Expanding the clinical phenotype of IARS2-related mitochondrial disease

Aminoacyl-tRNA synthetases (ARSs) are evolutionarily conserved enzymes that catalyze amino acid attachment to their cognate tRNA. This catalytic process, termed tRNA charging, is a prerequisite for the translation of genetic sequences into polypeptide chains [1]. Two distinct translation pathways take place in the cytoplasm and in the mitochondria each requiring a separate set of ARSs for the translation of nuclear and mitochondrial genes respectively; in total, there are 37 members of the ARS gene family. Two groups of 17 ARSs are each associated with cytoplasmic or mitochondrial translation, while three so-called bifunctional proteins act in both cellular locations [2, 3]. Based on their cellular localization and function, nomenclature for ARSs follows a systematic scheme that entails the recognised amino acid, followed by ARS for both cytoplasmic and bifunctional enzymes (i.e. IARS for cytoplasmic isoleucyl-tRNA synthetase), and a “2” is added to distinguish mitochondrial ARSs (i.e. IARS2 for mitochondrial isoleucyl-tRNA synthetase) [2, 3]. Although expression of ARSs is ubiquitous and protein synthesis is expected to be systemically impaired, a number of highly diverse clinical phenotypes can emerge from mutations in genes encoding ARSs that affect a wide range of tissues with particularly high metabolic demands [3].

IARS2 is a nuclear-encoded mitochondrial isoleucyl-tRNA synthetase that is imported from the cytosol into the mitochondria where it catalyzes the attachment of an isoleucine residue to a cognate mt-tRNA^Ile [4]. Genetic mutations in IARS2 (OMIM: 612801) on chromosome 1q41 were first described in an extended French-Canadian family with a syndrome abbreviated CAGSSS that is characterised by cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, and skeletal dysplasia [5]. A detailed follow-up on two previously published first-cousin probands from a genealogically related French-Canadian family dating back to the nineteenth century was also explored [5, 6]. The three probands in this extended family were homozygous for a pathogenic missense variant (c.2726C > T, p.Pro909Leu) in exon 21 of the 23 exon IARS2 gene. The proline residue exchange affected a predicted anticodon binding domain. An additional Danish proband presenting CAGSSS features was identified with a homozygous pathogenic variant (c.2620C > A, p.Gly874Arg) also affecting exon 21 [7]. One further proband with compound heterozygous mutations (c.1821G > A, p.Trp607*; c.2122G > A, p.Glu708Lys) was diagnosed with Leigh syndrome and died at the age of 18 months [5]. Furthermore, compound heterozygous variants in IARS2 (case 6: c.607G > C; p.(Gly203Arg) and c.2575 T > C; p.(Phe859Leu)); family 10: c.2446C > T; p.(Arg816*) and c.2575 T > C; p.(Phe859Leu)) have been found in two probands from China with sporadic pediatric cataract which characterised by far the mildest clinical presentation of IARS2-related disorders [8]. Moreover, recently identified novel compound missense variants in IARS2 (c.680 T > C; p.(Phe227Ser) and c.2450G > A; p.(Arg817His)) have been reported in one family with two Japanese siblings showing milder symptoms of CAGSSS and West syndrome concomitant with Leigh syndrome [9]. Thus, these reports assert that mutations in IARS2 are responsible for a phenotypic spectrum of rare autosomal recessive disorders that require further clinical characterisation.

