PMID_35617426-html.html

Supplementary Information

Identification of

PSMB5

as a genetic modifier of Fragile X-associated Tremor/Ataxia

Syndrome

Ha Eun Kong

, Junghwa Lim

, Alexander Linsalata

, Yunhee Kang

, Indranil Malik

, Emily G

Allen

, Yiqu Cao

, Lisa Shubeck

, Rich Johnston

, Yanting Huang

, Yanghong Gu

, Xiangxue

Guo

, Michael E Zwick

, Zhaohui Qin

, Thomas S Wingo

1, 5

, Jorge Juncos

, David L Nelson

Michael Epstein

, David J Cutler

, Peter K Todd

, Stephanie L Sherman

, Stephen T Warren

1, 6

and Peng Jin

1, *

Correspondence should be addressed to Peng Jin (peng.jin@emory.edu).

This PDF file includes:

Figures S1 to S5
Tables S1 to S4

DOI:10.1073/pnas.2118124119

Figure S1. Distribution of (CGG)

repeats in 108

FMR1

premutation carriers.

The relative

frequency distribution of (CGG)

repeats in the

FMR1

premuation carriers is shown, with each bar

illustrating a bin centered at (CGG)

repeats. Mean= 88.55, Median = 85, Standard

deviation=23.011.

Figure S2

PSMB5

rs11543947-A

is an eQTL that correlates with decreased expression of

PSMB5

in a spectrum of tissues.

Multi-tissue eQTL comparison (The Genotype-Tissue Expression

Project) shows the single-tissue eQTL data for 48 tissues that harbor

PSMB5

rs11543947-A

. In 46 out

of 48, the Normalized Effect Size (NES), computed as the effect of the alternative allele (ALT)
relative to the reference allele (REF), is negative, demonstrating that the expression of the gene is
diminished with the alternative allele. This negative NES is most robustly observed in tissues with
higher sample size (n>300); but the trend is apparent in all most tissues other than the hippocampus
and ovary, which have NES > 0.

Brain - Hippocampus
Ovary
Uterus
Brain - Nucleus accumbens (basal ganglia)
Colon - Sigmoid
Brain - Hypothalamus
Brain - Cerebellum
Esophagus - Muscularis
Nerve - Tibial
Brain - Cerebellar Hemisphere
Brain - Substantia nigra
Breast - Mammary Tissue
Small Intestine - Terminal Ileum
Colon - Transverse
Artery - Coronary
Brain - Caudate (basal ganglia)
Vagina
Brain - Amygdala
Lung
Prostate
Spleen
Thyroid
Cells - Transformed fibroblasts
Muscle - Skeletal
Artery - Tibial
Esophagus - Gastroesophageal Junction
Heart - Atrial Appendage
Whole Blood
Skin - Not Sun Exposed (Suprapubic)
Adipose - Subcutaneous
Skin - Sun Exposed (Lower leg)
Adipose - Visceral (Omentum)
Liver
Heart - Left Ventricle
Cells - EBV-transformed lymphocytes
Brain - Putamen (basal ganglia)
Pituitary
Stomach
Artery - Aorta
Esophagus - Mucosa
Brain - Frontal Cortex (BA9)
Testis
Brain - Anterior cingulate cortex (BA24)
Brain - Spinal cord (cervical c-1)
Adrenal Gland
Minor Salivary Gland
Pancreas
Brain - Cortex

Tissue

111
122
101
130
203
108
154
335
361
125
80
251
122
246
152
144
106
88
383
132
146
399
300
491
388
213
264
369
335
385
414
313
153
272
117
111
157
237
267
358
118
225
109
83
175
85
220
136

Samples

0.0317
0.000374
-0.0353
-0.0712
-0.0937
-0.101
-0.110
-0.115
-0.129
-0.132
-0.132
-0.135
-0.139
-0.141
-0.145
-0.159
-0.163
-0.164
-0.187
-0.191
-0.195
-0.195
-0.200
-0.205
-0.208
-0.209
-0.210
-0.223
-0.226
-0.228
-0.235
-0.235
-0.236
-0.240
-0.250
-0.257
-0.282
-0.288
-0.294
-0.311
-0.344
-0.355
-0.413
-0.415
-0.433
-0.463
-0.464
-0.476

