This study was approved by the Institutional Review Board of the George Washington University and Children’s National Medical Center and was conducted in accordance with the ethical principles of the Declaration of Helsinki of 1975 (revised 1983). Patient skin biopsy was performed after receiving written informed consent from the legally authorized representatives (parents of the proband) with permission to study the derived dermal fibroblasts.

A 3 mm skin biopsy was performed on the 5-year-old proband, from which dermal fibroblasts were derived as described (Uittenbogaard et al., 2018a). Derived fibroblasts were frozen at passage two and never used beyond passage 10. Human primary dermal fibroblasts from a healthy subject (Cat# GM03377E) were obtained from the Coriell Cell Repositories (Camden, NJ).

Fibroblasts from the proband and a control subject were fixed in 2.5% glutaraldehyde, 1% paraformaldehyde in 0.12 M sodium cacodylate buffer as described (Uittenbogaard et al., 2018b). Samples were imaged with a FEI Talos F200X-transmission electron microscope (Thermo Fisher).

Total genomic DNA was isolated from blood samples from the proband to perform WES and LR-PCR-MPS of the mitochondrial genome by the Medical Genetics Laboratories at Baylor College of Medicine, as described (Cui et al., 2013; Uittenbogaard et al., 2019). Reads were aligned to the human reference genome (UCSC hg19) using the NextGEN software (SoftGenetics; State College, PA). Variants were identified and annotated using an in-house bioinformatic pipeline with our filtering strategy summarized in Figure 2 of Uittenbogaard et al. (2019). The pathogenicity of variants was evaluated using the American College of Medical Genetics and Genomics guidelines by board-certified molecular geneticists. Computational analysis of nuclear variant’s pathogenicity was performed using PolyPhen-2 and SIFT.

The bioenergetic status of the proband’s fibroblasts was measured using the Seahorse Extracellular Flux XFp Analyzer (Agilent Technologies; Santa Clara, CA), as described (Gropman et al., 2020). Optimal cell density (5,000/well) and the concentration of FCCP (fluoro 3-carbonyl cyanide-methoxyphenyl hydrazine; 2 µM) were determined using the Cell Energy Phenotype Test kit. Using the XFp Mito Stress Test kit, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured under basal conditions and after sequential injections of oligomycin (1 µM), FCCP (2 µM), and a mix of rotenone and antimycin A (1 µM). Using the Seahorse XFp real-time ATP Rate assay, we simultaneously quantified the rate of ATP produced by OXPHOS and glycolysis according to the manufacturer’s recommendations. OCR and ECAR were measured under basal conditions and after sequential injections of oligomycin (1.5 µM) and a mix of rotenone and antimycin A (0.5 µM). Using the XFp Glycolytic Rate Assay, we analyzed the glycolytic rate by quantifying the total proton efflux and the glycolytic proton efflux as described (Uittenbogaard et al., 2018a).

All the data from three independent experiments, each including three technical replicates, were normalized to cell numbers after the assay and plotted as OCR (pmol/min/cell ± S.E.M.), and ECAR (mpH/min/cell ± S.E.M.) using the Seahorse MultiReport Generator software. Statistical analyses were performed using the unpaired student t-test with p-value of less than 0.05 considered statistically significant.

The 6-year-old male proband has an unclear clinical diagnosis exhibiting developmental delay, hypotonia, spasticity, ptosis, bilateral cataracts and sensorineural hearing loss. His global symptomatology is consistent with a suspected inborn error of metabolism of uncertain etiology. The proband’s parents, a 46-year-old father and a 39-year-old mother, are of African-American descent and nonconsanguineous. Maternal history was significant with five miscarriages in the setting of hypercoagulability due to a factor 2 mutation for which she is treated with Lovenox. She had gestational diabetes that was treated with diet.

The proband was born at 35 weeks’ gestation by C-section due to fetal deceleration and weighed 2.4 kg. During the neonatal period, he had hypoglycemia, requiring admission to the intensive care unit. He passed his newborn hearing screen and was discharged 1 week after delivery. At 5 months, a brain CT was performed to investigate anterior fontanelle bulging, which showed enlarged subarachnoid spaces, an absent thalamic massa intermedia, and a residual cavum septum pellucidum/vergae, but otherwise, normal brain density, texture, and volume. At 6 months, the proband had his first ophthalmology evaluation due to concerns for corneal opacities. He was diagnosed with bilateral infantile cataracts and congenital ptosis of the right upper lid. He underwent a surgical procedure for extraction of his right cataract and a sling for his right upper lid. He was diagnosed with plagiocephaly and hospitalized due to a bulging fontanelle and irritability. Due to his staring episodes, an electroencephalogram was performed, which failed to show any epileptiform discharges.

