Pathological variants in nuclear genes causing mitochondrial complex III deficiency: An update

Abstract

Mitochondrial disorders are a group of clinically and biochemically heterogeneous genetic diseases within the group of inborn errors of metabolism. Primary mitochondrial diseases are mainly caused by defects in one or several components of the oxidative phosphorylation system (complexes I–V). Within these disorders, those associated with complex III deficiencies are the least common. However, thanks to a deeper knowledge about complex III biogenesis, improved clinical diagnosis and the implementation of next‐generation sequencing techniques, the number of pathological variants identified in nuclear genes causing complex III deficiency has expanded significantly. This updated review summarizes the current knowledge concerning the genetic basis of complex III deficiency, and the main clinical features associated with these conditions.

1. INTRODUCTION

Mitochondrial diseases are caused by defects in the oxidative phosphorylation system (OXPHOS) and constitute a group of syndromes with variable severity, age of onset, and clinical presentation. ¹ , ² OXPHOS is formed of the four complexes of the mitochondrial electron transport system (ETS; cI–cIV), two mobile electron carriers connecting them (coenzyme Q [CoQ] and cytochrome c) and the ATP synthase (or cV). Defects in any of these constituents can be the cause of mitochondrial disease. ² Within the cases of mitochondrial disease, complex III (cIII) deficiency is the least frequently diagnosed, ² , ³ possibly because of its heterogeneous clinical presentation and lack of specific histochemical assays. ⁴ CIII deficiency was first found in patients harboring disease‐associated variants in MT‐CYB, the only cIII subunit encoded in the mitochondrial DNA (mtDNA), typically presenting with exercise intolerance, encephalomyopathy, and cardiomyopathy (reviewed in Ref. 2). The first pathogenic variants in a nuclear gene associated with cIII deficiency were found in BCS1L, ⁵ encoding a protein homologous to the yeast cIII assembly factor (AF) Bcs1. ⁶ Only a few years later, a pathogenic variant in a nuclear‐encoded structural subunit (UQCRB) was described. ⁷ Since then, a number of additional variants in the same and in other genes have been identified. Currently, there are known disease‐associated variants in nuclear genes encoding six cIII structural subunits (UQCRB, UQCRQ, CYC1, UQCRH, UQCRC2, and UQCRFS1) and five ancillary proteins involved in cIII maturation (UQCC2, UQCC3, LYRM7, BCS1L, and TTC19). This review will present an updated outlook on the nuclear genetic basis of mitochondrial cIII deficiency.

2. COMPLEX III FUNCTION, STRUCTURE, AND ASSEMBLY

CIII, or CoQ: cytochrome c‐oxidoreductase (EC 1.10.2.2), is the center of the ETS receiving electrons from CoQ, which is reduced by ETS complexes I and II, as well as by other dehydrogenases involved in different metabolic pathways, ⁸ and donating electrons to cytochrome c via a Q‐cycle mechanism. ⁹ This electronic transfer is coupled to proton pumping, contributing to the proton‐motive force utilized by the ATP synthase.

Mammalian cIII is an obligate dimer (cIII₂), where each protomer is composed of one mtDNA‐encoded subunit (MT‐CYB) and nine nuclear DNA‐encoded subunits (UQCRC1, UQCRC2, CYC1, UQCRH, UQCRB, UQCRQ, UQCRFS1, UQCR10, and UQCR11). ¹⁰ , ¹¹ MT‐CYB, the Rieske Fe‐S protein (UQCRFS1), and cytochrome c1 (CYC1) are the catalytic subunits. Human cIII₂ assembly happens through an ordered and sequential incorporation of catalytic and supernumerary subunits, with the assistance of several ancillary proteins serving as assembly or quality control factors (Figure 1). The process seems to be mostly conserved from yeast to humans, ³ , ¹² although it might differ in the middle steps. ¹³ , ¹⁴ Several human cIII AFs are homologous to the yeast proteins (UQCC1, UQCC2, UQCC3, LYRM7/MZM1L, and BCS1L). However, a growing number of cIII AFs encoded by genes present only in metazoans are being identified, including: TTC19, ¹⁵ , ¹⁶ OCIAD1, ¹⁷ OCIAD2, ¹⁸ STMP1, ¹⁹ C12ORF73/BRAWNIN, ²⁰ , ²¹ , ²² C16ORF91/UQCC4, ²³ and SMIM4. ²¹ , ²³ In addition to the above‐mentioned factors, the mitochondrial serine transporter SFXN1 was found to be involved in cIII₂ biogenesis. ²⁴ Therefore, the complete understanding of human cIII₂ assembly and maintenance will only be achieved by studying these processes specifically in human, when possible, or at least in other mammalian models.

