Chromosome anomalies in bone marrow as primary cause of aplastic or hypoplastic conditions and peripheral cytopenia: disorders due to secondary impairment of RUNX1 and MPL genes

Abstract

Background: Chromosome changes in the bone marrow (BM) of patients with persistent cytopenia are often considered diagnostic for a myelodysplastic syndrome (MDS). Comprehensive cytogenetic evaluations may give evidence of the real pathogenetic role of these changes in cases with cytopenia without morphological signs of MDS.

Results: Chromosome anomalies were found in the BM of three patients, without any morphological evidence of MDS: 1) an acquired complex rearrangement of chromosome 21 in a boy with severe aplastic anaemia (SAA); the rearrangement caused the loss of exons 2-8 of the RUNX1 gene with subsequent hypoexpression. 2) a constitutional complex rearrangement of chromosome 21 in a girl with congenital thrombocytopenia; the rearrangement led to RUNX1 disruption and hypoexpression. 3) an acquired paracentric inversion of chromosome 1, in which two regions at the breakpoints were shown to be lost, in a boy with aplastic anaemia; the MPL gene, localized in chromosome 1 short arms was not mutated neither disrupted, but its expression was severely reduced: we postulate that the aplastic anaemia was due to position effects acting both in cis and in trans, and causing Congenital Amegakaryocytic Thrombocytopenia (CAMT).

Conclusions: A clonal anomaly in BM does not imply per se a diagnosis of MDS: a subgroup of BM hypoplastic disorders is directly due to chromosome structural anomalies with effects on specific genes, as was the case of RUNX1 and MPL in the patients here reported with diagnosis of SAA, thrombocytopenia, and CAMT. The anomaly may be either acquired or constitutional, and it may act by deletion/disruption of the gene, or by position effects. Full cytogenetic investigations, including a-CGH, should always be part of the diagnostic evaluation of patients with BM aplasia/hypoplasia and peripheral cytopenias.

Figure 1

Ideograms of the normal chromosome 21, and of the rearranged 21s. The der(21) of patient 1 (A), and the inv(21) of patient 2 (B), summarizing the most informative FISH results. The following symbols represent the probes related to RUNX1 sequences used, and the corresponding exons are indicated in the figure, with signals present, not present or weak: # WI2-1915K14, ◍ CTD-2349F18, ★ WI2-942D2, ♦ WI2-605D9, ■ CTD-2235K24, □ WI2-847D7, ❂ WI2-542L2, ⬇ weak signal, NP signal not present. The other BAC probes used and mentioned in the text, but not related to RUNX1 sequences, are shown in the figure with other symbols

Figure 2

a-CGH profiles of the regions of imbalance of patients 1 and 2.A-B: Profiles of chromosomes 13 and 21 of patient 1 on DNA from BM sampled in June 2007; C: Profile of chromosome 21 of patient 2 on DNA from PB. The profiles shown were obtained with the 244 K genome-wide system

Figure 3

Relative expression of RUNX1 in the BM of patient 1. Results two years (A), and four years (B) after disease onset (2006 and 2008, respectively). The dark grey bars refer to the patient and the light grey bars to controls’ average values. Housekeeping control genes were ACTB in A, and UBC in B

Figure 4

Relative expression of RUNX1 in the BM of patient 2. The dark grey bar refer to the patient and the light grey bar to controls’ average values, UBC was used as control

Figure 5

Mitosis cut-outs with FISH results of patient 3.A: the arrow indicates the signal of probe RP11-125P23 (red), at the normal localization, but smaller than the one on the normal 1; the red signal on the long arms, both on the normal 1 and on the inv(1), is due do cross-hybridization, and the green signal is the one of probe RP11-90B12, flanking the MPL gene, used as internal control and displaced towards the centromere on the inv(1); B: the arrow indicates the signal of probe RP11-46G23 (red), moved towards the telomere on the inv(1); C: the arrow indicates the signal of probe RP11-690E2 (green), moved towards the centromere on the inv(1); D: the arrow indicates the inv(1) lacking the signal of probe RP11-372C15 (green, on the normal 1); the red signal is the one of probe RP11-113C10, flanking the MPL gene, used as internal control and displaced towards the centromere on the inv(1)

Figure 6

Patient 3: FISH for MPL gene and a-CGH results. Probes flanking the MPL gene indicate its localization on the inverted chromosome 1, at the right (A); a-CGH profile of the short arms of chromosome 1 shows the two deleted regions (B)

Figure 7

Patient 3: relative expression of MPL in BM. BM sampled at five different dates (light grey columns 1–5), compared to five control subjects (dark grey column 6: mean value ± standard deviation)