Spectrum of combined respiratory chain defects

Biochemical investigation of mitochondrial energy metabolism in patient samples dates back to the 1960s, and distinct defects in OXPHOS have been identified affecting either single enzyme complexes or combinations of complexes.

In general, defects of mitochondrial energy metabolism can be grouped into the following five categories (Fig. 1):

Furthermore, defects due to inhibition, e.g. by H₂S in the case of ETHE1 deficiency and inhibition of cytochrome c oxidase (Tiranti et al 2009) or inhibition of mitochondrial protein import by mutated huntingtin (Yano et al 2014), have been reported.

Combined OXPHOS defects are a very common finding in the diagnosis of disorders of mitochondrial energy metabolism (Scaglia et al 2004; Gibson et al 2008; Honzik et al 2012). In the patients seen by the diagnostic centre at the Department of Paediatrics in Salzburg, combined OXPHOS defects are by far the most frequent cause of disorders of mitochondrial energy metabolism, with a proportion of 57.3 % (Table 1).

It is important to point out that combined OXPHOS defects are often picked up in enzymatic measurements as isolated defects. A well-known example is the most frequent m.3243A > G ‘MELAS’ (mitochondrial encephalopathy lactic acidosis and stroke-like episodes) mutation that affects the mitochondrial tRNA^Leu(UUR). In muscle biopsies of these patients, an isolated complex I deficiency is a common biochemical finding; however, cytochrome c oxidase-deficient fibres can also be detected (Zierz et al 2014). Other defects are detected mainly as cytochrome c oxidase deficiency (Santorelli et al 1997). Complex I and cytochrome c oxidase seem to be the most vulnerable enzymes. This could be due to their larger number of mitochondrially encoded subunits, especially in the case of complex I (7 subunits, 2117 codons encoded in mtDNA) or cytochrome c oxidase (3 subunits, 1003 codons) versus ATP synthase (2 subunits, 296 codons) and complex III (1 subunit, 380 codons) (Anderson et al 1981). Alternatively, it could be due to different codon distributions; for example, there is a much higher abundance of codons for tRNA^Leu(UUR) in ND3 (8.7 % of all codons) and ND6 (9.1 % of all codons) of complex I compared to other mtDNA-encoded proteins, which contain less than 3 % of codons for tRNA^Leu(UUR). Finally, the different sensitivities of the OXPHOS complexes might be due to differences in the in vitro assay conditions in different laboratories (Gellerich et al 2004) resulting in experimental bias, since ATP synthesis cannot be quantified in frozen samples.

Therefore, classification as a combined OXPHOS defect in Table 1 was made on the basis of the genetic defect, which was available in 81 % of these patients, in addition to the results of biochemical measurements.

By investigation of oxidative phosphorylation enzymes in patient samples, different types of combined defects have been identified: e.g. complex I (CI) + complex IV (CIV), CI + CIII + IV + V, CI + CII + CIII, CI + III/CII + III, CIII + CIV or involvement of all complexes (Fig. 2).

More than one enzyme can be affected due to the following molecular mechanisms:

The mammalian mitochondrial genome is a circular molecule encoding 13 proteins (subunits of complexes I, III, IV and V), two ribosomal RNAs and 22 transfer RNAs. Depending on cell function and size, the number of mitochondria can vary, with copy numbers of mtDNA ranging from just a few to hundreds of thousands per nuclear genome. In contrast to the nuclear genome, mtDNA is replicated in a cell cycle-independent manner. Genetic defects in nuclear genes involved in mtDNA replication, its transcription or translation typically affect only the four OXPHOS enzymes that contain mitochondrially encoded subunits (complexes I, III, IV, and V) but spare complex II and citrate synthase; the latter is commonly used in biochemical analyses as a mitochondrial housekeeping enzyme.

Numerous cofactors play an essential role in mitochondrial energy metabolism. Some of these cofactors are required for several of the respiratory chain enzymes like coenzyme Q, iron-sulphur clusters, riboflavin and haem. Their deficiency typically results in defects of more than one respiratory enzyme.

Mitochondrial homeostasis involves several essential aspects of mitochondrial biogenesis, lipid synthesis, protein import, fission and fusion, quality control and targeted degradation.

