Analysis and interpretation of genetic NEUROLOGICAL DISORDERS

Transcription

1 , PH 1, POLG M, RAPSN, R CA, SGCB, SGC A3, SLC25A4, SLC5 1, SYNE2, SYT2, TAC NNT1, TNNT3, T N 4, TRIM63, TRIP4, AS39, VMA21, VPS S, ACADVL, ACTA1, AD 1, ANO5, ASCC1, ATP2A CACNA1S, CAPN3, CASQ P1, COL12A1, COL13A1, COL6A COX15, CPT2, CRYAB, CHAT, CHCHD10 HRNE, CHRNG, CHST14, DAG1, DES, DMD, DNA PM2, DPM3, DYSF, EARS2, ECEL1, ECHS1, EMD, ENO ARS2, FBN1, FBN2, FBXL4, FDX2, FHL1, FKRP, FKTN, FLAD1, FLNC, FOXRED1, G FER, GFM1, GFPT1, GLDN, GLE1, GMPPB, GNE, GYG1, GYS1, HACD1, HADHA, H HNRNPDL, HRAS, HSPG2, IARS2, ISCU, ISPD, ITGA7, KBTBD13, KCNJ2, KCNJ5, K KLHL40, K E1, LDB3, LDHA, LIMS2, LIPT1, L MTM1, MUSK, ND Analysis and interpretation of genetic NEUROLOGICAL DISORDERS FL HACD1 KCNJ2, KCN LIPT1, LMNA, LM YBPC1, MYF6, MYH2, NDUFA4, NDUFA9, N UFS8, NDUFV1, NEB PGK1, PGM1, PH OGLUT1, PO RBCK Portfolio

2 Why neurohic arises with the aim of providing analysis and interpretation of neurological disorders with a genetic basis as part of the services offered by Health in Code. Health in Code specializes in the diagnosis of inherited diseases that focuses on clinical practice and patient-oriented medicine. We are a technology-based company with headquarters in A Coruña and with an international scope, born after years of clinical experience and worldwide scientific collaborations. KNOWLEDGE MANAGEMENT CUTTING-EDGE TECHNOLOGY MULTIDISCIPLINARY TEAM During our more than 10 years dedicated to the field of genetic diagnosis of cardiovascular diseases, we have developed a knowledge management model that relies on a unique database able to integrate clinical information of the studied individuals, as well as molecular biology data from scientific publications and research groups we collaborate with, currently containing information from over individuals. We have a unique database model that is able to integrate clinical and molecular information from thousands of individuals This database is essential for the development of patientoriented medicine, allowing us to generate detailed reports with all the available clinical information about the detected variants. The quality and relevance of this information are assessed by experts at our laboratory who give advice on practical applications and recommendations for the clinical management of each patient. Our laboratory is equipped with the latest technology for massive DNA sequencing (NGS). The company has made a great effort to automate key laboratory processes (both in terms of equipment and software) in order to guarantee their traceability and minimize the probability of human errors, in compliance with the requirements of UNE-EN ISO as the highest quality standard for clinical laboratories. We are members of the European Molecular Genetics Quality Network (EMQN, UK), and we participate periodically in their external quality assessment (EQA) and comparative testing, both technical and interpretational, for neurological disorders. We are a team of medical specialists in the field of neurology, clinical geneticists, biologists, cardiologists, pharmacists, documentalists, nurses, epidemiologists, and bioinformaticians, among others. The development of neurohic services is complemented by close collaboration of the company with different research groups, as well as by our international network of experts who act as scientific advisors. This allows us to offer top quality services based on the most complete and updated knowledge.

3 3 PERSONALIZED COUNSELING: Our experts can offer support regarding study indication and interpretation of findings through direct and personal contact with our client. I I OUR CLINICAL REPORT WIDE PORTFOLIO OF NGS PANELS STUDY OF CNVs neurohic provides physicians with the necessary tools and information to make the best decisions based on the available information about each variant detected in the patient. The report is not intended to substitute for evaluation by a physician. The main aim is to guarantee the correct interpretation of the results, a fundamental tool to provide appropriate genetic counseling. neurohic offers the possibility to provide interpretation services for studies sequenced by our clients. We have created a series of specific services aimed at enabling the molecular diagnosis of genetic neurological disorders through the implementation of the latest NGS technologies in combination with other more traditional molecular techniques (Sanger, MLPA, nucleotide repeat expansion analysis). Therefore we have designed a series of panels, both general and specific, with the purpose of serving as tools to support diagnosis in the clinical practice. neurohic panels include a comprehensive selection of genes described in clinical practice guidelines, as well as those that have been associated with disease in recent literature. These panels allow analyzing the main genes involved in each group of diseases in a single study (more than 630 related genes are included). To detect large duplications and deletions (CNVs), we have developed an algorithm based on comparative analysis of coverage data generated by our NGS studies. This approach allows us to maximize the diagnostic yield of our panels without increasing their cost.

4 Services 264 genes genetic muscle disorders [GMD] 107 genes hereditary neuropathies Genetic Muscle Disorders Comprehensive Panel [264] Hereditary Neuropathies Comprehensive Panel [107] Structural Myopathies: Structural GMD Comprehensive Panel [107] Congenital Structural Genetic Muscle Disorders [58] Child- and Adult-Onset Structural Genetic Muscle Disorders [56] Limb-Girdle Muscular Dystrophies [34] Distal Myopathies [31] Myofibrillar Myopathies [13] Emery-Dreifuss Muscular Dystrophies [7] Dystrophinopaties [DMD] MLPA Sequencing NGS Oculopharyngeal Muscular Dystrophy [PABPN1] Metabolic Myopathies: Metabolic Myopathies Comprehensive Panel [113] Glycogen Storage Myopathies [19] Lipid Storage Myopathies [15] Mitochondrial Myopathies Nuclear Genes [79] Hereditary Motor and Sensory Neuropathy / Charcot- Marie-Tooth disease: CMT Comprehensive Panel [63] CMT - Demyelinating / Intermediate [30] CMT - Axonal / Intermediate [46] CMT - Deafness [21] CMT - Roma Population [3] CMT - Core Panel [4] CMT1A/HNPP gene dosage analysis by MLPA method [PMP22] Motor Neuropathy / SMN1-Negative Spinal Muscular Atrophy [30] Hereditary Sensory and Autonomic Neuropathy [22] Metabolic Neuropathy [18] Myotonia Non-Dystrophic Myotonias [8] Myotonic Dystrophy type 1 [DMPK] Congenital Myasthenic Syndromes: Congenital Myasthenic Syndromes Comprehensive Panel [23] Congenital Myasthenic Syndromes Core Panel [6] Arthrogryposis: Arthrogryposis Comprehensive Panel [51] Multiple Pterygium Syndrome, Escobar Variant and Related Disorders [15] Distal Arthrogryposis [10]

5 5 123 genes movement disordes 76 genes hereditary spastic paraplegia Movement Disordes Comprehensive Panel [123] Dystonia: Dystonia Comprehensive Panel [24] Primary Dystonia [8] Dopa-Responsive Dystonia [3] Myoclonus-Dystonia [3] Parkinson s Disease: Parkinson and Related Disorders Comprehensive Panel [14] Parkinson s Disease [6] Young-Onset Parkinson s Disease [6] Chorea and Huntington-like Disorders [19] Basal Ganglia Calcification [11] Aicardi-Goutières Syndrome Specific Panel [7] Neurodegeneration with Brain Iron Accumulation Syndromes (NBIAS) [10] Paroxysmal Movement Disorders [18] Metabolic Movement Disorders [32] Neuronal Ceroid Lipofuscinosis Specific Panel [11] Spastic Paraplegia Comprehensive Panel [76] Pure Spastic Paraplegia [28] Complicated Spastic Paraplegia [65] Spastic Paraplegia Core Panel [8] ALS/PLS [28] Alzheimer s Disease and Other Dementia [28] Genes mitocondriales nucleares: Mitochondrial Nuclear Genes Comprehensive Panel [174] Mitochondrial Respiratory Chain Complex Deficiency [45] mtdna Depletion [16] Nuclear Gene-Encoded Leigh Syndrome Core Panel [14] Pyruvate Dehydrogenase (PDH) Deficiency [12] Primary Coenzyme Q Deficiency [11] 28 genes amyotrophic lateral sclerosis / primary lateral sclerosis 28 genes Alzheimer s disease and other Dementia 211 genes mitochondrial disorders Mitochondrial Genome [37]

6 Genetic muscle disorders [GMD] GMD COMPREHENSIVE PANEL [264 genes] STRUCTURAL MYOPATHIES METABOLIC MYOPATHIES MYOTONIA CONGENITAL MYASTHENIC SYNDROMES ARTHRO- GRYPOSIS Structural GMD comprehensive panel [107 genes] Metabolic myopathies comprehensive panel [113 genes] Non-dystrophic myotonias [8 genes] Congenital myasthenic comprehensive panel [23 genes] Arthrogryposis comprehensive panel [51 genes] Child- and adult-onset structural genetic muscle disorders [56 genes] Congenital structural GMD [58 genes] Glycogen storage myopathies [19 genes] Myotonic dystrophy type 1 [DMPK] Congenital myasthenic syndromes core panel [6 genes] Multiple pterygium syndrome, Escobar variant and related disorders [15 genes] Limb-girdle muscular dystrophies [34 genes] Lipid storage myopathies [15 genes] Distal arthrogryposis [10 genes] Distal myopathies [31 genes] Mitochondrial myopathies nuclear genes [79 genes] Myofibrillar myopathies [13 genes] Emery-Dreifuss muscular dystrophies [7genes] Dystrophinopaties [DMD] Oculopharyngeal muscular dystrophy [PABPN1]

7 genetic muscle disorders I The broad range of muscular disorders has greatly expanded in the last decades thanks to advances in the field of genetics. Although there are acquired causes of muscle disease (immunologic, toxic, endocrine-metabolic), genetic causes account for 80% of cases, with more than 200 known genes so far. As a whole, they are considered rare diseases, with an overall prevalence of 1/3 500 live births. The increase in survival and the discovery of new therapeutic targets have contributed to the growing importance of this group of pathologies. It is worth noting that, although they are chronic and usually progressive degenerative diseases, over 50% have their onset in childhood. The GMD comprehensive panel constitutes a global approach to the most relevant genes related to genetic muscle disorders, namely: structural and metabolic myopathies, congenital myasthenic syndromes, myotonia, and arthrogryposis. 7 Genetic muscle disorders comprehensive panel [264 genes] ABHD5 ACAD9 ACADM ACADS ACADVL ACTA1 ADCY6 ADGRG6 ADSSL1 AGK AGL AGRN ALG14 ALG2 AMPD1 ANO5 ASCC1 ATP2A1 B3GALNT2 B4GAT1 BAG3 BCS1L BIN1 BVES C12orf65 CACNA1S CAPN3 CASQ1 CAV3 CAVIN1* CCDC78 CFL2 CLCN1 CNTN1 CNTNAP1 COL12A1 COL13A1 COL6A1 COL6A2 COL6A3 COLQ COQ2 COQ9 COX10 COX15 CPT2 CRYAB CHAT CHCHD10 CHKB CHRNA1 CHRNB1 CHRND CHRNE CHRNG CHST14 DAG1 DES DMD DNAJB6 DNM2 DOK7 DOLK DPAGT1 DPM1 DPM2 DPM3 DYSF EARS2 ECEL1 ECHS1 EMD ENO3 ETFA ETFB ETFDH ETHE1 FARS2 FBN1 FBN2 FBXL4 FDX2* FHL1 FKRP FKTN FLAD1 FLNC FOXRED1 GAA GBE1 GFER GFM1 GFPT1 GLDN GLE1 GMPPB GNE GYG1 GYS1 HACD1 HADHA HADHB HNRNPDL HRAS HSPG2 IARS2 ISCU ISPD ITGA7 KBTBD13 KCNJ18 KCNJ2 KCNJ5 KLHL24 KLHL40 KLHL41 KLHL9 LAMA2 LAMP2 LARGE1* LDB3 LDHA LIMS2 LIPT1 LMNA LMOD3 LPIN1 LRP4 LRPPRC MATR3 MEGF10 MICU1 MTFMT MTM1 MUSK MYBPC1 MYF6 MYH2 MYH3 MYH7 MYH8 MYOT MYPN NALCN NDUFA1 NDUFA10 NDUFA12 NDUFA2 NDUFA4 NDUFA9 NDUFAF2 NDUFAF5 NDUFAF6 NDUFS1 NDUFS2 NDUFS3 NDUFS4 NDUFS7 NDUFS8 NDUFV1 NEB OPA1 ORAI1 PABPN1 PDHA1 PDHB PDHX PDSS2 PET100 PFKM PGAM2 PGK1 PGM1 PHKA1 PHKA2 PHKB PHKG2 PIEZO2 PLEC PMM2 PNPLA2 PNPLA8 PNPT1 POGLUT1 POLG POLG2 POMGNT1 POMGNT2 POMK POMT1 POMT2 PREPL PUS1 PYGM RAPSN RBCK1 RYR1 SCN4A SCO1 SCO2 SDHA SDHAF1 SELENON* SERAC1 SGCA SGCB SGCD SGCG SIL1 SLC18A3 SLC19A3 SLC22A5 SLC25A20 SLC25A3 SLC25A4 SLC5A7 SNAP25 SPEG STAC3 STIM1 SUCLA2 SUCLG1 SURF1 SYNE1 SYNE2 SYT2 TACO1 TAZ TCAP TIA1 TK2 TMEM126B TMEM43 TMEM5 TNNI2 TNNT1 TNNT3 TNPO3 TOR1AIP1 TPK1 TPM2 TPM3 TRAPPC11 TRIM32 TRIM54 TRIM63 TRIP4 TRMU TRPV4 TSFM TTC19 TTN TWNK* TYMP UQCRQ VARS2 VCP VIPAS39 VMA21 VPS33B XK YARS2 ZBTB42 ZC4H2 *CAVIN1 (PTRF); FDX2 (FDX1L); LARGE1 (LARGE); SELENON (SEPN1); TWNK (C10orf2) customercare@healthincode.com I I

