PHENOTYPE-BASED NEXT GENERATION SEQUENCING FOR

Muscular Dystrophy Congenital Emery-Dreifuss Muscular Dystrophy Limb Girdle Muscular Dystrophy Duchenne Muscular Dystrophy Congenital Distal Myopathy Myofibrillar Myopathy Myotonic Syndromes Congenital Myasthenic Syndrome Periodic Malignant

A Phenotype-Based Next Generation Sequencing Approach

ATHENA DIAGNOSTICS NEXTGEN TESTING The More You Know, The More You Can Do

Phenotype-based evaluations for neuromuscular disease can allow for earlier diagnosis and treatment.

Teresa Canney, Medical Technologist, Next Generation Sequencing

When your patient presents with symptoms of The Value of muscle disease, the specific cause is not always clear. Using Next Generation Sequencing (NGS) to • Provide a definitive diagnosis that helps guide uncover a genetic cause of disease may lead to patient treatment and care decisions answers that direct the most appropriate • Save the patient and family from stressful, treatment and care for your patient. invasive, and expensive diagnostic procedures like muscle Athena Diagnostics® now offers NGS evaluations for • Help counsel the patient and relatives neuromuscular disease arranged by clinical concerning possible recurrence risk phenotype, testing only the most relevant , which streamlines the diagnostic process and saves • Provide key genetic information for family time and money. planning • Better understand the cause of disease with By combining diagnostic technology, an increase in genotype-specific trials fast turnaround time, and Athena Insight™, Athena Diagnostics offers a uniquely powerful toolset that can make an important difference in the care of your patient. It may also help predict the risk of disease in family members.

An accurate and early diagnosis can be essential for both evaluation and treatment because specific interventions are increasingly being recognized. Three Reasons Why Next Generation Sequencing from Athena Diagnostics Can Make a Difference

1. Phenotype-Guided Evaluations • Athena Diagnostics Neuromuscular Advanced Evaluations are organized by phenotype, making test selection easy and Phenotype- avoiding costly and unnecessary testing. Based NGS Diagnostics • Evaluations are based on peer-reviewed published literature* to facilitate clinically-meaningful diagnostic and treatment decisions. ATHENA DIAGNOSTICS NEXTGEN 2. Fast, Cost-Efficient Testing with Powerful TESTING Fast, In-Depth Proprietary Technology Cost-Efficient Results • Concordance with Sanger sequencing is achieved for excellent NGS Technology Interpretation sensitivity and specificity.

• Our hybridization-based enrichment technology allows removal of duplicate fragments, reducing amplification bias, providing excellent allele balance, more confident variant calls, and better sensitivity.

• Regions with known pseudogenes or other sequence homology are sequenced at no additional charge using Sanger technology if interference with NGS accuracy is detected.

• Each evaluation is accomplished with one blood draw and a 6-week turnaround.

3. Athena Insight™, In-Depth Variant Interpretation and Reporting • Created by scientists and geneticists at Athena Diagnostics, Athena Insight™ incorporates a standardized, evidence-based pathogenicity assessment/scoring process that stratifies variants based on the relative likelihood of pathogenicity.

• Our team of variant scientists collaborate on evaluations to provide the best possible results and optimal analytic value.

• Assessments of variants are based on multiple independent types of evidence. This provides the confidence of pathogenicity assessment from accumulated evidence in the medical literature, as well as variant databases.

• The clinician receives a complete synopsis and interpretation of findings with a determination of the likelihood of variants being benign or pathogenic. Results are presented in clear, concise terms suitable for use during discussions with patients and family members.

• Over 19,000 pathogenicity assessments on more than 12,000 unique variants have been successfully performed to date, focused on genetic disorders in , endocrinology, and nephrology. This allows a more accurate definition of the region of interest.

• In many cases, it is beneficial to test other family members to enhance interpretations of variants of unknown significance. Please call an Athena Diagnostics genetic counselor for details.

* Kaplan JC. The 2012 version of the table of monogenic neuromuscular disorders. Neuromuscular disorders : NMD 2011;21:833-61. Phenotype-Based Next Generation Sequencing for Neuromuscular Disease

Comprehensive Services New Billing Solutions that that Go Beyond Results Simplify Testing

Genetic Counseling Services Introducing the Athena Alliance Program Our team of genetic counselors is readily available to Athena Diagnostics is committed to providing testing provide in-depth information on the nature, inheritance, that makes a difference in patient care. As a part of and implications of genetic test results. that commitment, we are pleased to announce the • can help the physician guide the Athena Alliance Program, a new solution dedicated to patient in making informed medical and personal simplifying the billing process for you and your patients. decisions for themselves and their family.