Beta

0.8
1
0.7
0.5
0.2
0.3
0.4
0.01
0.005
0.1
0.4
1.6e-3
0.06
0.02
0.04
0.2
0.09
0.2
2.9e-7
0.03
0.02
1.8e-9
1.8e-6
6.4e-11
2.6e-7
1.3e-3
1.4e-4
2.1e-9
1.4e-5
8.7e-6
3.6e-8
1.1e-7
8.5e-4
9.9e-6
0.04
0.02
1.5e-3
8.2e-6
6.8e-5
1.0e-9
2.8e-3
6.6e-5
8.0e-4
1.3e-3
3.5e-6
1.5e-4
1.1e-5
2.7e-5

p-value

0.818
0.934
0.883
0.958
0.951
0.943
0.938
0.981
1.00
0.952
0.867
0.999
0.991
0.985
1.00
0.993
1.00
0.971
1.00
1.00
0.998
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.952
1.00
1.00
1.00
1.00
1.00
0.990
0.996
1.00
1.00
1.00
1.00
1.00
1.00

post-prob

Single-tissue eQTL

Effect Size (with 95% CI)

-0.6

-0.4

-0.2

-0.0

0.2

Effect Size (beta)

Single-tissue eQTL p-value

versus Multi-tissue Posterior Probability

0.0

0.2

0.4

0.6

0.8

1.0

m-value (Posterior Probability from METASOFT)

-l

(

-ti

-v

)

Figure S3. Expression of PSMB5.

A. The expression of

Psmb5

is not altered in FXTAS mouse

model. Psmb5 mRNA level was determined using 3-month-old FMRpolyG+RNA mice (1). B.
The expression of

PSMB5

is not altered in

FMR1

premutation iPSC-derived NPCs. C. Expression

Prosbeta5

was significantly reduced in Prosbeta5 TRiP line. Prosbeta5 TRiP line was crossed

with

elav-GAL4

and the expression of

Prosbeta5

was determined by RT-PCR using brain RNA.

D. siRNA knockdown of

Psmb5

in Neuro2A cells. RT-PCR shows diminished level of

Psmb5

mRNA in Neuro2A cells transfected with

Psmb5

siRNA vs. control siRNA, along with 5’UTR

CGG 99x FMR1-EGFP and empty vector control normalized to housekeeping genes Rpl and Tbp.

Figure S4. Titration of Ixazomib Citrate in Neuro2A cells transfected with 5’UTR CGG 99x
FMR1-EGFP.

Neuro2A cells transfected with 5’UTR CGG 99x FMR1-EGFP plasmid were

treated with various concentrations of Ixazomib Citrate for 84 hrs. Concentrations of Ixazomib
Citrate greater than 0.25 nM showed significant alleviation of CGG-associated toxicity.

Figure S5. RAN translation is diminished upon

PSMB5

knockdown in HEK293 cells.

Western

blotting  demonstrates  diminished  levels  of  RAN  translation  in  cells  transfected  with  PSMB5
siRNA  compared  to  siNT  (multiple  t-test  p-value  =  2.89  x10^-5).  +1  CGG  RAN  translation
reporters characteristically produce high molecular weight proteins (2).

Table S1.

FXTAS phenotype in premutation carriers.

Clinical phenotypes of 108

FMR1

premutation carriers.

Phenotype

Number of

samples

Repeat size (Mean

± S.D.)

