Biallelic variants in LARS2 and KARS cause deafness and (ovario)leukodystrophy

Abstract

Objective

To describe the leukodystrophy caused by pathogenic variants in LARS2 and KARS, encoding mitochondrial leucyl transfer RNA (tRNA) synthase and mitochondrial and cytoplasmic lysyl tRNA synthase, respectively.

Methods

We composed a group of 5 patients with leukodystrophy, in whom whole-genome or whole-exome sequencing revealed pathogenic variants in LARS2 or KARS. Clinical information, brain MRIs, and postmortem brain autopsy data were collected. We assessed aminoacylation activities of purified mutant recombinant mitochondrial leucyl tRNA synthase and performed aminoacylation assays on patients' lymphoblasts and fibroblasts.

Results

Patients had a combination of early-onset deafness and later-onset neurologic deterioration caused by progressive brain white matter abnormalities on MRI. Female patients with LARS2 pathogenic variants had premature ovarian failure. In 2 patients, MRI showed additional signs of early-onset vascular abnormalities. In 2 other patients with LARS2 and KARS pathogenic variants, magnetic resonance spectroscopy revealed elevated white matter lactate, suggesting mitochondrial disease. Pathology in one patient with LARS2 pathogenic variants displayed evidence of primary disease of oligodendrocytes and astrocytes with lack of myelin and deficient astrogliosis. Aminoacylation activities of purified recombinant mutant leucyl tRNA synthase showed a 3-fold loss of catalytic efficiency. Aminoacylation assays on patients' lymphoblasts and fibroblasts showed about 50% reduction of enzyme activity.

Conclusion

This study adds LARS2 and KARS pathogenic variants as gene defects that may underlie deafness, ovarian failure, and leukodystrophy with mitochondrial signature. We discuss the specific MRI characteristics shared by leukodystrophies caused by mitochondrial tRNA synthase defects. We propose to add aminoacylation assays as biochemical diagnostic tools for leukodystrophies.

Leukodystrophies comprise a large group of rare genetic disorders that share abnormal white matter of the brain and spinal cord as the central feature.^1,2 With the introduction of whole-exome and whole-genome sequencing (WES and WGS, respectively), the proportion of patients with unclassified leukodystrophy decreased from 50% in 2010³ to 20%–30% in 2016.^3,4 Strikingly, defects in several specific biological processes were found to be relatively common, including the mitochondrial respiratory chain and the process of translating RNAs into proteins.³

These 2 pathways come together in mitochondrial translation. Aminoacyl transfer RNA (tRNA) synthases (aaRS) link amino acids with their cognate tRNAs, a critical step in the translation of messenger RNA into proteins. Except for KARS and GARS shared by the cytoplasmic and mitochondrial translation machineries, separate sets of aaRS, encoded by -ARS genes, are present in the cytoplasm for the translation of approximately 24,000 nuclear genes, and in the mitochondria, encoded by -ARS2 genes, for the translation of 13 mitochondria-encoded genes.⁵ DARS2 pathogenic variants were the first identified defect in a mitochondrial aaRS causing LBSL (leukodystrophy with brainstem and spinal cord involvement and lactate elevation).⁶ Subsequently, EARS2 pathogenic variants were found in LTBL (leukodystrophy with thalamus and brainstem involvement and lactate elevation),⁷ and AARS2 pathogenic variants in AARS2-related ovarioleukodystrophy.⁸ Defects in almost all mitochondrial and cytoplasmic aaRS have been associated with human diseases, often brain disorders or peripheral neuropathies.⁹

In the present report, we discuss 2 more leukodystrophies related to mitochondrial aaRS defects caused by pathogenic variants in LARS2 and KARS.

Methods

Standard protocol approvals, registrations, and consents

Patient 1 was investigated as part of a collaborative Dutch-Australian WGS study to identify the genetic cause in 111 patients from the Amsterdam Database of Unclassified Leukodystrophies. Patients 2–5 underwent diagnostic WES. In all participating centers, approval from the institutional ethical committees and written informed consent from patients or guardians were obtained. Affected individuals were examined by neurologists at their primary care centers.

MRI and magnetic resonance spectroscopy

We scored all available MRIs according to a standardized protocol.¹⁰ Proton magnetic resonance spectroscopy (MRS) was only assessed for the presence of lactate. Examinations were executed on different magnetic resonance machines at various field strengths; no attempts at quantification were made, as no comparable results would be obtained.

