Phenotypes and genotypes of mitochondrial aminoacyl‐tRNA synthetase deficiencies from a single neurometabolic clinic

Abstract Mitochondrial aminoacyl‐tRNA synthetases play a major role in protein translation, synthesis, and oxidative phosphorylation. We reviewed all patients diagnosed with mitochondrial aminoacyl‐tRNA synthetase deficiencies diagnosed in a single neurometabolic clinic. We report five patients with mitochondrial aminoacyl‐tRNA synthetase deficiencies including DARS2, EARS2, PARS2, and RARS2 deficiencies. Siblings with DARS2 deficiency presented with global developmental delay within the first year of life. DARS2, EARS2, PARS2, and RARS2 deficiencies were identified by whole exome sequencing. We report coagulation factor abnormalities in PARS2 deficiency for the first time. We also report symmetric increased signal intensity in globus pallidi in FLAIR images in brain MRI in EARS2 deficiency for the first time. One patient with RARS2 deficiency had compound heterozygous variants in RARS2. One of those variants was an intronic variant. We confirmed the pathogenicity by mRNA studies. Mitochondrial aminoacyl‐tRNA synthetase deficiencies are diagnosed by molecular genetic investigations. Clinically available non‐invasive biochemical investigations are non‐specific for the diagnosis of mitochondrial aminoacyl‐tRNA synthetase deficiencies. A combination of brain MRI features and molecular genetic investigations should be undertaken to confirm the diagnosis of mitochondrial aminoacyl‐tRNA synthetase deficiencies.


| INTRODUCTION
Mitochondrial aminoacyl-tRNA synthetases are a group of catalytic enzymes. They play a major role in protein translation by delivering amino acids to the nascent polypeptide chain. They contribute to the accuracy of protein synthesis and protein expression. They also contribute to the functions of oxidative phosphorylation enzymes. There are 19 mitochondrial aminoacyl-tRNA synthetase genes. 1  13561). The clinical phenotypes are variable. Neurological features include severe early onset central nervous system disease, sensorineural hearing loss and leukodystrophy in brain magnetic resonance imaging (MRI). Additionally, liver, kidney, eye and cardiac organ system involvements are reported. Currently, diagnosis of mitochondrial aminoacyl-tRNA synthetase deficiencies are based on the molecular genetic investigations. Characteristic brain MRI features may suggest DARS2, PARS2 and RARS2 deficiencies. Whole exome sequencing will likely identify more patients to increase our knowledge of mitochondrial aminoacyl-tRNA synthetase deficiencies in the future.
Here we report five patients from four unrelated families with four different mitochondrial aminoacyl-tRNA synthetase deficiencies including DARS2 (two siblings), EARS2, PARS2 and RARS2 deficiencies. The interpretation of pathogenicity of variants of unknown significance can be difficult. To assist physicians in confirming their patients' genetic diagnostic confirmation, we describe phenotypes, genotypes and neuroimaging features of new patients with mitochondrial aminoacyl-tRNA synthetase deficiencies.

| MATERIALS AND METHODS
The Institutional Research Ethics Board approved this retrospective cohort study (Approval# 1000061126). Additionally, all families signed institutional case report consent forms. We included all patients with confirmed molecular genetic diagnoses of mitochondrial aminoacyl-tRNA synthetase deficiencies from a single neurometabolic clinic. This is a retrospective chart review study.