FIGURE 1.

Human dimeric complex III (cIII₂) assembly model. Structural subunits are shown schematically and assembly factors in framed font. Red font indicates proteins related to mitochondrial cIII deficiency. The pathway starts with the synthesis of MT‐CYB inside the mitochondria, which is stabilized in the inner membrane by translation enhancers/assembly factors UQCC1‐4, SMIM4, and BRAWNIN. The nuclear encoded subunits then get incorporated sequentially, starting with UQCRQ and UQCRB binding directly to MT‐CYB, while releasing the chaperones. The complex dimerizes during the intermediate steps forming the “pre‐cIII₂,” lacking the catalytic UQCRFS1 and the small accessory UQCR11 subunit. UQCRFS1 stabilization in the matrix is mediated by LYRM7, and BCS1L translocates the subunit from the matrix to the inner membrane to be incorporated into the nascent complex and finish its maturation. Once UQCRFS1 is inserted, TTC19 binds to fully assembled cIII₂ contributing to the clearance of the N‐terminal UQCRFS1 fragments that remain bound inside the cavity defined by the interface of UQCRC1 and UQCRC2. The proposed role for the recently described metazoa‐specific cIII AFs is the stabilization of MT‐CYB/early cIII₂ assembly (SMIM4, BRAWNIN/C12ORF73, UQCC4), maturation of CYC1 (OCIAD1) and stabilization or assembly of cIII₂ and supercomplex (SC) III₂IV (OCIAD2, STMP1, SFXN1). IMS: Intermembrane space.

3. COMPLEX III DEFICIENCY DUE TO PATHOLOGICAL VARIANTS IN GENES ENCODING STRUCTURAL SUBUNITS

3.1. UQCRB

Pathological variants in UQCRB, encoding the human orthologue to yeast Qcr7, have been found in three pediatric patients (Figure 2, Tables 1, and S1). UQCRB binds and stabilizes hemylated MT‐CYB during the first steps of cIII₂ assembly. ²³ , ²⁹ In addition to the first case of a Turkish girl from a consanguineous family harboring a homozygous 4‐bp deletion (NM_006294.5:c.306_309del, NP_006285.1:p.Arg105fs), ⁷ two patients carrying homozygous deletions in UQCRB: NM_006294.5:c.309_313del, NP_006285.1:p.Glu104fs ³⁰ and one subject carrying the same 4‐bp deletion variant as the first reported patient in one allele, together with a deletion encompassing the whole UQCRB gene, ³¹ have now been described. Although the clinical presentations were somewhat different between the cases, all three patients showed hypoglycemia, hyperlactatemia, and metabolic acidosis during the metabolic crises. UQCRB is an essential gene as indicated by the embryonic lethality of a homozygous knock‐out (KO) mouse model. ³²

FIGURE 2.

Pathogenic variants in cIII structural subunits. (A) Cryo‐electron microscopy (Cryo‐EM) structure of human cIII₂ (PDB 5XTE). ²⁵ The ten different structural subunits are indicated with different colors on the left panel. The names of the subunits in which pathogenic variants have been identified are underlined. The amino acid changes and positions of the missense pathogenic variants are indicated on the right panel, highlighted within the structure in red color. The images were created with UCSF ChimeraX. ²⁶ , ²⁷ (B) Protter‐based ²⁸ model of human UQCRB, UQCRFS1 and UQCRC2 subunits indicating frameshift (yellow) and nonsense (red) pathogenic variants.

TABLE 1.

Nuclear genes with pathological variants causing mitochondrial complex III deficiency.

