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YARS2 congenital sideroblastic anemia
includes the first cases with isolated sideroblastic anemia and patients with biallelic YARS2 variants that have no clinically ascertainable phenotype. We identified ten novel YARS2 variants and three previously reported variants. In vitro amino-acylation assays of five novel missense variants showed that three had less effect on the catalytic activity of YARS2 than the most commonly reported variant, p.(Phe52Leu), associated with MLASA2, which may explain the milder phenotypes in patients with these variants. However, the other two missense variants had a more severe effect on YARS2 catalytic efficiency. Several patients carried the common YARS2 c.572 G>T, p.(Gly191Val) variant (minor allele frequency = 0.1259) in trans with a rare deleterious YARS2 variant. We have previously shown that the p.(Gly191Val) variant reduces YARS2 catalytic activity. Consequently, we suggest that biallelic YARS2 variants, includ- ing severe loss-of-function alleles in trans of the common p.(Gly191Val) variant, should be considered as a cause of isolated congenital sideroblastic anemia, as well as the MLASA syndromic phenotype.
Introduction
Sideroblastic anemia is defined by the presence of bone marrow ringed sideroblasts, which are erythroblasts con- taining pathological intramitochondrial iron deposits.1 Congenital sideroblastic anemias (CSAs) are caused by a growing list of genetic variants that affect mitochondrial pathways, including heme synthesis, iron-sulfur cluster biogenesis, mitochondrial protein synthesis, and oxidative phosphorylation.2,3 Variants in YARS2 have been associat- ed with myopathy, lactic acidosis, and sideroblastic ane- mia 2 (MLASA2; OMIM #613561),4-8 and recently cases of YARS2-related myopathy in the absence of sideroblastic anemia have been reported.9 YARS2 encodes the mito- chondrial tyrosyl-tRNA synthetase, YARS2, which is responsible for the ATP-dependent conjugation of tyro- sine to its cognate tRNA, required to support mitochondr- ial protein synthesis.10 YARS2 catalyses this reaction in a two-step process. In the first step, tyrosine and ATP bind to the catalytic domain to form the tyrosyl-adenylate intermediate. In the second step, cognate tRNATyr binds the synthetase and the tyrosyl moiety is transferred to the tRNA CCA-end. The resulting tyrosyl-tRNATyr will be delivered to the ribosome.
The most commonly reported YARS2 variant, p.(Phe52Leu), prevalent in patients of Lebanese Christian descent, has been shown to reduce YARS2 amino-acyla- tion catalytic efficiency by approximately 9-fold, and leads to a reduction in mitochondrial protein synthesis in patients with MLASA2.4 Here we report YARS2 variants, some of which were associated with milder effects on amino-acylation, in patients with isolated CSA, or CSA with mild myopathy and lactic acidosis. In addition, we describe two pairs of genotypically identical siblings with divergent, affected and unaffected, clinical phenotypes. Importantly, some patients carry a common YARS2 c.572 G>T, p.(Gly191Val), that we and others have previously shown has a mild effect on amino-acylation activity,5,11 and suggest that these milder alleles may be the basis of the reduced penetrance and expressivity.
Methods
Clinical data
The patients and their immediate family members were referred to MMH, MDF, NS or LA for clinical consultation. Written informed consent was obtained from participants in the study, as approved by the Institutional Review Boards of Boston Children’s
Hospital, USA, the Radboud University Medical Center, the Netherlands, and the Hospital Germans Trias i Pujol, Badalona, Spain. In each case, CSA was ascertained by complete blood counts (CBCs), and peripheral blood or bone marrow morpholo- gy. Detailed clinical histories are provided in the Online Supplementary Appendix.
Variant detection
Targeted sequencing of nuclear encoded CSA genes,12 and the mitochondrial genome as well as mitochondrial DNA deletion analysis was performed on the probands of families 1-3 and 5-9. Genomic DNA was isolated from peripheral blood or skin fibrob- lasts, using the Puregene DNA Purification Kit (Qiagen, Valencia, CA, USA). DNA templates for sequencing were amplified from genomic DNA by PCR, enzymatically cleaned, bidirectionally sequenced using fluorescent dye termination sequencing chem- istry, and analyzed with the Sequencher 5.3 DNA sequence assembly software (Gene Codes, Ann Arbor, MI, USA), as previ- ously described.12
Exome sequencing for Patient 4 was performed on genomic DNA isolated from whole blood. The experimental workflow was performed at BGI Europe (Bejing Genome Institute Europe, Copenhagen, Denmark) using an Illumina Hiseq (Illumina, CA, USA) platform. Variants in genes previously associated with Mendelian diseases (OMIM), including CSAs, were analyzed bioinformatically.
Patient 10 DNA was analyzed using a targeted gene panel for congenital and acquired sideroblastic anemias, including ABCB7, ALAS2, GLRX5, PUS1, SF3B1, SLC19A2, SLC25A38, STEAP3, TRNT1 and YARS2. The library was constructed using the Custom HaloPlexTM Target Enrichment System (Agilent, Santa Clara, CA, USA) and sequenced on a MiSeq platform (Illumina, San Diego, CA, USA). Data were analyzed with SureCall software (Agilent, Santa Clara, CA, USA).
Patient 11 DNA was analyzed using a targeted gene panel for sideroblastic anemia (ABCB7, ALAS2, GLRX5, HSCB, HSPA9, PUS1, SLC25A38, STEAP3, YARS2) and ion semiconductor sequencing as developed by Ion Torrent systems.13
In silico predictions of variant pathogenicity were performed using the Alamut Visual suite of genetic analysis software (Interactive Biosoftware, Rouen, France), and linking externally to the PolyPhen2 and SIFT analytical tools.14,15 Minor allele frequen- cies are reported as in gnomAD (gnomad.broadinstitute.org) current as of September 2017.16
Amino-acylation assays
Recombinant wild-type and the p.(Leu61Val), p.(Met195Ile), p.(Ser203Ile), p.(Tyr236Cys) and p.(Gly244Ala) YARS2 variants were expressed in E. coli, purified to homogeneity and assayed for
haematologica | 2018; 103(12)
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