8 Structural myopathies We use the term "structural" for those forms of muscle disease that primarily affect the structure of muscle fibers and lead to alterations in their function. To better assess this group of disorders, two main categories were considered based on the age of onset of symptoms: Congenital structural genetic muscle disorders (present from birth) Child- and adult-onset structural genetic muscle disorders For a general approach to structural genetic muscle disorders, including congenital, child- and adult-onset forms, a comprehensive panel of 107 related genes has been developed. Structural genetic muscle disorders comprehensive panel [107 genes] ACTA1 ADSSL1 AGL ANO5 B3GALNT2 B4GAT1 BAG3 BIN1 BVES CAPN3 CAV3 CCDC78 CFL2 CNTN1 COL12A1 COL6A1 COL6A2 COL6A3 CRYAB CHKB DAG1 DES DMD DNAJB6 DNM2 DOLK DPM1 DPM2 DPM3 DYSF EMD FHL1 FKRP FKTN FLNC GAA GBE1 GMPPB GNE HACD1 HNRNPDL HRAS ISPD ITGA7 KBTBD13 KLHL40 KLHL41 KLHL9 LAMA2 LAMP2 LARGE1* LDB3 LIMS2 LMNA LMOD3 MATR3 MEGF10 MTM1 MYF6 MYH2 MYH7 MYOT MYPN NEB ORAI1 PABPN1 PHKA1 PLEC PMM2 PNPLA2 POGLUT1 POMGNT1 POMGNT2 POMK POMT1 POMT2 RYR1 SELENON* SGCA SGCB SGCD SGCG SIL1 SPEG STAC3 STIM1 SYNE1 SYNE2 TCAP TIA1 TMEM43 TMEM5 TNNT1 TNPO3 TOR1AIP1 TPM2 TPM3 TRAPPC11 TRIM32 TRIM54 TRIM63 TRIP4 TRPV4 TTN VCP VMA21 XK *LARGE1 (LARGE); SELENON (SEPN1) I The most relevant genes are highlighted in bold Congenital structural genetic muscle disorders panel [58 genes] ACTA1 B3GALNT2 B4GAT1 BIN1 CCDC78 CFL2 CNTN1 COL12A1 COL6A1 COL6A2 COL6A3 CHKB DAG1 DNM2 DOLK DPM1 DPM2 DPM3 FKRP FKTN GMPPB HACD1 HRAS ISPD ITGA7 KBTBD13 KLHL40 KLHL41 LAMA2 LARGE1* LMNA LMOD3 MEGF10 MTM1 MYF6 MYH2 MYH7 MYPN NEB ORAI1 PMM2 POMGNT1 POMGNT2 POMK POMT1 POMT2 RYR1 SELENON* SIL1 SPEG STAC3 STIM1 TMEM5 TNNT1 TPM2 TPM3 TRIP4 TTN *LARGE1 (LARGE); SELENON (SEPN1) I The most relevant genes are highlighted in bold The group of congenital structural genetic muscle disorders includes all genes related to congenital myopathy and congenital muscular dystrophy. Congenital myopathies comprise a group of disorders characterized by non-dystrophic morphological abnormalities on muscular biopsy. An approximate prevalence of 3.5-5/ live births has been estimated (Sharma et al., 2009). Most of these diseases manifest at birth or shortly thereafter with hypotonia, delay in motor development, and static or non-progressive weakness. Although these symptoms may be present since birth, diagnosis is often not reached until well into childhood or even in adulthood, since many of these signs may go unnoticed. On the other hand, congenital muscular dystrophies, despite showing symptoms from birth, usually have a more severe and progressive development and are characterized by the presence of muscular dystrophy on biopsy.

9 genetic muscle disorders I RELATED PHENOTYPES: Centronuclear myopathy Central core / minicore / multiminicore myopathy Congenital fiber-type disproportion Nemaline myopathy Collagen type VI-related disorders / Bethlem myopathy / Ullrich congenital muscular dystrophy Merosin-deficient / LAMA2-related congenital muscular dystrophy Dystroglycanopathies (Walker-Warburg syndrome, muscle-eye-brain disease, Fukuyama-type congenital muscular dystrophy, other forms of congenital muscular dystrophy with / without CNS involvement) Rigid spine syndrome with respiratory failure 9 Child- and adult-onset structural genetic muscle disorders panel [56 genes] ADSSL1 ANO5 BAG3 BVES CAPN3 CAV3 CRYAB DES DMD DNAJB6 DNM2 DYSF EMD FHL1 FKRP FKTN FLNC GNE HNRNPDL ISPD KLHL9 LAMP2 LDB3 LIMS2 LMNA MATR3 MYH2 MYH7 MYOT NEB PABPN1 PLEC POMGNT1 POMT1 POMT2 SELENON* SGCA SGCB SGCD SGCG SYNE1 SYNE2 TCAP TIA1 TMEM43 TNPO3 TOR1AIP1 TRAPPC11 TRIM32 TRIM54 TRIM63 TRPV4 TTN VCP VMA21 XK *SELENON (SEPN1) I The most relevant genes are highlighted in bold The group of child- and adult-onset structural genetic muscle disorders encompasses the rest of muscular dystrophies: a group of inherited diseases that affect skeletal muscle, characterized by a progressive degeneration of muscle fibers determining loss of strength. This heterogeneous group of diseases has been a subject of clinical and molecular studies for decades, leading to increasingly complex classifications based on genotype-phenotype correlation attempts. So far, one of the most useful classifications for the clinical practice is still the prevailing weakness pattern, which allows identifying phenotypes to guide genetic studies. We have relied on this classification to guide clinical decision making aimed at selecting the most suitable panel for molecular diagnosis: Dystrophinopathies (DMD) Limb-girdle muscular dystrophies (both at the pelvic and shoulder level) Emery-Dreifuss muscular dystrophy (characterized by a scapulohumeral-peroneal distribution and early contractures, associated with heart disease) Distal myopathies (with a pattern of weakness predominantly involving distal muscles) Oculopharyngeal muscular dystrophy* Facioscapulohumeral muscular dystrophy** With the exception of oculopharyngeal muscular dystrophy*, whose main pathogenic mechanism is a triplet repeat expansion in the PABPN1 gene (analyzed in our laboratory on specific request), and facioscapulohumeral muscular dystrophy**, whose main pathogenic mechanism is the contraction of a repetitive region in the DUX4 gene (detected by a technique not performed in our laboratory), there are specific panels for the study of the remaining pathologies. We have developed an additional panel for the study of myofibrillar myopathies, selected for their characteristic findings on muscle biopsy. customercare@healthincode.com I I

10 Limb-girdle muscular dystrophies panel [34 genes] ANO5 BVES CAPN3 CAV3 DAG1 DES DMD DNAJB6 DYSF FKRP FKTN GAA GMPPB HNRNPDL ISPD LIMS2 LMNA MYOT PLEC POGLUT1 POMGNT1 POMK POMT1 POMT2 SGCA SGCB SGCD SGCG TCAP TNPO3 TOR1AIP1 TRAPPC11 TRIM32 TTN The most relevant genes are highlighted in bold RELATED PHENOTYPES: Anoctaminopathies Calpainopathies Caveolinopathies Desminopathies Dysferlinopathies Fukutinopathies Laminopathies Myotilinopathies Sarcoglycanopathies Titinopathies Distal myopathies panel [31 genes] ADSSL1 AGL ANO5 BAG3 CAV3 CRYAB DES DNM2 DYSF EMD FHL1 FLNC GAA GBE1 GNE KLHL9 LAMP2 LDB3 LMNA MATR3 MYH7 MYOT NEB PHKA1 PNPLA2 SELENON* TCAP TIA1 TRPV4 TTN VCP SELENON (SEPN1) I The most relevant genes are highlighted in bold RELATED PHENOTYPES: Laing distal myopathy Miyoshi distal myopathy Nonaka distal myopathy Udd distal myopathy Welander distal myopathy Myofibrillar myopathies panel [13 genes] ACTA1 BAG3 CRYAB DES DNAJB6 FHL1 FLNC LDB3 MYOT PLEC TRIM54 TRIM63 TTN The most relevant genes are highlighted in bold Emery-Dreifuss muscular dystrophies panel [7 genes] EMD FHL1 LMNA SYNE1 SYNE2 TMEM43 TTN The most relevant genes are highlighted in bold

11 genetic muscle disorders I Dystrophinopathies genetic study [DMD] 11 For DMD, the following techniques can be performed: MLPA (for the detection of deletions/duplications of one or more exons) NGS (able to detect CNVs, point mutations and small in/dels) Related phenotypes: Duchenne muscular dystrophy Becker muscular dystrophy X-linked dilated cardiomyopathy Other DMD-related phenotypes Oculopharyngeal muscular dystrophy study [PABPN1] Triplet repeat expansion analysis. REFERENCES 1. Darras BT, Menache-Stroninki CC, Hinton V, Kunkel LM. Neuromuscular Disorders of Infancy, Childhood and Adolescence: A Clinician s Approach, 2nd ed, Darras BT, Jones HR Jr, Ryan MM, De Vivo DC (Eds), Academic Press, San Diego Emery AE. The muscular dystrophies. Lancet 2002; 359: Puckelwartz M, McNally EM. Emery-Dreifuss muscular dystrophy. Handb Clin Neurol 2011; 101: Romero NB, Clarke NF. Congenital myopathies. Handb Clin Neurol 2013; 113: Sewry CA, Jimenez-Mallebrera C, Muntoni F. Congenital myopathies. Curr Opin Neurol 2008; 21: Selcen D. Myofibrillar myopathies. Neuromuscul Disord 2011; 21: Sharma MC, Jain D, Sarkar C, Goebel HH. Congenital myopathies--a comprehensive update of recent advancements. Acta Neurol Scand May;119(5): Wicklund MP. The muscular dystrophies. Continuum (Minneap Minn) 2013; 19: customercare@healthincode.com I I

