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OBJECTIVE: To harmonize terminology in mitochondrial medicine, we propose revised clinical criteria for primary mitochondrial syndromes. METHODS: The North American Mitochondrial Disease Consortium (NAMDC) established a Diagnostic Criteria Committee comprised of members with diverse expertise. It included clinicians, researchers, diagnostic laboratory directors, statisticians, and data managers. The Committee conducted a comprehensive literature review, an evaluation of current clinical practices and diagnostic modalities, surveys, and teleconferences to reach consensus on syndrome definitions for mitochondrial diseases. The criteria were refined after manual application to patients enrolled in the NAMDC Registry. RESULTS: By building upon published diagnostic criteria and integrating recent advances, NAMDC has generated updated consensus criteria for the clinical definition of classical mitochondrial syndromes. CONCLUSIONS: Mitochondrial diseases are clinically, biochemically, and genetically heterogeneous and therefore challenging to classify and diagnose. To harmonize terminology, we propose revised criteria for the clinical definition of mitochondrial disorders. These criteria are expected to standardize the diagnosis and categorization of mitochondrial diseases, which will facilitate future natural history studies and clinical trials.

At present, there is just one approved therapy for patients with mitochondrial diseases in Europe, another in Japan, and none in the United States. These facts reveal an important and significant unmet need for approved therapies for these debilitating and often fatal disorders. To fill this need, it is critical for clinicians and drug developers to work closely with regulatory agencies. In the United States, mitochondrial disease patients and clinicians, the United Mitochondrial Disease Foundation, and pharmaceutical industry members have engaged with the Food and Drug Administration to educate each other about these complex and heterogeneous diseases and about regulatory requirements to obtain approvals for novel therapies. Clinical development of therapies for rare diseases has been facilitated by the 1983 US Orphan Drug Act (ODA) and similar legislation in Japan and the European Union. Further legislation and regulatory guidance have expanded and refined regulatory flexibility. While regulatory and financial incentives of the ODA have augmented involvement of pharmaceutical companies, clinicians, with patient advocacy groups and industry, need to conduct natural history studies, develop clinical outcome measures, and identify potential supportive surrogate endpoints predictive of clinical benefit, which together are critical foundations for clinical trials. Thus, the regulatory environment for novel therapeutic development is conducive and offers flexibility for mitochondrial diseases. Nevertheless, flexibility does not mean lower standards, as well-controlled rigorous clinical trials of high quality are still required to establish the efficacy of potential therapies and to obtain regulatory agency approvals for their commercial use. This process is illustrated through the authors' ongoing efforts to develop therapy for thymidine kinase 2 deficiency.

