smchd1 mutations associated with a rare muscular dystrophy ... · wolfgang mühlbauer, klaus w...

NATURE GENETICS

CORRECT ION NOT ICE

Nat. Genet. 49, 238–248 (2017)

SMCHD1 mutations associated with a rare muscular dystrophy can also cause isolated arhinia and Bosma arhinia microphthalmia syndromeNatalie D Shaw, Harrison Brand, Zachary A Kupchinsky, Hemant Bengani, Lacey Plummer, Takako I Jones, Serkan Erdin, Kathleen A Williamson, Joe Rainger, Alexei Stortchevoi, Kaitlin Samocha, Benjamin B Currall, Donncha S Dunican, Ryan L Collins, Jason R Willer, Angela Lek, Monkol Lek, Malik Nassan, Shahrin Pereira, Tammy Kammin, Diane Lucente, Alexandra Silva, Catarina M Seabra, Colby Chiang, Yu An, Morad Ansari, Jacqueline K Rainger, Shelagh Joss, Jill Clayton Smith, Margaret F Lippincott, Sylvia S Singh, Nirav Patel, Jenny W Jing, Jennifer R Law, Nalton Ferraro, Alain Verloes, Anita Rauch, Katharina Steindl, Markus Zweier, Ianina Scheer, Daisuke Sato, Nobuhiko Okamoto, Christina Jacobsen, Jeanie Tryggestad, Steven Chernausek, Lisa A Schimmenti, Benjamin Brasseur, Claudia Cesaretti, Jose E García-Ortiz, Tatiana Pineda Buitrago, Orlando Perez Silva, Jodi D Hoffman, Wolfgang Mühlbauer, Klaus W Ruprecht, Bart L Loeys, Masato Shino, Angela M Kaindl, Chie-Hee Cho, Cynthia C Morton, Richard R Meehan, Veronica van Heyningen, Eric C Liao, Ravikumar Balasubramanian, Janet E Hall, Stephanie B Seminara, Daniel Macarthur, Steven A Moore, Koh-ichiro Yoshiura, James F Gusella, Joseph A Marsh, John M Graham Jr, Angela E Lin, Nicholas Katsanis, Peter L Jones, William F Crowley Jr, Erica E Davis, David R FitzPatrick & Michael E Talkowski

In the supplementary information originally posted online, conversion errors introduced during the production of the PDF of supplementary figures corrupted some of the text in Supplementary Figure 1, the figure legend for Supplementary Figure 3 and Supplementary Figure 9. The errors have been corrected in this file as of 20 March 2017.

1

Simplex families (n=32)

A1 N139H/-

-/- -/-

B1 N524S/-

C1 L141F/-

D1 D2 D3 D4 M129K/- -/- -/- -/- E1

L141F/-

-/-

H348R/-

-/-

-/- -/-

G1 -/-

WES

Targeted sequencing/WES

A B C D E F

N1 H348R/-

Q1 -/-

K1#

2 L107P/-

K

M1 S135C/-

M

J1 T523K/-

S1 L141F/-

L1 H348R/-

*Targeted sequencing only

b

A2 A3 E2

F1 F4* F3*

F2*

AJ1 R552Q/-

P1 D420V/-

-/- -/-

P

P2* P3* -/-

U1 T523K/-

U

U2

V1 L141F/-

-/- -/-

V

V2 V3

I

2

I1 I4 I5

S135N/- -/- -/-

-/- -/- I2 I3

AI1 -/-

-/-

X1 H348R/-

-/-

X

X2 X3

N139H/- 2

Y

Y1

3 H348R/-

Z

Z1 AE1 AE4 -/-

-/- -/-

H348R/-

AE

AE2 AE3

AF1 S135C/-

-/- -/-

AF

AF2 AF3

G137E/- AG1

-/-

AG AG2

W1** E473Q

-/-

W

W2*

AA1** AA4* A242G/- -/-

AA

AA2* AA3* -/-

-/- AA5*

-/-

AC1 AC4 -/-

-/- -/-

H348R/-

AC

AC2 AC3

AD1** AD4* -/-

AD

-/- -/- AD2* AD3*

-/-

Pedigree!structure!and!familial!DNA!not!available!for!these!subjects:!

