Enabling Informed Clinical Decisions with Deep Insights. Routine Multi-gene Testing for Inherited Neuromuscular Disorders

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1 Enabling Informed Clinical Decisions with Deep Insights Routine Multi-gene Testing for Inherited Neuromuscular Disorders

2 Introduction Multi-gene Testing for Inherited Neuromuscular Disorders Inherited Neuromuscular Disorders (NMDs) are a complex and heterogeneous group of disorders with the genetic cause of the disease remaining unknown for many patients. Conventional gene-by-gene molecular diagnostic approaches (single-gene testing) do not provide adequate information to accurately diagnose a case with complex phenotypes and unknown mutations. Multi-gene testing using Next-Generation Sequencing (NGS) can provide accurate, and rapid clinical diagnosis for complex and heterogeneous neuromuscular disorders, at a cost lesser than conventional single-gene testing. Highlights of NGS Testing for Neuromuscular Disorders High clinical utility for improved disease management and treatment decisions. Molecular confirmation of a clinical diagnosis. Recurrence risk assessment for future pregnancies and prenatal testing after confirmation of diagnosis. Aids in differential diagnosis. Facilitates carrier testing (for autosomal recessive disorders). Identification of at-risk family members. Accurate information for comprehensive genetic counseling.

3 Clinical Indications of Disorders Covered in the Neuromuscular Test Disorders Muscular Dystrophies Dystonia Ataxia Congenital Myopathy Spastic Paraplegia Neuropathies Amyotrophic Lateral Sclerosis Description Muscular dystrophies (MD) are a group of muscle diseases characterized by muscle weakness and wasting that is usually progressive, with continuous degeneration/regeneration of muscle fibers. Serum creatine kinase level is usually elevated. In this test, MD has been further classified as: collagen type VI-related disorders, dystroglycanopathy, dystrophinopathy, Emery-Dreifuss MD and limb-girdle MD depending on the underlying genetic cause. Dystonia is a neuromuscular disorder characterized by involuntary muscle contractions that cause slow repetitive movements or abnormal postures and may have other neurologic features. Dystonia can affect only one muscle, groups of muscles or muscles throughout the body. In this test, dystonia has been further classified as: focal and generalized dystonia, complex dystonia and parkinsonism associated dystonia. Ataxia is a term for a group of disorders that affect coordination of muscle movements that includes gait (movement of limbs) abnormality. Ataxia is a non-specific clinical manifestation implying dysfunction of the parts of the nervous system that coordinate movement, such as the cerebellum. The clinical manifestations are seen in childhood itself in many cases, and may result from one or a combination of dysfunction of the cerebellar systems, lesions in the spinal cord or peripheral sensory loss. In this test, ataxia has been further classified as: cerebellar ataxia, spinocerebellar ataxia, episodic ataxia, spastic paraplegia associated ataxia, neuronal ceroid lipofuscinosis, mitochondrial disorders associated ataxia and epilepsy associated ataxia. Congenital myopathy refers to a genetically and clinically heterogeneous group of progressive disorders characterized by muscle weakness and hypotonia at birth or in infancy. Typically, an infant with a congenital myopathy will be "floppy," have difficulty breathing or feeding, and will lag behind other babies in meeting normal developmental milestones such as turning over or sitting up. In this test, congenital myopathy has been further classified as: nemaline myopathy, distal myopathy, myofibrillar myopathy, centronuclear myopathy, Bethlem myopathy and congenital fiber-type disproportion. Hereditary spastic paraplegia (HSP) is a group of inherited neuromuscular disorders that cause weakness and stiffness of the lower part of the body. HSP is a progressive condition and is also known as familial spastic paraparesis or Strümpell-Lorrain syndrome. Neuropathies are a group of disorders affecting the peripheral nervous system. The symptoms of hereditary neuropathies may be congenital or appear in adulthood. Variable expression of clinical features is seen among different family members, with some family members being more severely affected than others. In this test, neuropathies have been further classified as: hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease, peripheral neuropathy, axonal neuropathy, Dejerine-Sottas disease, polyneuropathy, amyloidosis associated neuropathy and ataxia associated neuropathy. Amyotrophic Lateral Sclerosis is a progressive neurodegenerative disease involving the upper (UMN) and lower motor neurons (LMN). Symptoms of UMN include hyperreflexia (overactive or overresponsive reflexes), increased muscle tone, and weakness. LMN signs include weakness, muscle wasting, hyporeflexia (below normal or absent reflexes), muscle cramps and involuntary twitching of muscles. The presentation varies amongst individuals.

