Three types of aCGH profiles in CMML
Using genome-wide, high-density arrays we established the aCGH profiles of 30 samples from 29 patients, comprising 24 CMMLs and 6 AT-CMMLs. Examples of profiles are shown in Figure and results are summarized in Table . Three main types of profiles were observed. Type 1 profiles showed gains or losses visible on the karyotype and affecting large regions of the genome, such as trisomy 8 (10%: cases 5, 12 and 88), deletions of part of the 20q arm (10%: cases 3, 74, and 96), or deletion (case 106) or complex rearrangements of chromosome 7 (case 3). Type 2 profiles showed rare and limited gains or losses that affected few or single genes such as deletions encompassing NF1 at 17q11 (case 80), RB1 at 13q14 (case 74), RUNX1 at 21q21 (case 88), CALN1 at 7q11 (case 12), amplification of 7q21 including the CDK6 gene (case 3) or a series of short deletions on the 3q arm (case 1). A surprising deletion of the MYC locus was observed in case 106. The type 3 profile was said "normal-like" since no obvious alteration was detected. It occurred in two-thirds of the cases.
Figure 1 Examples of aCGH profiles. A: aCGH profile of chromosome 17 in case 80. Red arrow shows deletion including NF1. B: aCGH profile of chromosome 13 in case 74 showing RB1 deletion. For a and b, a zoom of the region is shown to the right of the profiles. (more ...)
Molecular features of the 30 studied CMML
Mutations of RAS and RUNX1 genes
We analyzed the sequences of the three RAS
genes. No mutation of HRAS
was found. NRAS
mutations were found in cases 12 and 78, and KRAS
mutations in cases 79 and 89 (Table ). One of these mutations affected codon 146 in coding exon 3, a rare type of RAS
mutation that has been found in 4% colorectal cancers and two hematopoietic cell lines [13
]. For patient 79 we determined that the mutation was present in a heterozygous state in the CD34-purified fraction of the BM cells, in the polynuclear neutrophils, monocytes and B lymphocytes but absent in the T cells (data not shown).
We examined the sequence of exons 3 and 13 of the PTPN11
gene. Mutations were found in three cases. No mutation was found in exon 7 of RAF1
, which is a hotspot for mutations in Noonan syndrome (NS) [14
were also sequenced in their most frequently mutated regions (exons 7–11 and kinase-encoding exons, respectively). One mutation was identified in SOS1
in a region involved in NS [16
], none in BRAF
. No mutation was found in SPRED1
The NF1 gene was analyzed for mutations in cases 79 and 80. A silent, so far unreported point mutation (c.2178G>C) was found in case 79 (Table ). The deletion of an RB1 allele was confirmed by sequencing in case 74 and the remaining RB1 allele was normal. There was no JAK2 p.Val617Phe mutant in our panel of CMML cases.
Mutations were found in the RUNX1 gene in 10 patients (30%). Mutation in case 90 is predicted to induce neither amino acid change nor splicing effect and thus was not considered as functionally deleterious. The nine other nucleotide variations would result in truncated or mutant proteins. RUNX1 mutations are described in Figures and .
Figure 2 Mutation of RAS, PTPN11 and RUNX1 genes in CMML. Examples of mutations in candidate genes. From top to bottom, sequence of the mutated KRAS, PTPN11, SOS1 and RUNX1 alleles, demonstrating base change in the forward sequence at the position indicated by (more ...)
Figure 3 Characterization of RUNX1 mutations in CMML patients. A: Genomic organization of RUNX1 gene at 21q22.12 and RUNX1 protein. Functional (i.e. RUNT and RUNXI [for RUNX Inhibitor domain], as defined by PFAM accession numbers PF00853 and PF08504, respectively) (more ...)
Finally, no mutation was found in the STK11/LKB1 and SYK kinase genes.
A novel, cryptic rearrangement of RUNX1 following inv(21q)
The aCGH profile of case 88 showed two losses at 21q21.3 and q22.12 of about 1.04 Mb and 0.82 Mb, respectively (Figure ). They spanned the 3' part of USP16
, including exons 2 to 19, CCT8
as well as the 5' part of RUNX1
(including exons 1 to 4), respectively. We hypothesized that such a peculiar pattern could be due to a cryptic inv(21)(q21q22) associated with a microdeletion at one of the breakpoints. Given the features and orientation of the various potentially-involved genes, we surmise that a fusion could involve RUNX1
(encoding a de-ubiquitinating enzyme). This was confirmed by nested PCR amplification of reverse-transcribed RNA from the patient's BM cells, which detected a 245 bp-long USP16-RUNX1
transcript (Figure ). No reciprocal transcript was detected. Sequence analysis showed that the result of the inversion/fusion generated a chimeric USP16-RUNX1
transcript. The break/fusion was not present in the germline since we did not find the USP16-RUNX1
transcript in buccal smear cells of the patient. The USP16-RUNX1
gene fuses exon 1 of USP16
to exon 5 of RUNX1
thus not preserving the canonical ATG codons. The chimeric transcript exhibited several stop codons in its 5' part but the presence of multiple ATG codons through exons 5 to 7 of RUNX1
sequence could be used as new start codons and generate putative truncated RUNX1 proteins. A similar USP16-RUNX1
fusion (without microdeletion) was found in CMML 34 (Table ). In the two cases, the USP16-RUNX1
fusion transcripts did not have an open reading frame using the canonical start codons of USP16
(Figure ). According to the SMART program http://smart.embl-heidelberg.de/
, functional domains (i.e. RUNT and RUNXI [for RUNX Inhibitor domain], as defined by PFAM accession numbers PF00853 and PF08504, respectively) should disappear in such putative truncated RUNX1 proteins. RUNT and RUNXI domains are encoded mainly by exons 3 to 5 and exon 8, respectively (Figure ). The partial conservation of RUNX1
transcript sequence (exons 5 to 8) and a new folding could explain conformational changes and the absence of RUNT and RUNXI domains.
Figure 4 Genomic rearrangements involve USP16 and RUNX1 genes in CMML patients. A: CMML 88 aCGH profile of chromosome 21 shows regional deletions in 21q21.3 and 21q22.12. Arrows point to USP16 and RUNX1 genes targeted by transition profiles located in these respective (more ...)
Figure 5 USP16-RUNX1 rearrangement in CMML 88. Organization of chromosomal region 21q21.3-q22.12 with the location of the breakpoints (BP) and deleted regions from centromere (cen) to telomere (tel). Mb scale of the corresponding 21q21.3, 21q22.11 and 21q22.12 (more ...)
In total, RUNX1 was altered by mutation (9) or break (2) in 11 patients (8 CMMLs and 3 AT-CMMLs) (Table ).
Unsurprisingly, the 11q inversion in case 52 and the balanced t(1;3)(p36;q21) in case 90 escaped aCGH detection. The 11q inversion was probably a case of NUP98-DDX10
] and the t(1;3) a case of PRDM16/MEL1-RPN1
Different alterations in MP- and MD-CMML
Excluding the 6 AT-CMMLs, RAS and PTPN11 mutations were found in 6 of the 13 MP-CMMLs (~46%) whereas no such mutation was found in the 11 MD-CMMLs (Table ). In contrast, RUNX1 mutations occurred in both MP- (5 cases) or MD-CMML (3 cases).