Congenital sideroblastic anemia due to mutations in the mitochondrial HSP70 homologue HSPA9

Abstract

The congenital sideroblastic anemias (CSAs) are relatively uncommon diseases characterized by defects in mitochondrial heme synthesis, iron-sulfur (Fe-S) cluster biogenesis, or protein synthesis. Here we demonstrate that mutations in HSPA9, a mitochondrial HSP70 homolog located in the chromosome 5q deletion syndrome 5q33 critical deletion interval and involved in mitochondrial Fe-S biogenesis, result in CSA inherited as an autosomal recessive trait. In a fraction of patients with just 1 severe loss-of-function allele, expression of the clinical phenotype is associated with a common coding single nucleotide polymorphism in trans that correlates with reduced messenger RNA expression and results in a pseudodominant pattern of inheritance.

Figure 1

HSPA9 mutations in CSA. (A) HSPA9 CSA pedigrees. Affected status is indicated by black shading on the left of the symbol. The genotype is indicated on the right side of the symbol, in which gray shading in the upper and/or lower right quadrants indicates a low-frequency HSPA9 variant predicted to have functional consequences. When known, the paternal allele is indicated by shading in the lower right quadrant. A-I-1, A-I-2, A-II-1, A-II-2, A-II-3, A-II-4, A-II-5, A-III-1, A-III-2, A-III-3, A-III-4, and A-III-5 refer to I.1, I.2, II.1, II.2, II.6, II.7, II.19, III.2, III.3, III.4, III.9, and III.10, respectively, in van Waveren Hogervorst et al. A-IV-1, A-IV-2, and A-IV-3 were not included in van Waveren Hogervorst et al. Patient D-II-2 was not available for genotyping but had a phenotype identical to that of her sibling. (B) Mutations in HSPA9 mapped on the structure of bacterial HSP70 (Protein Data Bank ID: 2KHO). The N- and C-termini (term.) of the structure are noted. Human HSPA9 residues Ser200, Ser212, Gly388, Glu415, IleAsn458_459, Thr539, Arg573, and Glu577 were mapped to equivalent bacterial residues Ala149, Ala161, Gly342, Glu369, Ile412-Ala413, Ser493, Arg527, and Glu531. (C) Analysis of HSPA9 expression. Total mRNA was harvested from leukocytes (rs10117 genotype: number of samples analyzed; C/C: n = 16; C/T: n = 23; and T/T: n = 12); HSPA9 mRNA was assessed by quantitative real-time polymerase chain reaction and was normalized to β-actin. P values were calculated by using the Mann-Whitney test. **P < .005; *P < .05. Western blot analysis of HSPA9 protein expression in healthy individuals was grouped by rs10117 allele (C/C: n = 10; C/T: n = 10; and T/T: n = 9). Equivalent loading of mitochondrial lysate was confirmed by immunoblot analysis using an anti-adenosine triphosphate synthase, beta-subunit (ATPB) antibody. Protein expression was determined by densitometry analysis on a Biorad Chemidoc MP instrument with Image Laboratory 4.1 software. (D) Haploid ssq1Δ (left) and ssc1∆ (right) strains having plasmids harboring the indicated mutants, wild-type (WT) gene, or in the case of the viable ssq1∆ strain, a plasmid-lacking insert (vector). Tenfold serial dilutions were plated on minimal media and incubated at the indicated temperatures. Whole-cell lysates of the indicated strains were subjected to immunoblot analysis using polyclonal antibodies specific for SSQ1 (left), SSC1 (right) and, as a loading control, Ydj1. Names of the alleles correspond to the human HSPA9 numbering. For details of homologous yeast mutations, see supplemental Figure 1.