Case Report and Review of the Literature: A New and a Recurrent Variant in the VARS2 Gene Are Associated With Isolated Lethal Hypertrophic Cardiomyopathy, Hyperlactatemia, and Pulmonary Hypertension in Early Infancy

Mitochondriopathies represent a wide spectrum of miscellaneous disorders with multisystem involvement, which are caused by various genetic changes. The establishment of the diagnosis of mitochondriopathy is often challenging. Recently, several mutations of the VARS2 gene encoding the mitochondrial valyl-tRNA synthetase were associated with early onset encephalomyopathies or encephalocardiomyopathies with major clinical features such as hypotonia, developmental delay, brain MRI changes, epilepsy, hypertrophic cardiomyopathy, and plasma lactate elevation. However, the correlation between genotype and phenotype still remains unclear. In this paper we present a male Caucasian patient with a recurrent c.1168G>A (p.Ala390Thr) and a new missense biallelic variant c.2758T>C (p.Tyr920His) in the VARS2 gene which were detected by whole exome sequencing (WES). VARS2 protein was reduced in the patient's muscle. A resulting defect of oxidative phosphorylation (OXPHOS) was proven by enzymatic assay, western blotting and immunohistochemistry from a homogenate of skeletal muscle tissue. Clinical signs of our patient included hyperlactatemia, hypertrophic cardiomyopathy (HCM) and pulmonary hypertension, which led to early death at the age of 47 days without any other known accompanying signs. The finding of novel variants in the VARS2 gene expands the spectrum of known mutations and phenotype presentation. Based on our findings we recommend to consider possible mitochondriopathy and to include the analysis of the VARS2 gene in the genetic diagnostic algorithm in cases with early manifesting and rapidly progressing HCM with hyperlactatemia.

Mitochondriopathies represent a wide spectrum of miscellaneous disorders with multisystem involvement, which are caused by various genetic changes. The establishment of the diagnosis of mitochondriopathy is often challenging. Recently, several mutations of the VARS2 gene encoding the mitochondrial valyl-tRNA synthetase were associated with early onset encephalomyopathies or encephalocardiomyopathies with major clinical features such as hypotonia, developmental delay, brain MRI changes, epilepsy, hypertrophic cardiomyopathy, and plasma lactate elevation. However, the correlation between genotype and phenotype still remains unclear. In this paper we present a male Caucasian patient with a recurrent c.1168G>A (p.Ala390Thr) and a new missense biallelic variant c.2758T>C (p.Tyr920His) in the VARS2 gene which were detected by whole exome sequencing (WES). VARS2 protein was reduced in the patient's muscle. A resulting defect of oxidative phosphorylation (OXPHOS) was proven by enzymatic assay, western blotting and immunohistochemistry from a homogenate of skeletal muscle tissue. Clinical signs of our patient included hyperlactatemia, hypertrophic cardiomyopathy (HCM) and pulmonary hypertension, which led to early death at the age of 47 days without any other known accompanying signs. The finding of novel variants in the VARS2 gene expands the spectrum of known mutations and phenotype presentation. Based on our findings we recommend to consider possible mitochondriopathy and to include the analysis of the VARS2 gene in the genetic diagnostic algorithm in cases with early manifesting and rapidly progressing HCM with hyperlactatemia.
Keywords: VARS2 gene, oxidative phosphorylation, mitochondriopathy, hyperlactatemia, lethal hypertrophic cardiomyopathy, pulmonary hypertension INTRODUCTION Mitochondria are organelles responsible for energy production in the form of adenosine triphosphate (ATP) through the oxidative phosphorylation system (OXPHOS). The process of energy production is performed by the mitochondrial respiratory chain and ATP synthase located in the inner membrane of mitochondria (1). Mitochondrial disorders represent a wide spectrum of heterogeneous syndromes predominantly presenting with symptoms as lactic acidosis, hypotonia, developmental delay, failure to thrive, and encephalopathy due to cell energy depletion. Mitochondrial disorders are associated with many causative genes (2)(3)(4), which are encoded by mitochondrial DNA (mtDNA) or nuclear DNA (5). Valyl-tRNA synthetase (Val-tRNA) involved in mitochondrial translation (the process of protein synthesis from the information contained in a molecule of messenger RNA -mRNA) is encoded by the VARS2 gene (6) and catalyzes the amino acid valine's attachment to its tRNA (7). Dysfunction of the protein product is responsible for combined oxidative phosphorylation deficiency 20 (OMIM: # 615917) inherited in an autosomal recessive manner. Rare biallelic variants in the VARS2 gene have been associated with severe clinical features as mitochondrial encephalomyopathy or encephalocardiomyopathy in 23 affected individuals from 19 families worldwide (1,4,(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). In the present paper we describe a compound heterozygous case with a recurrent c.1168G>A (p.Ala390Thr) and a novel missense variant c.2758T>C (p.Tyr920His) in the VARS2 gene causing isolated hypertrophic cardiomyopathy, hyperlactatemia, and pulmonary hypertension leading to early death. The pathogenicity of the detected variants is supported by immunohistochemistry, enzymatic analysis, and western blot in a muscle sample of the affected individual. Finally, we compare our findings with the case reports published so far.