Gene	OMIM #	Cases/families	Onset	Clinical presentation	Metabolic features
Complex III deficiency due to pathological variants in genes encoding structural subunits
UQCRB	615158	3/3	Infancy—childhood	Episodic metabolic decompensation	Hypoglycemia, hyperlactatemia, metabolic acidosis
UQCRQ	615159	25/1	Infancy	Neurological phenotype/psychomotor retardation, dystonia and ataxia	Mildly elevated blood lactate levels
CYC1	615453	4/4	Neonatal—childhood	Episodic metabolic decompensation associated with illness	Ketoacidosis, lactic acidosis, hyperammonemia
UQCRH	620137	2/1	Childhood	Episodic metabolic decompensation associated with illness, encephalopathy	Lactic acidosis, metabolic acidosis, hypoglycemia and hyperammonemia
UQCRC2	615160	12/6	Neonatal—childhood	Episodic metabolic decompensation associated with illness/encephalomyopathy	Hyperlactatemic hypoglycaemia, hyperammonemia
UQCRFS1	618775	2/2	Prenatal	Metabolic decompensation/hypertrophic cardiomyopathy	Lactic acidosis
Complex III deficiency due to pathological variants in genes encoding ancillary proteins
UQCC2	615824	2/2	Neonatal—infancy	Proximal renal tubular acidosis/respiratory distress syndrome	Lactic acidosis, metabolic acidosis
UQCC3	616111	1/1	Neonatal	Delayed psychomotor development, muscular weakness	Hypoglycemia, lactic acidosis
LYRM7	615838	19/16	Infancy—adulthood	Leukoencephalopathy, episodes induced by febrile illnesses	Lactic acidosis
BCS1L	124000	24/18	Neonatal—infancy	GRACILE syndrome	Lactic acidosis
		12/5	Neonatal—childhood	Encephalopathy	Lactic acidosis
		25/11	Childhood	Björnstad syndrome, pili torti, hearing impairment	NA
		62/≥45	Prenatal—childhood	‘BCS1L mitopathy’	Lactic acidosis
		1/1	Childhood	Hypertrophic cardiomyopathy	Lactic acidosis
TTC19	615157	22/17	Infancy—adulthood	Neurological phenotype/encephalopathy/Leigh syndrome/development delay	Elevated or normal levels of lactate

3.2. UQCRQ

UQCRQ (Qcr8 homolog) is another supernumerary subunit joining MT‐CYB and UQCRB during early cIII₂ assembly ²³ , ²⁹ (Figure 1). A single missense variant in UQCRQ (NM_014402.5:c.134C > T, NP_055217.2:p.Ser45Phe; Figure 2A) was found in a cohort of 25 individuals belonging to a consanguineous inbred Israeli Bedouin kindred. ³³ Affected individuals presented with a neurological phenotype characterized by delayed psychomotor development and severe mental retardation, associated with reduced cIII enzymatic activity.

3.3. CYC1

Cytochrome c1, or CYC1, is one of the two nuclear‐encoded catalytic core cIII subunits, with the heme‐binding C‐terminal domain facing the intermembrane space. Originally, two unrelated patients carrying disease‐associated variants in CYC1 were reported (NM_001916.5:c.288G > T, NP_001907.3:p.Trp96Cys and NM_001916.5:c.643C > T, NP_001907.3:p.Leu215Phe) ³⁴ and later a third case with the same homozygous NM_001916.5:c.643C > T variant (NP_001907.3:p.Leu215Phe; Figure 2A) was reported. ³⁵ These patients presented with recurrent episodes of ketoacidosis, insulin‐responsive hyperglycemia, and neurodegenerative lesions with subsequently normal psychomotor development. A third missense variant in CYC1, NM_001916.5:c.949C > T (NP_001907.3:p.Arg317Trp), was described in a pediatric patient with a different manifestation of recurrent episodes of profound visual loss, partially responsive to corticosteroids and peripheral motor‐sensory neuropathy. ³⁶

3.4. UQCRH

UQCRH is a supernumerary cIII subunit located in the intermembrane space containing a CX₉C domain (Figure 2A), allowing the redox regulation of its import. ³⁷ Recently, a deletion of UQCRH exons 2 and 3 (NM_006004.4:c.55‐527_243 + 48del) was found in two related patients presenting with episodes of metabolic crisis, lactic acidosis, hypoglycemia, and brain abnormalities triggered by mild viral infection. ³⁸ Patient fibroblasts showed loss of UQCRH protein, decreased levels of cIII subunits, and impaired cIII activity. A Uqcrh KO mouse model recapitulated the reported symptoms showed by the patients and the biochemical findings. ³⁸ In a follow‐up study, the Uqcrh KO mice were described to have smaller heart size and cardiac contractile malfunction, which was possibly related with the lower mitochondrial respiratory capacity and redox imbalance. ³⁹

