Valentini Two new mutations of the P 5 ’ N - 1 gene found in Italian patients with hereditary hemolytic anemia : the molecular basis of the red cell enzyme disorder

Hereditary pyrimidine 5'-nucleotidase type-1 (P5’N-1) deficiency is the most frequent disorder of nucleotide metabolism causing hereditary non-spherocytic hemolytic anemia. It is an autosomal recessive disease and is characterized by mild-to moderate hemolytic anemia, reticulocytosis and hyperbilirubinemia. The hallmark of this red cell enzyme disorder is the presence of pronounced basophilic stippling in red blood cells (RBC) on a peripheral blood smear, associated with accumulation of pyrimidine nucleotides within the erythrocytes. P5’N-1 (EC 3.1.3.5) also known as uridine monophosphate hydrolase type-1 (UMPH-1) catalyzes the dephosphorylation of pyrimidine 5'-ribose monophosphates, especially UMP and CMP, allowing them to diffuse out of the cell as nucleosides. The enzyme has no activity on purine nucleotides. Its major role is in the catabolism of the pyrimidine nucleotides, mainly resulting from ribosomal RNA degradation during final erythroid maturation. The enzyme also transfers phosphate groups from pyrimidine nucleotides to pyrimidine nucleosides, suggesting that it has an additional role in nucleotide metabolism. The P5’N-1 activity is dependent on magnesium ions (Mg) and is readily inhibited by heavy metal ions, and metal chelating and thiol reactive reagents. P5’N-1 is a 286 amino acid-long monomer with an apparent molecular mass of approximately 34 kDa. The gene encoding P5’N-1 (NT5C3, UMPH-1) has been mapped to chromosome 7p15-p14. It consists of 11 exons leading to the production of three alternatively spliced mRNA. Presently, only the 286 amino acid protein has been isolated from RBC. Eighteen different mutations of P5’N-1 gene have been so far described in 30 unrelated families. Recently, four pathological variants of the P5’N-1 were expressed in an E. coli system and characterized. The correlation between the molecular defects and the residual enzyme activity of the patients’ blood samples suggested that in vivo deficiency of P5’N-1 is at least in part compensated by other nucleotidases. In this study we investigated two unrelated Italian patients affected by hereditary hemolytic anemia associated with P5’N-1 deficiency. We found two novel mutations, a two bp deletion at the splice site junction between intron 7 and exon 8 (c.396-397del AG) and an in-frame deletion of one codon (c.427-429del CAA) corresponding to glutamine 143. The pathological variant resulting from the deletion of glutamine 143 (Q143del) was produced in a recombinant form, purified, and characterized.