Clinical Description
All clinical information about our patient was obtained from medical records retrospectively with the patient's parents' informed consent and was correlated with potential causative mutations detected by whole-exome sequencing (WES).

Genetic Investigation
Genetic testing was performed from the peripheral blood of the patient. Karyotyping was performed according to a standard cytogenetic protocol. SNP array was performed with Illumina Cyto 850Kv1.1 Bead Chip. In addition, DNA isolated from the patient's peripheral blood lymphocytes was analyzed by whole-exome sequencing. The library was prepared by SureSelect60Mbv6 (Agilent) and paired-end sequenced on a HiSeq 4000 platform (Illumina) with a readlength of 100 bases. In order to align reads to the human genome assembly hg19 Burrows-Wheeler Aligner (BWA, v.0.5.87.5) was applied and detection of genetic variation was performed using SAMtools (v 0.1.18), PINDEL (v 0.2.4t), and ExomeDepth (v 1.0.0). The cut-off for biallelic inheritance was set to <1% allele frequency, for monoallelic inheritance to <0.1%. The size of reference entries was >20,000 exomes in the database at the time of analysis (16). With this approach, 97.3% of the target sequences were covered >20-fold. The detected mutations were verified by Sanger sequencing, followed by examining the DNA from peripheral blood of the patient's parents.

Muscle Biopsy
The biopsy of the vastus lateralis muscle was performed under general anesthesia according to standard procedures. Muscle tissue was cut into several pieces for formalin fixation, glutaraldehyde fixation, and rapid freezing in isopentane cooled in liquid nitrogen for subsequent analyses.

Enzymatic Analysis
Muscle 600 × g homogenates were used for determination of enzymatic activities of the OXPHOS complexes. Enzyme activities of the OXPHOS complexes were determined as previously described (17). The rotenone-sensitive complex I activity was measured spectrophotometrically as NADH/decylubiquinoneoxidoreductase at 340 nm. The enzyme activities of citrate synthase, complex IV (ferrocytochrome c/oxygenoxidoreductase), and oligomycin-sensitive ATPase activity of the F 1 F O ATP synthase (complex V) were determined as previously described (18). The whole reaction mixture for the ATPase activity measurement was treated for 10 s with an ultra-sonifier (Bio cell disruptor 250, Branson, Vienna, Austria). The reaction mixture for the measurement of the complex III activity contained 50 mM potassium phosphate buffer pH 7.8, 2 mM EDTA, 0.3 mM KCN, 100 µM cytochrome c, 200 µM reduced decyl-ubiquinol. The reaction was started by addition of the 600 g homogenate. After 3-4 min the reaction was inhibited with 1 µM antimycin A. All spectrophotometric measurements (Uvicon 922, Kontron, Milan, Italy) were performed at 37 • C.

Substrate Oxidation Analysis
Muscle 600 × g homogenate was incubated with different 14 Clabeled substrates ( Table 1) for 20 min at 37 • C according to Bookelman et al. (19) in a reaction volume of 50 µl. The reactions were stopped by the addition of 20 µl of 15% HClO 4 and the 14 CO 2 was collected in 1M NaOH-wetted filter paper snippets (Whatman) placed in the screw cap of 2 ml reaction tubes (Sarstedt). After incubation for 15 min on ice, the filter papers were transferred to new 2 ml reaction tubes containing 1 ml of scintillation buffer Ultima Gold (Perkin Elmer) and counted in a scintillation counter (Packard 1600 TR). The scintillation counts of the different substrate combinations were related to the total activities per reaction and calculated as nmol/h/mg protein activity oxidation.