12 Metabolic myopathies Metabolic myopathies are a group of inherited muscular disorders secondary to enzymatic defects affecting metabolism and energy production in muscle. Some of them are considered inherited metabolic diseases and, although they are rare causes of myopathy, their diagnostic relevance lies in the fact that some of them are potentially treatable. Their symptomatology can mimic other forms of muscular dystrophy or inflammatory myopathies, and they often manifest with subtle symptoms such as asymptomatic CK elevation, muscle cramps, myalgia, or myoglobinuria. Their prevalence is unknown: Pompe disease (acid maltase deficiency) affects 1/ people, and McArdle disease 1/ people. Its etiopathogenesis is related to problems in the metabolism of glycogen, lipids or in mitochondrial oxidative phosphorylation. Therefore, we have designed three specific panels for each metabolic pathway and a comprehensive panel that includes the most relevant genes involved in this type of diseases. Metabolic myopathies comprehensive panel [113 genes] ABHD5 ACAD9 ACADM ACADS ACADVL AGK AGL AMPD1 BCS1L C12orf65 CASQ1 CAVIN1* COQ2 COQ9 COX10 COX15 CPT2 CHCHD10 CHKB EARS2 ECHS1 ENO3 ETFA ETFB ETFDH ETHE1 FARS2 FBXL4 FDX2* FLAD1 FOXRED1 GAA GBE1 GFER GFM1 GYG1 GYS1 HADHA HADHB IARS2 ISCU KLHL24 LAMP2 LDHA LIPT1 LPIN1 LRPPRC MICU1 MTFMT NDUFA1 NDUFA10 NDUFA12 NDUFA2 NDUFA4 NDUFA9 NDUFAF2 NDUFAF5 NDUFAF6 NDUFS1 NDUFS2 NDUFS3 NDUFS4 NDUFS7 NDUFS8 NDUFV1 OPA1 PDHA1 PDHB PDHX PDSS2 PET100 PFKM PGAM2 PGK1 PGM1 PHKA1 PHKA2 PHKB PHKG2 PNPLA2 PNPLA8 PNPT1 POLG POLG2 PUS1 PYGM RBCK1 SCO1 SCO2 SDHA SDHAF1 SERAC1 SLC19A3 SLC22A5 SLC25A20 SLC25A3 SLC25A4 SUCLA2 SUCLG1 SURF1 TACO1 TAZ TK2 TMEM126B TPK1 TRMU TSFM TTC19 TWNK* TYMP UQCRQ VARS2 YARS2 *CAVIN1(PTRF); FDX2(FDX1L); TWNK(C10orf2) I The most relevant genes are highlighted in bold Glycogen storage myopathies panel [19 genes] AGL ENO3 GAA GBE1 GYG1 GYS1 KLHL24 LAMP2 LDHA PFKM PGAM2 PGK1 PGM1 PHKA1 PHKA2 PHKB PHKG2 PYGM RBCK1 The most relevant genes are highlighted in bold RELATED PHENOTYPES: GAA PYGM LAMP2 PFKM GYG1 / RBCK1 AGL / GBE1 PGAM2 PGK1 PGM1 PHKA1 / PHKA2 / PHKB / PHKG2 LDHA ENO3 Pompe disease (type II glycogenosis) McArdle disease (type V glycogenosis) Danon disease Muscle phosphofructokinase deficiency (type VII glycogenosis) Polyglucosan body myopathy Glycogen debranching / branching enzyme deficiency Phosphoglycerate mutase deficiency (type X glycogenosis) Phosphoglycerate kinase deficiency Congenital disorder of glycosylation type It Phosphorylase kinase deficiency (type IX glycogenosis) Lactate dehydrogenase deficiency (type XI glycogenosis) Enolase deficiency type III

13 genetic muscle disorders I Lipid storage myopathies panel [15 genes] 13 ABHD5 ACADM ACADS ACADVL AMPD1 CAVIN1* CPT2 ETFA ETFB ETFDH FLAD1 LPIN1 PNPLA2 SLC22A5 SLC25A20 *CAVIN1(PTRF) I The most relevant genes are highlighted in bold RELATED PHENOTYPES: ACADS / ACADM / ACADVL AMPD1 CPT2 Short-chain / Medium-chain / Very long-chain acyl-coa dehydrogenase deficiency Myoadenylate deaminase deficiency myopathy Carnitine palmitoyltransferase II deficiency ETFA / ETFB / ETFDH Glutaric aciduria type 2 FLAD1 LPIN1 PNPLA2 Flavin adenine dinucleotide synthetase deficiency myopathy Recurrent myoglobinuria Neutral lipid storage disease with myopathy CAVIN1 (PTRF) Congenital lipodystrophy type 4 SLC22A5 SLC25A20 ABHD5 Primary carnitine deficiency Carnitine-acylcarnitine translocase deficiency Chanarin-Dorfman syndrome Mitochondrial myopathies nuclear gene panel [79 genes] ACAD9 AGK BCS1L C12orf65 CASQ1 COQ2 COQ9 COX10 COX15 CHCHD10 CHKB EARS2 ECHS1 ETHE1 FARS2 FBXL4 FDX2* FOXRED1 GFER GFM1 HADHA HADHB IARS2 ISCU LIPT1 LRPPRC MICU1 MTFMT NDUFA1 NDUFA10 NDUFA12 NDUFA2 NDUFA4 NDUFA9 NDUFAF2 NDUFAF5 NDUFAF6 NDUFS1 NDUFS2 NDUFS3 NDUFS4 NDUFS7 NDUFS8 NDUFV1 OPA1 PDHA1 PDHB PDHX PDSS2 PET100 PNPLA8 PNPT1 POLG POLG2 PUS1 SCO1 SCO2 SDHA SDHAF1 SERAC1 SLC19A3 SLC25A3 SLC25A4 SUCLA2 SUCLG1 SURF1 TACO1 TAZ TK2 TMEM126B TPK1 TRMU TSFM TTC19 TWNK* TYMP UQCRQ VARS2 YARS2 *FDX2(FDX1L); TWNK(C10orf2) I The most relevant genes are highlighted in bold RELATED PHENOTYPES: Isolated mitochondrial respiratory chain complexes deficiency Combined oxidative phosphorylation (OXPHOS) deficiency Pyruvate dehydrogenase complex (PDH) deficiency Primary coenzyme Q deficiency Trifunctional protein (TFP) deficiency Progressive external ophthalmoplegia (PEO) / Hereditary optic atrophy Ethylmalonic/methylglutaconic aciduria Thiamine- and biotin-responsive encephalopathy Myopathy with lactic acidosis Alpers-Huttenlocher syndrome mtdna depletion syndrome Leigh syndrome Barth syndrome Perrault syndrome Sengers syndrome REFERENCES 1. Berardo A, DiMauro S, Hirano M. A diagnostic algorithm for metabolic myopathies. Curr Neurol Neurosci Rep 2010; 10: Darras BT, Friedman NR. Metabolic myopathies: a clinical approach; part I. Pediatr Neurol 2000; 22: van Adel BA, Tarnopolsky MA. Metabolic myopathies: update J Clin Neuromuscul Dis 2009; 10: customercare@healthincode.com I I

14 Myotonia Myotonia refers to a neurological symptom that describes difficulty in muscle relaxation after contraction. There may be an involvement pattern, although any muscle group can be affected. Among hereditary myotonias, there are mainly two groups: Dystrophic myotonia (or myotonic dystrophy, whose main form is also known as DM1 or Steinert disease): Its prevalence is estimated to be about 1/8 000 individuals. Clinically, it is a multisystem disease where myotonia is accompanied by muscle weakness, cardiac conduction problems, cataracts, and endocrine and gastrointestinal alterations. Its molecular mechanism consists of a trinucleotide expansion in the DMPK gene; therefore, its diagnosis requires a specific test. Non-dystrophic myotonias belong to the group of channelopathies, and their genetic defect determines the occurrence of symptoms including myotonia as well as weakness, myalgias, episodes of paralysis, etc. Non-dystrophic myotonias panel [8 genes] ATP2A1 CACNA1S CLCN1 HSPG2 KCNJ18 KCNJ2 KCNJ5 SCN4A The most relevant genes are highlighted in bold RELATED PHENOTYPES: CLCN1 SCN4A CACNA1S ATP2A1 KCNJ2, KCNJ18, KCNJ5 KCNJ18 HSPG2 Myotonia congenita (Thomsen / Becker) Myotonia congenita Paramyotonia congenita Hyper/hypokalemic periodic paralysis Hypokalemic periodic paralysis Brody myopathy Andersen-Tawil syndrome Thyrotoxic periodic paralysis Schwartz-Jampel syndrome Myotonic dystrophy type 1 study [DMPK] Triplet repeat expansion analysis. REFERENCES 1. Udd B, Krahe R. The myotonic dystrophies: molecular, clinical, and therapeutic challenges. Lancet Neurol 2012; 11: Miller TM. Differential diagnosis of myotonic disorders. Muscle Nerve 2008; 37:293-9.

15 genetic muscle disorders I Congenital myasthenic syndromes Congenital myasthenia is a group of disorders caused by a biochemical defect or a structural alteration of the neuromuscular junction that leads to a clinical picture of muscle weakness and fatigability from birth or early infancy. It is important to distinguish these forms of the disease from myasthenia gravis (a disorder of autoimmune origin) and neonatal myasthenia (in children of mothers with myasthenia gravis). The prevalence of congenital myasthenic syndromes has been estimated between 1/ (GeneReviews) and 9.2/ (Parr et al., 2014). Its etiology is largely genetic. As of today, several involved genes are known, allowing for two thirds of cases to have a positive genetic diagnosis (Jacob et al., 2009). The most frequently involved genes and their diagnostic yields are CHRNE (50%), RAPSN (15-20%), COLQ (10-15%), DOK7 (10-15%), CHAT (5%), and GFPT1 (2%). The core panel includes these six genes, and we have also developed a comprehensive panel with 23 related genes that will allow optimizing the diagnostic yield. 15 Congenital myasthenic syndromes comprehensive panel [23 genes] AGRN ALG14 ALG2 COL13A1 COLQ CHAT CHRNA1 CHRNB1 CHRND CHRNE DOK7 DPAGT1 GFPT1 GMPPB LRP4 MUSK PREPL RAPSN SCN4A SLC18A3 SLC5A7 SNAP25 SYT2 The most relevant genes are highlighted in bold Congenital myasthenic syndromes core panel [6 genes] COLQ CHAT CHRNE DOK7 GFPT1 RAPSN REFERENCES 1. Abicht A, Müller J S, Lochmüller H. Congenital Myasthenic Syndromes May 9 [updated 2016 Jul 14]. In: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJH, Bird TD, Ledbetter N, Mefford HC, Smith RJH, Stephens K, editors. GeneReviews [Internet]. Seattle (WA): University of Washington, Seattle; Jacob S, Viegas S, Lashley D, Hilton-Jones D. Myasthenia gravis and other neuromuscular junction disorders. Pract Neurol Dec;9(6): Parr JR, Andrew MJ, Finnis M, Beeson D, Vincent A, Jayawant S. How common is childhood myasthenia? The UK incidence and prevalence of autoimmune and congenital myasthenia. Arch Dis Child Jun;99(6): customercare@healthincode.com I I

16 Arthrogryposis Arthrogryposis, or arthrogryposis multiplex congenita (AMC), is characterized by the presence of non-progressive multiple joint contractures in different areas of the body present from birth. It is estimated that up to 1% of newborns are born with some type of congenital contracture, although the prevalence of arthrogryposis is estimated in 1/3 000 live births (Lowry et al., 2010). Approximately two thirds of affected individuals can be diagnosed by the age of two, and great progress is being made in the identification of specific genetic and non-genetic causes of arthrogryposis (Hall et al., 2014). It has been estimated that a genetic cause can be identified in approximately 30% of cases (Dimitraki et al., 2011). There are different forms that should be considered when selecting the genetic study: Amyoplasia or "classic arthrogryposis" accounts for one third of cases, with a prevalence of 1/ live births. They are sporadic cases with all four limbs affected, unaffected trunk, and no multisystem involvement. They usually do not have a genetic cause, and their etiology is due to maternal-fetal factors in pregnancy. However, some forms can clinically resemble distal forms of arthrogryposis. Therefore, prior to a genetic study request, cases must be carefully selected based on clinical suspicion and potential differential diagnoses. Those individuals with associated CNS involvement (malformations, intellectual disability, or other developmental disorders) or dysmorphic features require an assessment involving chromosomal and gene dosage studies (karyotype, SNP-arrays, etc.). Targeted genetic studies are more adequate and have a higher yield for distal arthrogryposis and multiple pterygium-associated conditions, for which we have developed specific panels. It is worth noting that up to 5% of arthrogryposis cases are secondary to myopathies and congenital myasthenic syndromes (Darras et al., 2015), which are targeted by the arthrogryposis comprehensive panel.