The precise mitochondrial functions of some proteins that cause combined OXPHOS defects are not yet clear. The X-chromosomally encoded AIFM1, well known as an apoptosis-inducing factor, seems to have a mitochondrial function as an NADH oxidoreductase; however, the association with OXPHOS deficiency is not well understood. Similarly the nature of the cytochrome c oxidase decrease in APOPT1 deficiency, a mitochondrial protein termed apoptogenic 1 and known from apoptosis studies, is not well understood (Melchionda et al 2014). CHCHD10 is a coiled-coil-helix-coiled-coil-helix domain-containing protein of unknown function localised to the intermembrane space of mitochondria, and its deficiency causes multiple deletions of mtDNA and combined OXPHOS deficiency (Bannwarth et al 2014). FBXL4, an F-box and leucine-rich repeat protein, is also an intermembrane space mitochondrial protein of unknown function. Deficiency of FBXL4 causes a decrease of all OXPHOS subunits but also of other mitochondrial proteins and mtDNA (Bonnen et al 2013; Gai et al 2013). Deficiency of the mitochondrial protein OPA3 causes 3-methylglutaconic aciduria, which has been found in several other defects of mitochondrial energy metabolism (Wortmann et al 2013), and fragmentation of the mitochondrial network (Grau et al 2013); however, the precise function of OPA3 remains unclear. Finally, multiple deletions of mtDNA have been reported in one study of a family with Aicardi-Goutieres syndrome 5 (MIM 612952) and SAMHD1 deficiency (Leshinsky-Silver et al 2011). The molecular link of SAMHD1 to mitochondrial DNA is not clear but could be related to its function in deoxynucleotide metabolism.

Accumulation of highly reactive metabolites like methacrylyl-CoA has been reported in defects of isoleucine catabolism, which takes place in mitochondria. This compound forms covalent bonds, e.g. with the sulphhydryl group of cysteine in proteins, which can destroy enzymes (Brown et al 1982). In fact, combined OXPHOS defects have been reported in HIBCH- (Loupatty et al 2007) and ECHS1- (Sakai et al 2014) deficient patients. In addition to these defects, combined OXPHOS deficiency has been reported in several forms of organic aciduria like propionic acidaemia and methylmalonic acidaemia (de Keyzer et al 2009).

In 2000 Schägger and Pfeiffer (Schagger and Pfeiffer 2000) introduced the concept of a respirasome with oligomerisation of the respiratory chain complexes and formation of domain structures on the inner mitochondrial membrane. In addition, oligomerisation of the ATP synthase has been shown, which is also integral for inner membrane structure (Wittig and Schagger 2008).

Mouse cells harbouring a high mutation load in cytochrome b, a mitochondrially encoded subunit of complex III, have been shown to be deficient in both complex III and complex I (Acin-Perez et al 2004). Homozygous loss-of-function mutations in cytochrome b have been reported in human oncocytic tumours with a complete loss of complex I (Gasparre et al 2008; Zimmermann et al 2011), which is clear evidence that assembled complex III is necessary for complex I assembly and supercomplex formation. Also a mutation in the UQCRC2 subunit resulted in aberrant supercomplex formation and deficiency of complex I in addition to complex III (Miyake et al 2013). Similar results were found in a knockdown cell line of Rieske iron-sulphur protein, another subunit of complex III (Diaz et al 2012). Furthermore, a deficiency of supercomplex formation was shown in SURF1 deficiency, which is known to be an assembly factor of cytochrome c oxidase (Kovarova et al 2012). Defective supercomplex formation (McKenzie et al 2006) and combined OXPHOS deficiency (Karkucinska-Wieckowska et al 2013) have also been found in patients with Barth syndrome and TAZ mutations leading to an increased lysocardiolipin pool in mitochondria.

This summary, although incomplete, demonstrates that defects in single subunits of OXPHOS enzymes and individual assembly factors but also in the lipid composition can result in deficiency of supercomplex formation and hence a combined OXPHOS deficiency.

The clinical phenotypes associated with combined OXPHOS defects are very heterogeneous, but in many cases encephalomyopathy is the main presentation. A very well-characterised example is the most common “MELAS” mutation m.3243A > G that can result in different clinical symptoms aside from MELAS, including sensorineural hearing loss, (isolated) myopathy, cardiomyopathy, seizures, migraine, ataxia, cognitive impairment, bowel dysmotility, short stature, diabetes, external ophthalmoplegia and Leigh syndrome (Nesbitt et al 2013). Since this mutation affects the mtDNA, the mutation load is variable and can be different in different tissues. Affected individuals usually carry this mutation in a high proportion; however, clinically unaffected or just mildly affected maternal relatives who carry a high mutation load are also found in these pedigrees (Dubeau et al 2000). Another well-studied example of clinical heterogeneity concerns patients with mutations in the POLG gene, encoding mitochondrial DNA polymerase γ. The clinical features of deficiencies in this gene include seizures and hepatopathy (Alpers disease), ataxia, neuropathy, myopathy, chronic progressive external ophthalmoplegia, ptosis, sensorineural deafness, parkinsonism and premature ovarian failure, hypogonadism and gastrointestinal dysmotility (Tchikviladze et al 2014). The same causative mutation can be either autosomal recessive or dominant, the latter usually resulting in delay of disease onset to adulthood.

