Abstract
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.
Keywords: Mitochondrial disease, Mitochondrial disorders, Oxidative-phosphorylation, Mitochondrial DNA
1. Introduction
Primary mitochondrial disorders are a group of clinically and genetically diverse conditions caused by defects in the metabolic pathways in mitochondria [1]. The pathogenesis of primary mitochondrial disorders is due to mutations in more than 350 genes, both nuclear and mitochondrial. It is often difficult - even for expert clinicians - to diagnose mitochondrial diseases (MtDs) because of their rarity; clinical, biochemical, and genetic heterogeneity; their phenotypic overlap with other disorders; and their lack of specific biomarkers[2]. As a consequence, patients are frequently misdiagnosed and consult, on average, more than eight physicians during their “diagnostic odyssey” [3].
Although individual MtDs are very rare, in toto, they are relatively frequent: the estimated prevalence of childhood-onset MtDs ranges from 5 to 15 cases per 100,000 individuals, and the prevalence of adult-onset forms has been estimated to be 9.6 cases per 100,000 individuals for mitochondrial DNA (mtDNA) related disorders and 2.9 cases per 100,000 individuals for nuclear DNA (nDNA) related disorders [4,5]. These disorders are often progressive and cause high morbidity and mortality among both pediatric and adult populations [6].
Specific clinical features can be suggestive of MtDs, especially when grouped in particular combinations that define recurrent and recognizable syndromes, which we refer to as classical mitochondrial syndromes (e.g., Leigh syndrome, Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS), or Myoclonic Epilepsy with Ragged-Red Fibers (MERRF). However, many mitochondrial disorders manifest phenotypes that do not conform to a classical mitochondrial syndrome. In addition to clinical characterization, definitive diagnosis is often achieved through histological, biochemical, and genetic testing.
To provide a tool to integrate clinical, biochemical, laboratory, and genetic findings, diagnostic criteria for adult with suspected mitochondrial disorders were published in 1996 (Adult Criteria, AC) [7], and modified for a pediatric population in 2002 (Modified Adult Criteria, MAC) [8]. A second scoring system for pediatric patients was presented in 2002 (Mitochondrial Disease Criteria, MDC), [9] and subsequently validated in 2006 [10]. These criteria have been demonstrated to retain utility in guiding the diagnosis of MtD [11].
In the 16 years since the last publication on MtD diagnostic criteria, there has been an outburst of new phenotypic and genotypic information about these disorders. New technologies in genetic and functional testing further require integration into the diagnostic criteria. These new data have facilitated the diagnosis and classification of these complex diseases in many patients. Investigators in the North American Mitochondrial Disease Consortium (NAMDC) recognized a need to revise the diagnostic criteria for mitochondrial disorders to incorporate this new information and to attain greater consistency and rigor in diagnostic criteria and terminology.
NAMDC assembled a committee of experts in the diagnosis of mitochondrial disorders to update the diagnostic criteria and achieve standardized definitions of clinical manifestations, laboratory abnormalities, and diagnoses. The primary goals were to standardize the clinical classification of MtDs. The development of research diagnostic criteria is not merely an academic exercise, but rather a critical step towards improving reproducibility in clinical research.
2. Materials and methods
The NAMDC Diagnostic Criteria Committee was composed of 11 members with diverse biomedical expertise including: pediatric and adult neurologists, clinical geneticists, clinical biochemical geneticists, and academic diagnostic laboratory directors. They reviewed relevant literature and sought to develop consensus recommendations. Their first iteration of the diagnostic criteria was completed in March 2012. This work built upon the original set of diagnostic criteria for adults (AC) published in 1996 by Walker et al. [7] and subsequent modifications by Bernier et al. [8] (MAC) as well as Wolf et al. (MDC) in 2002 [9] to include the pediatric population. Specifically, we have incorporated advances in diagnostic modalities to bring the criteria up-to-date with current knowledge. After the first draft was completed, the revised criteria were applied to MtD subjects enrolled with complete data in the NAMDC Registry. Since 2011, NAMDC has maintained this Registry, which has extensive clinical, biochemical, histological, and molecular genetic data on over 1300 patients with MtDs [12]. De-identified data extracted from the eCRFs of the 50 random patients were evaluated. Results of this analysis were then used to improve the criteria. Additional revisions to the criteria were discussed via email and teleconferences among the members of the Committee. The criteria were then applied to all subjects enrolled in the NAMDC Registry. The results from this process and subsequent modifications were used by the Committee to generate a second iteration of the Criteria. These were unanimously approved by the 11 members of the working group and the NAMDC site investigators, and finalized in Alexandria, VA, on June 25, 2019 during the NAMDC face-to-face meeting at the United Mitochondrial Disease Foundation (UMDF) 2019 Symposium.
3. Results
3.1. Important advances in the clinical classification of mitochondrial diseases
3.1.1. Recognition of multisystem disease
One of the major challenges for the diagnosis of MtDs is the clinical heterogeneity of these disorders. Most of the patients enrolled in the NAMDC Registry have multi-systemic disorders (≥3 affected organs), characteristically affecting tissues with high-energy demands, such as central nervous system, skeletal muscle, eye, and heart [13]. Their phenotypes often do not conform to widely recognized “classical” mitochondrial syndromes (20.1%) [12]. Moreover, many patients with MtDs report an average of 16 symptoms [14]. The NAMDC Diagnostic Criteria therefore recognize multisystem disease as a frequent mitochondrial clinical presentation.
