Megaloblastic Anemia and Mitochondriopathy Caused by a Homozygous Mutation in Sideroflexin-4.

Abstract 79

Megaloblastic anemias are characterized by impaired DNA metabolism, often due to deficiencies in vitamin B₁₂ or folate. Genes underlying hereditary forms of megaloblastic anemia not caused by vitamin B₁₂ or folate deficiencies, however, remain largely unknown. Here we characterize a genetic deficiency in a patient with infantile-onset megaloblastic anemia, developmental delays, and a mitochondrial disorder of unknown etiology. Analysis of peripheral blood smears from the patient revealed hypersegmented neutrophils and erythroid macrocytes, classic features of megaloblastic anemias. The patient's vitamin B₁₂ and folate levels are normal, eliminating their deficiency as potential causes of the disease.

Whole-exome sequencing of the proband cDNA identified a homozygous, single nucleotide deletion (c.231delC) in Sideroflexin-4 (SFXN4), a predicted mitochondrial multi-spanning transmembrane protein. We experimentally verified the mitochondrial localization of SFXN4 using a combination of western analyses on mitochondrial lysates and confocal fluorescence immunohistochemistry. Using trypsin-sensitivity assays on isolated mitoplasts, we further determined the submitochondrial localization of SFXN4 to the inner mitochondrial membrane.

Bioinformatic analyses predict that the mutation introduces a frame shift and a premature stop codon (p.Pro78Leufs*25), resulting in a severely truncated polypeptide. To determine whether the mutant mRNA were expressed in vivo, we used qRT-PCR to assess the steady state level of SFXN4 mRNA in cultured fibroblasts from the proband. qRT-PCR revealed a 92% reduction in SFXN4 expression, consistent with nonsense-mediated decay of the mutant transcript. Genotyping of the index patient and 3 generations of her nuclear family using both Sanger sequencing and allele-specific oligonucleotide hybridization showed that the mutant allele is inherited in an autosomal recessive manner (Fig. A), the result of a presumed founder effect.

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We used complementary zebrafish and human fibroblast systems to model the megaloblastic anemia and mitochondrial disease in the patient, respectively. Using splice-blocking antisense morpholino oligomers (MO) targeting sfxn4, we induced a loss-of-function phenotype in zebrafish embryos (hereafter, referred to as “morphants”). qRT-PCR confirmed the efficient knockdown of sfxn4, as morphants have <10% sfxn4 mRNA. Knockdown of sfxn4 in transgenic, Tg(globin LCR:eGFP) zebrafish showed a gross reduction in GFP⁺ erythrocytes and hemoglobinized cells stained by o-dianisidine (Fig. B, top), while quantification of the red cell population by flow cytometry showed a 60% reduction in the red cell mass. To characterize the anemia, we performed cytospins of flow-sorted erythroid cells from sfxn4 morphants and analyzed their morphology. Wright staining revealed that sfxn4 morphants have red cells with large nuclei containing non-condensed chromatin (Fig. B, bottom), consistent with the features of megaloblastic anemia observed in the index patient. Enumeration of the nuclear: cytoplasmic area ratios showed that red cells from sfxn4 morphants have a nearly 3-fold increase in the ratio of nuclear to cytoplasmic size.

We also investigated the mitochondrial disorder using patient fibroblasts, which showed a severe reduction in complex I (37%) and complex I+III (7%) activity. The over-expression of wild-type human SFXN4 in proband fibroblasts completely rescued the respiratory defect of complex I+III, while transfection of the mutant c.231delC SFXN4 construct failed to increase complex I+III activity. In a complementary strategy, the over-expression of wild-type SFXN4 cRNA from either zebrafish or human partially rescues the anemia in morphant embryos, validating their functional orthologous relationship.

In summary, a recessive loss-of-function mutation in SFXN4, a previously uncharacterized gene, causes the megaloblastic anemia and mitochondrial disorder described in the index patient. Genetic complementation studies in patient fibroblasts and sfxn4-silenced zebrafish morphants validate the pathogenicity of the mutation. Our findings: (1) demonstrate the requirement of SFXN4 for mitochondrial homeostasis and erythropoiesis, and (2) establish SFXN4 as a new candidate gene for mitochondriopathies and megaloblastic anemias.