In this study, we analysed whole exome sequencing data in two unrelated consanguineous Iranian patients presenting with clinical features overlapping with CAGSSS. Exome analysis revealed different novel homozygous variants in IARS2 in both families. Patient 1 disclosed a homozygous c.2725C > T, p.Pro909Ser variant which interestingly affects the same p.Pro909 amino acid residue that was described in the French-Canadian family. In the exome analysis of patient 2, whose clinical features were limited to only cataract and skeletal dysplasia, we identified a novel homozygous c.2282A > G, p.His761Arg variant in a region of homozygosity spanning 8 Mb. Additionally, we present a detailed and comparative clinical assessment of patients with a phenotypic spectrum of IARS2-related disorders and illustrate the predicted impact of the identified patient mutations on the protein structure. Moreover, cultured fibroblasts of patient 1 were analysed for mitochondrial enzymatic activity and protein extracts were immunoblotted to assess key mitochondrial oxidative phosphorylation (OXPHOS) proteins and steady-state IARS2 levels. Our patients, who have mild forms of CAGSSS, highlight the wide spectrum of clinical severity associated with IARS2 mutations. Furthermore, as proband 1 displays CAGSSS symptoms concomitant with growth hormone deficiency, central adrenal insufficiency, as well as type II esophageal achalasia, we propose that the phenotypic spectrum of CAGSSS-related disorders resulting from IARS2 variants could include this constellation of features.

Informed written consent was obtained from the families prior to their inclusion in the study. This study was performed under the tenets of the Declaration of Helsinki and approved by the Ethics Commission of the University of Würzburg (46/15), as well as the Ethical Commission of Sabzevar University of Medical Sciences (IR.medsab.rec.1395.120). Blood samples were collected from all participants. Genomic DNA was extracted from whole blood using a standard salting out method. DNA samples were quantified using Qubit 2.0 (Life Technologies, Carlsbad, CA, USA).

CAGSSS is a rare but highly distinctive syndrome with a unique constellation of features, arising from biallelic mutations in IARS2. The patients we described here show a considerable phenotypic overlap to previously described patients. However, not all cases fulfill all features of the CAGSSS acronym. We found significant variability regarding onset of symptoms and some individuals show additional symptoms not included in the acronym, suggesting a wider phenotypic spectrum. With only ten molecularly confirmed cases reported to date, genotype-phenotype correlations are difficult to make with certainty, but it seems possible that there is a predicted milder impact of the p.Pro909Ser variant on IARS2 protein function compared to the other identified alleles (Table 1).

Type II achalasia and adrenal insufficiency were observed in two individuals, patient 1 and patient 7 and interestingly both have a homozygous variant affecting the proline on position 909, p.Pro909Ser in patient 1 and p.Pro909Leu in patient 7 [5]. Due to the low cortisol values in patient 7, we also speculate an adrenal insufficiency that would require further studies. It would be particularly interesting for additional patients with IARS2 pathogenic variants to be monitored for adrenal insufficiency. The replicated finding of esophageal achalasia suggests an expansion of the CAGSSS phenotypic spectrum. Achalasia, a primary motor disorder of the esophagus, is typically diagnosed in adulthood [33]. Therefore, it is unsurprising that it was not mentioned as a clinical feature in the Danish proband [7]. This may also be an unlikely coincidence that achalasia was also described in a French-Canadian proband [5]. One could speculate that IARS2 could be one of many genetic factors for this phenotype; however, further reports are required to understand this observation.

In silico analysis suggests the c.2725C > T, p.Pro909Ser variant is likely benign with only one out of five in silico pathogenicity prediction tools ranking this variant as disease causing. Also, two out of five of these tools predicted the novel c.2282A > G, p.His761Arg variant in the current study as disease causing (Table 2). While these tools can be a helpful first insight to the pathogenicity of a variant, no single program provides an error-free prediction result and caution should be used with concluding pathogenicity based exclusively on in silico tools [34]. Based on previous studies, we assessed whether the p.Pro909Ser variant led to any demonstrable effects on OXPHOS function and steady-state IARS2 levels in patient fibroblasts although none were detected. This was somewhat surprising given the earlier report of the patient with the c.2726C > T, p.Pro909Leu variant documented decreased levels of IARS2 protein in patient cells [5]. Despite this, it is well documented that mutation of human mitochondrial ARSs are associated with marked clinical heterogeneity and tissue-specificity, and that cultured skin fibroblasts rarely replicate the functional mitochondrial deficit observed in post-mitotic and clinically-relevant tissues [35‐37].

