Evaluation of pathogenicity of WT1 intron variants by in vitro splicing analysis.

BACKGROUND: Wilms tumor 1 (WT1; NM_024426) causes Denys-Drash syndrome, Frasier syndrome, or isolated focal segmental glomerulosclerosis. Several WT1 intron variants are pathogenic; however, the pathogenicity of some variants remains undefined. Whether a candidate variant detected in a patient is pathogenic is very important for determining the therapeutic options for the patient. METHODS: In this study, we evaluated the pathogenicity of WT1 gene intron variants with undetermined pathogenicity by comparing their splicing patterns with those of the wild-type using an in vitro splicing assay using minigenes. The three variants registered as likely disease-causing genes: Mut1 (c.1017-9 T > C(IVS5)), Mut2 (c.1355-28C > T(IVS8)), Mut3 (c.1447 + 1G > C(IVS9)), were included as subjects along the 34 splicing variants registered in the Human Gene Mutation Database (HGMD)®. RESULTS: The results showed no significant differences in splicing patterns between Mut1 or Mut2 and the wild-type; however, significant differences were observed in Mut3. CONCLUSION: We concluded that Mut1 and Mut2 do not possess pathogenicity although they were registered as likely pathogenic, whereas Mut3 exhibits pathogenicity. Our results suggest that the pathogenicity of intronic variants detected in patients should be carefully evaluated.

Integration of measurable residual disease by WT1 gene expression and flow cytometry identifies pediatric patients with high risk of relapse in acute myeloid leukemia.

Background: Molecular testing plays a pivotal role in monitoring measurable residual disease (MRD) in acute myeloid leukemia (AML), aiding in the refinement of risk stratification and treatment guidance. Wilms tumor gene 1 (WT1) is frequently upregulated in pediatric AML and serves as a potential molecular marker for MRD. This study aimed to evaluate WT1 predictive value as an MRD marker and its impact on disease prognosis. Methods: Quantification of WT1 expression levels was analyzed using the standardized European Leukemia Network real-time quantitative polymerase chain reaction assay (qRT-PCR) among a cohort of 146 pediatric AML patients. Post-induction I and intensification I, MRD response by WT1 was assessed. Patients achieving a ≥2 log reduction in WT1MRD were categorized as good responders, while those failing to reach this threshold were classified as poor responders. Results: At diagnosis, WT1 overexpression was observed in 112 out of 146 (76.7%) patients. Significantly high levels were found in patients with M4- FAB subtype (p=0.018) and core binding fusion transcript (CBF) (RUNX1::RUNX1T1, p=0.018, CBFB::MYH11, p=0.016). Following induction treatment, good responders exhibited a reduced risk of relapse (2-year cumulative incidence of relapse [CIR] 7.9% vs 33.2%, p=0.008). Conversely, poor responders' post-intensification I showed significantly lower overall survival (OS) (51% vs 93.2%, p<0.001), event-free survival (EFS) (33.3% vs 82.6%, p<0.001), and higher CIR (66.6% vs 10.6%, p<0.001) at 24 months compared to good responders. Even after adjusting for potential confounders, it remained an independent adverse prognostic factor for OS (p=0.04) and EFS (p=0.008). High concordance rates between WT1-based MRD response and molecular MRD were observed in CBF patients. Furthermore, failure to achieve either a 3-log reduction by RT-PCR or a 2-log reduction by WT1 indicated a high risk of relapse. Combining MFC-based and WT1-based MRD results among the intermediate-risk group identified patients with unfavorable prognosis (positive predictive value [PPV] 100%, negative predictive value [NPV] 85%, and accuracy 87.5%). Conclusion: WT1MRD response post-intensification I serves as an independent prognostic factor for survival in pediatric AML. Integration of WT1 and MFC-based MRD results enhances the reliability of MRD-based prognostic stratification, particularly in patients lacking specific leukemic markers, thereby influencing treatment strategies.

Characterization of Two Gonadal Genes, zar1 and wt1b, in Hermaphroditic Fish Asian Seabass (Lates calcarifer).

