Molecular basis of Japanese variants of pyrimidine 5′‐nucleotidase deficiency

Abstract

Summary The type‐I isoform of pyrimidine 5′‐nucleotidase (P5N‐I) has an important role in the catabolism of pyrimidine mononucleotides during erythroid maturation. Two alternatively spliced forms of P5N‐I mRNA have been identified, and we found another alternatively spliced form in reticulocytes, which included an additional 87‐bp sequence. The sequence is located 6·2‐kb downstream of the exon 2 and 2·7‐kb upstream of the exon 3 sequence; consequently, the P5N‐I gene encodes 11 exons, which span approximately 48 kb. We identified five novel mutations in nine families with P5N‐I deficiency: two missense mutations (425C, 721C), one splice mutation (339C), one 1‐bp insertion (251‐insA‐252) and one 9‐bp deletion (del 192‐200). All patients were homozygous for each mutation. The mutant P5N‐I with 721C (G241R) had lower affinity for cytidine monophosphate, suggesting that Gly241 is important for substrate binding. Haplotype analysis showed that 721C, which had been identified in five unrelated families, was a founder mutation. The mutant P5N was then expressed in Cos‐7. The degradation of P5N with 425C (L142P) was significantly faster than a wild‐type control, and proteasome inhibitors restored the stability of L142P. These data suggest that L142P increases susceptibility to the degradation by the ubiquitin‐proteasome pathway.

Functional analysis of pyrimidine 5′-nucleotidase mutants causing nonspherocytic hemolytic anemia

“…Surprisingly, however, patients affected by the mutations L131P and G230S show only moderate alterations in enzyme activity of their red blood cells (Table 1) despite the substantial changes in the kinetic and thermostability parameters. A similar picture has been obtained for other mutations affecting P5ЈN-1, such as frameshift or nonsense mutations, 7,11,21 that are expected to lead to aberrant proteins likely to lack enzymatic activity. Thus, it appears that reductions in P5ЈN-1 activity caused by mutations can be compensated.…”

“…7 This mutant evidenced a drastic decay in the catalytic activity and a significant reduction in the thermostability. Clearly, position 230 cannot tolerate the large and charged arginine side chain.…”

“…5 An additional alternatively spliced form of P5ЈN-1 mRNA has been recently identified in reticulocytes, raising to 11 the total number of exons. 7 P5ЈN-1 deficiency is an autosomal recessive disorder characterized by hemolytic nonspherocytic anemia, heavy basophilic stippling in RBC peripheral blood smears, and accumulation of pyrimidine nucleotides within erythrocytes. 5,8,9 It has been suggested that P5ЈN-1 deficiency is the third most common RBC enzymopathy-after glucose-6-phosphate dehydrogenase and pyruvate kinase deficiency 10 -that causes hemolysis.…”

“…2,9 Fourteen different mutations have been identified at the DNA level to date 7 ; 4 of them are missense mutations and do not affect the protein length, and the remaining 10 (nonsense mutations, deletions, insertions, and alterations of the splicing sites) are expected to generate shorter or aberrant forms of the polypeptide. P5ЈN-1 deficiency is generally associated with mild to moderate hemolytic anemia, 7,11 although at present no correlation between the residual P5ЈN-1 activity and the severity of clinical manifestations has been documented.…”

Structure of Pyrimidine 5′-Nucleotidase Type 1

“…Similar although less pronounced accumulation of pyrimidine nucleotides may occur during severe lead poisoning (11). Four distinct missense mutations in hP5N-1 have been found in patients with NHA: D98V, L142P, N190S, and G241R (17)(18)(19)(20).…”