Bi-allelic Mutations in NDUFA6 Establish Its Role in Early-Onset Isolated Mitochondrial Complex I Deficiency

Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects’ fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects’ fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis. Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects’ fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects’ fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis. Isolated complex I deficiency (OMIM: 252010) is a common biochemical phenotype observed in pediatric mitochondrial disease. The associated clinical and genetic heterogeneity is vast—individuals can present with a plethora of clinical symptoms ranging from isolated myopathy to Leigh syndrome (OMIM: 256000). Complex I is the first and largest complex of the mitochondrial respiratory chain and comprises 45 structural subunits with a minimal contingent of 20 ancillary proteins required for assembly and/or biogenesis.1Formosa L.E. Dibley M.G. Stroud D.A. Ryan M.T. Building a complex complex: Assembly of mitochondrial respiratory chain complex I.Semin. Cell Dev. Biol. 2018; 76: 154-162Crossref PubMed Scopus (100) Google Scholar The genes encoding these proteins involve either the mitochondria’s own genetic material (mtDNA) or a nuclear-encoded gene of mitochondrial function,2Calvo S.E. Clauser K.R. Mootha V.K. MitoCarta2.0: An updated inventory of mammalian mitochondrial proteins.Nucleic Acids Res. 2016; 44: D1251-D1257Crossref PubMed Scopus (854) Google Scholar and to date, causative defects have been identified in 39 genes encoding either complex I structural subunits or assembly factors.3Frazier A.E. Thorburn D.R. Compton A.G. Mitochondrial energy generation disorders: Genes, mechanisms and clues to pathology.J. Biol. Chem. 2017; (Published online December 12, 2017)https://doi.org/10.1074/jbc.R117.809194Crossref PubMed Scopus (120) Google Scholar, 4Piekutowska-Abramczuk D. Assouline Z. Mataković L. Feichtinger R.G. Koňařiková E. Jurkiewicz E. Stawiński P. Gusic M. Koller A. Pollak A. et al.NDUFB8 mutations cause mitochondrial complex I deficiency in individuals with Leigh-like encephalomyopathy.Am. J. Hum. Genet. 2018; 102: 460-467Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Additionally, complex I deficiency can occur as a secondary consequence of dysfunction involving alternative mitochondrial processes.5Haack T.B. Kopajtich R. Freisinger P. Wieland T. Rorbach J. Nicholls T.J. Baruffini E. Walther A. Danhauser K. Zimmermann F.A. et al.ELAC2 mutations cause a mitochondrial RNA processing defect associated with hypertrophic cardiomyopathy.Am. J. Hum. Genet. 2013; 93: 211-223Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 6Freisinger P. Haack T. Kopajtich R. Johannes M. Ahting U. Sperl W. Plecko B. Wilichowski E. Meitinger T. Prokisch H. Mitochondriopathy due to mutations in MTFMT: A predominant neurologic phenotype.Neuropediatrics. 2013; 44 (FV12_03)Crossref Google Scholar Application of next-generation sequencing in the form of panel-based target capture or whole-exome sequencing has already demonstrated its utility in the diagnosis of heterogeneous conditions such as mitochondrial disease and has been the catalyst for disease-associated gene discovery through its application to undiagnosed cohorts.7Ohtake A. Murayama K. Mori M. Harashima H. Yamazaki T. Tamaru S. Yamashita Y. Kishita Y. Nakachi Y. Kohda M. et al.Diagnosis and molecular basis of mitochondrial respiratory chain disorders: Exome sequencing for disease gene identification.Biochim. Biophys. Acta. 2014; 1840: 1355-1359Crossref PubMed Scopus (43) Google Scholar Facilitated by the GeneMatcher tool,8Sobreira N. Schiettecatte F. Valle D. Hamosh A. GeneMatcher: A matching tool for connecting investigators with an interest in the same gene.Hum. Mutat. 2015; 36: 928-930Crossref PubMed Scopus (821) Google Scholar we report the findings from four unrelated, clinically affected children who presented with symptoms suggestive of mitochondrial disease. Subject 1, a female infant, was the first child of healthy non-consanguineous Hungarian parents. On antenatal anomaly scan, she was noted to be small for her gestational age and have intracranial ventriculomegaly. Assessment by fetal medicine confirmed symmetrical growth restriction on the 3rd percentile, and fetal MRI at 30 weeks of gestation confirmed ventriculomegaly with mild cerebellar hypoplasia. She was born at term (37 + 3 weeks) by elective caesarean section for intrauterine growth restriction. Her birth weight was 1,660 g (≪0.4th percentile [<−4 SD]), and head circumference was 32.5 cm (2nd–9th percentile). At delivery, she required five inflation breaths and was initially placed on continuous positive airway pressure. Venous cord gas showed a pH of 7.25 and a base excess (BE) of −5.00. Within 40 min, she