Carol A. Hoffman, Ph.D., M.S., LGC Genetic Counselor

Pre-Authorization Services Personalized Patient Services Pre-authorization for in-network orders eases the • Support from point of order, to specimen collection, workload in your office. to determination of insurance coverage, submission, and appeals Care360® • Regionally structured to provide local expertise The power and flexibility of this cloud-based, electronic • Decision support algorithms to order the most health record system developed by Quest Diagnostics useful testing for your patients enables 24/7 access and greater efficiencies in managing lab orders and results. Financial Assistance • Place test orders, analyze results, and gain • Income-based financial assistance is available to practice-wide insights quickly and easily. patients in all 50 U.S. states regardless of • Save time and improve access to critical insurance status. patient information. ATHENA DIAGNOSTICS NEXTGEN TESTING

Neuromuscular Genes Tested

5501 5502 5503 5504 5505 5506 5507 5508 5511 5518 5519 5530 Muscular Congenital Congenital Distal Myofibrillar Myotonic Periodic Malignant Congenital Emery- Limb Girdle DMD Dystrophy Muscular Myopathy Myopathy Myopathy Syndromes Paralysis Hyper- Myasthenic Dreifuss Muscular Evaluation Dystrophy thermia Syndrome* Muscular Dystrophy Dystrophy

ANO5 B3GALNT2 ACTA1 ANO5 BAG3 ATP2A1 CACNA1S RYR1 ARGN EMD ANO5 DMD †† CAPN3 †† B3GNT1 BIN1 CAV3 CRYAB CAV3 SCN4A CACNA1S CHAT FHL1 CAPN3 †† CAV3 CHKB CCDC78 DES DES CLCN1 KCNJ2 CHRNA1 LMNA CAV3 DAG1 COL6A1 CFL2 CRYAB FHL1 HSPG2 CHRNB1 SYNE1 DYSF CCDC78 COL6A2 CNTN1 DNM2 FLNC SCN4A CHRND SYNE2 FKRP DES COL6A3 DNM2 DYSF LDB3 DMP K †(DM1) CHRNE TMEM43 FKTN DMD †† DNM2 KBTBD13 FLNC MYOT CNBP †(DM2) COLQ LMNA DNAJB6 DPM2 KLHL40 GNE TTN DOK7 MYOT DYSF FHL1 MEGF10 KLHL9 SEPN1 DPAGT1 SGCA †† EMD FKRP MTM1 LDB3 GFPT1 SGCB FHL1 FKTN MYBPC3 MATR3 MUSK SGCD FKRP POMGNT2 MYH2 MYH7 RAPSN SGCG †† FKTN ISPD MYH7 MYOT SCN4A DNAJB6 ISPD ITGA7 NEB NEB DES LMNA LAMA2 RYR1 TTN TCAP MYOT LMNA SEPN1 VCP TRIM32 PLEC LARGE TNNT1 TIA1 TTN POMGNT1 POMGNT1 TPM2 POMT1 POMT1 POMT1 TPM3 POMT2 POMT2 POMT2 TRIM32 POMGNT1 PTRF SEPN1 TTN PLEC SGCA †† TCAP DAG1 SGCB TMEM5 TRAPPC11 SGCD SGCG †† SMCHD1 SYNE1 SYNE2 TCAP TMEM43 TRAPPC11 TRIM32 TTN

NOTE: *Test available in mid-2015 † Repeat Expansion Analysis †† Sequencing and Duplication/Deletion TEST CODE/ A T T t e h REPORTED SUBTYPES, FORMS OR GROUPS NMD PHENOTYPE CHARACTERIZATION PHENOTYPE h s i s e t

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The organization of Athena Diagnostics Evaluations is based on evidence from publications using NGS as a diagnostic tool for neuromuscular disorders and relevant variant databases. *

