Volker Straub

Disclosure statement • I am or have been a principal investigator for trials sponsored by Genzyme, GSK, Prosensa/Biomarin, ISIS Pharmceuticals, and Sarepta. • I received speaker honoraria from Genzyme, a Sanofi company. • I’m a member of the international Pompe advisory board of Genzyme, a Sanofi company, and have been on advisory boards for Acceleron Pharma, Audentes, Italfarmaco S.p.A., Nicox, Pfizer, Prosensa, Santhera, Summit Therapeutics and TrophyNOD. • I have a research collaboration with Ultragenyx and Genzyme/Sanofi.

Volker Straub

Disclosure statement Advances in the Limb-Girdle Muscular Dystrophies There’s nothing new under the sun, but there are still a lot of old things that we don’t know.

Bushby K, Beckmann JS. The limb-girdle muscular dystrophies -proposal for a new nomenclature. Neuromuscul Disord. 1995

LGMD2A Beckmann JS et al., C R Acad Sci III. 1991 CPN3 A for limb-girdle muscular dystrophy maps to 15 by linkage

LGMD1A Speer MC et al., Am J Hum Genet. 1992 MYOT Confirmation of genetic heterogeneity in limb-girdle muscular dystrophy: linkage of an autosomal dominant form to chromosome 5q.

LGMD2D Roberds SL et al., Cell. 1994 SGA Missense in the adhalin gene linked to autosomal recessive muscular dystrophy

LGMD2W Warman Chardon J et al., Neurology 2014 Pinch2 Exome Sequencing Identifies PINCH2 Mutations in Early Onset Autosomal Recessive LGMD with Severe Cardiomyopathy and Triangular Tongues What’s a limb girdle muscular dystrophy?

progressive weakness and wasting

genetically determined

primary damage of muscle fibres LGMD2I

 Is there another common denominator ? onset of symptoms? degree of progression? LGMD2A pattern of muscle involvement? mode of inheritance? muscle pathology? pathomechanism? LGMD1C What’s a limb girdle muscular dystrophy?

• clinically, Duchenne/Becker muscular dystrophy is the most common form of LGMD

 each male with limb girdle weakness and an elevated serum CK should first be tested for a dystrophinopathy

 each female with limb girdle weakness should first be tested for a dystrophinopathy -Glycoprotein Complex Laminin-2

ECM a-, b-

Sarcospan Caveolin-3

d a b g

Grb2

NOS1

Syntrophins

Dystrophin

F- •LGMD 2C LGMD 2I •LGMD 2D LGMD 2K, Laminin-2 •LGMD 2E LGMD 2M-P, T, U •LGMD 2F a-, b-Dystroglycan Sarcoglycans LGMD 1C

Dysferlin  LGMD 2B Caveolin-3

d a b g

Grb2

NOS1

Dystrophin  DMD / BMD

F-Actin pathogenetic aspects of LGMDs

LGMD1A, E LGMD2A LGMD2I LGMD1C LGMD2G, Q, R LGMD2K LGMD2B LGMD2J LGMD1B LGMD2M-P,, T, U LGMD2C-F

Sarcomer Nuclear Lamina Golgi Apparatus Sarcolemma

 cytoskeletal integrity  membrane damage  membrane repair  glycosylation

LGMD1A 5q31.2 Myotilin LGMD2A 15q15 Calpain-3 LGMD1B 1q22 A/C LGMD2B 2p13 Dysferlin LGMD1C 3p25.3 LGMD2C 13q12 g- LGMD1D 7q36 DNAJB6/Hsp40 LGMD2D 17q12 a-Sarcoglycan LGMD1E 2q35 LGMD2E 4q12 b-Sarcoglycan LGMD1F 7q32 Transportin 3 LGMD2F 5q33 d-Sarcoglycan LGMD1G 4q21 HNRPDL LGMD2G 17q11 LGMD1H 3p23 ? LGMD2H 9q31 TRIM32 FSHD LGMD2I 19q13 FKRP Bethlem LGMD2J 2q SMA3 LGMD2K 9q34 POMT1 Pompe LGMD2L 11p13 ANO5 MFMs LGMD2M 9q3 GNE LGMD2N 14q24 POMT2 RYR1 LGMD2O 1p3 POMGnT1 ACTA1 LGMD2P 3p21 Dystroglycan MYH7 LGMD2Q 8q24 ... LGMD2R 2q35 Desmin LGMD2S 4q35 TRAPPC11 LGMD2T 3p21 GMPPB LGMD2U 7p21 ISPD LGMD2V 17q25 GAA LGMD2W 2q14 PINCH2

