Rescue of respiratory failure in pulmonary alveolar proteinosis due to pathogenic MARS1 variants

Pulmonary alveolar proteinosis (PAP) is a heterogeneous condition with more than 100 different underlying disorders that need to be differentiated to target therapeutic options, which are generally limited.

hypoglycemia was detected, the pulmonary condition deteriorated, and the patient developed multiorgan failure. Six therapeutic whole lung lavages (WLL) were necessary to improve respiratory insufficiency. Methionine supplementation was started and a high protein diet ensured, leading to complete respiratory recovery.
The older brother, homozygous for the same MARS1 variant, had a long-known distinct eating preference of methionine-rich food and showed a less severe clinical phenotype. Decreased aminoacylation activity confirmed the pathogenicity of p.(Arg598Cys) in vitro. In agreement with our review of currently published ILLD patients, the presence of hepatopathy, developmental delay, muscular hypotonia, and anemia support the multisystemic character of the disease.
Conclusions: Catabolic events can provoke a severe deterioration of the pulmonary situation in ILLD with a need for repetitive WLL. Although the precise role of oral methionine supplementation and high protein intake are unknown, we observed an apparent treatment benefit, which needs to be evaluated systematically in controlled trials. catalyzing the aminoacylation of tRNA by their cognate amino acid, thereby linking it with the correct nucleotide anticodon. [5][6][7] While monoallelic pathogenic variants in cytosolic ARSs genes have been identified to cause neurodegenerative diseases, 8,9 biallelic pathogenic variants in cytosolic ARSs genes are typically associated with multisystemic phenotypes. Biallelic pathogenic variants in MARS1 lead to a disease characterized by an early onset PAP with rapid progression to pulmonary fibrosis and structural hepatopathy, 4,10 known as "interstitial lung and liver disease" (ILLD, MIM #615486). However, a systematic analysis of the clinical spectrum has not been performed till date. Of note, the introduction of pathogenic MARS1 variants into yeast caused attenuation of enzyme activity and growth retardation, which could be alleviated by methionine supplementation in cell culture. 4,11 In one patient diagnosed with PAP caused by pathogenic MARS1 variants, the respiratory condition improved after methionine supplementation was initiated. 12 No details, however, were given describing the further clinical course of the infant. We provide detailed data on the clinical course of two brothers affected by ILLD due to a novel homozygous MARS1 variant in comparison with the published patients suffering from this condition, along with functional data on MARS1 activity in fibroblasts of both brothers. Therapeutic options like WLL, a diet rich in proteins, and oral methionine supplementation are discussed. Our data expand the clinical and genetic phenotype of the disease and will be helpful for future treatment decisions of PAP caused by pathogenic variants in MARS1.

| Study design, recruitments of patients and data acquisition
Patients were studied in detail by thoroughly evaluating the medical history and re-evaluating liver histology. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2013. Informed consent to participate in the study was obtained from the parents. The study was approved by the ethical committee of the University Hospital Heidelberg. Regarding previously published patients, data were retrieved from the published reports identified by a comprehensive bibliographic search via PubMed and Google Scholar. Queries were based on the terms "MARS," "MARS1," "Interstitial lung and liver disease," and "ILLD." Phenotyping was performed according to Human Phenotype Ontology terminology.
The cut-off date for data analysis was 6 April 2020. build a generalized additive model by a quadratically penalized likelihood type approach (formula = y~s(x, bs = "cs")) of the geom_smooth function to enable improved pattern detection by plotting a smoothened line and the confidence interval around the calculated scores for each alternate amino acid position.

| In vitro studies in patient fibroblasts and controls
Fibroblasts were collected from affected individuals after informed consent was obtained. A fibroblast control cell line was purchased from Merck (SCC058, Darmstadt, Germany). All cells were tested for mycoplasma contamination. Fibroblasts were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO 2 . All studies were performed as biological triplicates. [D 3 ]-leucine and [ 13 C]-valine were added as internal standards, and the labeled amino acids were quantified by LC-MS/MS. Intra-assay and interassay variation were <15%. Isoleucyl-tRNA synthetase (IARS1), and leucyl-tRNA synthetase (LARS1) activities were simultaneously detected as control enzymes. Statistical significance of differences between activities of controls and individual patients were assessed using the Student t test, confidence interval (CI) 95% (SPSS 26.0.0.1).

