Recurrent genetic defects on chromosome 7q in myeloid neoplasms

Monosomy 7 (−7) and deletion of the long arm (del(7q)) of chromosome 7 (chr7) belong to the most commonly acquired karyotypic abnormalities in myeloid neoplasms and are associated with poor prognosis. Although somatic mutations or decreased expression of EZH2 have a role,^1–3 the molecular mechanisms behind chr7 alterations in most cases remain undefined. It is likely that other pivotal target genes contribute to −7/del(7q) pathogenesis. Somatic mutations may either be unique to del(7q) hemizygous inactivation, or be shared between 7q diploid and haploid cases. In addition to the hypothetical mutations in hemizygous configuration, del(7q) alone may result in haploinsufficient gene expression. Corresponding heterozygous hypomorphic mutations or epigenetic inactivation of genes located on chr7q may phenocopy the haploinsufficient expression resulting from del(7q). To identify somatic mutations and haploinsufficiently expressed genes located on chr7q, we applied whole-exome sequencing (WES) and expression analysis both in deletion cases and in those with an apparently normal diploid chr7. Here we report the recurrent genetic defects in CUX1, LUC7L2 and EZH2, and other results of the comprehensive molecular screen of chr7q.

Using metaphase cytogenetics and SNP-array-based karyotyping, loss of heterozygosity (LOH) lesions affecting 7q were identified in 171 of 1131 cases (15%), including low-risk myelodysplastic syndromes (MDS) (14%), high-risk MDS (34%), secondary acute myeloid leukemia (sAML) (18%), MDS/myeloproliferative neoplasms (MPN) (13%), MPN (6%) and primary acute myeloid leukemia (pAML) (8%) (Supplementary Table S1). Minimal commonly deleted regions (CDRs) were defined as 7q22 (100847518–101872055; CDR1), 7q34 (138190944–139672739; CDR2) and 7q35-q36 (144707068–148942012; CDR3), as previously described.⁴ Analysis of WES results for all exons on chr7 for 428 cases with different myeloid neoplasms (Figure 1a) identified 490 nonsilent alterations in 306 genes located on chr7 (5% of all alterations found; Supplementary Table S2). After stringent filtering and bioanalytic processing to avoid false positives (Supplementary Figure S1),⁵ we narrowed the focus of our investigations to ‘tier 1’ mutations; 155 mutated genes were found in 25% (17/68) of −7/del(7q) cases, in 100% (6/6) of uniparental disomy for chr7q (UPD7q) and in 30% (107/354) of diploid for chr7 (Figure 1b). All mutations were validated by Sanger sequencing and targeted deep sequencing of DNA from both tumor and germline (CD3⁺ T cells or buccal mucosa) cells. UPD7q was more frequently affected by somatic mutations than −7 or del(7q). Notably, 17% (39/228) of the somatic mutations were located in one of the CDRs with marker genes CUX1 in 7q22, LUC7L2 in 7q34, and CUL1 and EZH2 in 7q35–36.

Figure 1.

Mutations and LOH on chr7. (a) Mutations of chr7 detected by whole-exome sequencing in the whole cohort (N = 428) are shown in red. LOH on chr7 is demonstrated as follows: a normal diploid, blue and orange; a uniparental disomy (UPD7), double blue lines; and a deletion for chr7, dashed line. Three distinct CDRs, indicated by vertical rectangles, are identified on 7q by mapping of SNP-A karyotyping. (b) Frequency of the cases with mutations in each chr7 status (diploid, UPD and deletion), and mutational types (missense, nonsense, frameshift and splice site) are demonstrated by bar graph and pie chart, respectively (upper panel). Venn diagram shows the number of mutated genes categorized by their configuration of zygosity (heterozygous, homozygous and heterozygous) (lower panel).