3.1.2. Harmonization of classical clinical mitochondrial syndrome definitions
While many patients enrolled in the NAMDC Registry with MtDs were assigned to well-known classical syndromes due to the presence of cardinal signs or symptoms, these syndromes have lacked uniform definitions. In reviewing patient data entered into the NAMDC Registry, we have observed that these syndromes are frequently misdiagnosed, often based on the incorrect assumption that a specific genetic mutation always corresponds to a specific phenotype. This was the case, for example, with MELAS syndrome, diagnosed in 119 patients enrolled in the Registry before the definition of the diagnostic criteria, and confirmed only in 60 patients after their application [12]. Thus, we have specified criteria for syndromes based upon their original descriptions and subsequent reports that refined their distinctive features. Application of these criteria for classical mitochondrial syndromes is necessary to better define the clinical phenotype. The classical mitochondrial syndromes and defining criteria are enumerated in Table 1.
Table 1.
Classical mitochondrial syndromes.
| Alpers syndrome |
| All of A – C: |
| A. Psychomotor regression |
| B. Intractable seizures (confirmed by EEG and lack of response to at least two anti-seizure medications) |
| C. Hepatopathy (Liver disease/dysfunction) |
| Aminoglycoside-induced deafness |
| A. Sensorineural hearing loss in association with aminoglycoside exposure |
| Barth syndrome |
| Both A and B, plus at least one of C – E: |
| A. Male gender |
| B. Dilated cardiomyopathy or non-compaction cardiomyopathy, defined by echocardiogram or MRI |
| C. Neutropenia |
| D. At least one of 1–2: |
| 1. Growth delay |
| 2. Prolonged pubertal growth spurt |
| E. At least one of 1–3: |
| 1. Hypotonia |
| 2. Skeletal muscle weakness |
| 3. Chronic fatigue |
| Chronic Progressive External Ophthalmoplegia (CPEO) |
| A. Ptosis, Progressive External Ophthalmoplegia (PEO) or both (limb myopathy, exercise intolerance, and dysphagia may also be present) |
| CPEO-plus |
| Both A and B: |
| A. Ptosis, Progressive External Ophthalmoplegia (PEO) or both (limb myopathy, exercise intolerance, and dysphagia may also be present) |
| B. Any other manifestation listed in Table 2 but not fulfilling criteria for KSS, MNGIE, or SANDO |
| Kearns-Sayre syndrome (KSS) |
| All of A – D: |
| A. Ptosis, Progressive External Ophthalmoplegia (PEO) or both |
| B. Pigmentary retinopathy |
| C. Cardiac conduction block |
| D. Skeletal muscle involvement (see Table 2) |
| Leber hereditary optic neuropathy (LHON) |
| Both A and B: |
| A. Acute-onset central vision loss in one or both eyes |
| B. Family history compatible with maternal inheritance and not autosomal dominant inheritance (e.g., no male-to-male transmission) |
| Leigh syndrome |
| Both A and B: |
| A. Neurodevelopmental regression or delay |
| B. Bilateral lesions of the basal ganglia, midline brainstem or both by brain CT, brain MRI (T2/FLAIR hyperintense lesions) |
| Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS) |
| Both A and B: |
| A. Stroke-like episodes (sudden-onset focal neurological deficit with brain MRI or CT showing a cerebral lesion that does not conform to a large vessel territory and typically affects cortex and adjacent white matter) |
| B. Encephalomyopathy (see Table 3) |
| Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE) |
| All of A – E: |
| A. Ptosis, Progressive External Ophthalmoplegia (PEO) or both |
| B. Gastrointestinal dysmotility (see Table 2) |
| C. Neuropathy |
| D. Cachexia |
| E. Diffuse or patchy white matter lesions evident on brain MRI |
| Myoclonic Epilepsy with Ragged-Red Fibers (MERRF) |
| All of A – C: |
| A. Myoclonic seizures or myoclonus plus seizures |
| B. Ataxia |
| C. Mitochondrial myopathy (see Table 2) |
| Neuropathy Ataxia and Retinitis Pigmentosa (NARP) |
| All of A – C: |
| A. Peripheral neuropathy |
| B. Ataxia |
| C. Pigmentary retinopathy |
| Pearson syndrome |
| Both A and B: |
| A. Sideroblastic anemia |
| B. Exocrine pancreatic dysfunction |
| Reversible infantile myopathy with respiratory chain enzyme deficiencies |
| All of A – D: |
| A. Diffuse myopathic weakness |
| B. Hypotonia |
| C. Respiratory insufficiency (chronic shortness of breath) |
| D. Spontaneous improvement by age 3 years |
| Sensory Ataxic Neuropathy Dysarthria Ophthalmoplegia (SANDO) |
| All of A – D: |
| A. Sensory ataxia |
| B. Neuropathy |
| C. Dysarthria |
| D. Ophthalmoparesis |
3.1.3. Revising phenotypes of established clinical syndromes
One example of how the diagnostic criteria were refined is illustrated by the Chronic Progressive External Ophthalmoplegia (CPEO) and CPEO-plus syndromes. When the first set of criteria was applied to the NAMDC patients’ Registry, the diagnosis of “CPEO” required the presence of isolated ptosis, ophthalmoparesis, or both. Any additional clinical manifestations would have led to the assignment of a patient to the “CPEO-plus” category. However, in the stage one analysis of 878 NAMDC Registry patients, we observed that only one out of 28 individuals identified as CPEO fulfilled criteria for the diagnosis of isolated CPEO, while the other 27 patients with ptosis and/or ophthalmoparesis also had evidence of a more generalized myopathy with limb weakness, exercise intolerance, and/or dysphagia. Based upon these data and a review of the literature [15–18], we revised the definition of CPEO to include limb-myopathy, exercise intolerance, and dysphagia. Involvement of any other organ or system (e.g., non-skeletal muscle) would lead to the syndromic designation of “CPEO-plus”, unless the patient’s manifestations fulfilled criteria for Kearns-Sayre syndrome (KSS), Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE), or other classical mitochondrial syndrome.With these criteria, we are able to differentiate the pure myopathic syndrome (CPEO) from multisystem disorders (CPEO-plus, KSS, and MNGIE). Longitudinal studies are necessary in order to assess the frequency of developing multisystem disease in patients with CPEO.