3.5. UQCRC2

UQCRC2 is a matrix‐facing subunit interacting closely with UQCRC1 (Figure 1, Figure 2). Pathogenic variants in UQCRC2 (Tables 1 and S1) were first found in association with cIII deficiency in three consanguineous Mexican patients carrying the homozygous missense variant NM_003366.4:c.547C > T (NP_003357.2:p.Arg183Trp), ⁴⁰ which was also detected in two siblings born from French Caucasian parents and in an additional unrelated patient. ⁴¹ , ⁴² The main clinical presentation in all of these cases was metabolic decompensation with episodes of metabolic acidosis, hyperammonemia, hyperlactatemia, hypoglycemia and, remarkably, either none or only mild neurological involvement. ⁴⁰ , ⁴¹ , ⁴² , ⁴³ Other pathogenic variants in the same gene with similar clinical features have been identified, namely, homozygous NM_003366.4:c.266 T > C (NP_003357.2:p.Leu89Pro) in one case, compound heterozygous NM_003366.4:c.1330 T > A (NP_003357.2:p.Leu444Met); NM_003366.4:c.1087C > T (NP_003357.2:p.Gln363Ter) in another patient, and NM_003366.4:c.379C > T (NP_003357.2:p.Arg127Trp) allele together with total gene deletion in one other patient, ⁴² as well as compound heterozygous NM_003366.4:c.1189G > A (NP_003357.2:p.Gly397Arg); NM_003366.4:c.437 T > C (NP_003357.2:p.Phe146Ser) in the last subject. ⁴³ A different clinical picture was shown by a girl carrying the homozygous NM_003366.4:c.665G > C (NP_003357.2:p.Gly222Ala) variant in UQCRC2, presenting with severe encephalomyopathy and development delay, in addition to metabolic acidosis. ⁴⁴ The Gly222 residue is in the vicinity of the interface with UQCRC1 within the same protomer (Figure 2A). The change of this residue for an Ala was predicted to alter the protein–protein interaction between the two subunits, leading to the degradation of both proteins and a consequent loss of cIII₂ stability. ⁴⁴ This fact could explain the higher severity of the associated clinical presentation compared with the other cases, in which the changed residue might not be as crucial for the stability of the complex. Biochemical analyses showed decreased cIII₂ abundance and activity in patient skin fibroblasts ⁴⁰ , ⁴¹ , ⁴² , ⁴⁴ and muscle biopsies. ⁴² , ⁴⁴ In some patients, a combined cI and cIII deficiency was observed, ⁴¹ , ⁴⁴ which is a common occurrence in the case of severe cIII assembly defects. ¹⁴ , ⁴⁵

3.6. UQCRFS1

The Rieske iron–sulfur protein (UQCRFS1) is the last catalytic core subunit incorporated to pre‐cIII₂, finishing the maturation/activation of the enzyme (Figure 1). ⁴⁶ Despite the fact that most cases of cIII deficiency are due to defects in UQCRFS1 maturation, caused by disease‐associated variants in the accessory factors BCS1L, LYRM7, or TTC19 (see below), only two cases with pathogenic variants in UQCRFS1 itself have been so far reported. ⁴⁷ One of the two patients carried a homozygous NM_006003.3:c.215‐1G > C variant, predicting an in‐frame deletion of 10 amino acids (p.Val72_Thr81del10). The second individual harbored bi‐allelic variants NM_006003.3:c.41 T > A (NP_005994.2:p.Val14Asp) and NM_006003.3:c.610C > T (NP_005994.2:p.Arg204Ter) (Figure 2A,B). Both cases presented with fetal bradycardia, lactic acidosis, hypertrophic cardiomyopathy, and alopecia totalis, and one of the patients died at the age of three and a half months. ⁴⁷ Patient fibroblasts showed impaired mitochondrial respiration, decreased cIII₂ abundance and activity, and reduced steady‐state levels of UQCRFS1 protein, as well as localization of the NP_005994.2:p.Val14Asp UQCRFS1variant in the cytosol instead of in mitochondria. ⁴⁷

