ATM Gene Point Mutation and Frameshift Mutation Are Found In High-Risk, Untreated Chronic Lymphocytic Leukemia Patients with Interstitial Deletion of Chromosome 11q and Uniparental Disomy of Chromosome 11q

Interstitial deletion of chromosome 11q (11q-) detected by fluorescence in situ hybridization (FISH) is an unfavorable prognostic marker in chronic lymphocytic leukemia (CLL). CLL patients with 11q- often have younger age of onset, bulky lymphadenopathy, and poorer overall survival. The ataxia-telangiectasia mutated (ATM) gene, which encodes a protein kinase central to DNA damage response, is thought to be the critical gene in CLL 11q- through loss of ATM function. A subgroup of CLL patients with 11q- has been shown by other investigators to have a mutation in the remaining ATM allele, supporting a loss-of-function hypothesis. In an ongoing study, we are using SNP-based whole genome copy number variation (CNV) analysis to characterize in detail the 11q deletions in CLL patients. In addition, we have reported uniparental disomy (UPD) of 11q in CLL patients negative for FISH abnormalities. Here we report direct DNA sequence analysis of the ATM gene in CLL patients with 11q- and with UPD of 11q.

We performed CNV analysis and ATM sequencing on 10 patients aged 29–67 with early-intermediate stage, untreated CLL who had high risk for disease progression based on molecular and immunophenotypic markers. Eight patients had 11q- and 2 patients had UPD of 11q. CLL cells and normal cells were separated from patient peripheral blood samples by immunomagnetic beads. CNV analysis was performed on purified genomic DNA from CLL and normal cells for each patient to distinguish acquired copy number changes in malignant cells from polymorphic CNVs in the human genome. We used the Illumina 660w-quad beadchip, a SNP-based microarray for CNV analysis. CNV data was analyzed by CNV partition and PennCNV software. DNA sequence analysis of the ATM gene was performed by the dideoxy chain termination method using an ABI 3730 DNA Analyzer. All coding regions of the ATM gene (exons 4–65) and associated splice site junctions were amplified by PCR using purified genomic DNA from CLL cells. DNA sequence of the PCR products was determined on both strands. Sequence data was analyzed by Sequencher software.

In the 8 CLL patients with 11q−, CNV analysis demonstrates hemizygous interstitial deletions that vary from 980 Kb to 43.2 Mb. All deletions include the region of the ATM gene at 11q22.3. Two out of these 8 patients have a mutation in the residual ATM allele. One patient (CLL010) has a point mutation in exon 61 of ATM (c.8600G>A (gly2867glu)) that likely involves the substrate-binding region of the PI3-kinase domain. One patient (CLL029) has a frameshift mutation in exon 12 of ATM (c.1401_1405delCAAGA; asp467fs) that causes a premature stop signal at codon 484, presumably resulting in a truncated protein. In the 2 CLL patients with UPD of 11q, CNV analysis demonstrates copy-neutral loss of heterozygosity that extends from either 11q12.1 (CLL008) or 11q13.1 (CLL009) to the terminus of 11q. Both patients with UPD of 11q display homozygous mutation of the ATM gene. One patient (CLL008) has a point mutation in exon 58 of ATM (c.8161G>A (asp2721asn)) that likely involves the ATP binding site in the catalytic domain. One patient (CLL009) has a frameshift mutation in exon 24 of ATM (c.3238_3247del10bp/ins11bp; asp1080fs) that creates a premature stop signal at codon 1095, presumably resulting in a truncated protein.

We report a new finding that ATM gene mutation occurs in CLL patients with UPD of 11q. Our study further expands observations reported by others concerning the role of ATM mutations in CLL. ATM mutations that we characterized include point mutations that may interfere with kinase activity and frameshift mutations that may result in a truncated ATM protein. Our sequence analysis supports a loss-of-function mechanism for ATM in a subgroup of high-risk, untreated CLL patients. The absence of detectable ATM mutations in the majority of CLL patients with 11q- may imply the existence of hidden ATM mutations not found by our targeted exon sequencing approach or epigenetic mechanisms that result in loss of gene expression. The finding that untreated CLL patients with UPD of 11q have homozygous ATM mutation supports a clonal selection hypothesis for identical alleles of mutated ATM in the absence of mutagenic chemotherapy. Selection of a CLL subclone with mutated ATM may confer a proliferative advantage through increased cell division and/or resistance to apoptosis following DNA damage.

Zent: Genzyme: Research Funding; Genentech: Research Funding; Novartis: Research Funding; G.S.K.: Research Funding.