Germline RBBP6 Mutations In Myeloproliferative Neoplasms

The pathogenesis of myeloproliferative neoplasms (MPN) is determined by the acquisition of somatic mutations in several genes, such as JAK2, MPL, and TET2. Familial clustering of MPN is observed in 5-10% of cases following predominantly autosomal dominant inheritance with incomplete penetrance. Familial MPN patients carry somatically acquired mutations in JAK2, MPL and other genes similar to sporadic cases. It is thought that the germline mutations in familial cases of MPN predispose to acquisition of somatic mutations which in turn drive the disease. No gene has been implicated in familial MPN predisposition so far. The JAK2haplotype predisposing to MPN at population level does not explain familial MPN cases.

We studied a family with five MPN cases in four generations displaying an autosomal dominant pattern of inheritance with reduced penetrance. DNA was available from three MPN cases in the family. One of the patients carried a JAK2-V617F mutation and a deletion on chromosome 22q, while an MPL-W515L mutation was detected in another affected member of the family. The third patient did not have JAK2 or MPLmutations, but carried three chromosomal aberrations on chromosomes 2p, 7q and 15q. Chromosomal aberrations were detected using Affymetrix SNP 6.0 microarrays. The genotypic data from SNP 6.0 arrays was used to perform high-resolution nonparametric linkage analysis. In order to identify the germline mutation predisposing to MPN in the family we performed whole exome sequencing of the three affected members using Illumina next-generation sequencing technology. The mutations identified from exome sequencing were combined with the linkage analysis data.

We identified a germline mutation in the RBBP6 gene that segregates with the MPN phenotype in the studied pedigree. Further screening in familial and sporadic MPN cases identified several other germline RBBP6mutations in 5% of the familial and 0.6% of the sporadic MPN cases. Familial MPN cases mostly had the diagnosis of primary myelofibrosis. All the detected mutations cluster in putative p53-binding region of the protein, suggesting a possible link of the mutations and p53 function.

The functional effect of the mutations was assessed in a doxycycline-inducible HEK-flp-in cell line system. Upon doxycyline induction, the wild-type and mutant RBBP6 constructs were over-expressed and the consequent functional effect of mutation was assessed using RNA sequencing and global proteomic analyses. The combination of the two datasets yielded 109 genes/proteins that were significantly changed in both the RNA sequencing and the global proteomic results when comparing wild-type to mutant RBBP6, 87 of which were downregulated by the mutation, while 22 were upregulated. Several important cell cycle and apoptosis regulators were found among the deregulated genes, including TP53, BAX, BID and USP7 (downregulated) and CDK6 (upregulated). KEGG pathway analysis of the downregulated genes showed significant enrichment for the p53 and the apoptosis pathways.

We have identified germline mutations in RBBP6 that predispose to the development of MPN and are enriched in the families. Functional studies have shown that the mutations likely affect the p53 pathway and apoptosis and thus increase the risk of acquiring somatic mutations that lead to MPN. Our study implicates RBBP6 in MPN susceptibility and suggests that RBBP6 is an important cancer-associated gene.