Rare Germline Variant in NFATC4 Associated with Familial Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) has one of the highest familial risks of any cancer, with up to eightfold increased risk in first degree relatives of CLL patients. Genome-wide association studies (GWAS) have identified multiple common single nucleotide polymorphisms (SNPs) associated with risk, but these have modest effect. Similarly, identified germline variants associated with disease account for just a small minority of CLL patients, making familial CLL an attractive target for research, that could provide direct insight into pathobiology.

We used whole exome sequencing (WES; Illumina) to search for rare germline variants (<1% Minor Allele Frequency) in 36 patients from 17 families with Mendelian-like CLL inheritance, where at least two affected family members were sequenced. Median age at diagnosis was 56.4 (range 41-72), 61% were male, 10/23 (43%) were IGHV -unmutated, and 5/24 (21%) had del17p and/or TP53 mutation. We found 3819 rare variants shared within all affected members of a given family. Of those, 71 non-synonymous variants were shared across at least 2 families, and 39 of these were predicted to be pathogenic by at least two commonly-used phenotype prediction tools. None of these variants are within known somatic CLL driver genes, nor were any found in genes near known CLL GWAS SNPs. In order to prioritize these variants, we assessed whether they were enriched among CLL cases (n = 646) as compared to ethnically matched normal population controls (n=8,920), a dataset we have recently reported (Tiao, Improgo et al, Leukemia). Using Fisher's exact test with Benjamini-Hochberg (BH) correction for multiple tests, we found that 27 of the 71 shared variants across multiple families were also enriched in unrelated CLL patients compared to controls. Among those, a rare variant of nuclear factor of activated T-cells 4 (NFATC4), rs200328372, was the most highly enriched in CLL (OR 20.8, Fisher-q-BH 0.026). This variant affects a splice acceptor site predicted to have a deleterious effect on the protein (FATHMM for non-coding variants). In a pathway analysis (MSigDB, DAVID) of genes carrying shared variants across families, NFATC4 together with SOS1 and PTPN7 were associated with the T-cell receptor, B-cell receptor, and MAP-kinase pathways, suggesting a possible role in CLL pathogenesis.

The NFATC4 protein, also known as NFAT3, is part of a DNA-binding transcriptional complex that translocates to the nucleus after activation through dephosphorylation by calcineurin. This complex is involved in the inducible expression of cytokine genes in T cells, including interleukin (IL)-2 and IL-4. While NFATC1 has been implicated in CLL pathogenesis and was associated with more aggressive disease in a mouse model of CLL, the role of NFATC4 has not been elucidated. To further investigate this as well as its association with familial CLL, we compared gene expression profiling (GEP; Affymetrix U133 plus 2.0 array) data between familial and sporadic CLL patients and normal B-cells. While NFATC4 expression did not differ between normal B-cells (n=16) and CLL (n=143), it was significantly down-regulated in familial (n=47) as compared to sporadic (n=96) CLL cases (Mann-Whitney, p=0.005). In the 4 cases that harbor the shared risk variant, GEP results showed additional significant down-regulation of NFATC4 expression (Kruskal-Wallis, p=0.003 for all comparisons) (Figure 1).

RNA sequencing (Illumina) demonstrated NFATC4 down-regulation in all CLL samples (not specifically in familial CLL as with GEP; n=100) versus normal B-cells (n=6; Mann-Whitney, p=-0.00024), suggesting a putative role for NFATC4 as a tumor suppressor. Since NFATC4 has over 30 alternatively spliced transcripts potentially affected by the rs200328372 variant, we used Cufflinks software to look at isoform-level RNA sequencing data, available on two relatives who carried this variant, compared to CLL cases and controls. Both relatives had reduced expression of all isoforms (Figure 2), suggesting that this mutation may reduce NFATC4 expression altogether. Studies of protein expression are ongoing.

In conclusion, through rare germline variant analysis in familial CLL, we found a potentially deleterious variant of NFATC4 that is enriched in CLL. Subsequently, we used RNA sequencing data to show, for the first time, that NFATC4 expression is decreased in CLL versus normal B-cells, revealing a potential tumor suppressor mechanism in CLL.

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