Zygote arrest-1 (Zar1) and Wilms' tumor 1 (Wt1) play an important role in oogenesis, with the latter also involved in testicular development and gender differentiation. Here, Lczar1 and Lcwt1b were identified in Asian seabass (Lates calcarifer), a hermaphrodite fish, as the valuable model for studying sex differentiation. The cloned cDNA fragments of Lczar1 were 1192 bp, encoding 336 amino acids, and contained a zinc-binding domain, while those of Lcwt1b cDNA were 1521 bp, encoding a peptide of 423 amino acids with a Zn finger domain belonging to Wt1b family. RT-qPCR analysis showed that Lczar1 mRNA was exclusively expressed in the ovary, while Lcwt1b mRNA was majorly expressed in the gonads in a higher amount in the testis than in the ovary. In situ hybridization results showed that Lczar1 mRNA was mainly concentrated in oogonia and oocytes at early stages in the ovary, but were undetectable in the testis. Lcwt1b mRNA was localized not only in gonadal somatic cells (the testis and ovary), but also in female and male germ cells in the early developmental stages, such as those of previtellogenic oocytes, spermatogonia, spermatocytes and spermatids. These results indicated that Lczar1 and Lcwt1b possibly play roles in gonadal development. Therefore, the findings of this study will provide a basis for clarifying the mechanism of Lczar1 and Lcwt1b in regulating germ cell development and the sex reversal of Asian seabass and even other hermaphroditic species.

Familial focal segmental glomerulosclerosis associated with a WT1 gene missense mutation: A case report.

Focal s egmental glomerulosclerosis (F SGS) can cause protei nuria and loss o f k idney fun ction, leading to e ndstage renal di s ease (ESRD). Podocyte injury is the ce ntral pathophysiologi cal mechanis m of hereditary FSGS. Numerous mutations in genes e ncoding or affe cting the transcriptional regulation of podocyte cell compar tments have been detected in patients with genetic FSGS. Herein, we report a rare case of familial FSGS with an autosomal dominant WT1 mutation. A 63-year- old man developed pro teinuri a; his reading showed over 1g prote in/day. A pa thological diagn osis of FSG S was made after rena l biops y. H is elder brother an d a 36-year- old son also had ESRD. Heterozygous variant of WT1 (NM_024426.4) c.1373G>A (p.Arg458Gln ) mi s sense was dete cted in the patient a nd his son , by whole-exome sequen cing. Although genetic screening is not a par t of routine practice, it s hould be per for med in such cases to a id a ppropriate tre atment options sel ecting, revealing extra ren al symptoms, and family planning.

Genotype-Phenotype Correlation Analysis of WT1 Gene Variants in Denys-Drash Syndrome and Frasier Syndrome / 罕见病研究

ObjectiveThis study aims to explore the association between different genotypes of WT1 gene variations and the phenotypes of Denys-Drash syndrome (DDS) and Frasier syndrome (FS).MethodsThrough searching and summarizing the case information of WT1 gene variations recorded in NCBI PubMed and CNKI databases from January 1, 1991 to October 31, 2023, we analyzed the association between variation types, occurrence locations, and phenotypes such as progressive renal function impairment, genitourinary developmental abnormalities, nephroblastoma, and gonadal tumors between DDS and FS.ResultsA total of 128 articles, including 304 subjects, were included in this study, and 86 pathogenic variations of the WT1 gene were detected.The distribution characteristics of these variations were as follows: the most common occurrence was in exon 9(24/86, 27.9%) and exon 8 (23/86, 26.7%); the most common variation type was missense mutation(51/86, 59.3%), followed by splice site mutation (13/86, 15.1%).The disease types caused by WT1 gene variations were as follows: DDS had the highest number of cases (174/304, 57.2%), followed by FS (83/304, 27.3%); DDS was mainly caused by missense mutations on exon 9 and exon 8 (143/174, 82.2%), while FS was mainly caused by splice site mutations on intron 9 (76/83, 91.6%).ConclusionsThe missense variants in exon 9 and exon 8 on the WT1 gene mainly resulted in DDS, while the splice variants in intron 9 mainly resulted in FS. Infants and children with progressive renal injury should undergo a comprehensive evaluation of the genitourinary system, and early genetic diagnosis should be established to improve prognosis.

High-Throughput Splicing Assays Identify Known and Novel WT1 Exon 9 Variants in Nephrotic Syndrome.

