Nonsense mutations of the α-spectrin gene in hereditary pyropoikilocytosis

Hereditary pyropoikilocytosis (HPP) is an inherited hemolytic anemia characterized by peripheral blood smear findings reminiscent of those seen in patients suffering severe thermal burns.1,2 Erythrocytes from most HPP patients exhibit qualitative and quantitative abnormalities of the erythrocyte membrane protein spectrin, the principal structural component of the erythrocyte membrane skeleton. Qualitative spectrin defects are typically associated with missense mutations that lead to abnormal spectrin self-association, a process critical for membrane structure and function.3

The pathogenesis of qualitative spectrin defects, i.e. spectrin deficiency, in HPP erythrocytes is poorly understood. Whereas some HPP patients are compound heterozygotes or homozygotes for missense mutations in spectrin, others are heterozygotes for a missense mutaion and possess a second, thalassemia-like α-spectrin allele in trans to the missense mutation.4–6 This production-defective allele is associated with decreased or absent accumulation of α-spectrin on the erythrocyte membrane. With the exception of the original case described by Zarkowsky et al., the molecular basis of the production-defective α-spectrin allele in HPP is unknown.

We studied HPP probands from 2 HE/HPP kindreds. Both probands had typical hereditary pyropoikilocytosis. Laboratory findings included compensated hemolytic anemia, marked microcytosis (MCV<75 fL), and typical blood smears with erythrocyte morphology including elliptocytes, poikilocytes, microspherocytes, and fragmented cells. Informed consent was obtained in accordance with the Declaration of Helsinki.

One-dimensional SDS-PAGE analyses of erythrocyte membranes from both probands were qualitatively normal (data not shown). Quantitative analyses of spectrin content, measured by spectrin/band 3 ratios, demonstrated spectrin deficiency in both probands (Table 1). These are values typically seen in patients with HPP.

Limited tryptic digestion of normal spectrin followed by two-dimensional gel electrophoresis yields a pattern of five major proteolytically resistant domains of α-spectrin and 4 proteolytically resistant domains of β-spectrin.7 The 80kDa αI domain encodes the NH₂-terminus of α-spectrin which interacts with sequences from the 17 repeat of β-spectrin to form the αβ binding site for spectrin self-association. Most HPP-associated spectrin mutations affect the 80kDa αI domain and yield peptide maps containing one or more fragments of the domain.

Table 1.Biochemical and genetic studies in hereditary pyropoikilocytosis patients.

Both HPP probands exhibited abnormal tryptic spectrin maps, with the αI/50a kDa variant peptide. No normal αI 80kDa peptide was seen on maps from either of the HPP probands, implying homozygosity for the underlying spectrin mutation (Figure 1 and data not shown). Increased amounts of the αI/50akDa peptide have been associated with structural defects of spectrin in individuals with HE and HPP.

Mutations associated with the variant αI/50α peptide have been identified in exon 5 or 6 of the α-spectrin gene, part of the region encoding the spectrin self-association site. Exon 5 and 6 were amplified from genomic DNA romf the HPP probands and subjected to nucleotide sequence analysis. This revealed missense mutations in exon 5 (patient 1) or exon 6 (patient 2) of the α-spectrin gene (Table 1). These mutations have previously been associated with hereditary elliptocytosis or HPP.

To determine the basis of the observed disparity between spectrin tryptic maps and DNA sequence analyses, nucleotide sequence analysis of the remainder of the coding exons of the α-spectrin gene was performed on genomic DNA from the probands. Nonsense mutations were identified in trans in both patients, revealing the basis of the apparent homozygosity seen on spectrin tryptic maps (Table 1). The nonsense mutations found in these patients likely led to nonsense-mediated α-spectrin mRNA decay8 or protein proteolysis, as there was no evidence of a truncated α-spectrin peptide on one or two dimensional gel electrophoresis.

The presence or absence of the low expression allele, α, was determined in the probands and family members. Patient 1 was heterozygous for the α allele, in cis to the L207P mutation. Patient 2 did not carry the α allele on either allele.

Only a few production-defective alleles of the α-spectrin gene, α, α, α, and spectrin have been described,9–12 and only the α and spectrin alleles have been associated with the HPP phenotype. The α allele is characterized by a C-T mutation at −12 of intron 45 associated with partial in-frame skipping of exon 46. Deletion of the amino acids in exon 46 disrupts the folding of α-spectrin repeat 21, which participates in αβ spectrin nucleation. This inhibits assembly of the shortened peptide into spectrin dimers, leading to proteolytic degradation, with a resulting ~50% decrease in spectrin available for membrane assembly.12 In general, HE patients heterozygous for missense mutations of α-spectrin on one allele and α in trans are more severely affected, presumably because the decreased amount of α spectrin incorporated into the membrane increases the relative incorporation of spectrin containing the mutation in trans.

Figure 1.Limited tryptic digestion of spectrin. Erythrocyte spectrin extracted from erythrocytes of a normal control (A) and hereditary pyropoikilocytosis proband 2 (B) was partially digested with trypsin and fractionated by two-dimensional gel electrophoresis with IEF (SDS/PAGE with IEF). The proband’s spectrin digests demonstrate increased amounts of a 50kDa spectrin peptide (arrow) and no normal 80kDa αI domain peptide. In tryptic digests from the control (A), the 80kDa αI domain peptide (arrow) is normal.

There is abundant evidence that there are non-α production-defective α-spectrin alleles associated with HPP. In some patients, α in trans does not worsen clinical severity. In others, similar to patient 1, α is found in cis to an α-spectrin mutation, suggesting the inheritance of a non-α production-defective α-spectrin allele in trans. Non-α production defective α-spectrin alleles are characterized by reduced α-spectrin mRNA levels and diminished α-spectrin synthesis.4 In one HPP kindred, the molecular basis of the production defective allele is known.9 A mutation in the donor splice site of intron 22 of the α-spectrin gene, spectrin, leads to aberrant splicing producing α-spectrin cDNAs containing in-frame premature termination codons. These studies demonstrate that nonsense mutations cause spectrin deficiency in some HPP patients, and, together with the spectrin allele, reveal considerable genetic heterogeneity in the molecular basis of HPP.

Footnotes

• Funding: this work was supported in part by grants HL65448 (PGG) and DK019482 (BGF) from the National Institutes of Health.