A case report of congenital sideroblastic anemia caused by a novel ALAS2 mutation in conjunction with thalassemia

Abstract

Background

Congenital sideroblastic anemia (CSA) and thalassemia are both hereditary disorders of erythropoiesis, primarily affecting erythroid cells. Their typical manifestations include anemia and iron overload. In this study, we conducted clinical and molecular analyses on a male patient who was concurrently diagnosed with thalassemia and CSA.

Methods

The patient underwent a series of tests including complete blood count, bone marrow smear, and serum ferritin levels. Whole exome sequencing technology was employed for genetic mutation analysis, and bioinformatics methods were utilized to assess the functional impact of the predicted variants.

Results

The patient was previously diagnosed with alpha thalassemia (with genotype of --^SEA/-α^4.2), and has been receiving frequent blood transfusions over the past two years, presenting with severe anemia (Hb level of 44 g/L), iron overload, and 52% ring sideroblasts in the bone marrow. Whole exome sequencing revealed a hemizygous nonsense mutation (c.224 C > A) in the 5-aminolevulinate synthase (ALAS2) gene, which introduces a premature stop codon at the 75th amino acid position in the N-terminal region (P.S75X). Family analysis showed that the patient, her mother, and her sister all carry this variant, suggesting it is a de novo mutation. Computational analysis using various online software tools predicted the variant to be deleterious. The patient was treated with a combination of vitamin B₆, folic acid, and deferasirox. After six months, the Hb level increased to 104 g/L, while the serum ferritin level initially rose and subsequently decreased.

Conclusion

This study identified and reported a novel variant, ALAS2 c.224 C > A (P.S75X), which led to the co-occurrence of sideroblastic anemia in a male patient with thalassemia. The anemia symptoms induced by this variant were responsive to pyridoxine (vitamin B₆) supplementation therapy.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00277-026-06984-4.

Introduction

Congenital sideroblastic anemia (CSA) is a rare inherited hematological disorder. It is currently believed that CSA is primarily caused by mutations in genes associated with heme biosynthesis, iron-sulfur cluster biogenesis, and mitochondrial protein synthesis [1]. Among these, the most common type of CSA is X-linked sideroblastic anemia (XLSA), which is caused by mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene [2]. Thalassemia (Thal), on the other hand, is a group of heterogeneous genetic disorders resulting from reduced synthesis of Hb α or β chains [3]. In a patient exhibiting characteristics of both α-thalassemia and CSA, we identified a novel pathogenic variant in the ALAS2 gene.

Materials and methods

Patient information

The proband is a 46-year-old male patient who was admitted to our hospital for transfusion therapy due to a confirmed diagnosis of thalassemia, with a Hb level of 44 g/L. Thalassemia gene testing revealed a genotype of --^SEA/-α^4.2, β/β, which was inconsistent with the patient’s Hb level. Based on this, to further clarify the etiology, the patient underwent bone marrow aspiration and whole-exome sequencing. Peripheral blood samples were also collected from nine family members, and whole-exome sequencing and thalassemia gene testing were performed on all samples.

Whole-exome sequencing

Genomic DNA of high quality was extracted from whole blood samples, and target enrichment was performed using the Agilent SureSelect All Exon V5 capture kit as per the manufacturer’s protocols. Massively parallel sequencing was carried out on the HiSeq2000/2500 platform (Illumina) at Novogene (Beijing). In this study, 334,378 exons from 20,965 genes were captured using magnetic beads with streptomycin. Linear amplification by PCR was performed, and library quality was assessed for sequencing. The average sequencing depth was 100X, with 98.5% coverage at 30X and 99.84% coverage at 10X. The targeted regions encompassed exonic regions of all nuclear genes, including +/− 5 bp of exon-intron boundaries. The sequencing raw reads (Q30 > 90% quality level) were aligned to the human reference genome (accession no. hg19/GRCh37).

Variant calling was performed by an in-house pipeline following the GATK Best Practices [4]. Some software was used in this step, including bwa-0.7 [5], samtools-1.11 [6], and Genome Analysis TK-3.7 [7]. Then the variants were annotated by ANNOVAR (version 2019-10-24) [8] and given clinical interpretation by InterVar-2.0.2 according to ACMG/AMP 2015 guideline [9]. Finally, benign and likely benign variants were filtered out.

