To the editor:

GLRX5 is a 156-amino-acid mitochondrial protein that plays an essential role in the synthesis of Fe-S clusters.1  Sideroblastic anemia caused by GLRX5 deficiency has been reported in a single patient with GLRX5 messenger RNA splicing defect.2  No other mutations in GLRX5 have been identified in subsequent large-scale studies of patients with sideroblastic anemia.3,4  In this study, we report a Chinese congenital sideroblastic anemia (CSA) patient who is a compound heterozygote for 2 missense mutations in his GLRX5 gene.

Informed consent was obtained from the patient, his relatives, and healthy controls used in this study. The study was approved by the institutional review board of the Peking Union Medical College Hospital.

The patient, now a 46-year-old man, is the offspring of nonconsanguinous parents. Since the age of 29, he suffered from severe anemia (supplemental Table 1, available on the Blood Web site) and presented with dark skin and hepatosplenomegaly. A peripheral blood smear showed anisocytosis. A bone marrow smear showed mild erythroid hyperplasia and 19% ring sideroblasts. Transferrin saturation and serum ferritin were significantly elevated even in the absence of blood transfusions (supplemental Table 1), and liver biopsy revealed increased hepatic parenchymal cell stainable iron. In 2009, he presented with peripheral edema and developed diabetes, and he was then enrolled in our study for investigation of his anemia. The patient’s anemia was significantly alleviated after iron chelation (deferoxamine [DFO]) therapy and vitamin B6 supplementation for 6 months (supplemental Table 1). However, after discontinuation of DFO for 1 year, the patient again became moderately anemic (supplemental Table 1), indicating that DFO treatment, rather than vitamin B6 supplementation, was effective in increasing the hemoglobin levels in anemia. Iron chelation has been shown to improve anemia also in a proportion of acquired sideroblastic anemias.5  One potential mechanism for its action is that it reduces iron-dependent toxic reactive oxygen species formation.

The presence of >15% of ring sideroblasts in the bone marrow of the patient is consistent with a diagnosis of sideroblastic anemia. In order to confirm this diagnosis and identify the molecular defects, we screened mitochondrial DNA for deletions and directly sequenced genes known to be associated with sideroblastic anemias when mutated, including ALAS2, SLC19A2, ABCB7, SLC25A38, PUS1, YARS2, and GLRX5.6  In the differential diagnosis of microcytic anemia and congenital iron overload, we also excluded deficiencies in MFRN1, STEAP3, DMT1, HFE, TFR2, HAMP, HFE2, and SLC40A1. Two heterozygous missense mutations were found in the GLRX5 gene of the patient (Figure 1A), whereas no pathogenic nucleotide changes were detected in the other genes sequenced. The c. 301 A>C transversion resulted in a K101Q substitution in the GLRX5 protein. K101 in GLRX5 protein is highly conserved from yeast to humans (Figure 1B). The c. 443 T>C transition resulted in a L148S substitution. L148 in GLRX5 is conserved in Drosophila, zebrafish, chickens, and higher mammals, whereas in rodents, it is replaced by valine or isoleucine (Figure 1B).

Figure 1

Characterization of the GLRX5 mutations and functional studies in PBMCs of a healthy control, the patient, and the patient’s brother, who is heterozygous for the K101Q mutation. (A) Two compound heterozygous missense mutations were identified in the GLRX5 gene. (B) K101 is highly conserved from yeast to humans, whereas L148 in GLRX5 is conserved in Drosophila, zebrafish, chickens, and higher mammals and is replaced by amino acids with similar properties in mouse and rat. (C) Determination of IRP1 status by nondenaturing polyacrylamide gel electrophoresis. A decreased Fe-S-IRP1 protein level was observed in PBMCs of the proband. (D) Relative cytosolic aconitase activity (results expressed as the mean percentage of the aconitase activity in the PBMCs of the healthy control). (E) Western blotting of TfR1, H-ferritin, and FECH. Equal loading was confirmed by showing that COX IV and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were unchanged. C, healthy control; P, patient; BP, patient's brother.

Figure 1

Characterization of the GLRX5 mutations and functional studies in PBMCs of a healthy control, the patient, and the patient’s brother, who is heterozygous for the K101Q mutation. (A) Two compound heterozygous missense mutations were identified in the GLRX5 gene. (B) K101 is highly conserved from yeast to humans, whereas L148 in GLRX5 is conserved in Drosophila, zebrafish, chickens, and higher mammals and is replaced by amino acids with similar properties in mouse and rat. (C) Determination of IRP1 status by nondenaturing polyacrylamide gel electrophoresis. A decreased Fe-S-IRP1 protein level was observed in PBMCs of the proband. (D) Relative cytosolic aconitase activity (results expressed as the mean percentage of the aconitase activity in the PBMCs of the healthy control). (E) Western blotting of TfR1, H-ferritin, and FECH. Equal loading was confirmed by showing that COX IV and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were unchanged. C, healthy control; P, patient; BP, patient's brother.

The patient’s younger brother (who was heterozygous for the K101Q mutation) also had a history of mild anemia (supplemental Table 2). However, whether heterozygosity for the K101Q mutation predisposes to anemia or any related clinical condition remains uncertain, because the younger brother had abused alcohol, and his mother, who also has a heterozygous c. 301 A>C GLRX5 mutation, had normal blood counts (supplemental Table 2). The segregation of the mutations in family members indicates that the mutations are biallelic in the proband.

In the single GLRX5-deficient patient characterized, impair-ment of Fe-S biogenesis and disruption of iron homeostasis were demonstrated.2,7  In agreement, a decreased Fe-S-IRP1 protein level was observed in peripheral blood mononuclear cells (PBMCs) of the proband (Figure 1C). As a result, decreased activity of cytosolic aconitase was evident in PBMCs of the patient relative to that of his brother and a healthy control (Figure 1D). Consistent with these findings, TfR1 protein expression was increased and H-ferritin protein expression was reduced (Figure 1E). Mitochondrial Fe-S biogenesis was impaired in PBMCs of the patient as was revealed by the decreased ferrochelatase (FECH) level (Figure 1E).

In conclusion, these results confirm that mutations in the GLRX5 gene can lead to a rare form of sideroblastic anemia. CSA cases should be screened for mutations in GLRX5 when molecular defects in other more commonly involved genes have not been identified.

The online version of this article contains a data supplement.

Authorship

Acknowledgments: This work was supported by the grants from the National Basic Research Program of China, MOST 973 program (2012CB934000, G.N.), and the National Distinguished Youth Scholar of China (31325010, G.N.). G.N. also gratefully acknowledges the support of the Chinese Academy of Sciences Hundred Talents Program. G.J.A. is a Senior Research Fellow of the National Health and Medical Research Council of Australia.

Contribution: G.L. performed the experiments, analyzed data, and wrote the manuscript; S.G. helped with sequencing experiments; G.J.A. and C.C. provided guidance, analyzed data, and revised the manuscript; B.H. designed the studies and provided CSA patient information; and G.N. conceived and supervised the study, analyzed data, and revised the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Guangjun Nie, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, No. 11 Zhongguancun Beiyitiao, Beijing 100190, China; e-mail: niegj@nanoctr.cn; and Bing Han, Department of Hematology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100730, China; e-mail: hanbing_li@sina.com.cn.

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