Expanding the clinical and phenotypic heterogeneity associated with biallelic variants in ACO2

Patients

Our cohort consists of five newly described cases with a molecular diagnosis of ACO2 deficiency. The presentation and clinical course for each patient is summarized in Table 1. Two of these patients (P4 and P5) were briefly described in a large clinical exome sequencing (CES) study of >2200 Saudi families (cases REQ18-2038 and 17-6359, respectively), but are reported in detail here.⁸ Individuals were also identified and enrolled in the current study using GeneMatcher⁹ (Clinical data were retrospectively collected by each clinician participating in the study. The study was approved by our local institutional review board and in accordance with the Declaration of Helsinki. In addition, written informed consent was obtained to publish subject photographs.

Genetic analyses

All variants are reported based on NCBI reference sequences NM_001098.2 (Protein: NP_001089.1). Clinical trio whole-exome sequencing (WES, Mayo Clinic Laboratories, Rochester, MN) was performed and revealed two compound heterozygous variants of uncertain significance, c.2153T>C:p.(Ile718Thr) and c.2050C>T:p.(Arg684Trp), in ACO2 in P1. These variants were confirmed by familial mutation testing (Sanger sequencing confirmation) in P2 and established mode of inheritance in both parents. Similarly, research WES¹⁰ was performed in P3 who was found to carry two novel variants, c.719G>A: p.(Gly240Asp) and c.433-2_433-1delinsCT. Confirmatory sequencing to determine inheritance could only be performed for this patient’s mother (father deceased). Singleton clinical WES was performed in P4 and P5 as previously described and identified two private homozygous variants in ACO2 c.1187C>T: p.(Ser396Leu) and c.2338_2339delCA: p.(Gln780ValfsTer63), respectively.¹¹ Familial segregation was confirmed via Sanger sequencing in both individuals.

In silico predictions for nonsynonymous variants were performed using Variant Score Ranker (http://vsranker.broadinstitute.org/).¹² Simulations were performed using a homology model (PDB: 1b0j.1.A) of human mitochondrial aconitate hydratase (Q99798) from the SWISS-MODEL Repository (SMR) and generated by the SWISS-MODEL homology modeling pipeline.¹³ DynaMut was used to analyze and visualize changes in protein dynamics and stability resulting from vibrational entropy changes caused by missense mutations in ACO2 using normal-mode analysis (http://biosig.unimelb.edu.au/dynamut/).¹⁴ Variant allele frequencies were evaluated in gnomAD (https://gnomad.broadinstitute.org/).¹⁵ Missense Tolerance Ratio (MTR) scores and graphs were generated using MTR-Viewer (http://biosig.unimelb.edu.au/mtr-viewer/).¹⁶ Protein and transcript diagrams were generated using ProteinPaint (https://proteinpaint.stjude.org/).¹⁷

Clinical reports

In this study, we describe two brothers (P1 and P2), currently 12 and 15 years of age, who presented initially with ataxia at 12 and 20 months, respectively, in the setting of intercurrent febrile illnesses (Fig. 1A). Their ataxia resolved and recurred episodically, correlating to routine viral infections. Both brothers have mild developmental delays, severe expressive speech disorder with dysarthria, behavioral abnormalities including attention deficit hyperactivity disorder (ADHD), depressive symptoms with generalized anxiety, and impaired fine motor skills. Neither brother had significant dysmorphic features (Fig. 1B). Additionally, both brothers were initially thought to have susceptibility to infections but workup for suspected immune deficiencies was unrevealing. Both brothers have received monthly intravenous immunoglobulin (IVIg), however, which may have led to clinical improvement.

P3 is a 26-year-old female who presented with cerebellar hypoplasia, ataxia, spastic cerebral palsy, seizures, optic atrophy, and retinitis pigmentosa (Fig. 1A). This individual has moderate-to-severe intellectual disability, a vocabulary of 4–5 words, and is non-ambulatory.