Asymptomatic > age 69

70.33 ± 6.30

Late onset 1 phenotype (ataxia or tremor) >
age 65

83.61 ± 24.33

Mixed onset both phenotypes

97.90 ± 20.71

Early onset 1 phenotype < age 65

89.00 ± 25.38

Early onset both ataxia and tremor < age 65

98.58 ± 19.38

Total

108

88.60 ± 22.98

Table S2. Results of

Drosophila

screen

FXTAS r(CGG)

flies expressing GAL4 under the eye-specific gmr driver were crossed with flies

harboring an shRNA transgene against the target candidate gene, downstream from the UAS
sequence (UAS-TRiP). Progeny that express the shRNA against target gene in the eye were
collected, aged for 7 days and visualized using light microscopy and SEM. Results of the genetic
screen of 97 genes revealed 18 candidate genes that show significant modulation of the CGG-
toxicity associated rough eye phenotype in FXTAS

Drosophila

Human
Gene

Drosophila

Ortholog

UAS TRiP stock

Drosophila Phenotype

ACAD9

Egm

36739

Enhanced but n<10

ACO2

Acon

34028

No phenotype

CG4706

51359

No phenotype

58074

Slightly enhanced

ADPGK

CG6650

57528

No phenotype

AHCY

Ahcy

51477

No phenotype

AK2

Adk2

55320

Suppressed

ALDH1L2

CG8665

62266

No phenotype

AMHR2

wit

25949

No phenotype

41906

No phenotype

ANKHD1

mask

31574

No phenotype

34571

No phenotype

ANKS1A

CG4393

58087

Enhanced

AP1G2

AP-1gamma

27533

Enhanced

AREL1

CG4238

36086

No phenotype

ARHGEF3

RhoGEF64C

31130

No phenotype

ATF6B

Atf6

26211

No phenotype

ATRIP

mus304

61355

No phenotype

ATRNL1

dsd

53318

No phenotype

ATXN2L

Atx2

36114

No phenotype

44012

No phenotype

C1QBP

P32

34585

No phenotype

CCNB1

CycB

34544

No phenotype

38957

No phenotype

38979

Suppressed

CNOT2

Rga

35460

No phenotype

57549

No phenotype

DHX35

CG3225

55365

No phenotype

DICER

Dcr-1

28598

No phenotype

34826

Slightly Enhanced

42901

No phenotype

DMRTB1

dsx

26716

No phenotype

35645

No phenotype

41864

No phenotype

DUOX2

Duox

32903

Enhanced

33975

No phenotype

38907

No phenotype

DVL2

dsh

31306

Slightly enhanced

31307

No phenotype

ENDOU

CG2145

60036

No phenotype

61997

No phenotype

ENPEP

CG32473

43972

No phenotype

54013

Enhanced

58177

No phenotype

EPB41L3

cora

35003

No phenotype

ERCC3

hay

53345

Suppressed

EXOC7

Exo70

28041

Slightly enhanced

55234

No phenotype

FBLN1

CG31999

31587

No phenotype

31697

No phenotype

FPGS

CG2543

62271

No phenotype

FUS

caz

34839

Slightly enhanced

32990

Slightly enhanced

GCAT

CG10361

44517

No phenotype

GLI2

31320

Slightly enhanced

31321

Enhanced

GORASP1

Grasp65

34082

No phenotype

GPAM

mino

63587

No phenotype

HHAT

rasp

28921

No phenotype

HSPA4

Hsc70Cb

33742

Slightly suppressed

IGF2BP2

Imp

34977

No phenotype

38219

No phenotype

55645

No phenotype

ITGA10

scb

27545

Slightly enhanced

38959

No phenotype

ITGB4

mys

27735

Slightly enhanced

33642

Slightly enhanced

KCNJ16

lrk2

25820

No phenotype

41981

No phenotype

KHNYN

CG42360

60444

No phenotype

LAMA4

LanA

28071

Slightly enhanced

LGALS3BP

Tango10

57165

No phenotype

LIPN

CG6753

62921

No phenotype

LZTS2

CG15365

36856

No phenotype

MADD

Rab3-GEF

28954

No phenotype

MAGI3

Magi

25792

No phenotype

33411

No phenotype