**WES after failed targeted sequencing

Targeted sequencing

*Targeted sequencing only

#WES negative but detected in subsequent targeted sequencing

AK1 S135I/-

-/- -/-

AK

AK2 AK3

-/-

AL1 -/-

-/-

AL

AL2 AL3

Q345R/-

Multiplex Families (n=6) WES T

E136D/'(

-/- E136D/'(

3 2

Q345R/-

Q345R/- Q345R/-

-/-

O

AH AB

3 -/- -/-

H

4

S135N/'(

R WGS WES WES

Targeted sequencing

H1 H2

O1 O4* O5*

5

10

7

8 -/-

AB1**

Q400L/- Q400L/-

Q400L/- -/-

-/-

-/-

AH1

a

Legend Arhinia#

Anosmia Asymmetric nares Abnormal dentition !Coloboma Muscular dystrophy

Hypoplastic nose !Micropenis Cryptorchidism !

-/-

O3 O2

O6

O7* -/-

R1

T1

T2 T3

Targeted sequencing/WES

F171V/'(AB3* F171V/'(

AB2* AH2 AH3

AH6

AH5 AH4

**WES!a'er!failed!targeted!sequencing!

*targeted!sequencing!only!!!!

*targeted!sequencing!only!!!!Cleft lip /palate

proband #!Comorbid!phenotypes!can!be!found!in!supplementary!table!2!

Delayed Puberty

Nature Genetics: doi:10.1038/ng.3743

2

Supplementary Figure 1. Arhinia Cohort pedigrees. (a) Families were considered to be multiplex if the

proband had additional family members with arhinia or a hypoplastic nose. (b) Sporadic cases of arhina were

considered to be from simplex families. Arhinia probands are highlighted with black arrows in multiplex

famliies. Sequencing was performed using whole exome sequencing (WES), whole genome sequencing (WGS),

or targeted sequencing as indicated in the top left hand corner of each box. Family members who were

sequenced are indicated with a genotype and sequencing methods are uniform across pedigrees unless otherwise

specified.


3

Supplementary Figure 2. CADD score analyses for SMCHD1 variants in arhinia subjects compared to

ExAC controls and FSHD2 subjects

Combined Annotation Dependent Depletion (CADD) scores1 of deleteriousness were assigned to all unique,

rare (MAF<0.01), nonsynonymous SMCHD1 variants identified in arhinia subjects (n=20), ExAC controls

(n=379), and FSHD2 subjects (n=55) taken from the LOVD3 database (http://www.lovd.nl/3.0/). a. CADD

scores for SMCHD1 variants found in arhinia subjects are significantly more deleterious than variants in ExAC

controls (two sample t-test; p=1.3x10-5). This enrichment is largely driven by the higher frequency of less

deleterious variants that lie outside of the constrained 5’ region (exons 3-13, encompassing the ATPase domain)

in ExAC controls. b) Due to relatively high CADD scores of nonsense mutations, which are only observed in

FSHD2 patients, SMCHD1 variants in exons 3-13 demonstrate more deleterious CADD scores in FSHD2

subjects than variants in arhinia subjects (p=0.05). Missense-specific analyses, however, were not significantly

different (p=0.99).