MYO- MYOTONIC CONGENITAL - PERIODIC MALIGNANT FIBRILLAR SYNDROMES MYASTHENIC OPATHIES PARALYSIS HYPERTHERMIA SYNDROMES*

• Slowly progres - type 1 (DM1) • Muscle Spectrum of muscle • Episodes of associ - • In most cases, first signs sive weakness in and type 2 (DM2) are progressive (i.e. ocular, disease with X-linked ated with changes in serum ( and tachyp - distal and/or disorders, associated with cardiac bulbar, limb inheritance can present concentration nea) may appear when proximal defects, endocrine abnormalities, muscles, but no as: • Hyperkalemic PP (hyperPP) is charac - patient is given , muscles of neurological dysfunctions and cataracts cardiac or • Mild-muscle cramps terized by attacks of flaccid limb paraly - during anesthetization or individuals • DM1, the most common form of adult smooth muscle with sis with weakness associated with early post-op • Distal muscle onset MD, characterized by marked movement) and quadriceps abnormally elevated levels in blood • Affected patient can pres - weakness is more variability between and within family • Motor develop - myopathy potassium concentrations. Three clini - ent with metabolic , pronounced and members. The most severe form is mental delay • Severe-progressive cally distinct manifestations: (1) without , tachycardia, common (80%) congenital, showing developmental • Exercise muscle diseases , (2) with clinical or elec - hyperthermia, muscle rigid - than proximal delay, severe muscle weakness, intolerance classified as Duchenne tromyographic (EMG) myotonia, or (3) ity, with risk . with paramyotonia congenital (PMC). for cardiac or (25%) • Apneas (DMD)/Becker (BMD) • DM2 patients may have milder Attacks typically begin in the first cardiac arrest, muscle break - • Sensory symp - • Risk to infection w h e n s k e l e t al muscle toms, muscle clinical presentation than DM1, and is primarily affected decade of life, increase in down with CK increase, and 19 stiffness, aching, in mildest form, DM2 can be difficult • Earlier onset and as DMD-associ - frequency and severity over time. myoglobinuria with risk for or cramps to recognize. 16,17 cases typically ated dilated cardiomy - • Hypokalemic PP (hypoPP) is generally renal failure display: 21,22 may occur Non-dystrophic myotonic disorders are opathy (DCM) when characterized by reversible attacks of • AD inheritance • Peripheral neu - a heterogeneous group of skeletal – Difficulty heart is primarily muscle weakness associated with an • Clinical manifestation may ropathy present in muscle disorders. feeding affected abnormal decrease in blood potassium depend on genetic predis - 20% of cases • (MC) is the – Weak cry and • DMD usually presents concentrations. Recovery occurs when position, dose of trigger serum potassium normalizes. 20 • Overt cardiomy - most common suckling in early childhood with agents, or duration of expo - opathy present in . There are two forms: – Choking spells delayed milestones, • Andersen-Tawil syndrome (ATS) is sure. Almost all individuals 15-30% 15 autosomal dominant (Thomsen), and • Specific clinical including delays in characterized by a unique phenotype with MH appear normal autosomal recessive (Becker). Both course and sitting and standing consisting of , cardiac without any pathologic show a remarkable degree of pheno - severity are often independ ently; rapidly , and skeletal dysmorphic signs; it is impossible to typic and genotypic variability, and highly variable progressive abnormalities. Paralysis may occur with diagnose susceptibility either hyperkalemia or that without either the exposure are more severe in men than in • Subsets can be • BMD is characterized 18 exacerbate the cardiac arrhythmia with to triggering agents or by women. Variable age of onset. 23,24,25 by later-onset skeletal AD or AR 29 30 muscle weakness 28 sudden cardiac arrest in 10% of patients. specific diagnostic testing. 5505, Myofibrillar 5506, Myotonic 5511**, Congenital 5530, DMD 5507, Periodic Paralysis 5508, Malignant Myopathy Ad - Syndromes Advanced Myasthenic Syn - Evaluation Advanced Sequencing Hyperthermia Advanced vanced Sequencing Sequencing Evaluation drome Advanced 5531, DMD Evaluation Sequencing Evaluation Evaluation 7 g e nes tested Sequencing Duplication/Deletion 3 genes tested 2 genes tested 9 genes tested Evaluation** 13 genes tested

