Table 1.

Tiered Approach to Evaluation of Suspected Mitochondrial Disease

Tier 1	Early Screening
	Comprehensive metabolic profile, INR, alpha fetoprotein, CPK (creatine phosphokinase), phosphorus, complete blood count, and ammonia.
	Lactate/pyruvate, ideally obtained 1 hour after feeding (normal molar ratio is <20, normal postprandial lactate <2.8 mM).
	Serum ketone bodies: both quantitative 3-hydroxybutyrate and quantitative acetoacetate (3-hydroxybutyrate/acetoacetate ratio <4 is normal) and total free fatty acids to calculate ketone bodies: free fatty acid ratio.(13)
	Serum acylcarnitine profile; serum free and total carnitines.
	Urine organic acids (look for elevated lactate, succinate, fumarate, malate, 3- methyl-glutaconic or 2-hydroxyglutaric, 2-ketoglutaric, methylmalonic acid)
	Serum amino acids. (Look for elevation of alanine (abnormal > 500 μM, but more specific if > 600 μM).
Consider:	Quantitative 3-methylglutaconic acid (serum or urine).(14)
	Urine acylglycines and 2-ethylmalonic quantification (if multiple acyl-CoA dehydrogenase deficiency is suspected).
	Thymidine (plasma) (especially in cases with coexistent intestinal dysmotility concerning for MNGIE syndrome).
	Coenzyme-Q levels in leukocytes, not serum (for CoQ deficiency; leukocyte levels correlate better with tissue coenzyme Q levels, whereas serum levels reflect nutritional status).
	Quantitative serum methylmalonic acid (elevated in SUCLA and SUCLG1 deficiencies).
	CSF analysis: Lactate and pyruvate (if blood lactate is normal but evidence of CNS involvement), amino acids (especially elevated CSF alanine), and protein concentration (note CSF protein can be elevated in POLG1 disease early on, even when lactate is normal).
Tier 2	Genotyping for More Common Genes
	Most common with liver involvement: POLG1 (15, 16), DGUOK (17-19), MPV17 (20, 21) , SUCLG1 (22), C10ORF2/Twinkle (23), TRMU (see Tables 3 and 4).
	If neuromuscular features suggest MELAS (Mitochondrial Myopathy Encephalopathy, Lactic Acidosis and Stroke-like Episodes) or pancreatic insufficiency suggests Pearson’s marrow/pancreas syndrome: mitochondrial DNA point mutations/deletions.(24)
	If methylmalonic acid is elevated: SUCLG1.(22) If acylcarnitines and/or urine organic acids suggest specific fatty acid oxidation (FAO) defects: Genotyping for Long Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency (LCHAD) (25), Carnitine Palmitoyl Transferase Deficiency (CPTI & II deficiency) (26, 27), or Multiple Acyl-CoA Dehydrogenase Deficiency (MADD = Glutaric acidemia II = ETF & ETF-DH deficiency) (28), SLC25A20 for carnitine– acylcarnitine translocase (CACT deficiency).(29)
	For recurrent acute liver failure: TRMU, ACAD9, CPTI, SUCLGI.(22, 26, 30, 31) Identification of TRMU mutations is urgent in infants with acute liver failure since these patients frequently recover without the need for transplantation.(31, 32)
	Note: Next Generation Sequencing (e.g., exome sequencing) will allow for simultaneous evaluation of panels of all nuclear genes encoding mitochondrial proteins and all mitochondrial DNA genes at considerably lower cost in the future, encompassing the above gene tests, and will eventually replace single gene sequencing. It is already available in some countries.
Tier 3	Tissue Evaluation
	Liver biopsy: 1. Light microscopy, electron microscopy (place specimen in glutaraldehyde);  consider oil red O stain for fat on frozen section; consider iron stain if DGUOK  or BCS1L are suspected (Figure 2). 2. Frozen tissue for respiratory chain enzyme activity analysis. 3. Frozen tissue for DNA quantification (mitochondrial DNA depletion analysis). 4. Consider storing frozen tissue for future studies if amount adequate. Consider  blue native PAGE gel analysis with in-gel activity staining (Figure 3).
	Skin biopsy for fibroblast culture. Can be used for fatty acid oxidation enzyme activity, respiratory chain enzyme activities, blue native PAGE (BNP) testing.(33) FAO probe studies (especially if carnitine profile is abnormal), or high resolution respirometry.(34) Note: Because of heteroplasmy of mitochondrial genes or due to differential tissue expression of nuclear genes, abnormalities in patients with mitochondrial hepatopathy can sometimes only be confirmed in liver tissue.
	Muscle biopsy, especially if muscle involvement is present: light and electron microscopy. Consider histochemistry for respiratory chain complexes, respiratory chain enzyme assays, blue native PAGE (BNP) analysis, mtDNA depletion analysis, mtDNA whole genome sequencing and/or deletion analysis.
Tier 4	Further Molecular and Biochemical Evaluation
	Additional genes to consider: TRMU (31) BCS1L (35), SCO1 (36), TSFM (37), TWINKLE (5, 23), ACAD9 (30) (the latter especially if episodes of liver failure and fatty acid oxidation defect) (see Tables 3 and 4). Not all of these tests may be clinically available. A microarray is available to evaluate for large deletions or duplications in nuclear or mitochondrial genes.(38) Note: Next Generation Sequencing (e.g., exome sequencing) will allow for simultaneous evaluation of panels of all nuclear genes encoding mitochondrial proteins and many or all mitochondrial DNA genes at considerably lower cost in the future, and will eventually replace single gene sequencing. At present, its efficacy is highest in clinically and biochemically well characterized patients.
	Targeted molecular analyses based on the results of tissue-based respiratory chain enzyme assays and primary liver disease as presentation: 1. Isolated complex I deficiency: ACAD9 (30) 2. Isolated complex III deficiency: BCS1L (35) 3. Isolated complex IV deficiency: SCO1 (36) 4. Combined complex I, III, and IV deficiency with incompletely assembled  complex V bands on blue native PAGE: (This signifies a generic defect in the  processing of mtDNA encoded subunits.(39) It can be caused by a deficiency  of mtDNA (mtDNA depletion syndromes) or by a defect in transcription or  translation. A) If mtDNA content is <5% of normal in liver: mtDNA depletion syndrome:  POLG1, POLG2, TWINKLE, DGUOK, and TYMP.(40) B) With normal or mildly decreased mtDNA but with multiple deletions (assay  with long range PCR or with NextGen mtDNA analysis): Same as above,  but more often with POLG or TWINKLE or TYMP. C) With normal or (more common) elevated mtDNA: translation defects such  as EF-Tu, EGF-1, TSFM, TRMU, FARS2 (and other tRNA synthase genes,  tRNA modification genes, ribosomal genes, translation initiation,  elongation and termination factors).(31, 37, 41) Consider exome or mito  exome sequencing for the many genes associated with the translational  machinery. D) Deficiency of combined complex II-III assay but normal isolated assay II  and normal III indicates a likely coenzyme Q deficiency. Obtain coenzyme  Q levels, and review for causes of coenzyme Q deficiency.(42)