Introduction

Complex III (CIII) of the mitochondrial respiratory chain (RC) conveys electrons from ubiquinol to cytochrome c and forms the respirasome when associated with complexes I and IV (CI and CIV). CIII is composed of 11 subunits; one is encoded by mitochondrial DNA (CYB) and the others by nuclear DNA, as well as their assembly factors. CIII deficiency represents between 2 to 15% of primary mitochondrial diseases and its molecular origins have been elucidated in only approximately 50% of cases. Mutations in CYB and in BCS1L, TTC19 and LYRM7 that encode assembly factors are the most frequently identified to date.1, 2 Recently, defects in accessory proteins UQCC2 and UQCC3 have been associated with CIII deficiency.3, 4 Only five familial cases with mutations in nuclear genes encoding structural subunits (UQCRB, UQCRQ, UQCRC2 and CYC1) were reported.5, 6, 7,8 We report here a second family case with an UQCRC2 mutation leading to CIII deficiency and enlarge the clinical phenotype from lactic acidosis to hepatocellular insufficiency.

Case history

Clinical presentation

The patient was born at term following an uneventful pregnancy of French Caucasian parents presumably non-consanguinous. At few days of life, he presented poor feeding and lactic acidosis with hypoglycemia and hepatocellular deficiency (prothrombin time for the patient/control 52.6/13.1 s, factor V 5 UI dl−1). During the first year of life, he exhibited five episodes of hypoglycemia (about 1 mm, normal range N=2.8–4.4 mm) with ketonuria, hyperlactatemia (8–10 mm, N=0.6–2.4 mm), high lactate/pyruvate ratio (30–40, N=12–18) and elevated serum transaminases (AST 131 UI l−1, N=15–40 UI l−1, ALT 100 UI l−1, N=10–40 UI l−1) triggered by minor infections. Total recovery occurred under glucose perfusion after each recurrence. A fractioned−enriched diet and carnitine, biotin and thiamine supplementations were initiated.

Metabolic investigations during a decompensation episode showed elevated plasma alanine (1858 μm, N=234–390 μm) and proline (526 μm, N=133–227 μm), reflecting hyperlactatemia. Concomitant urine organic acid analysis revealed elevated 3-OH-butyrate (134 mmol per mmol creatinine, N<11 mmol per mmol creatinine) and dicarboxylic acid (adipate 85 mmol per mmol creatinine, N<35 mmol per mmol creatinine; suberate 86 mmol per mmol creatinine, N<10 mmol per mmol creatinine) levels, indicating activation of fatty acid oxidation and ketone production. Moreover, increased succinate (1772 mmol per mmol creatinine, N=17–80 mmol per mmol creatinine), a tricarboxylic acid (TCA) cycle intermediate and ethylmalonate (30 mmol per mmol creatinine, N<2 mmol per mmol creatinine) levels might suggest a defect in mitochondrial RC. The ammonemia was slightly elevated (60 μm, N<40 μm).

At 2.5 years of age, a metabolic work-up independent of decompensation episodes revealed a constant and moderate metabolic acidosis with elevated lactatemia (about 2.5 mm). Neurological, cardiac, muscular and psychomotor functions were normal. The patient presented a slight liver enlargement without splenomegaly and a discrete facial dysmorphia with epicanthus.

The patient is now 9 years old, with normal growth and neurological functions, with the exception of slight scholastic retardation. He is frequently hospitalized for episodes of vomiting and sleepiness with acute hepatocellular deficiency, cytolysis and hyperlactatemia without bleeding. He always recovers without sequelae with intravenous hydration. Standard laboratory tests are normal between crises.

Mitochondrial function analyses and molecular analyses

Specific investigations excluded tyrosinemia, fructosemia, galactosemia, fructose-1,6-diphosphatase deficiency and a beta-oxidation defect. Mitochondrial function was then explored in the patient’s fibroblasts.9, 10 The enzymatic activity of CIII and the rate of oxygen consumption in the presence of decyl-ubiquinol, which feeds electrons directly to CIII, were very low. The activity of CI was decreased slightly. Activities of CIV and TCA cycle enzymes were not affected. Immunoblot detection of native complexes showed a marked decrease of the quantities of CIII and CI (Table 1).