We reviewed electronic patient charts for clinical features, biochemical investigations, molecular genetic tests, brain MRI and brain magnetic resonance spectroscopy (MRS) results. Whole exome sequencing or targeted Sanger sequencing of DARS2 was performed according to molecular genetic laboratories' methods. We applied the recommendations for mutation nomenclature (www.hgvs.org/mutnomen) to name gene variations. We classified all variants using the Alamut variant interpretation software version 2.7-2 (http://www.interactivebiosoftware.com). We applied in silico analysis tools including Mutation Taster NCBI 37/Ensembler 69 (MutTaster), 2 Sorting Tolerant From Intolerant (SIFT) 4G annotator version 4.0.3 (Alamut variant interpretation software, http://www.interactivebiosoftware.com), 3 Polymorphism Phenotyping version 2.1.0 (PolyPhen-2) 4 and conservation species (Alamut variant interpretation software version 2.7-2). We also searched all variants in the Genome Aggregation Database (gnomAD) (http:// gnomad.broadinstitute.org/about) for their allele frequency in the general population. 5 We applied The American College of Medical Genetics and Genomics (ACMG) variant classification guidelines. 6

| RESULTS
There were five patients with four different mitochondrial aminoacyl-tRNA synthetase deficiencies including DARS2 (n = 2, two siblings, older sibling reported previously in 7 ), EARS2 (n = 1), PARS2 (n = 1) and RARS2 (n = 1) deficiencies. Four patients had whole exome sequencing including two research and two clinical whole exome sequencing. One patient had targeted Sanger sequencing of DARS2 (due to positive family history). About 200 patients underwent research or clinical whole exome sequencing in this neurometabolic clinic since 2014. These patients were the only patients diagnosed with mitochondrial aminoacyl-tRNA synthetase deficiencies in this neurometabolic clinic. The senior author received whole exome sequencing results from the research teams or clinical diagnostic laboratories with a single confirmatory genetic diagnosis.
Demographics, clinical features and genetic diagnosis of all patients were summarized in Table 1 patients with DARS2 and EARS2 deficiencies were consanguineous. All patients had normal plasma amino acids, homocysteine, acylcarnitine profile, total and free carnitine and urine organic acid analysis. In silico analysis results of all variants and their ACMG variant classification results were listed in Table 2. Brain MRS showed normal lactate peak in all patients. Brain MRI images of four patients were depicted in Figure 1. Brain MRI images of the older sibling with DARS2 deficiency was previously reported. 7 F I G U R E 1 Legend on next page.
Siblings with DARS2 deficiency presented with global developmental delay within the first year of life. Younger sibling had history of motor regression after a viral infection at the time of initial presentation. Additionally, older sibling had nystagmus and ataxia. In both siblings with DARS2 deficiency, the brain MRI revealed increased signal intensity in the dorsal columns, inferior and superior cerebellar peduncles, cerebellar white matter, the external and internal capsules and subcortical and periventricular white matter in T2 weighted images.
The patient with EARS2 deficiency had global developmental delay, generalized dystonia and dyskinesia within the first year of life. She had mild aspartate aminotransferase (AST) elevation. Plasma lactate level was mildly elevated (3.8 mmol/L; reference ranges <2.5 mmol/L) as well. Her brain MRI revealed symmetric increased signal intensity of the globus pallidus in FLAIR images.
The patient with PARS2 deficiency had seizures, nystagmus, ataxia, generalized dystonia and acquired microcephaly. She had coagulation abnormalities including prolonged partial thromboplastin time (PTT), and low factor IX and XI levels. Her brain MRI revealed markedly thinned corpus callosum in T1 weighted images and volume loss of the cortical and subcortical frontal lobes, and widening of the cortical sulci, Sylvian fissures and frontal horns in T2-weighted images. There was a transient delay in myelin maturation in T1-weighted brain MRIs performed at the age of 4 and 8 months, which was normalized at the age of 25 months.
The patient with RARS2 deficiency presented with neonatal epileptic encephalopathy in the first day of life. She was diagnosed at the age of 1.5 months. She had a hypoplastic proximal pulmonary artery branch in echocardiography. She had mild AST elevation. Her brain MRI revealed superior vermian and superior cerebellar atrophy in the T1 weighted images at the age of 5 days. Her repeat brain MRI showed a mild dilatation of the anterior horns of the lateral ventricles suggestive of a progressive supratentorial volume loss in T2 weighted images at the age of 6 weeks. She passed away at the age of 2 months.
