Congenital amegakaryocytic thrombocytopenia (CAMT) is an uncommon disorder, characterized by an isolated thrombocytopenia and the almost complete absence of megakaryocytes in the bone marrow. Several studies have indicated that the origin of CAMT is an intrinsic stem cell defect.1-3 Recently, we and others have demonstrated the presence of mutations in the thrombopoietin-receptor gene, c-mpl, as a possible cause of CAMT.4-7Although some mutations directly predict loss of Mpl function, it has not been established that others, notably those that lead to an amino acid substitution, also directly predict this loss.
To exclude that the mutations we found in our patients represent non–disease-related polymorphisms, we screened 50 healthy donors (100 alleles) for the presence of the different mutations by either sequence analysis or allele-specific restriction analysis.4 None of the healthy donors were carriers of our reported CAMT-associated mutations. In one new CAMT patient, 3 heterozygous mutations were observed: a G-to-C substitution at nucleotide 305 in exon 3, predicting an arginine-to-proline substitution at codon 102; a G-to-A transition at position 340, also in exon 3, leading to valine-to-methionine replacement at codon 114 (Mpl-114V/M); and a G-to-C substitution in the fifth nucleotide of intron 3, which leads to loss of the splice site 3′ of exon 3. Screening of 50 healthy donors revealed that 4 were heterozygous carriers of the G340A mutation. The other mutations were not observed in this population. The c-mpl-340A gene thus seems to have a frequency of 0.04 in a white Dutch population. Functional studies should reveal whether this Mpl-114V/M polymorphism influences the function of Mpl.
Recently, Ballmaier et al7 reportedc-mpl mutations in another series of patients with CAMT. One of their patients was a homozygous carrier for 2 different point mutations. One mutation predicted a stopcodon in exon 3. The second mutation was the G340A mutation, which we also found in healthy donors. Therefore, we propose that the first mutation plays a role in the development of CAMT in this patient. The G340A may not be involved in CAMT, and its presence in 2 CAMT patients may be incidental.
In conclusion, mutations that predict amino-acid substitutions found by genetic screening of patients with CAMT can be due to polymorphisms of the c-mpl gene. The relation of such mutations to disease should be proven by functional studies with the mutated protein.
Screening for c-mpl mutations in patients with congenital amegakaryocytic thrombocytopenia identifies a polymorphism
We agree with the hypothesis of van den Oudenrijn et al that a mutation with a frequency of 0.04 cannot be the cause for the rare disease congenital amegakaryocytic thrombocytopenia (CAMT). However, it remains to be investigated whether the detected polymorphism has an effect on the function of the c-Mpl protein and therefore can influence megakaryopoiesis and platelet formation in homozygous carriers. The presence of this mutation in healthy donors alone is not sufficient to exclude a correlation with diseases of megakaryopoiesis: all parents of patients with CAMT who bear c-mpl mutations in one allele have a normal hematopoiesis and are “healthy” donors.
As we found a homozygous nonsense mutation (C268T) upstream of the mutation G340A, we also were convinced that this C268T mutation was the cause for CAMT in this patient.1-1 Accordingly, we subsequently discussed this patient along with other patients bearing nonsense or frameshift mutations in the c-mpl gene and therefore are predicted to have a complete loss of c-Mpl function. As the patient CAMT-6 in our manuscript is homozygous for both mutations, which were inherited from her unrelated heterozygous parents, most likely the C268T mutation arose on the genetic background of the rare G340A polymorphism. This hypothesis can be tested easily with linkage analyses using polymorphic and informative markers.
Interestingly, the letter of van den Oudenrijn et al points to another fact concerning the prevalence of c-mpl mutations. Another patient is presented here bearing the previously reported (by Oudenrijn et al and us) G305C mutation predicted to lead to an arginine-to-proline substitution at amino acid position 102. This confirms our proposition that the mutation seems to occur more frequently in patients with CAMT, at least in Western Europe. In the combined group of 5 patients described by Oudenrijn et al;1-2 the 5 of our patients, who were from Western Europe; and the newly described patient in the above letter, the G305C mutation was found homo- or heterozygously in 4 different patients, corresponding to a frequency of 0.35 in Western European patients with CAMT. An initial screening for this mutation in newly diagnosed patients with CAMT therefore might be helpful. Thus far, besides the potential G340A polymorphism and the nonsense mutation A43X, which could be detected in 2 of our patients, the G304C mutation is the most frequent mutation described in patients with CAMT.
