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YARS2 congenital sideroblastic anemia
The five novel missense variants all lie in the catalytic domain of YARS2 (Figure 1A) and are rare in the gnomAD database (gnomad.broadinstitute.org) (Table 2). In silico pre- dictions of pathogenicity for p.(Leu61Val), p.(Met195Ile) and p.(Tyr236Cys) vary between the SIFT and PolyPhen2 prediction programs while p.(Ser203Ile) and p.(Gly244Ala) are predicted to be damaging to the YARS2 protein by both algorithms (Table 2 and Figure 1B). Conservation among species for each missense variant is shown in Online Supplementary Figure S1.
The nonsense variant, the splicing variant and three novel indels are likely to be deleterious. The splicing vari- ant c.1104-1G>A alters a canonical position in the 3ʹ splice acceptor site of intron 3 and it is predicted to result in skip- ping of exon 4. The YARS2 c.98C>A, p.(Ser33*) nonsense variant and the c.1165_1166insG, p.(Leu389Cysfs*6) frameshift variant both lie greater than 55 nucleotides upstream of the last exon-exon junction and are most like- ly targeted for nonsense mediated decay.18 The p.(Thr197_Leu208) in frame deletion results in loss of 12 residues in α-helical regions of the catalytic domain, and more precisely of cluster 1, which is important for tRNA acceptor end recognition19 (Figure 1B). The c.1360_1361insG, p.(Ile454Serfs*10) variant lies in the last exon of YARS2 and is not predicted to be targeted for non- sense mediated decay.18 This variant would cause a frameshift at position 454 in the S4-like domain, which is found in all prokaryotic and organellar tyrosyl-tRNA syn- thetases, and is thought to stabilize the interaction between the tRNA and YARS2.19,20
Amino-acylation activity of YARS2 missense variants
Amino-acylation assays are commonly used to evaluate the effect of variants on aminoacyl-tRNA synthetase activity, with reduced activity being a strong predictor of pathogenicity.21 Consequently, the effect of the five mis- sense variants, p.(Leu61Val), p.(Met195Ile), p.(Ser203Ile), p.(Tyr236Cys) and p.(Gly244Ala) on amino-acylation activity was measured by the incorporation of [14C]-tyro- sine into an E. coli tRNATyr substrate and compared to wild- type YARS2 activity. In vitro studies of the YARS2 variants revealed that amino-acylation efficiency was mildly reduced for p.(Leu61Val) and, p.(Met195Ile), while p.(Tyr236Cys) was not affected as compared to the wild- type enzyme (Table 3). YARS2 p.(Ser203Ile) and p.(Gly244Ala) demonstrated a 17-fold loss in catalytic effi- ciency. The reduced activity of YARS2 p.(Ser203Ile) is a consequence of an increased Km, indicating that its affinity for tRNATyr was reduced. On the other hand, the YARS2 p.(Gly244Ala) is characterized by a 13-fold lower kcat sug- gesting that the variant hinders efficient transfer of the tyrosyl moiety from the active site to the tRNA.
Discussion
Here we expand the clinical spectrum associated with YARS2 variants and describe patients with milder pheno- types who do not display all the features of MLASA2. Rather, most of the patients we describe presented princi- pally with a normo- or macro-cytic CSA; they are mostly non-syndromic and unlike the most common forms of non-syndromic sideroblastic anemia (e.g. ALAS2 or SLC25A38 deficiency), the anemia is not microcytic. Nevertheless, in addition to ringed sideroblasts, some of
Table 3. Kinetic parameters for tyrosylation of E. coli tRNATyr by YARS2 wild-type and novel missense variant recombinant proteins.
YARS2 variant
WT
p.(Leu61Val)
p.(Met195Ile)
p.(Ser203Ile)
p.(Tyr236Cys)
p.(Gly244Ala)
K k k /K m cat cat m
Lossof efficiency* (fold change)
1
4.2 2.0 17.3 0.9 17.8
-1 (mM) (min )
(efficiency)
0.75 20.0 26.7
1.45 9.1 6.3
1.90 25.5 13.4
18.60 28.6 1.5
0.70 16.5 23.6
1.00 1.5 1.5
*Loss of efficiency is calculated relative to wild-type (WT) YARS2.
these patients had vacuolization of marrow precursors and/or other cytopenias that are often seen in the syn- dromic sideroblastic anemias (e.g. Pearson syndrome), which may be a diagnostic clue.
Patients 1 and 3 had all the typical features of MLASA2, whereas Patients 2a, and 2b, who share homozygosity for the YARS2 Lebanese founder allele, p.(Phe52Leu), had only anemia. Patient 1 also had other features not typically associated with MLASA2, including neutropenia, throm- bocytopenia, pericardial effusion, and premature ovarian failure. Neutropenia and pericardial effusion have each been reported in one other patient homozygous for the p.(Phe52Leu) variant.5,22 Two other patients in the current series with other genotypes also had mild or intermittent neutropenia. Premature ovarian failure is associated with variants in several mitochondrial aminoacyl-tRNA syn- thetase-encoding genes including HARS2, LARS2 and AARS2,23-25 and thus may be a feature common to mito- chondrial protein synthesis defects. There are now 10 reported individuals homozygous for the YARS2 p.(Phe52Leu) variant5,22 and all have been symptomatic, supporting complete penetrance of this allele. However, the great range of phenotypic severity strongly suggests the presence of other genetic and environmental influ- ences that can modify the effects of YARS2 deficiency.
Patient 4 presented in late adolescence with sideroblas- tic anemia without myopathy and has a homozygous p.(Leu61Val) variant that diminished the amino-acylation catalytic efficiency 4-fold. Leu61 is located in a region of the catalytic domain specific to mitochondrial YARSs that was proposed to contact the tRNATyr acceptor helix (Figure 1B).19 In this case, HSCT appeared to be an effective treat- ment, restoring the patient’s hemoglobin levels to normal.
Patient 5 presented in infancy with CSA and was trans- fusion dependent other than a remission occurring between three and six years of age; she had no myopathy until her post-HSCT terminal illness. This patient had a YARS2 c.1165_1166insG variant predicted to result in a null allele, and a novel p.(Met195Ile) variant which lies within cluster 1, in a region involved in recognition of the tyrosine accepting arm of tRNATyr (Figure 1B).19 Some YARS proteins (e.g. yeast) have an isoleucine (Ile) at this position, suggesting that it might be a milder allele. Indeed, in vitro this mutant had little effect on YARS2 cat- alytic efficiency.
Patient 10 is a compound heterozygote for a splicing mutation (c.1104-1G>A) predicted to cause skipping of exon 4, and a missense variant p.(Ser203Ile), also located
haematologica | 2018; 103(12)
2013