Biallelic mutations in mitochondrial tryptophanyl‐tRNA synthetase cause Levodopa‐responsive infantile‐onset Parkinsonism

Mitochondrial aminoacyl‐tRNA synthetases (mtARSs) are essential, ubiquitously expressed enzymes that covalently attach amino acids to their corresponding tRNA molecules during translation of mitochondrial genes. Deleterious variants in the mtARS genes cause a diverse array of phenotypes, many of which involve the nervous system. Moreover, distinct mutations in mtARSs often cause different clinical manifestations. Recently, the gene encoding mitochondrial tryptophanyl tRNA synthetase (WARS2) was reported to cause 2 different neurological phenotypes, a form of autosomal recessive intellectual disability and a syndrome of severe infantile‐onset leukoencephalopathy. Here, we report the case of a 17‐year‐old boy with compound heterozygous mutations in WARS2 (p.Trp13Gly, p.Ser228Trp) who presented with infantile‐onset, Levodopa‐responsive Parkinsonism at the age of 2 years. Analysis of patient‐derived dermal fibroblasts revealed decreased steady‐state WARS2 protein and normal OXPHOS content. Muscle mitochondrial studies suggested mitochondrial proliferation without obvious respiratory chain deficiencies at the age of 9 years. This case expands the phenotypic spectrum of WARS2 deficiency and emphasizes the importance of mitochondrial protein synthesis in the pathogenesis of Parkinsonism.

subset of patients (5%-10%) manifest early-onset PD (EOPD) [1][2][3] and are more likely to harbor recognized genetic risk factors. 1,2,4 Levodopa-responsive Parkinsonism presenting in infancy or childhood is extraordinarily rare and may occur as a comorbidity to other diseases or genetic conditions. 5 From a neuropathology perspective, prototypical sporadic PD is regarded as a α-synucleopathy pathologically associated with Lewy bodies. Other conditions emerging from progressive nigrostriatal degeneration that manifest clinical features of PD but lack Lewy bodies are referred to as Parkinsonian syndromes or Parkinsonism.
Accumulating evidence indicates that the pathophysiology of Parkinsonism involves mitochondrial dysfunction. For example, mitochondrial toxins are known to induce Parkinsonism in human and animal models and several genes directly linked to mitochondrial function and mitophagy, including DJ-1 and PINK1, have strong associations with EOPD. [1][2][3] Although, the mitochondrial aminoacyl-tRNA synthetases (mtARSs) are key components of the mitochondrial translation machinery and are associated with disturbances of mitochondria function, they have not yet been associated with PD or Parkinsonian syndromes. Mutations in mtARS genes have been shown to cause a multitude of phenotypes, predominantly affecting the nervous system, 6,7 which can vary significantly depending on the specific mutations in a given mtARS. Recently, mitochondrial tryptophanyl-tRNA synthetase, encoded by WARS2, has been associated with the phenotypes of intellectual disability, leukoencephalopathy and seizures. 8,9 Here, we describe a patient with pathogenic WARS2 mutations presenting with infantile-onset, Levodoparesponsive Parkinsonism.

| Genetic analysis
High density whole genome SNP array methods were done as described in Reference 10.
Whole exome sequencing of both parents and the patient was performed at NIH Intramural Sequencing Center (NISC). SureSelectV5 (Agilent Technologies) was used for exome capture and subsequent sequencing was done on a HiSeq2000 instrument (Illumina Inc.). Sample library preparation, sequencing, and analysis were performed using the standard NISC pipeline 11 and Axeq Technologies (Seoul, South Korea). The analysis of whole exome sequencing data is described below.
Whole-exome sequence analysis was performed on genomic DNA that was isolated from the patient and his unaffected mother and father. The quality of whole exome data is shown in Table S1 (Supporting information).
The sequencing reads then were filtered for quality, and aligned to human reference genome NCBI build 37 (hg19) using in-house developed pipelines, one based on Novoalign (Novocraft Technologies), and separately a diploid aligner 12 run on a commercial platform (Appistry Inc.). Variants were called with HaplotypeCaller and GenotypeGVCFs. [12][13][14] Annotations utilized snpEff 15

| Analysis of dermal fibroblasts
A culture of dermal fibroblasts from the patient was established as previously described. 18 Control fibroblasts were purchased from Coriell Institute for Medical Research. For quantification of WARS2 and oxidative phosphorylation in dermal fibroblasts, human fibroblasts were trypsinized, pelleted, and resuspended in cell lysis buffer as previously described, 19