35279

No phenotype

MEOX2

btn

42530

No phenotype

MINA

CG2982

33596

Suppressed

MYO1C

Myo61F

41689

No phenotype

NANOG

bsh

29336

No phenotype

43994

Enhanced

NDE1

nudE

38954

No phenotype

41860

No phenotype

NIF3L1

CG4278

60362

No phenotype

OSGEPL1

CG14231

43269

No phenotype

54799

Enhanced

PABPC1L

pAbp

28821

Enhanced

36127

No phenotype

53247

No phenotype

PCK2

CG10924

36915

No phenotype

PDE11A

Pde6

25828

Slightly enhanced

35743

No phenotype

PDGFB

Pvf1

39038

Slightly enhanced

PDLIM5

Zasp52

31561

Slightly enhanced

PHTF1

phtf

43631

No phenotype

57270

No phenotype

PIK3R3

Pi3K21B

36810

No phenotype

38991

No phenotype

PKN3

pkn

28335

Enhanced

42567

No phenotype

55876

No phenotype

PLSCR1

CG9084

63599

No phenotype

PLXNB3

PlexB

28911

No phenotype

57813

No phenotype

PMS2

Pms2

55614

No phenotype

PPFIBP1

Liprin-beta

41672

No phenotype

64045

No phenotype

PRDM1

Blimp-1

36634

No phenotype

57479

No phenotype

PSMB5

Prosbeta5

34810

Suppressed

PTH2R

Dh44-R1

28780

No phenotype

PTPRN

IA-2

33672

Slightly enhanced

44099

No phenotype

PURG

Pur-alpha

36849

No phenotype

RAB27A

Rab27

31887

Slightly enhanced

35774

No phenotype

50537

Enhanced

RBM14

lark

27703

Enhanced

RGL3

Rgl

28938

No phenotype

33389

No phenotype

RNF157

CG9941

43234

No phenotype

RPE

CG30499

42816

Enhanced

62232

No phenotype

RYR3

RyR

28919

No phenotype

29445

Slightly enhanced

31540

No phenotype

SEPT10

Sep2

28004

No phenotype

31216

No phenotype

SGSM1

CG32506

28776

No phenotype

SH3GL2

EndoA

27679

Slightly enhanced

SLC2A6

Tret1-1

42880

No phenotype

SLC3A1

Mal-A5

41851

No phenotype

52910

No phenotype

Mal-B2

62253

No phenotype

SLC7A1

CG7255

58532

No phenotype

SND1

Tudor-SN

34865

No phenotype

SPINT1

CG13748

41623

No phenotype

CG6495

29452

No phenotype

STAT4

Stat92E

31318

No phenotype

33637

No phenotype

STX5

Syx5

29397

Enhanced but n<10

TAF6L

mia

57790

No phenotype

TAF9

e(y)1

32345

Enhanced

TIE1

Tie

54005

No phenotype

TLR5

Toll-9

30535

No phenotype

34853

No phenotype

TMC3

CG3280

50984

No phenotype

TNC

CG30280

53271

No phenotype

53693

No phenotype

TRPM6

Trpm

31291

Slightly enhanced

31672

No phenotype

35581

No phenotype

VRK3

ball

31350

No phenotype

55330

No phenotype

35571

No phenotype

Table S3.

Drosophila

screen of subunits of the Proteasome complex

FXTAS r(CGG)

flies expressing GAL4 under the eye-specific gmr driver were crossed with flies

harboring shRNA transgene against the target candidate gene, downstream from the UAS sequence
(UAS-TRiP). Progeny that express the shRNA against target gene in the eye were collected, aged
for 7 days and visualized using light microscopy and SEM. Results of the genetic screen of 35
genes revealed four proteasome subunits that show significant modulation of the CGG-toxicity
associated rough eye phenotype in FXTAS Drosophila. These four genes did not alter the eye
phenotype upon knockdown in wildtype GMR-GAL4 flies, but significantly enhanced the CGG-
associated rough eye phenotype in Gmr-GAL4, UAS-(CGG)

-EGFP

Drosophila

Human Gene

Drosophila

Ortholog

UAS TRiP
stock

Gmr-GAL4,

UAS-(CGG)