UK5A elevated serum CK activity + + +

 LGMD Sequence Sample enrichment • 2-10µg high quality • Solid/solution DNA • Targeted/exome

Sequencing • Targeted  LGMD2I & A • Exome • Genome Genomes and DNA variations

• Genomes are relatively conserved

89% shared 95-98% shared • Any 2 humans share 99.9% of their DNA • Remaining 0.1% is VARIABLE • Total of 3 million of variants • Majority of variants are inherited from one’s parents Not all variants are significant

silent • ≅99.9% are likely benign or silent

• ≅0.1% – traits (100-1000) Traits – risk factors (dozens) – disease-causing (a few) risk factors mutations Interpretation of NGS results

Is the variant novel or has been found before? • 90% not novel (dbSNPs databases with millions of known variants) If novel variants: • does the variant lie in a gene? Only ~1% • Does the variant change gene function? Only ~10% • Has the gene itself been associated with human disease? Only ~10% of human associated with human disease

The clinical context is paramount LGMD? Panel of used in immunoanalysis Know your population

• LGMD2I (FKRP) 21% • LGMD2L (anoctamin 5) 15-20% • LGMD2A (calpain 3) 15% • LGMD2C-F (sarcoglycans) 13% • LGMD1B (lamin A/C) 7% • LGMD2B (dysferlin) <2%

Norwood et al., Brain, 2009 Hicks, Sarkozy et al., Brain, 2010

Translational Research Pathway

Trials

Gene identification/ • Diagnosis/ Care pathophysiology Standards

• Natural history • Patient Registries •Regulatory affairs

• Trial sites •Ethics • Biomarkers • Outcome measures •Commissioning/ health • Animal models economics • Delivery mechanisms • Proof of principle studies

Therapy delivery Disease - gene Species Genotype

LGMD2A - CAPN3 Mouse Capn3-/- Capn3−/− (C3KO) LGMD2B - DYSF D. melanogaster mfr C. Elegans Fer-1 mutation Zebrafish Morpholino knockout of Dysf Morpholino knockout of Dysf and annexinA 6 Mouse SJL/J natural model A/J natural model

Dysf prmd LGMD2C - SGCG Zebrafish Morpholino knockout of delta-SG Mouse Sgcg-/- LGMD2D - SGCA Mouse Scga-/- LGMD2E - SGCB Mouse Sgcb-/-

LGMD2F - SGCD D. melanogaster Three different δ- sarcoglycan deletion mutants Mouse Scgcd-/- Scgcd-/- Hamster BIO14.6 LGMD2G -TCAP Zebrafish Morpholino knockout of Tcap Mouse Tcap-/- LGMD2H - TRIM32 D. melanogaster l(2)thin [l(2)tn] (Caenorhabditis elegans) (Drosophila melanogaster) Mouse Trim32-/- (T32KO) D489N KI LGMD2I - FKRP Zebrafish Morpholino knockout of Fkrp Morpholino knockout of Fkrp Mouse Fkrp-/- L276I KI P448L KI LGMD2J - TTN Zebrafish erzschlag mutant Mouse IG KO mice (lacking Ig TTN domain) Mouse Ttn tm1.1Isrd

LGMD2K - POMT1 D. melanogaster Pomt1 ko

rotated abdomen (rt) ko

Mouse Pomt1-/-

LGMD2M - FKTN Zebrafish Morpholino knockout of Fukutin Mouse Fukutin-/- LGMD2N - POMT2 D. melanogaster Pomt2 ko

D. melanogaster wisted (tw) ko Mouse Pomt2-/- LGMD2O - POMGNT1 Mouse POMGnT1-/-

LGMD2P - DAG1 Zebrafish V567D KI Mouse Dg–/– T190M KI mice (Danio rerio) (Mus musculus) LGMD2Q - PLEC Mouse Plec-/-

LGMD2R - DES Zebrafish Morpholino knokout of desmin Mouse Desmin−/−

LGMD2T - GMPPB Zebrafish Gmppb-/- LGMD2V - GAA Mouse GAA -/- Clinical Outcome Study (COS) for Dysferlinopathy

Medical, physiotherapy and MRI/MRS assessments will be performed on 6 occasions over 3 years

In total 15 centres are participating in this international study and to date 193 patients have been recruited

the stars correspond to: Barcelona, Berlin, Charlotte, Columbus, Marseille, Munich, Newcastle, Padova, Paris, Saint Louis, Sevilla, Stanford, Sydney, Tokyo, Washington.