4 Who to Test? Patient with clinical features (hypotonia, hypertonia, spasticity, increased CPK levels, movement disorders) indicative of one of the 7 neuromuscular conditions. Carrier testing for couples who have lost a child whose clinical features were indicative of one of these disorders. Single gene test or MLPA tests have returned inconclusive; to rule out point mutations. For differential diagnosis. Benefits of NGS/Multi-gene Testing for Neuromuscular Disorders 1. Rapid turnaround time and cost-effectiveness. 2. Unprecedented sequencing speed by massive parallel sequencing technology and ability to look at many genes at the same time. 3. NGS can detect different types of mutations in the DNA sequence, such as SNVs, indels and large deletions or duplications.. Diagnosis of complex phenotypes. Can the Neuromuscular Test Be Offered for Prenatal Diagnosis? This test is not offered directly for prenatal diagnosis. However, a prenatal diagnosis can be offered based on the identified variation(s) in the proband's sample or after carrier testing in the parents.

5 Neuromuscular Test Genes & Test Selection Disorders Disorders subtype Gene list Number of Genes Muscular dystrophy Collagen type VI-related disorders COL6A1, COL6A2, COL6A3, COL12A1 Dystroglycanopathy B3GNT1, FKRP *, FKTN, ISPD *, LARGE, POMGNT1, POMGNT2, POMT1, POMT2 9 Dystrophinopathy DMD, SGCA, SGCB 3 Emery-Dreifuss muscular dystrophy EMD *, FHL1, LAMP2, SUN2, SYNE1, SYNE2, TMEM3 7 Limb-girdle muscular dystrophy ANO5, CAPN3, CAV3 *, DES *, DNAJB6, DYSF, EMD *, FHL1, FKRP *, FKTN, GAA, LAMA2, MYOT, PLEC, POMGNT1, POMT1, POMT2, SGCA, SGCB, SGCD, SGCG, TCAP, TRIM32, TTN 2 ANO5, BAG3, BIN1, CHKB, CLCN1, DAG1, DES *, DPM1, DPM3, DYSF, FKTN, FRG1 *, GAA, ITGA7, MYOT, PABPN1 *, PHKA1, PLEC, POMGNT1, POMT2, PYGM, SCNA, SEPN1 *, TTN 2 Dystonia Focal and generalized dystonia DRD5, PRKRA, THAP1, TOR1A Complex dystonia ARSA, ATP7B, AUH, BCS1L, C19orf12, COX10, COX15, CP, DCAF17, FA2H, FOXRED1, FTL, GCDH, MECP2 *, MTFMT, NDUFA10 *, NDUFA12, NDUFA2, NDUFA9, NDUFAF2, NDUFAF6, NDUFS1, NDUFS3, NDUFS, NDUFS7, NDUFS8, NPC1, NPC2, PANK2 *, PARK2, PDGFB, PDHX *, PLA2G6, SDHA, SUCLA2, SURF1 *, VPS13A, WDR5, XK 39 Parkinsonism associated dystonia ATP13A2, ATP1A3, GCH1 *, PRKRA, SLC6A3, SPR, TH 7 ACTB, ADCY5 *, ARFGEF2, ATM *, ATP1A3, BCAP31, DRD2, FA2H, PNKD, PRRT2, SCP2, SLC2A1, SLC30A10, SUCLA2, TIMM8A 15 Ataxia Cerebellar ataxia ATCAY, ATP1A3, ATP8A2, CA8, CACNA1A, CLN6 *, CYP27A1, DNMT1, FASTKD2, KIAA0226, OPA3, PAX6, PLEKHG, SCN8A, VLDLR, WDR81, ZNF Spinocerebellar ataxia ADCK3, AFG3L2 *, ANO10, C10orf2, CA8, CACNA1A, CCDC88C, ELOVL, FGF1, GJB1, GRM1, IFRD1, ITPR1, KIAA0226, PDYN, PRKCG, SETX, SPTBN2, SYNE1, SYT1, TDP1, TGM6, TPP1, TRPC3, TTBK2, WWOX 26 Episodic ataxia CACNA1A, CACNB, KCNA1, SLC1A3 Spastic paraplegia associated ataxia KIF1A, PNPLA6, SPAST, SPG7 *, ZFYVE26, ZFYVE27 6 Neuronal ceroid lipofuscinosis CLN5 *, CLN6 *, KCTD7 *, TPP1 Mitochondrial disorders associated ataxia COQ2, COQ9 *, NDUFS7, NUBPL, PDSS2, SDHA 6 Epilepsy associated ataxia GOSR2, KCTD7 *, MTTP, POLG, PRICKLE1 *, WWOX 6 ABCB7, ABHD12, AFG3L2 *, APTX, ATM *, ATP1A3, C10orf2, CACNA1A, COQ2, CYP27A1, DARS2, DNAJC19, FLVCR1 *, FMR1 *, FXN *, GJB1, KCNJ10, KCTD7 *, MECP2 *, MRE11A, MTPAP, MTTP, NKX2-1 *, NPC1, NPC2, OPA1, PEX10 *, PEX16, PEX2, PEX7, PHYH, PIK3R5, PMM2, PNKP, PNPLA6, POLG, POLR3A, POLR3B, PRX *, PSEN1, RNF170, SACS, SCN1A, SIL1, SRD5A3, TTPA *, VLDLR, WFS1 8 Congenital myopathy Nemaline myopathy Distal myopathy ACTA1, CFL2, KBTBD13 *, NEB *, TNNT1, TPM2, TPM3 CAV3 *, DYSF, FLNC, KLHL9, MATR3, MYH7, MYH1 7 7 Myofibrillar myopathy BAG3, CRYAB, FLNC, LDB3 *, MYOT 5 Centronuclear myopathy BIN1, DNM2 *, MTMR1, MYF6, TTN 5 Bethlem myopathy COL6A1, COL6A2, COL6A3, COL12A1