At 16 months, he showed signs of developmental delay with mild hypotonia and delayed walking. He was referred to physical and speech therapists and diagnosed with sensorineural hearing loss. A temporal bone CT performed at 30 months showed normal appearing inner ear structures, but disclosed interval cerebellar atrophy. A brain MRI at 32 months confirmed cerebellar atrophy and absent massa intermedia (Figures 1A–G). It also revealed brainstem atrophy, hypoplasia of the anterior commissure, optic pathway, and olfactory system as well as cerebral malformations including abnormal parietoinsular gyration, a thick-walled cavum septum pellucidum/vergae, and deficiency of the anterior limb of the internal capsule. Furthermore, multifocal lesions of varying ages were present with diffusion abnormalities ranging from reduced to facilitated, involving the globi pallidi, midbrain (including but not limited to the periaqueductal gray matter, colliculi, and substantia nigra), pontine tegmentum, medulla, cerebellar gray and white matter, and imaged cervical spinal cord consistent with acute on chronic metabolic injury. Many of the small vessels within and along the brain surface were prominent and consistent with hypervascularity, especially in the basal ganglia. These findings were again demonstrated when MRIs were done at the age of five and six. Single voxel ¹H MRS performed concurrently over the left cerebral deep gray nuclei showed a small lactate peak, increased glutamate, and citrate elevation, elevating suspicion of a mitochondrial etiology. The proband was referred to our Neurogenetics clinic upon suspicion of a neurometabolic disease with a mitochondrial etiology based on his significant developmental regression and several lactic acidosis episodes.

At 5 years, he had a facial droop and underwent another brain MRI that showed recurrent small vessel hypervascularity and additional acute on chronic lesions in similar anatomic regions of the basal ganglia, brainstem, and cerebellum, consistent with metabolic disease progression. A single voxel ¹H MRS over the left cerebral deep gray nuclei confirmed mild lactate, increased glutamate, and citrate elevation (Figure 1H). The proband was treated with intravenous dextrose during episodes of metabolic decompensation and started on levocarnitine at three doses of 330 mg. Although the proband’s levels of alpha-aminoadipate and cystathionine were elevated, his acylcarnitine and very long chain fatty acid levels were normal. His screen for congenital disorder of glycosylation was negative. EKG showed normal sinus rhythm, and echocardiography showed no structural abnormalities. Following several episodes of choking, the proband underwent a swallow evaluation, which revealed prolonged oral phase of the swallow with no aspiration of solid food.

At 6 years, examination revealed dysmorphic features including bilateral ptosis, pendulous lips, small chin, and myopathic facies. His bilateral cataracts reduces his visual acuity. He only perceives movements. He has some axial hypotonia with bilateral spasticity in lower extremity. His walking is severely limited, being able to only take a few steps in his gait trainer without assistance.

The constellation of clinical features and brain MRI findings of the proband are consistent with an LS-like phenotype. He exhibits additional atypical features, including the clinical findings of dysmorphism, the neuroimaging findings of absent thalamic massa intermedia, residual cavum septum pellucidum/vergae, hypoplasia of the anterior commissure, optic pathway and olfactory system, as well as cerebral malformations including abnormal parietoinsular gyration, a thick-walled cavum septum pellucidum/vergae, and deficiency of the anterior limb of the internal capsule.