3.1.4. Updating diagnostic criteria to reflect current clinical practice
The often-cited historical criteria for KSS are an obligate triad of: CPEO, pigmentary retinopathy, and onset before age 20 years plus one of the following three manifestations: cardiac conduction block, ataxia, or elevated cerebrospinal fluid (CSF) protein greater than 100 mg/dL. We noted shortcomings with this definition including: the requirement of age-of-onset before 20 years, which is an arbitrary cut-off not well-supported by data, and the measurement of CSF protein levels, which are rarely obtained in current clinical practice for suspected KSS patients. Hence, we decided not to include these as criteria for the diagnosis of KSS. Accordingly, we replaced the original definition of KSS with the tetrad of: 1) ptosis and/or progressive external ophthalmoplegia, 2) pigmentary retinopathy, 3) cardiac conduction block, and 4) skeletal muscle involvement.
Myoclonic Epilepsy with Ragged-Red Fibers (MERRF) is a multisystem disorder clinically characterized by myoclonus, epilepsy, ataxia, and weakness [19]. The presence of ragged-red fibers (RRFs) in skeletal muscle biopsies has been a “classical” feature of the syndrome. Based on the observation that muscle biopsies have been performed less frequently after the introduction of next generation sequencing (NGS) techniques (whole mtDNA sequencing, gene panels, whole exome and genome sequencing) and that, occasionally, RRFs may not be observed in patients with MERRF [20] we now exclude the presence of RRFs from the clinical definition of the syndrome. Recently, Finsterer and colleagues used a standardized approach to clarify the definition of MERRF [21]. They based their definition on clinical manifestations, and did not include the presence of RRFs. In their study, the distinctive feature of this rare mitochondrial disorder was not surprisingly myoclonic epilepsy. Interestingly, they have also assessed the strength of gene-disease associations and have confirmed that MERRF is primarily an MT-TK disease, with pathogenic variants in this gene accounting for ~90% of MERRF patients, similar to the NAMDC cohort [12].
The syndrome originally described as “benign infantile mitochondrial myopathy due to reversible cytochrome c oxidase (COX) deficiency” also required re-assessment [22]. This syndrome was originally defined by early onset diffuse skeletal muscle weakness with COX-deficiency, hypotonia, and respiratory insufficiency that spontaneously improves by 3 years of age. However, this syndrome should be described as reversible infantile myopathy with respiratory chain deficiency because defects in multiple respiratory chain complexes in addition to COX have been reported [23,24].
Through these revisions, we have endeavored to generate the necessary strict well-delineated standardized criteria for accurate diagnoses of these syndromes.
3.1.5. Recognition of new clinical syndromes
Although the original development of the classification of MtD syndromes has greatly facilitated the recognition and diagnosis as well as classification of this heterogeneous group of disorders, the majority of patients, as illustrated in the cohort of patients in the NAMDC Registry, do not fulfill the criteria for a “classical” clinical syndrome [12]. Novel clinical syndromes continue to be reported in association with mitochondrial dysfunction, and at an increasing rate in the last decade due to the wider use of next-generation DNA sequencing techniques. To begin to address the conundrum of categorizing this large group of patients, we have tabulated the new clinical syndromes as well as organ/system manifestations strongly suggestive of mitochondrial disorders in Tables 2 and 3. Eventually, these and other well-defined and specific clinical phenotypes may be included with the “classical” syndromes, after a thorough analysis of defining criteria.
Table 2.
Clinical manifestations of mitochondrial disease by organ system
| Neurological | ||
| • Ataxia: cerebellar* • Ataxia: sensory* • Chorea* • Dementia* • Dystonia • Headaches (non-migraine) • Hyperreactive reflexes (incl. Babinski sign, Hoffman sign) |
• Hypotonia • Migraine headaches • Myoclonus • Neuropathy:axonal • Neuropathy: demyelinating • Ophthalmoparesis • Parkinsonism* |
• Seizures* • Spasticity • Strokes or Stroke-like lesions* • Encephalopathy (at least one neurological manifestation marked with an *) |
| Psychiatric | ||
| • Generalized anxiety disorder° • Depressive Disorder Due to Another Medical Condition° |
• Psychotic disorder due to a another medical condition° | |
| Developmental | ||
| • Fine motor disability • Gross motor disability • Language disability • Delayed social development • Cognitive delay or regression • Abnormal neuropsychological test (not including only behavioral abnormalities) |
• Growth delay • Prolonged pubertal growth spurt • Psychomotor regression (loss of acquired skills) • Congenital Hypotonia (floppy baby) • Autism spectrum disorder° • Intellectual disability |
|
| Muscular | ||
| • Dysarthria • Dysphagia • Exercise intolerance |
• Weakness • Myoglobinuria/Rhabdomyolysis |
• Myopathy • External ophthalmoparesis • Ptosis |
| Cardiac | ||
| • Cardiac conduction block • Dilated cardiomyopathy • Arrhythmia |
• Cardiomyopathy (Hypertrophic, Non-compaction, Restrictive) • Wolff-Parkinson-White |
|
| Renal | ||
| • Renal tubular acidosis (e.g. Fanconi syndrome) • Nephrotic syndrome |
• Focal segmental glomerulosclerosis • Other renal |
|
| Hepatic | ||
| • Hepatopathy (e.g. steatosis, oncocytic changes by liver biopsy) | • Cirrhosis • Hepatic failure (e.g. hepatic encephalopathy) |
|
| Endocrine | ||
| • Diabetes mellitus • Hypothyroidism • Hypogonadotropic hypogonadism |
• Hypoparathyroidism • Adrenal insufficiency • Other endocrine |
|
| Hematologic | ||
| • Sideroblastic anemia | • pancytopenia | |
| Otologic | ||
| • Sensorineural hearing loss | ||
| Ophthalmologic # | ||
| • Cataracts • Optic neuropathy |
• Pigmentary retinopathy | |
| Constitutional | ||
| • Cachexia/Thinness (BMI < 18.5) • Chronic fatigue • Chronic shortness of breath |
• Short stature (patient’s height is below the 3rd percentile) • Exercise intolerance |
|
| Gastrointestinal | ||
| • Recurrent nausea/vomiting • *Dysphagia • *Gastroparesis • *Gastrointestinal pseudo-obstruction |
• *Intestinal dysmotility • Gastrointestinal dysmotility (at least one gastrointestinal manifestation marked with an *) • Exocrine pancreatic dysfunction |
|
| Miscellaneous | ||
| • Multiple symmetric lipomatosis | • Idiopathic osteopenia/pathologic fractures | |
defined according to DSM-5; Black, Donald W., and Jon E. Grant. DSM-5® Guidebook: The Essential Companion to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, American Psychiatric Publishing, 2014.