6 Disorders Congenital myopathy Disorders subtype Congenital fiber-type disproportion Gene list ACTA1, RYR1, SEPN1 *, TPM2, TPM3 ACTA1, ANO5, ATP2A1, CAV3 *, CHKB, CNTN1, COL6A1, COL6A2, COL6A3, DES *, FHL1, FKBP1, GBE1, GFER *, GNE, HRAS, ISCU, ITGA7, LAMP2, MEGF10, MTM1, MYH1, MYH2, MYH7, MYOT, OPA1, ORAI1, PABPN1 *, PNPLA2 *, PUS1, RYR1, SEPN1 *, SIL1, TAZ, TPM2, TPM3, TTN, VCP, YARS2 Number of Genes 5 39 Spastic paraplegia Spastic paraplegia ABCD1, ALDH18A1, ALS2, APB1, APE1, APM1, APS1 *, AP5Z1, ATL1, ATP2B, BSCL2, C12orf65, C19orf12, CCT5, CYP7B1, ELOVL, ENTPD1, ERLIN2, EXOSC3, FA2H, GAD1, GBA, HSPD1, IFIH1, KDM5C, KIAA0196, KIF1A, KIF5A, L1CAM, PLP1, PNPLA6, PSEN1, RAB3GAP2, REEP1, RTN2, SACS, SLC16A2, SLC33A1, SPAST, SPG11, SPG20, SPG21, SPG7 *, VCP, ZFYVE26, ZFYVE27 7 Neuropathy Hereditary motor and sensory neuropathy AARS, ATL1, ATP7A, BSCL2, CCT5, DCTN1, DNMT1, FAM13B, GARS, GJB1, HADHB, HK1, HSPB1 *, HSPB3, HSPB8, IFRD1, IGHMBP2, IKBKAP, KIF1A, MFN2, MPZ, MTMR2, NDRG1, NEFL, NGF, NTRK1, PHYH, PRX *, RAB7A, REEP1, SACS, SCN9A, SETX, SH3TC2, SLC12A6, SLC5A7, SPTLC2, SYT2, TRPV, VRK1, WNK1 2 Charcot-Marie-Tooth disease AARS, AIFM1, ARHGEF10, BSCL2, CTDP1 *, DNAJB2, DNM2 *, DYNC1H1, EGR2 *, FBLN5, FGD, FIG, GAN, GARS, GDAP1, GJB1, HARS, HK1, HOXD10, HSPB1 *, HSPB8, IGHMBP2, INF2, KARS, KIF1B, KIF5A, LITAF, LRSAM1, MED25, MFN2, MPZ, MTMR2, NDRG1, NEFL, PMP22, PRPS1, PRX *, RAB7A, REEP1, SBF2, SETX, SH3TC2, SLC12A6, SOX10, SPTLC2, SYT2, TRPV, VCP, WNK1, YARS 50 Peripheral neuropathy AARS, ARHGEF10, GDAP1, GJB1, GJB3, KARS, MYH1, SCN10A, SLC12A6, SOX10, TRPV, YARS 12 Axonal neuropathy CD59, GAN, LRSAM1, MFN2, NEFL, SETX, TDP1, TRPV, VRK1 9 Dejerine-Sottas disease EGR2 *, MPZ, PMP22, PRX * Polyneuropathy ABHD12, APOA1, CD59, GJB1, MCM3AP, SLC25A19 *, TTR, TYMP 8 Amyloidosis associated neuropathy APOA1, TTR, TYMP 3 Ataxia associated neuropathy AFG3L2 *, IFRD1, SACS, SETX, TDP1 5 AAAS, AARS, ABCD1, AP1S1, ARHGEF10, C10orf2, CTDP1 *, DFNB59, DHH *, DIAPH3, DYNC1H1, EGR2 *, FBLN5, GDAP1, GJB1, GLA, HADHB, IGHMBP2, MPZ, MTMR2, NTRK1, OPA1, OTOF, PMP22, PNPLA6, POLG, PRF1, PRPS1, PRX *, RPIA, SCN10A, SCN9A, SCP2, SH3TC2, SLC12A6, SLC25A19 *, SNAP29, SOX10, SYT2, TSFM *, TTR, TYMP 2 Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis ALS2, ANG, APEX1, C9orf72, CHGB *, CHMP2B *, DAO, DCTN1, ERBB, FIG, FUS, GRN, MATR3, OPTN, PARK7, PFN1, PON1, PON2, PON3, PSEN1, SETX, SIGMAR1 *, SOD1, SPG11, SQSTM1 *, TAF15, TARDBP, TBK1, TRPM2, TRPM7, UBQLN2, VAPB, VCP 3 Footnote: * Genes having 85%-95% coverage. All other genes have >95% coverage in this test. Disclaimer: Gene coverage varies from sample to sample in the NGS runs. Spinocerebellar ataxia caused due to repeat expansions in the gene, are not covered in this panel. How to Select a Test for Your Patient? Based on the clinical indications of the patient and family history, select one appropriate disorder type/disorder subtype from the list provided above.