Brain pathology

Brain tissue from patient 1 was collected at autopsy with a postmortem delay of 6 hours. Formalin-fixed, paraffin-embedded tissue was sectioned at 4 μm and stained for hematoxylin & eosin or Kluver periodic acid–Schiff according to standard methods. Tissue sections were incubated with antibodies against proteolipid protein (myelin marker, 1:3,000; AbD Serotec, Kidlington, UK), glial fibrillary acidic protein (GFAP) (marker of astrocytes, 1:1,000; Millipore, Billerica, MA), CD68 (marker for macrophages and microglia, 1:400; Dako, Carpinteria, CA), neurofilament 70–200 kDa (marker for abnormal axons, 1:10; Mono), and CD45 (marker for lymphocytes, 1:100; Dako). Negative controls by omitting the primary antibody were included in each experiment. Immunoreactivity was visualized with secondary antibodies (Envision rabbit/mouse, Dako) and diaminobenzidine tetrachloride. Sections were counterstained with hematoxylin. Sections were photographed using a Leica DM6000B microscope (Leica, Wetzlar, Germany).

Molecular genetic analyses

In patient 1 and her parents, trio WGS was performed on genomic DNA using 2 × 150 nt paired-end reads on an Illumina X (Illumina Cambridge Ltd., Little Chesterford, UK). Read alignment was performed using BWA-mem; GATK HaplotypeCaller v3.7 was used for variant calling; SnpEff v4.3m for variant annotation. A custom pipeline was used for variant filtration and prioritization based on population allele frequency, anticipated inheritance pattern, and predicted functional effect of variants.

In patient 2 and parents, the coding regions of 4,813 genes associated with known phenotypes (TruSight One Illumina panel) were sequenced, processed, and analyzed as described.¹¹

In patient 3 and parents, trio WES analysis was performed as described¹² with the following modifications. Solo whole-exome capture was performed with the SureSelect Clinical Research Exome V1 kit (Agilent, Santa Clara, CA) and libraries were sequenced on a HiSeq 4000 according to the manufacturer's recommendations for paired-end 76 bp reads.

In patient 4, WES was performed on DNA using an Agilent SureSelectXT Human All Exon 50 Mb kit and sequenced on an Illumina HiSeq 2000. Data analysis was performed focusing on a mitochondrial disease gene panel, as previously described.¹³

In patient 5 and parents, trio WES was performed as described previously.¹⁴

All variants were confirmed by Sanger sequencing.

Aminoacylation assays

Recombinant mtLeuRS were synthesized to assess aminoacylation of the p.Asn124Ile and p.Arg663Trp variants (identified in patient 2). LARS2 variants were introduced into the pET22b/LARS2 construct¹⁵ using site-directed mutagenesis. Wild-type and variant LARS2 were expressed and purified as described.¹⁶ The sequence of Escherichia coli tRNA₅^Leu(UAA) was cloned, transcribed, and purified according to established procedures.¹⁷ In vitro leucylation of the tRNA^Leu transcript was performed in the presence of 30 µM L-[¹⁴C]leucine at 37°C, as described.¹⁸ Kinetic parameters were determined from Lineweaver-Burk plots in the presence of 3–30 nM wild-type or variant mtLeuRS and concentrations of E coli tRNA^Leu transcript ranging from 0.3 to 5.6 µM. Experimental errors for k_cat and K_m varied by ≤20%. Data were expressed as averages of at least 3 independent experiments.

To determine patients' mtLeuRS activity, steady-state aminoacylation assays were performed in mitochondria from lymphoblasts (kit from Qiagen, Hilden, Germany) following previously published methods.¹⁹ [¹³C₂]-leucine and [¹³C₆,¹⁵N]-isoleucine (Cambridge Isotopes, Tewksbury, MA) were used as substrates and [D₃]-leucine as internal standard. Intra-assay coefficient of variation was <15%. MtIleRS activity was used as control. To determine cytosolic LysRS activity, steady-state aminoacylation assays were performed in fibroblasts under similar conditions, with [D₄]-lysine (Sigma, Munich, Germany) as substrate and [¹³C₆]-arginine (Cambridge Isotopes) as internal standard. All samples were measured in triplicate.

Other studies on mtLeuRS

Immunoblotting and densitometry were performed on patient 2 muscle, as described,²⁰ with the following modifications: membranes were probed with a 1:250 dilution of anti-mtLeuRS (ab96221; Abcam, Cambridge, UK), 1:500 anti-OXPHOS (Abcam ab11041), or a 1:1,000 dilution of anti-VDAC1 (Abcam ab14734) overnight at 4°C.

Data availability

Anonymized data will be shared by request from any qualified investigator.

Results

Clinical characterization and first laboratory results

All patients are from nonconsanguineous families. Family histories were unrevealing, except for patient 5; he had a sibling with deafness, who declined testing. Pregnancy and delivery were normal or noncontributory in all.

In patient 1, a female, profound congenital sensorineural deafness was diagnosed shortly after birth. Development was somewhat delayed with unsupported walking at 18 months. She learned to speak and use sign language. She graduated from normal level high school and achieved employment in administrative work. She entered premature menopause due to ovarian failure at 29 years. Following a fall at age 33, motor decline set in with cerebellar ataxia and spasticity, left more than right. She developed supranuclear gaze palsy and left-sided central facial palsy. Increasing swallowing difficulties and weight loss prompted PEG (percutaneous endoscopic gastrostomy) tube placement. Pneumonia led to respiratory failure and death at 35 1/2 years. Autopsy was performed.

