Germline GAB2 Mutations in Childhood Acute Lymphoblastic Leukemia

Recent studies using next-generation sequencing of selected individuals, such as those with familial leukemia or congenital syndromes, have identified rare and highly penetrant germline mutations that predispose to childhood acute lymphoblastic leukemia (ALL). High hyperdiploidy (HD), the most common cytogenetic subtype of childhood ALL, is enriched in children with RASopathies who develop ALL and, similarly, a high proportion of ALL patients with germline ETV6 or IKZF1 mutations presented with the HD subtype. Here, we aimed to identify novel predisposition genes in a selected group of HD-ALL patients.

Targeted sequencing of 538 cancer-relevant genes was carried out using the UCSF500 Cancer Gene Panel in diagnostic bone marrow (i.e. tumor) DNA from 57 HD-ALL patients from the California Childhood Leukemia Study (CCLS). HD-ALL patients were selected based on absence of somatic KRAS or NRAS hotspot mutations detectable by Sanger sequencing, and absence of somatic copy number deletions from multiplex ligation-dependent probe amplification (MLPA) assays. After filtering out likely somatic mutations (mutant allele fraction <0.44), and restricting to variants with low frequency in unselected individuals (allele frequency <0.01% in the Exome Aggregation Consortium, ExAC) and with predicted functional effects (Combined Annotation Dependent Depletion, CADD score ≥20), we identified 151 putative predisposing mutations. Of 41 mutations of interest selected for validation, 37 (90.2%) were confirmed as germline in origin via Sanger sequencing of remission or newborn bloodspot DNA.

Rare and predicted functional germline mutations in known (NBN, SH2B3, ETV6, CREBBP, MSH6) or suspected (MLL, ABL1, FLT3, MYH9) ALL predisposition genes were identified in nine out of 57 patients (15.8%). Three additional patients harbored germline mutations in the GRB2-associated binding protein 2 (GAB2), a known binding partner of PTPN11-encoded SHP2 and activator of the ERK/MAPK and PI3K/AKT pathways. Two GAB2 mutations, a missense mutation S592F and frameshift mutation P621fs, were predicted to be highly functional (CADD scores = 34 and 36 respectively) and absent in ExAC. Frequency of rare and damaging GAB2 mutations was significantly higher in our patient set (2.6%) than in ExAC (0.28%, P = 2.70 x 10^-6). We replicated this finding in sequencing data from 309 ALL patients in the TARGET (Therapeutically Applicable Research to Generate Effective Treatments) project (0.81% vs. 0.28%, P = 0.015).

Patient GAB2 mutations were cloned into HEK293 cells and, following EGF stimulation, we found that the P621fs mutation reduced SHP2 binding and ERK1/2 phosphorylation but increased AKT phosphorylation. This suggested possible Ras-independent leukemogenic effects, supported by a lack of somatic Ras pathway mutations in the three GAB2 mutant patients. Additional functional analyses and sequencing of larger patient cohorts will be required to elucidate the role of germline GAB2 mutations in childhood ALL.