Sideroblastic anemia, iron overload, and <i>ALAS2</i> R452S in African‐American males: Phenotype and genotype features of five unrelated patients
Authors: Pauline L. Lee, Thomas J. Reid, Sylvia S. Bottomley, James C. Barton
Journal: American Journal of Hematology (2011)

There are few reports of X-linked sideroblastic anemia (XLSA), iron overload, and mutations in ALAS2 that encodes 5-aminolevulinate synthase 2 in persons of sub-Saharan African Native descent [1-3]. A 50-year-old African-American man presented with microcytic anemia and mild thrombocytopenia. He had sideroblastic anemia, severe iron overload, cirrhosis, mild cardiomyopathy, diabetes mellitus, and antiplatelet antibodies. He was hemizygous for ALAS2 R452S (exon 9; c.1406C→A) and heterozygous for HFE H63D, but had no coding region mutations in TFR2, HAMP, HJV, or SLC40A1. Specific activity of the R452S protein was 80% of that of the normal recombinant enzyme. Its kinetic characteristics did not differ from those of the wild-type protein. We identified four other probands with sideroblastic anemia and ALAS2 R452S; all were African-American males. The five individuals appear phenotypically homogeneous, presenting with moderate anemia; the four adults presented with iron overload. This report updates the occurrence of XLSA among race/ethnicity groups, and raises the possibility that ALAS2 R452S occurs only in persons of sub-Saharan African Native descent. X-linked sideroblastic anemia (XLSA; OMIM +301300) results from mutations in the ALAS2 gene that encodes 5-aminolevulinate synthase 2 [4, 5]. XLSA is phenotypically heterogeneous and is associated with iron overload due to ineffective erythropoiesis. Severity of anemia and its response to pyridoxine supplementation vary greatly with the location and type of amino acid alteration within the ALAS2 protein structure [6, 7]. Most reported XLSA cases occurred in European whites or in Japanese. A 50-year-old African-American man was referred for evaluation of microcytic anemia and neutropenia; he had not been treated with transfusion. He had had diabetes mellitus, Type I, for three years. For several years, he took zinc 300 mg daily for as long as six consecutive weeks for body building. He did not ingest iron supplements. There was no family history of anemia or iron overload. Hemoglobin (Hb) was 9.3 g/dL, mean corpuscular volume (MCV) 72.0 fL, red blood cell distribution width 18.6%, leukocytes 2.5 × 109/L, absolute neutrophil count 0.7 × 109/L, and platelets 88.0 × 1012/L. Erythrocytes were dimorphic; reticulocytes were 0.6%. Hb electrophoresis was normal. Transferrin saturation was 89%; serum ferritin was 1,895 μg/L. Serum pyridoxine, copper, and ceruloplasmin levels were mildly subnormal. HbA1C level was 10.7%. Alanine and aspartate aminotransferase levels were within reference limits. Prothrombin and partial thromboplastin times were normal. Serologic reactions for hepatitis A, B, and C were negative. Antiplatelet IgG test was positive (29%; reference 0–4.5%). The microcytic anemia and neutropenia were initially attributed to copper deficiency due to chronic zinc ingestion. After treatment with oral pyridoxine (50 mg daily) and oral and intravenous copper supplements, the leukocyte count and serum levels of copper and ceruloplasmin changed little, and further characterization of the abnormal iron measures was undertaken. On two occasions after diagnosis of sideroblastic anemia, his Hb level was <8.0 g/dL and he was treated with transfusion. A bone marrow examination showed 80% cellularity and many ring sideroblasts (15–20% of nucleated erythroid precursors). Karyotype was 46, XY. FISH analysis did not reveal abnormalities of myelodysplasia. Liver biopsy showed cirrhosis with markedly increased stainable iron in hepatocytes, macrophages, and biliary epithelium. Hepatic iron concentration was 31,775 μg/g dry weight (reference 200–2,400 μg/g dry weight); hepatic iron index was 11.3 μmol/g dry weight/y. T2* MRI detected low signal intensities in liver and spleen, consistent with iron overload. Echocardiography revealed left ventricular dysfunction, global hypokinesis, and ejection fraction of 45%. Coronary angiography was normal. T2* MRI study of the heart indicated normal signal intensity. Abnormalities in this case were interpreted as XLSA, with consequent iron overload. The causes of the diabetes mellitus and mild cardiomyopathy could not be determined. The hypoceruloplasminemia