CADD scores of Arhinia and Exac Nonsynomous Variants in SMCHD1

CADD score

Frequency

0 5 10 15 20 25 30 35

050

100

150

ExacArhinia

CADD scores of Arhinia and FSHD2 Nonsynomous Variants in SMCHD1

CADD scoreFrequency

10 20 30 40 50

05

1015

20 FSHD2Arhinia

a b

pall= 1.3x10-5

pexon3-13=0.004 pall=0.28 pexon3-13=0.05


4

a

1 2 3 4 5 6 7 49

ATPase

e3i3 MO e5i5 MO

SMC

Hinge

ATP-

ase

ENSDARP00000140355

1,983 aa

ENSDARG00000104374

chr7:71,345,081-71,469,452; GRCz10

bA A A G C G T T G C G T A A G T C T G A

A A A G C G T T G C C C T A T G C G T T

A A T G T G G C A T C C T A T G C G T T

G A G C T T T A C A C C T A T G C G T T

C A T T T C A A A G T G A A G A A G C C

C G A G A A A A C G T G A A G A A G C C

G T C T T T C C A A T G A A G A A G C C

exon 3 intron 3 ins91 bp

exon 3 exon 4

exon 3 del38 bp exon 4

exon 2 exon 4

exon 5 exon 6



β-actin

β-actin

smchd1

smchd1

smch

d1 e3

i3

MO

Control

smch

d1 e5

i5

MO

Control


5

Supplementary Figure 3. Efficiency of morpholinos (MO) targeting D. rerio smchd1

a. Schematic of the single ortholog of SMCHD1 in zebrafish. Top: the gene transcript is shown with exons

(green boxes); untranslated regions (white boxes); introns (dashed lines); MO target sites and primers used to

generate RT-PCR products shown in panel b (purple and yellow for e3i3 and e5i5, respectively). Bottom, a

schematic of zebrafish SMCHD1 protein with the ATPase domain (blue) and SMC Hinge domain (red) are

shown. b. Agarose gel images (left) and Sanger sequence traces (right) showing aberrantly spliced smchd1

transcripts produced after injection with 9 ng e3i3 or 9 ng e5i5 MOs. Both MOs induce splicing defects that

result in either retention of intronic sequence, frameshift deletions, or in-frame deletions with concomitant

reduction in wild-type transcript. beta-actin was used to control for RNA integrity.


6

Supplementary Figure 4. smchd1 morpholinos (MO)s induce dose-dependent effects on cartilage

structure in zebrafish

Three different doses of each of e3i3 or e5i5 MOs were injected into batches of -1.4col1a1:egfp embryos, and

imaged live at 3 days post-fertilization (dpf) with fluorescent imaging to detect GFP-positive cells. Cartilage

structures were assessed on ventral images, and pairwise statistical comparisons were made between each MO-

injected batch and controls. a. Quantification of ethmoid plate width measured on ventral images. The furthest

distal width (a), was normalized to the width at the ethmoid plate-trabecula junction (b), see Fig. 5a, top panel.

b. Quantification of ceratohyal angle in control and smchd1 MO larval batches. c. Number of ceratobranchial

arch pairs (see Fig. 5a, top) in control and MO-injected larval batches. Statistical significance is indicated with

*** (p<0.0001); NS, not significant. n=34-64 embryos/injection, with masked scoring; all experiments were

repeated. Error bars indicate standard error of the mean.

a b c

1

1.2

1.4

1.6

1.8

2

2.2

(a/b ratio)

e3i3

MO

e5i5

MO

--

3ng-

6ng-

9ng-

-3ng

-6ng

-9ng

ethmoid plate

******

******

******

60

70

80

90

100

110

120

(degrees)

e3i3

MO

e5i5

MO

--

3ng-

6ng-

9ng-

-3ng

-6ng

-9ng

ceratohyal angle

******

***

NS

******

0%

20%

40%

60%

80%

100%

e3i3

MO

e5i5

MO

--

3ng-

6ng-

9ng-

-3ng

-6ng

-9ng

ceratobranchial

arch pairs

0 1-2 3-4

******

***

NS

******


7

Supplementary Figure 5. In vivo modeling of smchd1 in zebrafish demonstrates craniofacial phenotypes

