Pathogenic Relevance of Germ Line TET2 Alterations

Several familial mutations predisposing to the development of leukemia have been identified, mostly in association with childhood or adolescent presentations of myeloid neoplasms or bone marrow failure states. However, many of these genetic syndromes have variable penetrance and latency. Thus, late presentations of familial diseases are possible as with DDX41 mutations associated with adult myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Growing evidence exists to support the notion that germ line (GL) alterations may play a greater than previously known role in otherwise typical spontaneous adult myeloid neoplasms. These alterations may include known predisposition leukemia genes (i.e.,. ETV6, RUNX1, CEBPa, and DDX41), heterozygous alterations known to generate recessive disorders, or new unidentified genes. The search for such predisposition genes is difficult but we hypothesized that genes frequently damaged by somatic mutations may also be affected by GL alterations, a similar principle as seen for CEBPa or TP53. Further, that a collection of rare variants (MAF<1%) within a gene could be associated with risk of MDS according to the allelic burden (AB) theory. To develop proof-of-principle of this theory we focused on the gene TET2. In comparisons of the AB of rare TET2 GL variants of patients vs. ethnically-matched controls (selected according to ethnic indicator SNPs), MDS patients examined by whole exome sequencing (WES, N=368) had more rare coding-sequence variants than controls (p<.001). On average, patients had 3 TET2 rare variants, while controls had only 1.5 variants. This suggests that the burden of TET2 rare variants could increase risks of myeloid neoplasms. This finding is in agreement with several other observations; 1.) TET2 is frequently mutated and extremely polymorphic; 2.) it is often associated with biallelic mutations, of which some may be a combination of GL and somatic lesions; and 3.) GL alterations in TET2 may predispose to acquisition of additional lesions.

We analyzed a cohort of 4159 patients with myeloid neoplasms using WES and targeted deep next generation sequencing (NGS). Extensive scoring algorithms, along with mutational databases were used to detect GL variants. Serial tumor samples were examined to discriminate GL from somatic defects. We identified 2518 TET2 SNPs in 1475 patients. Multiple SNPs were seen in the same patient, 28% of patients had 2 SNPs, 11% had 3 and 6% had 4 or more SNPs. Of those 52% were found in proximity to the catalytic domain, and 10% were homozygous. TET2 SNPs irrespective of their population frequency were found in 21% of MDS, 27% of MDS/MPN, 31% of MPN, and 33% of AML patients.

We focused our analysis on the top 10 canonical SNPs with the highest odds ratio (OR) when comparing frequencies in our population to that of healthy controls derived from the ExAC base of 60,000 individuals. The SNPs with the highest OR was present in 2% of patients (p.Val1718Leu) vs. 0.5% of controls. Combinations of SNPs (biallelic configuration) were then investigated; SNPs p.Leu1721Trp and p.Pro363Leu co-occurred in 8% of patients, and p.Val218Met, p.Leu34Phe and p.His1778Arg co-occurred in 3%. We also queried whether GL TET2 SNPs create a haplotype (i.e., increased the risk for acquisition of somatic TET2 mutations) or are mutually exclusive. Overall, 32% of cases with any TET2 SNP also acquired a TET2 somatic mutation vs the WT form for these (68%). Conversely, among the somatic mutant carriers, for example SNP p.Met1701Ile, SNP p.Tyr864His and SNP p.Gly355Asp had an OR vs. controls of 1.1. In particular, 55% of all TET2 mutants had SNP p.Leu1721Trp in a biallelic fashion vs. 46% of somatic WT cases. This suggests a predisposition haplotype. We also examined the relationship between unrelated somatic mutations and "top OR" TET2 SNPs. No correlation was seen between SNPs and other somatic mutations.

In sum, several GL TET2 variants or their combination may constitute complex, likely low penetrance predisposing factors for myeloid neoplasm that also interact with somatic lesions and lead to cancer decades later.