Aim The North American Mitochondrial Disease Consortium (NAMDC) comprises a network of 17 clinical centers with a mission to conduct translational research on mitochondrial diseases. NAMDC is a part of the Rare Disease Clinical Research Network (RDCRN) and is funded by the National Institutes of Health. To foster its mission, NAMDC has implemented a comprehensive Mitochondrial Disease Clinical Registry (hereafter NAMDC Registry), collected biosamples deposited into the NAMDC Biorepository, defined phenotypes and genotypes of specific disorders, collected natural history data, identified outcome measures, characterized safety and long-term toxicity and efficacy of promising therapies, and trained young investigators interested in patient-oriented research in mitochondrial disease. Methods Research conducted by NAMDC is built on the foundation of the Clinical Registry. Data within the registry are encrypted and maintained in a centralized database at Columbia University Medical Center. In addition to clinical data, NAMDC has established a mitochondrial disease biorepository, collecting DNA, plasma, cell, and tissue samples. Specimens are assigned coded identifiers in compliance with all relevant regulatory entities and with emerging NIH guidelines for biorepositories. NAMDC funds two pilot projects each year. Pilot grants are small grants typically supporting an early stage concept to obtain preliminary data. Pilot grants must enhance and address major issues in mitochondrial medicine and specific areas of need for the field and for the successful outcome of NAMDC. The grant selection process is facilitated by input from multiple stakeholders including patient organizations and the strategic leadership of NAMDC. To train new mitochondrial disease investigators, NAMDC has established a Fellowship Program which offers a unique training opportunity to senior postdoctoral clinical fellows. The fellowship includes a 6-month period of intensive training in clinical trial methodology through the Clinical Research Enhancement through Supplemental Training program and equivalent programs at the other sites, along with rotations up to 3 months each to two additional consortium sites where a rich and varied training experience is provided. Nine core educational sites participate in this training program, each offering a summer grant program in mitochondrial medicine funded by our NAMDC partner the United Mitochondrial Disease Foundation (www.umdf.org). All clinical research in NAMDC depends on the participation of mitochondrial disease patients. Since individual mitochondrial disorders are often extremely rare, major communication and recruitment efforts are required. Therefore, NAMDC has forged a very close partnership with the premier patient advocacy group for mitochondrial diseases in North America, the United Mitochondrial Disease Foundation (UMDF). Results The NAMDC Registry has confirmed the clinical and genetical heterogeneity of mitochondrial diseases due to primary mutations in mitochondrial DNA or nuclear DNA. During the 8 years of this NIH-U54 grant, this consortium, acting in close collaboration with a patient advocacy group, the UMDF, has effectively addressed these complex diseases. NAMDC has expanded a powerful patient registry with more than 1600 patients enrolled to date, a website for education and recruitment of patients (www.namdc.org), a NAMDC biorepository housed at the Mayo Clinic in Rochester, MN, and essential diagnostic guidelines for consensus research. In addition, eight clinical studies have been initiated and the NAMDC fellowship program has been actively training the next generation of mitochondrial disease clinical investigators, of which six have completed the program and remain actively involved in mitochondrial disease research. Conclusion The NAMDC Patient Registry and Biorepository is actively facilitating mitochondrial disease research, and accelerating progress in the understanding and treatment of mitochondrial diseases.

ObjectiveTo describe clinical, biochemical, and genetic features of participants with mitochondrial diseases (MtDs) enrolled in the North American Mitochondrial Disease Consortium (NAMDC) Registry.MethodsThis cross-sectional, multicenter, retrospective database analysis evaluates the phenotypic and molecular characteristics of participants enrolled in the NAMDC Registry from September 2011 to December 2018. The NAMDC is a network of 17 centers with expertise in MtDs and includes both adult and pediatric specialists.ResultsOne thousand four hundred ten of 1,553 participants had sufficient clinical data for analysis. For this study, we included only participants with molecular genetic diagnoses (n = 666). Age at onset ranged from infancy to adulthood. The most common diagnosis was multisystemic disorder (113 participants), and only a minority of participants were diagnosed with a classical mitochondrial syndrome. The most frequent classical syndromes were Leigh syndrome (97 individuals) and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (71 individuals). Pathogenic variants in the mitochondrial DNA were more frequently observed (414 participants) than pathogenic nuclear gene variants (252 participants). Pathogenic variants in 65 nuclear genes were identified, with POLG1 and PDHA1 being the most commonly affected. Pathogenic variants in 38 genes were reported only in single participants.ConclusionsThe NAMDC Registry data confirm the high variability of clinical, biochemical, and genetic features of participants with MtDs. This study serves as an important resource for future enhancement of MtD research and clinical care by providing the first comprehensive description of participant with MtD in North America.

Duchenne muscular dystrophy (DMD) is a progressive, lethal neuromuscular disorder caused by the absence of dystrophin protein due to mutations of the dystrophin gene. Drisapersen is a 2′-O-methyl-phosphorothioate oligonucleotide designed to skip exon 51 in dystrophin pre-mRNA to restore the reading frame of the mRNA. This study assessed safety, tolerability, and pharmacokinetics of drisapersen after a single subcutaneous administration in non-ambulatory subjects. Eligible subjects were non-ambulant boys aged ≥9 years, in wheelchairs for ≥1 to ≤4 years, with a diagnosis of DMD resulting from a mutation correctable by drisapersen treatment. Four dose cohorts were planned (3, 6, 9 and 12 mg/kg), but study objectives were met with the 9 mg/kg dose. Less than proportional increase in exposure was demonstrated over the 3–9 mg/kg dose range, though post hoc analysis showed dose proportionality was more feasible over the 3–6 mg/kg range. Single doses of drisapersen at 3 and 6 mg/kg did not result in significant safety or tolerability concerns; however, at the 9 mg/kg dose, pyrexia and transient elevations in inflammatory parameters were seen. The maximum tolerated dose of 6 mg/kg drisapersen was identified for further characterization in multiple dose studies in the non-ambulant DMD population.

