CT=1/TT=65
02
0.00000814
5
benign
variants of
unclear
significance
21116830
21116830
exon
21
missense
G/A
c.3052G>
A
p.A1018T
T=2/C=13
004
0.0233/0.0/0.0154
TT=0/TC=2/CC=65
01
0.00004047
possibly
damaging
variants of
unclear
significance
21116700
21116700
exon
21
missense
T/C
c.3182T>C
p.I1061T
G=5/A=13
0.0465/0.0227/0.0384
GG=0/GA=5/AA=6
0.0002457
possibly
pathogenic
001
498
damaging
21116698
21116698
exon
21
missense
G/A
c.3184G>
A
p.A1062T
T=1/C=13
005
0.0116/0.0/0.0077
TT=0/TC=1/CC=65
02
n/a
possibly
damaging
variants of
unclear
significance
21116628
21116628
intron
21
unknown,
noncoding
T/C
c.3245+9t>
c
IVS21+9t>c
G=1/A=13
005
0.0/0.0227/0.0077
GG=0/GA=1/AA=6
502
0.00001897
na
variants of
unclear
significance
21116627
21116627
intron
21
unknown,
noncoding
G/A
c.3245+10
g>a
IVS21+10g>
a
T=1/C=13
005
0.0116/0.0/0.0077
TT=0/TC=1/CC=65
02
0.00000964
7
na
variants of
unclear
significance
21115645
21115645
exon
22
missense
G/A
c.3265G>
A
p.E1089K
T=1/C=13
005
0.0116/0.0/0.0077
TT=0/TC=1/CC=65
02
n/a
probably
damaging
likely
pathogenic
21115488
21115488
exon
22
missense
T/G
c.3422T>G
p.V1141G
C=1/A=13
005
0.0116/0.0/0.0077
CC=0/CA=1/AA=65
02
n/a
probably
damaging
variants of
unclear
significance
21114495
21114495
exon
23
missense
G/T
c.3506G>T
p.S1169I
A=2/C=13
004
0.0/0.0454/0.0154
AA=0/AC=2/CC=65
01
n/a
probably
damaging
variants of
unclear
significance
21114453
21114453
exon
23
missense
G/A
c.3548G>
A
p.R1183H
T=7/C=12
999
0.0/0.1589/0.0538
TT=0/TC=7/CC=64
96
0.0001516
probably
damaging
variants of
unclear
significance
21114445
21114445
exon
23
missense
C/T
c.3556C>T
p.R1186C
A=2/G=13
004
0.0233/0.0/0.0154
AA=0/AG=2/GG=6
501
0.00002395
probably
damaging
variants of
unclear
significance
21114444
21114444
exon
23
missense
G/A
c.3557G>
A
p.R1186H
T=1/C=13
005
0.0116/0.0/0.0077
TT=0/TC=1/CC=65
02
0.00003991
probably
damaging
likely
pathogenic
21114435
21114435
exon
23
missense
A/G
c.3566A>
G
p.E1189G
C=1/T=13
005
0.0116/0.0/0.0077
CC=0/CT=1/TT=65
02
0.00001599
possibly
damaging
likely
pathogenic
21113475
21113475
exon
24
missense
A/G
c.3598A>
G
p.S1200G
C=61/T=1
2945
0.0/1.3845/0.469
CC=2/CT=57/TT=6
444
0.0009578
probably
damaging
variants of
unclear
significance
21113458
21113458
exon
24
frameshift
na
c.3611_36
14delTTA
C
p.L1204fs
A1=1/R=1
2519
0.0/0.0234/0.008
A1A1=0/A1R=1/RR
=6259
0.00000790
6
na
likely
pathogenic
21113454
21113454
exon
24
missense
T/C
c.3619T>C
p.F1207L
G=1/A=13
0.0/0.0227/0.0077
GG=0/GA=1/AA=6
n/a
possibly
variants of
unclear
005
502
damaging
significance
21113384
21113384
exon
24
missense
T/C
c.3689T>C
p.L1230S
G=1/A=13
005
0.0116/0.0/0.0077
GG=0/GA=1/AA=6
502
0.00003161
probably
damaging
variants of
unclear
significance
21113331
21113331
exon
24
frameshift
na
c.3742_37
45delCTC
A
p.L1248Vfs1
3
A1=2/R=1
2518
0.0242/0.0/0.016
A1A1=0/A1R=2/RR
=6258
n/a
na
likely
pathogenic
21112207
21112207
exon
25
stop
mutation
C/T
c.3796C>T
p.R1266X
A=1/G=13
005
0.0116/0.0/0.0077
AA=0/AG=1/GG=6
502
0.00001579
na
pathogenic
21112158
21112158
UTR3
UTR3
unknown,
noncoding
G/T
c.3838+8G
>T
IVS25+8G>
T
A=1/C=13
005
0.0/0.0227/0.0077
AA=0/AC=1/CC=65
02
0.00000789
3
na
variants of
unclear
significance
Position
(start)
Position
(end)
Exon/
intron
Exon/
intron
no.