Patients with DARS2, EARS2, and PARS2 deficiencies had either homozygous or compound heterozygous missense variants. Missense variants in DARS2 and EARS2 were classified as variant of unknown significance by ACMG variant classification guidelines. Both variants were classified as disease causing and possibly damaging in two out three in silico prediction tools and both of them were not reported in public databases in the general population or as polymorphisms in the dbSNP database. Variant in DARS2 resulted in the similar phenotypic and brain MRI features in both siblings. Variant in EARS2 was highly conserved in species. Patient with RARS2 deficiency had two novel truncating variants, c.633_636delAGAA (p.Glu212Glnfs*7) and c.1113-21A>C in RARS2. To investigate pathogenicity of the intronic variant, RNA was isolated from lymphoblasts and reverse transcriptase PCR and Sanger sequencing of the RARS2 cDNA were performed. A deletion r.633_636delAGAA was detected and a splice error harboring a deletion of exon 14 (r.1113_1273del). Parental testing confirmed the carrier status in the parents in all patients.

| DISCUSSION
We report the phenotypes and genotypes of five patients with mitochondrial aminoacyl-tRNA synthetase deficiencies from a single neurometabolic clinic to expand our current knowledge for these disorders. Gross motor delay with relatively normal cognitive functions and spasticity were prominent features in patients with DARS2 deficiency, while the patients with EARS2 and PARS2 deficiencies had cognitive dysfunction and movement disorders. Seizures were well controlled with a single anti-epileptic medication in patients with EARS2 and PARS2 deficiencies. Mild elevation of AST was present in patients with EARS2 and RARS2 deficiencies and synthetic liver dysfunction was present in the patient with PARS2 deficiency. To the best of our knowledge, we report for the first time prolonged PTT, and low factor IX and XI levels in PARS2 deficiency.
F I G U R E 1 Brain MRI images of four patients with mitochondrial aminoacyl-tRNA synthetase deficiencies were depicted. A,B,C, Brain MRI of the patient (patient 2) with DARS2 deficiency at the age of 56 week. T2 weighted sagittal image, A, of the spine showed increased signal intensity in the dorsal column (arrows). T2 weighted axial image, B, demonstrated increased signal intensity in the cerebellar white matter (short arrow) and in the superior cerebellar peduncles white matter (arrow). Axial T2 weighted image, C, revealed involvement of the subcortical and periventricular white matter and the external capsule, anterior and posterior (arrow) limbs of the internal capsules. D,E,F, Brain MRI of the patient with EARS2 deficiency at the age of 4 years 6 months old. There was bilateral symmetric increased signal in globus pallidus (arrows) on axial, E, and coronal, F, in FLAIR images. G,H,I, Brain MRI of the patient with PARS2 deficiency at the age of 8 months. Sagittal T1 weighted image, G, demonstrated a markedly thinned corpus callosum. Incomplete myelin maturation of the frontal lobes was shown in axial T1 weighted image, H. Axial T2 weighted image revealed prominent frontal, I, and Sylvian CSF spaces and widened cortical sulci and anterior horns of the lateral ventricles reflecting frontal lobe volume loss. J,K,L, Brain MRI of the patient with RARS2 deficiency at the age of 6 weeks. Superior vermian atrophy (arrow) was demonstrated in sagittal T1 weighted image. J, Marked superior vermian and superior cerebellar atrophy (arrows) was shown in axial, K, and coronal, L, T2 weighted images DARS2 deficiency has been first reported as leukoencephalopathy with brainstem and spinal cord involvement in brain MRI and elevated lactate in brain MRS in 2007. 8 More than 75% of patients had elevated lactate in brain MRS. Serum and cerebrospinal fluid lactate levels were elevated in less than 20% of patients. Only one family with three affected children were reported with an intrafamilial phenotypic variability. Two younger siblings had infantile onset global developmental delay and the oldest sibling presented with a mild lower limb spasticity at the age of 20 years. The older sibling was diagnosed due to the diagnosis of the younger siblings. 9 In our study, both siblings with DARS2 deficiency had global developmental delay within the first year of life. Younger sibling presented with motor regression after a viral infection at the age of 6 months.
