Molecular pathogenesis

In view of the above, it is clear that any explanation of the pathogenesis of megaloblastic anemia at the molecular level must, on the one hand, rationalize it as the final common pathway of a variety of underlying lesions and, on the other hand, account for the phenomenon of ineffective erythropoi-esis. Since the inherited causes of megaloblastic anemia are defects in the purine or pyrimidine biosynthetic pathways, and folate is the coenzyme of these pathways, it is natural to focus on this area of metabolism. It has been held for a long time that in megaloblastic anemia a decreased concentration of nucleotide precursors becomes rate-limiting for DNA synthesis, and as a result cell proliferation is curtailed and there fore few cells are produced. However, since cell proliferation is most active, this model can be rejected out of hand: the marrow is hypercellular rather than hypocellular, suggesting that the underlying defect may be qualitative and not merely quantitative.

Since the hematological consequences of the deficiency of either folate or Cbl are indistinguishable, it seems reasonable to surmise that there must be at least one point in common in their action, or that one depends on the other (Figure 12.1). Indeed, Cbl is required for the conversion of methyltetrahy-drofolate (methylTHF), the main form of folate in the serum, to THF, the active form in bone marrow cells. Various derivatives of THF intervene in several steps of the biosynthesis of purine and pyrimidine nitrogen bases, but when folate is in

The molecular basis of anemia 127

Small bowel lumen

Enterocytes

Plasma

Pte Glun

Pte Glun

Pte Glu1

CH3H4Pte Glu t

Pte Glu

•CH3H4Pte Glu1

^^S-adenosyltransferase

Homocysteine Methionine i Methyl-

Cbl1

Folate transporter folate transporter

Jejunal enterocyte

I Cbl3+

Cbl3+

Cbl3+ +TCII

* I Cbl receptor

Ileal enterocyte

5, 10 Methylene tetrahydrofolate reductase

5, 10 Methylene tetrahydrofolate reductase

5, 10-CH2-H4Pte Glun

Thymidylate synthetase

Normal DNA synthesis

Dihydrofolate reductase

C 3+ I Cbl reductase

Mitochondrion

Methionine \ \

synthase H4Pte Glu1

Folate polyglutamate synthetase

CUAAGUCGU GAUUCAGCC

Serine

Hydroxymethyl transferase

DNA synthesis in folate and Cbl deficiency

Dihydrofolate reductase dUMP

C 3+ I Cbl reductase

5, 10-CH2-H4Pte Glun

Thymidylate synthetase

Normal DNA synthesis

Mitochondrion

CoA MMCoAmutase

ATP-Cb transferase

SuccinylCoA

CTAAGTCGT GATTCAGCC

Hematopoietic cell

Fig. 12.1 The metabolic basis of megaloblastosis in folate and vitamin B12 (cobalamin, Cbl) deficiency

The absorption of folates takes place in the proximal small bowel while Cbl bound to intrinsic factor (IF) is absorbed in the ileum. Folate enters the cells in the form of methyltetrahydrofolate (methylTHF). Cbl is transferred to and enters the cells bound to transcobalamin II (TCII). In the cytoplasm, Cbl is necessary for the reaction catalysed by methionine synthase, whereby the CH3 group of methylTHF is transferred to homocysteine; as a result, THF and methionine respectively are produced. The polyglutamated form of THF is converted to 5,10-methyleneTHF, which donates the single carbon group CH2- to the reaction catalysed by thymidylate synthetase, whereby dUMP is converted to dTMP, which is used in DNA synthesis. Under conditions of folate and/or Cbl deficiency, there is a shortage of 5,10-methyleneTHF. The result of this is, on the one hand, that dTMP is drastically reduced and not available for DNA synthesis and, on the other hand, that dUMP is in excess. Strong evidence exists suggesting that, under such circumstances, dUMP is misincorporated in the DNA, leading eventually to changes characteristic of megaloblastosis (see text). Note also that under Cbl-replete conditions the conversion of 5,10-methyleneTHF to methylTHF is inhibited by S-adenosylmethionine (SAM); by contrast, in Cbl deficiency SAM is in short supply and consequently this inhibition is relaxed, diverting the formation of 5,10-methyleneTHF to methylTHF, thus exacerbating the shortage of dTMP and the accumulation of dUMP. Cbl also plays a significant role in the mitochondrial metabolic pathways necessary for the conversion of the products of propionate metabolism (i.e. methylmalonyl-CoA) into easily metabolized products. As is evident from the metabolic inter-relationships of folate and Cbl, in folate deficiency homocysteine levels will increase; in Cbl deficiency, not only homocysteine but also methylmalonyl-CoA (and methylmalonic acid) will be increased: indeed, measurement of the serum and urine levels of homocysteine and methylmalonic acid is used in clinical practice for the diagnosis of folate and Cbl deficiency, especially at early stages. Explanatory notes