P4 was delivered at term to a 27-year-old primigravida mother. His parents are first cousins (Fig. 1A). Antenatal follow up was unremarkable. He was admitted to neonatal intensive care unit (NICU) 2 h after birth because of cyanosis. He was noticed to have poor respiratory drive and respiratory acidosis. Neurologic examination showed generalized hypotonia with diminished reflexes. He developed abnormal episodes characterized by twisting movements involving his arms and seizure disorder that was controlled by clonazepam. An electroencephalographic study (EEG) revealed gross abnormalities consistent with multifocal epilepsy. He had absent visual tracking and protective blinking. Ophthalmologic examination showed bilateral retinal degeneration and optic atrophy. He was overall nondysmorphic (Fig. 1B). Cardiovascular workup was normal. He had central apnea requiring ventilator support and failed several attempts of weaning and extubation until his death at the age of 9 months.

P5 is an 11-year-old female with progressive spastic quadriplegia, severe hypotonia, absent visually evoked potentials, and severe developmental and motor delays (Fig. 1A). She was born at term following an uneventful pregnancy via spontaneous vaginal delivery. She had her first recognized seizure at 3 months of age and has had at least one seizure per week since then despite being on three antiepileptic medications (Levetiracetam, topiramate, and lamotrigine).

Additional details can be found in the supplemental materials and in Table 1.

Laboratory investigations

P1 had a normal karyotype and normal array CGH testing. His older brother (P2) had more extensive evaluations including a normal karyotype, normal array CGH, normal Prader-Willi/Angelman syndrome methylation testing, and normal MECP2, FMR1, and KCNA1 gene testing. Extensive metabolic evaluations were unrevealing as well. These included normal ammonia, creatine kinase, carbohydrate-deficient transferrin, alpha fetoprotein, hexosaminidase A, biotinidase, coenzyme Q10 quantification, amino acids (plasma, CSF, urine), 7-dehydrocholesterol, peroxisomal panel, urine amino acids, oligosaccharide screen, acylglycines, glycosaminoglycans, urine purine and pyrimidine panel, hexosaminidase A, biotinidase, sphingomyelinase, and mitochondrial respiratory chain complex probe. He also had normal mitochondrial DNA (mtDNA) sequencing and normal ataxia evaluation through Athena Diagnostics. This included normal analyses of SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA10, SCA17, DRPLA, FRDA1, SCA14, SETX, POLG1, SCA5, SIL1, TTPA, and KCNC3.

P3 had a normal microarray, normal MECP2 gene testing, normal PWS/Angelman methylation studies, and normal mitochondrial testing (including deletions/duplications). Biochemical testing including total and free carnitine, acylcarnitine profile, carbohydrate-deficient transferrin, urine organic acids, lactate, biotin, and peroxisomal studies were all normal.

P4 had normal 46, XY male karyotype. His creatine kinase was normal (152 U/L). Muscle biopsy revealed nonspecific myopathy and there was no evidence of congenital muscular dystrophy, nemaline myopathy, dystrophinopathy, or sarcoglycanopathy. Merosin was normally expressed. Testing for spinal muscular atrophy (SMA) was normal. Very long-chain fatty acids for peroxisomal disorders were negative. Additional metabolic screening was unremarkable.

A limited clinical history for P5 showed acylcarnitine testing, plasma amino acids, and urine organic acids were normal.

Additional details can be found in the supplemental materials and in Table 1.

Brain imaging

A comparison of brain MRI findings in each patient is shown in Figure 1C. P1 had noncontrast MRI of the brain and lumbar spine, without comparison performed at 2 years and 3 months of age. MRI of the head was normal with normal myelination for age with bilateral terminal zones in the periatrial white matter. The pons and superior cerebellar vermis were slightly small for age (Fig. 1C). Lumbar spine, from T7 vertebral body through the sacrum showed a normal spinal cord, conus, and filum terminale without evidence of tethered cord (not shown). P2 had his most recent brain MRI at 6 years of age. Midbrain, pons, and middle cerebellar peduncles were noted to be small in size (Fig. 1C). MRI of the cerebellar hemispheres was normal, although the superior cerebellar vermis was felt to be mildly atrophic. There was mildly prominent T2 signal surrounding the 4th ventricle, but no unusual enhancement. The corpus callosum was within normal limits. In P3, brain MRI at 16 years of age showed mild cerebellar atrophy but was otherwise normal (Fig. 1C). A brain CT at age 18 revealed similar findings (not shown). In P4, brain MRI at 5 months and 11 days of age showed dilatation of the ventricles and prominent subarachnoid spaces, thinning of the corpus callosum, hypoplastic cerebellar vermis, and hypoplastic pons (Fig. 1C), while brain CT at the age of 1 day was unremarkable. Brain MRI in P5 was performed around 4 years of age and showed global hypomyelination and other nonspecific findings (Fig. 1C).