ALAS2 gene mutation analysis

To analyze and predict the impact of the nonsense mutation (P.S75X) on the function of the ALAS2 gene, we employed bioinformatics software. For this purpose, we utilized bioinformatics programs such as CADD Score (https://cadd.gs.washington.edu/), DANN Score (https://genome.ucsc.edu/), and Mutation Assessor (http://mutationassessor.org). To investigate the effect of the c.224 C > A mutation on the three-dimensional crystal structure of the protein, the online software program AlphaFold (https://alphafold.ebi.ac.uk/) was used to computationally model the normal ALAS2 molecule. The template protein structure was obtained from the Protein Data Bank (https://www.rcsb.org/pdb/home/home.do).

Results

Clinical features

The patient showed no signs of hepatosplenomegaly, and all other clinical examination indicators were normal. The results of the complete blood count are as follows: White Blood Cells (WBC): 3.91 × 10⁹/L (reference range: 3.50–9.50 × 10⁹/L); Red Blood Cells (RBC): 0.96 × 10¹²/L (reference range: 4.30–5.80 × 10¹²/L); Hb: 44 g/L (reference range: 130–175 g/L); Mean Corpuscular Volume (MCV): 86.5 fL (reference range: 82.0–100.0 fL); Mean Corpuscular Hemoglobin (MCH): 45.8 pg (reference range: 27.0–34.0 pg); Mean Corpuscular Hemoglobin Concentration (MCHC): 530.0 g/L (reference range: 316.0–354.0 g/L); Red Cell Distribution Width (RDW): 17.1% (reference range: 11.5–14.5%); Reticulocytes (RET): 0.10% (reference range: 0.50% − 1.50%); Platelets (PLT): 267 × 10⁹/L (reference range: 125–350 × 10⁹/L); The results of serum iron-related indicators are as follows: Serum Iron Concentration: 56.80 µmol/L (reference range: 8.10–28.30 µmol/L); Unsaturated Iron-Binding Capacity: 25.20 µmol/L (reference range: 19.70–66.20 µmol/L); Total Iron-Binding Capacity: 82.00 µmol/L (reference range: 45.00–75.00 µmol/L); Ferritin Concentration: 1022.50 ng/mL (reference range: 23.90–336.20 ng/mL), with a historical peak of 3340.96 ng/mL; Erythropoietin Level: 77.20 mIU/mL (reference range: 4.3–29 mIU/mL).

Results of peripheral blood smear and bone marrow aspiration

Peripheral blood smear revealed anisocytosis of mature erythrocytes, with enlarged central pallor observed in a subset of cells (Fig. 1Ac). Bone marrow aspiration demonstrated normocellular granulopoiesis without significant dysplastic changes, while erythropoiesis was predominantly characterized by proliferation of intermediate and late-stage erythroblasts (Fig. 1Aa). Iron staining of the marrow showed: Extracellular iron (++), 80% of erythroblasts with positive intracellular iron granules, Ring sideroblasts comprising 52% of erythroid precursors (Fig. 1Ab). These morphological and iron metabolic findings collectively support a diagnosis of sideroblastic anemia.

Fig. 1.

The patient underwent bone marrow cytological examination, family pedigree analysis, whole-exome ALAS2 sequencing of all family members, and ALAS2 modeling. A Bone marrow cytology: a Polychromatic erythroblast (red arrow), the cells appear round or oval in shape, with a round nucleus exhibiting deeply purple-stained chromatin arranged in strands or small clumps. Nucleoli are absent. The cytoplasm is abundant, and binucleated intermediate erythroblasts may be observed. b Ring sideroblasts (blue arrow). c Peripheral blood smear. B Family tree. a Mother: ALAS2 c.224 C > A, thalassemia -α4.2/αα. b Eldest sister: ALAS2 c.224 C > A, thalassemia --SEA/-α4.2. c Second sister: ALAS2 c.224 C > A, thalassemia -α4.2/αα. d Patient: ALAS2 c.224 C > A, thalassemia --SEA/-α4.2. e Younger sister: ALAS2 c.224 C > A, thalassemia --SEA/-α4.2. f Eldest sister’s son: ALAS2 wild type (WT), thalassemia -α4.2/αα. g Son: ALAS2 WT, thalassemia αα/-α4.2. h Younger sister’s son: ALAS2 WT, thalassemia Negative. i Eldest sister’s grandson: ALAS2 WT, thalassemia --SEA/αα. C Genetic testing results of sideroblastic anemia in the patient’s family. D AlphaFold Prediction of ALAS2 Gene Structure: The red arrow indicates that the mutation introduces a premature stop codon at this position, leading to premature termination of protein synthesis, corresponding to the protein S75X. AlphaFold produces a per-residue model confidence score (pLDDT) between 0 and 100. Some regions below 50 pLDDT may be unstructured in isolation.Very high (pLDDT > 90),High (90 > pLDDT > 70),Low (70 > pLDDT > 50)Very low (pLDDT < 50).