Introduction Recent in vitro studies suggest that CAPN3 deficiency leads initially to accelerated myofiber formation followed by depletion of satellite cells (SC). In normal muscle, upregulation of miR-1 and miR-206 facilitates transition from proliferating SCs to differentiating myogenic progenitors.

Sporadic inclusion-body myositis (sIBM) is the most common idiopathic inflammatory myopathy affecting those >50 years of age.1,2 Typically, onset is characterized by insidious and frequently asymmetrical weakness of the arms and legs, which is often erroneously attributed to aging and can therefore remain undiagnosed for years. Progressive weakness of the quadriceps muscles in sIBM is an early finding, followed by a decline in functional abilities, progressive loss of ambulation, and wheelchair dependence. The typical course of decline is 10–15 years after onset of the disease.3,4 Currently, no treatments have been found to slow or reverse the progression of muscle weakness and deterioration in sIBM.5–16 Recent opportunities for treatment of sIBM have emerged using strategies to inhibit the myostatin pathway to increase quadriceps muscle strength. Contrasting approaches include an inhibitory myostatin antibody that binds to the activin IIB receptor (BYM338; Novartis Pharmaceuticals; ClinicalTrials.gov) or an alternatively spliced product of the follistatin gene delivered by adeno-associated virus (AAV) directly into muscle.17,18 The quadriceps muscles are targets for these clinical trials because of their preferential weakness in sIBM, their suspected contribution to gait impairment, and their accessibility as a treatment target. In the BYM338 trial, the size of the quadriceps will be measured at 8 weeks and, in the follistatin gene therapy trial, quadriceps muscle strength is the intended primary outcome variable. Despite these laudatory goals, an important unaddressed issue is the absence of any previous study demonstrating a correlation between quadriceps muscle strength and function. In this study our aim was to directly assess knee extensor strength using the highest standards of measurement recognized by academic and regulatory authorities,19 including the 2- and 6-minute walk tests (2MWT and 6MWT)20; the IBM Functional Rating Scale (IBMFRS)21; and timed tests22–24 of stair climbing, standing from a chair, and stepping up on curbs.

This study tested the hypothesis that gamma-glutamyl transferase (GGT) can be used as a reliable biomarker to distinguish skeletal muscle from liver damage. Twenty-eight Duchenne muscular dystrophy subjects with proven dystrophin gene mutations were enrolled. Included were 14 ambulatory and 14 nonambulatory patients with approximately half of each cohort taking corticosteroids. Twenty normal males served as controls. Initial blood samples for serum GGT and creatine kinase were taken between 8AM and 9AM and redrawn 8 hours later to test for variability. Between blood draws, subjects resumed normal activities in a play environment or could leave the clinic. Not a single duchenne muscular dystrophy patient showed a GGT outside the control range at any time point, while creatine kinase levels were 14 to 200 times normal. Validation of this finding is essential for management of patients with muscle disorders exposed to potentially hepatotoxic drugs for clinical management or monitoring subjects participating in clinical trials.

Becker muscular dystrophy (BMD) is a variant of dystrophin deficiency resulting from DMD gene mutations. Phenotype is variable with loss of ambulation in late teenage or late mid-life years. There is currently no treatment for this condition. In this BMD proof-of-principle clinical trial, a potent myostatin antagonist, follistatin (FS), was used to inhibit the myostatin pathway. Extensive preclinical studies, using adeno-associated virus (AAV) to deliver follistatin, demonstrated an increase in strength. For this trial, we used the alternatively spliced FS344 to avoid potential binding to off target sites. AAV1.CMV.FS344 was delivered to six BMD patients by direct bilateral intramuscular quadriceps injections. Cohort 1 included three subjects receiving 3 × 10(11) vg/kg/leg. The distance walked on the 6MWT was the primary outcome measure. Patients 01 and 02 improved 58 meters (m) and 125 m, respectively. Patient 03 showed no change. In Cohort 2, Patients 05 and 06 received 6 × 10(11) vg/kg/leg with improved 6MWT by 108 m and 29 m, whereas, Patient 04 showed no improvement. No adverse effects were encountered. Histological changes corroborated benefit showing reduced endomysial fibrosis, reduced central nucleation, more normal fiber size distribution with muscle hypertrophy, especially at high dose. The results are encouraging for treatment of dystrophin-deficient muscle diseases.