in 3 day post-fertilization (dpf) -1.4col1a1:egfp larvae

a. Quantification of ceratohyal angle in control and smchd1 morphant and CRISPR/Cas9 F0 mutant larval

batches (ch; dashed line in Fig. 5a). b. Number of ceratobranchial arch pairs (cb, indicated with * in Figure 5a)

in control, smchd1 MO-injected, and CRISPR/Cas9 F0 mutant larval batches. Statistical significance is

indicated with *** (p<0.0001), ** (p<0.01), or *(p<0.05); g, guide RNA; NS, not significant. n=19-50

embryos/injection with masked scoring; all experiments were repeated. Error bars indicate standard error of the

mean.

60

70

80

90

100

110

120

0%

20%

40%

60%

80%

100%

MO

RN

A

--

e3i3-

e3i3WT

e5i5-

e5i5WT

-g

-g

Ca

s9

-

-

-

-

-

+

-

(degrees)

ceratohyal angle

MO

RN

A

--

e3i3-

e3i3WT

e5i5-

e5i5WT

-g

-g

Ca

s9

-

-

-

-

-

+

-

ceratobranchial

arch pairs

0 1-2 3-4

******

**

****

***

******

**

***

a b


8

a

1 2 3 4 5 6 7 49ATPase

gRNA

ENSDARG00000104374chr7:71,345,081-71,469,452; GRCz10

b

Con

trol 1

500 bp400 bp300 bp

200 bp

smchd1homoduplex

smchd1heteroduplex

Con

trol 2

F0-1

F0-2

F0-3

F0-4

*

F0-5

*

F0-6

F0-7

*

F0-8

*

F0-9

*

F0-1

0

Control CGGCCCGGCAGCAGCGCTGCTCCACCG------------------CGGACTTTCGACATCTCCAACGCAG

F0-7-01 CGGCCCGGCAGCAGCGCT-----------------------------GACTTTCGACATCTCCAACGCAGF0-7-02 CGGCCCGGCAGCAGCGCTGCTCCACCGACTGCTCCACCGACTCCACGGACTTTCGACATCTCCAACGCAGF0-7-03 CGGCCCGGCAGCAGCGCTGCTCCACCG--------------------GACTTTCGACATCTCCAACGCAGF0-7-04 CGGCCCGGCAGCAGCGCTGCTCCACCG--------------------GACTTTCGACATCTCCAACGCAGF0-7-05 CGGCCCGGCAGCAGCGCTGCTCCACCGACTGCTCCACCGACTCCACGGACTTTCGACATCTCCAACGCAGF0-7-06 CGGCCCGGCAGCAGCG-------------------------------GACTTTCGACATCTCCAACGCAGF0-7-07 CGGCCCGGCAGCAGCG-------------------------------GACTTTCGACATCTCCAACGCAGF0-7-08 CGGCCCGGCAGCAGCGCTGCTCCACCG------------------C----------------------AGF0-7-09 CGGCCCGGCAGCAGCGCTGCTCCACCG------------------C----------------------AGF0-7-10 CGGCCCGGCAGCAGCGCTGCTCCAC-------------------------TTTCGACATCTCCAACGCAGF0-7-11 CGGCCCGGCAGCAGCGCTGCTCCACCG------------------C----------------------AGF0-7-12 CGGCCCGGC-------------------------------------GGACTTTCGACATCTCCAACGCAG

c PAM smchd1 guide RNA


9

Supplementary Figure 6. Genome editing of smchd1 using CRISPR/Cas9 to generate F0 zebrafish

mutants

a. Schematic of the smchd1 ortholog in zebrafish. The locus is shown with exons (green boxes); untranslated

regions (white boxes); introns (dashed lines); guide (g)RNA target site and primers used to generate PCR

products shown in panel b (red box and triangles, respectively); exons 3-7 encode the ATPase domain (blue). b.

