The RUNX1 gene is involved in several different and frequently recurring translocations with various partner genes in leukaemia, and point mutations or deletions of this gene are relevant as well in the pathogenesis of different types of MDS and leukaemia subtypes [6–9]. Germline heterozygous mutations of RUNX1 cause the autosomal dominant disease “familial platelet disorder with propensity to AML” (FPD/AML) (OMIM #601399). Therefore, it is not surprising that RUNX1 haploinsufficiency due to constitutional chromosome deletions may cause a clinical phenotype which includes thrombocytopenia besides intellectual disability and other symptoms . Only one case is reported in the literature with a constitutional RUNX1 deletion leading to non-syndromic thrombocytopenia with MDS . Patients 1 and 2 reported here demonstrate that thrombocytopenia or BM hypoplasia with consequent SAA may in fact be due to structural anomalies of chromosome 21 involving RUNX1 which are not deletions, but complex rearrangements which may also be acquired instead of constitutional.
In detail, the chromosome anomaly in patient 1 was acquired and clonal in BM as shown by chromosome analyses and FISH on interphase nuclei. The fact that the der(21) was already present at the onset of the SAA provides support for its relevance in the disease aetiology. Altogether, our results demonstrate that the der(21) implies a deletion of most of the RUNX1 gene, besides the inverted duplication of a more distal 10.332 Mb region (Figures 1A-2B): the expression of RUNX1 is consequently reduced (Figure 3) and causes defective maturation/proliferation of haematopoietic cells and SAA, what does not exclude the possible action also of other concomitant factors. During the disease course the abnormal clone varied in size within a range from 5% to 50%, as shown from chromosome and FISH analyses, without any specific clinico-haematological variation. RUNX1 expression in BM varied in parallel with the different size of the clone with the der(21) (Figure 3). Since November 2006 to November 2008, this abnormal clone decreased in size (to 9%-5%) at the same time of the appearance of a new independent clone with the int del(13)(q12-13q21) which, on the contrary, increased progressively in size (5%-32%). Worthy of note, similar deletions of chromosome 13 are recurrent in myeloproliferative disorders, in particular in polycythaemia vera and primary myelofibrosis, but also in MDS and AML [12, 13]: no morphological signs of overt MDS, however, was ever noticed in our patient 1.
In patient 2 the complex rearrangement of chromosome 21 led to three regions of imbalance, two duplications and one deletion, besides the benign CNV inherited from the father. The RUNX1 gene was disrupted by the rearrangement, and a tiny segment including exon 5 was duplicated. During the diagnostic procedures, a mutation analysis of RUNX1 had been performed, but failed to reveal any change, obviously because no mutation was present in exon sequences. So, in this patient the disruption of RUNX1 led to decreased expression (Figure 4) and to thrombocytopenia. Being the rearrangement constitutional, we searched accurately for possible clinical findings, other than haematological, which might be related to the imbalances: when she was 8-year-old, height and weight were between the 25th and the 50th centile, there was no developmental delay neither mental retardation, and none of the dysmorphic/malformative signs described in cases of 21q22 overlapping deletions , or any other relevant symptoms, were present.
The definite diagnosis of patient 3 is CAMT. CAMT is an autosomal recessive disorder characterized by absent or reduced number of Mks in the BM since birth, extremely elevated serum levels of TPO, and very low platelet count that in some cases increases transiently during the first year of life . Prognosis of CAMT patients is poor, because all of them develop in childhood a tri-linear marrow aplasia that is always fatal when left untreated. In our patient, pancytopenia, severely reduced BM cellularity, increased TPO levels and reduced platelet expression of TPO receptor, suggested a diagnosis of CAMT. However, homozygous or compound heterozygous mutations of the MPL gene, coding for the TPO receptor, that are responsible for CAMT , were not identified: nevertheless, expression analysis showed a very low level of MPL transcript (Figure 7). Moreover, polymorphism analysis showed that the patient had inherited from the parents the same alleles of his healthy sister.
The locus MPL is on the short arms of chromosome 1, in band 34.2, and the BM clonal structural anomaly just of the short arms of chromosome 1 may hardly be considered as fortuitous. FISH with probes flanking MPL and a-CGH results excluded the disruption of the MPL gene (Figure 6).
We postulate the following pathogenetic mechanism for CAMT in our patient. The paracentric inversions are usually considered balanced anomalies, but it has been shown that a-CGH applied to search for constitutional anomalies is able to detect a number of imbalances undetected with conventional cytogenetic methods, as was the case, e. g., of 10% of 13,926 patients with mental disability and apparently normal karyotype reviewed by Sagoo et al., 2009 . In patients with apparently balanced constitutional structural rearrangements, it has been demonstrated that many cryptic deletions may be found by a-CGH, more frequently at the breakpoint, with consequent phenotypic abnormalities . So, the finding of two cryptic deletions at the breakpoints of the inversion of our patient is not too surprising: it is conceivable that this kind of unexpected imbalances may be found in acquired anomalies as well, and that position effects may derive from these cryptic imbalances. A well-known example of these cryptic rearrangements in an acquired apparently balanced translocation, regards chronic myeloid leukaemia with the Ph chromosome by translocation t(9;22)(q34;q11), in which deletions in regions flanking the translocation breakpoints are frequent, and imply a poor outcome . In constitutional anomalies, firm evidence is available of the deregulation of transcripts due to the dissociation from long-range regulatory elements, phenomenon usually called position effect: structural anomalies were shown to be able to act on specific genes which may be tens of megabases apart from the breakpoints identified [19, 20]. The a-CGH results in patient 3 showed a distance of about 36.5 Mb from the MPL gene to the telomeric breakpoint of the inversion and of about 76.5 Mb to the centromeric one. The evidences from the literature on constitutional rearrangements concern position effects acting even at distance, but always on the chromosome where the breakage has took place, in cis, not on its homologue, but in mammals long-range DNA interactions were demonstrated acting both in cis and in trans, and causing variable gene expression level . So, we postulate that in patient 3 the primary event causing the disease was the acquired clonal paracentric inversion, with the loss of the two regions near the breakpoints, and that some sequences in the lacking segments impaired the function of the MPL gene by position effect, both on the inv(1) and on the normal 1, thus leading to highly reduced expression and to acquired CAMT.