Loss of heterozygosity 4q24 and TET2 mutations associated with myelodysplastic/myeloproliferative neoplasms

Abstract

Chromosomal abnormalities are frequent in myeloid malignancies, but in most cases of myelodysplasia (MDS) and myeloproliferative neoplasms (MPN), underlying pathogenic molecular lesions are unknown. We identified recurrent areas of somatic copy number-neutral loss of heterozygosity (LOH) and deletions of chromosome 4q24 in a large cohort of patients with myeloid malignancies including MDS and related mixed MDS/MPN syndromes using single nucleotide polymorphism arrays. We then investigated genes in the commonly affected area for mutations. When we sequenced TET2, we found homozygous and hemizygous mutations. Heterozygous and compound heterozygous mutations were found in patients with similar clinical phenotypes without LOH4q24. Clinical analysis showed most TET2 mutations were present in patients with MDS/MPN (58%), including CMML (6/17) or sAML (32%) evolved from MDS/MPN and typical MDS (10%), suggesting they may play a ubiquitous role in malignant evolution. TET2 mutations affected conserved domains and the N terminus. TET2 is widely expressed in hematopoietic cells but its function is unknown, and it lacks homology to other known genes. The frequency of mutations in this candidate myeloid regulatory gene suggests an important role in the pathogenesis of poor prognosis MDS/MPN and sAML and may act as a disease gene marker for these often cytogenetically normal disorders.

Figure 1

LOH 4q24 mapping and TET2 sequencing. (A) Topographical map illustrating overlapping regions of UPD4q24 (purple bars) and del4q24 (orange bars) in patients with myeloid malignancies. A minimally affected region on 4q24 (0.35 Mb), containing TET2 and PPA2, was identified in 18 of 398 patients studied (* indicates that some patients' DNA was not available for mutational screening; n = 3). (B) Representative examples of SNP-A karyograms (CNAG version 3.0) of chromosome 4 of both whole bone marrow cells and paired CD3⁺ lymphocytes in 2 patients, demonstrating the somatic nature of acquired LOH4q24 and TET2 mutations, which are shown in corresponding ferrograms in the right portion of the figure. SNP-A karyograms: rows a and b correspond to the copy number as measured by the intensity of individual hybridization signals; row c represents frequency of heterozygous calls; and row d indicates the allelic imbalance with parental allele copy numbers.

Figure 2

Identification of mutations in TET2 gene (4q24). (A) Genomic sequencing of all coding TET2 exons revealed the presence of nonsense, missense, and frameshift mutations. These mutations were homozygous and associated with UPD4q (top left), hemizygous associated with deletion (top right), or heterozygous associated with no chromosomal lesion (bottom right). Unique compound heterozygosity was found in 1 patient (bottom left). Detailed description of the SNP-A karyograms is provided in Figure 1B. (B) Schematic representation of the topographic distribution of the individual mutations in the TET2 protein (Isoform A NM_001127208). Amino acid sequences of TET family members are aligned (TET2, TET1, and TET3). *Highly conserved residues among all 3 proteins (LCXH domains). Most mutations were found in 2 conserved domains, LCXH 1 and 2 (50%, missense changes, frameshifts and codon stop changes are in red and underlined) while nonsense and frameshift mutations were more prominent in the N terminus (62%). Q1084P and Y867H are novel, unannotated SNPs (see “Note added in proof”).

Figure 3

TET2 mRNA expression. (A) TET2 mRNA expression in fractions of healthy bone marrows (n = 5). Error bars represent SD of 3 replicates used for each group. (B) TET2 mRNA expression in CD34⁺ cells derived from controls (n = 6) and patients with TET2 WT and TET2 mutants (n = 16). ● represent TET2 WT patients; ○, TET2-mutated patients; and —, mean for each group.

Figure 4

Frequency of TET2 mutations in studied patients. (A) Frequency of TET2 mutations among various clinical subtypes of myeloid disorders. The frequencies of TET2 mutations are calculated for each disease category including CMML-1/2 (n = 17), MDS/MPN-sAML (n = 14), MDS/MPN (n = 30), and MDS (n = 14). (B) Distribution of various disease subtypes among patients with TET2 mutation (n = 19 or 100% TET2 mutations).

Figure 5

Survival of TET2-mutated and WT patients with myeloid malignancies. Each dot represents 1 patient.