External ophthalmoparesis under “muscular” and “neurological”; ptosis under “muscular”.
Table 3.
Criteria for mitochondrial clinical syndromes.
| Cardiomyopathy |
| At least one of A – D: |
| A. Dilated cardiac muscle abnormality defined by echocardiogram or MRI |
| B. Hypertrophic cardiac muscle abnormality defined by echocardiogram or MRI |
| C. Restrictive cardiac muscle abnormality defined by echocardiogram or MRI |
| D. Left ventricular non-compaction defined by echocardiogram or MRI |
| Diabetes and deafness (DAD) |
| Both A and B: |
| A. Diabetes mellitus |
| B. Sensorineural hearing loss |
| Encephalomyopathy |
| All of A – C: |
| A. Clinical manifestation in skeletal muscle (see Table 2) |
| B. Encephalopathy (see Table 2) |
| C. Does not fulfill criteria for any other mitochondrial syndrome |
| Hepatocerebral syndrome |
| All of A-C |
| A. Progressive or persistent liver dysfunction |
| B. Encephalopathy (see Table 2) |
| C. Absence of confirmed Alpers-Huttenlocher syndrome Leukoencephalopathy |
| At least one of A – C, plus D: |
| A. Cognitive impairment |
| B. Increased skeletal muscle tone (spasticity) |
| C. Hyperactive reflexes |
| D. Diffuse, patchy, or cystic white matter lesions evident on brain MRI |
| Maternally inherited deafness |
| Both A and B: |
| A. Sensorineural hearing loss |
| B. Maternally inherited hearing loss |
| Multi-systemic syndrome |
| Both A and B: |
| A. Clinical manifestations must include at least three of the organ system presentations (including neurologic, developmental, muscular, cardiac, renal, hepatic, endocrine, hematologic, otologic, ophthalmologic, and gastrointenstinal- see Table 2). |
| B. A progressive clinical course with episodes of exacerbation (e.g. following intercurrent illnesses). |
Cardiomyopathy
3.2. Beyond clinical classification: The importance of diagnostic studies (biochemistry, genetics, histology, metabolic profile, and neuroimaging)
The diagnostic evaluation of patients with a mitochondrial disorder typically begins with the identification of the clinical features that can either be part of a well-defined mitochondrial syndrome or involve organs in specific patterns that suggest MtDs. Although recognition of hallmark manifestions of MtDs is critical to initiate the diagnostic process, clinical features alone are often insufficient to make a definite diagnosis of MtD. Data from the following five laboratory analyses are usually necessary to definitively diagnose MtDs: biochemistry, histology (skeletal muscle, liver, and heart), metabolic markers, neuroimaging, and molecular genetic defects (nuclear or mitochondrial).
3.2.1. Biochemistry
Prior to the identification of causative genetic mutations, mitochondrial diseases were typically recognized by detection of defects in mitochondrial oxidative phosphorylation enzymes. Robust quality standards must be met when considering biochemical data, including specimen collection, handling, and shipment. [25] Most commonly collected tissues include skeletal muscle, liver, heart, brain, cultured fibroblasts, and peripheral white blood cells. A normal protein/activity marker (e.g., citrate synthase) has to be concomitantly measured in order to avoid the occurrence of false positive results.
3.2.2. Histology
Biopsy of an affected tissue in patients with suspected MtDs often provides important diagnostic information on the structure and function of mitochondria. Systematic analysis of frozen skeletal muscle sections is an important diagnostic tool for evaluating MtDs. The use of modified Gomori trichrome, succinate dehydrogenase (SDH), cytochrome c oxidase (COX), and SDH/COX combined staining often reveal mitochondrial defects, such as COX deficiency or mitochondrial proliferation evident as ragged-red fibers (RRFs) or ragged-blue fibers (RBFs), [26] Although not entirely specific, these findings have been considered classical features of MtD pathology. [27]
3.2.3. Metabolic profile
Accumulations of metabolites caused by mitochondrial dysfunction such as lactate, pyruvate, and intermediates of the Krebs cycle have been used as a diagnostic clue in the clinical investigation of MtDs. A persistent elevation of plasma lactate, elevated CFS lactate, and serum lactate/pyruvate ratio > 20 are considered highly suspicion of MtDs. Moreover, elevated urine 3-methylglutaconic acid at least twice above normal reference range is strongly associated with specific MtDs (e.g. Barth’s syndrome and TMEM70 defects). [28]
Nevertheless, those alterations often lack both specificity and sensitivity to definitively diagnose mitochondrial disorders, and can be found in conditions causing secondary mitochondrial dysfunction. [29–31] Recently, serum fibroblast growth factor-21 (FGF-21) and serum growth and differentiation factor-15 (GDF-15) have emerged as novel, potential biomarkers of MtDs, particularly mitochondrial myopathies. [32,33] Both markers may be more sensitive and specific than currently used metabolites [34]; however, elevations of FGF-21 and GDF-15 have been observed with a range of non-mitochondrial disorders (e.g., obesity, diabetes, and liver disease) [35–37] and need to be better validated.