Table 1 Analyses of mitochondrial function in the patient’s fibroblasts
Full size table

Mitochondrial DNA Sanger sequencing of patient’s fibroblasts first excluded mitochondrial DNA origin (especially CYB mutation). Targeted resequencing of 150 nuclear genes involved in mitochondrial defects (including all nuclear genes encoding CIII subunits) revealed a homozygous mutation c.547C>T, p.Arg183Trp in the UQCRC2 gene (the list of the sequenced genes and the targeted sequencing methods are in Supplementary Table S1). The c.547T>C homozygous mutation and parental alleles segregation were then confirmed by Sanger sequencing of exon 7 (Ref Seq NM_003366.2, electrophoregrams in Supplementary Figure S1). The minor allele frequency of this variant is 1/121054 in ExAC (http://exac.broadinstitute.org) database and 0/8598 in ESP (http://evs.gs.washington.edu/EVS) database and the Polyphen score11 predicted a probably damaging amino-acid substitution (score=1.0). This mutation is the same as that found in a Mexican consanguineous family (patients P1, P2 and P3) described by Miyake et al. in the first and only description of UQCRC2 deficiency.7 This previous study proved the pathogenicity of this mutation by its absence in control alleles, the high conservation of the Arg183 residue across species, and the deleterious structural consequences of the p.Arg183Trp mutation.

Discussion

We present here the second reported case of mutation in the UQCRC2 gene, causing mitochondrial defect. Our study confirmed that the p.Arg183Trp mutation in core protein 2 leads to a marked decrease in CIII activity and quantity in fibroblasts, as observed for P1 in the study of Miyake et al.7 Immunoblot analysis revealed severe reduction in the quantity of free CI in our own and in the previous study (respectively −76 and −50% of control). These results confirm that the mutation in core protein 2 alters the stability of free CI.

Based on the increase in urinary dicarboxylic acid levels and TCA cycle intermediates, additional roles of CIII core protein 2 in beta-oxidation or TCA cycle function were suggested.7 A comparable metabolic pattern was observed in our patient. However, beta-oxidation function was normal in our patient’s fibroblasts and we showed that TCA cycle enzyme activities were normal, along with oxygen consumption in the presence of pyruvate or succinate (Table 1). Our investigations suggested therefore that the urinary metabolite profile observed in UQCRC2 deficiencies reflected probably the RC blockade.

The clinical presentations of our patient and the three related patients previously reported were all characterized by recurrent access of lactic acidosis with glycemic disturbance. However, transient but severe hepatocellular deficiency observed in our patient was not reported in the other cases. Interestingly, the patients had a normal neurological function at 9, 5 and 4 years (patient P3 is too young to determine long-term neurological outcome). The other three cases with CIII deficiency that we previously described, due to mutations in structural subunits cytochrome c1 (CYC1) and protein Hum-QPC (UQCRB), all presented neonatal lactic acidosis also and a normal cognition at 7, 22 and 19 years, respectively.5, 8 Intriguingly, the 20 familial cases with mutations in assembly genes LYRM7 and TTC19 were almost all marked by long-term neurological impairment.2, 12 The number of patients is too small to determine if this results from random difference in prevention of toxic encephalopathy during decompensation or if the defect in CYC1, UQCRC2 and UQCRB-encoded proteins is less deleterious for brain development. However, LYRM7 and TTC19 mutation-induced encephalopathy was not systematically preceded by metabolic crisis, leading some authors to suggest that the LYRM7 and TTC19 proteins play additional roles in brain function. CIII deficiency observed in these cases may not constitute the primary cause of encephalopathy.12

In conclusion, our study reports a new case of primary deficiency of CIII due to a mutation in a structural subunit and characterized by a neonatal lactic acidosis without long-term neurological impairment.