17 genetic muscle disorders I Arthrogryposis comprehensive panel [51 genes] 17 ACTA1 ADCY6 ADGRG6 ASCC1 BIN1 CNTNAP1 COL6A1 COL6A2 COL6A3 CHAT CHRNA1 CHRNB1 CHRND CHRNE CHRNG CHST14 DNM2 DOK7 ECEL1 FBN1 FBN2 FKRP FKTN GLDN GLE1 KLHL40 KLHL41 LAMA2 LMNA MUSK MYBPC1 MYH2 MYH3 MYH8 NALCN NEB PIEZO2 POMGNT2 RAPSN RYR1 SELENON* SYNE1 TNNI2 TNNT3 TPM2 TPM3 TRIP4 VIPAS39 VPS33B ZBTB42 ZC4H2 *SELENON (SEPN1) Multiple pterygium syndrome, Escobar variant and related disorders panel [15 genes] ADCY6 ADGRG6 CNTNAP1 CHRNA1 CHRNB1 CHRND CHRNG DOK7 GLDN GLE1 MUSK MYBPC1 PIEZO2 RAPSN ZBTB42 Distal arthrogryposis panel [10 genes] ECEL1 FBN2 MYBPC1 MYH2 MYH3 MYH8 PIEZO2 TNNI2 TNNT3 TPM2 The most relevant genes are highlighted in bold REFERENCES 1. Dimitraki M, Tsikouras P, Bouchlariotou S, Dafopoulos A, Konstantou E, Liberis V. Prenatal assessment of arthrogryposis. A review of the literature. J Matern Fetal Neonatal Med Jan;24(1): Hall JG. Arthrogryposis (multiple congenital contractures): diagnostic approach to etiology, classification, genetics, and general principles. Eur J Med Genet Aug;57(8): Lowry RB, Sibbald B, Bedard T, Hall JG. Prevalence of multiple congenital contractures including arthrogryposis multiplex congenita in Alberta, Canada, and a strategy for classification and coding. Birth Defects Res A Clin Mol Teratol Dec;88(12): Darras BT, Menache-Stroninki CC, Hinton V, Kunkel LM. Neuromuscular Disorders of Infancy, Childhood and Adolescence: A Clinician s Approach, 2nd ed, Darras BT, Jones HR Jr, Ryan MM, De Vivo DC (Eds), Academic Press, San Diego customercare@healthincode.com I I

18 Hereditary neuropathies HEREDITARY NEUROPATHIES COMPREHENSIVE PANEL [107 genes] CHARCOT-MARIE- TOOTH (CMT) MOTOR SENSORY AND AUTONOMIC METABOLIC CMT comprehensive panel [63 genes] Motor neuropathy / SMN1-negative spinal muscular atrophy [30 genes] Hereditary sensory and autonomic neuropathy [22 genes] Metabolic neuropathy [18 genes] CMT - Demyelinating / intermediate [30 genes] CMT - Axonal / intermediate [46 genes] CMT - Deafness [21 genes] CMT - Roma population [3 genes] CMT - Core panel [4 genes] CMT1A / HNPP gene dosage analysis by MLPA [PMP22]

19 hereditary neuropathies I Charcot-Marie-Tooth (CMT) disease or hereditary motor and sensory neuropathy is the most frequent inherited neuromuscular disease, with a prevalence of 1/2 500 individuals (Suter and Sherer, 2003). 19 CMT is a complex disorder at the molecular level, with at least genetic variants associated with about 80 genes (Timmerman et al., 2014). In the wide series described, molecular alteration is identified in 60%-70% of patients (80% of demyelinating forms and 25% of axonal forms) (Rossor et al., 2015). Approximately 90% of alterations are found in genes PMP22, MPZ, GJB1, and MFN2 (DiVicenzo et al., 2015), although this number varies among populations and is particularly reduced in regions with a high prevalence of recessive inheritance forms. 40%-50% of CMT cases are type 1 (CMT1, demyelinating form), of which 70-80% are caused by a duplication of a region of about 1.5 Mb in 17p12 containing the PMP22 gene (CMT1A). Hereditary motor neuropathy (HMN) comprises 10% of all hereditary neuropathies, with a diagnosis rate of 20%-32% (Bansagi et al., 2017). Hereditary neuropathies comprehensive panel [107 genes] AAAS AARS ABCD1 AIFM1 APTX ASAH1 ATL1 ATL3 BICD2 BSCL2 CCT5 CHCHD10 COX6A1 CYP27A1 DCTN1 DHTKD1 DNAJB2 DNM2 DNMT1 DST DYNC1H1 EGR2 ELP1* FBLN5 FBXO38 FGD4 FIG4 FXN GAN GARS GDAP1 GJB1 GJB3 GM2A GNB4 HARS HEXA HEXB HINT1 HK1 HSPB1 HSPB3 HSPB8 IGHMBP2 INF2 KARS KIF1A KIF5A L1CAM LITAF LMNA LRSAM1 MARS MED25 MFN2 MME MORC2 MPV17 MPZ MTMR2 NAGLU NDRG1 NEFH NEFL NGF NTRK1 PDK3 PHYH PLEKHG5 PLP1 PMP22 PNKP POLG PRPS1 PRX RAB7A REEP1 RETREG1* SBF1 SBF2 SCN10A SCN11A SCN9A SEPT9 SETX SGPL1 SH3TC2 SIGMAR1 SLC12A6 SLC52A2 SLC52A3 SLC5A7 SOX10 SPG11 SPTLC1 SPTLC2 SURF1 TFG TRIM2 TRPA1 TRPV4 TTR UBA1 VAPB VCP WNK1 YARS *ELP1(IKBKAP); RETREG1 (FAM134B) RELATED PHENOTYPES: PMP22 PMP22 GAN SEPT9 ELP1(IKBKAP) Charcot-Marie-Tooth type 1A (CMT1A or duplication of the 17p12 region) Tomacular neuropathy / hereditary neuropathy with liability to pressure palsies (HNPP or deletion of the 17p12 region) Giant axonal neuropathy Hereditary neuralgic amyotrophy Familial dysautonomia Loci included: CMT1A, CMT1B, CMT1C, CMT1D, CMT1E, CMT1F, CMT2A2A, CMT2A2B, CMT2B, CMT2B1, CMT2B2, CMT2C, CMT2CC, CMT2D, CMT2E, CMT2F, CMT2I, CMT2J, CMT2K, CMT2L, CMT2M, CMT2N, CMT2O, CMT2P, CMT2R, CMT2S, CMT2T, CMT2U, CMT2V, CMT2V, CMT2W, CMT2Z, CMT4A, CMT4B1, CMT4B2, CMT4B3, CMT4C, CMT4D, CMT4F, CMT4G, CMT4H, CMT4J, CMT4K, CMTDIB, CMTDIC, CMTDIE, CMTDIF, CMTRIA, CMTRIB, CMTRIC, CMTRID, CMTX1, CMTX6, DSMA1, DSMA2, DSMA3, DSMA4, DSMA5, FEPS1, FEPS2, FEPS3, HMN2A, HMN2B, HMN2D, HMN5A, HMN5B, HMN7B, HMN8, HSAN1A, HSAN2B, HSAN2D, HSAN3, HSAN4, HSAN5, HSAN7, HSN1C, HSN1D, HSN1E, HSN1F, HSN2A, HSN2C, SMALED1, SMALED2 customercare@healthincode.com I I

20 Charcot-Marie-Tooth Charcot-Marie-Tooth comprehensive panel [63 genes] AARS AIFM1 BICD2 BSCL2 COX6A1 DCTN1 DNAJB2 DNM2 DYNC1H1 EGR2 FBLN5 FBXO38 FGD4 FIG4 FXN GAN GARS GDAP1 GJB1 GJB3 GNB4 HARS HINT1 HK1 HSPB1 HSPB8 IGHMBP2 INF2 KARS LITAF LMNA LRSAM1 MARS MED25 MFN2 MME MORC2 MPZ MTMR2 NAGLU NDRG1 NEFH NEFL PDK3 PLEKHG5 PMP22 PNKP PRX RAB7A SBF1 SBF2 SGPL1 SH3TC2 SLC12A6 SLC52A2 SLC52A3 SPG11 SURF1 TFG TRIM2 TRPV4 VCP YARS The most relevant genes are highlighted in bold CMT - Demyelinating / intermediate [30 genes] AIFM1 COX6A1 DNM2 EGR2 FBLN5 FGD4 FIG4 GDAP1 GJB1 GJB3 GNB4 HARS HK1 INF2 KARS LITAF LRSAM1 MPV17 MPZ MTMR2 NDRG1 NEFL PLEKHG5 PMP22 PRX SBF1 SBF2 SH3TC2 SURF1 YARS The most relevant genes are highlighted in bold RELATED PHENOTYPES: AIFM1 FBLN5 GJB3 INF2 MPV17 SH3TC2 Cowchock syndrome Neuropathy with/without age-related macular degeneration Peripheral neuropathy with hearing loss Sensorimotor neuropathy associated with focal segmental glomerulosclerosis Mitochondrial DNA depletion syndrome type 6 (hepatocerebral type) Mononeuropathy of the median nerve CMT - Axonal / intermediate panel [46 genes] AARS AIFM1 COX6A1 CHCHD10 DHTKD1 DNAJB2 DNM2 DYNC1H1 GAN GARS GDAP1 GJB1 GNB4 HARS HINT1 HSPB1 HSPB8 IGHMBP2 INF2 KARS KIF5A LMNA LRSAM1 MARS MED25 MFN2 MME MORC2 MPZ NAGLU NEFH NEFL PDK3 PLEKHG5 RAB7A SGPL1 SLC12A6 SLC52A2 SLC52A3 SPG11 SURF1 TFG TRIM2 TRPV4 VCP YARS The most relevant genes are highlighted in bold RELATED PHENOTYPES: HINT1 KIF5A SLC12A6 TFG Axonal neuropathy with neuromyotonia Spastic paraplegia Agenesis of the corpus callosum with peripheral neuropathy Hereditary motor and sensory neuropathy, Okinawa type CMT - Deafness panel [21 genes] AARS AIFM1 EGR2 GJB1 GJB3 INF2 LITAF LRSAM1 MFN2 MPZ MTMR2 NDRG1 NEFL PDK3 PMP22 PRPS1 PRX SBF2 SH3TC2 SURF1 TFG

21 hereditary neuropathies I CMT - Roma population panel [3 genes] 21 HK1 NDRG1 SH3TC2 RELATED PHENOTYPES: HK1 NDRG1 Hereditary motor and sensory neuropathy, Russe type Hereditary motor and sensory neuropathy, Lom type CMT - Core panel [4 genes] GJB1 MFN2 MPZ PMP22 CMT1A / HNPP gene dosage analysis (17p12 region of PMP22) by MLPA Motor neuropathy Motor neuropathy /SMN1-negative spinal muscular atrophy [30 genes] AARS ASAH1 BICD2 BSCL2 CHCHD10 DCTN1 DNAJB2 DYNC1H1 FBXO38 GARS HARS HEXB HINT1 HSPB1 HSPB3 HSPB8 IGHMBP2 MFN2 MORC2 PLEKHG5 REEP1 SETX SIGMAR1 SLC52A2 SLC52A3 SLC5A7 TFG TRPV4 UBA1 VAPB The most relevant genes are highlighted in bold RELATED PHENOTYPES: ASAH1 BICD2, DYNC1H1 BSCL2 CHCHD10 HEXB SETX SLC52A2, SLC52A3 UBA1 VAPB Spinal muscular atrophy with progressive myoclonic epilepsy Spinal muscular atrophy with lower extremity predominance Silver syndrome Jokela-type spinal muscular atrophy GM2 gangliosidosis Juvenile amyotrophic lateral sclerosis Brown-Vialetto-Van Laere / Fazio-Londe syndrome X-linked spinal muscular atrophy Spinal muscular atrophy, Finkel type customercare@healthincode.com I I

