Ferrochelatase Mutation Screening by Denaturing High Performance Liquid Chomatography in Idiopathic Acquired Sideroblastic Anemia (Refractory Anemia with Ringed Sideroblasts) and Erythrocytic Protoporphyria.

BACKGROUND: The molecular etiology of idiopathic acquired sideroblastic anemia (IASA), now considered a form of myelodysplastic syndrome, is currently unknown. Romslo et al (Blood 1982) reported a patient with IASA who had moderately elevated free erythrocytic protoporphyrin (FEP); this observation is now frequently made in IASA, helping distinguish IASA (high FEP) from inherited sideroblastosis (low FEP). In addition, rare patients with erythropoietic protoporphyria (EPP)—defined by cutaneous photosensitivity, dramatically elevated FEP levels, and germline point mutations in the ferrochelatase (FECH) gene at 18q21.3—have ringed sideroblasts in their marrow. We hypothesized that patients with IASA might have acquired somatic FECH mutations.

METHODS AND RESULTS: We designed a denaturing high performance liquid chromatography (DHPLC) assay to explore this possibility and to avoid problems related to mutation screening in the setting of mixed clonality. To validate the DHPLC assay, the coding region of the FECH gene from 2 molecularly undiagnosed EPP patients without liver disease was analyzed. In one patient, both a heterozygous 69delG mutation (GenBank Accession NM_000140) and heterozygosity for the FECH expression-modulating IVS3-48C/T polymorphism were detected. In the other patient, no FECH coding mutations or polymorphisms were detected, either by DHPLC or conventional dye sequencing. FEP measurements were then obtained on 2 IASA patients and were elevated (65 and 115 mcg/dL; normal 1–10 mcg/dL) with normal urine and fecal porphyrins. Genomic DNA obtained from these 2 patients as well as archival DNA from 30 other patients with IASA was amplified and tested for FECH mutations. No coding region mutations were detected. Synonymous polymorphisms in exon 7 (rs536765) and 9 (rs536560) were found in 6/32 (19%; normal heterozygosity 0.398) and 14/32 (44%; normal heterozygosity 0.402) samples, respectively. The IVS3-48C/T polymorphism (rs2272783) was found in 3/32 (9%) (prevalence in the general population is 11%, Gouya Nat Genet 2002). In addition, 3 intronic polymorphisms not in the refSNP database were detected: IVS8+34 C/T (2/32), IVS8-61delG (5/32), and IVS9-59delA (2/32).

CONCLUSION: IASA is not associated with coding mutations in FECH. Elevation of FEP in ASA must instead be due to the lack of mitochondrial iron in the proper form for incorporation into the porphyrin ring; attention should instead focus on factors responsible for maintaining the appropriate redox state and compartmentalization of iron. DHPLC can detect FECH mutations and polymorphisms, but because of the high frequency of the latter and the consequent need to sequence multiple exons, it is not a practical screening tool for studying undiagnosed EPP patients.