3.2.4. Neuroimaging
Diagnostic utility of brain imaging with Magnetic Resonance Imaging (MRI) as well as detection of tissue metabolites using Magnetic Resonance Spectroscopy (MRS) have been well-documented in mitochondrial disorders. [38–41] Common structural findings include bilateral increased T2- and FLAIR-signals in the deep gray matter, stroke-like lesions in non-large vessel territories, leukoencephalopathy, and cerebral and cerebellar atrophy. [39] MRS with 31P and 1H spectra is commonly used to evaluate MtDs, and allows for the identification of lactate, choline, N-acetylaspartate, and phosphorus metabolites.
3.2.5. Molecular genetics
Since the initial identification of the first mtDNA mutations in 1988, [42–44] detection of pathogenic molecular genetic variants has become the gold standard for diagnosis of MtDs. The most recent American College of Medical Genetics and Genomics (ACMG) guidelines [45] must be followed for the interpretation of nuclear genome sequence variants. Regarding mtDNA point mutations, not only the position of the variants and the nucleotide change must be considered, but also the heteroplasmic levels. Rapid advances in molecular genetic testing and clinical availability of Next Generation Sequencing (NGS) techniques have enhanced the diagnostic ability of clinicians. [46–48] For example, it is now possible to detect single-deletion by NGS or long-range polymerase chain reaction (PCR) in urine, blood, or buccal swab of patients. [49,50]
4. Conclusions
The identification and recognition of clinical phenotypes are typically the first steps to achieve specific diagnoses of mitochondrial patients and to focus confirmatory genetic testing. Advances in the field of mitochondrial medicine have driven the need to revise the definition of MtDs, to improve patient diagnosis and classification as well as clinical trial readiness through natural history studies.
The issue of clinical syndromic phenotypes has been addressed by this paper, with delineation of specific criteria for classic mitochondrial syndromes. We have based our criteria on review of the literature, expert opinion, and real-world data present in our NAMDC registry, followed by rigorous analysis and revisions by the members of the Committee.
We recognized limitations of the proposed approach, including analyses of retrospective data and potential biases of existing clinical classification systems. Prospective use of these syndromic definitions will be necessary for validation and further refinement. We plan to optimize the diagnosis of mitochondrial disorders by implementing an automated algorithm into the NAMDC Registry that will assign a diagnosis in real time, based on pre-programmed clinical criteria fulfilling the NAMDC criteria. Moreover, a data-driven machine-learning approach may be applied not only to refine diagnostic guidelines for assigning patients to current diagnostic entities, but potentially more interestingly to identify novel phenotypic presentations among the currently clinically unclassified mitochondrial patients.
It will be necessary to periodically review definitions of the syndromes according to the advances in our knowledge. It is important to emphasize that the current criteria are not intended for clinical diagnoses, but rather meant to be used for classification of patients in research studies. Nevertheless, we have noted the importance of clinical laboratory tools to diagnose MtDs. It is also critical to bear in mind that mitochondrial disease typically evolve over years before reaching the full classical syndrome, as some symptoms naturally precede others. Although all the signs/symptoms are necessary to classify a patient within a specific syndrome, this classification should not delay patients’ diagnosis and treatment.
Further research is needed to better understand this important group of diseases and develop new therapeutic strategies. Although MtDs taken together are frequent, [51] the incidence of each individual disorder is rare. As with other rare disorders, the small numbers of patient, complex and often delayed diagnoses, clinical heterogeneity, and geographic dispersion, represent additional challenges to the development of clinical trials and subsequent evidence-based clinical care approaches. We believe that the proposed clinical NAMDC criteria will contribute to streamlining nomenclature and diagnoses across centers. This will, in turn, help collaborations within the US and internationally to simplify translational research efforts with the ultimate goal to facilitate clinical development of promising novel therapies.
Funding support
The North American Mitochondrial Disease Consortium (NAMDC) is part of Rare Diseases Clinical Research Network (RDCRN), an initiative of the Office of Rare Diseases Research (ORDR), National Center for Advancing Translational Sciences (NCATS). The project was funded by NIH U54 NS078059 sponsored by the National Institute of Neurological Disorders and Stroke (NINDS), the Eunice Kennedy Shriver National Institute of Child Health and Development (NICHD), the Office of Dietary Supplements (ODS), and NCATS. In addition, NAMDC has received private funding from the United Mitochondrial Disease Foundation (UMDF), a Patient Advocacy Group (PAG) for individuals with mitochondrial disease and their family members, as well as the J. Willard and Alice S. Marriott Family Foundation.
Role of the funder/sponsor
The funder had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Abbreviations:
- MtD
Mitochondrial disease
- mtDNA
mitochondrial DNA
- MELAS
Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes
- MERRF
Myoclonic Epilepsy with Ragged-Red Fibers
- CPEO
Chronic Progressive External Ophthalmoplegia
- KSS
Kearns-Sayre syndrome
- MNGIE
Mitochondrial NeuroGastroIntestinal Encephalomyopathy
- NAMDC
North American Mitochondrial Disease Consortium
- MAC
Modified Adult Criteria
- MDC
Mitochondrial Disease Criteria
- AC
Adult Criteria
- COX
cyto-chrome c oxidase
- NGS
next generation sequencing
- RRFs
ragged-red fibers
- SDH
succinate dehydrogenase
- RBFs
ragged-blue fibers
- FGF-21
fibroblast growth factor-21
- GFDF-15
growth and differentiation factor-15
- MRI
Magnetic Resonance Imaging
- MRS
Magnetic Resonance Spectroscopy
- ACMG
American College of Medical Genetics and Genomics
Footnotes
Declaration of Competing Interest
None reported.
