Publication Date:
2012-11-16
Description:
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. 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
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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