Molecular basis of spectrin deficiency in hereditary pyropoikilocytosis

Abstract

Hereditary pyropoikilocytosis (HPP) is a recessively inherited hemolytic anemia characterized by severe poikilocytosis and red blood cell fragmentation. HPP red blood cells are partially deficient in spectrin and contain a mutant alpha or beta-spectrin that is defective in terms of spectrin self-association. Although the nature of the latter defect has been studied in considerable detail and many mutations of alpha-spectrin and beta spectrin have been identified, the molecular basis of spectrin deficiency is unknown. Here we report two mechanisms underlying spectrin deficiency in HPP. The first mechanism involves a thalassemia-like defect characterized by a reduced synthesis of alpha-spectrin as shown by studies involving synthesis of spectrin in two unrelated HPP probands and their parents: One parent carries the elliptocytogenic spectrin mutation, whereas the other parent is fully asymptomatic. Peripheral blood mononuclear cells as a source of erythroid burst-forming unit (BFUe) were cultured in a two-phase liquid culture system that gives rise to terminally differentiated erythroblasts. Pulse-labeling studies of an equal number of erythroblasts or morphologically identical maturity showed that the synthesis of alpha-spectrin as well as the mRNA levels as measured by the competitive polymerase chain reaction (PCR) method are markedly reduced in the presumed asymptomatic carriers and the HPP probands. In contrast, the synthesis and mRNA levels of beta-spectrin were normal. These results constitute a direct demonstration of an alpha-spectrin synthetic defect in a subset of asymptomatic carriers of HPP and HPP probands. The second mechanism underlying spectrin deficiency involves increased degradation of mutant spectrin before its assembly on the membrane. This is evidenced by pulse labeling studies of erythroblasts from a patient with HPP associated with a homozygous state for spectrin alpha I/46 mutation (leu-pro mutation at AA 207 of alpha-spectrin). These studies showed that although spectrin is synthesized in the cytosol in normal amounts, the rate of turnover of alpha-spectrin is faster resulting in about 40% to 50% reduced assembly of alpha-spectrin and beta-spectrin on the membrane. Thus, spectrin deficiency in this case is at least in part caused by increased susceptibility of the mutant spectrin to degradation before its assembly on the membrane. We conclude that at least two separate mechanisms underlie the molecular basis of spectrin deficiency in HPP.

Anemia lurking in introns

“…Exome sequencing and wholegenome sequencing are very helpful for defining the molecular basis for human diseases in well-characterized protein coding and RNA splice junction regions. However, the present study shows that association, (b) those who may be compound heterozygous for structural variants of the self-association site, and finally, (c) those who may be heterozygous for a single structural variant of spectrin self-association and who possess a second, uncharacterized defect (2,10,11). The last group of patients exhibits marked spectrin deficiency, suggesting the presence of a production-defective or a thalassemia-like α-spectrin allele in trans with the structural variant.…”

β spectrinPRAGUE: a truncated β spectrin producing spectrin deficiency, defective spectrin heterodimer self‐association and a phenotype of spherocytic elliptocytosis

“…However, in contrast to spherocytic HE, in which the spectrin deficiency, and the spectrin self-association defect are both caused by a single genetic defect and, consequently, are dominantly inherited, HPP red cells contain two distinct genetic defects, each of which is inherited from one of the parents. These two distinct defects include either homozygous or compound heterozygous state for one or two spectrin mutations or a compound heterozygous state for a mutant cy spectrin and a defect involving reduced spectrin synthesis (Hanspal et al, 1993). Consequently, the spectrin deficiency of HPP cells is characteristically more profound than in the spherocytic HE and greater amounts of mutant spectrin are present in HPP red cells underlying marked instability of the cells as manifested by the presence of fragments of the peripheral blood film.…”

“…Two-phase liquid culture for early and late erythroblasts. Erythroblasts were prepared from patient peripheral blood in a two-phase liquid culture system as described (Hanspal et al, 1993). Erythroblasts RNA was prepared using the guanidine isothiocyanate technique (Sambrook et al, 1989), reverse transcribed, and PCR amplified using primers P285 and P284 (1 min at 94°C; 1 min at 64°C and 1 min at 72°C) for 25, 30 and 35 cycles.…”

“…decreased amount of the truncated p' spectrin is detected in erythroblasts prepared from peripheral blood. Erythroblasts were prepared from patient peripheral blood in a two-phase liquid culture system as described (Hanspal et al, 1993). (A) mRNA was isolated, reverse-transcribed using random hexamers and PCR-amplified using primers P285 and P284 flanking the deleted exon X.…”

“…reported membrane protein mutations in common HE include mutations of (Y or / 3 spectrin, affecting the selfassociation of spectrin heterodimers to tetramers (Liu rt al, 1981a). HPP represents a homozygous or doubly heterozygous state for such mutations, or a compound heterozygosity for a mutant spectrin and for another genetic defect involving reduced synthesis of a spectrin (Hanspal et al, 1993). The second form ofHE designated as Southeast Asian ovalocytosis (SAO) is highly prevalent in certain ethnic groups of Southeast Asia (Aniato & Booth, 1977;Holt ct al, 1981).…”