Patient 2 is a male in whom hypotonia, macrocephaly, and inguinal hernia were noted neonatally. Early development was delayed. He was able to walk unaided at 36 months; his gait remained instable. From 18 months, he used hearing aids for sensorineural deafness. He displayed autistic behavior with hyperactivity and later also aggression. From age 17 years, he developed atypical seizures with atonic and hypertonic crises, responsive to gabapentin and carbamazepine, but EEGs, including long-term video-EEG, did not reveal epileptic activity. Following commencement of neuroleptics he became overweight and developed a metabolic syndrome with hepatic steatosis, mildly raised transaminases, and insulin-requiring diabetes mellitus in his 20s. In his 30s, his gait became ataxic. At age 33, he developed a supranuclear gaze palsy and extrapyramidal dysfunction with resting tremor and dystonia. At age 36, he developed acute hemiparesis, ascribed to stroke. Following another stroke at age 37, he became very spastic. At present, he is wheelchair-bound and requires assistance for all activities of daily living.

Patient 3 is a male, who was small for gestational age and hypotonic at birth. Early development was delayed. At age 1 1/2 years, he was diagnosed with profound sensorineural deafness; he received a unilateral cochlear implant at 2 1/2 years. At age 4, he displayed episodes of abnormal movements, but EEG did not reveal epilepsy. He developed behavioral problems including hyperkinesia, self-mutilation, temper tantrums, and aggression. At present, he is 8 years old. His behavior has improved. He does not speak, but uses sign language. He has mild pyramidal signs with brisk reflexes but no ataxia. No regression has occurred until now.

Patient 4 is a female, in whom congenital sensorineural deafness was diagnosed. She received a unilateral cochlear implant at 38 years. Early development was normal and she attended normal school. She had her menarche at 16 years, soon followed by amenorrhea. Ovarian failure was diagnosed and hormonal replacement was started. At age 45, she developed right-sided coordination problems. At age 46, she experienced a subacute right-sided weakness along with orientation and concentration problems. Neurologic examination revealed pyramidal dysfunction on the right and some axial ataxia. CT was unrevealing; MRI could not reliably be assessed because of cochlear implant-related artifacts. She experienced further decline and presently, at 48 years, she cannot walk without support because of bilateral spasticity.

Patient 5 is a male, in whom early development was uneventful. He walked unaided at 11 months. At 7 years, he developed sensorineural deafness. He followed special education because of learning difficulties but could undertake simple professional work. He married and had 2 children. At 35 years, he became completely deaf. He then developed increasing behavioral and cognitive problems. On neurologic examination, signs of pyramidal and cerebellar dysfunction were found. His cognitive and motor decline was rapid and he died at age 36 years.

In patients 1 and 4, luteinizing hormone and follicle-stimulating hormone were high, while estrogen was low, indicative of ovarian failure. In patient 1, thyroid hormone, thyroid-stimulating hormone, cortisol, adrenocorticotrophic hormone, and prolactin were normal. In patient 4, ultrasound revealed streak ovaries. Nerve conduction studies (patients 1 and 2) and electroretinogram (patient 2) were unremarkable.

The patients underwent numerous genetic and metabolic tests, which were unrevealing, except for lactate levels. In patients 1 and 2, CSF lactate was mildly elevated (2.7 mmol/L, upper limit 1.2; and 2.6 mmol/L, upper limit 1.8, respectively). In patients 1–4, blood lactate was normal. In patient 5, lactate was slightly elevated in blood and CSF (1.8 and 1.7 mmol/L, respectively, upper limit for both 1.2). Studies focused on mitochondrial respiratory chain defects were unrevealing. Assessment of substrate oxidation rates and activities of individual respiratory chain complexes showed normal results or borderline reductions in mitochondria isolated from muscle (patients 1 and 2), fibroblasts (patient 1), and liver (patient 2). Western blotting revealed no reduction in any of the respiratory chain complexes in fibroblasts; in muscle, a slightly reduced complex I expression was found (patient 2). Sequence analysis of the mitochondrial genome revealed no abnormalities in patients 1, 2, and 4.

Brain MRI and MRS findings

Patient 1 underwent MRIs at 32, 33, and 34 years of age (figure 1A). The first MRI showed extensive cerebral white matter abnormalities with partial sparing of directly subcortical and frontal white matter, corpus callosum, and anterior limb of the internal capsule. Numerous spots of more normal signal were seen within the abnormal white matter. The posterior limb of the internal capsule and splenium of the corpus callosum were affected. The thalamus contained areas of abnormal signal. Pyramidal tracts and medial lemniscus at the level of the pons were affected. The hilum of the dentate nucleus and an area in the middle cerebellar peduncle on the right had abnormal signal. The cerebellum was slightly atrophic. Two subsequent MRIs showed progression with increasing involvement of frontal white matter and larger parts of the corpus callosum. The right pyramidal tract became affected from motor cortex to pyramid in the medulla, whereas involvement on the left was more limited (figure 1A). Diffusion restriction was observed in the pyramidal tract, right more than left (figure 1A) and the splenium of the corpus callosum. Some contrast enhancement was present in the right pyramidal tract in the centrum semiovale (figure 1A). Fluid-attenuated inversion recovery images did not show rarefaction or cystic degeneration. MRS revealed highly elevated lactate in the abnormal white matter (figure 1A). Spinal imaging was normal.

