Proteomics reveals reduced expression of transketolase in pyrimidine 5′-nucleotidase deficient patients

Purpose: To date, it remains a challenge to correctly and timely diagnose red blood cell (RBC) enzymopathies that result in hereditary nonspherocytic hemolytic anemia (HNSHA), the third most common of which is pyrimidine 5′-nucleotidase (P5N) deficiency with just over 100 cases recognized and confirmed worldwide. Experimental design: We have investigated the RBC proteome of a patient with P5N deficiency due to a homozygous frameshift mutation in the NT5C3A gene. Protein expression levels were analyzed against healthy controls and against patients with hemolytic anemia of different origin, to account for the patient's elevated reticulocyte versus RBC ratio. Results: Stringent relative quantification of the patient's protein levels revealed reduced levels of P5N, and unexpectedly, also decreased levels of transketolase, an enzyme involved in the nonoxidative phase of the pentose phosphate pathway, one of the few key pathways active in RBCs. Immunoblotting of whole blood samples from this and other P5N-deficient patients with dissimilar mutations indicated that P5N deficiency was correlated with reduced transketolase levels. Conclusions and clinical relevance: Consequently, insight into patient RBC proteomes illustrates potential benefit of coupling quantitative proteomics strategies with routine HNSHA diagnostic procedures. Proteomics facilitates finding novel biomarkers for HNSHA patients, for example, suffering from P5N deficiency, providing new prospects for future diagnosis and therapy.