References
- [1].DiMauro S, Schon EA, Carelli V, Hirano M, The clinical maze of mitochondrial neurology, Nat. Rev. Neurol 9 (2013) 429–444, 10.1038/nrneurol.2013.126. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [2].Chinnery PF, Mitochondrial disorders overview, in: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mefford HC, Stephens K, Amemiya A, Ledbetter N (Eds.),GeneReviews((R)), Seattle (WA), 1993. [Google Scholar]
- [3].Grier J, Hirano M, Karaa A, Shepard E, Thompson JLP, Diagnostic odyssey of patients with mitochondrial disease: Results of a survey, Neurol. Genet 4 (2018) e230, 10.1212/NXG.0000000000000230. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [4].Gorman GS, Schaefer AM, Ng Y, Gomez N, Blakely EL, Alston CL, Feeney C, Horvath R, Yu-Wai-Man P, Chinnery PF, Taylor RW, Turnbull DM, McFarland R, Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease, Ann. Neurol 77 (2015) 753–759, 10.1002/ana.24362. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [5].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. Prim 2 (2016) 16080, 10.1038/nrdp.2016.80. [DOI] [PubMed] [Google Scholar]
- [6].Pfeffer G, Horvath R, Klopstock T, Mootha VK, Suomalainen A, Koene S, Hirano M, Zeviani M, Bindoff LA, Yu-Wai-Man P, Hanna M, Carelli V, McFarland R, Majamaa K, Turnbull DM, Smeitink J, Chinnery PF, New treatments for mitochondrial disease-no time to drop our standards, Nat. Rev. Neurol 9 (2013) 474–481, 10.1038/nrneurol.2013.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [7].Walker UA, Collins S, Byrne E, Respiratory chain encephalomyopathies: a diagnostic classification, Eur. Neurol 36 (1996) 260–267. [DOI] [PubMed] [Google Scholar]
- [8].Bernier FP, Boneh A, Dennett X, Chow CW, Cleary MA, Thorburn DR, Diagnostic criteria for respiratory chain disorders in adults and children, Neurology 59 (2002) 1406–1411. [DOI] [PubMed] [Google Scholar]
- [9].Wolf NI, Smeitink JA, Mitochondrial disorders: a proposal for consensus diagnostic criteria in infants and children, Neurology 59 (2002) 1402–1405. [DOI] [PubMed] [Google Scholar]
- [10].Morava E, van den Heuvel L, Hol F, de Vries MC, Hogeveen M, Rodenburg RJ, Smeitink JA, Mitochondrial disease criteria: diagnostic applications in children, Neurology 67 (2006) 1823–1826, 10.1212/01.wnl.0000244435.27645.54. [DOI] [PubMed] [Google Scholar]
- [11].Witters P, Saada A, Honzik T, Tesarova M, Kleinle S, Horvath R, Goldstein A, Morava E, Revisiting mitochondrial diagnostic criteria in the new era of genomics, Gene. Med 20 (2018) 444–451, 10.1038/gim.2017.125. [DOI] [PubMed] [Google Scholar]
- [12].Barca E, Long Y, Cooley V, Schoenaker R, Emmanuele V, DiMauro S, Cohen BH, Karaa A, Vladutiu GD, Haas R, Van Hove JLK, Scaglia F, Parikh S, Bedoyan JK, DeBrosse SD, Gavrilova RH, Saneto RP, Enns GM, Stacpoole PW, Ganesh J, Larson A, Zolkipli-Cunningham Z, Falk MJ, Goldstein AC, Tarnopolsky M, Gropman A, Camp K, Krotoski D, Engelstad K, Rosales XQ, Kriger J, Grier J, Buchsbaum R, Thompson JLP, Hirano M, Mitochondrial diseases in North America: An analysis of the NAMDC Registry, Neurol. Genet 6 (2020) e402, 10.1212/NXG.0000000000000402. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [13].Parikh S, Goldstein A, Karaa A, Koenig MK, Anselm I, Brunel-Guitton C, Christodoulou J, Cohen BH, Dimmock D, Enns GM, Falk MJ, Feigenbaum A, Frye RE, Ganesh J, Griesemer D, Haas R, Horvath R, Korson M, Kruer MC, Mancuso M, McCormack S, Raboisson MJ, Reimschisel T, Salvarinova R, Saneto RP, Scaglia F, Shoffner J, Stacpoole PW, Sue CM, Tarnopolsky M, Van Karnebeek C, Wolfe LA, Cunningham ZZ, Rahman S, Chinnery PF, Patient care standards for primary mitochondrial disease: a consensus statement from the Mitochondrial Medicine Society, Genet. Med 19 (2017) 10.1038/gim.2017.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [14].Zolkipli-Cunningham Z, Xiao R, Stoddart A, McCormick EM, Holberts A, Burrill N, McCormack S, Williams L, Wang X, Thompson JLP, Falk MJ, Mitochondrial disease patient motivations and barriers to participate in clinical trials, PLoS One 13 (2018) e0197513, 10.1371/journal.pone.0197513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [15].Mancuso M, McFarland R, Klopstock T, Hirano M, consortium M on Trial Readiness in Mitochondrial, International Workshop:: Outcome measures and clinical trial readiness in primary mitochondrial myopathies in children and adults. Consensus recommendations. 16–18 November 2016, Rome, Italy, Neuromuscul. Disord 27 (2017) 1126–1137, 10.1016/j.nmd.2017.08.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [16].Pitceathly RD, Rahman S, Hanna MG, Single deletions in mitochondrial DNA–molecular mechanisms and disease phenotypes in clinical practice, Neuromuscul. Disord 22 (2012) 577–586, 10.1016/j.nmd.2012.03.009. [DOI] [PubMed] [Google Scholar]