MFM consists of Myotonia comprises a small group 13 subsets are Forms include: Periodic paralyses are classified several genetically of muscle diseases: identified by gene: • Duchenne muscular into groups: distinct subtypes: • M y o t onic dystrophies • ARGN dystrophy (DMD) • Hyperkalemic PP (hyperPP) • Myofibrillar My - Myotonic dystrophy type 1 (DM1), • CHAT • Becker muscular • Hypokalemic PP (hypoPP) Myotonic dystrophy type 2 (DM2) dystrophy (BMD) opathy 1 (MFM1) • COLQ • Andersen-Tawil syndrome -related • Non-dystrophic myotonic • DMD-associated • CHRNE • Thyrotoxic PP • Myofibrillar My - disorders, including dilated cardiomyopa - opathy 2 (MFM2) Myotonia Congenita (MC), • CHRNA1 thy (DCM) 28 • Myofibrillar My - Thomsen's Disease and • CHRNB1 opathy 3 (MFM3) Becker's Disease • CHRND Myotilinopathy • RAPSN • Myofibrillar My - • DOK7 opathy 4 (MFM4) • DPAGT1 ZASP-related • MUSK • Myofibrillar My - opathy 5 (MFM5) • GFPT1 • Myofibrillar My - • SCN4A opathy 6 (MFM6) • Congenital Myopa - thy with Fiber-Type Disproportion • Early-onset myopa - thy with fatal car - diomyopathy • FHL1-related my - ofibrillar myopa - thy 15

* Data on file. ** Testing available in mid-2015. Test Menu for Neuromuscular Disease from Athena Diagnostics

Whole Blood, Test Test Lavender Top Tube/ Turnaround Code Name Minimum Volume* (mL) Time 5501 Muscular Dystrophy Advanced Evaluation 6-8 Adult, 1-2 Pediatric 6 weeks CPT Codes**: 81404 x4, 81405 x9, 81406 x8, 81408 x2, 81161, 81479 5502 Congenital Muscular Dystrophy Advanced Sequencing Evaluation 6-8 Adult, 1-2 Pediatric 6 weeks CPT Codes: 81404 x2, 81405 x2, 81406 x3, 81407 x3, 81408, 81479 5503 Advanced Sequencing Evaluation 6-8 Adult, 1-2 Pediatric 6 weeks CPT Codes: 81406 x2, 81407 x2, 81408 x2, 81479 5504 Distal Myopathy Advanced Sequencing Evaluation 6-8 Adult, 1-2 Pediatric 6 weeks CPT Codes: 81404, 81405 x2, 81406 x3, 81407, 81408 x2, 81479 5505 Myofibrillar Myopathy Advanced Sequencing Evaluation 6-8 Adult, 1-2 Pediatric 6 weeks CPT Codes: 81404, 81405 X2, 81406, 81479 5506 Myotonic Syndromes Advanced Sequencing Evaluation 6-8 Adult, 1-2 Pediatric 6 weeks CPT Codes: 81401, 81404, 81406, 81479 5507 Periodic Paralysis Advanced Sequencing Evaluation 6-8 Adult, 1-2 Pediatric 6 weeks CPT Codes: 81403, 81406, 81479 5508 Advanced Sequencing Evaluation 6-8 Adult, 1-2 Pediatric 6 weeks CPT Codes: 81408, 81479 5518 Emery-Dreifuss Muscular Dystrophy Advanced Sequencing Evaluation 6-8 Adult, 1-2 Pediatric 6 weeks CPT Codes: 81404, 81405, 81406 X2, 81479 5519 Limb Girdle Muscular Dystrophy Advanced Evaluation 6-8 Adult, 1-2 Pediatric 6 weeks CPT Codes: 81404 x3, 81405 x7, 81406 x6, 81408, 81479 5511 Congenital Myasthenic Syndrome Advanced Sequencing Evaluation*** 6-8 Adult, 1-2 Pediatric 6 weeks 5530 DMD Evaluation (new code, existing test) 6-8 Adult, 1-2 Pediatric 6 weeks CPT Codes: 81161, 81408 5531 DMD Duplication/Deletion (new code, existing test) 6-8 Adult, 1-2 Pediatric 6 weeks CPT Code: 81161 *We require whole blood in EDTA tubes (lavender top tubes). Adults: 10 mL; Children: 4 mL; Infants: 2 mL. Outside DNA is discouraged; however, high quality extracted DNA that meet our standards can be accepted. The test requires a minimum of 20 ug of DNA at a concentration of 50 ng/ul with a minimum volume of 400 ul. **The CPT codes provided are based on AMA guidelines and are for informational purposes only. CPT coding is the sole responsibility of the billing party. ***Available in mid-2015. Please contact one of our genetic counselors regarding specific acceptance policies and specimen requirements for at 800-394-4493.

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Quest, Quest Diagnostics, any associated logos, and all associated Quest Diagnostics registered or unregistered trademarks are the property of Quest Diagnostics. Unless otherwise noted, any person depicted in this material is a model. ADX555SG-10/14-REV00