Among many rare somatic events, eight genes (EZH2, CUX1, LUC7L2, CFTR, PLXNA4, DYNC1l1, NRCAM and CUL1) corresponding to regions affecting del(7q) were affected by multiple mutations, totaling 44 events (Supplementary Figure S2). For example, mutations of a core component of E3 ubiquitin ligase complex CUL1^6,7 were detected only in cases with −7/del(7q) (N = 2, hemizygous mutations). Conversely, somatic mutations of PLXNA4, encoding a component of the neuropilin–plexin complex,⁸ were observed only in cases with diploid 7q. In cases with diploid 7q, we observed 23 different heterozygous alterations, of which 63% (five of the eight genes) were also affected in −7/del(7q) or UPD7q (hemizygous/homozygous) (Figure 1b). Previously described EZH2 (7q36.1) mutations, located in CDR3, were seen in either heterozygous, homozygous or hemizygous configurations. However, EZH2 mutations were most common in UPD7q cases (67%, 4/6), and in only 5% (4/88) of −7/del(7q) cases. The other CDRs also contained genes recurrently affected by mutations including CUX1 (7q22, N = 5) and LUC7L2 (7q34, N = 8). Somatic mutations were also identified on 7p (HDAC9, IKZF1 and EGFR), which may account for the differences between −7 and del(7q).

In addition to somatic defects in the genes on CDRs of 7q, copy number loss of the wild-type genes might result in haploinsufficiency and the resultant effects may be similar to the heterozygous hypomorphic or loss of function mutations. To investigate haploinsufficient gene expression due to −7/del(7q), we compared relative mRNA levels between cases with and without −7/del(7q). Underexpressed genes (< −2 s.d. of 17 healthy controls) were more common in −7/del(7q), in particular within the three CDRs (Supplementary Figure S3). Furthermore, cases with deletion of CUX1, LUC7L2 and EZH2 (all located in CDRs) showed a significantly lower mRNA expression compared with those with diploid 7q (P<0.001, <0.001 and <0.05, respectively, Figure 2a). When mutated in case diploid for 7q, these genes were commonly affected by frameshift and nonsense mutations (Supplementary Table S3), implying loss of function. Thus, it is likely that either a hypomorphic mutation, and/or haploinsufficiency may lead to the downstream pathological consequences. Conversely, there was no significant haploinsufficiency identified in the genes located outside CDRs (NRCAM (7q31.1), PLXNA4 (7q32.3) and CFTR (7q31.2) (data not shown).

Figure 2.

Gene expression of CUX1, LUC7L2 and EZH2. (a) By expression array analysis, −7/del(7q) patients’ samples (n = 9) show significantly decreased expressions of all CUX1, LUC7L2 and EZH2 genes relative to those from MDS patients who possess normal diploid chr7 (n = 174). Statistics analysis was performed using Student’s t-test with a two-sided test. *P<0.05, **P<0.001, N.S.; not significant. (b) Frequency of deletion and somatic mutation in CUX1, LUC7L2 and EZH2. In the cohort of patients with myeloid malignancies, SNP array karyotyping and metaphase cytogenetics (n = 1559) were applied for the detection of 7q deletion affecting CUX1, LUC7L2 and EZH2 loci. Mutations of each gene were searched in 428 cases by whole-exome and validation sequencing. Frequency of the deletions and mutations was calculated in each disease phenotypes. MDS-low includes refractory cytopenia with unilineage dysplasia, refractory cytopenia with multilineage dysplasia, MDS with isolated del5q, MDS unclassifiable and refractory anemia with ring sideroblasts. MDS-high includes refractory anemia with excess blasts. *P<0.001 (χ²-test). (c) Overall survival was compared among patients with mutations (mt) of CUX1, LUC7L2 or EZH2, wild type (wt) and del(7q). (d) Overall survival was compared between AML patients with low expression of EZH2, CUX1 and EZH2 and those with normal expression of the corresponding genes. P-values presented correspond to the Cox regression between the groups indicated. n.s.; not significant.