Introduction: Frasier syndrome (FS) is a rare Mendelian form of nephrotic syndrome (NS) caused by variants which disrupt the proper splicing of WT1. This key transcription factor gene is alternatively spliced at exon 9 to produce 2 isoforms ("KTS+" and "KTS-"), which are normally expressed in the kidney at a ∼2:1 (KTS+:KTS-) ratio. FS results from variants that reduce this ratio by disrupting the splice donor of the KTS+ isoform. FS is extremely rare, and it is unclear whether any variants beyond the 8 already known could cause FS. Methods: To prospectively identify other splicing-disruptive variants, we leveraged a massively parallel splicing assay. We tested every possible single nucleotide variant (n = 519) in and around WT1 exon 9 for effects upon exon inclusion and KTS+/- ratio. Results: Splice disruptive variants (SDVs) made up 11% of the tested point variants overall and were tightly concentrated near the canonical acceptor and the KTS+/- alternate donors. Our map successfully identified all 8 known FS or focal segmental glomerulosclerosis (FSGS) variants and 16 additional novel variants which were comparably disruptive to these known pathogenic variants. We also identified 19 variants that, conversely, increased the KTS+/KTS- ratio, of which 2 are observed in unrelated individuals with 46,XX ovotesticular disorder of sex development (46,XX OTDSD). Conclusion: This splicing effect map can serve as functional evidence to guide the clinical interpretation of newly observed variants in and around WT1 exon 9.

Genotype-phenotype correlation of WT1 mutation-related nephropathy in Chinese children.

Introduction: This study aimed to analyze the clinical characteristics of nephropathy associated with WT1 gene mutations in Chinese children and explore the relationship between genotype and clinical phenotype. Methods: Cases diagnosed at the Guangzhou Women and Children's Medical Center, were combined with those retrieved from PubMed and China National Knowledge Infrastructure (CNKI) databases from January 2015 to June 2022 and integrated into a study cohort; grouped according to gene mutation sites, clinical phenotype, and renal pathological types. The clinical characteristics between groups were compared, and the relationship between genotype and age of onset, clinical phenotype, and pathological type were retrospectively analyzed. Results: The center enrolled 15 confirmed children: seven cases of non-simple nephropathy, including Denys-Drash syndrome (DDS) and Frasier syndrome (FS); eight cases of isolated steroid-resistant nephrotic syndrome (ISRNS); and 13 cases (86.7%) that progressed to end-stage renal disease (ESRD). The initial hemoglobin and bicarbonate levels of patients with clinical non-simple nephropathy were significantly lower than those with simple nephropathy, whereas the serum creatinine levels were higher than those of patients with simple nephropathy. A total of 75 cases of nephropathy associated with WT1 mutations in the study cohort met the inclusion and exclusion criteria. The most common clinical manifestations of WT1 mutations in this cohort were DDS (29/75, 38.7%) and ISRNS (37/75, 49.3%). A renal biopsy was performed in 43 patients, and the common types of renal pathology were focal segmental glomerulosclerosis (23/43, 53.5%) and DMS (13/43, 30.2%). Within the cohort, there were 12 cases (16.0%) in the exon 8 mutation group, 32 (42.6%) in the exon 9 group, 19 (25.3%) in the intron 9 group, and 12 (16.0%) in other gene site mutation groups. Common sites of WT1 mutations in Chinese children were exons 9 and intron 9. Exon 8 mutations were uniquely correlated with the age of onset within three months [5/7; 71.4%; Adjusted standardized residual (AR) = 4.2]. The renal survival time in the exon 8 mutation group was the shortest (P = 0.003). Discussion: The molecular and biological characteristics of WT1 mutation-related nephropathy determine the clinical type, pathological features, and renal survival time of the disease; and there was a strong correlation between the genotype and clinical phenotype.

Prognostic value of the WT-1 gene combined with recurrent cytogenetic genes in acute myeloid leukemia.

Wilms tumor gene 1 (WT-1 gene) is overexpressed in most patients with acute myeloid leukemia (AML) and is an indicator for minimal residual disease (MRD) monitoring, but because the WT-1 gene has relatively low specificity, further studies of the prognostic value of a combination of the WT-1 and other genes are needed. The aim of this study was to explore the prognostic value of the WT-1 gene combined with recurrent cytogenetic genes in AML. In AML, the transcript expression of the WT-1 gene was closely related to leukemic tumor burden and acted as an accurate molecular indicator for MRD detection. Most patients with low expression levels of the WT-1 gene after induction and consolidation therapy were significantly associated with favorable relapse-free survival (RFS) and overall survival (OS), but 17.6% of patients relapsed and died of primary disease. However, when analyzing the WT-1 gene combined with recurrent cytogenetic genes, none of the patients with low expression levels of the WT-1 gene and recurrent cytogenetic genes negative relapsed and died in the median follow-up time of 19 months (range: 3-94 months). Thus, the combination of the WT-1 gene and recurrent cytogenetic genes is a more accurate indicator for MRD monitoring and prognosis evaluation in AML patients.