4. COMPLEX III DEFICIENCY DUE TO PATHOLOGICAL VARIANTS IN GENES ENCODING ANCILLARY PROTEINS

4.1. UQCC2

UQCC2 is the human orthologue of yeast Cbp6, which by interacting with UQCC1 (Cbp3) coordinates MT‐CYB translation and the start of cIII₂ assembly. ²³ , ⁴⁸ , ⁴⁹ A homozygous splice site variant in UQCC2 (NM_032340.4:c.214‐3C > G) was first reported in a patient presenting with cIII deficiency and severe metabolic acidosis, dysmorphic features, delayed development, and autistic features (Figure 3A, Tables 1, and S1). ⁴⁸ Later, two homozygous missense variants (NM_032340.4:c.[23G > C;28C > T]), both affecting highly conserved residues (NP_115716.1:p.[Arg8Pro;Leu10Phe]; Figure 3A), were found in one patient showing severe cIII deficiency associated with lactic acidosis, increased urinary pyruvate, respiratory distress syndrome and developed seizures progressing to status epilepticus (Tables 1 and S1). The patient died at the age of 33 days due to respiratory failure. ⁵² In both cases, analysis of patient cells showed profound decrease of UQCC2 protein and combined cI and cIII deficiency. ⁴⁸ , ⁵²

FIGURE 3.

Pathogenic variants in cIII ancillary proteins. AlphaFold ⁵⁰ , ⁵¹ models of human UQCC2 (A) and UQCC3 (B) indicating the amino acid changes and positions of the missense pathogenic variants, highlighted within the structure in red color. The images were created with UCSF ChimeraX. ²⁶ , ²⁷ (C) AlphaFold ⁵⁰ , ⁵¹ (left) and Protter‐based ²⁸ (right) models of human LYRM7, indicating the amino acid changes of the identified missense pathogenic variants, highlighted in red color within the structure. Frameshift, nonsense and duplication pathogenic variants are indicated in the right panel in yellow, red and blue color, respectively. The images of the AlphaFold models were created with UCSF ChimeraX. ²⁶ , ²⁷

4.2. UQCC3

UQCC3 is the predicted orthologue of yeast Cbp4, also involved in early cIII₂ assembly and MT‐CYB maturation (Figure 1). So far, a single patient was reported harboring a homozygous missense variant (NM_001085372.3:c.59 T > A, NP_001078841.1:p.Val20Glu) in UQCC3 (Tables 1, S1, and Figure 3B), presenting with lactic acidosis, hypoglycemia, hypotonia, and delayed development without dysmorphic features. ⁵³

4.3. LYRM7

LYRM7 encodes the human orthologue to yeast Mzm1 (MZM1L), which is a chaperone stabilizing UQCRFS1 in the mitochondrial matrix also involved in the delivery of the Fe‐S clusters to the catalytic center (Figure 1). ⁵⁴ , ⁵⁵ , ⁵⁶ Pathological variants in LYRM7 (Figure 3C, Tables 1, and S1) have been found associated with mitochondrial cIII deficiency and a particular type of cavitating leukoencephalopathy, with a course of the disease often leading to death. ⁵⁷ , ⁵⁸ , ⁵⁹ , ⁶⁰ , ⁶¹ , ⁶² , ⁶³ , ⁶⁴ , ⁶⁵ In most of the reported patients, diagnostic evaluation of respiratory chain enzyme activity revealed isolated cIII deficiency in muscle biopsies, whereas the activities were normal in skin fibroblasts. ⁵⁸ , ⁵⁹ , ⁶⁰ Interestingly enough, the neurological phenotype in most of these patients is triggered and/or worsened by febrile illness. ⁵⁸ , ⁶⁰ , ⁶⁵ In another case, a homozygous truncating variant in LYRM7 (NM_181705.3:c.52delA, NP_859056.2:p.Arg18fs) was found in a patient showing a metabolic clinical phenotype and cIII deficiency in liver and skin fibroblasts. ⁶⁶ Even though the same patient also carried a homozygous truncating variant in MTO1, encoding a mitochondrial tRNA modifying enzyme, ⁶⁷ functional complementation of the patient‐derived cells with wild‐type LYRM7, indicated the defect in LYRM7 as causative.

