Summary of Recommendations for the Genetic Diagnosis of Mitochondrial Disorders
Diagnostic work-up for suspected mitochondrial disorder (MID) is a stepwise procedure. The first step comprises a comprehensive individual and family history and clinical investigations by specialists in neurology, ophthalmology, otology, endocrinology, cardiology, gastroenterology, nephrology, haematology or dermatology. Important instrumental procedures include chemical investigations of the serum, cerebrospinal fluid (CSF) and urine, electrophysiological investigations, functional and imaging studies of the cerebrum, and muscle biopsy. Based on their results the probability for the presence of a MID can be assessed according to the Nijmegen, Bernier or Walker diagnostic criteria.
In a second step, clinicians need to decide whether an individual phenotype conforms to any of the syndromic MIDs or represents a non-syndromic MID and need to determine if the phenotype occurred sporadically or followed a Mendelian or maternal trait of inheritance.
Genetic testing is the third step and depends on step 1 and 2. If the phenotype suggests syndromic MID due to mitochondrial DNA (mtDNA) point mutations (MELAS, MERRF, NARP, LHON) DNA-microarrays using allele-specific oligo-nucleotide hybridisation, real-time-polymerase chain reaction (PCR) or single-gene-sequencing are indicated. If the phenotype suggests syndromic MID due to mtDNA deletion (mtPEO, KSS, Pearson's syndrome) mtDNA analysis starts with restriction fragment length polymorphism (RFLP) or Southern-blot from appropriate tissues (see Fig. 2 in the original guideline document). mtDNA deletions with low heteroplasmy rate may be detected only by long-range PCR. If neither a single deletion nor multiple deletions are found, mtDNA sequencing is recommended (Level B).
If RFLP or Southern-blot of muscle mtDNA detects multiple mtDNA deletions (breakage syndromes), sequencing of POLG1 (adPEO, arPEO, SANDO, SCAE, AHS), POLG2, PEO1 (adPEO), ANT1 (adPEO), TYMP (MNGIE) or OPA1 (ADOAD) genes should follow (Level B). Sequencing should start with the POLG1 gene, since it is the most likely to carry a mutation. Sequencing of TYMP should be performed only if serum thymidine is elevated. If the phenotype suggests LS appropriate nuclear DNA (nDNA) genes encoding for respiratory chain complex (RCC) subunits or assembly factors (SURF1, ATP12, SCO1, SCO2, COX10, COX15, NDUFS, NDUFV, SDH) need to be sequenced. Sequencing of appropriate nDNA genes is also required if the phenotype suggests GRACILE (TMEM70), IOSCA (PEO1), MLASA (PUS1), Barth syndrome (TAZ/G4.5), DDS/Mohr-Tranebjaerg syndrome (DDP1), MIRAS (POLG1) or CMT2A (mitofusin-2) (Level B).
If an individual presents with a non-syndromic phenotype, biochemical investigations of the most affected tissues (muscle, liver, brain, skin) should clarify if a single or multiple RCCs are defective (Level B). If a single autosomally inherited defect is present, sequencing of appropriate structural subunits or assembly-factors of RCCI, III, IV or V is necessary (Level B). If biochemical investigations in muscle tissue suggest coenzyme-Q (CoQ)-deficiency, sequencing of genes involved in the Co-Q biosynthesis (most frequently ETFDH) should be carried out (Level B). If the single RCC defect is maternally inherited, one should proceed with mtDNA sequencing of appropriate mtDNA genes (Level B).
If multiple autosomally inherited biochemical defects are found, a Southern-blot or quantitative PCR should clarify if there is mtDNA depletion or not. If these investigations detect mtDNA depletion, sequencing of the TK2 or RRM2B genes is indicated if the skeletal muscle is the predominantly affected organ. Elevated creatine-kinase may help to prioritise testing for TK2 or RRM2B. If muscle and cerebrum are the most affected organs, sequencing of the SUCLG1, SUCLA2 genes or if the liver is the predominantly affected organ sequencing of the POLG1, PEO1, DGUOK or MPV17 genes is recommended (Level B, see Fig. 2 in the original guideline document).
If Southern-blot fails to detect mtDNA depletion, sequencing of genes involved in the mitochondrial protein synthesis machinery (PUS1, ETF, EFG1, ETFs, MRPS16, MRPS22, RARS2, DARS2) is recommended (Level B, see Fig. 2 in the original guideline). Corresponding proteins of the MRPS22, ETF and ETFs genes are involved in the initiation and elongation of peptides during protein synthesis and are responsible for the group of elongation-factor-disorders. In case of pontocerebellar hypoplasia (PCH) and severe psychomotor impairment, the RARS2 gene should be sequenced for splice-site mutations. Sequencing of the FASTKD2 gene is required in cases with developmental delay, asymmetric cerebral atrophy, epilepsy, hemiplegia and myopathy with COX-deficiency after exclusion of other causes of COX-deficiency.