Limb-girdle muscular dystrophy type 2C (LGMD2C) is considered one of the severe forms of childhood-onset muscular dystrophy. The geographical distribution of founder mutations in the SGCG gene has a prominent effect on the prevalence of LGMD2C in certain populations. The aim of this study was to confirm the hypothesis that the c.787G>A (p.E263K) mutation in the SGCG gene is a founder mutation among Puerto Rican Hispanics and to characterize the associated clinical and immunohistochemical phenotype. Genotyping of six polymorphic microsatellite markers internal to (D13S232) and flanking (D13S175, D13S292, D13S787, D13S1243, D13S283) the SGCG gene was performed on four unrelated Puerto Rican patients with LGMD2C. Preserved ambulation to the second decade of life was observed in at least two subjects. Immunostaining of skeletal muscle demonstrated absence of γ-sarcoglycan in all affected subjects. Two markers, D13S232 and D13S292, were highly informative and confirmed that all four families share the haplotype of the mutant allele. Our findings confirm that the E263K missense mutation in the SGCG gene is a founder mutation in Puerto Rican Hispanics. A slowly progressive disease course with prolonged preservation of ambulation can be seen in association with this mutation, providing evidence for phenotypic variability.

Recent in vitro studies suggest that CAPN3 deficiency leads initially to accelerated myofiber formation followed by depletion of satellite cells (SC). In normal muscle, up-regulation of miR-1 and miR-206 facilitates transition from proliferating SCs to differentiating myogenic progenitors.We examined the histopathological stages, Pax7 SC content, and muscle-specific microRNA expression in biopsy specimens from well-characterized LGMD 2A patients to gain insight into disease pathogenesis.Three distinct stages of pathological changes were identified that represented the continuum of the dystrophic process from prominent inflammation with necrosis and regeneration to prominent fibrosis, which correlated with age and disease duration. Pax7-positive SCs were highest in the fibrotic group and correlated with down-regulation of miR-1, miR-133a, and miR-206.These observations, and other published reports, are consistent with microRNA dysregulation leading to inability of Pax7-positive SCs to transit from proliferation to differentiation. This results in impaired regeneration and fibrosis.

Background Limb girdle muscular dystrophies (LGMD) are inclusive of 7 autosomal dominant and 14 autosomal recessive disorders featuring progressive muscle weakness and atrophy. Studies of cardiac function have not yet been well-defined in deficiencies of dysferlin (LGMD2B) and fukutin related protein (LGMD2I). In this study of patients with these two forms of limb girdle muscular dystrophy, cardiovascular magnetic resonance (CMR) was used to more specifically define markers of cardiomyopathy including systolic dysfunction, myocardial fibrosis, and diastolic dysfunction. Methods Consecutive patients with genetically-proven LGMD types 2I (n = 7) and 2B (n = 9) and 8 control subjects were enrolled. All subjects underwent cardiac magnetic resonance (CMR) on a standard 1.5 Tesla clinical scanner with cine imaging for left ventricular (LV) volume and ejection fraction (EF) measurement, vector velocity analysis of cine data to calculate myocardial strain, and late post-gadolinium enhancement imaging (LGE) to assess for myocardial fibrosis. Results Sixteen LGMD patients (7 LGMD2I, 9 LGMD2B), and 8 control subjects completed CMR. All but one patient had normal LV size and systolic function; one (type 2I) had severe dilated cardiomyopathy. Of 15 LGMD patients with normal systolic function, LGE imaging revealed focal myocardial fibrosis in 7 (47%). Peak systolic circumferential strain rates were similar in patients vs. controls: εendo was -23.8 ± 8.5vs. -23.9 ± 4.2%, εepi was -11.5 ± 1.7% vs. -10.1 ± 4.2% (p = NS for all). Five of 7 LGE-positive patients had grade I diastolic dysfunction [2I (n = 2), 2B (n = 3)]. that was not present in any LGE-negative patients or controls. Conclusions LGMD2I and LGMD2B generally result in mild structural and functional cardiac abnormalities, though severe dilated cardiomyopathy may occur. Long-term studies are warranted to evaluate the prognostic significance of subclinical fibrosis detected by CMR in these patients.