Figure 1. MRI of patients with LARS2 and KARS pathogenic variants.

(A) MRI in patient 1 at age 33 years shows extensive cerebral white matter abnormalities with partial sparing of directly subcortical white matter, especially in the frontal region (white arrowheads). Areas of abnormal signal are present in the thalamus (green arrowhead) and to a lesser degree the basal nuclei (blue arrowhead). Pyramidal tracts in the posterior limb of the internal capsule, midbrain, pons, and medulla are affected, right (red arrows) more than left. Spinal cord is normal; cerebellum is slightly atrophic. Diffusion restriction is observed in the pyramidal tract, right (white arrow) more than left, and splenium of the corpus callosum. Some contrast enhancement is present in the right pyramidal tract in the centrum semiovale (white open arrow). MRS reveals highly elevated lactate (doublet at 1.33 ppm, orange arrow) in the abnormal white matter. (B) MRIs in patient 2 at 36 (upper row) and 37 (lower row) years of age. The earlier MRI shows extensive cerebral white matter abnormalities, sparing frontal and directly subcortical white matter (white arrowheads). Thalamus (green arrowhead), hilum of the dentate nucleus, and peridentate white matter (yellow arrowheads) are affected. Small cystic lesions with elongated shape are present, suggesting enlarged perivascular spaces (white arrows). A few foci of diffusion restriction are seen in the centrum semiovale (white open arrows). The later MRI reveals small cystic spaces in the same areas (white open arrows), compatible with lacunar infarctions. The white matter abnormalities have progressed and involve the frontal white matter and entire pyramidal tracts (red arrows). Numerous microbleeds are seen as black dots in the area of the dentate nucleus, thalamus, basal nuclei, and cerebral white matter. (C) MRI of patient 5 at 36 years. Sagittal T2-weighted image shows involvement of the genu and splenium of the corpus callosum (white arrows) and cerebellar atrophy. The axial images show abnormal signal in the frontal and parieto-occipito-temporal white matter, sparing the segment in between. The anterior limb of the internal capsule (yellow arrow), fronto-pontine tracts (yellow arrows) and parieto-occipito-temporo-pontine tracts (green arrows) are affected. Areas of diffusion restriction are present within the abnormal white matter. MRS reveals highly elevated lactate (doublet at 1.33 ppm, orange arrow) in the abnormal white matter. N-acetyl aspartate (2.02 ppm) is close to zero. MRS = magnetic resonance spectroscopy.

Patient 2 underwent MRIs at 24, 32, 36, and 37 years (figure 1B). The first MRI showed extensive white matter abnormalities in the parieto-occipital white matter, sparing the directly subcortical white matter. Numerous spots of more normal signal were seen within the abnormal white matter (figure 1B). The posterior limb of the internal capsule was partially affected; corpus callosum and anterior limb were intact. The thalamus, hilum of the dentate nucleus, and peridentate white matter contained signal abnormalities (figure 1B). The brainstem was spared. There was no cerebellar atrophy and no abnormal enhancement after contrast. The subsequent 3 MRIs showed progression with increasing involvement of frontal white matter. Thalamic abnormalities increased and basal nuclei became abnormal. Within the abnormal cerebral white matter, small cystic lesions appeared with elongated shape, suggesting enlarged perivascular spaces (figure 1B). Numerous microbleeds arose in the area of the dentate nucleus and increased over time; the latest MRI also showed microbleeds in the area of the thalamus, basal nuclei, and the cerebral hemispheres (figure 1B). Multiple small areas of diffusion restriction were seen spread over the cerebral hemispheric white matter at age 36, which had disappeared at age 37, leaving behind small cystic spaces, compatible with lacunar infarctions (figure 1B). MRS revealed no lactate.

In patient 3, MRI at age 3 years was normal. MRS was not obtained.

In patient 4, MRI at age 46 years revealed extensive cerebral white matter abnormalities with multiple small cystic spaces, either lacunar infarctions or enlarged perivascular spaces. The thalamus contained signal abnormalities. Anterior and especially posterior limbs of the internal capsule, and pyramidal tracts and medial lemniscus in the brainstem were affected. Images were deteriorated because of artifacts related to the cochlear implant. MRS was not obtained.