Assessment of genome-editing efficiency using polyacrylamide gel electrophoresis (PAGE). Genomic DNA

was extracted from single embryos at 2 days post fertilization (dpf), and PCR amplified. PCR products were

denatured, annealed slowly and migrated on a 15% polyacrylamide gel. All ten F0 embryos displayed

heteroduplexes not present in two uninjected controls. Asterisks (*) indicate embryos assessed for percent

mosaicism with TOPO-TA cloning and Sanger sequencing. c. Representative sequence alignments from one

embryo to estimate percent mosaicism. One control and five F0 embryos were assessed (n=10-12

clones/embryo); all F0 clones harbored insertions (green) or deletions (red), suggesting ~100% efficiency.

gRNA sequence (yellow) and protospacer adjacent motif (PAM, orange) are shown.


10

Supplementary Figure 7. Ectopic expression of human SMCHD1 mRNA produces no detectable effects

on cartilage structure in zebrafish

Human SMCHD1 message was injected into batches of -1.4col1a1:egfp embryos, and imaged live at 3 days

post-fertilization (dpf) with fluorescent imaging to detect GFP-positive cells. Cartilage structures were assessed

on ventral images. a-c. 25pg wild type (WT) or mutant RNA was injected and pairwise statistical comparisons

were made between each variant RNA-injected batch and WT RNA; p.Ser135Cys, p.Leu141Phe, and

60

70

80

90

100

-

WT

S135C

L141F

H348R

P690S

V708I

(degrees)

ceratohyal angle

25 p

g

RN

A1

1.2

1.4

1.6

1.8

2

2.2

2.4

-

WT

S135C

L141F

H348R

P690S

V708I

25 p

g

RN

A ethmoid plate

(a/b ratio)

1

1.2

1.4

1.6

1.8

2

2.2

2.4

-

50pg

-

50pg

100pg

-

100pg

WT

RN

A

-

-

50pg

50pg

-

100pg

100pg

H348R

RN

A

ethmoid plate

(a/b ratio)

60

70

80

90

100

-

50pg

-

50pg

100pg

-

100pg

WT

RN

A

-

-

50pg

50pg

-

100pg

100pg

H348R

RN

A

(degrees)

ceratohyal angle

0%

20%

40%

60%

80%

100%

-

WT

S135C

L141F

H348R

P690S

V708I

25 p

g

RN

A ceratobranchial

arch pairs

1-2 3-4

0%

20%

40%

60%

80%

100%

-

50pg

-

50pg

100pg

-

100pg

WT

RN

A

-

-

50pg

50pg

-

100pg

100pg

H348R

RN

A ceratobranchial

arch pairs

1-2 3-4

Supplementary Figure 4

a b c

d e f


11

p.His348Arg are recurrent mutations in arhinia cases; p.Pro690Ser is associated with FSHD2; p.Val708Ile

(rs2276092) is a common variant in ExAC. d-f. WT or p.His348Arg encoding RNA was injected either alone or

combined in equimolar ratios at higher doses (50pg and 100pg) a, d. Quantification of ethmoid plate width

measured on ventral images. The furthest distal width (a), was normalized to the width at the ethmoid plate-

trabecula junction (b), see Fig. 5a, top panel. b, e. Quantification of ceratohyal angle in control and SMCHD1

RNA-injected larval batches. c, f. Number of ceratobranchial arch pairs (see Fig. 5a, top) in control and RNA-

injected larval batches. Pairwise statistical comparisons between each variant and WT at the same dose were not

significant. n=28-41 embryos/injection (panels a-c); n=42-56 embryos/injection (panels d-f) with masked

scoring; all experiments were repeated. Error bars indicate standard error of the mean.