Limb-girdle muscular dystrophies comprise a rare heterogeneous group of genetic muscular dystrophies, involving 15 autosomal recessive subtypes and seven autosomal dominant subtypes. Autosomal recessive dystrophy is far more common than autosomal dominant dystrophy. Typical clinical features include progressive limb muscle weakness and atrophy (proximal greater than distal), varying from very mild to severe. Significant overlap of clinical phenotypes, with genetic and clinical heterogeneity, constitutes the rule for this group of diseases. Muscle biopsies are useful for histopathologic and immunolabeling studies, and DNA analysis is the gold standard to establish the specific form of muscular dystrophy. A definitive diagnosis among various subtypes is challenging, and the data presented here provide neuromuscular clinicians with additional information to help attain that goal.

Reports of dysferlinopathy have suggested a clinically heterogeneous group of patients. We identified specific novel molecular and phenotypic features that help distinguish dysferlinopathies from other forms of limb-girdle muscular dystrophy (LGMD). A detailed history, physical exam, and protein and mutation analysis of genomic DNA was done for all subjects. Five of 21 confirmed DYSF gene mutations were not previously reported. A distinct "bulge" of the deltoid muscle in combination with other findings was a striking feature in all patients. Six subjects had atypical calf enlargement, and 3 of these exhibited a paradoxical pattern of dysferlin expression: severely reduced by direct immunofluorescence with overexpression on Western blots. Six patients showed amyloid deposits in muscle that extended these findings to new domains of the dysferlin gene, including the C2G domain. Correlative studies showed colocalization of amyloid with deposition of dysferlin. The present data further serve to guide clinicians facing the expensive task of molecular characterization of patients with an LGMD phenotype.

Becker muscular dystrophy (BMD), a dystrophinopathy with milder skeletal muscle manifestations compared to Duchenne muscular dystrophy, characteristically demonstrates weakness of knee extensors (KE), accompanied by muscle atrophy and fibrosis. The weakness predisposes to gait impairment and loss of ambulation. Myostatin, a member of the TGF-β superfamily of signal peptides, is expressed in skeletal muscle, acting through the activin type IIB (ACTRIIB) receptor. Follistatin, a myostatin antagonist, inhibits this pathway resulting in muscle hypertrophy, and improved muscle strength. FS315 and FS288 are two isoforms of follistatin generated by alternative splicing. FS315 has no cell-surface affinity providing a safety margin avoiding pituitary–gonadal axis complications. FS344 is the precursor cDNA to FS315. When FS344 is delivered to mdx mice in adeno-associated virus (AAV1. CMV. FS344), muscle mass and strength increase. Similar findings are seen in non-human primates without safety concerns. Six BMD patients, 18 years and older with proven in-frame DMD gene mutations were enrolled in a clinical trial. The first 3 subjects received 12 intramuscular (IM) injections of AAV1. CMV. FS344 at a total dose of 6e11 vg/kg in the quadriceps muscles bilaterally. A second cohort received the same number of IM injections at higher total dose (1.2e12 vg/kg). Studies extending beyond one year in Cohort 1 and for six months in Cohort 2 showed no adverse events. The distance walked (meters) on the 6-min walk test (6MWT) was the primary functional outcome measure. At one year the distance walked by two subjects in Cohort 1 increased by 125 m and 58 m; subject 3 improved modestly by 9 m. In Cohort 2, one patient reaching the 6-month time point increased by 108 meters. For the entire group of BMD patients only a single patient declined in distance. Preliminary analysis of muscle biopsies favor increased muscle regeneration contributing to improved function.