In patient 5, MRIs were obtained at 35 and 36 years (figure 1C). Sagittal T2-weighted images revealed involvement of the anterior segment and splenium of the corpus callosum and cerebellar atrophy. The axial images revealed abnormal signal in the frontal and parieto-occipito-temporal white matter, sparing the segment in between. The anterior limb of the internal capsule, frontopontine tracts, and parieto-occipito-temporo-pontine tracts were affected over their entire extent in the brainstem up to the level of the pons. Areas of diffusion restriction were present within the abnormal white matter. There was no abnormal contrast enhancement. MRS revealed highly elevated lactate in the abnormal white matter. Spinal imaging revealed no abnormalities.

Autopsy findings

Brain weight of patient 1 was normal (1,200 gr); external inspection was unremarkable. On coronal cut (figure 2A), lateral ventricles were slightly enlarged. Cerebral white matter was inhomogeneous in color and consistency, partly gray and gelatinous, partly white and firm-elastic (figure 2, B and C). There were no stroke-like lesions or hemorrhages.

Figure 2. Neuropathology of LARS2 leukodystrophy.

(A) Whole coronal brain slice shows mild dilatation of the lateral ventricles, indicating mild white matter atrophy, and discoloration of the hemispheric white matter with better preservation of the U-fibers. (B and C) Luxol fast blue periodic acid–Schiff (LFB-PAS, B) and stain against the major myelin protein proteolipid protein (PLP, C) of whole mounts of the right cerebral hemisphere show lack of myelin most evident in the deep white matter with relative sparing of the U-fibers. The PLP stain also shows that the lack of myelin has a patchy appearance (arrow). (D) Hematoxylin & eosin (H&E) stain of the frontal lobe confirms that the U-fibers are better preserved. (E and F) LFB-PAS and H&E stains of the deep frontal white matter show variable degrees of tissue rarefaction and microcystic degeneration of the tissue. Note the relatively low numbers of oligodendrocytes, as identified by their small, deeply basophilic, round nucleus. (G) The vacuoles are crossed by PLP-immunopositive strands, indicative of intramyelinic edema. (H and I) Stain against the astrocytic glial fibrillary acidic protein (GFAP) (H) and LFB-PAS (I) demonstrate that the degree of reactive gliosis is meagre compared to the tissue damage and that astrocytes contain GFAP-positive intracytoplasmic inclusions (H and I, inset). (J) Stain against CD68 of the deep parietal white matter shows absence of macrophages and scanty microglial reaction. (K) Stain against neurofilaments 200 KDa (NF) of the frontal lobe shows an axonal spheroid. (L) H&E stain of the capsula externa shows relative preservation of the tissue with near-normal amounts of myelin and oligodendrocyte numbers. (M) H&E stain of the midbrain shows microcystic appearance of the white matter of the corticospinal tracts with myelin pallor and reduced oligodendrocytes numbers. (N) In the same region, NF-positive axonal spheroids are numerous. (O) H&E stain of cerebellar folium shows loss of cells in the cortical granular layer and of Purkinje cells. The cell density in the molecular layer is near to normal. Original magnifications: D and O, ×50; E–N, ×200.

Microscopic examination of the cerebral hemispheres revealed inhomogeneously pale white matter with rarefaction to cystic degeneration of periventricular and deep white matter and relative sparing of U-fibers (figure 2, D–F). There was lack of myelin; remaining myelin sheaths displayed intramyelinic edema and some blebbing. In most affected areas, oligodendrocytes were decreased in number. Reactive astrocytes were locally seen, sometimes clustered around blood vessels; reactive gliosis was meagre compared to the degree of tissue damage (figure 2H). At the edge of the areas of severe damage, reaching the better preserved U-fibers, astrocytes had gemistocytic morphology with ample eosinophilic cytoplasm, in which round and elongated inclusions were appreciable that were immunoreactive for GFAP. Throughout the white matter, scattered CD68-positive macrophages were visible and few activated microglia without signs of active myelin breakdown (figure 2J). In the areas without myelin, uncovered axons were present together with some spheroids, especially in long tracts (figure 2K). There were no infiltrates of inflammatory cells or abnormalities of blood vessel walls. Occasional hemosiderin-containing or weak Oil red O-positive macrophages were seen in perivascular spaces. The white matter of the U-fibers was more cellular with increased numbers of cells with morphology of oligodendrocytes or oligodendrocyte progenitors (figure 2L). The neocortex was normal without loss of neurons or myelin.

The internal and external capsules showed loss of myelin and reactive gliosis. In the thalami, moderate tissue rarefaction was accompanied by reactive gliosis and mild loss of neurons. The basal nuclei displayed only a slight decrease in the number of larger neurons in the neostriatum. Pituitary gland, hypothalamus, and hippocampus were not affected.

In the brainstem, pyramidal tracts showed variable lack of myelin and axons and numerous axonal spheroids, especially in the midbrain. Neuronal loss in gray matter structures was at most mild.