Tissues most frequently chosen are blood (MELAS, MERRF, NARP, LHON, Pearson's syndrome) or muscle (KSS). Availability of muscle tissue for mtDNA-analysis is mandatory in patients with phenotypes associated with large-scale mtDNA rearrangements, which can be easily missed in leukocytes except for Pearson syndrome. Alternative tissues for DNA analysis are cells from the urine sediment, buccal mucosa, or hair follicles. mtDNA point mutations can be also detected in blood samples. However, in maternally inherited phenotypes associated with heteroplasmic point mutations, the percentage of mutant mtDNA is often significantly higher in muscle than other tissues.
- ADOAD = autosomal dominant optic atrophy
- adPEO = autosomal dominant progressive external ophthalmoplegia
- AHS = Alpers-Huttenlocher syndrome
- ANT = adenine nucleotide translocase
- arPEO = autosomal recessive progressive external ophthalmoplegia
- ATP = adenosine-5'-triphosphate
- COX = cytochrome c oxidase
- CMT = Charcot-Marie-Tooth disease type 2A
- DARS2 = aspartyl-tRNA synthetase 2
- DDP = deafness dystonia peptide 1
- DDS = deafness-dystonia syndrome
- DGUOK = deoxyguanosine kinase
- DNA = deoxyribonucleic acid
- EFG = elongation factor G
- ETF = electron transfer flavoprotein
- ETFDH = electron-transferring flavoprotein dehydrogenase
- FASTKD2 = Fas-activated serine/threonine kinase (FAST) kinase domains 2
- GRACILE = growth retardation, aminoaciduria, cholestasis, iron overload, lactic acidosis, and early death
- IOSCA = infantile-onset spinocerebellar ataxia
- KSS = Kearns-Sayre syndrome
- LHON = Leber's hereditary optic neuropathy
- LS = Leigh syndrome
- MELAS = mitochondrial encephalopathy, lactic acidosis and stroke-like-episodes syndrome
- MERRF = myoclonus epilepsy with ragged-red fibres
- MIRAS = mitochondrial recessive ataxia syndrome
- MLASA = mitochondrial myopathy and sideroblastic anaemia
- MNGIE = Myo-neuro-gastro-intestinal encephalomyopathy
- MPV17 = mitochondrial inner membrane protein
- MRPs = mitochondrial ribosomal proteins
- mtPEO = chronic progressive external ophthalmoplegia
- NADH = nicotinamide adenine dinucleotide (reduced form)
- NARP = neurogenic weakness, ataxia and retinitis pigmentosa
- NDUFS = NADH dehydrogenase (ubiquinone) Fe-S protein
- NDUFV = NADH dehydrogenase (ubiquinone) flavoprotein
- OPA1 = optic atrophy type 1
- PEO1 = progressive external ophthalmoplegia 1
- POLG1 = polymerase (DNA directed), gamma
- PUS1 = pseudouridylate synthase 1
- RARS2 = arginyl-transfer (t)RNA synthetase 2
- RRM2B = ribonucleotide reductase M2 B (TP53 inducible)
- SANDO = sensory ataxic neuropathy, dysarthria and ophthalmoparesis
- SCAE = spino-cerebellar ataxia and epilepsy with or without ophthalmoplegia
- SCO1 and SCO2 = human cytochrome c oxidase assembly factors 1 and 2
- SDH = succinate dehydrogenase
- SUCLA2 = succinate-CoA ligase, beta subunit
- SUCLG1 = succinate-CoA ligase, alpha subunit
- SURF1 = surfeit locus protein 1
- TAZ/G4.5= tafazzin/G4.5 gene
- TK2 = thymidine kinase 2
- TMEM70 = transmembrane protein 70
- TYMP = thymidine phosphorylase
Evidence Classification Scheme for a Diagnostic Measure
Class I: A prospective study in a broad spectrum of persons with the suspected condition, using a "gold standard" for case definition, where the test is applied in a blinded evaluation, and enabling the assessment of appropriate tests of diagnostic accuracy.
Class II: A prospective study of a narrow spectrum of persons with the suspected condition, or a well-designed retrospective study of a broad spectrum of persons with an established condition (by "gold standard") compared to a broad spectrum of controls, where test is applied in a blinded evaluation, and enabling the assessment of appropriate tests of diagnostic accuracy.
Class III: Evidence provided by a retrospective study where either persons with the established condition or controls are of a narrow spectrum, and where test is applied in a blinded evaluation.
Class IV: Any design where test is not applied in blinded evaluation OR evidence provided by expert opinion alone or in descriptive case series (without controls).
Rating of Recommendations
Level A rating (established as useful/predictive or not useful/predictive) requires at least one convincing class I study or at least two consistent, convincing class II studies.
Level B rating (established as probably useful/predictive or not useful/predictive) requires at least one convincing class II study or overwhelming class III evidence.
Level C rating (established as possibly useful/predictive or not useful/predictive) requires at least two convincing class III studies.