| Western blot
For immunoblotting, cells were washed in phosphate buffered saline with tween (0.1%), and resolved in radioimmunoprecipitation assay buffer.
Twenty μg of protein of every sample were separated on a 10% polyacrylamide gel. Primary antibodies against MARS1 (Sigma, Darmstadt, Germany) and β-actin (Santa Cruz Biotechnology, Heidelberg, Germany) were incubated overnight. Secondary hrp-coupled antibodies (goatantirabbit or rabbit-antimouse) were obtained from Dianova (Hamburg, Germany). Enhanced chemiluminescence of proteins was detected using Vilberscan Fusion FX7.

| Case presentations including liver histology
The index patient was born to healthy, consanguineous parents (firstgrade cousins) as the third child of four siblings ( Figure 1A). Birth of the index patient was at term (38 + 4 weeks of gestational age) via Cesarean section with low body weight (2120 g; z − 2.97) and height (44 cm; z − 3.29), 15 hence presenting small for gestational age (SGA). He was admitted to the neonatology ward due to hypoglycemia (min. 2.17 mmol/L). He was discharged in good clinical conditions after some days of intensive feeding. Aged 4 months, the boy was admitted to hospital with pneumonia ( Figure    He was born at 35 + 1 weeks of gestation via Cesarean section due to a HELPP-syndrome of the mother (birthweight 2040 g, z − 1.39). 15 In the neonatal period, the boy developed apnea, tachypnea, and hypoglycemia  Figure 4A,B), impaired lung function, and a slight gross and fine motor coordination disorder. However, no signs of liver disease were detected. Based on the positive experience in his younger brother, a protein-rich diet was assured (2-3 g/kg/d) and methionine supplementation initiated (see below).
Find more details on the clinical course of both patients in the supplement.

| Methionine supplementation and monitoring in both brothers
The plasma levels of methionine of the two brothers were closely   Table S1. The lung was the predominantly affected organ present in all 36/36 individuals, whereas liver affection was reported for 31/36, ranging from hepatomegaly and elevated hepatic transaminases to cholestasis and early cirrhosis. Other organ affections were noticed when data was available: at least 17 patients were born small for gestational age, 29 of 35 presented with failure to thrive, anemia was present in 7/7. There is limited information on the neurological phenotype, such as developmental delay or muscular hypotonia (the latter found in 4/5 patients). Three patients were reported to have hypothyroidism. 14/36 patients died at a median age of 16.4 months (range 5 months-25 years) which was associated to respiratory failure in all cases.

| Phenotypic and genetic specturm of ILLD
Altogether, these 36 patients carried 13 different variants, including 11 missense variants, 1 nonsense variant, and 1 duplication triplet insertion leading to an aminoacid duplication. Variants were distributed throughout the gene with a clearly increased density in the catalytic domain, in line with higher prediction scores compared to variants affecting other domains of the protein (Figures 1C,D). As the variants carried by the patients from the Réunion Island are attributed to one founder, 4 they were counted only once for the density plot ( Figure 1C).

| In-vitro analyses
Western blot analysis showed normal levels of MARS1 in fibroblasts of patient F1:II.3 ( Figure 6A). However, the aminoacylation activity was significantly reduced in F1:II.3 to about 5% of controls (P < .01).
In the older brother (F1:II.1), MARS1 activity was reduced to 22% of that of controls, still significant (P < .01). To demonstrate that the reduced enzyme activity was specific for MARS1, we analyzed the activity of IARS1 and LARS1 in parallel and found no difference to the control cells ( Figure 6B).