The expression of WT1 gene and its clinical characteristics in children with acute lymphoblastic leukemia / 中国医师进修杂志

Objective:To investigate the expression of WT1 gene in children with acute lymphoblastic leukemia (ALL), and explore its clinical characteristics and correlation with the prognosis of ALL.Methods:The clinical data of 183 children with newly diagnosed ALL in Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from January 2015 to May 2019 were retrospectively analyzed. The expression level of WT1 gene in bone marrow samples was detected by real-time fluorescence quantitative polymerase chain reaction. The children were followed up to June 2021 with a median follow-up time of 46 months (0 to 63 months).Results:Among 183 children with ALL, the WT1 gene positive was in 130 cases (71.04%), and the expression level was 1.41% (0.26%, 6.73%); WT1 gene negative was in 53 cases (28.96%). The expression levels of WT1 gene in children with T-cell lymphoblastic leukemia (T-ALL), non-hyperdiploid and middle/high-risk were significantly increased, and there were statistical differences ( P<0.05 or <0.01); however, there were no statistical differences in the expression levels of WT1 gene between children with different gender, chromosome karyotype, hepatosplenomegaly and the first diagnosis white blood cell count ( P>0.05). There were no statistical differences in complete remission rate and recurrence rate after induction chemotherapy between WT1 gene positive children and WT1 gene negative children: 87.69% (114/130) vs. 86.79% (46/53) and 16.15% (21/130) vs. 18.87% (10/53), P>0.05. By the end of follow-up, 179 children were followed up, and there was no statistical difference in survival rate between WT1 gene positive children and WT1 gene negative children: 89.68% (113/126) vs. 86.79% (46/53), P>0.05. Among the children with WT1 gene positive, relapse was in 21 cases, and there was no statistical difference in the expression level of WT1 gene after complete remission or after relapse, compared with that while the first diagnosis ( P>0.05); among non-relapse children, 96 completed the detection, the expression level of WT1 gene after complete remission was significantly lower than the first diagnosis: 0.17% (0.04%, 0.49%) vs. 2.01% (0.41%, 8.82%), and there was statistical difference ( P<0.01). Kaplan-Meier survival curve analysis result showed there was no statistical difference in survival time between WT1 gene positive children and WT1 gene negative children ( P>0.05). According to the median expression level of WT1 gene (1.41%), the children with WT1 gene positive were divided into high expression (66 cases) and low expression (64 cases), there was no statistical difference in survival time between high expression children and low expression children ( P>0.05). Conclusions:WT1 gene is commonly expressed in children with ALL and is associated with some clinical features and prognosis of the children. Decreased WT1 gene expression may result in better prognosis.

Desmoplastic Small Round Cell Tumor of the Head and Neck: A Potential Diagnostic Pitfall.

Primary desmoplastic small round cell tumor (DSRCT) in the head and neck region is extremely rare. There is limited information about its clinicopathological characteristics, prognosis, and treatment modalities. The purpose of this study is to provide a comprehensive review of DSRCT occurring primarily in the head and neck, to demonstrate its peculiar morphology and immunohistochemical expression, and to address the differential diagnoses. A total of 25 cases were collected after a thorough review of the relevant literature. DSRCT was most frequently reported in the major salivary glands, followed by the eyes. Furthermore, some cases were misinterpreted as poorly differentiated carcinoma, Ewing sarcoma, and olfactory neuroblastoma. Diagnosing DSRCTs in the head and neck can be very challenging due to their rarity in this location, overlapping morphology, and immunohistochemistry. In these cases, following a systemic approach helps to solve diagnostic problems.

[Expression of Wilms' Tumor 1 Gene in Bone Marrow of Patients with Myelodysplastic Syndrome and Its Clinical Significance].