As illustrated by these two examples, it is not possible to describe a general clinical picture of combined OXPHOS defects. In the following, some clinical features and syndromes are summarised that are associated with certain types of combined OXPHOS and can be helpful in the diagnosis of patients:

Hepatopathy is found only in certain defects of mitochondrial energy metabolism but especially in a number of combined OXPHOS disorders (Table 2). Hepatic presentation is frequently encountered in disorders of mitochondrial replication associated with POLG or C10orf2 (Twinkle); in disorders of mitochondrial nucleotide metabolism involving DGUOK, MPV, SUCLG1 and TRMU (usually transient infantile manifestation); in aberrant translation regulation by TSFM (Vedrenne et al 2012a) and in some cases of GFM1 deficiency, and was also reported in patients with EARS2 (1 patient) and FARS2 deficiency (Rahman 2013). Furthermore, hepatopathy is also a relatively common feature in MEGDEL syndrome with SERAC1 deficiency, which involves lipid metabolism (Wortmann et al 1993).

Nephropathy may be an underdiagnosed sign of mitochondrial disease but it has been reported in several combined OXPHOS defects. Proximal tubulopathy is a typical finding in early onset mitochondrial DNA depletion syndrome caused by RRM2B deficiency (Bourdon et al 2007) and was also reported in a family with C10orf2 (Twinkle)-deficient patients (Prasad et al 2013). Renal tubulopathy is further found in translational defects involving SARS2, MRPS22 and TSFM (O'Toole 2014). In coenzyme Q synthesis defects, nephrotic syndrome (ADCK4, PDSS2, COQ2, COQ6) and tubulopathy (COQ9) are leading features (Desbats et al 2014). Patients with XPNPEP3 deficiency, encoding X-prolyl aminopeptidase 3, develop a nephronophthisis-like nephropathy but can also involve other organs (O'Toole et al 2010). Furthermore, tubulointerstitial nephritis and focal segmental glomerulosclerosis have been associated with various mitochondrial tRNA mutations, and single deletions of mtDNA have been reported to cause proximal as well as distal tubulopathy (O'Toole 2014).

Perrault syndrome is an autosomal recessive disorder characterised by sensorineural hearing loss in males and females and ovarian dysfunction in females. Neurologic features have been described in some affected women (Newman et al 1993). To date, mutations in five genes (CLPP, HARS2, LARS2, C10orf2 [Twinkle] (Morino et al 2014), HSD17B4) have been reported, with all but the last causing combined OXPHOS deficiency.

Haematologic manifestations of combined OXPHOS defects include aplastic, macrocytic or sideroblastic anaemia, leukopenia, neutropenia, thrombocytopenia or pancytopenia. Sideroblastic anaemia is characterised by the presence of ringed sideroblasts in the bone marrow and can be caused by PUS1 deficiency (affecting mitochondrial pseudouridine synthase) and presenting clinically as myopathy, lactic acidosis and sideroblastic anaemia (MLASA) (Bykhovskaya et al 2004). Deficiency of YARS2, the mitochondrial tyrosyl-tRNA synthetase, also results in a MLASA phenotype (Riley et al 2010). Recently, patients with mutations in TRNT1 (tRNA CCA-adding nucleotidyl transferase) have been reported. Clinically they present with congenital sideroblastic anaemia with immunodeficiency, fever and developmental delay (SIFD) (Chakraborty et al 2014). Two patients with either homozygous (Camaschella et al 2007) or compound heterozygous (Liu et al 2014) mutations in GLRX5, a mitochondrial enzyme of iron sulphur cluster maturation also needed for haem biosynthesis, have been described. Macrocytic anaemia with megaloblastic features has been reported in patients with SFXN4 deficiency, an inner mitochondrial membrane protein with a presumed iron transport function (Hildick-Smith et al 2013). Thrombocytopenia has been reported as a major feature in patients with autosomal dominant CYCS deficiency of cytochrome c (Morison et al 2008; De Rocco et al 2014). Neutropenia is associated in male patients with Barth syndrome, caused by TAZ mutations, in addition to cardiomyopathy, skeletal myopathy, prepubertal growth delay and a distinctive facial gestalt (Ferreira et al 1993). In addition to global developmental delay, hypotonia and other clinical features, neutropenia has been identified in patients deficient in FBXL4, an intermembrane space mitochondrial protein involved in mitochondrial biogenesis (Gai et al 2013). Furthermore, neutropenia has also been reported in a patient with the common m.3243A > G mutation in the MT-TL1 gene (De Kremer et al 2001). Pearson marrow–pancreas syndrome (MIM 557000) is caused by deletions of mtDNA, with variable generalised clinical manifestations, including haematologic presentation with early transfusion-dependent anaemia, neutropenia, thrombocytopenia, and, less abundant, also ringed sideroblasts in bone marrow aspirates (Broomfield et al 2014).

Leigh syndrome (LS, MIM 256000) is characterised by progressive neurologic disease with motor and intellectual developmental delay, signs and symptoms of brain stem and/or basal ganglia disease, and raised lactate (Thorburn and Rahman 1993). LS or Leigh-like presentation is most prevalent in defects of OXPHOS subunits and assembly factors, but also several combined OXPHOS defects present with this neurologic manifestation and involve either mitochondrial nucleotide or RNA metabolism, translation, a form of coenzyme Q deficiency, and a few defects in mitochondrial homeostasis (Table 2).