DNA analysis

This testing project covers the entire exome region of the individual, encompassing a total of 25,030 genes. The coverage of this region reached 98.40%, with an average sequencing depth of 156X in the target region. The proportion of the target region with a sequencing depth greater than 30X was 98.85%. The average sequencing depth of the full-length mitochondrial genome exceeded 1000X. We identified one novel variant ALAS2:NM_000032.5: c.224 C > T (p.Ser75Leu), upon querying the ClinVar database, this variant was not recorded. We confirmed this mutation with Sanger sequencing (Fig. 1Cd), and we have uploaded this mutation site to the database (SUB15285727).

In the family analysis, the individuals involved include: (a) the patient’s mother, (b) the patient’s eldest sister, (c) the patient’s second sister, (d) the patient, and (e) the patient’s younger sister. A novel variant c.224 C > A in exon 3 of the ALAS2 gene was identified in these five individuals. Additionally, the thalassemia genotypes of the patient’s mother and second sister were -α^4.2/αα, while the genotypes of the patient, her eldest sister, and younger sister were --^SEA/-α^4.2 (Fig. 1B, C).

Online protein structure prediction and analysis

We used online software to predict the pathogenicity of the ALAS2 variant (c.224 C > A), and the results showed that this variant is a harmful variant. The relevant prediction results are shown in Table S.

The online software program AlphaFold was used to perform computational modeling of both the normal ALAS2 molecule and the mutant ALAS2 molecule. The ALAS2 template protein structure was obtained from the Protein Data Bank. In the normal ALAS2 protein, the P.S75X mutation (i.e., the insertion of a stop codon at the 75th amino acid residue in the N-terminus) results in the interruption of protein synthesis, as indicated by the red arrow in Fig. 1D. Additionally, the N-terminus of this template protein is missing 142 amino acid residues, and the structural prediction shows a pLDDT value of less than 70, indicating low confidence in the model’s structural reliability. (Fig. 1D).

Treatment outcome

The patient was treated with vitamin B6 (20 mg/d), folic acid (10 mg/d), and deferasirox (1250 mg/d). The patient responded well to the treatment, with a gradual increase in Hb levels and a gradual decrease in ferritin levels. After 6 months of treatment, the Hb level rose to 104 g/L, and the ferritin level was 3096.00 ng/mL. (Table 1).

Table 1.

The comparison of various indicators before and after treatment

Reference Range	RBC (1012/L)	Hb (g/L)	MCV (fL)	MCH (pg)	MCHC (g/L)	Ferritin (ng/mL)
	4.30–5.80	130–175	82.0-100.0	27.0–34.0	316–354	23.9-336.2
At Diagnosis	0.96	44	86.5	45.8	530	1022.5
1 Month Later	2.27	68	85.0	30.0	352	/
4 Months Later	6.48	105	58.2	16.2	279	3325
6 Months Later	6.36	104	59.0	16.4	277	3096

Discussion

Sideroblastic anemia (SA) is a group of hereditary and acquired bone marrow disorders characterized by pathological iron accumulation in the mitochondria of erythroid precursor cells [1]. XLSA is the most common type of SA, caused by germline mutations in the erythroid-specific ALAS2 gene, which encodes the first rate-limiting enzyme in heme biosynthesis [10]. The diagnostic criterion for sideroblastic anemia is the presence of ring sideroblasts accounting for more than 15% of the cells in the bone marrow [11].

The patient in this case was initially diagnosed with thalassemia, and consequently underwent thalassemia genetic testing, which revealed a thalassemia genotype of --^SEA/-α^4.2. However, the patient’s Hb levels and transfusion frequency were markedly inconsistent with this thalassemia genotype. Upon further investigation, including bone marrow aspiration, it was discovered that the bone marrow contained a significant proportion of ring sideroblasts, accounting for 52% of the cells. Whole exome sequencing identified a nonsense mutation in exon 3 of the ALAS2 gene (c.224 C > A: p. S75X). Previously reported truncating mutations in ALAS2 (p.P561X, p.V562X, p.H563X, p.E569X, and p.F575X) have been associated with impaired Hb synthesis [12]. The patient had previously been diagnosed with α-thalassemia and received occasional transfusions due to the condition, but sideroblastic anemia had not manifested. The factors that trigger this disease have not yet been identified. Among nearly 200 patients diagnosed with CSA based on bone marrow examination results, less than 3% carried a β-thalassemia genotype [13]. Given that most disruptions in gene synthesis are deleterious, we validated the pathogenicity of the P.S75X variant based on online simulation analysis results and the significant improvement observed in the patient after vitamin B6 supplementation, which confirmed the variant’s pathogenicity. ALAS2 is the first key enzyme in the heme biosynthesis pathway in erythroid cells. It uses pyridoxal phosphate (PLP) as a coenzyme to catalyze the synthesis of δ-aminolevulinic acid (ALA) from glycine and succinyl-CoA [14]. ALAS2 consists of 587 amino acids, including an N-terminal region (aa 1–142), a conserved catalytic core (aa 143–544), and a C-terminal extension (aa 545–587) [15]. During structural prediction, the absence of a template for the N-terminal 1–142 region made it impossible to accurately predict the structure of the protein at the P.S75X variant site. Studies have shown that most pathogenic variants are located in exon 5 and exon 9, the latter of which contains the PLP-binding site at lysine 391 [16].