4.4. BCS1L

BCS1L is a member of the AAA+ (ATPases associated with diverse cellular activities) family of proteins, ⁶⁸ forming a homo‐heptameric structure ⁶⁹ , ⁷⁰ securing the ATP‐dependent translocation of UQCRFS1 from the matrix to the inner mitochondrial membrane, incorporating the third catalytic subunit during the last steps of cIII₂ assembly ⁷¹ (Figure 1). Disease‐associated variants in BCS1L are the most frequent cause of mitochondrial cIII deficiency showing a wide range of clinical phenotypes and severity (Figure 4, Tables 1, and S1). Disease manifestations associated with BCS1L pathogenic variants can be either: (i) purely visceral, as shown in the cases of severe growth retardation, aminoaciduria, cholestasis, iron overload, lactic acidosis, and early death (GRACILE) syndrome (Figures 4B and S1) ⁷¹ , ⁷² , ⁷³ , ⁷⁴ , ⁷⁵ ; (ii) cIII deficiency and encephalopathy (Figures 4C and S1B) ⁷⁶ , ⁷⁷ , ⁷⁸ ; (iii) Björnstad syndrome, which is characterized by pili torti and sensorineural hearing loss (Figures 4D and S1) ⁷⁸ , ⁷⁹ , ⁸⁰ , ⁸¹ , ⁸² , ⁸³ , ⁸⁴ ; (iv) a combination of symptoms associated with cIII enzymatic deficiency, in a phenotypic continuum that has been denominated as “BCS1L mitopathies” (Figures 4E and S1), ⁸⁵ involving GRACILE‐like features, renal pathology (including Fanconi syndrome), hepatopathy, hypotonia, encephalopathy, movement disorders, seizures, or developmental delay ⁵ , ⁷¹ , ⁸⁵ , ⁸⁶ , ⁸⁷ , ⁸⁸ , ⁸⁹ , ⁹⁰ , ⁹¹ , ⁹² , ⁹³ , ⁹⁴ , ⁹⁵ , ⁹⁶ , ⁹⁷ , ⁹⁸ , ⁹⁹ , ¹⁰⁰ , ¹⁰¹ ; and more recently, (v) hypertrophic cardiomyopathy. ¹⁰² Correlation between the BCS1L pathogenic variants and the observed heterogeneous clinical phenotypes is not fully understood yet, ⁷⁸ , ⁸⁵ , ⁹¹ but it seems to be related with the localization of the variants in the tertiary and/or quaternary BCS1L protein structure (Figure 4A), since only considering the localization within the different domains in the primary structure was inconclusive. ⁸⁵ However, once the structure of the mouse Bcs1l was solved, it was possible to map the pathogenic variants within the 3D structure of the heptameric complex. ⁶⁸ Thus, there is an indication that the severity of the disease depends on the integrity of the Bcs1‐specific region (Figure 4), since most of the variants located in that domain are associated with the most severe phenotypes of GRACILE syndrome and "BCS1L‐mitopathy" (Figure 4B,E). These amino acid changes fall within the interface of the protomers and might alter the stability of the whole complex and could lead also to proton leak. ⁶⁸ On the other hand, pathogenic variants in the AAA region would only affect BCS1L activity but not the stability of the complex. ⁶⁸ CIII deficiency in most of the BCS1L‐related cases is isolated, although slightly reduced cI and/or cIV activity has been reported in some cases. ⁷⁶ , ⁷⁸ , ⁹¹ Compatible with the role of BCS1L in the incorporation of UQCRFS1, accumulation of pre‐cIII₂ is prominent in patient‐derived cells and skeletal muscle. ⁷⁶ , ⁷⁸ , ⁹¹

FIGURE 4.

BCS1L pathogenic variants related to various disease manifestation. (A) Cryo‐electron microscopy (Cryo‐EM)‐based structure of mouse BCS1L homo‐heptamer (PDB 6UKO) ⁶⁹ showing the organization of the individual protomers within the complex. The localization of the three distinct functional BCS1L regions within the tertiary and quaternary structures is indicated. AlphaFold ⁵⁰ , ⁵¹ models of human BCS1L indicating the amino acid changes of the missense pathogenic variants and highlighted in red color within the structures, related with various disorders: (B) GRACILE syndrome, (C) encephalopathy, (D) Björnstad syndrome, and (E) ‘BCS1L mitopathy’ and hypertrophic cardiomyopathy (underlined variant). The images were created with UCSF ChimeraX. ²⁶ , ²⁷