- [17].Mancuso M, Orsucci D, Angelini C, Bertini E, Carelli V, Comi GP, Donati MA, Federico A, Minetti C, Moggio M, Mongini T, Santorelli FM, Servidei S, Tonin P, Toscano A, Bruno C, Bello L, Caldarazzo Ienco E, Cardaioli E, Catteruccia M, Da Pozzo P, Filosto M, Lamperti C, Moroni I, Musumeci O, Pegoraro E, Ronchi D, Sauchelli D, Scarpelli M, Sciacco M, Valentino ML, Vercelli L, Zeviani M, Siciliano G, Redefining phenotypes associated with mitochondrial DNA single deletion, J. Neurol 262 (2015) 1301–1309, 10.1007/s00415-015-7710-y. [DOI] [PubMed] [Google Scholar]
- [18].Heighton JN, Brady LI, Newman MC, Tarnopolsky MA, Clinical and demographic features of chronic progressive external ophthalmoplegia in a large adult-onset cohort, Mitochondrion (2017) 10.1016/j.mito.2017.12.006. [DOI] [PubMed]
- [19].DiMauro S, Hirano M, Merrf, in: Pagon RA, Adam MP, Ardinger HH, Wallace SE, Amemiya A, Bean LJH, Bird TD, Ledbetter N, Mefford HC, Smith RJH, Stephens K (Eds.),GeneReviews(R), Seattle (WA), 1993. [Google Scholar]
- [20].Mancuso M, Petrozzi L, Filosto M, Nesti C, Rocchi A, Choub A, Pistolesi S, Massetani R, Fontanini G, Siciliano G, MERRF syndrome without ragged-red fibers: the need for molecular diagnosis, Biochem. Biophys. Res. Commun 354 (2007) 1058–1060, 10.1016/j.bbrc.2007.01.099. [DOI] [PubMed] [Google Scholar]
- [21].Finsterer J, Zarrouk-Mahjoub S, Shoffner JM, MERRF classification: implications for diagnosis and clinical, Trials Pediatr. Neurol 80 (2018) 8–23, 10.1016/j.pediatrneurol.2017.12.005. [DOI] [PubMed] [Google Scholar]
- [22].DiMauro S, Nicholson JF, Hays AP, Eastwood AB, Koenigsberger R, DeVivo DC, Benign infantile mitochondrial myopathy due to reversible cytochrome c oxidase deficiency, Trans. Am. Neurol. Assoc 106 (1981) 205–207. [PubMed] [Google Scholar]
- [23].Horvath R, Kemp JP, Tuppen HA, Hudson G, Oldfors A, Marie SK, Moslemi AR, Servidei S, Holme E, Shanske S, Kollberg G, Jayakar P, Pyle A, Marks HM, Holinski-Feder E, Scavina M, Walter MC, Coku J, Gunther-Scholz A, Smith PM, McFarland R, Chrzanowska-Lightowlers ZM, Lightowlers RN, Hirano M, Lochmuller H, Taylor RW, Chinnery PF, Tulinius M, DiMauro S, Molecular basis of infantile reversible cytochrome c oxidase deficiency myopathy, Brain 132 (2009) 3165–3174, 10.1093/brain/awp221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [24].Uusimaa J, Jungbluth H, Fratter C, Crisponi G, Feng L, Zeviani M, Hughes I, Treacy EP, Birks J, Brown GK, Sewry CA, McDermott M, Muntoni F, Poulton J, Reversible infantile respiratory chain deficiency is a unique, genetically heterogenous mitochondrial disease, J. Med. Genet 48 (2011) 660–668, 10.1136/jmg.2011.089995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [25].Parikh S, Goldstein A, Koenig MK, Scaglia F, Enns GM, Saneto R, Anselm I, Cohen BH, Falk MJ, Greene C, Gropman AL, Haas R, Hirano M, Morgan P, Sims K, Tarnopolsky M, Van Hove JL, Wolfe L, DiMauro S, Diagnosis and management of mitochondrial disease: a consensus statement from the Mitochondrial Medicine Society, Genet. Med 17 (2015) 689–701, 10.1038/gim.2014.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [26].Tanji K, Bonilla E, Light microscopic methods to visualize mitochondria on tissue sections, Methods 46 (2008) 274–280, 10.1016/j.ymeth.2008.09.027. [DOI] [PubMed] [Google Scholar]
- [27].Phadke R, Myopathology of adult and paediatric mitochondrial diseases, J. Clin. Med 6 (2017) 10.3390/jcm6070064. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [28].Wortmann SB, Duran M, Anikster Y, Barth PG, Sperl W, Zschocke J, Morava E, Wevers RA, Inborn errors of metabolism with 3-methylglutaconic aciduria as discriminative feature: proper classification and nomenclature, J. Inherit. Metab. Dis 36 (2013) 923–928, 10.1007/s10545-012-9580-0. [DOI] [PubMed] [Google Scholar]
- [29].Jackson MJ, Schaefer JA, Johnson MA, Morris AA, Turnbull DM, Bindoff LA, Presentation and clinical investigation of mitochondrial respiratory chain disease. A study of 51 patients, Brain 118 (Pt 2) (1995) 339–357. [DOI] [PubMed] [Google Scholar]
- [30].Sim KG, Carpenter K, Hammond J, Christodoulou J, Wilcken B, Acylcarnitine profiles in fibroblasts from patients with respiratory chain defects can resemble those from patients with mitochondrial fatty acid beta-oxidation disorders, Metabolism 51 (2002) 366–371. [DOI] [PubMed] [Google Scholar]
