While deletion of the long arm of chromosome 7 (del(7q)) along with monosomy 7 (-7) are common in myeloid neoplasms and especially MDS, their associated pathogenetic consequences and the genes responsible for the clinico-morphologic phenotypes remain unknown. To characterize the molecular defects resultant from del(7q), we applied a combined analysis of SNP-array karyotyping, whole exome NGS, targeted deep NGS and deep whole RNA NGS to facilitate identification of somatic mutations, loss of heterozygosity (LOH) and haploinsufficiency.
First, using a cohort of 1595 patients, we precisely defined 3 commonly deleted regions in 7q22, 7q34, and 7q35-36. To identify genes involved in the pathogenesis of del(7q), we applied whole exome NGS to 428 patients with MDS and related conditions, including 72 cases of -7/del(7q) or UPD7. We found that both recurrent deletions with haploinsufficient expression and somatic nonsense/frameshift mutations were present in the genes CUX1 (7q22), LUC7L2 (7q34) and EZH2 (7q36), located on 7q. For this project we focused our study on the LUC7L2 gene encoding a spliceosomal protein that interacts with U1 snRNP to recognize 5’ splice sites. This function is in contrast to other spliceosomal genes mutated in MDS, such as SF3B1, U2AF1, SRSF2 and SF3A1that interact with the U2 snRNP complex and the 3’ splice site.
Initially, we found 8 cases of LUC7L2 mutation including 2 cases of hemizygous and 1 case of homozygous mutations; all LUC7L2 mutations result in premature stop codons. The concomitant mutational spectrum in LUC7L2 mutant cases differed from those with EZH2 or CUX1 mutations. For example, TP53 and TET2 are mostly mutually exclusive with LUC7L2 mutations. While LUC7L2 mutant cases were low grade-MDS (RCUD or RCMD) or CMML, the survival impact of LUC7L2 mutation was similar to that seen in -7/del(7q) (LUC7L2; HR=2.36). Most of the mutations are heterozygous (diploid chr7) or homozygous (UPD7q). However, in addition to a few hemizygous mutations, wild type expression of LUC7L2 is haploinsufficient in del(7q). In total we found 117 cases (9%) with del(7q) and resultant decreased expression of LUC7L2, showing comparably poor survival as in the mutant cases (HR=1.99).
Next, we hypothesized that dysfunction of LUC7L2 causes splicing defects (missplicing) in specific genes involved in leukemogenesis. Using deep RNA NGS we compared splicing patterns of 201,837 exons between the cases with deficient function of LUC7L2 (mutation/low-expression; n=11) versus wild-type (n=11), and identified concordant alternative splicing patterns in 44 genes (increased exon skipping in 27 genes; increased exon retention in 17 genes). For example, we found abnormal splicing of genes involved in functionally important pathways including the RAS pathway (NF1) and the TGF-β pathway (SMAD5). In cases of mutation/low-expression of LUC7L2, as a result of increased skipping of NF1 exon 31(NM_001042492), the type I isoform of NF1 predominates as compared to wild type (types I/II ratio; 0.76 vs. 0.45, p<.01, respectively). Missplicing also occurred in SMAD5 exon 2 (5’UTR site) resulting in a significantly lower SMAD5 expression. In agreement with RNA sequencing results, lentivirus-mediated shRNA knockdown of LUC7L2 in K562 cells resulted in the concordant effects on NF1 type 1 isoform and SMAD5 exon 2 skipping. Similar splicing patterns were observed in MDS cases with -7/del(7q), involving the LUC7L2gene.
In conclusion, novel somatic mutations of LUC7L2 suggest that it could be a candidate gene associated with the poor prognosis of -7/del(7q) and UPD7. Loss of function or low expression of LUC7L2 results in distinctly altered splicing patterns involving genes associated with proliferation or leukemogenesis.
Disclosures:
Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding.
Low-grade oncocytic renal tumor (LOT): mutations in mTOR pathway genes and low expression of FOXI1