was self-ventilating in room air, but a capillary blood gas at that time revealed metabolic acidosis (pH = 7.12, lactate = 5.9 mmol/L, BE = −10.2) and hypoglycaemia (blood glucose = 1.2 mmol/L). A 10% dextrose infusion (60 mL/kg/day) was commenced, and benzylpenicillin and gentamicin were administered. Subsequent capillary blood gas analyses revealed an improvement in the metabolic acidosis but persistent hypoglycaemia (pH = 7.31, glucose = 1.8 mmol/L). A further bolus of 10% dextrose was administered before the infusion rate was increased to 90 mL/kg/day. Although the metabolic acidosis showed some improvement, the capillary blood lactate remained elevated (despite boluses of 10% dextrose and 0.9% saline). Repeat blood analyses revealed clotting abnormalities, hyperammonaemia (129 μM; normal < 100 μM), and a rising blood lactate (9.3 mmol/L; normal < 2.2 mmol/L). An abdominal ultrasound showed echogenic linear areas in the liver and normal kidneys. A second dose of vitamin K, followed by fresh-frozen plasma (FFP), sodium bicarbonate, 0.9% saline bolus, and frusemide, was administered. Apnoea at around 24 hr of age necessitated intubation and artificial ventilation before transfer to the regional neonatal intensive-care unit. Clinical examination demonstrated severe generalized hypotonia and absent primitive reflexes. Cerebral function monitoring (amplitude-integrated electroencephalography) showed a significantly suppressed baseline and no detectable seizure pattern. Cranial MRI undertaken at just over 36 hr of age demonstrated a diffusely abnormal signal intensity of the entire supratenterial white matter and decreased cortical folding for her age (Figure 1A), as well as of the brainstem (Figure 1B) and cerebellar white matter. Over the next 12 hr, despite extensive resuscitative efforts, including concomitant administration of three different inotropes (dopamine, dobutamine, and noradrenaline) and further FFP and packed red cell transfusion, the pupils became fixed and dilated, and she continued to deteriorate. Clotting remained abnormal (international normalized ratio = 3.2; normal < 1.1), although with the exception of gamma-glutamyl transpeptidase (1,225 IU; normal < 271 IU), liver-function tests were normal. Lactic acidosis exhibited relentless progression with a blood lactate reading in excess of 20 mM immediately before death at approximately 51 hr of age. Subject 2 is the first son of healthy second-cousin Kurdish parents, who have a healthy younger daughter. He was born by normal vaginal delivery at 36 weeks of gestation (birth weight = 2.8 kg [50th percentile]) after an uneventful pregnancy. He spoke his first words at 12 months and at 16 months was walking independently. By 2 years, his parents noted a general physical weakness, and at 25 months, shortly after an unexplained fever, his gait deteriorated. Further motor regression and rapid onset of spasticity of all four limbs followed. He developed swallowing difficulties and required a gastrostomy tube for feeding. At 2.5 years, was and was to or He showed some over months, which and by 3 was walking with Although have been no further of rapid has and has and from of age. Over the 2 years, has all in his is just and but his is He has an for and 3 is as a of is normal. investigations revealed normal lactate in blood mmol/L; normal < 2.2 and mmol/L; normal < mmol/L). and of not MRI showed extensive and in including and the in the and some of which with (Figure revealed elevated lactate in On the were not and and (Figure Subject a infant, was born to parents after a by and intrauterine growth restriction. was by caesarean section at 36 and although not at was noted to be small for his gestational age kg percentile]) and including an mild and At 3 weeks became but continued to 3 months, an onset of At months after to the for was that and was hypotonia with and of his seizure revealed elevated lactate mmol/L; normal < and increased signal in the on remained despite and also with of to the that by 2.5 years, developed first noted at 2 years, was with By years, his and diffusely on his although head growth was and remained in all At his age of years, but is to or He is but to is significantly in to of his symptoms which confirmed involving the cortical matter of the as well as a A second showed elevated lactate mmol/L; normal < and of revealed of = Subject 3 has a healthy younger parents also at the of and from were to also have but no were for analysis. Subject a female infant, was the first child of non-consanguineous white parents, was The was born at term by vaginal delivery, and the required no (birth weight = kg head circumference = At she was to the after a of suggestive of a seizure On was but blood gas lactate was She as a of and persistent intensive-care in a her acidosis continued her death at weeks of age. pH