Evolutionary conservation of structurally and functionally important regions is typically a criterion for inferring pathogenicity. Conserved regions have been subjected to negative selection and disease causing variants tend to occur disproportionately in highly conserved amino acids [38]. The homology model of human IARS2 allows the observation that the Glu708Lys variant, a variant associated with Leigh syndrome, affects a glutamic acid directly involved in the binding of mt-tRNA^Ile (at the level of nucleotide G69 as inferred from the crystal structure of the complex of tRNA^Ile with a bacterial isoleucine tRNA ligase, PDB code 1FFY), and is also very close to the catalytically important KMSKS motif and also nearby the binding site of Ile-AMP (Fig. 3b). The location of Glu708 inside the protein can be reliably assessed from the sequence alignment with the bacterial template (Additional file 1). Thus, it could be expected that the conserved Glu708 might have roles in the correct function of IARS2 and that the Glu708Lys change, which implies a reversal of the electric charge at this site, would somehow influence the enzyme activity. However, the possible clinical importance of the Glu708Lys variant expected from modelling does not appear to be supported by population frequency data, which show a combined eight homozygous (five South Asian individuals, two non-Finnish Europeans, and one Central Asian (GME) individual) and 374 heterozygous carriers from the five population frequency databases used (Table 2) and yielded a combined calculated MAF of 0.00131. In combination with the null Trp607* truncating mutation in the Leigh syndrome patient, [5], we speculate that the Glu708Lys variant alone has moderate severity but may still be functional and provide a viable amount of enzymatic activity but not enough to overcome a null allele. Supported by population frequency data, a homozygous null Trp607* variant is likely to be lethal. We assert that a homozygous Glu708Lys orientation in IARS2 may provide enough enzymatic activity to be viable, and in light of the population frequency data, phenotypically normal. Thus, it remains to be seen whether an association with Leigh syndrome is correct. Phenotypic presentation is also likely absent in the context of variants that mildly disrupt synthetase activity over a certain threshold, since sufficient synthetase activity can be maintained by even low functional activity [39]. Further studies are necessary to understand the role of this variant.

The His761Arg and Gly874Arg amino acid exchanges occur very close to each other (Fig. 3c). The Gly874Arg exchange affects a conserved glycine in the N-terminus of a helix involved in the binding of mt-tRNA^Ile (Fig. 3c) and might influence this interaction through conformational changes. As a matter of fact, the Gly874Arg change introduces a large and cationic residue very close to Lys872, thus producing both hindrance and repulsive electric forces promoting conformational changes in the helix. The interactions between the latter helix and the helix bearing His761 might be altered by the replacement of this conserved histidine with an arginine. The Pro909Leu and Pro909Ser variants affect a conserved proline in a solvent exposed loop. Both replacements modify the flexibility of the loop as proline residues provide unique conformational restraints among amino acids. In the case of the Pro909Leu variant, since Pro909 is surrounded by the hydrophobic Ile907, Leu911, and Ile954 residues (Fig. 3c), we foresee that the additional hydrophobic residue introduced by mutation would favour clustering of these hydrophobic residues to minimise their exposure to water. Therefore, the Pro909Leu change is expected to cause more prominent conformational changes compared to the Pro909Ser variant wherein the hydrophilic nature of the loop is preserved by the serine residue. This is consistent with the more severe phenotype associated with the Pro909Leu variant compared to the Pro909Ser variant.

Presently, most of the CAGSSS pathogenic missense variants that have been identified reside in exon 21 (Gly874Arg, Pro909Leu, Pro909Ser) of IARS2. It remains unknown whether homozygous variants in this exon tend to be associated with CAGSSS and variants of unknown significance in other affected exons are responsible for Leigh syndrome, a syndrome that is attributed to the death of a patient at 18 months of age that was proposed to be due to compound heterozygous IARS2 variants (c.1821G > A, p.Trp607* in exon 14 and c.2122G > A, p.Glu708Lys in exon 17) [5]. Japanese siblings who were diagnosed with CAGSSS, Leigh, and West syndrome showed compound heterozygous variants in IARS2 that affected exons 4 (c.680 T > C, p.Phe227Ser) and 20 (c.2450G > A, p.Arg817His). Patients 2 and 3 presented with ophthalmological and skeletal deficits and had a homozygous variant in exon 18. It can be reasoned that loss-of-function variants result in a more severe phenotype, since tRNA-charging activity would be abolished. This effect is likely independent of the affected exon.

