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Mitochondrial diseases in North America: An analysis of the NAMDC Registry.

Objective To describe clinical, biochemical, and genetic features of participants with mitochondrial diseases (MtDs) enrolled in the North American Mitochondrial Disease Consortium (NAMDC) Registry. Methods This 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. Results One 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. Conclusions The 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.

Mitochondria are the organelles that generate cellular energy (adenosine triphosphate) via oxidative phosphorylation (OxPhos). OxPhos is an elaborate multiprotein machine composed of a series of enzyme complexes embedded in the inner mitochondrial membrane (complex I-V). 1 Because virtually all tissues require mitochondria to function, mitochondrial dysfunction manifests commonly as multisystem disorders, with frequent involvement of high-energy demand tissues, such as brain, muscle, and heart. 2 The OxPhos system consists of 85 subunits and is under the control of 2 genomes, the nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Mitochondrial diseases (MtDs) can arise due to pathogenic variants in either of the 2 genomes. 3 Because of the genetic complexity and the multiple biochemical functions of these organelles, MtDs are phenotypically and genetically heterogeneous. Because of this vast diversity, these disorders are challenging to diagnose, manage clinically, and investigate scientifically. Epidemiologically, MtDs are considered rare diseases, but among rare disorders, they are relatively frequent, with an overall estimated prevalence of 11.5:100,000. 3 In totality, MtDs represent the most prevalent group of inherited neurologic disorders. 4 Disease registries constitute a cornerstone of MtDs patient care because they help to establish a reliable picture of the distribution and characteristics of the participants, focus resources, and gather accurate data for efficient clinical mechanistic studies and therapeutic trials. 5 This article reviews the spectrum of clinical, biochemical, and molecular genetic features of MtD participants enrolled in the North American Mitochondrial Disease Consortium (NAMDC) Registry (hereafter referred to as the Registry).

Standard protocol approvals, registrations, and patient consents
The NAMDC is an NIH-funded collaborative effort of 17 North American medical centers, which work in close partnership with the United Mitochondrial Disease Foundation and other patient advocacy groups. Since 2011, the NAMDC has maintained a Registry that has collected clinical, biochemical, histologic, and molecular genetic data on participants with MtDs. The Registry protocol was approved by the NIH and the NAMDC central Institutional Review Board (IRB) with local context review at each NAMDC site (NCT01694940). To enroll, participants had to visit an experienced clinician at one of the sites for a review of medical history, physical examination, and laboratory tests and provide written consent using IRB-approved forms. Clinicians input data using a secure web-based data entry system. Data were collected through December 1, 2018.

Data availability
The NAMDC Clinical Registry data are stored in a secure database and are available to other investigators on submission of a data use application and approval by the NAMDC Data Use Committee.

Clinical diagnosis
Participants are enrolled in the Registry based on the NAMDC site investigators' clinical diagnoses of MtDs. Diagnoses are based on established and published clinical criteria. 6,7 Twenty-five different clinical diagnoses were coded: 15 classical mitochondrial clinical syndromes and 8 with an association of symptoms and signs commonly observed in mitochondrial participants (table 1). [8][9][10][11][12] Some of the syndromes require an association of clinical and radiologic findings, e.g., for the diagnosis of leukoencephalopathy, white matter lesions evident on brain MRI are typically associated with cognitive impairment, long-tract signs, or both. Encephalopathy was defined by the presence of dementia, seizures, corticospinal tract dysfunction, movement disorders, or combinations of these manifestations due to cerebral pathology.
Two nonspecific groups of participants with complex clinical manifestations that do not fall in any of the other categories are (1) multisystemic and (2) other clinical disorders. For multisystemic disease, clinical manifestations must involve at least 3 organs with a progressive clinical course that does not otherwise conform to a classical phenotype. Participants without clinical manifestations were classified as asymptomatic carriers, which are not included in this analysis.

Statistical analysis
The Fisher exact test (SAS 9.4: SAS Institute Inc., Cary, NC) was used in the comparisons of participants with different genetic features, and Holm adjustment (R 3.5.1: Bell Laboratories, New Providence, NJ) for multiple comparisons.

Results
Of the 1,533 total participants enrolled in the Registry (January 2011-December 2018), 1,410 had complete data on clinical manifestations, biochemistry, and genetics. Genetic diagnosis was available for 722 participants. After the exclusion of 56 asymptomatic carriers, 666 genetically diagnosed symptomatic individuals remained for analysis (table 1).  (table 2). Apart from key clinical findings in CNS and skeletal muscle involvement, heart abnormalities were recorded (21 participants), with cardiac arrhythmias being the most prevalent (table 2, table e (table 2, table e-2). In addition to stroke-like episodes (SLEs), the most common CNS manifestations were seizures, migraine, and ataxia (tables e-1 and e-2). Thirty-nine participants had constitutional symptoms (short stature and low body mass index) (table 2). Genetically, m.3243A>G in the mitochondrial MTTL1 gene was the most frequently observed pathogenic variant. In addition, 10 of 69 participants (14.5%) with POLG pathogenic variants manifested SLEs, but only 1 was diagnosed with MELAS.