First symptoms of disease manifestation

• Lower limb weakness (72%) – proximal (15%) – distal (32%) – both (25%) • 7% upper limb weakness as a first symptom – most commonly distal lower limbs • 27% Muscle wasting patients – most often distal lower limbs • 13%Muscle pain, stiffness or cramps • 6% pseudohypertrophy Data being collected

Screening Baseline 6 months 12 months 24 months 36 months (Visit 1) (Visit 2) (Visit 3) (Visit 4) (Visit 5) (Visit 6) Blood draw – for laboratory tests (all) X X X X Cardiac echo X X Electrocardiogram (ECG) X X

Ongoing medical assessment: X X X X X anamnesis and physical examination Exercise assessment X X X X Myometry & manual muscle testing X X X X X X

Functional/Activity assessment X X X X X X (physio) Timed tests (physio) X X X X X X MRI x x x x MRS (certain sites only) x x x x LGMD2B – dysferlinopathy

Jain COS Study Consortium Longitudinal study of 38 LGMD2I patients with the common FKRP mutation(Leu276Ile) across 4 centres Assessments . FVC in sitting and lying . Myometry: hip flexors, abductors, adductors, knee flexors, extensors, ankle dorsiflexors . Timed tests including 6 point graded quality of movement: Timed up and go, 10 m walk/run, stair climb and descend and the timed rise from a chair, 6 minute walk distance

. An adapted Northstar scale for LGMD2I Willis T et al., PLoS One 2013 Willis T et al., PLoS One 2014 muscle assessment in LGMD2I

Willis T et al., PLoS One 2013 no significant changes in muscle strength and function over 12 months time in LGMD2I

except for FVC!

Willis T et al., PLoS One 2013 muscle MRI: change in fat % over 12 months time in LGMD2I

Willis T et al., PLOS ONE 2013

Baseline 12 months LGMD2A – LGMD2A Patient Registry (Coalition to Cure Calpain -3) www.lgmd2a.org

LGMD2B – The International Dysferlinopathy Registry http://dysferlinregistry.org/ (Jain Foundation)

LGMD2C – LGMD2C Patient Registry (Kurt and Peter Foundation) www.lgmd2cregistry.org

LGMD2D – LGMD2D Patient Registry (LGMD2D Foundation) www.lgmd2d.org/patient-resources

LGMD2I – Global FKRP Registry (LGMD2I Research Fund) www.fkrp-registry.org . LGMD2K/M/N/O - Congenital Muscle Disease Internat. Registry www.cmdir.org (Cure CMD)

The more we learn about the natural history of LGMD, the easier it will be to develop care standards. Translational Research Pathway

Trials

Gene identification/ • Diagnosis/ Care pathophysiology Standards

• Natural history • Patient Registries •Regulatory affairs

• Trial sites •Ethics • Biomarkers • Outcome measures •Commissioning/ health • Animal models economics • Delivery mechanisms • Proof of principle studies

Therapy delivery • Gene transfer • Surrogate gene transfer • Exon skipping • Gene knockdown • Protease inhibition • Non-molecular approaches LGMD2 Interventional drug / Natural hystory A B C D E F G H I J K L M N O P Q R S T U V W

TRIM3 POMT POMT POMG TRAP GMPP CAPN3 DYSF SCGG SCGA SCGB SCGD TCAP FKRP TTN ANO5 FKTN DAG1 PLEC DES ISPD GAA LIMS2 2 1 2 nT1 PC11 B Natural hystory X X X