Cerebellar white matter, especially the hilum of the dentate nucleus, showed lack of myelin, intramyelinic edema, and astrogliosis. Cerebellar cortex showed inhomogeneous loss of neurons in granular and Purkinje cell layers accompanied by reactive Bergmann gliosis and some macrophages.

Genetic analysis identifies LARS2 and KARS as candidate genes

WGS in patient 1 identified biallelic heterozygous variants in LARS2 (NM_015340.3): c.462delT (p.Lys155Asnfs*3) and c.1120A>C (p.Ile374Leu). WES identified biallelic heterozygous LARS2 variants c.371A>T (p.Asn124Ile) and c.1987C>T (p.Arg663Trp) in patient 2, c.516G>T (p.Arg172Ser) and c.1028C>T (p.Thr343Met) in patient 3, and c.683G>A (p.Arg228His) and c.880G>A (p.Glu294Lys) in patient 4. In patient 5, WES revealed biallelic heterozygous variants in KARS (NM_001130089.1): c.323G>A (p.Arg108His) and c.1426G>T (p.Val476Phe). For patients 1, 2, 3, and 5, both parents were carriers of one of the variants. For patient 4, the mother carried the c.683G>A variant; the father was not available for testing.

All mutations were predicted to be damaging to protein function and all mutations were absent or very rare in the Genome Aggregation Database with no homozygotes reported (table 1). When comparing the phenotype of our patients, as well as patients from the literature, to their LARS2 or KARS genotype, we did not find clear genotype-phenotype correlations (figure 3).

Table 1.

LARS2^a and KARS(2)^b variants with frequency and pathogenicity predictions

Figure 3. LARS2 and KARS variants.

Schematic representation of the variants identified in this study (in red) or in the literature (in black) on the KARS and LARS2 genes and proteins, and their corresponding phenotypes. Variants identified in this study and previously reported^{1,12–18,30,35–46,48} appear in purple. The mutation nomenclature for KARS variants is based on the mitochondrial isoform (NM_001130089.1). DD = developmental delay.

LARS2 and KARS expression profiles

To gain insights into factors possibly accounting for variable phenotypic expression, we assessed expression profiles of KARS and LARS2 using the Genotype-Tissue Expression (GTEx) and BrainSpan resources.²¹ Of note, KARS, and to a lesser degree LARS2, showed the lowest expression levels in brain tissue from adult individuals (GTEx database). Expression was highly variable for both genes from one individual to the other in the normal population.

Functional analyses of LARS2 and KARS variants

The mtLeuRS protein has been cloned and expressed in E coli with a C-terminal 6-His Tag, purified to homogeneity higher than 95%, and shown to be active in the aminoacylation of E coli tRNA^Leu.^16,22 We measured in vitro aminoacylation activities of purified recombinant mtLeuRS p.Asn124Ile and p.Arg663Trp variants, observed in patient 2, by the incorporation of [¹⁴C]-leucine into an E coli tRNA^Leu substrate. These variants demonstrated a 3-fold loss of catalytic efficiency (table 2).

Table 2.

Kinetic parameters for leucylation of Escherichia coli tRNA^Leu_(UAA) transcript by wild-type and variant recombinant mtLeuRS

MtLeuRS protein is 903 amino acids in length and contains a mitochondrial signal sequence comprising the first 39 amino acids.¹⁵ Variant p.Asn124Ile is located in the catalytic domain and variant p.Arg663Trp at the interphase between the catalytic and tRNA-binding domains of mtLeuRS (figure 3). The crystal structure of both Thermus thermophilus LeuRS²³ and E coli LeuRS²⁴ complexed to its cognate tRNA suggests that neither of these residues is in direct contact with ATP, leucine, or the tRNA substrates, although p.Arg663Trp is strictly conserved in all bacterial-like LeuRS. In agreement, we found that neither p.Asn124Ile nor p.Arg663Trp mtLeuRS mutants affect tRNA binding to mtLeuRS, as shown by constant K_m values. Therefore, the 3-fold efficiency losses are mainly the result of a decreased aminoacylation rate (k_cat).

In mitochondria isolated from lymphoblasts of patients 2–4, mtLeuRS activity was assessed and compared with mitochondria from 3 unaffected cell lines, using mtIleRS activity as control (figure 4A). MtLeuRS activity was 41%–45% of average control values (figure 4B). Cytosolic LysRS activity in fibroblasts of patient 5, harboring KARS mutations, showed 52% residual activity compared to controls (figure 4B). No lymphoblasts were available to assess mtLysRS activity.

Figure 4. Aminoacylation assay.

(A) MtLeuRS activity normalized to average control cells at 100% of patients (n = 3) and controls (n = 3) as a group in mitochondria isolated from lymphoblast cell lines. MtIleRS activity is shown as simultaneously measured control enzyme. (B) MtLeuRS activity normalized to average control cells at 100% of individual patients and controls in mitochondria isolated from lymphoblast cell lines. Samples were measured in triplicate; error bars represent SEM.

