Treatment of Mitochondrial Phenylalanyl-tRNa-Synthetase Deficiency (FARS2) with Oral Phenylalanine

Abstract

Objective By loading transfer RNAs with their cognate amino acids, aminoacyl-tRNA synthetases (ARS) are essential for protein translation. Both cytosolic ARS1-deficiencies and mitochondrial ARS2 deficiencies can cause severe diseases. Amino acid supplementation has shown to positively influence the clinical course of four individuals with cytosolic ARS1 deficiencies. We hypothesize that this intervention could also benefit individuals with mitochondrial ARS2 deficiencies.

Methods This study was designed as a N-of-1 trial. Daily oral L-phenylalanine supplementation was used in a 3-year-old girl with FARS2 deficiency. A period without supplementation was implemented to discriminate the effects of treatment from age-related developments and continuing physiotherapy. Treatment effects were measured through a physiotherapeutic testing battery, including movement assessment battery for children, dynamic gait index, gross motor function measure 66, and quality of life questionnaires.

Results The individual showed clear improvement in all areas tested, especially in gross motor skills, movement abilities, and postural stability. In the period without supplementation, she lost newly acquired motor skills but regained these upon restarting supplementation. No adverse effects and good tolerance of treatment were observed.

Interpretation and Conclusion Our positive results encourage further studies both on L-phenylalanine for other individuals with FARS2 deficiency and the exploration of this treatment rationale for other ARS2 deficiencies. Additionally, treatment costs were relatively low at 1.10 €/day.

Keywords

aminoacyl-tRNA-synthetase - individual treatment - ataxia - mitochondrial disorder - mitochondrial diseases

Introduction

Mitochondrial diseases (MDs) represent a heterogeneous group of inborn metabolic diseases with a wide spectrum of signs and symptoms.[1] While no curative treatment is available, pathomechanism-based treatments that alleviate symptoms are increasingly recognized.[2] [3] [4]

The aminoacyl tRNA synthetases (ARSs) are an essential and universally distributed family of enzymes that play a critical role in protein synthesis by coupling tRNAs with their cognate amino acids for decoding mRNAs according to the genetic code.[5] The 19 ARSs located in the cytosol are usually referred to as ARS1 (e.g., alanyl-ARS = AARS1) and the respective mitochondrial counterparts as ARS2 (e.g., alanyl-ARS2 = AARS2).

Only recently, pathogenic variants in the 19 genes encoding the different mitochondrial ARSs have been linked to human disease.[5] As known from other MDs, the phenotypic spectrum is diverse and can vary substantially between affected individuals. How exactly ARS2 deficiencies lead to human disease is unknown. Currently, no specific treatment is available.

Mitochondrial FARS2 deficiency has been described in at least 37 affected individuals from 25 families.[6] The most common clinical features are seizures, developmental delay especially in motor skills, and truncal hypotonia. Roughly two subgroups can be distinguished within the spectrum: one with an infantile-onset (early onset seizures, developmental delay, failure to thrive, liver disease) and another with later-onset (spastic paraplegia, developmental delay, seizures, and cerebellar syndrome with ataxia, tremor, bradykinesia, dystonia, and dysarthria).[6]

For some of the cytosolic ARS1 deficiencies (IARS1-, LARS1-, FARSB-, SARS1-deficiency), it was recently shown that aminoacylation in patient-derived fibroblasts was diminished and fibroblasts growth was severely deprived when amino acid concentrations were low. This observation led to an individual (oral) treatment in four affected individuals with the corresponding amino acid (e.g., phenylalanine for FARSB). All patients showed a beneficial response with regard to growth, head circumference, development, coping with infection, and different individual other signs and symptoms.[7]

Diminished aminoacylation was also predicted for mitochondrial FARS2 deficiency.[8] Therefore, we envisioned that oral L-phenylalanine (L-Phe) could also alleviate the symptoms of the mitochondrial FARS2 deficiency in a 3-year-old girl presenting with muscular hypotonia, ataxia, intention tremor, and dysarthria.

Patient and Methods

Case Report

This girl was born as the second child to Austrian-Slovakian parents. Her parents and her older brother have no medical complaints. The pregnancy was uneventful and the child was born by natural vaginal delivery at term. Anthropometric data at birth and postnatal adaptation were within normal range.