Although no other endocrine disorders have been connected to other patients with mutations in IARS2, mitochondrial disease itself represents a high risk for a variety of endocrine diseases. GHD has been related to multiple patients with mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS), mtDNA deletions disorders, and nuclear encoded defects [40]. The hypothalamic-pituitary axis dysfunction has been proposed as the underlying pathophysiological mechanism, including chronic ischemia and energy deficiency of the diencephalon, associated with the mitochondrial genetic abnormality of the hypothalamus. This may correlate with our patient, since he shows a co-existing central adrenal insufficiency. Adrenal insufficiency has also been characterised in several patients with other forms of mitochondrial dysfunction, mostly with Kearns-Sayre syndrome, Person syndrome, MELAS, and POLG-related disease [41]. The most accepted pathophysiological mechanism is associated with the high energy demands of endocrine glands; therefore, the impaired mitochondrial ATP production and/or oxidative stress may greatly reduce the ability to secrete hormone or maintain normal feedback [40].

Whilst this manuscript was in peer review, two additional publications described IARS2 variants in patients. One of the reports described a Japanese family with two siblings who were diagnosed with Leigh syndrome that was concomitant with some of the features of CAGSSS, as well as West syndrome (Table 1 patients 4 and 5) [9]. Both siblings had novel, compound heterozygous IARS2 [(c.680 T > C; p.[(Phe227Ser)] and c.2450G > A; p. [(Arg817His)] variants in exons 4 and 20, respectively, and exhibited delayed motor development, as well as infantile spasms and abnormal brain MRI diagnostic imaging leading to a diagnosis of Leigh syndrome. One of the two siblings had cataracts and the other sibling had a neonatal hearing screening result requiring follow-up. Both female children were in their first decade of life at the time of publication.

The second publication which characterised by far the mildest clinical presentation characterized to date described two probands from China with sporadic pediatric cataract [8]. Both probands were identified with compound heterozygous variants in IARS2 (case 6: c.607G > C; p.(Gly203Arg) in exon 4 and c.2575 T > C; p.(Phe859Leu) in exon 21; family 10: c.2446C > T; p.(Arg816*) in exon 20 and c.2575 T > C; p.(Phe859Leu) in exon 21). Both probands shared the Phe859Leu exchange, which affects the anticodon-binding domain and can be reasoned to exhibit only mild/moderate effects. One of the reasons for this hypothesis is that the amino acid exchange conserves hydrophobicity. Another being that the Gly203Arg that is located in the aminoacyl-tRNA synthetase domain and the Arg816* null allele can reasonably be predicted as causing more severe effects on the protein. If the Phe859Leu exchange were to cause a severe protein change, we would expect a severe or even lethal phenotype in the presence of the null allele. Thus, the allelic protein product with the Phe859Leu exchange should still maintain functionality.

In conclusion, we describe two additional independent families with a total of three affected individuals displaying clinical features overlapping with CAGSSS and novel IARS2 variants, expanding the clinical and mutational spectrum. Patient 1 also presented with type II esophageal achalasia, as well as growth hormone deficiency and central adrenal insufficiency, which could result from dysfunction of the hypothalamic-pituitary axis. The unusual combination of findings suggest that other endocrine disorders in patients with an IARS2-associated mitochondriopathy could likewise be possible and should be excluded. We propose that these additional phenotypes expand the syndromic constellation of symptoms in adults. Early recognition of IARS2-related pathophysiology has potentially important implications in clinical management, seeing that undetected adrenal insufficiency could lead to a life threatening crisis.