- [31].D. Mitochondrial Medicine Society’s Committee on, Haas RH, Parikh S, Falk MJ, Saneto RP, Wolf NI, Darin N, Wong LJ, Cohen BH, Naviaux RK, The in-depth evaluation of suspected mitochondrial disease, Mol. Genet. Metab 94 (2008) 16–37, 10.1016/j.ymgme.2007.11.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [32].Suomalainen A, Elo JM, Pietilainen KH, Hakonen AH, Sevastianova K, Korpela M, Isohanni P, Marjavaara SK, Tyni T, Kiuru-Enari S, Pihko H, Darin N, Ounap K, Kluijtmans LA, Paetau A, Buzkova J, Bindoff LA, Annunen-Rasila J, Uusimaa J, Rissanen A, Yki-Jarvinen H, Hirano M, Tulinius M, Smeitink J, Tyynismaa H, FGF-21 as a biomarker for muscle-manifesting mitochondrial respiratory chain deficiencies: a diagnostic study, Lancet Neurol 10 (2011) 806–818, 10.1016/S1474-4422(11)70155-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [33].Yatsuga S, Fujita Y, Ishii A, Fukumoto Y, Arahata H, Kakuma T, Kojima T, Ito M, Tanaka M, Saiki R, Koga Y, Growth differentiation factor 15 as a useful biomarker for mitochondrial disorders, Ann. Neurol 78 (2015) 814–823, 10.1002/ana.24506. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [34].Davis RL, Liang C, Sue CM, A comparison of current serum biomarkers as diagnostic indicators of mitochondrial diseases, Neurology 86 (2016) 2010–2015, 10.1212/WNL.0000000000002705. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [35].Semba RD, Sun K, Egan JM, Crasto C, Carlson OD, Ferrucci L, Relationship of serum fibroblast growth factor 21 with abnormal glucose metabolism and insulin resistance: the Baltimore longitudinal study of aging, J. Clin. Endocrinol. Metab 97 (2012) 1375–1382, 10.1210/jc.2011-2823. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [36].Chow WS, Xu A, Woo YC, Tso AW, Cheung SC, Fong CH, Tse HF, Chau MT, Cheung BM, Lam KS, Serum fibroblast growth factor-21 levels are associated with carotid atherosclerosis independent of established cardiovascular risk factors, Arterioscler. Thromb. Vasc. Biol 33 (2013) 2454–2459, 10.1161/ATVBAHA.113.301599. [DOI] [PubMed] [Google Scholar]
- [37].Koo BK, Um SH, Seo DS, Joo SK, Bae JM, Park JH, Chang MS, Kim JH, Lee J, Jeong WI, Kim W, Growth differentiation factor 15 predicts advanced fibrosis in biopsy-proven non-alcoholic fatty liver disease, Liver Int (2017) 10.1111/liv.13587. [DOI] [PubMed]
- [38].Kaufmann P, Shungu DC, Sano MC, Jhung S, Engelstad K, Mitsis E, Mao X, Shanske S, Hirano M, DiMauro S, De Vivo DC, Cerebral lactic acidosis correlates with neurological impairment in MELAS, Neurology 62 (2004) 1297–1302. [DOI] [PubMed] [Google Scholar]
- [39].Saneto RP, Friedman SD, Shaw DW, Neuroimaging of mitochondrial disease, Mitochondrion 8 (2008) 396–413, 10.1016/j.mito.2008.05.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [40].Finsterer J, Central nervous system imaging in mitochondrial disorders, Can. J. Neurol. Sci 36 (2009) 143–153. [DOI] [PubMed] [Google Scholar]
- [41].Mascalchi M, Montomoli M, Guerrini R, Neuroimaging in mitochondrial disorders, Essays Biochem 62 (2018) 409–421, 10.1042/EBC20170109. [DOI] [PubMed] [Google Scholar]
- [42].Holt IJ, Harding AE, Morgan-Hughes JA, Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies, Nature 331 (1988) 717–719, 10.1038/331717a0. [DOI] [PubMed] [Google Scholar]
- [43].Wallace DC, Singh G, Lott MT, Hodge JA, Schurr TG, Lezza AM, Elsas LJ 2nd, E.K. Nikoskelainen, Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy, Science 242 (1988) 1427–1430, 10.1126/science.3201231. [DOI] [PubMed] [Google Scholar]
- [44].Zeviani M, Moraes CT, DiMauro S, Nakase H, Bonilla E, Schon EA, Rowland LP, Deletions of mitochondrial DNA in Kearns-Sayre syndrome, Neurology 38 (1988) 1339–1346, 10.1212/wnl.38.9.1339. [DOI] [PubMed] [Google Scholar]
- [45].Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL, Committee ALQA, Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, Genet. Med 17 (2015) 405–424, 10.1038/gim.2015.30. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [46].Cui H, Li F, Chen D, Wang G, Truong CK, Enns GM, Graham B, Milone M, Landsverk ML, Wang J, Zhang W, Wong LJ, Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders, Genet. Med 15 (2013) 388–394, 10.1038/gim.2012.144. [DOI] [PubMed] [Google Scholar]
- [47].Whittaker RG, Blackwood JK, Alston CL, Blakely EL, Elson JL, McFarland R, Chinnery PF, Turnbull DM, Taylor RW, Urine heteroplasmy is the best predictor of clinical outcome in the m.3243A>G mtDNA mutation, Neurology 72 (2009) 568–569, 10.1212/01.wnl.0000342121.91336.4d. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [48].Wong LJ, Next generation molecular diagnosis of mitochondrial disorders, Mitochondrion 13 (2013) 379–387, 10.1016/j.mito.2013.02.001. [DOI] [PubMed] [Google Scholar]
- [49].McCormick E, Place E, Falk MJ, Molecular genetic testing for mitochondrial disease: from one generation to the next, Neurotherapeutics 10 (2013) 251–261, 10.1007/s13311-012-0174-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [50].Aure K, Ogier de Baulny H, Laforet P, Jardel C, Eymard B, Lombes A, Chronic progressive ophthalmoplegia with large-scale mtDNA rearrangement: can we predict progression? Brain 130 (2007) 1516–1524, 10.1093/brain/awm067. [DOI] [PubMed] [Google Scholar]
- [51].Gorman GS, Grady JP, Ng Y, Schaefer AM, McNally RJ, Chinnery PF, Yu-Wai-Man P, Herbert M, Taylor RW, McFarland R, Turnbull DM, Mitochondrial donation–how many women could benefit? N. Engl. J. Med 372 (2015) 885–887, 10.1056/NEJMc1500960. [DOI] [PMC free article] [PubMed] [Google Scholar]