EARS2 deficiency has been first reported as leukoencephalopathy with thalamus and brainstem involvement in brain MRI and elevated lactate in brain MRS in 2012. 10 Seizures were reported in 15% of patients. Infantile spasms were the most common seizure type. Elevated serum lactate levels were reported in 15% of patients ranging from 2.6 to 7.4 mmoL/L. Two patients had intermittent elevation of liver enzymes up to five times. Brain MRI features included symmetric signal changes in the deep white matter (usually sparing the periventricular rim), thalami and brainstem. Increased lactate in brain MRS was also reported. 11 One patient had typical brain MRI features at the age of 8 months, which partially improved at the age of 29 months. A repeat brain MRI demonstrated new increased signal intensity in the left caudate and left globus pallidus in T2 weighted images at the age of 5 years and 10 months. 12 Our patient had low apgar scores at birth and was resuscitated at the time of delivery. Based on this history, she was diagnosed with hypoxic-ischemic encephalopathy. Her brain MRI showed bilateral increased signal intensity in globus pallidus in FLAIR images, but there were no neuroimaging features of hypoxia or other typical MRI features of EARS2 deficiency. To the best of our knowledge, we report isolated bilateral globus pallidus changes in FLAIR images in brain MRI in EARS2 deficiency for the first time.
PARS2 deficiency was first described by Sofou et al. 13 The phenotypic spectrum ranged from infantile onset neurodegenerative disease, called Alpers syndrome, to global developmental delay and ataxia. All patients had infantile onset seizures. Infantile spasms were the most common seizure type in more than 60% of patients. Hepatocellular dysfunction was reported in few patients with PARS2 deficiency. 13,14 None of the patients had synthetic liver dysfunction in that study. To the best of our knowledge, we report synthetic liver dysfunction characterized by prolonged PTT, low factor IX and XI levels for the first time. Typical brain MRI features included leukoencephalopathy, cerebral atrophy especially in the perisylvian and fronto-temporal brain regions and abnormal signal intensities in the basal ganglia. 15,16 Interestingly, perisylvian and fronto-temporal atrophy in brain MRI resembles brain MRI features of glutaric aciduria type I. We think that it is important to include PARS2 deficiency in the differential diagnosis of perisylvian and fronto-temporal brain atrophy in brain MRI in addition to glutaric aciduria type 1.
RARS2 deficiency was first described as pontocerebellar hypoplasia 6 disease by Edvardson et al. 17 The majority of patients with RARS2 deficiency had pontocerebellar hypoplasia or progressive cerebral and pontocerebellar atrophy. 18 About one-third of the variants in RARS2 are truncating variants. 18 Interestingly, we also identified compound heterozygous truncating variants, a 4 base pair deletion and an intronic variant in RARS2. Based on the phenotype and suggestive neuroimaging features as well as functional studies, we confirmed the diagnosis of RARS2 deficiency. We report for the first time an intronic variant in RARS2.
Reports of variant of unknown significance in molecular genetic investigations pose difficulties for clinicians. Identification of these variants require extensive phenotypic and neuroimaging analysis as well as application of in silico analysis tools to confirm a genetic diagnosis. Functional analysis of variants in mitochondrial aminoacyl-tRNA synthetase genes will be essential to guide clinicians for their diagnostic odyssey in the future.
In summary, we report five patients with mitochondrial aminoacyl-tRNA synthetase deficiencies from a single neurometabolic clinic. Combination of characteristic brain MRI features and molecular genetic investigations should be undertaken to confirm the diagnosis of mitochondrial aminoacyl-tRNA synthetase deficiencies.