was attributed to iron overload and decreased hepatic synthesis. Because this patient's anemia did not respond to pyridoxine supplementation, it was not possible to treat the iron overload with phlebotomy, and he was prescribed deferoxamine. The thrombocytopenia, attributed to IgG anti-platelet antibodies, resolved without specific treatment. The patient was hemizygous for ALAS2 R452S (exon 9; c.1406C→A). He was also heterozygous for the HFE polymorphism H63D, but did not have coding region mutations in TFR2, HAMP, HJV, or SLC40A1. The specific activity of the mutant ALAS2 protein was 80% of that of the normal recombinant enzyme (16.7 μmol NADH/h/mg). Its kinetic characteristics did not differ from those of the wild-type protein: Km for substrate glycine, 5 mM, for substrate succinyl CoA, 1.4 μM; thermotransition temperature, 46.5°C. Two other Arg452 mutants, R452C and R452H, which we expressed in the same system, had specific activities of 113% and 80%, respectively, and their substrate kinetics and thermal stability were also similar to those of the normal enzyme [8]. These observations agree with those of Furuyama et al. [9]. We identified four other unrelated patients with ALAS2 R452S, each an African–American male with sideroblastic anemia [1, 3, 4]. Previously unpublished details of two cases [4] were available from one of the authors (SSB). Features of the five patients at diagnosis are compiled in Table I. Ages ranged from 10 to 50 years. Hb concentrations were relatively uniform (mean 9.6 ± 0.6 (1 SD) g/dL). MCV values varied moderately (mean 67.0 ± 8.0 fL). Values of TS and SF were generally higher in older patients. Three of four patients treated with pyridoxine supplements had either partial or complete responses of anemia. Three patients, ages 33–50 y, presented with evidence of cirrhosis due to iron overload. A 38-year-old man presented with necrotizing fascitis and sepsis due to group A Streptococcus sp. Mutations in other genes that would account for iron overload phenotypes were not detected in any patient. Family members with anemia consistent with XLSA were reported in two kinships. ALAS2 R452 is encoded by a triplet that is conserved among higher organisms [3, 6]. ALAS2 R452H and R452C have been reported only in white kinships with XLSA [6, 9, 11, 12]. We found that the probands reported to have ALAS2 R452S are five apparently unrelated African-American men (Table I). It was not possible to determine whether R452S was inherited by the proband reported here from a sub-Saharan African Native ancestor, was introduced into his kinship by white admixture, or was a de novo mutation. Two of the probands had family members with ALAS2 R452S or sideroblastic anemia (Table I). We investigated ALAS2 haplotypes of Patients 1, 2, 4, and 5 using 10 single nucleotide polymorphisms that occur in ALAS2 introns 1-2 [13]. Each individual had the reference allele at each of the ten positions (Supporting Information Table I). We detected no abnormality other than R452S within any ALAS2 exon in the four patients we studied. Thus, with the available data, it cannot be excluded that the four patients are related. Severity of anemia in the five ALAS2 R452S probands was moderate, and all but the child had marked iron overload. Three patients, the youngest aged 33 years, presented with evidence of cirrhosis; two others, ages 47 and 50 years, also had cardiomyopathy and diabetes mellitus attributed to severe iron overload. Although the cause of necrotizing fascitis and Group A Streptococcus sepsis in one patient was not explained, this bacterium can acquire iron from ferritin or hemoglobin, but not from transferrin [14]. Pathogenic mutations in other iron-related genes were not detected in the five probands. The four patients we studied had neither the −3.7-kilobase α- thalassemia deletion common in African Americans [15, 16] nor abnormal hemoglobin electrophoreses. Thus, there are insufficient observations to determine whether such attributes affect the anemia and iron phenotypes of persons with ALAS2 R452S. Arginine 452 in ALAS2 is the most frequently altered amino acid residue that causes XLSA, accounting for approximately one-quarter of probands or kinships reported to date [9, 17]. This positively charged amino acid is located on the protein's surface and would not affect substrate or cofactor binding directly [7], consistent with the in vitro enzymatic activity and kinetic measurements