Hereditary sideroblastic anemia (SA) is characterized by anemia with ringed sideroblasts in the bone marrow (BM) [1]. Phenotypic expression is highly variable, and the most common hereditary SA is X-linked sideroblastic anemia (XLSA, OMIM #300751) caused by mutation of the erythroid-specific δ-aminolevulinate synthase gene, ALAS2 [234].

We present a case of a 12-month-old boy with severe congenital anemia who was finally diagnosed as having XLSA after simultaneous BM study and molecular genetic analysis by massively parallel sequencing (MPS). The boy was referred from an outpatient pediatric clinic because of severe anemia and progressive pallor. He was a full-term baby, and no one in his family had been diagnosed as having anemia. The pallor was considered pathological, and his weight gain of 7.9 kg was insufficient, leaving him below the third percentile for his age. He was transfused packed red blood cells (RBCs) to correct his Hb level of 59 g/L; however, his pallor persisted, and Hb level recovered only temporarily. He was subsequently referred to the Department of Pediatrics at Kangbuk Samsung Hospital. The initial complete blood count, as determined at our institute, was as follows: Hb, 70 g/L; hematocrit, 27.5%; mean corpuscular volume, 63.2 fL; mean corpuscular hemoglobin, 15.9 pg; mean corpuscular hemoglobin concentration, 25.1 g/dL; red cell distribution width, 31.3%; white blood cells, 7.0×10⁹/L; and platelets, 417×10⁹/L. A peripheral blood smear revealed a dimorphic red cell population due to transfusion (Fig. 1A).

His iron profile suggested plentiful body iron stores, as indicated by the following parameter values: elevated serum iron, 49.8 µmol/L (laboratory reference range, 11.6–43.3); normal ferritin, 267.1 pmol/L (36.0–898.8); and total iron-binding capacity, 52.4 µmol/L (42.2–68.0). Hemolytic anemia was excluded by negative direct and indirect Coombs' tests, and he had normal lactate dehydrogenase (324 IU/L [0–250]) and haptoglobin (4.76 µmol/L [3.00–20.00]) levels.

BM aspirate smear revealed an estimated myeloid:erythroid ratio of 1.6:1 and showed small erythroblasts with abnormal condensation of nuclear chromatin and ragged cytoplasm (Fig. 1B, C). Iron staining on the BM aspirate slide showed ringed sideroblasts, which comprised 40% of erythroid precursors (Fig. 1D).

For molecular diagnosis, we conducted MPS after obtaining written informed consent from the patient's parents. Genomic DNA was extracted from peripheral blood. We used the TruSight One sequencing panel (Illumina, San Diego, CA, USA), which includes more than 125,395 probes targeting a 12-Mb region spanning 4,813 genes, including the six known hereditary SA-related genes (ALAS2, SLC25A38, GLRX5, HSPA9, PUS1, and ABCB7) for library preparation and target enrichment. We performed MPS using the NextSeq platform (Illumina) with 150-bp paired-end sequencing, using the pipeline of the Burrows-Wheeler Aligner MEM algorithm, version 0.7.12 [5]; Picard tools, version 1.96 (https://broadinstitute.github.io/picard/); and Genome Analysis Toolkit (GATK), version 3.5 [6] for data analysis. Variant calling and annotation were performed with GATK Haplotype-Caller, Variant Effect Predictor, and dbNSFP. We identified a hemizygous, known mutation, NM_000032.4:c.508C>A (p.Arg170Ser), in exon 5 of ALAS2, and validated this variant by Sanger sequencing. According to the guidelines of the American College of Medical Genetics and Genomics [7], this variant is classified as likely pathogenic based on two moderate (PM1 and 2) and four supporting (PP2-5) pathogenic evidences. Finally, we diagnosed the patient as having XLSA with an ALAS2 mutation, and found his mother to be a heterozygous carrier by using Sanger sequencing (Fig. 2).

The patient was started on pyridoxine therapy (100 mg/day orally) as standard treatment for the goals of maintaining Hb above 100 g/L and normal growth for his age with minimal RBC transfusions [89]. Because his Hb level did not show improvement with pyridoxine treatment alone at his one-month follow-up visit, we added a 1-mg oral folate supplement daily. Subsequently, his growth improved, as his weight increased to the 12th percentile at 18 months of age. This study was approved by the Institutional Review Board of Kangbuk Samsung Hospital (KBSMC 2018-02-018).

To date, 64 distinct disease-causing mutations have been described in individuals or families with XLSA [10]. One-by-one Sanger sequencing of candidate genes is generally used to identify pathogenic mutations in hereditary diseases. In our case, MPS enabled the simultaneous analysis of multiple genes, which allowed immediate appropriate treatment. To the best of our knowledge, this is the second case of XLSA caused by missense ALAS2 p.Arg170Ser mutation, which was reported for the first time by Harigae et al in 2010 [6]. Edgar et al [11] reported a different ALAS2 nucleotide substitution, p.Arg170Leu, in a 31-year-old male patient who showed favorable outcome.

Hereditary anemias are often misdiagnosed as iron deficiency anemia, and this can have dangerous clinical consequences, including parenchymal iron overload. In this case, we identified early-onset hereditary SA with severe anemia. Immediate and accurate diagnosis of XLSA led to effective treatment and a favorable clinical course. Therefore, MPS is a useful method for the rapid diagnosis of hereditary anemia in newborns or infants with severe anemia.