She presented at the age of 10 months with delayed motor development (unable to sit independently, no crawling) that led to initiation of physiotherapy. She walked independently by the age of 2 years but frequently stumbled and fell and was easily fatigued. Upon neurological examination at the same age, truncal muscular hypotonia with hypertonia of the lower extremities, gait ataxia, intention tremor, and dysarthria were noticed. The deep tendon reflexes at all extremities were normal. Her language and cognitive development were age appropriate.

Laboratory testing revealed intermittently elevated serum lactate levels (2.7; 1.7; 1.8 and 4.1 mmol/L, ref.: 0.5–1.8 mmol/L), serum amino acid profile, and urinary organic acid analysis were unremarkable. Brain electroencephalography and brain magnetic resonance imaging at the age of 1 year were unremarkable.

Exome sequencing from leucocyte DNA revealed two variants in FARS2 (NM_006567.3): c.422G > A; p.(Gly141Glu) and c.461C > T; p.(Ala154Val). Sanger sequencing of the parents confirmed the maternal origin of the c.422G > A, p.(Gly141Glu) variant, the c.461C > T; p.(Ala154Val) variant was not found in the father. Both variants were absent from gnomAD in homozygous state. The c.422G > A has not been reported before, the c.422G > A was proven to disturb mt-tRNA^Phe aminoacylation when found in compound heterozygous state with c.1082C > T p.(Pro361Leu).[9]

N-of-1 Trial with Phenylalanine and Outcome Measures

Baseline testing (T0 at the age of 2 11/12 years) consisted of physical examination, movement assessment battery for children (MABC-2), dynamic gait index (DGI), gross motor function measure 66 (GMFM-66), PEDSQL quality of life questionnaire (for details see supplementary data), and laboratory testing (plasma amino acid profiling [4 hours postprandial]), complete blood count, liver enzymes, kidney function;). Thereafter, treatment with oral L-Phe (3 × 150 mg/d, Phe minis, MetaX, Friedberg, Germany) was started. This dose equals the daily recommended intake of L-Phe based upon a total recommend protein intake of 1 g/kg/day. The costs for L-Phe were 1.10 €/day.

After 21 weeks (T1), the test battery was repeated and L-Phe paused for 8 weeks. After another examination (T2) L-Phe was restarted and testing repeated after another 13 weeks (T3). Additionally, the parents noted their observations in a diary.

Ethical Considerations

This study was conducted in accordance with the Declaration of Helsinki.[10] The parents gave written consent for genetic testing and further research and publication within the “mitoNET”-study/database (approved by ethical committee Land Salzburg, Austria, 415-E/1317/20-2022).

Results

As shown in [Fig. 1], an improvement in all subtests of the test battery was seen at the end of the two L-Phe treatment periods. During pausing of treatment, a worsening in all parameters was documented (videos available on request).

Fig. 1 Results of physiotherapy testing batteries and quality of life questionnaire. Results of the different tests (movement assessment battery for children [MABC-2], dynamic gait index [DGI], gross motor function measure 66 [GMFM-66], quality of life [QoL]) in relation to supplementation with L-phenylalanine (L-Phe) and other therapies. PR, Percentile Rank; SD, standard deviation.

Upon neurological examination at T0, the proband showed age-adequate fine motor skills that were compromised by intention tremor. Muscular strength was globally reduced. Barefoot walking on toes or heels as well as standing on one leg was impossible. She showed a broad-based gait and slurred speech. At T1, her speech was clearer and more fluid. Her steps were more stable and the intention tremor reduced. Barefoot walking on toes and heels as well as standing on one leg was impossible.

At T2, the girl was fatigued and difficult to motivate. She fell more frequently. Parents reported that she had had abdominal complaints suggesting a gastroenteritis (diarrhea, vomiting and subfebrile temperature for 6 days duration) 2 weeks before, but that this had not led to noticeable acute deterioration. Also, all family members were tested positive for severe acute respiratory syndrome coronavirus 2, but the proband did not show any other symptoms. Her speech was dysarthric. Her gait was broader and she was more instable compared to T1.

At T3, gait had improved again and her speech was rated age-adequate for clarity and fluidity. In contrast to earlier testing, she was able to climb stairs while alternating between legs at each step.

The parental observations (diary): During the first treatment period, the parents reported that her daughter had much more energy and was more active especially with respect to walking and running. Before treatment, she had not been able to carry her backpack for school as she fell backward when putting it on, she acquired this skill within several weeks of the first treatment period. The parents also provided videos showing her improved skills of driving a scooter. When treatment was paused, parents reported an increased instability and frequent falls especially when playing with peers. Without knowing about the treatment trial, the kindergarten echoed the parental observations. They additionally reported that she lost interest in physically strenuous activities during the treatment pause. For the second treatment period, the parents especially reported regained motor skills, improved confidence in movements, and interest in learning new motor skills, for example, the girl wanted to learn swimming.