Genetic and in silico analysis

P1 and P2 both carry compound heterozygous variants, paternally inherited c.2153T>C: p.(Ile718Thr) and maternally inherited c.2050C>T: p.(Arg684Trp) (Fig. 2A and B). The p.(Ile718Thr) variant has not been reported previously, or has it been observed in population databases (gnomAD). The variant falls in a highly conserved residue (to yeast) and is predicted to be damaging by in silico prediction software. ΔΔG predictions (a measure of the change in monomeric protein stability when a point mutation is introduced) show a destabilizing effect of the p.(Ile718Thr) variant (ΔΔG: −3.151 kcal/mol) with an increase in molecule flexibility (ΔΔSVib ENCoM: 0.275 kcal/mol/K) (Fig. 2C). The p.(Arg684Trp) variant was previously observed in two recently described patients in trans with the c.1787A>G: p.(His596Arg) missense variant.⁷ Both residues are evolutionary conserved and were predicted to impact substrate binding. Functional studies in the Δaco1 yeast strain showed a 25% reduction in enzymatic activity for the homologous Arg684Trp variant, Arg681Trp, suggesting that Arg684Trp is a hypomorphic ACO2 variant.^{7, 18, 19}

P3 was found to carry two variants, c.719G>A: p.(Gly240Asp) and c.433-2_433-1delinsCT in trans, with the c.433-2_433-1delinsCT splice-site mutation confirmed in the patient’s mother (Fig. 2A and B). Her father was deceased at the time of testing. In silico splice prediction tools (SpliceSiteFinder-like, MaxEntScan, GeneSplicer, and NNSPLICE) show complete loss of the exon 4 splice acceptor site with the c.433-2_433-1delinsCT variant. The p.(Gly240Asp) variant has been reported rarely in gnomAD (0.000007953% or 2/251480 total alleles) and falls in a highly conserved residue (to yeast). ΔΔG predictions show a destabilizing effect of the p.(Gly240Asp) variant (ΔΔG: −0.057 kcal/mol) with a net decrease in molecule flexibility (ΔΔSVib ENCoM: −0.168 kcal/mol/K) (Fig. 2C).

In P4, the c.1187C>T: p.(Ser396Leu) homozygous variant falls in a moderately conserved residue and has not been reported in gnomAD or other population databases (Fig. 2A and B). In silico prediction algorithms consistently show a deleterious effect of this mutation and ΔΔG predictions show a stabilizing effect (ΔΔG: 1.519 kcal/mol) with a net decrease in molecule flexibility (ΔΔSVib ENCoM: −0.375 kcal/mol/K) (Fig. 2C). This novel variant falls near the c.1181G>A: p.(Gly394Glu) variant that has been observed in several patients including in association with infantile cerebellar-retinal degeneration in trans with a p.(Trp574Cys) variant.²⁰

In P5, the c.2338_2339delCA: p.(Gln780ValfsTer63) homozygous variant falls in the last amino acid before the TGA stop codon (Fig. 2A) and is a predicted stop loss alteration resulting in inclusion of a novel peptide sequence (VRAVPPRPAAGVKFSSTCAISGS DPSSHGFLFQDGVTRHASCSPLSPRSDCGCGGGVLKITF*) that is 62 amino acids in length. It is possible that this novel peptide could impair localization of this protein, lead to aggregation, or disrupt normal protein structure, thereby affecting overall stability. The same homozygous p.(Gln780ValfsTer63) variant was found in another Arab family (Family 046) in two affected children (phenotype similar to reported).^{3, 21} In addition, two other cases have been described with a similar frameshift mutation, c.2328_2331delGGAA: p.(Lys776Asnfs*49) that is also predicted to lead to the addition of a novel peptide sequence at the end of ACO2.⁴