Severe congenital lactic acidosis and hypertrophic cardiomyopathy caused by an intronic variant in NDUFB7

Mutations in structural subunits and assembly factors of complex I of the oxidative phosphorylation system constitute the most common cause of mitochondrial respiratory chain defects. Such mutations can present a wide range of clinical manifestations, varying from mild deficiencies to severe, lethal disorders. We describe a patient presenting intrauterine growth restriction and anemia, which displayed postpartum hypertrophic cardiomyopathy, lactic acidosis, encephalopathy, and a severe complex I defect with fatal outcome. Whole genome sequencing revealed an intronic biallelic mutation in the NDUFB7 gene (c.113‐10C>G) and splicing pattern alterations in NDUFB7 messenger RNA were confirmed by RNA Sequencing. The detected variant resulted in a significant reduction of the NDUFB7 protein and reduced complex I activity. Complementation studies with expression of wild‐type NDUFB7 in patient fibroblasts normalized complex I function. Here we report a case with a primary complex I defect due to a homozygous mutation in an intron region of the NDUFB7 gene.

Next generation sequencing, with whole genome sequencing (WGS) and more recently RNA sequencing (RNA-Seq), has revolutionized the diagnosis of mitochondrial diseases. It has allowed the discovery of numerous genetic mutations underlying defects in the oxidative phosphorylation system (OXPHOS). Mutations exclusively restricted to complex I (proton-pumping NADH-ubiquinone oxidoreductase) together constitute the most common cause of respiratory chain deficiencies (MIM# 252010). These genetic alterations are responsible for a broad spectrum of clinical features, ranging from syndromes affecting multiple organs like Leigh syndrome and mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), to single organ diseases including Leber hereditary optic neuropathy (LHON) or hypertrophic cardiomyopathy (Fassone & Rahman, 2012;Fiedorczuk & Sazanov, 2018;Rodenburg, 2016). In addition, complex I defects have been associated with adult onset of neurodegenerative disorders such as Parkinson disease (Fassone & Rahman, 2012;Fiedorczuk & Sazanov, 2018). Mitochondrial respiratory chain complex I is the largest complex of OXPHOS. It is composed of 14 central subunits, conserved from bacteria to humans, at least 30 accessory subunits, and 14 or 15 assembly factors (Formosa et al., 2018;Guerrero-Castillo et al., 2017). Significant advances have recently been made on the elucidation of the structure and assembly steps of complex I. The structure resembles an L shaped form, where one arm is hydrophilic and oriented into the mitochondrial matrix, while the other is hydrophobic and integrated into the mitochondrial inner membrane. The hydrophilic arm is divided into an N module (NADH oxidation) and a Q module (ubiquinone reduction), while the hydrophobic arm is constituted by a P module (proton pumping). Due to the existence of four pumping sites, the P module can be divided in proximal, a and b (P P -a and P P -b), and distal, a and b (P D -a and P D -b). The last module P D -b, in fully assembled complex I, is composed of the central subunit ND5 and accessory subunits NDUFB2, NDUFB3, NDUFB7, NDUFB8, and NDUFB9 (Formosa et al., 2018(Formosa et al., , 2020Galemou Yoga et al., 2020;Guerrero-Castillo et al., 2017;Parey et al., 2018;Wirth et al., 2016).
Deleterious alterations in proteins of the P D -b module have been described and related with complex I disorders. Like other defects in this complex, these mutations could cause a wide range of clinical phenotypes. Mutations in MT-ND5 have been linked with distinct mitochondrial disorders such as Leigh syndrome (MIM# 256000), MELAS (MIM# 540000), and LHON (MIM# 535000; Danhelovska et al., 2020). The single pathogenic mutation described for NDUFB9 is characterized by progressive hypotonia and increased serum lactate (Haack et al., 2012b). Pathogenic variants in NDUFB3 are associated with intrauterine growth retardation, encephalopathy, myopathy, hypotonia, and lactic acidosis (Calvo et al., 2012;Haack et al., 2012a).
Similarly, deleterious mutations in NDUFB8 have been reported in individuals with a progressive disease characterized by encephalomyopathy, cardiac hypertrophy, respiratory failure, hypotonia, and lactic acidosis. These patients displayed abnormalities in brain magnetic resonance imaging (MRI) consistent with Leigh syndrome (Piekutowska-Abramczuk et al., 2018). Regarding NDUFB7 and NDUFB2, no pathogenic mutations have been identified in patients. However, a critical role for NDUFB7 in complex I assembly in Yarrowia lipolytica has been suggested (Dröse et al., 2011). Furthermore, in vitro experiments have suggested that the absence of any of the accessory subunits of the P D -b module may critically affect the assembly and function of human complex I (Dröse et al., 2011;Formosa et al., 2020;Stroud et al., 2016). Here we report the first case of a mitochondrial disorder caused by an intronic mutation leading to a cryptic splice site in the NDUFB7 gene. The affected patient was born in 2015 of consanguineous Iranian parents with an unaffected 2 years older sister. Pre-natal features included cardiomegaly detected at the gestational Week 25, intrauterine anemia (that required two transfusions) and intrauterine growth restriction. showed widely distributed cysts, likely due to prenatal events and possible dysplasia of the corpus callosum. MR spectroscopy revealed increased lactate and decreased N-acetylaspartate in the basal ganglia. The patient suffered from repeated desaturations and was treated in hospital until his death at 55 days of age from cardiorespiratory failure. Newborn screening did not reveal any underlying causes for the patient's phenotype. Urinary organic acids were normal, except for a transiently increased excretion of lactate. Amino acids, acylcarnitines, and carnitine levels in plasma were also normal.
Analysis of alpha-glucosidase enzyme activity was normal, excluding Pompe disease as a cause of disease. The PDHA1 gene was sequenced, since pathogenic mutations in this gene are a common cause of early lactic acidosis in children. Mitochondrial ATP production ( Figure S1) and respiratory chain enzyme activities ( Figure 1a) in mitochondria isolated from skeletal muscle were determined as previously described (Wibom et al., 2002). These analyses revealed a severe complex I defect that was later confirmed by Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE;  (Calvo et al., 2012;Danhelovska et al., 2020;Haack et al., 2012a;2012b;Piekutowska-Abramczuk et al., 2018). It is also noteworthy that the case reported herein is a prime example of the F I G U R E 2 Protein status in NDUFB7 patient and complementation studies with retroviral transfection of WT-NDUFB7. (a) Representative western blot analysis of NDUFB7 protein in primary (p) and immortalized (i) fibroblasts from patient (P), control (C) and father (F). VDAC (Porin) was used as loading control. (b) Representative Western blot analysis of transduced and non-transduced immortalized fibroblasts. VDAC (Porin) was used as loading control and protein signal was determined as described with appropriate antibodies. Complex I modules are given between brackets.
(c) In-gel activity of Complex I, IV, and V in BN-PAGE. Mitochondrial extracts were separated by BN-PAGE and gels were incubated with corresponding substrates as described. (d) Oxygen consumption in permeabilized cells from controls (white) and patient (black) and respective NDUFB7 transduced cells (diagonal bars). Measurements were performed in the presence of complex I substrates GMP (Glutamate, Malate, and Pyruvate) with ADP (State III) and without ADP (State IV); complex II substrates GMP, ADP, and Succinate; Complex II activity was measured in the presence of Rotenone. Bars represent mean ± SD (n = 4). BN-PAGE, Blue Native Polyacrylamide Gel Electrophoresis are now possible to detect by WGS and evaluate by RNA-Seq. This