22 Sensory and autonomic neuropathy Hereditary sensory and autonomic neuropathy panel [22 genes] ATL1 ATL3 CCT5 DNMT1 DST ELP1* KIF1A KIF5A NAGLU NGF NTRK1 RAB7A RETREG1* SCN10A SCN11A SCN9A SEPT9 SPTLC1 SPTLC2 TRPA1 TTR WNK1 *ELP1(IKBKAP); RETREG1(FAM134B) I The most relevant genes are highlighted in bold RELATED PHENOTYPES: NTRK1 SCN9A SCN10A, SCN11A, TRPA1 Congenital insensitivity to pain with anhydrosis Erythromelalgia, paroxysmal pain, insensitivity to pain Episodic pain syndrome Metabolic neuropathy Metabolic neuropathy panel [18 genes] AAAS ABCD1 APTX CYP27A1 FXN GAN GJB3 GM2A HEXA HEXB L1CAM PHYH PLP1 PNKP POLG PRPS1 SOX10 TTR RELATED PHENOTYPES: AAAS ABCD1 APTX, PNKP CYP27A1 HEXA, HEXB, GM2A PHYH PLP1 POLG SOX10 TTR Triple-A / Allgrove / Achalasia-addisonianism-alacrima syndrome Adrenoleukodystrophy Ataxia with oculomotor apraxia Cerebrotendinous xanthomatosis GM2 gangliosidosis Refsum disease Pelizaeus-Merzbacher disease POLG-related neuropathy Demyelinating neuropathy, central demyelination, Waardenburg / Hirschsprung disease Amyloid neuropathy REFERENCES 1. Bansagi B, Griffin H, Whittaker RG, Antoniadi T, Evangelista T, Miller J, Greenslade M, Forester N, Duff J, Bradshaw A, Kleinle S, Boczonadi V, Steele H, Ramesh V, Franko E, Pyle A, Lochmüller H, Chinnery PF, Horvath R. Genetic heterogeneity of motor neuropathies. Neurology Mar 28;88(13): DiVincenzo C, Elzinga CD, Medeiros AC, Karbassi I, Jones JR, Evans MC, Braastad CD, Bishop CM, Jaremko M, Wang Z, Liaquat K, Hoffman CA, York MD, Batish SD, Lupski JR, Higgins JJ. The allelic spectrum of Charcot-Marie-Tooth disease in over 17,000 individuals with neuropathy. Mol Genet Genomic Med Nov;2(6): Rossor AM, Evans MR, Reilly MM. A practical approach to the genetic neuropathies. Pract Neurol Jun;15(3): Suter U, Scherer SS. Disease mechanisms in inherited neuropathies. Nat Rev Neurosci Sep;4(9): Timmerman V, Strickland AV, Züchner S. Genetics of Charcot-Marie-Tooth (CMT) Disease within the Frame of the Human Genome Project Success. Genes (Basel) Jan 22;5(1):13-32.

23 hereditary neuropathies I Genotype-phenotype correlation in neuropathies. Most frequently associated symptoms 23 CMT+ spinal deformity CMT+ optic atrophy CMT+ diaphragm / vocal cord paralysis CMT+ upper limb predominance CMT- congenital / early childhood Dejerine-Sottas / congenital hypomyelination PNP+ pyramidal signs PNP+ CNS involvement Small fiber / painful neuropathy EGR2 GDAP1 EGR2 BSCL2 EGR2 EGR2 ALS2 AIFM1 NTRK1 FGD4 MFN2 GDAP1 GARS FGD4 MPZ ATL1 ALS2 SCN9A FIG4 PRPS1 IGHMBP2 HSPB8 GDAP1 PMP22 B4GALNT1 ATL1 SCN10A GARS MFN2 REEP1 MFN2 PRX BICD2 B4GALNT1 SCN11A GDAP1 MPZ TFG MPZ BSCL2 BICD2 TRPA1 HK1 MTMR2 MTMR2 C12orf65 BSCL2 HSPB8 PMP22 NEFL CCT5 C12orf65 LMNA TRPV4 PMP22 DDHD1 CCT5 MFN2 PRX DYNC1H1 DDHD1 MPZ SBF2 EGR2 DDHD1 MTMR2 SH3TC2 FA2H DYNC1H1 NDRG1 TRPV4 KIF5A EGR2 PMP22 MARS FA2H PRX MFN2 FXN SBF2 NEFL GAN SH3TC2 NIPA1 GARS SLC12A6 PLP1 GJB1 TRPV4 PNPLA6 INF2 REEP1 KIF5A SETX MARS SPG11 MFN2 SPG20 NEFL SPG7 NIPA1 TFG NTRK1 VCP PLP1 ZFYVE26 PNPLA6 REEP1 SACS SETX SH3TC2 SLC12A6 SPG11 SPG20 SPG7 TFG VCP ZFYVE26 customercare@healthincode.com I I

24 Movement disorders MOVEMENT DISORDERS COMPREHENSIVE PANEL [123 genes] DYSTONIA PARKINSON CHOREA BG NBIAS PAROXYSMAL METABOLIC CALCIFICATION Dystonia comprehensive panel [24 genes] Primary dystonia [8 genes] Parkinson and related disorders comprehensive panel [14 genes] Parkinson s disease [6 genes] Chorea and Huntington-like disordes [19 genes] Basal ganglia calcification [11 genes] Aicardi- Goutières syndrome specific panel [7 genes] Neurodegeneration with brain Iron accumulation syndromes (NBIAS) [10 genes] Paroxysmal movement disorders [18 genes] Metabolic movement disorders [32 genes] Neuronal ceroid lipofuscinosis specific panel [11 genes] Doparesponsive dystonia [3 genes] Young-onset Parkinson s disease [6 genes] Myoclonusdystonia [3 genes]

25 movement disorders I General diagnostic approach for all types of movement disorders considered for study, regardless of age. 25 Movement disorders comprehensive panel [123 genes] ADAR ADCY5 ANO3 APTX ARSA ATP13A2 ATP1A2 ATP1A3 ATP6AP2 ATP7B BCAP31 C19orf12 CACNA1A CACNA1B CACNB4 CIZ1 CLN3 CLN5 CLN6 CLN8 COASY COL6A3 CP CTSD CTSF CYP27A1 DCAF17 DCTN1 DLAT DNAJC5 DNAJC6 FA2H FBXO7 FOLR1 FTL GALC GBA GCDH GCH1 GLB1 GLRA1 GLRB GM2A GNAL GRN HEXA HEXB HPCA HPRT1 IFIH1 KCNA1 KCNMA1 KCNQ2 KCTD17 KIF1C KMT2B L2HGDH LRRK2 MAPT MECR MFSD8 NKX2-1 NPC1 NPC2 NUP62 PANK2 PARK7* PDE10A PDE8B PDGFB PDGFRB PDHA1 PINK1 PLA2G6 PLP1 PNKD POLG PPT1 PRKN* PRKRA PRNP PRRT2 PTS QDPR RNASEH2A RNASEH2B RNASEH2C RNF216 SAMHD1 SCN1A SCN9A SGCE SLC19A3 SLC1A3 SLC20A2 SLC25A19 SLC2A1 SLC30A10 SLC39A14 SLC6A3 SLC6A5 SMPD1 SNCA SPG11 SPR SQSTM1 SYNJ1 TENM4 TH THAP1 TIMM8A TOR1A TPK1 TPP1 TREX1 TUBB4A VAC14 VPS13A VPS35 WDR45 XK XPR1 ZFYVE26 *PRKN(PARK2); PARK7(DJ1) In addition to encompassing all the genes present in the different specific panels, some other genes belonging to rarer clinical pictures and with more complex phenotypes have been included. Among the ones covered by this panel, it is worth highlighting: RELATED PHENOTYPES: ADCY5 BCAP31 KIF1C, APTX, SQSTM1 PLP1 POLG SPG11, ZFYVE26 TENM4 TIMM8A Familial dyskinesia and facial myokymia Deafness, dystonia, and cerebral hypomyelination Neurodegenerative clinical condition with ataxia and oculomotor disorders Pelizaeus-Merzbacher disease Mitochondrial ataxic syndrome Complex spastic paraplegia Hereditary essential tremor Mohr-Tranebjaerg syndrome (deafness-dystonia) customercare@healthincode.com I I

26 Dystonia Dystonia is characterized by sustained or intermittent muscular contractions that lead to abnormal postures and movements, often repetitive. Symptoms of dystonia may appear from early childhood to late adulthood, affecting one (focal), several (multifocal, segmental), or numerous parts of the body (generalized). Generalized dystonia usually includes adolescent-onset progressive and incapacitating disorders with a known genetic basis. The group of primary focal dystonia generally appears in adults and usually involves the neck, face, or arms; the number of known genes is lower, although up to 25% of cases tend to have a positive family history (Steeves et al., 2012). It is worth noting that dystonias are diseases with great phenotypic and genotypic heterogeneity, often with incomplete penetrance even within the same family (Albanese et al., 2013). A prevalence of approximately 16/ individuals is estimated (Steeves et al., 2012). Although numerous risk factors have been described by association studies, the diagnostic-genetic approach is aimed at performing diagnoses with a clinical application for the patient. For this, four phenotype groups have been established: Primary dystonia (isolated, focal, or generalized, as the main symptom): 8-gene core panel, with the particularly relevant genes TOR1A (DYT1), THAP1 (DYT6), and GNAL (DYT25, adult-onset craniocervical dystonia). Dopa-responsive dystonia (Segawa syndrome): 3-gene specific panel (GCH1, TH, and SPR). Myoclonus-dystonia: 3-gene specific panel (SGCE [DYT11], whose yield is up to 50% in familial cases, and TOR1A or [DYT1] and KCTD17, which are included as differential diagnoses). Combined dystonia dystonia-plus or complex dystonia phenotypes: 24-gene extended panel for a more comprehensive etiologic approach and for complex cases. Dystonia comprehensive panel [24 genes] ANO3 ATP1A3 CACNA1B CIZ1 COL6A3 GCH1 GNAL HPCA KCTD17 KMT2B MECR PNKD PRKRA PRRT2 SGCE SLC2A1 SLC30A10 SLC39A14 SLC6A3 SPR TH THAP1 TOR1A TUBB4A Primary dystonia panel [8 genes] ANO3 CIZ1 GNAL HPCA PRKRA THAP1 TOR1A TUBB4A The most relevant genes are highlighted in bold Dopa-responsive dystonia panel [3 genes] GCH1 SPR TH The most relevant genes are highlighted in bold Myoclonus-dystonia panel KCTD17 SGCE TOR1A [3 genes] The most relevant genes are highlighted in bold REFERENCES 1. Albanese A, Bhatia K, Bressman SB, Delong MR, Fahn S, Fung VS, Hallett M, Jankovic J, Jinnah HA, Klein C, Lang AE, Mink JW, Teller JK. Phenomenology and classification of dystonia: a consensus update. Mov Disord Jun 15;28(7): Steeves TD, Day L, Dykeman J, Jette N, Pringsheim T. The prevalence of primary dystonia: a systematic review and meta-analysis. Mov Disord Dec;27(14):

27 movement disorders I Parkinson Parkinsonism is a group of neurological diseases with symptoms including bradykinesia, muscle stiffness, resting tremor, and postural instability. Parkinson's disease, the most common form of parkinsonism and the second most frequent neurodegenerative disease (after Alzheimer's disease), is clinically characterized by these four cardinal motor symptoms, as well as by a good response to treatment with levodopa. Its prevalence is estimated to be between 1%-2% of the population at age 65 and about 4% at age 85. Approximately 15% of cases are familial, although some studies suggest that up to 60% of sporadic cases could be explained by genetic factors (Hamza et al., 2010). It is currently considered a multifactorial disease, with numerous environmental and genetic factors involved. The most commonly involved monogenic determinants have been selected for a core study panel for this disease, including SNCA, LRRK2, and VPS35 (with autosomal dominant transmission and generally late onset), and PRKN (PARK2), PINK1, and PARK7 (DJ1) (with autosomal recessive transmission and an earlier onset). For the study of early-onset parkinsonism, usually appearing during adolescence (<20 years), we have developed a specific panel with the most common causative genes. Other types of atypical parkinsonism and Parkinson-plus disorders (such as progressive supranuclear palsy, corticobasal degeneration, multiple system atrophy, or Lewy body dementia) are covered by the comprehensive panel. 27 Parkinson and related disorders comprehensive panel [14 genes] ATP13A2 ATP6AP2 DCTN1 DNAJC6 FBXO7 LRRK2 MAPT PARK7* PINK1 PLA2G6 PRKN* SNCA SYNJ1 VPS35 *PARK7 (DJ1); PRKN (PARK2) I The most relevant genes are highlighted in bold Parkinson's disease panel [6 genes] LRRK2 PARK7* PINK1 PRKN* SNCA VPS35 *PARK7 (DJ1); PRKN (PARK2) I The most relevant genes are highlighted in bold Young-onset Parkinson's disease panel [6 genes] ATP13A2 ATP6AP2 DNAJC6 FBXO7 PLA2G6 SYNJ1 The most relevant genes are highlighted in bold REFERENCES 1. Hamza TH, Payami H. The heritability of risk and age at onset of Parkinson's disease after accounting for known genetic risk factors. J Hum Genet Apr;55(4): customercare@healthincode.com I I

