Biallelic Loss‐of‐Function NDUFA12 Variants Cause a Wide Phenotypic Spectrum from Leigh/Leigh‐Like Syndrome to Isolated Optic Atrophy

Abstract Background Biallelic loss‐of‐function NDUFA12 variants have hitherto been linked to mitochondrial complex I deficiency presenting with heterogeneous clinical and radiological features in nine cases only. Objectives To fully characterize, both phenotypically and genotypically, NDUFA12‐related mitochondrial disease. Methods We collected data from cases identified by screening genetic databases of several laboratories worldwide and systematically reviewed the literature. Results Nine unreported NDUFA12 cases from six pedigrees were identified, with presentation ranging from movement disorder phenotypes (dystonia and/or spasticity) to isolated optic atrophy. MRI showed basal ganglia abnormalities (n = 6), optic atrophy (n = 2), or was unremarkable (n = 1). All carried homozygous truncating NDUFA12 variants, three of which are novel. Conclusions Our case series expands phenotype–genotype correlations in NDUFA12‐associated mitochondrial disease, providing evidence of intra‐ and inter‐familial clinical heterogeneity for the same variant. It confirms NDUFA12 variants should be included in the diagnostic workup of Leigh/Leigh‐like syndromes – particularly with dystonia – as well as isolated optic atrophy.

NDUFA12 variants should be included in the diagnostic workup of Leigh/Leigh-like syndromesparticularly with dystoniaas well as isolated optic atrophy.
NDUFA12 is a nuclear gene encoding the supernumerary subunit A12 of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase), the foremost multimeric enzyme of the respiratory chain which contributes 40% of the proton driving force for ATP synthesis. 1 Subunit A12 is proposed to act in assembling and stabilizing the extramembrane arm of CI. 2 The first reported case of NDUFA12-related mitochondrial disease was a Pakistani child with Leigh syndrome (LS) carrying a homozygous nonsense variant. 1 Few NDUFA12 cases have been described since then, with clinical manifestations ranging from complex neurological syndromes with prominent dystonia/ spasticity and MRI evidence of basal ganglia (BG) changes to isolated optic atrophy (OA) in one case. 3,4 We report nine additional cases from six kindreds (Fig. 1A), all carrying biallelic NDUFA12 variants, three of which are novel, and review cases previously reported.

Methods
To fully characterize the clinical spectrum and course of NDUFA12-related mitochondrial disease, we collected phenotypic and genetic data from cases identified by retrieving databases of several diagnostic and research genetic laboratories worldwide. Variants were prioritized according to the following criteria: (1) variant in NDUFA12 coding regions or at exonintron boundaries; (2) gnomAD v3.1 frequency < 0.001; (3) no phenotype restriction. Among resulting variants, we selected pathogenic and likely pathogenic variants according to the American College of Medical Genetics and Genomics (ACMG) standards and guidelines, 5 in particular for variants with unequivocal or highly predicted loss-of-function effect. A systematic literature review of NDUFA12 cases previously reported was performed by consulting PubMed ® and Google Scholar using "NDUFA12" as search term on 31/05/2021. Predetermined clinical and genetic features were extracted and tabulated. Finally, genes associated with both dystonia and optic neuropathy were identified through Genomics England PanelApp (https://panelapp. genomicsengland.co.uk/) and searched on PubMed ® using the gene name as search term on the same date. PubMed ® results were filtered for article type "review" and/or "systematic review". "Mutation" and "patient" were occasionally used as additional search terms to rule out non-clinical studies. Phenotypes associated with the above-mentioned genes were outlined to delineate the differential diagnosis of NDUFA12-associated mitochondrial disease.

Clinicogenetic Characterization of New Cases
Pedigree A Case 1: This 10-year-old Pakistani female, born to healthy consanguineous parents, was delivered at 37 weeks following labor induction due to intrauterine growth restriction (birth weight: 1.9 kg). She had unremarkable developmental milestones. She experienced a febrile seizure at age 4 and developed progressive gait and posture impairment as well as left arm weakness since age 6.5, when brain MRI revealed symmetrical T2-hyperintensity of the posterior putamen ( Fig. 1B), with lesions demonstrating a mixed pattern of chronic gliosis and more acute features of cytotoxic oedema. She experienced an episode of status dystonicus at age 8. One year later, she was admitted to the hospital due to an episode of prolonged lethargy which was ultimately attributed to accidental baclofen overdosing. Paired lactate values in CSF and blood were normal. Repetitive nerve conduction studies (NCS)/electromyography (EMG) were unremarkable. Muscle biopsy revealed non-specific mild predominance of slow fibers and mild myopathic features, whereas assessment of respiratory chain enzymes (RCE) showed low CI activity (0.034; normal: 0.104-0.268) and normal activity of complexes II, III, and IV. Genetic testing for common mitochondrial and nuclear genes associated with Leigh syndrome came back to be normal. She was started on coenzyme Q10, thiamine, biotin, and anticholinergics and lost to follow-up between the age of 7 and 10 years. On examination at age 10, she was wheelchair bound with scoliosis and truncal hypotonia, limb flexor spasticity and dystonic posturing of the extremities. Her cognitive functions were unremarkable. Due to recent deterioration of her visual function in the absence of any identifiable traumatic, inflammatory, or infectious trigger, the patient underwent an ophthalmological assessment and was diagnosed with significant visual deficit and severe OA. An extensive next-generation sequencing panel for nuclear mitochondrial genes revealed a homozygous NM_018838.5(NDUFA12):c.178C > T (p.Arg60*) variant.