7 Genetic Testing Process Test Prescription Provide prescription to your patient for testing. Risk Assessment Risk assessment (to identify the risk, based on patient s family history). Pre-test Genetic Counseling Pre-test genetic counseling (pros and cons of the test will be explained to patient). Sample Collection The hospital or our product specialist collects sample from patient in kits provided by us. Sample is sent to our lab for processing. Sample Processing & Analysis Samples are processed through state-of-the art analysis, interpretation, and reporting platforms by our team of expert scientists. Report Receive your clinical report securely through . The final results are delivered in an easy-to-read report containing actionable genomic variant information. Our genetic counselors are available to help you review the results and answer any questions you may have. Post-test Genetic Counseling Post-test counseling (the results of the report will be explained to the patient by the genetic counselor). Sample Requirements (any one of the following) Saliva in collection kits provided by Strand Blood in EDTA (purple top tube) DNA isolated from blood Turnaround Time (TAT) -6 weeks from receipt of sample in the lab

8 About Strand A History of Innovative Genomic Research Strand Life Sciences is a global genomic profiling company aimed at empowering cancer care and genetic testing for inherited diseases. Strand works with oncologists, pathologists, and community hospitals to enable faster clinical decision support for accurate molecular diagnosis, prognosis, therapy recommendations, and clinical trials. Strand s central reference laboratories are located in Bangalore, India and Colorado, USA. SCGPM-MKTG V2 A Trusted Partner to Companies Worldwide For 15 years, our genomics products and solutions have facilitated the work of leading researchers and medical geneticists in over 2,000 laboratories and 100 hospitals around the world. Strand Center for Genomics & Personalized Medicine (A unit of Strand Life Sciences Pvt. Ltd.) UAS Alumni Association Building, Veterinary College Campus, Bellary Road, Hebbal, Bangalore Phone: , support.strandx@strandls.com, Strand Life Sciences