28 Chorea and Huntington-like syndromes Although Huntington's disease is the most common form of chorea, it is estimated that 1%-7% of suspected cases have a negative HTT triplet expansion study (Martino et al., 2012), and other differential diagnoses must be considered. In addition to other non-genetic conditions (infectious, immune, toxic, or vascular), there are several phenocopies of Huntington's chorea that must be taken into account, for which this panel has been designed. Chorea and Huntington-like disorders panel [19 genes] ATP7B C19orf12 CP DCAF17 FTL HPRT1 NKX2-1 NUP62 PANK2 PDE10A PDE8B PDGFRB PLA2G6 PRNP RNF216 SLC20A2 VAC14 VPS13A XK The most relevant genes are highlighted in bold RELATED PHENOTYPES: ATP7B CP HPRT1 NKX2-1 NUP62, PDE8B, VAC14 PANK2, PLAG2G6, C19orf12, FTL PRNP SLC20A2, PDGFRB VPS13A, XK Wilson's disease Aceruloplasminemia Lesch-Nyhan syndrome Benign hereditary chorea Striatonigral degeneration NBIAS Prion diseases Basal ganglia calcification Neuroacanthocytosis and McLeod syndrome REFERENCES 1. Martino D, Stamelou M, Bhatia KP. The differential diagnosis of Huntington's disease-like syndromes: 'red flags' for the clinician. J Neurol Neurosurg Psychiatry Jun;84(6):650-6.

29 movement disorders I Basal ganglia calcification Basal ganglia calcification is a nonspecific finding that can occur in the context of certain infectious, metabolic, and genetic syndromes. It can simply constitute a benign incidental finding (around 1% of CT scans performed for other reasons in patients over age 60), a sequel of a connatal infection. However, genetic study must be considered if clinical symptoms of unknown origin are present, particularly in patients with a positive family history. 29 Two conditions have been taken into account when developing this panel: In children, Aicardi-Goutières syndrome (AGS) is an early-onset encephalopathy presenting with cerebral atrophy, leukodystrophy, and typically basal ganglia calcifications and high interferon levels in CSF. Several related genes have been described (RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, TREX1, ADAR, and IFIH1), which reach a diagnostic yield of 90%-95% in cases with suspected AGS (Crow et al., 2015). Since its original description (Aicardi and Goutières, 1984), its phenotypic spectrum has been expanded, which must be taken into account particularly in late-onset forms. In adults in their third and fifth decades of life, idiopathic basal ganglia calcification (Fahr's disease) should be considered: it is an autosomal dominant condition, usually of familial presentation, characterized by the symmetrical calcification of basal ganglia and other regions of the brain. Its clinical picture can range from asymptomatic stages to a wide spectrum of neuropsychiatric symptoms. The genes involved so far are SLC20A2, XPR1, PDGFB, and PDGFRB. Basal ganglia calcification comprehensive panel [11 genes] ADAR IFIH1 PDGFB PDGFRB RNASEH2A RNASEH2B RNASEH2C SAMHD1 SLC20A2 TREX1 XPR1 The most relevant genes are highlighted in bold Aicardi-Goutières syndrome specific panel [7 genes] ADAR IFIH1 RNASEH2A RNASEH2B RNASEH2C SAMHD1 TREX1 The most relevant genes are highlighted in bold REFERENCES 1. Crow YJ, Chase DS, Lowenstein Schmidt J, Szynkiewicz M, Forte GM, Gornall HL, et al. Characterization of human disease phenotypes associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR, and IFIH1. Am J Med Genet A Feb;167A(2): customercare@healthincode.com I I

30 NBIAS (Neurodegeneration with brain iron accumulation syndromes) NBIAS stands for neurodegeneration with brain iron accumulation syndromes and covers a series of heterogeneous, often overlapping entities whose main characteristic is the accumulation of iron in the brain. This accumulation is predominantly observed at the level of basal ganglia on brain MRI (T2, spin-echo, and gradient echo sequences). One of its most frequent forms is pantothenate kinase-associated neurodegeneration (PKAN, formerly known as Hallervorden-Spatz syndrome), which is generally recognized on MRI by the "eye of the tiger" sign (a hypointense area with a hyperintense center in the globus pallidus). The prevalence of these syndromes is estimated at around 1-3/ individuals. They present clinically as neurodegenerative diseases with movement disorders and pyramidal, cerebellar, autonomic, and eventually cognitive and psychiatric signs. Our NBIAS panel includes several causative genes, allowing an approximate diagnostic yield of 65% (Schneider et al., 2016). In order of importance, it is worth highlighting the following genes: PANK2 (35%-50%), PLA2G6 (20%), C19orf12 (6%-10%), and WDR45 (1%-2%) (Gregory and Hayflick, 2014). Neurodegeneration with brain iron accumulation syndromes (NBIAS) [10 genes] ATP13A2 C19orf12 COASY CP DCAF17 FA2H FTL PANK2 PLA2G6 WDR45 The most relevant genes are highlighted in bold REFERENCES 1. Gregory A, Hayflick S. Neurodegeneration with Brain Iron Accumulation Disorders Overview Feb 28 [updated 2014 Apr 24]. In: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJH, Bird TD, Ledbetter N, Mefford HC, Smith RJH, Stephens K, editors. GeneReviews [Internet]. Seattle (WA): University of Washington, Seattle;

31 movement disorders I Paroxysmal movement disorders Paroxysmal movement disorders are a heterogeneous group of diseases with recurring episodes of symptoms related to involuntary movements characterized by a sudden onset and a more or less abrupt disappearance after a variable period. Strictly speaking, this definition includes several known forms of episodic dyskinesia, classified below according to their triggering factor. Paroxysmal kinesigenic dyskinesia (particularly associated with pathogenic variants in the PRRT2 gene) Paroxysmal non-kinesigenic dyskinesia (particularly associated with pathogenic variants in PNKD and KCNMA1) Exercise-induced dyskinesia (particularly associated with pathogenic variants in the SLC2A1 gene, also known as glucose transporter deficiency or GLUT-1 deficiency). There are other clinical pictures that do not exactly constitute movement disorders, but they share the common characteristic of paroxysmal neurological symptoms with a fairly similar pathophysiological basis (many of them are channel-mediated) that, on occasion, can overlap to some degree in common clinical practice. 31 Paroxysmal movement disorders panel [18 genes] ATP1A2 ATP1A3 CACNA1A CACNB4 DLAT GLRA1 GLRB KCNA1 KCNMA1 KCNQ2 PDHA1 PNKD PRRT2 SCN1A SCN9A SLC1A3 SLC2A1 SLC6A5 RELATED PHENOTYPES: ATP1A3, ATP1A2 CACNA1A, ATP1A2, SCN1A CACNA1A, CACNB4, KCNA1, SLC1A3 GLRA1, GLRB, SLC6A5 SCN9A Alternating hemiplegia Hemiplegic migraine Episodic ataxia Hereditary hyperekplexia / Startle syndromes Paroxysmal extreme pain disorder customercare@healthincode.com I I

32 Metabolic movement disorders Inherited metabolic disorders are a group of diseases that include defects affecting enzymes or proteins involved in cellular metabolism. Many of these diseases have neurological manifestations and can present with complex clinical pictures, combining cognitive and muscular symptoms, ataxia, epilepsy, or movement disorders. Most commonly, movement disorders are not so much a predominant symptom as one of the manifestations of the disease. However, some metabolic disorders can start with some type of abnormal involuntary movement as their first symptom. Particularly, dystonia, myoclonus, chorea, stereotypies, and parkinsonism may be a part of this spectrum of manifestations. The importance of these diseases lies in the fact that many of them can be effectively treated and that their early identification can prevent neurological damage. In a cohort of patients with movement disorders studied by Gouider-Khouja et al. (2010), up to 29% were found to have a movement disorder secondary to metabolic disease, with dystonia and myoclonus as the most frequent symptoms (54% and 28%, respectively). One of the metabolic disease groups with the highest overall prevalence of movement disorders are mitochondrial diseases. Suspicion of any of these diseases should lead to the study of the mitochondrial genome or of nuclear genes involved in mitochondrial metabolism (see specific panel). On the other hand, we have selected some phenotypes that should be included in the differential clinical diagnosis due to the occurrence of a movement disorder as the first symptom: Metabolic movement disorders comprehensive panel [32 genes] ARSA ATP7B CLN3 CLN5 CLN6 CLN8 CP CTSD CTSF CYP27A1 DNAJC5 FOLR1 GALC GBA GCDH GLB1 GM2A GRN HEXA HEXB L2HGDH MFSD8 NPC1 NPC2 PPT1 PTS QDPR SLC19A3 SLC25A19 SMPD1 TPK1 TPP1 Neuronal ceroid lipofuscinosis specific panel [11 genes] CLN3 CLN5 CLN6 CLN8 CTSD CTSF DNAJC5 GRN MFSD8 PPT1 TPP1

33 movement disorders I RELATED PHENOTYPES: CLN3, CLN5, CLN6, CLN8, CTSD, CTSF, DNAJC5, GRN, MFSD8, PPT1, TPP1 ARSA Metachromatic leukodystrophy ATP7B Wilson's disease Neuronal ceroid lipofuscinosis (NCL) CP Aceruloplasminemia / systemic hemosiderosis CYP27A1 Cerebrotendinous xanthomatosis FOLR1 Cerebral folate transport deficiency GALC Krabbe disease GBA Gaucher disease GCDH / L2HGDH Glutaric aciduria type 1 and L-2-hydroxyglutaric aciduria GLB1, GM2A, HEXA, HEXB Gangliosidosis NPC1, NPC2, SMPD1 Niemann-Pick disease type C and types A & B PTS, QDPR Tetrahydrobiopterin deficiency / Hyperphenylalaninemia SLC19A3, SLC25A19, TPK1 Thiamine- and biotin-responsive encephalopathies 33 REFERENCES 1. Gouider-Khouja N, Kraoua I, Benrhouma H, Fraj N, Rouissi A. Movement disorders in neuro-metabolic diseases. Eur J Paediatr Neurol Jul;14(4): customercare@healthincode.com I I

34 Hereditary spastic paraplegia SPASTIC PARAPLEGIA COMPREHENSIVE PANEL [76 genes] Pure spastic paraplegia [28 genes] Complicated spastic paraplegia [65 genes] Spastic paraplegia core panel [8 genes]

