Molecular mechanisms of disease in hereditary red blood cell enzymopathies

Metabolically defective red blood cells are old before their time, and suffer from metabolic progeria. The focus of this thesis was to identify the molecular mechanisms by which inherited enzymopathies of the red blood cell lead to impaired enzyme function and, consequently, shorten red blood cell survival. We studied patients with deficiencies of key enzymes of the Embden-Meyerhof Pathway (hexokinase HK and pyruvate kinase PK) and the Hexose Monophosphate Shunt (glucose-6-phosphate dehydrogenase G6PD). The clinical hallmark of these patients is chronic hemolysis as a result of a depletion of the cellular ATP content (HK or PK deficieny) or increased susceptibility to oxidative damage (G6PD deficiency). The investigated mutations caused disease by means of a variety of mechanisms and affected several aspects of human gene expression. In chapter 2 we described a severely PK-deficient patient. In reticulocyte RNA from this patient we detected a monoallelic pattern of (mutant) PKLR gene expression, associated with three in cis mutations in the erythroid-specific promoter of PKLR. Ultimately, this led to the identification of a novel transcriptional regulatory element in the proximal promoter that was actively involved in DNA-protein interaction. The synthesis of only one species of mutant PK was also demonstrated in another patient with severe PK deficiency. In this case, the production of mutant PK was due to aberrant processing of PKLR pre-mRNA. The concerning patient, described in chapter 3, was compound heterozygous for two mutations which were both likely to affect correct splicing. A novel ex vivo approach was employed to study the effects of both mutations on pre-mRNA processing and multiple aberrant transcripts were characterized. On the protein level, once a mutant enzyme is synthesized it may be unstable because of significant structural perturbation. In chapter 4 we studied 28 PK-deficient patients, classified according to their phenotype. Twenty-four different mutant alleles were identified, including 14 novel mutations. Of the novel mutations, 12 predicted a single amino acid substitution. We used the recently elucidated tetrameric crystal structure of human erythrocyte PK to study the consequences of the amino acid changes on structure and function of PK. The location and interactions of the affected amino acid, as well as the nature of the introduced substitution were important determinants for the resulting molecular perturbation and its impact on protein function. This was further illustrated by the two patients with severe G6PD deficiency, described in chapter 6. Both patients were hemizygous for a different G6PD variant caused by a de novo missense mutation. Both mutations affected the interface of the G6PD dimeric enzyme, a region crucial for G6PD enzymatic activity. The patients displayed, however, quite distinct clinical pictures. In chapter 7 we showed that also subtle alterations in crucial areas of the enzyme can cause a severe impairment of enzyme function. In this case, a patient was diagnosed with severe HK deficiency caused by a conservative missense mutation in the active site of HK. Biochemical analysis of mutant HK from the patient displayed only a relatively modest effect of the amino acid substitution on enzymatic properties in vitro. However, we showed that its reasonable to assume a more significant effect in vivo. The variety of clinical features associated with the various enzymopathies, regardless of the underlying molecular mechanism, have unequivocally made clear that the phenotype of hereditary red blood cell enzymopathies is not solely dependent on the molecular properties of mutant proteins but rather reflects a complex interplay between physiological, environmental and other (genetic) factors