Western blotting

Immunoblotting of mtLeuRS from patient 2 muscle showed reduced levels compared to age-matched controls, while no significant differences were observed in muscle respiratory chain complex levels (figure 5). Immunoblotting of fibroblasts from patient 2 showed no change in mtLeuRS protein level and no effect on mitochondrial respiratory chain complex protein levels relative to control fibroblasts (figure 5).

Figure 5. Immunoblot.

Immunoblot analysis of LARS2 and the respiratory chain complexes (I–V) in patient 2 (P2) and age-matched control (C) muscle. VDAC1 was used as a loading control.

Discussion

In this report, we present 5 patients with leukodystrophy and deafness, related to LARS2 or KARS pathogenic variants. Apart from the cerebral hemispheric white matter, the corpus callosum is affected, connecting the abnormalities on both sides. The leukodystrophy is characterized by long tract involvement, in patients with LARS2 mutations especially the pyramidal tracts, and in the patient with KARS mutations especially the fronto-pontine and parieto-occipito-temporo-pontine tracts. Leukodystrophies caused by mitochondrial tRNA synthase defects (AARS2, DARS2, EARS2, KARS, and LARS2) share certain features. The pattern seen in the patient with KARS mutations is similar to that seen in the leukodystrophy caused by AARS2 pathogenic variants.⁸ Of note, long tract involvement is also a typical feature of LBSL caused by DARS2 mutations.⁶ The consistent thalamus involvement is shared by the diseases caused by LARS2 and EARS2 pathogenic variants.⁷ All these disorders share a mitochondrial signature on MRI with often lactate elevations within the abnormal white matter in MRS, while blood and CSF lactate is normal or borderline.^6–8 Areas of diffusion restriction within the abnormal white matter on MRI suggest myelin microvacuolization, a feature of mitochondrial leukoencephalopathies.^7,8,25 Distinct features for LARS2 pathogenic variants, seen in patients 2 and 4, both with clinically manifest strokes, are signs of a vasculopathy, with microbleeds, highly enlarged perivascular spaces, and lacunar infarctions. In leukodystrophies, earlier disease onset is generally associated with more rapid decline than later disease onset.²⁶ In AARS2-, LARS2-, and KARS-related disease, however, limited disease signs may be present from early childhood, followed in adulthood by rapid decline over the course of a few years. Unlike other leukodystrophies, including mitochondrial leukodystrophies, we did not identify any precipitating factors in our patients, except for a head trauma in patient 1.

The leukodystrophy caused by LARS2 pathogenic variants is novel. LARS2 encodes mitochondrial leucyl-tRNA synthase (mtLeuRS), which is dedicated to the charging of mitochondrial tRNA^Leu. LARS2 variants have previously been associated with infantile multisystem disease²⁷ and Perrault syndrome.²⁸ KARS encodes lysyl-tRNA synthase (LysRS) that aminoacylates tRNA^Lys in the cytosol or mitochondria, the cytoplasmic and mitochondrial isoforms resulting from alternative splicing. KARS variants have been associated with hypertrophic cardiomyopathy, deafness, peripheral neuropathy,^29–32 and recently also leukodystrophy.³³ In one study, calcium deposits in addition to white matter abnormalities were reported in brain and spinal cord,³¹ not observed in patient 5. Although the distribution of the white matter abnormalities was not described in detail, the figures suggest a similar distribution as observed in patient 5, indicating a consistent AARS2-like pattern,⁸ with the exception of absence of spinal cord abnormalities in our patient.

Pathology of patient 1 shows a widespread loss of oligodendrocytes, correlating with profound lack of myelin. The affected white matter displays cystic degeneration, seen on some MRIs, and intramyelinic edema, confirming diffusion imaging findings. Astrocytes are affected, with lack of proper reactive gliosis and presence of GFAP-positive intracytoplasmic inclusions, suggesting a deranged cytoskeletal intermediate filament network, which could explain the meagre reactive gliosis, disproportional to the degree of tissue damage.³⁴ This is similar to findings in Vanishing White Matter and Alexander disease astrocytes.¹ Taken together, these data suggest a combined primary involvement of oligodendrocytes and astrocytes in LARS2-related leukodystrophy. Patient 1 did not have strokes and brain autopsy did not reveal vascular pathology. It is therefore not possible to speculate about the origin of the early-onset vascular pathology in patients 2 and 4. The axonal spheroids are probably a secondary phenomenon. Individual leukodystrophies are each characterized by specific findings.^1,26 The above pathology is specific of LARS2-related leukodystrophy. Metachromatic leukodystrophy, Krabbe disease, and X-linked adrenoleukodystrophy show active demyelination with abundant myelin debris and macrophages. Vanishing White Matter displays lack of myelin with abnormal astrocytes and meagre reactive gliosis, but floridly increased numbers of immature oligodendrocytes. Hypomyelinating conditions such as Pelizaeus-Merzbacher disease feature lack of oligodendrocytes and myelin, but strongly reactive astrocytes and microglia. Prototypic mitochondrial diseases such as Leigh syndrome show loss of tissue due to necrosis, and vascular proliferation, reactive astrogliosis, and microgliosis. Megalencephalic leukoencephalopathy with subcortical cysts features intramyelinic edema and no reactive gliosis, changes in oligodendrocyte cell density, or lack of myelin.