of the known R452 mutants. Regardless, the mutations may destabilize the protein in vivo, or arginine 452 may be important for the essential interaction of ALAS2 with a protein partner [7]. Further knowledge regarding fundamental aspects of the kinetics and protein dynamics of ALAS is incomplete [18]. Anemia in many XLSA patients responds variably to supplementation with pyridoxine which is converted in vivo to pyridoxal 5′-phosphate (PLP), the active cofactor essential for ALAS activity. The likelihood of a response is related to the location of the mutated amino acid in the ALAS2 protein [6, 7]. Pyridoxine-responsive mutations in the vicinity of the PLP binding site alter the affinity of the enzyme for the cofactor to varying degrees, whereas mutations involving substrate binding are refractory to pyridoxine supplements [7]. Mutations distant from such sites, such as R452S, may exert indirect effects on enzyme function or stability, in which instances structural explanations for a response to pyridoxine are speculative [7]. In some cases, pyridoxine response occurred after reversal of the iron overload [11, 17], presumably through improved mitochondrial function in erythroblasts. Hepatic iron overload, largely in hepatocytes, is typical of patients with untreated XLSA and is morphologically indistinguishable from that of HFE hemochromatosis [19]. Cirrhosis develops in some patients [12, 17, 20, 21], and this complication is related to the individual's rate of iron absorption dictated by ineffective erythropoiesis [22], and by confounding factors, e.g., consumption of iron supplements, coinheritance of a hemochromatosis gene(s), and alcoholism. Late in the course, cardiac and pancreatic damage from iron overload further contribute to morbidity and reduced survival. These consequences of the disorder are demonstrated in Cases 3–5 (Table I). In conclusion, the five case studies of African–American males represent the only reported probands with ALAS2 R542S. A distant common ancestor cannot be excluded. The hematologic phenotype of the R452S mutation is moderately severe and is similar to that associated with other R452 mutations in most cases. The severity of the iron overload associated with the three known R452 mutations cannot be predicted from the degree of anemia. This is manuscript number 21182-MEM from The Scripps Research Institute. This work was supported in part by a grant from the National Institutes of Health DK53505-12, the Stein Endowment Fund, and The Beutler Foundation (P.L.L.); The U.S. Department of Veterans Affairs, University of Oklahoma Health Sciences Center Provost's Fund, and the Oklahoma Center for Advancement of Science and Technology (S.S.B.); and Southern Iron Disorders Center (J.C.B.). The research studies in this report were approved by the Institutional Review Boards of The Scripps Research Institute, the University of Oklahoma Health Sciences Center, Brookwood Medical Center, and the University of Alabama at Birmingham. Genomic DNA of the proband was isolated from buffy coat pellets. Amplification and direct sequencing to detect mutations of ALAS2 [2], HFE [23], TFR2 [24], HAMP [25], HJV [26], and SLC40A1 [25] were performed as previously reported. The orangutan nucleotide sequence used to number the ALAS2 gene region containing the single nucleotide polymorphisms (AY532109) described by Nachman et al. [13] was used to BLAST against the human ALAS2 gene (NG_008983); the homologous region is located within introns 1-3. Haplotyping of the polymorphic region of human ALAS2 introns 1-2 was performed by standard amplification methods and direct sequencing. The single nucleotide polymorphisms are displayed in Supporting Information Table I. Primer sequences used for haplotyping are available upon request. The mutant ALAS2 protein was expressed in E. coli and its specific activity and kinetic characteristics were determined as previously described [8, 27]. We performed a manual and computerized search to identify other unrelated patients with ALAS2 R452S. Pauline L. Lee*, Thomas J. Reid III , Sylvia S. Bottomley , James C. Barton [email protected]§ ¶, * Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, Conway Hematology-Oncology, Conway, Arkansas, Hematology-Oncology Section, University of Oklahoma College of Medicine, Oklahoma City, Oklahoma, § Southern Iron Disorders Center, Birmingham, Alabama, ¶ Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama.