Erythrocytes have an average lifespan of 120 days, after which they are typically removed by macrophages in the reticuloendothelial system. Hemolytic anemia can shorten erythrocyte lifespan, leading to varying clinical presentations depending on whether hemolysis occurs intravascularly or extravascularly. Among intrinsic causes of hemolysis, pyrimidine 5'-nucleotidase (P5N) deficiency is a notable condition, often presenting as nonspherocytic hemolytic anemia. We report a case of a 65-year-old female patient with systemic lupus erythematosus and a history of splenectomy, who was admitted for evaluation of persistent hemolytic crises. Clinical examination, peripheral blood smear analysis, and genetic testing were performed, including next-generation sequencing to identify mutations in the NT5C3A gene associated with P5N deficiency. The patient exhibited macrocytic anemia and basophilic stippling on peripheral blood smear, with normal results from osmotic fragility tests and G6PD levels. Genetic testing revealed a homozygous c.693+1G>A variant in the NT5C3A gene, classified as possibly pathogenic based on ACMG criteria. This variant is linked to P5N deficiency, which aligns with the patient's clinical presentation of non-immune hemolytic anemia. The identification of the NT5C3A gene mutation through next-generation sequencing highlights the significance of molecular technologies in diagnosing rare forms of hemolytic anemia. This case underscores the necessity for genetic counseling for affected individuals and their families, as well as the importance of continued follow-up and supportive care in managing hemolytic anemia related to enzyme deficiencies.

Pyrimidine 5'-nucleotidase deficiency is a rare erythrocyte enzymopathy. Here we report two cases of hemolytic anemia in brothers of Polish origin that are associated with a very rare mutation. Heterozygous deletion in the NT5C3A gene (c.444_446delGTT), inherited most likely from their asymptomatic mother, resulted in a single amino acid residue deletion (p.F149del) in cytosolic pyrimidine 5'-nucleotidase. However, only the mutated transcript was present in the reticulocyte transcriptome of both patients. Only residual activity of pyrimidine 5'-nucleotidase in the brothers' erythrocytes could be observed when compared with the controls, including their asymptomatic father and sister. Western blot showed no sign of the presence of 5'-nucleotidase protein in the erythrocytes of both studied patients. The 2.5-fold reduction of the purine/pyrimidine ratio observed only in the brothers' erythrocytes confirms the correlation of the results of molecular analysis, including whole-exome sequencing, with the phenotype of the pyrimidine 5'-nucleotidase deficiency. Altogether, our results may substantiate the hypothesis of the heterogeneity of the molecular basis of the defect involving both the mutation presented here and negative regulation of expression of the "normal" allele.

Congenital hemolytic anemias (CHAs) comprise defects of the erythrocyte membrane proteins and of red blood cell enzymes metabolism, along with alterations of erythropoiesis. These rare and heterogeneous conditions may generate several difficulties from the diagnostic point of view. Membrane defects include hereditary spherocytosis and elliptocytosis, and the group of hereditary stomatocytosis; glucose-6-phosphate dehydrogenase and pyruvate kinase, are the most common enzyme deficiencies. Among ultra-rare forms, it is worth reminding other enzyme defects (glucosephosphate isomerase, phosphofructokinase, adenylate kinase, triosephosphate isomerase, phosphoglycerate kinase, hexokinase, and pyrimidine 5'-nucleotidase), and congenital dyserythropoietic anemias. Family history, clinical findings (anemia, hemolysis, splenomegaly, gallstones, and iron overload), red cells morphology, and biochemical tests are well recognized diagnostic tools. Molecular findings are increasingly used, particularly in recessive and de novo cases, and may be fundamental in unraveling the diagnosis. Notably, several confounders may further challenge the diagnostic workup, including concomitant blood loss, nutrients deficiency, alterations of hemolytic markers due to other causes (alloimmunization, infectious agents, rare metabolic disorders), coexistence of other hemolytic disorders (autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria, etc.). Additional factors to be considered are the possible association with bone marrow, renal or hepatic diseases, other causes of iron overload (hereditary hemochromatosis, hemoglobinopathies, metabolic diseases), and the presence of extra-hematological signs/symptoms. In this review we provide some instructive clinical vignettes that highlight the difficulties and confounders encountered in the diagnosis and clinical management of CHAs.

At least 16 genetically determined conditions qualify as red blood cell enzymopathies. They range in frequency from ultrarare to rare, with the exception of glucose-6-phosphate dehydrogenase deficiency, which is very common. Nearly all these enzymopathies manifest as chronic hemolytic anemias, with an onset often in the neonatal period. The diagnosis can be quite easy, such as when a child presents with dark urine after eating fava beans, or it can be quite difficult, such as when an adult presents with mild anemia and gallstones. In general, 4 steps are recommended: (1) recognizing chronic hemolytic anemia; (2) excluding acquired causes; (3) excluding hemoglobinopathies and membranopathies; (4) pinpointing which red blood cell enzyme is deficient. Step 4 requires 1 or many enzyme assays; alternatively, DNA testing against an appropriate gene panel can combine steps 3 and 4. Most patients with a red blood cell enzymopathy can be managed by good supportive care, including blood transfusion, iron chelation when necessary, and splenectomy in selected cases; however, some patients have serious extraerythrocytic manifestations that are difficult to manage. In the absence of these, red blood cell enzymopathies are in principle amenable to hematopoietic stem cell transplantation and gene therapy/gene editing.