Western Blot
Muscle tissue homogenates from the patient and controls (centrifuged at 600 × g) were analyzed on 10% acrylamide/bisacrylamide gels and transferred to nitrocellulose membranes. The membranes were washed in Tris-buffered saline (TBS) for 5 min, air-dried for 30 min, washed 10 min in TBS, and blocked 1 h at room temperature in 1x western blocking solution in TBS-T (Roche, Mannheim, Germany). After washing with TBS-Tween 20 (0.5%; TBS-T), the membranes were incubated with the primary antibody diluted in 1x western blocking solution in TBS-T. The indicated primary antibody dilutions and incubation times were used for western blot analysis: polyclonal rabbit VARS2

Case Report -Clinical Features of the Patient
The male patient was born at term (at 39+3 weeks of gestation) by vaginal delivery using vacuum extraction, as the first child of non-consanguineous Caucasian parents. Due to the prenatally suspected aortic stenosis, the patient was immediately transferred to the Neonatal Intensive Care Unit. Birth weight (3140 g; 21st percentile), birth length (51 cm; 48th percentile), head circumference (33.5 cm; 17th percentile), Apgar scores 9/10/10, and pH from the umbilical artery were normal (pH: 7.27). The newborn had marks after vacuum extraction over the parietal region (cephalohematoma); muscle tone was normal, and no dysmorphic features were present. Postnatal echocardiography showed borderline width of the isthmus and aortic arch (but still within normal range), bicuspid aortic valve, and an open ductus arteriosus Botalli. No surgery or prostaglandin treatment was necessary, but the patient developed a need for oxygen therapy. On the third day after birth blood gas analysis showed an uncompensated metabolic acidosis with hyperlactatemia: plasma lactate: 6.7 mmol/l (normal range < 2.1 mmol/l), pH: 7.28 (N: 7.35-7.45), pCO 2 : 42.1 mmHg (N: 35-45 mmHg), HCO − 3 : 18.2 mmol/l (N: 21-28), Base excess: −7.3 (N: 0 ± 3 mmol/l) with normoglycemia (Blood glucose: 59 mg/dl, N: 50-114). Anion gap was slightly elevated (AG: 16.9, N: 7-16). Since an inherited disorder of metabolism was suspected, metabolic screening was performed, which showed repeatedly lactate elevation: preprandial lactate: 6 mmol/l (N: 0.5-1.6), 3-OH-butyrate: 0.1 mmol/l (N: 0.03-0.3) glucose: 57 mg/dl (N: 50-114); postprandial lactate: 4 mmol/l, 3-OHbutyrate: 0.3 mmol/l, glucose: 84 mg/dl; plasmatic alanine elevation: 539.8 µmol/l (N: 116-376) and normal level of lactate in urine (19 mmol/mol creatinine, N 51 (1-156) mmol/mol creatinine). Other parameters (acylcarnitine profile, creatine kinase, aminotransferases, ammonemia, electrolytes, blood count) were normal. Glucose infusion (5 mg/kg/min.) was administered. The patient could have oral intake (suction) and neurological status was normal, with no signs of lethargy or hypotonia. Sonography of the abdomen did not show any pathology, especially no hepatosplenomegaly. Ophthalmologic examination showed normal findings without signs of cataract. The patient developed progressive symptoms of heart failure over the next few days. Echocardiography on the 16th day of life revealed primary pulmonary hypertension and hypertrophic cardiomyopathy of the left heart. Investigation of alphaglucosidase excluded Pompe disease (19 nmol/mg-normal value). Cardiac catheterization showed pulmonary hypertension (PHT) and hemodynamic failure of the right heart. The myocardial biopsy was contraindicated due to the patient's severe clinical condition. Dobutamine, nitric oxide therapy, sildenafil, and oxygen therapy, with 60% FiO2 was administered. Based on suspicion of a mitochondrial disorder, muscle biopsy from the vastus lateralis muscle was performed for histopathological purposes. No changes for a primary muscle disorder were found. Based on specific enzyme histochemical staining (e.g., combined COX-SDH), no evidence for the presence of a mitochondrial myopathy could be demonstrated. Genetic testing of the ACAD9 gene associated with mitochondrial complex I deficiency, nuclear type 20 (OMIM: # 611126) did not reveal any pathogenic mutation, and therefore whole-exome sequencing was indicated. Because of lasting suspicion of a mitochondrial disorder, treatment with riboflavin (20 mg/kg/d), coenzyme Q10 (30 mg/kg/d), and continuous intravenous glucose was administered. Under this treatment, lactate levels declined but still were above normal values. Levels were elevated during PHT crises (values during PHT crises were 11-13.3 mmol/l). On day 22, cardiac insufficiency worsened, and a first cardiopulmonary resuscitation was necessary. MRI of the brain and thorax performed on day 23 showed a focal hemorrhage in the right frontal subcortical region. The degree of myelination was appropriate for age. Cardiomegaly was evident. Low pleural effusions on both sides of the thorax and dystelectasia/atelectasia in both lungs were seen. On day 31, because of recurrent PHT crisis, dilatation and stent treatment of the foramen ovale was performed but was not successful. The patient was intubated and sedated with ketamine, midazolam, sufentanil, and myorelaxed. Cardiomyopathy progressed despite intensive care and the patient died on day 47 due to cardiopulmonary failure. A few days after the patient's death, the result of the WES analysis confirmed the state of compound heterozygosity for variants in the VARS2 gene, supporting the assumption of a mitochondrial disorder in the child. Following the wish of the parents, no autopsy was performed.