35 hereditary spastic paraplegia I Hereditary spastic paraplegia has an estimated prevalence of 1.8/ Genetic cause is identified in 33%-55% of families with autosomal dominant inheritance (AD-SP) and in 18%-29% of families with autosomal recessive inheritance (AR-SP). The most frequent form of AD-SP is SPG4 (SPAST), accounting for 40% of AD-SP forms and 20% of sporadic forms (Ruano et al., 2014). SPG3A (ATL1) is the cause of 10%-15% of AD-SP cases (up to 40% in SPG4-negative cohorts), with the most frequent form starting in the first decade of life (Giudice et al., 2014). SPG11 is the most common cause of AR-SP (20%-50%) (Stevanin et al., 2008). Anita Harding's historical description distinguishes pure and complicated forms (Harding, 1983). The pure form presents isolated pyramidal signs such as spasticity, hyperreflexia, Babinski sign, and motor deficits, which can be associated with sphincter disorder and deep sensitivity alterations. Complicated forms comprise several clinical entities combining spastic paraplegia with other neurological/non-neurological signs such as cerebellar ataxia, optic atrophy, retinitis pigmentosa, thinning of the corpus callosum, neuropathy, or epilepsy, among others. 35 Spastic paraplegia comprehensive panel [76 genes] ABCD1 ADAR ALDH18A1 ALDH3A2 ALS2 AMPD2 AP4B1 AP4E1 AP4M1 AP4S1 AP5Z1 ARL6IP1 ARSI ATL1 ATP2B4 B4GALNT1 BICD2 BSCL2 C12orf65 C19orf12 CCT5 CSF1R CYP27A1 CYP2U1 CYP7B1 DARS2 DDHD1 DDHD2 ENTPD1 ERLIN1 ERLIN2 FA2H FLRT1 GBA2 GFAP GJC2 HSPD1 IBA57 IFIH1 KCNA2 KIF1A KIF1C KIF5A L1CAM MARS MARS2 NIPA1 NT5C2 PGAP1 PLP1 PNPLA6 RAB3GAP2 REEP1 REEP2 RNASEH2B RTN2 SACS SETX SLC16A2 SLC2A1 SLC33A1 SPAST SPG11 SPG20 SPG21 SPG7 TECPR2 TFG USP8 VAMP1 VPS37A WASHC5* WDR48 ZFR ZFYVE26 ZFYVE27 *WASHC5 (KIAA0196) RELATED PHENOTYPES: ABCD1 ADAR, IFIH1, RNASEH2B ALS2, FIG4, SETX ALDH3A2 CCT5 CSF1R CYP27A1 DARS2 GFAP KIF1C, KCNA2, MARS2, VAMP1 SACS SLC2A1 Adrenoleukodystrophy Aicardi-Goutières syndrome Sjögren-Larsson syndrome Amyotrophic lateral sclerosis Sensory neuropathy in hereditary spastic paraplegia Hereditary diffuse leukoencephalopathy with spheroids Cerebrotendinous xanthomatosis Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation Alexander disease Spastic ataxia Spastic ataxia, Charlevoix-Saguenay-type GLUT1 deficiency Loci included: SPG1, SPG2, SPG3A, SPG4, SPG5A, SPG6, SPG7, SPG8, SPG9A, SPG10, SPG11, SPG12, SPG13, SPG15, SPG17, SPG18, SPG20, SPG21, SPG22, SPG26, SPG28, SPG30, SPG31, SPG33, SPG35, SPG39, SPG42, SPG43, SPG44, SPG45, SPG46, SPG47, SPG48, SPG49, SPG50, SPG51, SPG52, SPG53, SPG54, SPG55, SPG56, SPG57, SPG58, SPG59, SPG60, SPG61, SPG62, SPG63, SPG64, SPG65 SPG66, SPG67, SPG68, SPG69, SPG70, SPG71, SPG72, SPG74. customercare@healthincode.com I I

36 Pure spastic paraplegia panel [28 genes] ABCD1 AP5Z1 ATL1 ATP2B4 BSCL2 CYP2U1 CYP7B1 DDHD1 ERLIN1 HSPD1 IFIH1 KIF1A KIF1C KIF5A NIPA1 NT5C2 PLP1 REEP1 REEP2 RNASEH2B RTN2 SLC33A1 SPAST SPG11 SPG7 WASHC5* ZFR ZFYVE27 *WASHC5 (KIAA0196) I En negrita, se señalan los genes más relevantes Complicated spastic paraplegia panel [65 genes] ADAR ALDH18A1 ALS2 AMPD2 AP4B1 AP4E1 AP4M1 AP4S1 AP5Z1 ARL6IP1 ARSI ATL1 B4GALNT1 BICD2 BSCL2 C12orf65 C19orf12 CCT5 CSF1R CYP27A1 CYP2U1 CYP7B1 DARS2 DDHD1 DDHD2 ENTPD1 ERLIN2 FA2H FLRT1 GBA2 GFAP GJC2 IBA57 IFIH1 KCNA2 KIF1A KIF1C KIF5A L1CAM MARS MARS2 NIPA1 NT5C2 PGAP1 PLP1 PNPLA6 RAB3GAP2 REEP1 RNASEH2B SACS SETX SLC16A2 SLC2A1 SPAST SPG11 SPG20 SPG21 SPG7 TECPR2 TFG USP8 VAMP1 VPS37A WDR48 ZFYVE26 The most relevant genes are highlighted in bold Spastic paraplegia core panel [8 genes] ATL1 CYP7B1 KIF5A REEP1 SPAST SPG11 SPG7 ZFYVE26 REFERENCES 1. Harding AE. Classification of the hereditary ataxias and paraplegias. Lancet May 21;1(8334): Lo Giudice T, Lombardi F, Santorelli FM, Kawarai T, Orlacchio A. Hereditary spastic paraplegia: clinical-genetic characteristics and evolving molecular mechanisms. Exp Neurol Nov;261: Ruano L, Melo C, Silva MC, Coutinho P. The global epidemiology of hereditary ataxia and spastic paraplegia: a systematic review of prevalence studies. Neuroepidemiology. 2014;42(3): Stevanin G, Azzedine H, Denora P, Boukhris A, Tazir M et al. SPATAX consortium. Mutations in SPG11 are frequent in autosomal recessive spastic paraplegia with thin corpus callosum, cognitive decline and lower motor neuron degeneration. Brain Mar;131(Pt 3):

37 hereditary spastic paraplegia I Genetic guide to complicated forms of spastic paraplegia (SP) according to associated neurological signs. 37 SP-cerebellar signs SP-ocular involvement SP-epilepsy SP-neuropathy SP-intellectual disability / Cognitive impairment SP-leukoencephalopathy SP-thinning of the corpus callosum SP-MRI abnormalities ALDH18A1 CYP7B1 ALDH3A2 ALDH18A1 ALDH3A2 ADAR ALDH18A1 ADAR AP4M1 ALDH18A1 AP4B1 ARL6IP1 AP4B1 ALDH3A2 AMPD2 ALDH18A1 ARSI ATL1 AP4E1 ARSI AP4E1 AP4B1 AP4B1 AMPD2 ATL1 C12orf65 ATL1 ATL1 AP4M1 AP5Z1 ATL1 AP4B1 B4GALNT1 C19orf12 ERLIN2 B4GALNT1 AP4S1 B4GALNT1 CYP2U1 AP5Z1 CYP7B1 DDHD2 FA2H BICD2 ATL1 CSF1R DDHD2 ATL1 DARS2 FLRT1 GFAP BSCL2 B4GALNT1 CYP7B1 ERLIN2 B4GALNT1 GBA2 GBA2 KCNA2 C12orf65 CSF1R DARS2 GBA2 CSF1R GFAP IBA57 NIPA C19orf12 CYP2U1 FA2H L1CAM CYP7B1 GJC2 KIF5A PLP1 CCT5 DDHD2 GFAP NT5C2 DARS2 KCNA2 NT5C2 SLC16A2 CYP2U1 ERLIN2 GJC2 SPG11 DDHD2 KIF1A RAB3GAP2 SLC2A1 CYP7B1 FA2H KIF5A SPG21 ERLIN2 KIF1C SACS SPAST DARS2 GBA2 MARS2 SPG7 FA2H MARS2 SPAST SPG11 DDHD1 GJC2 PLP1 TECPR2 GBA2 NIPA SPG11 ZFYVE26 FLRT1 IFIH1 SPAST ZFR GFAP REEP1 SPG7 IBA57 KCNA2 SPG11 ZFYVE26 GJC2 SACS TFG KIF1A KIF1C SPG20 IFIH1 SETX VAMP1 KIF5A KIF5A SPG21 KIF5A SLC16A2 ZFYVE26 L1CAM L1CAM ZFYVE26 L1CAM SLC2A1 MARS MARS2 MARS2 SPAST NIPA NIPA NT5C2 SPG20 PLP1 NT5C2 PLP1 SPG21 PNPLA6 PLP1 SPAST SPG7 REEP1 RAB3GAP2 SPG11 TECPR2 SACS REEP1 SPG20 VAMP1 SETX SLC16A2 SPG21 ZFYVE26 SPAST SLC2A1 SPG7 SPG11 SPAST TECPR2 SPG7 SPG11 ZFR TFG SPG20 ZFYVE26 WDR48 SPG21 ZFYVE26 TECPR2 VPS37A ZFYVE26 customercare@healthincode.com I I

38 Amyotrophic lateral sclerosis/ Primary lateral sclerosis ALS/PLS [28 genes]

39 amyotrophic lateral sclerosis / primary lateral sclerosis I Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with a prevalence of 5.4/ (Chiò et al., 2013). Although most cases are sporadic, 10% of patients have a positive family history. Pathogenic variants in genes C9orf72, SOD1, TARDBP, and FUS explain almost two thirds of the familial forms (>25%, 20%, 5%, and 5% respectively). Only one genetic alteration has been identified in the C9orf72 gene. It consists of a GGGGCC hexanucleotide expansion, which is not detectable by next-generation-sequencing. 39 Primary lateral sclerosis (PLS) is characterized by the isolated involvement of the upper motor neuron, which distinguishes it from amyotrophic lateral sclerosis, in which involvement of the lower motor neuron also occurs. The diagnosis of PLS is reached by excluding other causes of disease such as spastic paraplegia, multiple sclerosis, metabolic disease, or myelopathy. Amyotrophic lateral sclerosis / Primary lateral sclerosis panel [28 genes] ALS2 ANG CCNF CHCHD10 CHMP2B DAO DCTN1 ERBB4 FIG4 FUS HNRNPA1 MATR3 NEFH OPTN PFN1 PRPH SETX SIGMAR1 SLC52A2 SLC52A3 SOD1 SPG11 SQSTM1 TARDBP TBK1 UBQLN2 VAPB VCP The most relevant genes are highlighted in bold RELATED PHENOTYPES: ALS2, FIG4, SPG11, TBK1 CHCHD10, SQSTM1, TBK1 SLC52A2, SLC52A3 Primary lateral sclerosis Amyotrophic lateral sclerosis with / without frontotemporal dementia Brown-Vialetto-Van Laere and Fazio-Londe syndrome Loci included: ALS1, ALS2, ALS4, ALS5, ALS6, ALS8, ALS9, ALS10, ALS11, ALS12, ALS14, ALS15, ALS16, ALS17, ALS18, ALS19, ALS20, ALS21, FTDALS2, FTDALS3, FTDALS4. REFERENCES 1. Chiò A, Logroscino G, Traynor BJ, Collins J, Simeone JC, Goldstein LA, White LA. Global epidemiology of amyotrophic lateral sclerosis: a systematic review of the published literature. Neuroepidemiology. 2013;41(2): customercare@healthincode.com I I

40 Alzheimer's disease and other Dementia ALZHEIMER'S DISEASE AND OTHER DEMENTIA [28 genes]

41 Alzheimer's disease and other Dementia Dementia constitutes an important social, health, and economic problem. Twenty five percent of people over age 55 have a family history of dementia. In 2016, it affected 46.8 million people worldwide. With an exponential growth, this number is estimated to reach million by 2050 (World Alzheimer Report 2016). Alzheimer's disease (AD) is the most common type of primary neurodegenerative dementia (60%-80% of cases). Approximately 25% of patients with AD have two or more affected relatives. Among familial forms, only 5% have an early onset (age <65 years). In these cases, transmission is autosomal dominant and is caused by pathogenic variants in genes PSEN1 (30%-70%), PSEN2 (<5%), and APP (10%-15%) (Loy et al., 2014). Allele 4 of the APOE gene represents a risk factor for AD (OR=2-3 in heterozygosis, OR=14.9 in homozygosis; Farrer et al., 1997), although the presence of the allele 4 of APOE is not necessary or sufficient to develop disease. Therefore, although genotyping of APOE can be clinically useful to support diagnosis in the context of other data suggesting AD, particularly in late-onset forms (age >65), its use is not indicated in asymptomatic individuals. Between 5% and 15% of pre-senile dementia cases (<65 years) are frontotemporal type, with a prevalence of 10-15/ in the age group between 45 and 65 years. 25%-50% of patients with frontotemporal dementia have a family history of dementia or psychiatric disease, and 10%-30% are compatible with an autosomal dominant inheritance pattern (Rohrer et al., 2009). Pathogenic variants in C9orf72, GRN, and MAPT are involved in 80% of families with autosomal dominant forms. A single genetic alteration in the C9orf72 gene has been identified, consisting in a GGGGCC hexanucleotide expansion, not detectable by next-generation-sequencing. 41 Alzheimer's disease and other Dementia comprehensive panel [28 genes] APOE APP ATP13A2 CSF1R CHCHD10 CHMP2B FUS GBA GRN HNRNPA1 HNRNPA2B1 ITM2B LRRK2 MAPT PINK1 PLA2G6 PRNP PSEN1 PSEN2 SNCA SNCB SQSTM1 TARDBP TBK1 TIMM8A TREM2 UBQLN2 VCP The most relevant genes are highlighted in bold RELATED PHENOTYPES: APP, PRNP ATP13A2, GRN, LRRK2, MAPT, PINK2, PLA2G6, SNCA, SNCB CHCHD10, SQSTM1, TBK1 CSF1R FUS, TARDBP, UBQLN, VCP GBA GRN HRNPA2B1, VCP MAPT MAPT, GRN, CHMP2B, DCTN1, TREM2 PRNP SNCA, SNCB TIMM8A TREM2 Cerebral amyloid angiopathy Frontotemporal dementia-parkinson Frontotemporal dementia with/without amyotrophic lateral sclerosis Hereditary diffuse leukoencephalopathy with spheroids Amyotrophic lateral sclerosis with frontotemporal dementia Gaucher disease Primary progressive aphasia Dementia associated with inclusion body myopathy and Paget disease Pick disease Progressive supranuclear palsy/ Corticobasal degeneration Dementia associated with prion disease Lewy body dementia Mohr-Tranebjaerg syndrome Nasu-Hakola disease REFERENCES 1. World Alzheimer Report 2016; 2. Loy CT, Schofield PR, Turner AM, Kwok JB. Genetics of dementia. Lancet Mar 1;383(9919): doi: /S (13) Farrer LA, Cupples LA, Haines JL, Hyman B, Kukull WA, Mayeux R, Myers RH, Pericak-Vance MA, Risch N, van Duijn CM. Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium. JAMA Oct 22-29;278(16): Rohrer JD, Guerreiro R, Vandrovcova J, Uphill J, Reiman D, Beck J, Isaacs AM, Authier A, Ferrari R, Fox NC, Mackenzie IR, Warren JD, de Silva R, Holton J, Revesz T, Hardy J, Mead S, Rossor MN. The heritability and genetics of frontotemporal lobar degeneration. Neurology Nov 3;73(18): customercare@healthincode.com I I