| DISCUSSION
Recently we have described biallelic pathogenic variants in MARS1 to cause an early-onset and severe disease named interstitial lung and liver disease (MIM #615486). 4 In this study, we present two brothers homozygous for a previously unreported variant in MARS1 and further detail the phenotype in particular with respect to metabolic vulnerability and additional organ-involvement of this condition.
Following a catabolic situation, the index patient experienced hypoglycemia and in a second episode a respiratory deterioration with liver and kidney failure. This observation is of specific interest, as acute deterioration triggered by catabolism has not been reported in ILLD patients so far, although acute deterioration is known in other cytosolic ARS deficiencies. [19][20][21] To stabilize the boy temporarily, several WLLs were necessary. Such symptomatic treatment is sometimes necessary for many years. 17 Therefore, alternative and less invasive treatments are urgently needed. Based on mutationally reduced MARS1 enzymatic activity which can be overcome in vitro by methionine substrate supply, and anecdotal evidence from patients on La Réunion and surrounding islands indicating a benefit from the consumption of large amounts of eggs, we supplemented methionine and ensured a high protein intake. We argue that this intervention contributed to a significant improvement in growth, pulmonary, and neurodevelopmental state. Of note, MARS1-04 (Table S1) was also reported to improve his clinical condition after methionine supplementation, but no details on dosage or methionine levels were given, nor was follow-up data available. 12 However, methionine supplementation should be handled carefully with monitoring of methionine plasma concentrations (suggested target range 80-150 μmol/L), as high levels (>600 μmol/L) are neurotoxic leading to severe brain edema. 22 The index patient showed a considerable fluctuation of methionine concentrations despite standardized measurement regimens. Interestingly, high daily protein intake is also  20,21 and isoleucine supplementation appeared to be beneficial in IARS1 deficiency. 19 However, data are lacking to judge this effect and further studies are needed to evaluate the benefit of nutritional therapeutic approaches in the group of ARS deficiencies.
The homozygous variant of the index patient was also found in his older brother. As his clinical course was milder the question was raised whether disease severity was influenced by environmental factors. In fact, the older brother had special dietary habits, for example, the intensive consumption of eggs in everyday life since early childhood and hence an increased protein and methionine intake. In addition, the aminoacylation activity in fibroblasts was higher in this brother than in the index patient, an observation that cannot be explained by nutritional habits but pointing at a higher residual activity as a potential cause of the milder clinical phenotype. Scrutinizing exome data of the index patient did not reveal further suspicious variants neither in MARS1 nor in genes encoding for direct interaction partners of MARS1 in the multi synthetase complex. This complex consists of eight cytosolic ARSs together with three auxiliary proteins and regulates transcription, translation, and various signaling pathways. 23,24 Analysis of the distribution of genetic variants found in ILLD patients demonstrates a clear accumulation of variants in the region of the catalytic domain, a finding which well reflects the in silico analysis of disease severity scores related to variant location. This provides further evidence that the pathomechanism in ILLD is linked to defective aminoacylation. MARS1 belongs to the group of cytosolic aminoacyl-tRNA synthetases playing a pivotal role in protein translation in all kinds of cells. In line with this, defects of those enzymes affect growth and multiple organ systems, mainly lungs, liver, nervous system, hematopoietic system, and musculature. 25 Besides intense pulmonary affection and progressive cirrhotic liver disease, our index patient F1:II.3 suffered from severe global developmental delay, dystrophy, muscular hypotonia, and anemia.
Systematically analyzing all currently published patients with biallelic pathogenic MARS1 variants, our study emphasizes that human disease related to biallelic MARS1 variants is not restricted to lung and liver, but also comprises abnormalities of the nervous system, growth, and hematopoietic system. Deep phenotyping is crucial to understand the natural history of the disease and to improve care for affected individuals and families. We recommend that patients with biallelic MARS1 variants should be thoroughly analyzed regarding multisystemic signs and symptoms, especially monitoring psychomotor development, growth, and blood abnormalities, even if lung disease with recurrent respiratory infections is the clinically predominant and leading sign. As in other forms of PAP, we assume that those respiratory infections, in particular when they affect the alveolar region, result in disturbance of alveolar macrophage surfactant clearance, leading to the accumulation of alveolar surfactant, that is, PAP, if there is a predisposition from genetic or other factors for PAP. In conclusion, our study points to the fact that biallelic pathogenic variants in MARS1 not only provoke interstitial lung and cholestatic liver disease, but cause a multisystemic phenotype including growth retardation, neurodevelopmental delay, anemia, and muscular hypotonia. Catabolic events like infections or surgery can lead to acute deterioration and multiorgan failure. WLL can stabilize the pulmonary condition, if PAP is predominant. High protein intake together with methionine supplementation may improve PAP, but also other organ manifestations in ILLD and should be considered to be started early in life. Controlled clinical trials are necessary to investigate the role of protein and methionine supplementation in individuals with biallelic pathogenic variants in MARS1.