Next generation sequencing technologies present opportunities for improved clinical molecular diagnosis of neuromuscular disorders (NMD). We sought to determine the feasibility of using whole exome sequencing to detect mutations in 347 isoforms of 143 NMD genes listed in the World Muscle Society Muscle Gene Table ( www.musclegenetable.org ). We initially tested DNA samples from eight subjects with limb-girdle muscular dystrophies (LGMD) and known mutations, using exome capture with one of three commercially available kits. We detected the same mutations as gene-specific Sanger sequencing had previously detected in seven of eight, missing 1 MYOT mutation due to a lack of representation of this specific exon in the capture kit. This led us to compare commercially available exome capture kits for coverage of NMD genes, by both predicted and by empiric coverage, in normal control genomic DNA. Based on manufacturer targeted region files, the three kits each targeted 92–94% of the known NMD exons; however, our actual capture results demonstrated that at best ∼60% of these exons obtained 100% coverage. In our hands the best performing kit, Agilent SureSelect v3, captures an average of 92.7% of the bases in the NMD gene exons, but only 58% of the NMD isoforms had all exons captured at 100% (e.g., 42% of the NMD genes had at least one exon with one base insufficiently captured to make a genotyping call). Illumina TruSeq captured 89.2% of exons but only 36.5% of genes had 100% coverage, and Nimblegen v2 captured 90.5% of genes but only 46.3% genes had 100% sequence coverage. We predict that by utilizing all three kits, ∼70% of the known NMD isoforms would have sufficient sequence coverage to make a genotyping call for 100% of all exons. When considering adoption of this approach clinically, this is an important limitation to be aware of but continued improvements in commercially available capture kits should allow economical and rapid NMD gene sequence analysis.

Objective: Establish safety of rAAV.CMV.FS gene transfer by intramuscular (i.m.) injection for improving quadriceps strength in preparation for phase I clinical trial. Background FS is a potent myostatin inhibitor that increases muscle size and strength in mice and non-human primates when secreted by muscle following delivery by AAV. Safety concerns have included impeded release of pituitary-gonadal axis hormones and general organ toxicity. In this study we used a non-tissue binding FS isoform (FS344) to address these safety concerns in mice at viral doses 10-fold greater than those planned for clinical use. Design/Methods: Mice were randomized to three experimental arms with bilateral i.m. injections to quadriceps muscle of rAAV1.CMV.FS344 equal to highest clinical dose (2.0e12 vg/kg) or 10-fold higher (2.0e13 vg/kg). Animals were followed to sacrifice time points at 6-,12-,24-, and 36-weeks (5 animals/sex/cohort/timepoint) post-injection (p.i.). Outcomes included: bodyweight, hematology, chemistry panels, pituitary-gonadal hormones; immune studies, and histopathology of 24 organs from each animal. Results: No treatment-related adverse clinical or toxicology signs were observed and viral DNA in non-injected tissues dissipated by 24 weeks. Within 6 weeks, high dose animals showed scattered focal infiltrates of inflammatory cells corresponding to T-cell immunity to AAV1 identified by IFN-γ ELISpot, but no other pathology was observed. Wet weights of quadriceps increased by 3.5-fold at 36 weeks p.i. in high dose animals. Muscle fiber hypertrophy was observed at 6 weeks and fiber diameter increased by 50% compared to controls by 36 weeks. Hypertrophic-multinucleated fibers were observed at 12 weeks p.i. with increased density of PAX7 positive nuclei. Conclusions: This study shows rAAV1.CMV.FS344 is safe and well tolerated at the proposed clinical doses and causes satellite cell proliferation and significant muscle hypertrophy by fusion with differentiated satellite cells. Early transient inflammation in muscle is related to immune response to AAV1. These results support this approach for clinical trial use. Supported by: Grants from The Myositis Association and Parent Project Muscular Dystrophy, Inc; and support from the Foundation at Nationwide Children9s Hospital. Disclosure: Dr. Kota has nothing to disclose. Dr. Shilling has nothing to disclose. Dr. Montgomery has nothing to disclose. Dr. Lewis has nothing to disclose. Dr. Bevan has nothing to disclose. Dr. Shontz has nothing to disclose. Dr. Kaminoh has nothing to disclose. Dr. Rosales has nothing to disclose. Dr. Viollet has nothing to disclose. Dr. Flanigan has received personal compensaiton for activities with AVI Therapeutics, Prosensa Therapeutics, and PTC, Inc. Dr. Flanigan has received research support from PTC Therapeutics. Dr. Clark has nothing to disclose. Dr. Kaspar has nothing to disclose. Dr. Sahenk has nothing to disclose. Dr. Mendell has received research support from AVI BioPharmaceuticals Inc.