OBJECTIVE: To investigate the expression level and clinical significance of Wilms' tumor 1 (WT1) in bone marrow of patients with myelodysplastic syndromes (MDS). METHODS: The clinical data of 147 MDS patients who accepted real-time quantitative polymerase chain reaction (RT-PCR) to detect the expression level of WT1 in bone marrow before treated in Nanfang Hospital, Southern Medical University from January 2017 to April 2021 were retrospectively analyzed. According to the expression level of WT1, the patients were divided into WT1+ group and WT1- group, their clinical characteristics and prognosis were analyzed. RESULTS: The positive rate of WT1 in 147 MDS patients was 82.3%. There were significant differences in bone marrow blast count, aberrant karyotypes, WHO 2016 classification, and IPSS-R stratification between WT1+ group and WT1- group (all P<0.05). Furthermore, the higher the malignant degree of MDS subtype and the risk stratification of IPSS-R, the higher expression level of WT1. Compared with WT1- group, there were no differences in overall survival (OS) time and the time of transformation to AML in WT1+ group (both P>0.05). In patients who did not accept transplantation, the median OS time of WT1+ patients was significantly shorter than that of WT1- patients (P=0.049). Besides, regarding WT1+ group, patients who underwent transplantation had longer OS time and lower mortality than those who received hypomethylating agents (P=0.002, P=0.005). CONCLUSION: WT1 expression level directly reflects the disease progression, and it is also associated with prognosis of MDS patients.

Isolated steroid-resistant nephrotic syndrome in a Chinese child carrying a de novo mutation in WT1 gene:a case report and literature review.

BACKGROUND: Isolated steroid-resistant nephrotic syndrome (ISRNS) is caused by mutations in the Wilms' tumor-1 (WT1) gene, which encodes glomerular podocytes and podocyte slit diaphragm.We report a novel 8-year-old female patient with ISRNS carrying a de novo missense mutation in WT1 gene and presenting a new type of pathology, have never been reported.We also systematically review previous reports of ISRNS in Chinese children. CASE PRESENTATION: A 8-year-old Chinese patient who had steroid-resistant nephrotic syndrome,responded poorly to immunosuppressant, and had no extrarenal manifestations. The patient had a female phenotype and karyotype of 46, XX. A new type of renal pathology, proliferative sclerosing glomerulonephritis (PSG),and a de novo missense mutation in WT1 gene, c.748C > T (p.R250W),which have not yet been reported, were identified. She was diagnosed with ISRNS.The patient progressed to end-stage renal disease at the age of 10 years,underwent dialysis and kidney transplant. Renal function and urine protein were normal during 4-year follow-up. CONCLUSIONS: WT1 gene testing should be performed to guide treatment for patients with steroid-resistant nephrotic syndrome, especially for isolated cases and female patients.

WT1 Gene Mutations, rs16754 Variant, and WT1 Overexpression as Prognostic Factors in Acute Myeloid Leukemia Patients.

(1) Background: The aim of our study was the complex assessment of WT1 variants and their expression in relation to chromosomal changes and molecular prognostic markers in acute myeloid leukemia (AML). It is the first multidimensional study in Polish AML patients; (2) Methods: Bone marrow aspirates of 90 AML patients were used for cell cultures (banding techniques and fluorescence in situ hybridization), and to isolate DNA (WT1 genotyping, array comparative genomic hybridization), and RNA (WT1 expression). Peripheral blood samples from 100 healthy blood donors were used to analyze WT1 rs16754; (3) Results: Allele frequency and distribution of WT1 variant rs16754 (A;G) did not differ significantly among AML patients and controls. Higher expression of WT1 gene was observed in AA genotype (of rs16754) in comparison with GA or GG genotypes­10,556.7 vs. 25,836.5 copies (p = 0.01), respectively. WT1 mutations were more frequent in AML patients under 65 years of age (p < 0.0001) and affected relapse-free survival (RFS). The presence of NPM1 or CEBPA mutations decreased the risk of WT1 mutation presence, odds ratio (OR) = 0.11, 95% CI 0.02−0.46, p = 0.002 or OR = 0.05, 95% CI 0.006−0.46, p = 0.002, respectively. We observed significantly higher WT1 expression in AML CD34+ vs. CD34−, −20,985 vs. 8304 (p = 0.039), respectively. The difference in WT1 expression between patients with normal and abnormal karyotype was statistically insignificant; (4) Conclusions: WT1 gene expression and its rs16754 variant at diagnosis did not affect AML outcome. WT1 mutation may affect RFS in AML.

Significance of WT1 and multiparameter flow cytometry assessment in patients with chronic myelomonocytic leukemia receiving allogeneic hematopoietic stem cell transplantation.