Molecular Genetics Findings
Cytogenetic examination showed a normal male karyotype 46, XY. Genomic imbalances were ruled out by the SNP array. The whole-exome sequencing revealed the following biallelic variants in VARS2 gene: allele 1 [NM_020442.6:exon13: c.1168G>A (p.Ala390Thr)] (https://www.ncbi.nlm.nih.gov/ clinvar/variation/522814/); allele 2 [NM_020442.6:exon27: c.2758T>C (p.Tyr920His)] (https://www.ncbi.nlm.nih.gov/ clinvar/variation/997679/) (Figures 1A,B). The finding was verified by Sanger sequencing. Examination of the parents showed the paternal VARS2 variant c.1168G>A (p.Ala390Thr) in a heterozygous form and the maternal VARS2 variant c.2758T>C (p.Tyr920His) in a heterozygous form ( Figure 1C). Both variants involve residues that are highly conserved among phylogenetically distant organisms. At position 920, tyrosine is most frequently found in some organisms, also the amino acid phenylalanine, both with a phenyl group. In plants, this position is not conserved with serine or threonine in the alignment. Position 390 is highly conserved (Figure 1D). Position 390 corresponds to the tRNA-synthetase domain and position 920 corresponds to the anticodon binding domain of the protein VARS2 (1,12). These findings support the pathogenicity of the variants found in our patient.

Histopathology, Immunohistochemistry, Enzymatic Analysis, Substrate Oxidation, and Western Blot
Western blot analysis revealed a reduction of VARS2 protein amount in muscle homogenates of the affected patient compared to seven controls (Figures 2A,C; Supplementary Table 1). VARS2 was normalized to four loading proteins namely VDAC1, CS, GPI, and SDHA. Residual VARS2 protein levels were between 16 and 34% of the control levels. No obvious histopathological findings pointing toward a mitochondrial disorder were found. The enzymatic analysis showed values in or above the normal range for the mitochondrial marker enzyme citrate synthase (summary of the results is shown in Table 1). Absolute complex I activity was significantly reduced in patient muscle compared to controls. Consistently, the complex I activities normalized to the citrate synthase and complex II were significantly lower in patients muscle ( Table 1). Normalized to complex II which is not dependent on mitochondrial translation a downregulation of complex IV was found. An increase in combined normalized complex II-III activity was present. A compensatory upregulation of unaffected complexes is a well-known phenomenon in mitochondrial disorders. In substrate oxidation analysis some activities were in relation to the protein content in the normal range, others were elevated. Also in relation to citrate synthase similar activities were high. In relation to complex II the oxidation rate of pyruvate + carnitine was slightly elevated. These elevated activities are likely due to compensatory upregulation of mitochondria. In contrast, the ratio of the long oxidation path via pyruvate + malate compared to pyruvate + carnitine was somewhat reduced with a value of 0.63 (normal 0.68-1.09). This is consistent with a defect in the respiratory chain, while the reaction from pyruvate to acetyl-carnitine works better. This shortcut of the full oxidation route forms only one NADH, while the whole path creates 4 NADH and 1 FADH 2 . Consistently, western blot analysis revealed a reduction of complexes I, III and IV in patient homogenate compared to controls (Figure 2). The reduction was present compared to several normalization proteins VDAC1 (outer mitochondrial membrane), CS (mitochondrial matrix), GPI (cytosol) and SDHA (inner mitochondrial membrane and independent from mitochondrial translation) (Figure 2;  Supplementary Figure 1; Supplementary Table 1). Also in immunohistochemical staining of VDAC1 (Voltage-dependent anion-selective channel 1) and subunits of the OXPHOS complexes in skeletal muscle showed a slight reduction of NDUFB8 (complex I subunit) and UQCRC2 (complex III subunit) in the affected individual compared to controls (Supplementary Figure 2). A compensatory upregulation of complex II was also present in immunohistochemical staining.

