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-tRNAPhe 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]