12

CTCTAGTTAAAAGTGGCATGTATGAGTATTATGCGAGTGAAGGACAGAATCCTTTGCgtaagtaacctgctcccgcacgttttgaaagttgttagtctcctttggtcacatacg

gRNA

Mutated base

CTCTAGTTAAAAGTGGCATGTATGAGTATTATGCGAGTGAAGGACAGAATCCTTTCCgtaagtaacctgctcccgcacgttttgaaagttgttagtctcctttggtcacatacg

chr17:71,463,705-71,463,818 GRCm38

EXON 3 intron 3

Smchd1

repair template

nono no no no

nono no

p.Leu141Phe/null embryo 1

p.Leu141Phe/nullembryo 2

p.Leu141Phe/p.Leu141Phenull/nullwild type

nono

Supplementary Figure 8. CRISPR/Cas9 targeting of Smchd1 in mouse embryos

The top panel indicates the genomic location of the targeted region and the sequences represent

the wild-type (WT; bottom) and mutant (top) repair templates used to model the p.Leu141Phe variant. The

position of the guide RNA spanning the exon 3/intron 3 boundary is indicated by the orange box. The 63

embryos recovered following two zygotic injection sessions displayed a range of variants including WT,

homozygous knock-ins (KI) of p.Leu141Phe, homozygous knock-outs (KO), compound heterozygotes

(p.Leu141Phe/null), and complex compound heterozygous deletions (Supplementary Table 3). Digital

transverse sections from optical projection tomography through the head of five such embryos collected at 13.5

dpc are shown in the lower panel. The genotype of each embryo is given above the sections. 3D representation


13

of the heads of the homozygous null and homozygous missense embryo are shown below the cognate section.

Each embryo demonstrated patent nasal passages with clear evidence of formed nostrils. No evidence was

found for full or partial arhinia in any of these embryos. As we were unable to generate non-mosaic

p.Leu141Phe heterozygotes, the experiments were repeated with a different repair template in an attempt to

knock-in the p.Glu136Asp variant (Supplementary Table 3). Fourteen of the 20 genotyped embryos showed

evidence of gRNA-targeted mutation but only one heterozygous p.Glu136Asp mutation was recovered and this

was in cis with an essential spice site mutation. This embryo was not imaged.


14

!!!

!!!

! !!

!!!!!!!!!!!!!!!!!!!

!!!!!!!!!!!

Anti-SMCHD1 Abcam 250 kDa

Anti-Tubulin Abcam-50 kDa Anti-b-Actin Abcam-42 kDA

Anti-SMCHD1 Bethyl 250 kDa

R²#=#0.83311#

0#0.5#1#

1.5#2#

2.5#3#

3.5#4#

0# 0.5# 1# 1.5# 2# 2.5# 3# 3.5#Normalize*Protein*Expression

*Bethyl*

Normalized*Protein*Expression*Abcam*

Correla:on*between*Abcam*and*Bethyl*An:bodies*with*Ac:n*Normaliza:on*

R²#=#0.77257#

0#

0.5#

1#

1.5#

2#

2.5#

3#

3.5#

0# 0.5# 1# 1.5# 2# 2.5# 3#Normalize*Protein*Expression

*Bethyl*

Normalized*Protein*Expression*Abcam*

Correla:on*between*Abcam*and*Bethyl*An:bodies*with*Tublin*Normaliza:on*

gel1% gel2%A1%%%AH1%AH3%%AH5%AH2*%AH4%AH6%%A2%

gel3%C1%%%B1%%%AG1%%D1%%%%D2%%%%D3%%%%D4%%%AG2% W1%%%Y1%%%%E1#%%AE1%%AE3%AE2%%AE4%%

Arhinia%case%with%SMCHD1'MutaAon%Familial%control%with%Arhinia%related%phenotype%and%SMCHD1'MutaAon%Familial%unaffected%control%%

a!

b!

c!

