A Novel WT1 Mutation Identified in a 46,XX Testicular/Ovotesticular DSD Patient Results in the Retention of Intron 9

The 46,XX testicular DSD (disorder/difference of sexual development) and 46,XX ovotesticular DSD (46,XX TDSD and 46,XX OTDSD) phenotypes are caused by genetic rearrangements or point mutations resulting in imbalance between components of the two antagonistic, pro-testicular and pro-ovarian pathways; however, the genetic causes of 46,XX TDSD/OTDSD are not fully understood, and molecular diagnosis for many patients with the conditions is unavailable. Only recently few mutations in the WT1 (WT1 transcription factor; 11p13) gene were described in a group of 46,XX TDSD and 46,XX OTDSD individuals. The WT1 protein contains a DNA/RNA binding domain consisting of four zinc fingers (ZnF) and a three-amino acid (KTS) motif that is present or absent, as a result of alternative splicing, between ZnF3 and ZnF4 (±KTS isoforms). Here, we present a patient with 46,XX TDSD/OTDSD in whom whole exome sequencing revealed a heterozygous de novo WT1 c.1437A>G mutation within an alternative donor splice site which is used for −KTS WT1 isoform formation. So far, no mutation in this splice site has been identified in any patient group. We demonstrated that the mutation results in the retention of intron 9 in the mature mRNA of the 46,XX TDSD/OTDSD patient. In cases when the erroneous mRNA is translated, exclusively the expression of a truncated WT1 +KTS protein lacking ZnF4 and no −KTS protein occurs from the mutated allele of the patient. We discuss potential mechanisms and pathways which can be disturbed upon two conditions: Absence of Zn4F and altered +KTS/−KTS ratio.

The Molecular Characteristics and Clinical Relevance of NUP98-Other Translocations in Pediatric Acute Myeloid Leukemia

Background Cytogenetic and molecular aberrations are important prognostic factors in pediatric acute myeloid leukemia (AML). NUP98 translocations with more than 30 different partner genes have been identified in pediatric AML. The 2 most common fusions, NUP98-NSD1 and NUP98-KDM5A, have been shown to have distinct characteristics and are both associated with adverse outcomes. Although NUP98 fusions with less common fusion partners have been identified, the biological and clinical implications of these variants are unknown. Methods To determine the biological and clinical implications of the less common "other" NUP98 translocations (NUP98-X), we evaluated the clinical characteristics and transcriptome and genome sequencing data from 2396 children and young adults with AML within 4 consecutive Children's Cancer Group (CCG) and Children's Oncology Group (COG) trials CCG-2961, AAML03P1, AAML0531 and AAML1031. All NUP98-X translocations were confirmed by RNA sequencing. Results Of the 2396 patients screened, 164 patients (6.8%) had a NUP98 translocation. We identified 20 patients with a NUP98-X fusion (0.83%) and compared them with those with NUP98-NSD1 (n=110, 4.5%), NUP98-KDM5A(n=34, 1.4%), and a reference cohort without NUP98 translocations (n=2232). Translocation partners identified in the NUP98-X group were HOXA9 (n=4), HOXD13 (n=3), PHF15 (n=2), PHF23 (n=2), and single cases of BPTF, BRWD3, DDX10, HMGB3, HOXA13, KAT7, PRRX1, SET and TOP1. Besides the distinct characteristics of NUP98-NSD1 and NUP98-KDM5A, the NUP98-X group showed high inter-patient variance in clinical characteristics compared to our reference cohort. NUP98-X patients showed a clear bimodal age distribution with half of the patients being in the older age category and a similar number in the category <3 years (Figure 1). White blood cell count and blast percentages were in line with those of the reference cohort. We investigated the co-occurrence of common pediatric AML mutations within our cohort (Figure 2). There was a lack of additional driving mutations with the exception of WT1 mutations that are significantly more prevalent in the NUP98-X group compared to the reference cohort (25% vs 8%, p=0.018). NUP98-X patients with a WT1 mutation have a median age of 16.3 years compared to 2.3 years for those without a WT1 mutation. Also, in contrast to NUP98-NSD1 translocated patients, the co-occurrence of FLT3-ITD did not occur in NUP98-X translocated patients. Further, NUP98-X patients had a more varied transcriptome profile than did patients with NSD1 or KDM5A translocations (Smith J, et al. ASH abstract 2020). We evaluated the impact of NUP98-X translocations in response to the initial induction therapy. The morphological complete remission (CR) rate after course 1 was 65% in the NUP98-X cohort versus 76% in the reference cohort (p=0.266). NUP98-NSD1 patients had an inferior CR rate (36%, p<0.001), but the CR rate in NUP98-KDM5A patients was similar (77%) to the reference cohort. Of all NUP98-X patients, 11% underwent stem cell transplantation (SCT), compared to 17% in the reference cohort, 40% in the NUP98-NSD1 and 23% in the NUP98-KDM5A cohort. Outcome analysis for NUP98-X patients demonstrated that despite differences in disease characteristics, they have similar adverse outcomes as patients with NSD1 or KDM5A translocations (Figure 3). Overall survival (OS) at 5 years from diagnosis was 35% compared to 65% in the group without NUP98 translocations (p=0.006, Figure 3). Event-free survival was 35% for NUP98-X patients, compared to 48% for those without NUP98 translocations (p=0.282). These adverse outcome rates are comparable to those reported in studies on high-risk pediatric AML patients. Conclusion NUP98-X translocated pediatric AML patients represent a rare cohort with a high variability in both translocation partners and other clinical characteristics. Despite this heterogeneity, the OS of these patients is comparable to high-risk pediatric AML patients, which justifies a high-risk stratification of these patients and emphasizes the need for developing new treatment strategies. Further research within large patient cohorts is needed to investigate the biologic and clinical characteristics of these rare translocations and potential differences between the different NUP98-X fusion partners. Disclosures Kaspers: AbbVie: Ended employment in the past 24 months; Helsinn Healthcare: Ended employment in the past 24 months; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Janssen R&D: Ended employment in the past 24 months. Cooper:Celgene: Other: Spouse was an employee of Celgene (through August 2019).

Nucleotide Transition 390C-T in the Wilms' Tumor 1 Gene: A Risk Factor of Hypospadias?

Introduction: The gene Wilms' tumor 1 (WT1) encodes a unique transcription factor. Its defects are known to cause a wide range of complex genitourinary malformations and may contribute to non-syndromic forms of hypospadias. Materials and Methods: We performed WT1 mutation analysis and copy number analysis of WT1-interacting protein in 13 Hungarian patients diagnosed with isolated hypospadias. Results: Sequencing of WT1 revealed a high frequency of heterozygosity for transition 390C-T (5 heterozygotes out of 13 patients, including 2 brothers). WT1-interacting protein had a normal copy number in all patients. Conclusion: Nucleotide substitution 390C-T may play a role in the pathogenesis of non-syndromic hypospadias. The genotype-phenotype correlation should be confirmed by a larger-scale analysis.