The sideroblastic anemias (SAs) are disorders of ineffective erythropoiesis, collectively characterized by abnormal Prussian blue-positive granules (i.e., iron-stuffed mitochondria) that encircle marrow erythroblast nuclei to form ringed sideroblast cells.1 SAs are usually acquired, but occasionally congenital. While the causes of the common acquired forms of SA remain largely unknown, the molecular genetics of several of the inherited forms of SA is now well understood. 2,3 For instance, X-linked SA is often associated with germline mutations in the erythroid-specfic isoform of 5-aminolevulinate synthase (ALAS2), and several mitochondrial metabolic defects have also been linked to inherited SAs. However, there are still many congenital SA cases of unknown molecular origin.
The precise relationship between SA and erythropoietic protoporphyria (EPP, MIM #177000) is unclear. A substantial fraction of patients with EPP have anemia (48% of women and 33% of men in the largest series), which is usually mild and associated with diminished iron stores.4 Ferrochelatase, the enzyme deficient in EPP, is encoded by the FECH locus at 18q21.3 and catalyzes the final step in heme biosynthesis: addition of ferrous iron to the protoporphyrin ring.5 In one analysis of 9 EPP patients, scattered ringed sideroblasts were observed by light microscopy in the bone marrows of 7 patients, while mitochondrial electron energy-loss spectroscopy (EELS) indicated SA-like iron compounds in all 9 samples.6 Additionally, a 1973 report described a case of EPP with fatal liver disease associated with SA-like features.7 Despite these observations, most idiopathic acquired SA cases do not have FECH mutations, even though modest elevations of erythrocyte protoporphyrin levels are common in this group.3,8
Here we describe a child who presented with congenital SA of unclear etiology, in whom we detected markedly elevated protoporphyrin and reduced FECH mRNA expression compared to healthy controls.
A boy of mixed European descent (age 2 years, 11 months) with an unremarkable perinatal history was noted to be anemic during a well-child evaluation (hemoglobin 9.6 g/dL, mean corpuscular volume 89 fL, and RDW 23.9%). Peripheral smear showed only anisocytosis; white count, leukocyte differential, platelet count, iron studies, and hemoglobin electrophoresis were all unremarkable. Bone marrow examination revealed mild erythroid hyperplasia and 30% ringed sideroblasts. The patient’s mother was healthy, and the father was unavailable for study. During a follow-up visit at age 7 years, 8 months, the patient reported a burning pain in his hands with sun exposure, but without any erythroderma or blistering. Both plasma and free RBC protoporphyrin was measured and found to be markedly elevated (7.7 μg/dL [normal <1.0 μg/dL]; and 1460 μg/dL [normal <60 μg/dL] respectively). Marrow examination at age 14 showed 80% ringed sideroblasts (Figure 1) with a normal karyotype. Mitomycin C chromosome stress testing was negative. Peripheral blood indices from the date of last follow-up at age 15 revealed a hemoglobin of 9.5 g/dL, mean corpuscular volume of 89.3 fL, and a RDW of 32.9%. Iron studies revealed a serum iron of 138 μg/dL, a total iron-binding capacity of 312 mg/dL, and a ferritin of 45 mg/L (normal 14–336 μg/L).
After obtaining the consent of the patient and his mother for analysis of their biological material and case report, genomic DNA was extracted from peripheral blood mononuclear cells and FECH was analyzed as described.3,9
Total RNA was isolated from whole peripheral blood from the patient, his mother (maternity confirmed using a VNTR panel), and 2 healthy controls. We performed real-time quantitative polymerase chain reaction (RQ-PCR) using TaqMan Universal Master Mix, an ABI 7900 Fast RT-PCR system, and the Hs00164616_m1 FECH FAM primer-probe set (all Applied Biosystems, Foster City, CA, USA). Assays were performed in triplicate, with expression ratios calculated using the 2^-ΔΔC method.
Genomic DNA analysis of the patient revealed heterozygosity for the common promoter −251G, IVS1-23C>T and IVS3-48T>C polymorphisms (GTC haplotype) that down-regulate FECH expression, as well as a neutral 425G/A (p.R96Q, SNP rs1041951) polymorphism (GenBank accession NP_000131, Figure 2). The patient’s mother demonstrated the IVS1-23C>T and IVS3-48T>C polymorphisms. FECH mRNA expression in the patient was approximately 40% of that of his mother (20% of normal), whose gene expression in turn was approximately 50% that of healthy controls.
The mutations responsible for the clinical phenotype of EPP are diverse, with no clear correlation between genotype and either protoporphyrin levels, disease severity, clinical phenotype (i.e., liver versus cutaneous disease) or FECH enzyme activity. Inheritance patterns of EPP are complex. For an individual to manifest clinical symptoms of EPP, inheritance of either two mutant alleles (recessive pattern) or both a mutated allele and a low-expression normal allele (e.g., the GTC haplotype in this case; dominant pattern with incomplete penetrance) appears to be necessary.10,11 This case is of interest because of the unusual clinical presentation of EPP dominated by SA (30% and later 80% ringed sideroblasts), rather than photosensitivity (minimal) or hepatic abnormality (absent). To our knowledge this is the first description of a FECH mutation presenting initially as isolated congenital SA, suggesting that EPP should be considered in the differential diagnosis of SA without other features. Similar phenotypic diversity has been described with germline mutations in many other genes. One example that includes porphyria is the GATA1 erythroid transcription factor, where different mutations can lead to thalassemia, macrothrombocytopenia, or congenital erythropoietic porphyria (CEP).12
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