from to blood gas lactate from to = and the lactate was at = mmol/L). MRI shortly after identified in the and clinical were with a diagnosis of mitochondrial prompting genetic analysis. and were from and to for metabolic was for 3 but revealed no analyses of confirmed isolated complex I deficiency in and 2 and of of from 3 revealed normal complex I despite clinically suggestive of mitochondrial disease. of complex assembly were undertaken in fibroblasts from 3 and revealed a marked complex I assembly defect in a clinical diagnosis of complex I for and was for all in with the of and by genetic investigations were for all four of the entire mitochondrial (mtDNA) revealed only suggestive of an underlying genetic for Next-generation sequencing was undertaken in an to genetic a was for 1, as M. He L. S. et mitochondrial variant a and a mild biochemical and clinical Genet. 2016; PubMed Scopus Google Scholar whole-exome sequencing was for 2 and the was with the Exome and on a were to the was as R. Pollak A. S. M. E. J. P. M. 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A highly for and of mitochondrial variants in 2014; PubMed Scopus Google Scholar structural and variants were for < in including and the and for or variants to be pathogenic in or variants were to with a in excess of causative variants in an subunit of complex NDUFA6 (Figure Subject variants NDUFA6 a to cause a and a to cause a Subject 2 was to a 2 to cause a 3 a to cause the of the Subject was to and in of of NDUFA6 the of 2 was for the NDUFA6 The variant is in individuals in = The variant is in one in = as is the variant The variant is in 16 individuals in = The and variants in and of the subjects’ NDUFA6 variants in the in with a and support for a NDUFA6 variants have been to The and NDUFA6 variants can be as pathogenic to of and S. N. S. D. S. J. M. E. E. et and for the interpretation of A consensus of the of and and the for 2015; Full Text Full Text PDF PubMed Scopus Google Scholar the variant is to be The variant of 12 and in of = = = = = = often as the of and a of is of a M. P. 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T. et diagnosis of RNA 2017; PubMed Scopus Google Scholar support as the predominant and RNA data with the of (Figure These data have to be associated with the accession number to further the consequence of the subjects’ NDUFA6 we investigations with fibroblast cell lines from for were of fibroblasts was with the as D. E. for and from small of Biol. PubMed Scopus Google Scholar and revealed a of cell in cell of 2 and 3 was to that of fibroblasts (Figure of mitochondrial from fibroblasts and cell lines was as M. J. Haack T.B. He L. E. et mutations cause mitochondrial disease of the 2015; PubMed Scopus Google Scholar using structural subunits from complex I complex complex III complex IV and complex revealed a reduction of assembled complex I levels, all other were Complex I with assembly were in the subjects’ cell but not in (Figure Subject 1, who presented with the clinical the marked reduction in assembled complex 2 and 3 were also complex I although to a of complex I structural were with of and fibroblast cell to M. J. Haack T.B. He L. E. et mutations cause mitochondrial disease of the 2015; PubMed Scopus Google Scholar using structural subunits of complex I and revealed a marked reduction in the of complex I subunits for 1, whereby the and subunits were affected (Figure A but was observed for 2 and with analyses (Figure of complex and were as of fibroblasts with as a of complex I revealed of complex I in 2 in (Figure that pathogenic variants have not been in NDUFA6, fibroblast cell lines from were to as D.A. A.E. E. H. M. S. et is a mitochondrial complex IV assembly for of Genet. 2015; PubMed Scopus Google Scholar Subsequent by confirmed that transduction of all three cell lines with a NDUFA6 transcript the complex I assembly the subjects’ NDUFA6 variants as the cause of disease (Figure of from the fibroblast cell lines from and 3 the with of complex I (Figure of the fibroblast cell from 2 was but was to further the consequence of the subjects’ NDUFA6 variants on mitochondrial complex I we complexome of (Figure and fibroblasts (Figure as I. S. T. N. B. of the mitochondrial complex I assembly 2018; PubMed Scopus Google Scholar, H. L. M. J. S. B. M. I. I. U. as a of the mitochondrial complex I assembly Full Text Full Text PDF PubMed Scopus Google Scholar of complexome data the assembly defect observed after of fibroblasts from and A reduction of subunits was in the affected (Figure the assembly defect with the observed A of and from the is in 1, and to with the of NDUFA6 is that NDUFA6 is a for and the complex I and that of NDUFA6 or its function in and with the subunits of the N-module. or marked reduction 2 and and of the mitochondrial from complex I is also from complexome is