In patient 2, in vitro aminoacylation activities of purified recombinant mtLeuRS showed a 3-fold loss of catalytic efficiency. Aminoacylation assays performed on lymphoblasts and fibroblast of patients 2–4 showed about 50% reduction of mtLeuRS and LysRS activity. Given the growing number of leukodystrophies related to -ARS and -ARS2 variants, functional assessment of aaRS activities could be a useful biochemical assay as second-line investigation (i.e., after plasma lactate, pyruvate, amino acids, homocysteine, very long-chain fatty acids, cholestanol; urinary organic acids, sulfatides and sialic acid; and activity of lysosomal enzymes in cells) in patients with leukodystrophies, especially if associated with deafness.

All defects in mitochondrial tRNA synthases affect the same process and one would expect similar disease phenotypes. Considering the housekeeping nature of the affected process, one would expect a multisystem disease. The associated disorders, however, are different. Most may cause a disorder of the CNS (AARS2, CARS2, DARS2, EARS2, FARS2, KARS, LARS2, MARS2, NARS2, PARS2, QARS, RARS2, TARS2, VARS2, and WARS2) followed by ovarian failure (AARS2, HARS2, LARS2) and deafness (HARS2, IARS2, LARS2, and KARS).⁹ Within the CNS, the disorders show distinct patterns of structural abnormalities, involving gray and/or white matter, leading to recognizable patterns on MRI. However, mutations in the same gene may also lead to different disorders. For instance, LARS2 mutations have been associated with multisystem disease, Perrault syndrome, intellectual disability, premature ovarian failure, and leukodystrophy^{28,29,35–39} (current report), while KARS mutations have been associated with peripheral neuropathy, cardiomyopathy, renal tubular acidosis, anemia, and leukodystrophy^{30–33,40–46} (current report) We did not find evidence of genotype-phenotype correlation. The variability in clinical phenotype may be related to the overall residual enzyme activity, which would depend on the nature and association of the variants found on each allele, but also on the expression of the gene in the affected tissue. We would expect the disorder to mainly affect the tissue where the expression and residual activity are lowest. Of interest, both genes, especially KARS, have the lowest expression in affected tissues including the brain. The individual expression of both LARS2 and KARS appears variable from one individual to the other, also regarding different tissues. Therefore, residual enzyme activity and individual variation in tissue expression may explain which tissue is most affected. The selective involvement of some specific white matter tracts could be related to the greater effects of some -ARS2/-ARS mutations in certain cell types, as shown in neuronal cell lines for DARS2.⁴⁷ Complete loss of function of cytoplasmic and mitochondrial aaRS is likely incompatible with life, and the combination of variants observed in living individuals only leads to a moderate decrease of the tRNA synthase activity, as shown in this study.

We add LARS2 mutations to the spectrum of gene defects that may underlie deafness, ovarian failure, and a distinct leukodystrophy. We demonstrate that KARS mutations cause a leukodystrophy that is very similar to the one associated with AARS2 mutations. We propose aminoacylation assays as a biochemical diagnostic tool for leukodystrophies.

Glossary

aaRS

aminoacyl transfer RNA synthase

GFAP

glial fibrillary acidic protein

GTEx

Genotype-Tissue Expression

LBSL

leukodystrophy with brainstem and spinal cord involvement and lactate elevation

MRS

magnetic resonance spectroscopy

tRNA

transfer RNA

WES

whole-exome sequencing

WGS

whole-genome sequencing

Appendix. Authors

Study funding

This study was in part financed by the Australian National Health and Medical Research Council (NHMRC 1068278), the Victorian Government's Operational Infrastructure Support Program, and the ZonMw grant 40-00812-98-11005. The Genotype-Tissue Expression (GTEx) Project was supported by the Common Fund of the Office of the Director of the NIH, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS.

Disclosure

M. van der Knaap, M. Bugiani, M. Mendes, L. Riley, J. Rudinger-Thirion, M. Frugier, M. Breur, J. Crawford, J. van Gaalen, M. Schouten, M. Willems, Q. Waisfisz, F. Tran Mau-Them, and R. Rodenburg report no disclosures relevant to the manuscript. R. Taft is an employee of Illumina, Inc. B. Keren, J. Christodoulou, C. Depienne, C. Simons, G. Salomons, and F. Mochel report no disclosures relevant to the manuscript. Go to Neurology.org/N for full disclosures.