| Clinical description
The patient was the first and only child born to non-consanguineous parents of European descent with no relevant family history. He was born at 37 weeks of gestation as a product of in vitro fertilization that was necessary because of tubal factor infertility. Due to premature labor that started at 23 weeks gestation and maternal bleeding related to placenta previa, he was delivered via cesarean section.
Apgar scores were 9 and 9 out of 10 (at 1 and 5 minutes), with birth weight of 3302 g, length of 50.8 cm, and unknown occipital frontal circumference or OFC. Following delivery, he was noted to have pneumothorax and received a chest tube and was monitored on a ventilator for 4 days. He also developed a mild form of jaundice, but did not require phototherapy and was discharged at 5 days.
Early development was normal until the age of 1 when he was The patient was evaluated at the NIH at age 9 and had advanced Levodopa-responsive Parkinsonism with prominent and unpredictable "on-off" fluctuations, peak-dose dyskinesia, stooped posture and disabling off-dystonia. Chronic headaches occurred due to retrocollic dystonic spasms. A brain magnetic resonance imaging (MRI) suggested progressive generalized brain atrophy, but there was no dysmyelination, leukoencephalopathy, or abnormalities of the basal ganglia ( Figure 1A-C At age 10, he underwent implantation of deep brain stimulator (DBS) leads that resulted in substantial improvement in the motor complications of PD.

| Clinical data and samples
The patient, following informed and written consent, was admitted in the NIH Clinical Center under the protocol 76-HG-0238, "Diagnosis

| Genetic analysis
Pathogenic SNPs, small insertions/deletions, and copy number and structural variants in the PD-associated genes PINK1, PRKN, PARK7, and SCNA were ruled out by whole exome sequencing and whole genome single nucleotide polymorphism (SNP) array analysis.

| Analysis of dermal fibroblasts
Western blot analysis of patient fibroblasts revealed a marked decrease in WARS2 steady-state protein levels relative to controls, demonstrating a functional consequence of the identified WARS2 variants ( Figure 2C). The steady state levels of OXPHOS subunits NDUFB8 (CI), UQCRC2 (CIII), COXI (CIV) and ATP6 (CV) were Increased and swollen mitochondria seen in muscle EM Reference 9 8 This paper relatively unaffected in the patient as compared to controls ( Figure 2C), using SDHA (CII) as a loading control since CII is entirely nuclear-encoded.

| Muscle biopsy analysis
A quadriceps muscle biopsy had non-specific changes including mild variation in fiber size with rare atrophic fiber, and normal COX and The latest mtARS to be linked to a disease is WARS2, based upon 2 independent case reports. Musante et al 9  This difference likely reflects the unique impact that a specific variant in WARS2 can have on the resulting cellular and clinical phenotype, or different methodologies. In general, it is not uncommon for mtARS defects to have no effect on RC subunit expression in patient fibroblasts. 23 Interestingly, the mtDNA content of the muscle is still lower than age-matched control; this, together with the finding of increased mitochondrial CoQ10 content may suggest some compensation for the mtDNA depletion.
WES and whole genome SNP array data analysis for known variants and copy number variations associated with PD did not yield significant findings. The association of the patient's WARS2 variants to Parkinsonism or PD may be further clarified by systematically interrogating available cohorts for such mutations. MRI imaging at the time of our evaluation revealed a structurally normal study without evidence of abnormal mineral or metal deposition in the deep brain nuclei or leukoencephalopathy, but rather minimal nonspecific atrophy. The temporal pattern of our patient's clinical progression has followed the prototypical course of adult idiopathic PD, except that it began 6 decades earlier. Specifically, our patient exhibited the typical early "honeymoon" response to Levodopa supplementation allowing for several years of excellent clinical response, followed by increasingly severe motor fluctuation associated with progressive nigrostriatal denervation that eventually led to the need for DBS at age 10 to achieve control of motor complications.
In conclusion, we expand the phenotypic spectrum of disorders caused by biallelic mutations in WARS2 gene to include infantileonset, Levodopa-responsive Parkinsonism and implicate WARS2 dysfunction as a potential driver of mitochondrial dysfunction leading to nigrostriatal degeneration and Parkinsonism.