*Indicates outlier sample #Indicates sample with abnormal Bethyl antibody binding

●

●

●●

−1

0

1

2

3

4

x1 x2 x3 x4 x5 x6 x7 x8

Rel

ativ

e pr

otei

n ex

pres

sion

!!!!!!+!!!!!!!!!!!!!!!!&!!!!!!!!!!!!!+!!!!!!!!!!!!!!!!&!!!!!!!!!!!!!!+!!!!!!!!!!!!!!!!&!!!!!!!!!!!!!!+!!!!!!!!!!!!!!!&!!!!!!!!!!!Tublin!!!!!!!!!!!!!!!!!!!!!!Actin!!!!!!!!!!!!!!!!!!!!!!Tublin!!!!!!!!!!!!!!!!!!!!!!Actin!!!!

Abcam!! ! ! ! !!!Bethyl!

SMCHD1 Mutation: Protein Control: Antibody:

pall=0.14 pno-outliers=0.29

!!


!!


!!pall=0.44 pno-outliers=0.48

!!


15

Supplementary Figure 9. Western blotting of SMCHD1 in arhinia cases and familial controls

Western blotting was performed on 10 arhinia cases, 11 unaffected familial controls, and 2 family members

(AH3, AHG5) with mutations in SMCHD1 and an arhinia-related phenotype (anosmia and hypoplastic nose).

To measure protein levels of SMCHD1 from lymphoblastoid cell lines (LCLs), we used two different anti-

SMHD1 antibodies (Bethyl, Abcam), which were normalized against both anti-tublin and anti-beta actin

loading controls. b) SMCHD1 protein levels were consistently lower in subjects with an SMCHD1 mutation

versus familial controls but none of the comparisons across different anti-SMCHD1 antibodies and loading

controls showed a statistically significant difference. Boxplots were created with BoxPlotR2

(http://boxplot.tyerslab.com c) We found a high correlation (r=0.77,r=0.83) between the different anti-

SMCHD1 antibodies regardless of loading control.


16

Supplemental Figure 10. QQ Plot of differential expression analysis between arhinia cases and familial

controls.

Expression of transcription was compared between 10 arhinia cases and 10 familial controls using a two-sample

permutated test. P-values from this analysis largely match an expected distribution under the null hypothesis

(red line).


17

Supplemental Figure 11. Repeat expression analysis in normal and affected SMCHD1 mutant subjects.

RNAseq libraries were filtered for uniquely mapped reads and counts overlapping the hg19 repeatmasker

annotation were computed. The R packages egdeR3 and DeSeq24 were implemented to perform library

normalization and differential expression. Data was filtered for individual repeat classes (LTR, L1, L2, Satellite

& SINE) and median read counts were computed per normal/affected individual. The R package beeswarm5

was used to plot the range of median read counts. Student’s t-tests were used to compare controls and patients.

Controls (green); patients (red)

References

1. Kircher, M. et al. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet 46, 310-5 (2014).

2. Spitzer, M., Wildenhain, J., Rappsilber, J. & Tyers, M. BoxPlotR: a web tool for generation of box plots. Nat Methods 11, 121-2 (2014).

3. Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-40 (2010).

4. Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550 (2014).

5. Eklund, A. Beeswarm: the bee swarm plot, an alternative to stripchart. R package version 0.1. 1, 2011. (2015).

Read

cou

nts

Control

LTR

Patient

L1 L2 Satellite SINE200

0

p > 0.05 p > 0.05 p > 0.05 p > 0.05 p > 0.05

Figure x. Repeat expression analysis in normal and affected SMCHD1 mutant cases. RNAseqlibraries were filtered for uniquely mapped reads and counts overlapping the hg19 repeatmasker annotationwere computed. The R packages ‘egdeR’ and ‘DeSeq2’ were implemented to perform library normalisationand differential expression. Data was filtered for individual repeat classes (LTR, L1, L2, Satellite & SINE) and medianread counts were computed per normal/affected individual. The R package ‘beeswarm’ was used to plotthe range of median read counts. Student’s t-tests were used to compare controls and patients.


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