Natural hystory X X X X X X X

Natural hystory x

Natural hystory • natural history X Natural hystory X X X X

• phase I Interventional drug - Phase II/III X X X X X

Interventional drug - • phase II Phase II/III x

Interventional drug - Phase I x Interventional drug - Phase I/IIa X

Interventional drug - Phase I X

Interventional drug - Phase I/II X X X X X X

Interventional drug - Phase I X

VitD X

rituximab X

deflazacort X

deflazacort X

prednisolone X

MRI X

MRI X X

Registries X X X X X X X X Exome sequencing in 1000 patient with limb girdle weakness of unknown origin - Older than 10 years - elevated serum CK activity PhenoTips http://PhenoTips.org

• Software tool for collecting and analyzing phenotypic information for patients with genetic disorders – Free and open source – Web-based

• PhenoTips is used in several projects, including Neuromics

• Specific Muscular Dystrophy form: – Detailed phenotypic data – Gender, ethnicity, mode of inheritance, age of onset – Pedigree function – No identifiable data

41 MYO-SEQ – batch#1 (n=83)

9x TTN 7x CAPN3 7x DYSF 2x LAMA2 2x GAA DMD DES ANO5 TRIM32 59% CAV3 POLG PLEC

MYO-SEQ – batch#2 (n=192)

7x CAPN3 ACTA1 7x DYSF ANO5 7x RYR1 (AD/AR) BAG3 3x COL6A2 CAV3 3x DMD (carriers) COL6A3 3x LMNA DOK7 2x DES FKRP 2x GMPPB GFPT1 GYG1 72% ISPD LAMA2 MHY7 MTM1 POMT2 RAPSN SEPN1 SGCA TNNT3 TTN VMA21 MYO-SEQ – overall (n=271)

14x CAPN3 ACTA1 14x DYSF BAG3 10x TTN COL6A3 8x RYR1 DMD 3x DMD (carrier) DOK7 3x COL6A2 FKRP 3x DES GFPT1 3x LAMA2 GYG1 3x LMNA ISPD 2x ANO5 MHY7 2x CAV3 MTM1 2x GAA PLEC 2x GMPPB POLG

POMT2 191 RAPSN Samples received SEPN1

SGCA Samples sent for exome 781 SMCHD1 sequencing 568 TNNT3 Samples pending to be TRIM32 sent VMA21 Case history: AB

Mild non-progressive proximal and distal 25 weakness years old Rigid spine + elbow and finger contractures Ophthalmoplegia FVC 78%. Normal cardiac function

Muscle biopsy: type 1 fibre predominance Muscle MRI: suggestive of Bethlem Negative genetic investigations: COL6A1/2/3, RYR1, FHL1, LMNA

Exome sequencing result (SeqNMD)

Missense variant in STIM1 c.242G>A p.(Gly81Asp) Segregation: de novo Absent in Exac / other control databases Predicted pathogenic by in silico tools

From Shim (2015) Known condition associated to STIM1 mutations:

Mutation in the CC1 domain (p.R304W): heterozygous gain of function mutation Tubular aggregate myopathy identified in all patients with Stormorken syndrome Hypocalcaemia Platelet dysfunction Hypo-/Asplenia Miosis Icthyosis Learning difficulties

Mutations in the EF-hand domain identified in patients with Tubular Aggregate Myopathy +/- miosis

From Nesin (2014) Review of patient in light of WES findings:

Hypocalcaemia Normal appearance of spleen on USS Howell Jolly Bodies on blood film (basophilic nuclear remnants in erythrocytes) Reports marked prolonged bleeding since childhood – awaiting haematology assessment Muscle biopsy reviewed: tubular aggregates detected using STIM1 – not previously noted

Networking

• Data will feed into RD-connect: – A platform for the harmonisation of omics data from rare disease research projects and supports the International Rare Disease Research Consortium (IRDiRC) goals

49 Data in the RD-Connect platform

Clinical data Genomic data (registries, phenotypic databases) (WES, WGS)

Other omics data Sample data (metabolomics, (biobank databases) transcriptomics, proteomics…) Advances in the Limb-Girdle Muscular Dystrophies

Bringing therapies to patients with LGMD and other rare diseases needs a cultural shift on the part of academics, diagnostic laboratories, industry, research institutions and research funders towards a research paradigm that is more open to networking and data sharing

L. Johnston, R. Thompson et al., The impact of integrated omics technologies for patients with rare diseases Expert Opinion on Orphan Drugs, November 2014