4.5. TTC19

Tetratricopeptide repeat domain 19 (TTC19) binds cIII₂ once it is completely assembled, being involved in the proteolytic removal of processed UQCRFS1 N‐terminal fragments that remain bound to cIII₂. ¹⁵ , ⁴⁶ Nevertheless, the exact molecular mechanisms by which this process is necessary to maintain cIII₂ integrity and function still need to be defined. Pathological variants in TTC19 (Figure 5, Tables 1, and S1) appear almost exclusively associated with encephalopathy with variable age of onset and disease progression. ¹⁶ , ¹⁰³ , ¹⁰⁴ , ¹⁰⁵ , ¹⁰⁶ , ¹⁰⁷ , ¹⁰⁸ , ¹⁰⁹ , ¹¹⁰ , ¹¹¹ , ¹¹² , ¹¹³ , ¹¹⁴ Most of the cases are associated with loss‐of‐function variants in TTC19, but for still unknown reasons, lactic acidosis is reported in only about 50% of the cases (Tables 1, S1 and Figure 5). ¹¹² The presence or absence of high blood lactate could depend on the stage of the disease, ¹⁰⁴ and in some cases even though blood lactate was reported normal, ¹H‐MRS revealed the presence of lactate in the putamina of the patients. ¹⁶ Patient‐derived fibroblasts cultured in standard conditions frequently do not express the biochemical cIII defect, whereas cells grown in a galactose‐containing medium reveal the cIII activity decrease. This indicates a lack of adaptation to higher metabolic demands, which may be even more pronounced in the patient neuronal cells. In addition, measurements in skeletal muscle biopsies allowed the diagnosis of isolated cIII deficiency in most of these patients. ¹⁶ This suggests a different impact of TTC19 loss‐of‐function in different human cell types, although the mechanisms underlying this variable tissue susceptibility are still unknown.

FIGURE 5.

TTC19 pathogenic variants. AlphaFold ⁵⁰ , ⁵¹ (left) and Protter‐based ²⁸ (right) models of human TTC19 indicating the amino acid changes of the identified missense pathogenic variants, highlighted in red color within the structure. Frameshift and nonsense pathogenic variants are indicated in the right panel in yellow and red color, respectively. The images of the AlphaFold models were created with UCSF ChimeraX. ²⁶ , ²⁷

5. DIAGNOSIS AND TREATMENT OF MITOCHONDRIAL COMPLEX III DEFICIENCY

Currently, there are no specific and reliable biomarkers for the direct and unequivocal diagnosis of mitochondrial diseases in general, and of cIII deficiency in particular. Therefore, the diagnostic workflow is complex and comprises multiple tests. If the clinical evaluation raises the suspicion of a mitochondrial disorder with the presence of symptoms such as muscle weakness, multiorgan involvement, developmental delay, and neurological abnormalities, then biological samples are taken from the patient to continue the diagnostic path. Usually blood, cerebrospinal fluid (CSF), as well as skin and muscle biopsies are collected for biochemical and functional analyses. ¹¹⁵ Biochemical analyses include determination of lactate and pyruvate levels in blood and CSF, as well as organic acid and amino acid levels in blood and urine, which can serve as secondary metabolic markers of mitochondrial dysfunction. Moreover, functional studies in muscle biopsies and/or cultured skin fibroblasts, such as cIII enzymatic activity and mitochondrial respiration measurements are normally employed to determine the presence and extent of the functional deficiencies. In the last years, there has been a tendency to decrease the invasiveness in the diagnostic procedures, for example measuring mitochondrial respiration in blood lymphocytes as an alternative to performing a muscle biopsy. ¹¹⁶ In addition, the implementation of next‐generation sequencing in the diagnostic clinics, i.e., whole exome or genome sequencing, has allowed to directly find disease‐causing genetic variants in DNA extracted from blood samples, ¹¹⁷ with no need to perform functional analyses using biopsy material. In the case of variants with uncertain pathogenicity, other “omic” analyses, i.e., transcriptomics, proteomics and metabolomics, have been employed to determine the origin of the disease. ¹¹⁷ Finally, in the case of neurological involvement, the genetic and biochemical tests are usually complemented with neuroimaging, using magnetic resonance imaging and/or computerized tomography, ¹¹⁵ which can directly indicate cIII deficiency as in the case of LYRM7‐associated disease, which appears with a characteristic brain imaging pattern. ⁵⁸ , ⁵⁹ , ⁶⁵ , ¹¹⁵