42 Mitochondrial disorders MITOCHONDRIAL NUCLEAR GENES COMPREHENSIVE PANEL [174 genes] MITOCHONDRIAL GENOME [37 genes] Mitochondrial respiratory chain complex deficiency [45 genes] mtdna depletion [16 genes] Nuclear gene-encoded Leigh syndrome [14 genes] Pyruvate dehydrogenase (PDH) deficiency [12 genes] Primary coenzyme Q deficiency [11 genes]

43 mitochondrial disorders I Mitochondrial disorders are overall the most common group of inherited metabolic diseases, and they are among the most frequent hereditary neurological diseases. They are clinically heterogeneous, can occur at any age, and generally manifest with a wide array of clinical symptoms, usually being defined as multisystem diseases. Some mitochondrial disorders can be grouped into specific syndromes such as Leigh syndrome (subacute necrotizing encephalomyelopathy), MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), or Alpers-Huttenlocher syndrome. Diagnosis is usually based on clinical criteria, as well as on biochemical and histochemical analysis of biopsies. Genetic study contributes to the improved clinical and molecular characterization of these patients and allows providing adequate genetic advice for the individuals and their families. The prevalence of childhood-onset mitochondrial diseases is 5-15/ individuals (Gorman et al., 2014). The most common form of presentation is Leigh syndrome (2.5/ ). The prevalence in adults is estimated in 9.6/ (due to mtdna mutations) and 2.9/ (due to mutations in nuclear genes). The mitochondrial defect can be caused by mutations in nuclear DNA (ndna) or mitochondrial DNA (mtdna) genes. With regard to genetic suspicion, it is worth noting that 80% of mitochondrial diseases in adults are due to pathogenic variants in mtdna; however, these variants only account for 20%-25% of childhood-onset cases (in which nuclear genes have a greater importance) (Gorman et al., 2014). We have developed a selection of specific panels for the study of mitochondrial diseases based on the main biochemical findings, as well as a core panel for the study of nuclear gene-encoded Leigh syndrome, with a comprehensive panel including 174 known genes for mitochondrial diseases. 43 Mitochondrial nuclear genes comprehensive panel [174 genes] AARS2 ABCB7 ACAD9 ACAT1 ACO2 AFG3L2 AGK AIFM1 APOPT1 APTX ATAD3A ATP5E BCS1L BOLA3 C12orf65 C19orf12 CARS2 CLPP COA3 COQ2 COQ4 COQ6 COQ8A* COQ8B* COQ9 COX10 COX14 COX15 COX20 COX6A1 COX6B1 COX8A CPS1 CYC1 CHCHD10 CHKB DARS2 DGUOK DLAT DLD DNA2 DNAJC19 DNM1L EARS2 ECHS1 ELAC2 ETFA ETFB ETFDH ETHE1 FARS2 FASTKD2 FBXL4 FDX2* FLAD1 FOXRED1 GFER GFM1 GLRX5 GTPBP3 HADHA HADHB HARS2 HIBCH HSD17B10 IBA57 ISCA2 ISCU KIF5A LARS2 LIAS LRPPRC LYRM4 LYRM7 MARS2 MFF MGME1 MIPEP MPC1 MPV17 MRPS16 MRPS22 MTFMT MTO1 MTPAP NADK2 NARS2 NDUFA1 NDUFA10 NDUFA11 NDUFA12 NDUFA2 NDUFA8 NDUFA9 NDUFAF1 NDUFAF2 NDUFAF3 NDUFAF4 NDUFAF5 NDUFAF6 NDUFB11 NDUFB3 NDUFB9 NDUFS1 NDUFS2 NDUFS3 NDUFS4 NDUFS6 NDUFS7 NDUFS8 NDUFV1 NDUFV2 NFU1 NUBPL OPA1 OPA3 PDHA1 PDHB PDHX PDP1 PDSS1 PDSS2 PET100 PNPLA8 PNPT1 POLG POLG2 PUS1 RARS2 RMND1 RNASEH1 RRM2B SCO1 SCO2 SDHA SDHAF1 SERAC1 SLC19A3 SLC25A1 SLC25A12 SLC25A19 SLC25A20 SLC25A22 SLC25A26 SLC25A3 SLC25A4 SUCLA2 SUCLG1 SURF1 TACO1 TARS2 TAZ TFAM TIMM8A TK2 TMEM126A TMEM126B TMEM70 TRMT10C TRMT5 TRMU TRNT1 TSFM TTC19 TUFM TWNK* TXN2 TYMP UQCRB UQCRC2 UQCRQ VARS2 YARS2 YME1L1 *COQ8A (ADCK3); COQ8B (ADCK4); FDX2 (FDX1L); TWNK (C10orf2) customercare@healthincode.com I I

44 RELATED PHENOTYPES: ABCB7 ACO2 Ataxia and sideroblastic anemia ADAR, IFIH1, RNASEH2B Glutaric aciduria type 2 AFG3L2 AGK ATAD3A C19orf12, GLRX5 CHKB CLLP, HARS2 COQ8B (ADCK4) ETFDH ETHE1 HADHA, HADHB POLG RARS2 SLC19A3, SLC25A19 SLC25A1 TIMM8A TYMP Optic atrophy and cerebellar-retinal degeneration Spinocerebellar ataxia Sengers syndrome (cardiomyopathy, acidosis, hypotonia) Harel-Yoon syndrome (psychomotor retardation, hypotonia, spasticity, neuropathy) Spastic paraplegia - Neurodegeneration with brain iron accumulation syndromes (NBIAS) Congenital muscular dystrophy with mtdna depletion Perrault syndrome (deafness) Nephrotic syndrome Methylglutaconic aciduria (ataxia, dilated cardiomyopathy) Ethylmalonic encephalopathy Trifunctional protein (TFP) deficiency: cholestasis, myopathy, rhabdomyolysis Alpers-Huttenlocher syndrome (progressive neurodegeneration with liver disease) Pontocerebellar hypoplasia Thiamine-responsive encephalopathy (mitochondrial-like) Glutaric aciduria (corpus callosum agenesis, optic nerve atrophy) Mohr-Tranebjaerg syndrome (deafness-dystonia-optic neuropathy) Mitochondrial neurogastrointestinal encephalopathy (MNGIE) Mitochondrial respiratory chain complex deficiency panel [45 genes] ACAD9 APOPT1 BCS1L COA3 COX10 COX14 COX15 COX20 COX8A CYC1 FASTKD2 FOXRED1 LRPPRC LYRM7 NDUFA12 NDUFA2 NDUFA8 NDUFA9 NDUFAF1 NDUFAF2 NDUFAF3 NDUFAF4 NDUFAF5 NDUFAF6 NDUFB11 NDUFB9 NDUFS1 NDUFS2 NDUFS3 NDUFS4 NDUFS7 NDUFS8 NDUFV1 NFU1 NUBPL PET100 SCO1 SDHA SDHAF1 SURF1 TACO1 TMEM126B TTC19 UQCRC2 UQCRQ mtdna depletion panel [16 genes] AGK DGUOK DNA2 FBXL4 MGME1 MPV17 OPA1 POLG POLG2 RRM2B SLC25A4 SUCLA2 SUCLG1 TFAM TK2 TWNK* *TWNK (C10orf2) Nuclear gene-encoded Leigh syndrome core panel [14 genes] ETHE1 FOXRED1 LRPPRC MTFMT NDUFS4 PDHA1 PDHX PDSS2 PET100 SCO2 SERAC1 SLC19A3 SUCLA2 SURF1

45 mitochondrial disorders I Pyruvate dehydrogenase (PDH) deficiency panel [12 genes] 45 DLAT DLD GLRX5 LIAS NADK2 NFU1 PDHA1 PDHB PDHX PDP1 SLC19A3 SLC25A19 Primary coenzyme Q deficiency panel [11 genes] APTX COQ2 COQ4 COQ6 COQ8A* COQ8B* COQ9 ETFDH FDX2* PDSS1 PDSS2 *COQ8A (ADCK3); COQ8B (ADCK4); FDX2 (FDX1L) Mitochondrial genome [37 genes] Analysis of the 37 mtdna genes and their disease-associated variants. The detection of point mutations and large deletions, as well as the possibility to determine the degree of heteroplasmy in the submitted sample, are included in this study. RELATED PHENOTYPES: Myoclonic epilepsy with ragged-red fibers (MERRF) Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (MELAS) Leber hereditary optic neuropathy (LHON) Neuropathy, ataxia, and retinitis pigmentosa (NARP) Chronic progressive external ophthalmoplegia (CPEO) Kearns-Sayre syndrome Leigh syndrome Pearson syndrome REFERENCES 1. Gorman GS, Chinnery PF, DiMauro S, Hirano M, Koga Y, McFarland R, Suomalainen A, Thorburn DR, Zeviani M, Turnbull DM. Mitochondrial diseases. Nat Rev Dis Primers Oct 20;2: customercare@healthincode.com I I

46 Sample shipment STUDY REQUEST The sample for genetic testing must be sent together with a correctly filled request form (including clinical data and statement on the existence of informed consent). Please download it from or contact us. SAMPLE COLLECTION Peripheral blood* Genomic DNA* Saliva 3 to 5 ml in EDTA tubes Recommended amount: Please use the indicated kit for sample collection. NGS > 5-10 μg (A260/280 = ) Sanger > 1 μg (A260/280 = ) You can request it at customercare@healthincode.com *For delivery in over 48 h, controlled-temperature shipment (4-8 ºC) is recommended SAMPLE PACKAGING The sample collection tube must be placed inside a secondary package of the appropriate size for the number of samples shipped. Sufficient absorbent material should be included to absorb all the contents of the primary recipient in case of leakage. Both containers will preferably be placed inside a rigid box for blood and tissue samples. SAMPLE SHIPMENT Schedule your shipment so that sample reception takes place Monday to Thursday from 8:00 to 17:00. HEALTH IN CODE S. L. Edificio El Fortín, As Xubias s/n A Coruña, Spain If you wish, you can request a pick-up service at customercare@healthincode.com RESULTS We will deliver our report via: Certified Health in Code Client Portal

47 sample shipment I 47 PRE-TEST AND POST-TEST COUNSELLING Our studies include the possibility of pre-test and post-test counselling: Contact customercare@healthincode.com neurohic@healthincode.com Ed. El Fortín As Xubias s/n A Coruña Spain customercare@healthincode.com I I

48 Ed El Fortín s/n A Coruña Spain