Next Generation Exome Sequencing for Identification of the Gene Mutations Associated with Loss of Heterozygozity on Chromosome 7 In Myeloid Malignancies

Abstract 297

Loss of heterozygosity (LOH) involving chromosome-7 is one of the most common unbalanced chromosomal defects found in chronic and acute myeloid malignancies. Recent application of single nucleotide polymorphism arrays (SNP-A) led to realization that recurrent areas of LOH may be not only due to deletion of the whole chromosome-7, its long arm but due to copy-neutral LOH, most often involving 7q. We hypothesized that deletions and uniparental disomy (UPD) involving long arm of chromosome-7 may be associated with pathogenic hemizygous or homozygous mutations, respectively. Such mutations may affect tumor suppressor genes and likely contribute to/drive malignant evolution in myeloid disorders. We identified a large number of patients with lesions of chromosome-7 (monosomy-7, del(7q), and UPD(7q)), including 38 patients with microdeletions 7q. After unsuccessful targeted Sanger sequencing of large numbers of genes on chromosome-7 we set up to apply the next generation sequencing (NGS) of the exome libraries generated from patients with LOH7. Exome chromosome-7 libraries were enriched for the content of coding sequences using the SureSelect capture synthetic biotinylated RNA probes, tiling all the coding regions from chromosome-7. For NGS exome sequencing we selected 6 cases of monosomy-7 (2 sAML, 1 MDS, 1 chronic myelomonocytic leukemia (CMML), 1 juvenile myelomonocytic leukemia (JMML), and 1 aplastic anemia (AA)), 2 cases of del(7q) with MDS, 3 cases of UPD(7q) (2 MDS and 1 MDS/MPN) and also studied paired germline samples when possible. We treated monosomy-7 and del(7q) as one class and analyzed them together. Cases with UPD(7q) were analyzed separately. Averaged sequencing depth was 79. Each monosomy-7 sample had 3788, 3792, 5306, 4013, 5237, 4016 potential alterations (average; 4359/sample), and each del(7q) sample had 5431, 4521 observations (average; 4976/sample). After exclusion of observations outside of affected regions del(7q), we eliminated previously reported SNPs and non-coding lesions and selected 658 observations of which 422 were nonsynonymous. For further analysis, the alteration leading to stop codons were chosen as tier 1 candidates (N=16). After elimination of false positives due to the accumulation of reading errors at specific locations were discarded, 3 candidates were left and verified by Sanger sequencing. All of 3 were confirmed as new SNPs. After elimination of false positives, the alteration present in multiple samples were designated as tier 2 group (N=101). All of these sequence changes were shown to be new SNPs; 2 were not confirmed by traditional sequencing. Mutations for which the non-reference base occurred greater than 50% were designated tier 3 candidates (N=73), of which 25 candidates have been already checked by resequencing. To date, we have identified 2 somatic point mutations confirmed by Sanger sequencing, including NRCAM1Q1040K and LMTK2A1147T and each identified in different monosomy-7 samples. Screening of 30 monosomy-7 or del(7q) samples showed that observed mutations were not recurrent. The similar stepwise analytic approach was applied to 3 cases of UPD(7q) (average; 4312 observations per sample). After exclusion of reported SNPs and synonymous alterations, we selected a total of 147 alterations. The number was further reduced to 5 potential pathogenic changes after elimination of false positive NGS artifacts. By verification with Sanger sequencing, recurrent EZH2 homozygous mutations (both were R690H) were confirmed in 2 cases with UPD7q. Then we sequenced whole the exons in additional 12 cases of UPD(7q) were sequenced yielding 2 EZH2 mutations. In addition, EZH2 mutations were identified in 2 cases of microdeletion 7q36.1 and 4 cases without LOH7q occurring in heterozygous constellation. No mutations of EZH2 were found in monosomy-7 or del(7q) sequenced (N=28). In conclusion, to date, using NGS strategy, we have identified 3 new mutations including NRCAM1Q1040K, LMTK2A1147T, and recurrently occurring mutation of EZH2 (EZH2R690H). Several additional candidate mutations are currently screened.