Whole Exome Sequencing (“mutatome”) Of Deletion 5q

Interstitial deletion of chromosome 5q (del(5q)) is one of the most common karyotypic abnormalities in MDS. While a relatively small fraction of patients with del(5q) and 5q- syndrome show a relatively uniform clinical phenotype and a low rate of progression, the majority of del(5q) myeloid neoplasms are more heterogeneous. Prognosis correlates with the size and location of the deletion, with large deletions spanning subtelomeric and/or subcentromeric region correlating with a poor prognosis. Survival differences may relate to still undefined pathogenetic mechanisms underlying del(5q), which may involve hemizygous mutations or haploinsufficiency. With the latter scenario, it is possible that heterozygous mutations of genes located on 5q may phenocopy the deletion. To further elucidate the molecular mechanisms underlying del(5q), we preformed a comprehensive analysis of myeloid neoplasms using single nucleotide polymorphism array (SNP-A) and next generation whole exome sequencing (NGS) of paired DNA samples (germline/tumor) from 55 cases characterized by del(5q) among a total 428 patients with MDS and related disorders; we focused on mutations located on 5q in both diploid and deletion cases.

In the total cohort, we identified 243 somatic mutations in 158 genes on chr5q, including well-known NPM1 or novel recurrent DDX41 mutations; 147 mutations were heterozygous, 11 hemizygous (in del(5q)). No homozygous mutations were found. Applying SNP-A-based karyotyping, we defined the commonly deleted region (CDR) as between 5q32 and 5q33.2 (145299747-153828955). In patients with 5q- syndrome, the proximal and terminal regions of chr5q were always retained; therefore we defined commonly retained regions (CRR) as CRR1 (proximal, 5q11.1 to 5q14.2, 48400001-81634579) and CRR2 (distal, 5q34 to 5q35.3, 164213764-180915260). The deletions of CRRs consequently contributed to worse prognosis in the aggressive types of MDS with longer del(5q).

First we focused our study on the genes located on CRRs. We identified 120 heterozygous alterations in CRRs, including CWC27 (5q12.3), MAP1B (n=2, 5q13.2), NPM (n=50, 5q35.1), C5orf25 (n=4, 5q35.2) and DDX41 (n=4, 5q35.3); these mutations occurred only in a heterozygous configuration. Interestingly, spliceosome-associated gene CWC27 and RNA helicase DDX41 showed haploinsufficient expression in haploid cases without mutation, suggesting that mutated genes located on CRRs can be pathogenic due to both haploinsufficiency of WT genes and heterozygous mutations. Furthermore, patients with decreased expression of these genes had a poor survival (CWC27; HR=2.48, DDX41; HR=1.98).

In positions corresponding to CDR and its proximal regions, we found 123 heterozygous alterations in 97 genes (50% of all alterations on 5q found), including recurrently mutated genes (FAT2: n=4; G3BP1: n=2) and hemizygous mutations of KDM3B (n=3, 5q31.2) and MCC (n=1, 5q22.2). In GPR98, associated with Usher syndrome, we detected both recurrent heterozygous and hemizygous mutations (each n=1). Also, minor alleles (frequencies were .002 and .004) of non-synonymous variants of GPR98 were selectively retained and wild-type alleles were deleted in del(5q) cases (n=2).

We also searched accessory genetic events observed on other chromosomes in del(5q) cases. By SNP-A, deletions of CRRs (longer del(5q)) were significantly more associated with additional chromosomal defects. Similarly, some specific genes, including the splicing machinery genes and IDH family genes, were uniquely observed in the longer del(5q) cohort.

In conclusion, we detected multiple pathogenic mutations in whole chr5q which might stratify del(5q) patients at risk for disease progression, though no single mutations could explain a majority of cases. Decreased expression or mutation of CWC27 and DDX41, located on CRRs, may exemplify the common pathophysiology shared by heterozygous mutations and haploinsufficient expressions on chr5q. Consequently, it is possible that deletion alone, through decreased expression, may be pathogenic.