No adverse effects were noticed, or reported by the patient or her family. Safety laboratory values (whole blood count, liver transaminases, and kidney function) were controlled every 3 months and were within normal limits. Plasma phenylalanine level ([Fig. 1]) was elevated above the reference range (116.2 µmol/L, ref.: 36.2–85.7 µmol/L) once but far below the level that would require treatment in case of a phenylketonuria patient (> 360 μmol/L[11]).

Discussion

In this n-of-1 trial, we document the beneficial effects of L-Phe supplementation for an individual with mitochondrial phenylalanine-tRNA-synthetase (FARS2) deficiency.

During both treatment cycles, we documented an improvement in gross motor function, movement abilities, postural stability as well as quality of life ([Fig. 1]). As we saw a loss of newly acquired skills during the treatment pause, we were able to discriminate the effects of supplementation from age-related development as well as from effects of continuing physiotherapy and occupational therapy. During the end of the treatment pause, the patient underwent gastrointestinal complaints and subfebrile temperature during 6 days. While we formally cannot exclude that this contributed to the observed worsening, the family denies any acute deterioration during this period.

Furthermore, we can report a good tolerance without adverse effects. Additionally, the monthly costs for L-Phe supplementation were low.

Our findings are in line with the recent report of the treatment of—cytosolic—ARS1 deficiencies with their respective cognate amino acids in four affected individuals.[7]

Due to the rarity of FARS2 deficiency, no data on natural history are available, only the clinical phenotype until the genetic diagnosis was reached is reported in the literature.[6] Hence, we cannot directly compare the clinical course of our proband with other individuals affected by FARS2 deficiency. Obviously, our study has some limitations (n = 1, no blinded observers with exception of the kindergarten teacher). To further strengthen our findings, blinding of the patient/family and treating team as well as more treatment cycles with more treatment pauses would be desirable. However, the parents denied this. Naturally, also a longer follow-up is needed to judge the long-term treatment effect and to exclude other factors influencing the course of disease. Nonetheless, we choose to publish our data now in order to inform other affected individuals and their treating team to encourage further studies. We can only speculate that the young age of our patient having the more mild end of the spectrum of clinical findings described with FARS2 deficiency is a favorable prerequisite for the response to L-Phe. If this treatment will also work in patients with a more severe phenotype and older age/longer disease duration will need further studies.

Based on the observed positive effects with good tolerance, no adverse events, and low costs, we conclude that further studies including more individuals with FARS2 deficiency performed in a double-blinded manner are needed. In principle, expansion to other mitochondrial ARS2 deficiencies seems reasonable.

Appendix

Movement Assessment Battery for Children

The MABC-2 is a widely used standardized clinical assessment to evaluate milder movement disorders. This validated version has been standardized for children and adolescents aged 3 to 16 years.[12] The tasks and normative samples are divided into three age bands (3–6, 7–10, and 11–16 years). There are eight tasks per age band, divided into three domains: (a) manual dexterity, (b) balls skills, and (c) balance.

Standard scores for each domain can be compared to normative data and interpreted in terms of percentile equivalents (a) less than or equal to 5th percentile reflecting definite motor impairment, (b) less than or equal to 15th percentile reflecting borderline motor impairment, or (c) more than 15th percentile reflecting no motor impairment, where higher standard scores represent greater impairment. These cutoffs are most commonly used in interpreting results following assessment, particularly when it comes to making referrals for specialized education programming.[12]

There is also a 60-question checklist that requires a parent or teacher to make a qualitative judgment as to how a variety of movement skills are performed in natural contexts. It is scored according to how well the child can perform each item and identifies whether or not the child should be further assessed using the complete MABC-2.