To date, among the 104 ALAS2 mutations registered in the HGMD (http://www.hgmd.cf.ac.uk/ac/index.php), most are associated with XLSA, and these mutations are predominantly located in the highly conserved regions of exons 5 to 11 [17]. The mutation site we discovered is in exon 3, and reports on mutations in this exon are relatively rare. ALAS2 mutations cause sideroblastic anemia (CSA) with an X-linked inheritance pattern. Most males carrying the XLSA allele will develop the disease, and heterozygous female carriers may also exhibit sideroblastic anemia due to skewed X-chromosome inactivation [18]. However, the majority of heterozygous females do not show clinical manifestations of anemia because the normal ALAS2 protein can maintain the necessary level of red blood cell production [19].

In the case of this study, we found that the patient’s mother and her three sisters all carried the pathogenic variant of the ALAS2 gene, yet none of them developed the disease. Clinically, XLSA predominantly affects hemizygous males, with the onset typically occurring before the age of 40. These patients exhibit varying degrees of microcytic hypochromic anemia accompanied by systemic iron overload. However, there are also a few male patients who do not develop the disease. The patient in this case was 46 years old at the time of onset. To date, approximately 122 ALAS2 pathogenic variants have been reported in XLSA patients.

ALAS2 gene variants at different mutation sites exhibit distinct therapeutic responses. Studies suggest that nonsense mutations—particularly those introducing premature stop codons or compromising ALAS2 protein stability—often render patients unresponsive to vitamin B6 therapy [20]. Typically, mutations proximal to the PLP-binding site correlate with favorable pyridoxine responses, whereas those distal to the binding site are associated with refractory congenital sideroblastic anemia (CSA) [2].

Notably, this patient harbors a distal nonsense mutation in ALAS2 yet demonstrates a robust response to vitamin B6, challenging the conventional genotype-phenotype correlation. This observation supports the structural model of human ALAS2 (hsALAS2) as an obligate homodimer, wherein each catalytic active site is formed by two monomeric subunits, each binding one PLP molecule [15].

Iron overload is partly due to ineffective erythropoiesis, which promotes intestinal iron absorption, leading to a subsequent decrease in the level of hepcidin [21]. This patient had been receiving continuous blood transfusions for 2 years due to an undiagnosed SA, and his serum ferritin level continued to rise. Regardless of whether red blood cells are transfused, patients with SA are prone to iron overload [22].

In summary, This study reports a novel pathogenic variant of the ALAS2 gene, c.224 C > A: P.S75X, which caused XLSA in a male patient who responded well to pyridoxine supplementation. Rapid and accurate diagnosis facilitates effective treatment, promotes favorable clinical outcomes, and ultimately reduces the risk of long-term complications.

Limitations of the Study: (1) As a case report, its inherent nature determines that the sample size is singular, and the described experience and outcomes may not be generalizable to other patients. (2) Due to the absence of a template for the N-terminal 142 amino acid residues of the protein, the reliability of N-terminal modeling is low. 3.When collecting samples for pedigree analysis, the researchers did not include all family members for hematological testing.

CADD Score Interpretation: Phred Score: If the Phred score ≥ 20, the variant is among the top 1% of deleterious variants. If the Phred score ≥ 30, the variant is highly likely to be pathogenic. MutationAssessor Score: The score ranges from − 5.76 to 5.37. If the score < 1.938, the variant is associated with polymorphism. If the score > 1.938, the variant is associated with disease.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

This work was supported by the High Level and Key Health Research Plan of Yangjiang (No. 2023001).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was reviewed and approved by the Ethics Committee of People’s Hospital of Yangjiang (Approval No.20230001).

Conflict of interest

The authors affirm no conflicts of interest.