DISCUSSION
Mitochondriopathies associated with mutations in mitochondrial aminoacyl-tRNA synthetases present a wide spectrum of miscellaneous disorders based on various genetic changes. Recently, several mutations in VARS2 gene were associated with clinical features such as hypotonia, psychomotor delay, encephalopathy, cardiomyopathy, hyperlactatemia, but the correlation between genotype and phenotype remains unclear (1,15). To date, 19 families with more than 23 affected individuals have been described in the literature worldwide (1,4,(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) whereby c.1100C>T (p.Thr367Ile) is the most common variant in the VARS2 gene present in 60.8% (14/23) of the published cases (see Table 2). Common features of homozygous carriers for variant c.1100C>T (p.Thr367Ile) include microcephaly, global psychomotor delay, and hypotonia, less often nystagmus, limb spasticity, and difficulties with feeding. In the first 12 months of life, ataxia, dystonic movements, and seizures can occur, which later (from 2 to 4 years) almost always lead to refractory epilepsy and status epilepticus based on mitochondrial encephalopathy (1,6,13). Nevertheless, hypertrophic cardiomyopathy (HCM) has never been observed in these patients (eight patients of which five had no HCM, in three cases the results of cardiologic examination are not available) (1,6,13,15). However, in compound heterozygotes (for variant c.1100C>T (p.Thr367Ile) and another, or two other pathogenic variants, hypertrophic cardiomyopathy was present in 14/15 cases (1,4,(7)(8)(9)(10)(11)(12)14). These additional findings support Bruni et al. (1) assumption that "c.1100C>T variant could have a lesser effect to the heart." In the present paper we describe a compound heterozygous case with a recurrent c.1168G>A    Figure 1D) (15). However, OXPHOS activity was decreased only in six of them (the first patient had low complex IV activity, the second patient had combined complex I + complex IV deficiencies, the third patient had low complex IV activity, the fourth had low complex I activity, the fifth had low complex IV activity, and the sixth showed reduced complex I and III activity). In our case the results showed a strong reduction of complex I, a moderate reduction of complex III and IV, and compensatory upregulation in complex II and V. A review of all published patients so far is shown in Table 2. Clinical manifestations, disease severity, and life expectancy vary significantly between published cases. We assume that the different phenotypic manifestations in the reported cases could result from the variable expression of valyl-tRNA synthetase in different tissues at different time points after birth, and/or due to unknown epigenetic effects, but further investigations in this field are needed.

CONCLUSION
The data and finding of a novel variants in the VARS2 gene of our case expands the spectrum of known mutations in humans and the spectrum of associated clinical features. Nowadays, thanks to increasing number of publish data we can assume a reduced impact of the homozygous c.1100C>T (p.Thr367Ile) mutation on the myocardium, but we cannot postulate that specific OXPHOS complexes or organ systems are impaired due to specific mutations in the VARS2 gene. Further investigations to find specific genotype-phenotype correlations are needed in this field. It is known that biallelic mutations in the VARS2 gene cause systemic impairment. Structural cardiac abnormalities and hypertrophic cardiomyopathy (HCM) could be the first manifestation of the disease leading to early death in the newborn period or in early infancy before developing other clinical features. That is why we think that it is of utmost importance to consider the presence of a possible mitochondriopathy in these patients and to include the analysis of the VARS2 gene in the genetic diagnostic algorithm in cases with early manifesting and rapidly progressing HCM with hyperlactatemia.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of the Land Salzburg (number 415-E/2552/10-2019). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.