The proband’s mitochondrial genome, which was sequenced by LR-PCR-MPS, does not harbor any pathogenic mitochondrial variants. WES was performed using blood sample from the proband applying the filering strategy as described in Figure 2 of Uittenbogaard et al. (2019). After filtering steps were applied to 30,000 variants, we removed artifacts and false positive variants, and performed our analysis on the 25,000 remaining variants that revealed the presence of three heterozygous nuclear variants in the EARS2, MTFMT, and NARS2 genes, all linked to combined oxidative phosphorylation deficiency (COXPD) (Table 1). The EARS2 c.368T > A (p.L123Q) variant maps in the exon three of the EARS2 protein, which regulates mitochondrial protein translation by attaching glutamate to the cognate mitochondrial t-RNA (Mai et al., 2017). It is reported at a low allele frequency in the population databases dbSNP (www.ncbi.nlm.nih.gov/snp/) and gnomAD (http://gnomad.broadinstitute.org/variant). Both the SIFT and PolyPhen2 algorithms predicted the p. L123Q variant to be deleterious. Exon 3, which encodes the one of the four catalytic domains of mitochondrial glutamyl-tRNA synthetase, is a hotspot for nuclear variants (Steenweg et al., 2012). Pathogenic EARS2 variants are associated with the rare mitochondrial disease COXPD 12 (OMIM No. 614924) (Talim et al., 2013). The novel MTFMT variant c.20G > A (p.R7H) is predicted pathogenic and maps in exon 1 of the mitochondrial methionyl-tRNA formyltransferase (MTFMT) protein (Table 1). Several pathogenic MTFMT variants are associated with COXPD 15 (OMIM No 614947) (Neeve et al., 2013). The pathogenic NARS2 variant c.791C > G (p.S264C) maps in the exon 7 that in part encodes the catalytic domain of the mitochondrial asparaginyl-tRNA synthetase 2 (Table 1). Several NARS2 variants are linked to COXPD 24 (OMIM No. 616239) (Simon et al., 2015; Valander et al., 2015; Seaver et al., 2018).

Based on the presence of these variants, we investigated the ATP metabolism using the proband’s fibroblasts from a skin biopsy performed at the age of five. As a control subject, we used commercially available dermal fibroblasts from a healthy subject of similar age range whose metabolic profile has already been characterized and comparable to two other healthy subjects (Uittenbogaard et al., 2018b). We measured OCR, a key functional indicator of the mitochondrial energy metabolism, to accurately assess OXPHOS parameters using the Mitochondrial Stress Test assay. The proband’s OXPHOS parameters were greatly reduced, when compared to those of a healthy subject (Figure 2). We found a 65% decline of the basal respiration and a 69% reduction of ATP-linked respiration (Figure 2C). The maximal respiratory capacity evoked by the protonophore FCCP dropped by 62% (Figure 2C). Finally, the spare respiratory capacity decreased by 55%, hindering the ability to sustain an energy crisis (Figure 2C).

We next examined the rate of ATP production in the proband’s fibroblasts from glycolysis and OXPHOS using the XFp Real-Time ATP rate assay. Both OCR and ECAR were simultaneously measured upon injection of oligomycin followed by injection of rotenone and antimycin A to fully inhibit mitochondrial ATP production (Figure 3A). The proband’s mitochondrial rate of ATP production decreased by 63% (Figure 3B), corrobating the overall OXPHOS decline. The 16% increase in glycolysis-mediated ATP production rate was not enough to offset the 31% decrease in total ATP rate production.

We next investigated the glycolytic metabolism of the proband using the Glycolysis Rate assay, which accurately assesses glycolytic activity by correlating one-to-one with lactate accumulation. The total Proton Efflux Rate (PER) and the Glycolytic Proton Efflux Rate (GlycoPER) were measured using both OCR and ECAR values to account for mitochondrial (CO₂) acidification from the mitochondrial TCA cycle (Figures 4A,B) (Mookerjee et al., 2017). Basal glycolysis increased by 34%, which was confirmed by an increased PER to 94%, compared to 79% in healthy fibroblasts (Figure 4C). We measured the compensatory glycolysis response as an indication of metabolic reprogramming toward glycolysis following a provoked energy crisis by rotenone and antimycin A. The proband’s fibroblasts failed to increase glycolysis as a mean to compensate for this acute mitochondrial ATP crisis (Figure 4C).

We then conducted a mitochondrial morphometric analysis using transmission electron microscopy to investigate whether the proband’s mitochondria showed ultrastructural defects. The proband’s fibroblasts contained a decreased mitochondrial population, when compared to that of healthy fibroblasts (Figure 5). The proband’s mitochondria were small with rare and swollen cristae (Figure 5). These morphometric results confirm the proband’s dysfunctional mitochondrial energy metabolism (Figures 2–4).