Complex Patterns of Chromosome 11 Aberrations in Myeloid Malignancies Target <i>CBL, MLL, DDB1</i> and <i>LMO2</i>

<div><p>Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of <i>CBL</i>. In one patient the 11qUPD amplified somatic mutations in both <i>CBL</i> and the DNA repair gene <i>DDB1</i>. A duplication within <i>MLL</i> exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with <i>de novo</i> acute myeloid leukemia (P=0.013). One patient with a deletion at the <i>LMO2</i> locus harbored an additional point mutation on the other allele indicating that <i>LMO2</i> might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized.</p> </div

Summary of chromosome 11 aberrations.

<p>Large chromosomal aberrations are indicated with colored bars around the ideogram of chromosome 11. Green – gains; red – deletions; blue – uniparental disomies. The position of the bars relative to the chromosome ideogram indicates the position and size of the aberration. For the two patients of whom two samples were analyzed (UPN 23 and UPN 42 – see Table S1) recurrent aberrations are depicted only once. The positions of <i>CBL, MLL, EED, SF1</i>, <i>DDB1</i> and <i>LMO2</i> are indicated by vertical lines. Mutations in these genes are depicted by orange circles along these lines. Common deleted regions are indicated at the bottom of Figure 2 listing the genes they cover. The ideogram depicts G-banding pattern at ~850-band resolution level.</p

Mutational patterns in <i>CBL</i>.

<p><b>A</b>: Sample 45 had a 6 bp tandem duplication in CBL leading to the insertion of the amino acids valine (V) and aspartic acid (D) after position 390. <b>B</b>: One sample identified in a cohort screen for mutations in <i>CBL</i> exons 8 and 9 carried two mutations, one in exon 8 (W408C) and a second one in intron 8 at the splice acceptor site (G to A). PCR subcloning and analysis of colony DNA revealed that the two mutations are on different alleles. Depicted are two representative colonies. Colony 43 has the mutation in exon 8 but not in intron 8 whereas colony 17 shows the opposite case. <b>A</b>, <b>B</b>: Depicted are the genomic (letters) as well as the respective amino acid (box chains) sequences. Numbers indicate amino acid positions in the Cbl protein. Amino acids, which are substituted due to mutations are in red boxes. The splice site alteration is a red circle. Black arrows indicate the positions of the mutations below the Sanger sequencing traces. <b>C</b>: Overview of <i>CBL</i> mutagenesis in MPN. Different genetic mechanisms are involved in increasing mutant gene dosage of <i>CBL</i>. Each panel shows schematically the two parental copies of chromosomes 11 (blue and yellow) and the <i>CBL</i> gene (white rectangles). Mutations are indicated with asterisks. From left to right: heterozygous mutation in <i>CBL</i>; uniparental disomy introduces homozygous <i>CBL</i> mutations; gain of a part of chromosome 11q leads to a duplication of the <i>CBL</i> mutation while one wild type allele is still present; compound heterozygosity established by two different mutations on the different alleles of the <i>CBL</i> gene in one cell. In addition, the loss of a part of chromosome 11q deleting one <i>CBL</i> allele and leaving the other allele unaffected (wild type <i>CBL</i>) is likely to introduce phenotypes due to haploinsufficiency.</p

Mutations detected in <i>DDB1, MLL</i> and <i>LMO2</i>.

<p><b>A</b>: Sample 36 harbored an 11q UPD as indicated by the blue bar below the chromosome 11 ideogram. We found two somatic mutations in <i>DDB1</i> and <i>CBL</i>. As can be seen in the Sanger sequencing traces, both mutations are homozygous due to amplification by the UPD. <b>B</b>: In sample 50 a tandem duplication in <i>MLL</i> exon 3 was detected. The top graph shows whole exome coverage data across <i>MLL</i> exon 3. The data is plotted as the log₂ ratio of the normalized exome sequencing coverage in the patient sample divided by the median normalized coverage of 8 independent control samples at each genomic position (X-axis). The position of the duplication is indicated by the red bar. Sanger sequencing confirmed an in-frame tandem duplication of 171 amino acids as shown at the bottom. <b>C</b>: A common deleted region on chromosome 11p targets <i>LMO2</i>. All deletions in the analyzed cohort that span the <i>LMO2</i> locus are depicted next to the chromosome 11 ideogram. Red bars indicate deletions, green bars indicate gains. In sample 42, which harbored a deletion spanning the LMO2 locus, we also detected a point mutation in <i>LMO2</i>. The middle section shows a signal intensity plot measuring copy number from Affymetrix microarrays. The plot depicts signal intensity (log₂ scale) differences between the patient and a healthy control pool for each probe (as implemented in the Affymetrix Genotyping Console software). The deletion in sample 42 can be seen as the deviation from 0 for all probes in the deleted genomic region (X-axis). The point mutation in <i>LMO2</i> as identified by Sanger sequencing is depicted at the bottom of panel C. <b>A</b>, <b>B</b> and <b>C</b>: Depicted are the genomic (letters) as well as the respective amino acid (box chains) sequences. Numbers above the boxes indicate amino acid positions in the proteins. Amino acids substituted in the patient samples are indicated by red boxes. The red circle indicates a splice site mutation. Reference and mutant sequences are shown. The arrows indicate the site of mutations below the Sanger sequencing traces.</p

Cohort distribution.

<p><b>A</b>: Distribution of the 813 samples analyzed by Affymetrix microarrays according to diagnosis. <b>B</b>: Fraction of samples that harbor chromosome 11 aberrations (black bars) for each disease entity in percent. The P-value indicates an association of chromosome 11 aberrations with disease progression in MPN. MPN, myeloproliferative neoplasm; CML, chronic myeloid leukemia; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome.</p