Pedigree B
Case 2: This 21-year-old Turkish male, product of a third-degree consanguineous marriage, developed progressive kyphoscoliosis and gait difficulty with left foot in-turning since age 7. Dystonia did not respond to levodopa or anticholinergics and became generalized over 2 years. The patient was wheelchair bound at age 11 and experienced intractable focal seizures since age 12. Although there was no history of intellectual disability, he showed cognitive deterioration with disease progression. On examination, he showed generalized dystonia, with dysarthria and feeding difficulty due to oromandibular involvement, kyphoscoliosis, left hand clenching, lower-limb hyperreflexia, and diffuse muscle atrophy ( Fig. 1C; Video 1). There was no visual impairment. walking (Video 1). Lower-limb hyperreflexia and kyphoscoliosis were also detected. His cognitive functions were normal, and he graduated from university.

Pedigree C
Case 4: This 9-year-old Egyptian male, born to first cousins, had normal development until age 2, when he started walking on his tiptoes and falling. Achilles tenotomy surgery provided transient improvement. After age 4, he developed right hand tremor, scoliosis, and progressive stiffness in his lower limbs, with loss of independent walking. He was reported having school problems, but he had never undergone intelligence testing nor shown any deterioration of his cognitive function on follow-up. On examination, he showed kyphoscoliosis, acral dystonia with dystonic hand tremor, and lower-limb spasticity ( Fig. 1C; Video 1). His visual function was unremarkable. Serum ceruloplasmin, creatine kinase, and amino acids and acylcarnitines were normal. He had increased urine lactate. Brain MRI revealed T2/FLAIR-hyperintensity and T1hypointensity with cystic areas in the BG (Fig. 1B).
Case 5: One of Case 4's elder sisters developed right hand grip weakness since age 16. Her past medical history was otherwise unremarkable, including absence of cognitive and visual issues. Brain MRI showed bilateral T2/FLAIR-hyperintensity of globi pallidi.

Pedigree D
Case 6: This 16-year-old Saudi male, son of first cousins, experienced motor developmental delay and progressive gait unsteadiness with frequent falls since age 2. He developed fixed flexion of the right hand which progressed to right hemiplegia, and severe visual impairment. He was reported with mild attention deficit disorder, but there was no history of intellectual disability nor cognitive deterioration. Brain MRI detected symmetrical T2-hyperintensity of the BG, with cystic degeneration on the left. Neurological examination revealed dysarthria and limb spasticity. Ophthalmological assessment revealed OA. Plasma lactate was normal on two occasions, whereas plasma pyruvate was increased (3.07 mg/dl; normal: 0.3-0.7). Case 7: Case 6's 12-year-old brother had a history of severe global neurodevelopmental delay, balance difficulties and falls since age 6. There was no history of intellectual disability nor cognitive impairment. On examination, he was dysarthric and wheelchair bound due to spastic quadriplegia. Brain MRI detected symmetrical T2-hyperintensity of the BG. Plasma lactate was 4.9 mmol/L (normal: 0.5-1).
WES revealed a homozygous NM_018838.5(NDUFA12): c.4G > T (p.Glu2*) variant in both siblings. Segregation analysis revealed that their younger sister, who was asymptomatic and did not present any neurological or ocular manifestations on examination at age 9, was homozygote for the same mutant allele.

Pedigree E
Case 8: This 16-year-old Syrian male, product of consanguineous parents, had a one-year history of progressive bilateral visual loss without any identifiable trigger. He had an asymptomatic sibling. On examination, visual acuity (VA) was 20/1600 bilaterally, with normal intraocular pressure and anterior segment US biomicroscopy. Direct and consensual pupillary light reflexes were absent. Fundoscopy disclosed mild optic disc pallor bilaterally, with cup-to-disc ratios 0.2 (right eye) and 0.4 (left eye), narrow temporal rim of the left optic disc, and retinal arterial tortuosity bilaterally. Fundus autofluorescence was normal (Fig. 1D). His neurological assessment was otherwise unremarkable, his cognitive functions were normal, and his mood was low. Serum lactate was normal. Serological testing for HIV, syphilis and HTLV was negative. Paraneoplastic antibodies and rheumatological workup including serum anti-AQP4, anti-MOG, and anti-CRMP5 antibodies were unremarkable, as well as serum thiamin, cyanocobalamin, and folate levels. He had normal brain and orbit MRI, and slightly increased CSF protein levels (63.9 mg/dL; normal: <45). WES revealed a homozygous NM_018838.5(NDUFA12): c.253G > T (p.Glu85*) variant.