To elucidate the clinical impact of recurrent deficiency in specific genes on chr7, we correlated clinical characteristics to either deletion or corresponding somatic inactivating mutations. Mutations and deletions in CUX1, LUC7L2 and EZH2 were observed in 11%, 11% and 12% of cases, respectively (Figure 2b) and were less prevalent in cases with MPN compared with MDS (P = 0.02, <0.01 and = 0.02, respectively) and sAML (P = 0.01, <0.01 and <0.01, respectively). Deletions involving these three gene loci were more prevalent in high-risk MDS than in low-risk MDS (P<0.001) and in sAML than in pAML (P<0.001). Consistent with this finding, mutations in any one of three genes were significantly associated with shorter survival (P<0.0001; HR = 2.55; 95% CI, 1.98 to 7.64; Figure 2c): CUX1 mutations were linked to compromised survival, as were mutations in LUC7L2 or EZH2 (HR = 2.32 and HR 3.16, respectively). Furthermore, the presence of low CUX1, LUC7L2 or EZH2 expression correlated with a significantly shorter survival (P = 0.001; HR = 2.05, 95% CI, 1.45 to 4.45; Figure 2d) in pAML patients. We performed additional clinical investigations, but no relationship between the low expression of these three genes and other clinical parameters (for example, age, cytogenetics and blood counts) was found (data not shown).

We also investigated the potential relationship of somatic mutation events observed on other chromosomes to concomitant −7/del(7q) and UPD7 (Supplementary Figure S4a). There were clear differences between both LOH7 groups, in which −7/del(7q) was more associated with accessory chromosomal events (that is, del(5q), del(17p) or trisomy 8) than cases with UPD7q. Although well-known, frequent mutations in U2AF1, TET2 and TP53 were commonly found in both LOH7 groups, some specific genes, including the CSMD family, were uniquely observed in −7/del(7q). LOH7 was also associated with somatic mutations in SETBP1 (P = 0.02, Supplementary Figure S4b). When we investigated concomitant mutations and genetic events in mutation cases with CUX1, LUC7L2 and EZH2, these three most common mutations were not mutually exclusive (Supplementary Figure S4c). It is possible that concomitant mutations exert synergetic effects analogous to the defect created by a long deletion on chr7q (involving multiple CDRs). TET2 mutations were most prevalent in CUX1 or EZH2 mutant cases (P<0.001, Supplementary Figure S4d), suggesting that TET2 lesions constitute the driver events. In addition to LUC7L2, we found mutations in other spliceosomal genes (U2AF1 or U2AF2) in CUX1 or EZH2 mutation cases, which were also mutated in cases of LOH7q without these mutations. Of note is that we could not find any concomitant TP53 mutations in cases with mutated CUX1, LUC7L2 and EZH2. In sum, our findings indicate that diverse additional molecular events might have a significant role in hypomorphic gene defects on chr7.

Our results demonstrate the various associations of the LOH7q (including UPD) with or without concomitant mutations involving genes located in 7q region. Regardless of their causes (deletion or normal diploid chr7) or heterozygous hypomorphic mutation, haploinsufficient expression of CUX1, LUC7L2 and EZH2 is associated with a poor survival suggesting that hypomorphic mutations and haploinsufficiency both lead to deficient function of these genes in −7/del(7q) myeloid neoplasms. Hypomorphic EZH2 mutations (located in SET domain) found in myeloid neoplasms have been implicated in premature termination or direct abrogation of histone methyltransferase activity. It remains unclear why EZH2 mutations occur more commonly in the context of UPD7q rather than −7/del(7q),² but likely, as reported here and recently,⁹ loss of EZH2 function may occur through multiple pathways, including homozygous mutations with UPD7q, haploinsufficiency and through phenocopy by splicesome mutations. The function and likely the consequences of LUC7L2 mutations are distinct from EZH2; LUC7L2 is one of the splicesome subunits interacting with U1 snRNP to recognize non-consensus splice donor sites.^10,11 We identified eight mutant cases, interestingly, mostly LUC7L2 mutations resulted in premature stop codons. The biological effect of wild-type and mutant LUC7L2 on myeloid neoplasms has not been studied. CUX1 as a transcription factor regulates a large number of genes and microRNAs involved in DNA replication, DNA damage response and cell cycle progression.^12,13 In agreement with clinical observations, including not only MDS/MPN overlap but also MPN cases,¹⁴ Cux1 transgenic mice developed an MPN-like myeloid leukemia with massive expansion of neutrophils.¹⁵

In conclusion, no single gene defect explains the pathogenesis of LOH, but some specific molecular events, including recurrent somatic mutations in CUX1, LUC7L2 and EZH2, are recurrent in −7/del(7q). While del(7q) results in low expression in these genes, down modulation and hypomorphic mutations are also seen in some cases diploid for 7q and all are uniformly associated with poor survival.