Similar to mitochondrial disorders associated with other OXPHOS deficiencies, there is still no cure for cIII deficiency‐associated disease. Currently, treatment only involves clinical management of the symptoms administering supplements like vitamin cocktails and CoQ, physical therapy, nutritional support, and, when appearing, management of decompensation episodes. ¹¹⁵ , ¹¹⁸ However, currently there is active research to find a cure for mitochondrial disorders due nuclear genetic defects by tackling the very origin of the disease through the development of personalized medicine strategies, such as gene therapy, cell therapy, and organ replacement, ¹¹⁸ , ¹¹⁹ while still searching for pharmacological approaches to boost mitochondrial biogenesis and/or activity. ¹²⁰

6. CONCLUDING REMARKS

Since the last comprehensive overview of the nuclear genetic bases of mitochondrial cIII deficiency, which was published in 2015, ³ the amount of cases that have been reported has increased significantly. Thus, the description of new cases and novel variants in genes already known to cause these conditions has broadened our knowledge concerning the clinical phenotypes associated with cIII deficiency of different genetic origin. In addition, pathogenic variants have been identified for the first time in UQCRH ³⁸ and UQCRFS1. ⁴⁷ Moreover, novel metazoan‐specific cIII ancillary proteins have been discovered, ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ²¹ , ²³ improving our understanding of the human cIII₂ assembly process and possibly also of the molecular basis of cIII deficiency, if disease‐associated variants will be found in these factors by a targeted search in yet unresolved cases. However, despite these developments, the molecular pathological mechanisms underlying the clinical heterogeneity in different cases of mitochondrial cIII deficiency are still not fully comprehended. Even though mitochondria are ubiquitous organelles essential for the function of all cells, one or more organs can be affected, while other tissues do not show signs of disease, even within a group of patients with pathogenic variants in the same gene. This could be related to the observations that respiratory chain activities vary depending on the tissue ¹²¹ , ¹²² and/or that there is a different impact of cIII deficiency on the bioenergetics of mitochondria from different organs, being significant in brain but not in liver. ¹²³ Additionally, mitochondrial dysfunction triggers a series of cellular responses that we are just starting to understand, which might affect various cell types and tissues differently. ¹²⁴ , ¹²⁵ , ¹²⁶ With the growing evidence on the importance of these phenomena in the pathogenesis of mitochondrial disorders in general, and of complex III deficiency in particular, the tissue specificity of disease manifestations deserves to be a main topic of investigation in the future.

AUTHOR CONTRIBUTIONS

Writing—original draft and visualization: KC. Writing—review and editing: EF‐V.

FUNDING INFORMATION

Our research is funded by an EMBO Postdoctoral Fellowship (ALTF 710–2022 to KC), Telethon/Cariplo Foundation (GJC21014 to EF‐V), Department of Biomedical Sciences—UNIPD (FERN_FAR22_01 to EF‐V), and Association Française contre les Myopathies (AFM)‐Telethon (24962 to EF‐V).

CONFLICT OF INTEREST STATEMENT

Kristýna Čunátová and Erika Fernández‐Vizarra declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supporting information

Figure S1: BCS1L pathogenic variants related to various syndromes. Protter²⁸‐based model of human BCS1L indicating (A) frameshift, nonsense and other pathogenic variants and (B) missense pathogenic variants characterized in patients with diverse disease manifestations.

Table S1: Compilation of the pathogenic variants and clinical findings associated with nuclear genetic defects causing cIII deficiency. The position of the variants within the cDNA and protein sequences are indicated using the latest versions of the reference sequences in the NCBI databases (https://www.ncbi.nlm.nih.gov/). This does not necessarily coincide with the numbering that was reported originally. When available, hyperlinks to the ClinVar or dbSNP databases are included in the table. The numbering in the cDNA sequences considers the A nucleotide in the starting codon (ATG) as position number 1. NA, Not available.

Čunátová K, Fernández‐Vizarra E. Pathological variants in nuclear genes causing mitochondrial complex III deficiency: An update. J Inherit Metab Dis. 2024;47(6):1278‐1291. doi: 10.1002/jimd.12751