Dynamic Gait Index

The DGI is a performance-based tool used to quantify a child's ability to execute efficient balance and postural control within active movement. It measures balance abilities within altering dynamic movement and the response to changing gait task demands. This test can be used to predict a tendency to fall in an individual. There is a maximum score of 24 points. A reduction in the score by three points is considered clinically meaningful.[13]

Gross Motor Function Measure 66

The GMFM-66 was originally developed to monitor and adjust therapies for the rehabilitation of children with cerebral palsy. It consists of 66 items that can be divided into five categories (lying and rolling, sitting, crawling and kneeling, standing, walking, running, and jumping). The gross motor ability estimator is a software required to calculate and interpret the final scores.[14]

Quality of Life

The pediatric quality of life questionnaire is a semiquantitative approach to assess the progression of a child throughout their development. The test can be used for children between the age of 2 and 18 years. Depending on their age, the questions can be answered in part by the parents. It consists of 15 items regarding physical as well as psychological health. Each item is then scored on a scale from 0 to 4 which is then transformed into a score from 100 to 0 (100 = 0, 75 = 1, 50 = 2, 25 = 3, 0 = 4). The total score then is calculated within a specialized program.[15]

Conflict of Interest

None declared.

Authors' Contributions

S.L.O., K.S., M.T.A., E.G., R.E.B., W.M.S., E.T., E.H., A.E., D.M., and J.A.M. made substantial contributions to acquisition, analysis, or interpretation of data for the work and drafted the work. SBW conceptualized and designed the study and contributed substantially to acquisition and analysis of data, drafting a significant portion of the manuscript or figures.

All authors revising it critically for important intellectual content and approved the final version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

^* These authors contributed equally.

• References

• 1 Gorman GS, Chinnery PF, DiMauro S. et al. Mitochondrial diseases. Nat Rev Dis Primers 2016; 2: 16080
• 2 Hoytema van Konijnenburg EMM, Wortmann SB, Koelewijn MJ. et al. Treatable inherited metabolic disorders causing intellectual disability: 2021 review and digital app. Orphanet J Rare Dis 2021; 16 (01) 170
• 3 Repp BM, Mastantuono E, Alston CL. et al. Clinical, biochemical and genetic spectrum of 70 patients with ACAD9 deficiency: is riboflavin supplementation effective?. Orphanet J Rare Dis 2018; 13 (01) 120
• 4 Zweers H, van Wegberg AMJ, Janssen MCH, Wortmann SB. Ketogenic diet for mitochondrial disease: a systematic review on efficacy and safety. Orphanet J Rare Dis 2021; 16 (01) 295
• 5 Konovalova S, Tyynismaa H. Mitochondrial aminoacyl-tRNA synthetases in human disease. Mol Genet Metab 2013; 108 (04) 206-211
• 6 Almannai M, Faqeih E, El-Hattab AW. et al. FARS2 deficiency. In: Adam MP, Everman DB, Mirzaa GM. et al., eds. GeneReviews((R)). Seattle (WA, USA): 1993
• 7 Kok G, Tseng L, Schene IF. et al. Treatment of ARS deficiencies with specific amino acids. Genet Med 2021; 23 (11) 2202-2207
• 8 Elo JM, Yadavalli SS, Euro L. et al. Mitochondrial phenylalanyl-tRNA synthetase mutations underlie fatal infantile Alpers encephalopathy. Hum Mol Genet 2012; 21 (20) 4521-4529
• 9 Vantroys E, Larson A, Friederich M. et al. New insights into the phenotype of FARS2 deficiency. Mol Genet Metab 2017; 122 (04) 172-181
• 10 [Anonymous] . World Medical Association declaration of Helsinki. Recommendations guiding physicians in biomedical research involving human subjects. JAMA 1997; 277 (11) 925-926
• 11 van Wegberg AMJ, MacDonald A, Ahring K. et al. The complete European guidelines on phenylketonuria: diagnosis and treatment. Orphanet J Rare Dis 2017; 12 (01) 162
• 12 Psotta R, Abdollahipour R. Factorial validity of the movement assessment battery for children-2nd Edition (MABC-2) in 7-16-year-olds. Percept Mot Skills 2017; 124 (06) 1051-1068
• 13 Lubetzky-Vilnai A, Jirikowic TL, McCoy SW. Investigation of the dynamic gait index in children: a pilot study. Pediatr Phys Ther 2011; 23 (03) 268-273
• 14 Beckers LW, Bastiaenen CH. Application of the gross motor function measure-66 (GMFM-66) in Dutch clinical practice: a survey study. BMC Pediatr 2015; 15: 146
• 15 Varni JW, Seid M, Rode CA. The PedsQL: measurement model for the pediatric quality of life inventory. Med Care 1999; 37 (02) 126-139

Publication History

Received: 01 December 2022

Accepted: 04 January 2023

Accepted Manuscript online:
05 January 2023

Article published online:
20 February 2023