-EGFP

Drosophila

phenotype

Gmr-GAL4 only

phenotype

PSMA1

Prosalpha6T

55243

Enhanced

No phenotype

Prosalpha6

34811

No phenotype

53974

Few Progeny

No phenotype

PSMA2

Prosalpha2

36898

No phenotype

44560

No phenotype

PSMA3

Prosalpha7

33660

Enhanced

No phenotype

PSMA4

Prosalpha3

55217

Enhanced

No phenotype

77145

Slightly enhanced

No phenotype

PSMA5

Prosalpha5

34786

No Progeny

Cell death

PSMA6

CG30382

27557

Slightly enhanced

No phenotype

PSMA7

Prosalpha4

36063

No Pheotype

No phenotype

65161

No Progeny

No phenotype

Prosalpha4T1

61980

No Pheotype

No phenotype

PSMB1

Prosbeta6

34801

No Progeny

Cell death

PSMB2

Prosbeta4

32390

No Progeny

No phenotype

Prosbeta4R2

67790

No Pheotype

No phenotype

PSMB3

Prosbeta3

34868

No Progeny

Cell death

PSMB4

Prosbeta7

34812

No Progeny

No phenotype

PSMB6/9

Prosbeta1

34824

Few Progeny

No phenotype

PSMB7/10

Prosbeta2

67363

No Phenotype

No phenotype

PSMB5/PSMB8

Prosbeta5R1

77351

No Phenotype

No phenotype

PSMC1

Rpt2

34795

No Progeny

Cell death

PSMC2

Rpt1

33930

Enhanced

Cell death

PSMC3

Rpt5

32422

No Phenotype

No phenotype

PSMC4

Rpt3

34917

No Phenotype

No phenotype

PSMC5

Rpt6

34712

No Phenotype

No phenotype

PSMC6

Rpt4

32874

No Phenotype

No phenotype

PSMD1

Rpn2

34961

Slightly enhanced

No phenotype

PSMD2

Rpn1

34348

No Progeny

No phenotype

PSMD3

Rpn3

30503

Few Progeny

Cell death

34561

Enhanced

No phenotype

PSMD4

Rpn10

34566

Slightly enhanced

No phenotype

PSMD6

Rpn7

34787

No Progeny

No phenotype

PSMD7

Rpn8

31567

No Progeny

No phenotype

PSMD8

Rpn12

34560

Few Progeny

No phenotype

PSMD9

CG9588

28527

No Phenotype

No phenotype

PSMD11

Rpn6

29385

No Progeny

No phenotype

PSMD12

Rpn5

34532

Slightly enhanced

No phenotype

PSMD13

Rpn9

34034

No Progeny

No phenotype

PSMD14

Rpn11

33662

No Progeny

No phenotype

PSME3

REG

55248

No phenotype

ADRM1

Rpn13

42785

Slightly enhanced

No phenotype

UCHL5

Uch-L5

35433

No phenotype

64681

No phenotype

Table S4.

Primers for RT-PCR

Gene

Forward

Reverse

Psmb5
(mouse)

CTTCGCAATAAGGAACGCATC TTATCCCAGCCACAGATCATG

Tbp (mouse-
housekeeping)

AAGAAGGGAGAATCATGGAC
C

GAGTAAGTCCTGTGCCGTAAG

Rpl15 (mouse-
housekeeping)

TAAACGCCCAGTTCCTAAGG

ACTCTCAAAGCCCCACAATG

PSMB5
(human)

ACAGTCACCCCAAGAAACAC

CATCTACCAAGCCACCTACAG

U16 (human)

TGCCTGCTGTCAGTAAGCTG

TGCTCAGTAAGAATTTTCGTCAA

References:

C. Sellier

et al.

, Translation of Expanded CGG Repeats into FMRpolyG Is Pathogenic

and May Contribute to Fragile X Tremor Ataxia Syndrome.

Neuron

, 331-347 (2017).

M. G. Kearse

et al.

, CGG Repeat-Associated Non-AUG Translation Utilizes a Cap-

Dependent Scanning Mechanism of Initiation to Produce Toxic Proteins.

Mol Cell

314-322 (2016).