Pedigree F
Case 9: This 33-year-old Turkish male born to consanguineous parents presented at age 28 with sudden bilateral painless visual loss (VA 20/40), which slowly progressed over the following years. No triggers were identified at symptom onset. There was a history of mild intellectual disability. His VA was 20/200 bilaterally at age 30 and remained relatively stable ever since. Perimetry revealed central scotomas spanning most of the central 30 degrees of the visual field bilaterally, more pronounced in the left eye. Intraocular pressure was normal, and anterior segment US biomicroscopy was unremarkable aside from mild cataract. Pupils were isochoric, with the left one showing relative afferent deficit. Fundoscopy revealed pale optic discs with cup-to-disc ratios 0.7 (right eye) and 0.8 (left eye), whereas macula, peripheral retina, and vessels were unremarkable. Bilateral optical coherence tomography detected markedly reduced thickness of the peripapillary retinal nerve fiber layer and microcystic macular edema (left>right; Fig. 1E). Neurological assessment was otherwise unremarkable, with cognitive functions being unchanged since early childhood. Serum lactate levels were not tested. MRI brain orbits detected optic chiasm atrophy. Screening for cardiovascular risk factors, including sleep apnea, was unremarkable. An NGS panel for nuclear and mitochondrial OA-associated genes detected a heterozygous ENST00000304511.2(TMEM126A):c.314G > A (p.Arg105Gln) variant, whose causal relationship was excluded based on high frequency in population databases and in silico prediction tools. Whole-genome sequencing revealed a homozygous variant NM_018838.5(NDUFA12):c.83del (p.Phe28Serfs*11).  Fig. 1G). Intriguingly, the asymptomatic sibling of two affected individuals (Pedigree D) carried the mutant allele in the homozygous state, thus suggesting either incomplete penetrance or further intra-familial variability with respect to age at symptom onset. 6 Discussion CI deficiency is the commonest biochemical defect in children with mitochondrial diseases, including LS/Leigh-like syndrome (LLS). 7-9 Diagnosis of LS requires progressive neurological deterioration with clinical evidence of BG and/or brainstem dysfunction, developmental delay, and elevated serum or CSF lactate, along with neuroradiological or neuropathological evidence of BG and/or brainstem lesions. 8 When these stringent criteria are not fulfilled (e.g., atypical neuroimaging, normal lactate levels), a diagnosis of LLS can be considered. 8 Six cases in our series fulfilled the diagnostic criteria for LS/LLS, thus providing further evidence that biallelic loss-of-function NDUFA12 variants are a rare cause of these phenotypes. 1,3 Dystonia, either isolated or combined with pyramidal features, emerges as the most common motor feature in NDUFA12related LS/LLS. As dystonia was always associated with MRI evidence of BG damage in our series and previous cases, a secondary etiology (structural damage caused by mitochondrial dysfunction) most likely explains its occurrence in NDUFA12-related LS/LLS. 10 By contrast, we confirmed that biallelic NDUFA12 variants are associated with isolated OA, even in the absence of MRI findings, as previously reported in one case. 3 In the original case reported by Ostergaard et al. 1 and Case 1, visual impairment occurred some years after disease onset, thus suggesting visual function should be assessed on follow-up of patients harboring biallelic NDUFA12 variants with pure motor presentations. Overall, this adds to the observation that the best-established nuclear genes linked to OA (Table S1) are involved in mitochondrial pathways, as are pathogenic variants in mitochondrial DNA accounting for Leber hereditary optic neuropathy. 11 An overview of genes associated with both dystonia and OA is provided in Table S1. Unlike several of these genes, which are recognized with both dominant and recessive inheritance, NDUFA12 is more likely associated with recessive inheritance only since the observed/expected ratio for both missense and loss-of-function variants is close to 1 according to gnomAD. Finally, we highlight that increased lactate levels were detected in only two out eight cases of our new cohort when this testing was performed, whereas plasma pyruvate level was increased in one case with normal plasma lactate. This proportion is lower than in previously reported cases, which is in keeping with a higher prevalence of LLS phenotype in our series. 8 Our series expands the age of onset of NDUFA12-related mitochondrial disease to as late as 28 years. Furthermore, it demonstrates significant intra-familial variability (e.g., Cases 2-3, 4-5, 6-7) and occurrence of the same NDUFA12 variant in patients/ kindreds with different phenotypes (inter-familial heterogeneity, e.g., p.Glu85* associated with OA in Case 8 and LS in Pedigree N). This suggests that yet undetermined genetic, epigenetic, and environmental factors modulate the variable expression of mutant NDUFA12 alleles at the phenotypic level. 6 In conclusion, our case series expands the phenotypegenotype spectrum of NDUFA12-associated mitochondrial disease and provides evidence of inter-and intra-familial clinical heterogeneity associated with the same variant. It supports the inclusion of NDUFA12 variants in the diagnostic workup of not only LS/LLS, particularly when dystonia is the prominent motor manifestation, but also isolated OA.