Variant
effect
Alleles
cDNA
change
Protein
change
All Allele
ESP
MAF (%) (EA/AA/All)
All Genotype ESP
ExAC_allele
_freq
Polyphen
Prediction
Disease
relevance
classification
74946943
74946943
UTR3/
Exon
5
untran
slated
UTR-3
unknown,
noncoding
A/G
c.*34a>g
NA
C=1/T=13
005
0.0/0.0227/0.0077
CC=0/CT=1/TT=65
02
na
variant of
unknown
significance
74946947
74946947
UTR3/
Exon
5
untran
slated
UTR-3
unknown,
noncoding
A/G
c.*30a>g
NA
C=1/T=13
005
0.0116/0.0/0.0077
CC=0/CT=1/TT=65
02
na
variant of
unknown
significance
74946948
74946948
UTR3/
Exon
5
untran
slated
UTR-3
unknown,
noncoding
C/T
c.*29c>t
NA
A=1/G=13
005
0.0116/0.0/0.0077
AA=0/AG=1/GG=6
502
na
variant of
unknown
significance
74951141
74951141
Exon
3
missense
C/G
c.340C>G
p.P114A
C=1/G=13
005
0.0116/0.0/0.0077
CC=0/CG=1/GG=6
502
probably
damaging
variant of
unknown
significance
74951189
74951189
Exon
3
missense
A/C
c.292A>C
p.N98H
G=11/T=1
2995
0.1163/0.0227/0.0846
GG=0/GT=11/TT=6
492
benign
variant of
unknown
NPC2
significance
74951203
74951203
Exon
3
missense
G/T
c.278G>T
p.C93F
A=1/C=13
005
0.0116/0.0/0.0077
AA=0/AC=1/CC=65
02
probably
damaging
likely
pathogenic
74951210
74951210
Exon
3
missense
G/A
c.271G>A
p.D91N
T=8/C=12
998
0.093/0.0/0.0615
TT=0/TC=8/CC=64
95
possibly
damaging
variant of
unknown
significance
74951257
74951257
Exon
3
missense
A/T
c.224A>T
p.H75L
A=1/T=13
005
0.0/0.0227/0.0077
AA=0/AT=1/TT=65
02
probably
damaging
variant of
unknown
significance
74951269
74951269
Exon
3
missense
A/G
c.212A>G
p.K71R
C=2/T=13
004
0.0233/0.0/0.0154
CC=0/CT=2/TT=65
01
probably
damaging
variant of
unknown
significance
74953107
74953107
Exon
2
missense
G/A
c.115G>A
p.V39M
T=1/C=13
005
0.0116/0.0/0.0077
TT=0/TC=1/CC=65
02
probably
damaging
likely
pathogenic
74953135
74953135
Exon
2
missense
G/A
c.88G>A
p.V30M
T=25/C=1
2979
0.2907/0.0/0.1922
TT=0/TC=25/CC=6
477
possibly
damaging
likely
pathogenic
74959921
74959921
Exon
1
stop
mutation
G/T
c.58G>T
p.E20X
A=1/C=13
001
0.0116/0.0/0.0077
AA=0/AC=1/CC=65
00
na
pathogenic
74959922
74959922
Exon
1
missense
C/A
c.56C>A
p.A19D
T=1/G=12
999
0.0/0.0227/0.0077
TT=0/TG=1/GG=64
99
probably
damaging
variant of
unknown
significance
74959929
74959929
Exon
1
missense
G/A
c.49G>A
p.A17T
T=1/C=12
999
0.0/0.0227/0.0077
TT=0/TC=1/CC=64
99
benign
variant of
unknown
significance
74959940
74959940
Exon
1
missense
T/C
c.38T>C
p.L13P
G=4/A=13
000
0.0465/0.0/0.0308
GG=0/GA=4/AA=6
498
possibly
damaging
variant of
unknown
significance
NPC1 and NPC2 variants VUS class 3 and higher [9] identified in the EVS. EVS data were filtered using the following criteria: MAF all <1%, nonsynonymous, exonic/splicing, target region: exons ±20bp, non-synonymous; variants VUS class 3 and higher. Last column presents the predicted
variant disease classification according to the NP-C disease gene variation database (Stampfer et al.; in preparation).