that the of NDUFA6 has been demonstrated to be for in the H. M. M. M. K. H. I. U. The subunit of mitochondrial complex I an and is for 2014; PubMed Scopus Google Scholar Interestingly, the complexome data evidence of a complex I assembly to the assembly intermediate (Figure has been observed as a consequence of pathogenic mutations affecting subunits (OMIM: and (OMIM: I. A molecular for mitochondrial complex I assembly is in a PubMed Scopus Google Scholar The of to from the assembly intermediate after of either or Q-module subunits the that of a in complex I M. Ryan M.T. Assembly of mitochondrial complex I and defects in PubMed Scopus Google Scholar the of subunits, complexome data support an of the kDa intermediate to in supercomplex formation, whereby assembled III and IV in complex with the P- and of complex I NDUFA6, NDUFA7, and the Q-module associated of This supercomplex does not a its of but is with other findings that subunits not to as supercomplex M. M. A. Thorburn D.R. Ryan M.T. of the assembly for and subunits complex Biol. PubMed Scopus Google Scholar, D.A. L.E. B. A.E. Dibley M.G. T. Thorburn D.R. et subunits for assembly and function of mitochondrial complex 2016; PubMed Scopus Google Scholar This is also by the that not a subunit of the with of the structural subunits of either complex III or IV in the M. J. R. Y. M. of mammalian respiratory supercomplex 2016; Full Text Full Text PDF PubMed Scopus Google Scholar identified genes associated with a biochemical of complex I deficiency the assembly (OMIM: Kopajtich R. A. Haack T.B. E. T. et diagnosis of RNA 2017; PubMed Scopus Google Scholar and (OMIM: Compton A.G. L.E. M. Haack T.B. J. K. P. E. et mutations in cause severe complex I deficiency with a clinical J. Hum. Genet. 2016; Full Text Full Text PDF PubMed Scopus (43) Google Scholar, L. B. L. Assouline Z. M. B. S. et in complex I assembly in and isolated complex I J. Hum. Genet. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar the (OMIM: M. A. P. A. R. L. et mutations in with complex I Genet. 2018; PubMed Scopus Google Scholar and (OMIM: a subunit of the RNA S. E. W. M. in with mitochondrial dysfunction and 2017; PubMed Scopus Google Scholar The an isolated complex I deficiency and the genetic defect is often not and can in occur as a secondary consequence of mitochondrial dysfunction in bi-allelic pathogenic variants in (OMIM: encoding a mitochondrial that for in mitochondrial often in an isolated complex I deficiency despite affecting T.B. B. Wieland T. A. M. E. U. et diagnosis in mitochondrial complex I deficiency using Genet. PubMed Scopus Google Scholar The isolated complex I deficiency observed in fibroblasts from and 2 is with the bi-allelic NDUFA6 variants identified that NDUFA6 an structural subunit of complex I. NDUFA6 is one of subunits that have been to be for complex I assembly and D.A. L.E. B. A.E. Dibley M.G. T. Thorburn D.R. et subunits for assembly and function of mitochondrial complex 2016; PubMed Scopus Google Scholar Subject who biochemical evidence of complex I that his NDUFA6 variant is to the is common for fibroblasts to normal complex I despite a marked deficiency in a clinically relevant H. A. R. Compton A.G. et chain complex I deficiency by mitochondrial J. Hum. Genet. PubMed Scopus Google Scholar but the for in despite a complex I assembly Although the fibroblast cell from 3 also demonstrated normal complex I not a growth defect was demonstrated was with the to be for (Figure The of the NDUFA6 the normal complex I for variant as a variant in the encoding transcript that the variant be = = = = = The is due to the of alternative transcripts in the and is by the associated with the in the alternative to an alternative was for in have been of of proteins complexome was and have to with the target in and not is that NDUFA6 is to and in of for in the of and NDUFA6 in the in the of the pathogenic The is not to the of the NDUFA6 variants by or variants to the same in an This report bi-allelic NDUFA6 variants in four pediatric who have a clinical diagnosis of mitochondrial disease. the three with tissues, associated complex I assembly defects were demonstrated with of the biochemical This NDUFA6 as the structural subunit of complex I to be associated with that one normal complex I in report the importance of an to genetic diagnosis. variant interpretation and assessment of appropriate transcripts an and a for the in The no in is by the for Mitochondrial the for the in and to the the the for Mitochondrial the the of the to and to the of Mitochondrial and the through the for Mitochondrial the the the and the a is by the and the is by and is by the Mitochondrial is by the and and by the and We for for to and for the The of the and not of the or of The accession for variants and and with and

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