Chronic progressive external ophthalmoplegia
Fifty-five participants were diagnosed with cPEO/cPEO+ (18 cPEO and 37 cPEO+). The majority were adults (table 2). Muscle involvement in cPEO was not confined to the extrinsic ocular muscles, as skeletal myopathy (10 participants) and dysphagia (4 participants) were also observed. Participants with cPEO+ frequently had CNS symptoms (24 participants), with ataxia and hearing loss being the most prevalent (    Age at onset <2 y 6 1 - Other clinical diagnosis --  and ophthalmoparesis (38 participants) were frequently reported (table e-2). Hearing loss was less common (18 individuals).

Participants with POLG1 pathogenic variants
Seventy participants (25 males and 45 females) had pathogenic variants in POLG1 (figure 2). Onset of symptoms occurred from infancy through late adulthood (range <1-53 years). Twenty-four pathogenic variants were observed, distributed along the entire gene and with a high frequency in the linker region. Clinical presentation encompassed 11 different syndromes. Sensory ataxic neuropathy with dysarthria and    (2), and dilated cardiomyopathy (1); hence, those patients were given non-autosomal dominant optic atrophy diagnoses (multisystemic disease, cPEO plus, and encephalomyopathy).

Discussion
Clinical registries are essential for collecting data on rare diseases; those data can be critical for guiding their diagnoses and care. This study, using data from the NAMDC Registry, is the first to depict the spectrum of mitochondrial disorders in North America.
Although participants in the Registry can be enrolled based on clinical manifestations (a genetic diagnosis is not required), this analysis considers only participants with a definite genetic diagnosis. Nonetheless, it is worth highlighting the complexity of the diagnostic pathway in these patients. Indeed, even in the era of next-generation sequencing, 13 and similarly to other large cohort studies, 14 Because MtDs are typically due to defects in energy metabolism, the Registry includes data on biochemical defects. Enzyme activity assay helps to direct molecular analyses and to identify participants eligible for future therapies targeting specific enzymatic defects. 18,19 Among the many such defects, OxPhos deficiency has historically been considered the hallmark of MtDs. 7 In the Registry, the majority of the participants had an OxPhos defect, followed by PDC deficiency. Participants with OxPhos defects most frequently presented with multisystemic diseases. This may reflect the complex genetic control of the respiratory chain machinery. 20,21 Remarkably few participants had complex V defects, possibly due to low frequency, detection difficulty, or both.
To portray the features of classic mitochondrial syndromes is extremely important as new therapeutic strategies approach the patient bedside.
Participants with MELAS were prevalent in our cohort. Although previous studies have suggested that male sex may represent a risk factor for development of a full-blown MELAS syndrome, we did not observe a difference by sex in our population. Moreover, the ratio of males was similar among participants with the m.3243A>G variant (35%) and those with a diagnosis of MELAS (40%) (table e-2, links.lww. com/NXG/A226). In contrast to data obtained in other studies, 22 MELAS was the most common diagnosis in participants carrying the m.3243A>G variant. The high proportion of participants with MELAS in the NAMDC may be due to ascertainment bias because some of the NAMDC sites have research projects on this disorder. 23 Our data also confirm the relatively low prevalence of complete MERRF syndrome. Previous reports from the German and Italian registries showed that only half of the participants with the m.8344A>G pathogenic variant have classic MERRF syndrome. 24 The same proportion is observed in our cohort. Of interest, the age at onset in our MERRF cohort is younger than reported elsewhere, with an age at onset before 12 years of age in 6 of 9 participants with available data. We identified hearing loss in 56.5% of our participants with the m.8344A>G variant and 46.2% with MERRF (table e-2, links.lww.com/ NXG/A226). This is in line with some other studies but lower than the German registry data. 25,26 cPEO/cPEO+ diagnoses are common but less frequent than in previous reports. 15 This discrepancy may have arisen because the NAMDC Registry includes the multisystemic syndrome category into which multiple complex cPEO+ participants were classified.
The Registry data highlight the vast genetic heterogeneity of the patients with MtDs. We observed a higher frequency of mtDNA pathogenic variants over nDNA (table 4). The combination of pediatric and adult clinics in the NAMDC facilitates a comprehensive analysis across the MtD population. This may help explain why the proportions of mtDNA and nDNA variants in the Registry differ from those in other recent reports. 27 Other factors such as accessibility and feasibility of mtDNA testing may also have an impact. In the Registry, participants with nDNA pathogenic variants had earlier disease onset than participants with mtDNA pathogenic variants, with onset before age 2 years in 50.4% individuals (table 4). We observed no differences in ptosis and ophthalmoparesis between the 2 groups, in contrast to what was observed in the Italian registry. 15 High frequencies of SLE, diabetes mellitus, and deafness in mtDNA participants were present, probably due to common mtDNA pathogenic variant-associated phenotypes. The higher frequency of developmental abnormalities in the nDNA subgroup confirms previous data 27 and is concordant with the younger age at onset.
The identification in our cohort of several genes mutated only in single individuals corroborates the ultra-rare nature of many MtDs genotypes.
This study provides the first wide-ranging look at the MtD population in North America. The data also identify areas of the Registry that require further improvement. Given the estimated prevalence of MtDs, 28 it is likely that only a small proportion of North American participants has been captured. To address this limitation, the NAMDC has developed a remote enrollment system to reach patients living in areas distant from a NAMDC center. In addition, the NAMDC has created disease-specific natural history subregistries and other studies that will improve the acquisition of longitudinal data and help to identify outcome measures for future clinical trials.