Submicroscopic deletion of 5q involving tumor suppressor genes (CTNNA1, HSPA9) and copy neutral loss of heterozygosity associated with TET2 and EZH2 mutations in a case of MDS with normal chromosome and FISH results

Abstract

Advances in genome-wide molecular cytogenetics allow identification of novel submicroscopic DNA copy number alterations (aCNAs) and copy-neutral loss of heterozygosity (cnLOH) resulting in homozygosity for known gene mutations in myeloid neoplasms. We describe the use of an oligo-SNP array for genomic profiling of aCNA and cnLOH, together with sequence analysis of recurrently mutated genes, in a patient with myelodysplastic syndrome (MDS) presenting with normal karyotype and FISH results. Oligo-SNP array analysis revealed a hemizygous deletion of 896 kb at chromosome 5q31.2, representing the smallest 5q deletion reported to date. The deletion involved multiple genes, including two tumor suppressor candidate genes (CTNNA1 and HSPA9) that are associated with MDS/AML. The SNP-array study also detected 3 segments of somatic cnLOH: one involved the entire long arm of chromosome 4; the second involved the distal half of the long arm of chromosome 7, and the third encompassed the entire chromosome 22 (UPD 22). Sequence analysis revealed mutations in TET2 (4q), EZH2 (7q), ASXL1 (20q11.21), and RUNX1 (21q22.3). Coincidently, TET2 and EZH2 were located at segments of cnLOH resulting in their homozygosity. Loss of heterozygosity affecting these two chromosomes and mutations in TET2 and EZH2 are indicative of a myelodysplastic syndrome with a poor prognosis. Deletion of the tumor suppressor genes CTNNA1 and HSPA9 is also likely to contribute to a poor prognosis. Furthermore, the original cnLOHs in multiple chromosomes and additional cnLOH 14q in the follow-up study suggest genetic evolution of the disease and poor prognosis. This study attests to the fact that some patients with a myelodysplastic syndrome who exhibit a normal karyotype may have underlying genetic abnormalities detectable by chromosomal microarray and/or targeted mutation analyses.

Keywords: ASXL1; CTNNA1; Copy neutral loss of heterozygosity (cnLOH); EZH2; HSPA9; MDS; RUNX1; TET2; Uniparental disomy (UPD).

Figure 1

Morphologic and immunophenotypic findings. A) At presentation, there was granulocytic predominance with progressive maturation and no significant increase in blasts or monocytes. The core biopsy shows hypercellular bone marrow with left-shifted granulocytic predominance. Flow cytometry reveals granulocytic predominance (in green) but no significant increase in monocytes (in blue) or myeloblasts (in red). B) At follow-up, the morphologic and immunophenotypic findings are similar to those at presentation.

Figure 2

Routine cytogenetic and fluorescence insitu hybridization (FISH) results. A) G-banded chromosome analysis shows a normal female karyotype. B-D) FISH analyses show normal hybridization with an MDS panel using probes for EGR1 (5q31, red signal) and its control probe D5S23 (5p15.2, green signal) in B; D7S486 (7q31, red signal) and its control probe D7Z1 (7centromere, green signal) in C; CEP8 (green signal) for chromosome 8 centromere and D20S108 (20q12, red signal) in D.

Figure 3

5q31.2 deletion. A) Chromosome 5 with deletion at q31.2. B) SNP-array results, including the weighted log2 ratio, copy number state, and allele peaks at the deleted region. C) Database of genomic variants showing an 896-kb deletion in the short arm of chromosome 5 within band q31.2 (position 137,821,899 to 138,718,504), including the CTNNA1 and HSPA9 genes.

Figure 4

FISH inverted DAPI image showing deletion of the CTNNA1 gene using the BlueGnome FISH probe RP11-114B12 (5q31.2, red signal). The deleted chromosome 5 is indicated by an arrow.

Figure 5

SNP-array results at diagnosis showing loss of heterozygosity (LOH) at the long arm of chromosomes 4, 7, and 22 as purple bars next to the corresponding chromosomes.

Figure 6

SNP-array results at the follow-up study showing loss of heterozygosity (LOH) of chromosome 14, in addition to the LOH chromosomes 4, 7, and 22 found at initial diagnosis. LOH is shown as purple bar next to the corresponding chromosome.