Supplementary Online Content

Zukosky K, Meilleur K, Traynor BJ, et al. Association of a novel ACTA1 mutation with a dominant progressive scapuloperoneal myopathy in an extended family. JAMA Neurol. Published online May 4, 2015. doi:10.1001/jamaneurol.2015.37.

eFigure 1. Muscle Magnetic Resonance Imaging and Ultrasonography eFigure 2. Immunohistochemistry in Patients IV-23 and V-6 eFigure 3. Immunohistochemistry in Patient IV-23 eFigure 4. Sanger Sequencing Chromatogram of the c.591C>A Mutation in 4 of ACTA1 eFigure 5. Transfection of WT-, E197D- and D286G-GFP Constructs Into COS-7 Cells eFigure 6. Transfection of Additional Cell Lines With E197D-GFP and E197D-HA eFigure 7. Zebrafish Injected With WT-ACTA1 or E197D-ACTA1 eTable. The 49 Open Reading Frames in the Linkage Region: rs1340867 to rs1043909

This supplementary material has been provided by the authors to give readers additional information about their work.

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Downloaded From: https://jamanetwork.com/ on 10/01/2021 eFigure 1. Muscle Magnetic Resonance Imaging and Ultrasonography

a c

b d

Muscle ultrasonography (12-Mhz linear probe) of the thigh in patients IV-23 and V-6, 12 Mhz linear probe. Image in patient IV-23 shows advanced muscle involvement of the vastus lateralis with uniform increase in echogenicity (C). In patient V-6 there is a mixed pattern in the rectus femoris with both granular and streak-like features, suggesting the presence of clusters of atrophic fibers (D).

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Downloaded From: https://jamanetwork.com/ on 10/01/2021 eFigure 2. Immunohistochemistry in patients IV-23 and V-6: alpha sarcomeric actin staining reveals no aggregates (a, b, c)

Alpha-actinin 1 staining (d, e, f) shows no rod formation but accentuates some of the trabeculation in the biopsy from IV-23 (e). Laminin alpha2 (LamaA2) staining (g, h, i) accentuates the atrophic fibers (h, i) and reveals normal membrane integrity.

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Downloaded From: https://jamanetwork.com/ on 10/01/2021 eFigure 3. Immunohistochemistry in patient IV-23: nNOS staining shows that atrophic fibers are staining robustly, in keeping with a myogenic but not a neurogenic cause of the atrophy.

63x, DAPI counterstain.

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eFigure 4. Sanger sequencing chromatogram of the c.591C>A mutation in exon 4 of ACTA1, demonstrating its heterozygous nature (reverse primer tracing)

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eFigure 5. Transfection of WT-, E197D- and D286G-GFP constructs into COS-7 cells

(a) The nemaline myopathy disease control D286G-GFP results in the expected rod formation, while E197D shows no rod formation. Occasional aggregate formation, as shown, was no different in frequency from the wild-type transfection. (b) GFP Western blot analysis reveals no significant differences in the expression levels after transfection.

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eFigure 6. Transfection of additional cell lines with E197D-GFP and E197D-HA.

(a) No rod formation was observed in Cos-7 cells transfected with E197D-GFP at all time points examined. (b) The nemaline myopathy disease control D286G-GFP results in the expected rod formation in C2C12 cells, while E197D-GFP shows no rod formation at 48 hours post transfection. (c) C2C12 cells tranfected with E197D- HA showed no signs of rod formation at 72hrs. (d) Immunofluorescent staining of E197D-HA transfected C2C12 and HEK 293 cells with 5C5 (Santa Cruz, Dallas, Tx) and HSP47 (Stressgen, Ann Arbor, MI) antibodies showed no rod formation or abnormalities of the actin cytoskeleton. Scale bar = 100m

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eFigure 7. Zebrafish injected with WT-ACTA1 or E197D-ACTA1

a) Zebrafish embryos injected with 400pg RNA encoding wild-type or E197D ACTA1 had normal morphology and did not display phenotypes associated with myopathies, such as curved bodies and decreased muscle mass. (b) Whole-mount immunostaining of 2-day-old zebrafish, injected with E197D RNA, using Acti- stain (Cytoskeleton, Inc) demonstrated normal myofibrillar organization and absence of nemaline rods in the muscle. (c) Percentage of fish that hatched out of their chorion by 54 hpf were calculated to evaluate changes in motility. The percentage of fish able to hatch out of their chorion was not significantly different between the groups that were injected with 400pg wild-type ACTA1 or the E197D variant. Control represents uninjected fish. Data presented are an average of 3 experiments (97-103 fish) ± SEM.

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eTable. The 49 open reading frames in the linkage region: rs1340867 to rs1043909 (Chr1:225,611,595-230,417,121). ACTA1 is highlighted in red.

LBR. ENAH, SRP9, EPHX1, TMEM63A, EPHX1, LEFTY1, PYCR2, LEFTY2, SDE2, H3F3A, ACBD3, MIXL1, LIN9, PARP1, C1orf95, ITPKB, CABC1, ADCK3, PSEN2, ADCK3, CDC42BPA, ZNF678, SNAP47, JMJD4, PRSS38, WNT9A, WNT3A, ARF1, GJC2, MRPL55, IBA57, C1orf145, OBSCN, ARF1, GUK1, TRIM11, TRIM17, RNF187, DUSP5P1, RHOU, RAB4A, SPHAR, CCSAP, ACTA1, ABCB10, TAF5L, URB2, GALNT2

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