In the above figure, the various forms of Cbl are shown in red. PteGlu1/n, mono- or polyglutamated forms of folate; CH3H4PteGlu, methylTHF; 5,10-CH2H4PteGlun, 5,10-methyleneTHF; H2PteGlun, dihydrofolate. Enzymes shown in italics are those the hereditary deficiency of which causes megaloblastic anemia. Asterisks indicate steps in folate and Cbl metabolism whose defects can also cause hereditary megaloblastic anemia.

CH3H4Pte Glun short supply these steps can be bypassed by using preformed bases (the so-called salvage pathway). The one reaction for which folate (in the form of 5,10-methyleneTHF) is irreplaceable is the conversion of dUMP to dTMP, for which it is the methyl group donor; as a result, this conversion will be impaired when either folate or Cbl is deficient. This reaction is crucially important because thymidine is, of course, the one base in which DNA normally differs from RNA. In principle, one might have expected that a block of this reaction would prevent DNA synthesis, but we have seen that this is not the case. On the other hand, it is well established that in vitro DNA polymerase is able to incorporate dUTP into DNA, especially if the dUTP concentration is much higher than that of dTTP, which will be the case when the conversion of the former to the latter is impeded. Several studies indicate that this also takes place in vivo; indeed, the major molecular lesion in megaloblastic anemia may be this misincorporation of dUridine (dU) instead of T into DNA. The cell's DNA replicating machinery includes an enzyme, uracil glucosidase, which has the specific function of removing dU, should it be occasionally and illegitimately incorporated into DNA. Therefore dU will be retained in newly synthesized DNA only when the capacity of uracil glucosidase to remove it has been exceeded. For this reason, very little dU is actually found in megaloblastic DNA, but even that may have a disruptive effect on chromatin structure. Perhaps more importantly, if dU incorporation has been rampant rather than occasional, the numerous strand breaks produced by uracil glucosidase may exceed the capacity of other repair enzymes, thus causing the accumulation of damaged DNA and eventually cell death. Surprisingly, a careful study has not detected in megaloblastic bone marrow features regarded as characteristic of apoptosis. We must therefore presume that cell death takes place by a different pathway or that phagocytosis of dead cells is rapid and highly efficient.

If shortage of dTTP is the fundamental metabolic defect underlying megaloblastic anemia, since its production is 5,10-methyleneTHFA-dependent, the formation of which is in turn Cbl-dependent, it is clear that this same mechanism explains why megaloblastic anemia is the common manifestation not only of nutritional folate and Cbl deficiency but also of all genetically determined lesions of the transport and metabolism of folate or Cbl (Table 12.1). It is not clear why megaloblastic anemia should occur in other inherited conditions, such as HPRT deficiency and orotic aciduria, in which it is the salvage pathway rather than the de novo pathway of the nitrogen bases that is compromised. At the moment we can only speculate by analogy. Perhaps the metabolic blocks in these conditions also entail serious alterations in the absolute and/or relative pool sizes of the various deoxynucleoside triphosphates. Once again, this could cause misincorporation followed by repair attempts that are not always successful. In summary, although the rate of uracil incorporation into DNA has been controversial for years, a recent review validates its role in the pathogenesis of megaloblastic anemia.

Finally, despite the significant advances in the understanding of megaloblastosis, the molecular basis of demyelination that underlies the neurological complications of advanced Cbl deficiency remains elusive.

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