INTRODUCTION: The objective of this study was to investigate the clinical significance of minimal residual disease (MRD) monitoring through Wilms tumor 1 (WT1) gene expression and multicolor flow cytometry (FCM) in patients with chronic myelomonocytic leukemia (CMML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: For this purpose, WT1 gene expression and the CMML-related abnormal immunophenotype were examined using real-time quantitative polymerase chain reaction and FCM, respectively. RESULTS: In total, 59 patients with CMML who underwent allo-HSCT were enrolled in this study. Thirteen cases (22.0%) developed hematological relapse, and 15 patients (25.4%) expired during the follow-up period. Thirty-four patients (37.6%) were positive for WT1 (WT1+), and 44 patients (74.6%) were positive for FCM prior to allo-HSCT. After allo-HSCT, there were 21 WT1+ patients (35.6%) and 10 patients (16.9%) who were positive in FCM (FCM+). Post-transplant WT1+ (post-WT1 0.6+; 50.7% vs. 7.6%, p < .001) and post-transplant FCM+ (post-FCM+; 90.0% vs. 8.8%, p < .001) indicated a higher 3-year cumulative incidence of relapse (CIR) compared with the WT1- or FCM-patients. Multivariate analysis of event-free survival (EFS), overall survival (OS), and CIR showed that the FCM status after transplantation was an independent prognostic factor for relapse (p < .05). CONCLUSION: Both FCM and WT1 after HSCT were identified as important predictors of recurrence of CMML following transplantation and may be useful in guiding interventions against disease relapse.

WT1 Pathogenic Variants are Associated with a Broad Spectrum of Differences in Sex Development Phenotypes and Heterogeneous Progression of Renal Disease.

Wilms' tumor suppressor gene 1 (WT1) plays an essential role in urogenital and kidney development. Heterozygous germline pathogenic allelic variants of WT1 have been classically associated with Denys-Drash syndrome (DDS) and Frasier syndrome (FS). Usually, exonic pathogenic missense variants in the zinc finger region are the cause of DDS, whereas pathogenic variants affecting the canonic donor lysine-threonine-serine splice site in intron 9 cause FS. Phenotypic overlap between WT1 disorders has been frequently observed. New WT1 variant-associated phenotypes, such as 46,XX testicular/ovarian-testicular disorders of sex development (DSD) and primary ovarian insufficiency, have been reported. In this report, we describe the phenotypes and genotypes of 7 Brazilian patients with pathogenic WT1 variants. The molecular study involved Sanger sequencing and massively parallel targeted sequencing using a DSD-associated gene panel. Six patients (5 with a 46,XY karyotype and 1 with a 46,XX karyotype) were initially evaluated for atypical genitalia, and a 46,XY patient with normal female genitalia sought medical attention for primary amenorrhea. Germ cell tumors were identified in 2 patients, both with variants affecting alternative splicing of WT1 between exons 9 and 10. Two pathogenic missense WT1 variants were identified in two 46,XY individuals with Wilms' tumors; both patients were <1 year of age at the time of diagnosis. A novel WT1 variant, c.1453_1456 (p.Arg485Glyfs*14), was identified in a 46,XX patient with testicular DSD. Nephrotic proteinuria was diagnosed in all patients, including 3 who underwent renal transplantation after progressing to end-stage kidney disease. The expanding phenotypic spectrum associated with WT1 variants in XY and XX individuals confirms their pivotal role in gonadal and renal development as well as in tumorigenesis, emphasizing the clinical implications of these variants in genetic diagnosis.

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.

Exonic WT1 pathogenic variants in 46,XY DSD associated with gonadoblastoma.

OBJECTIVE: The literature regarding gonadoblastoma risk in exonic Wilms' tumor suppressor gene (WT1) pathogenic variants is sparse. The aim of this study is to describe the phenotypic and genotypic characteristics of Asian-Indian patients with WT1 pathogenic variants and systematically review the literature on association of exonic WT1 pathogenic variants and gonadoblastoma. DESIGN: Combined retrospective-prospective analysis. METHODS: In this study, 46,XY DSD patients with WT1 pathogenic variants detected by clinical exome sequencing from a cohort of 150 index patients and their affected relatives were included. The PubMed database was searched for the literature on gonadoblastoma with exonic WT1 pathogenic variants. RESULTS: The prevalence of WT1 pathogenic variants among 46,XY DSD index patients was 2.7% (4/150). All the four patients had atypical genitalia and cryptorchidism. None of them had Wilms' tumor till the last follow-up, whereas one patient had late-onset nephropathy. 11p13 deletion was present in one patient with aniridia. The family with p.Arg458Gln pathogenic variant had varied phenotypic spectrum of Frasier syndrome; two siblings had gonadoblastoma, one of them had growing teratoma syndrome (first to report with WT1). On literature review, of >100 exonic point pathogenic variants, only eight variants (p.Arg462Trp, p.Tyr177*, p.Arg434His, p.Met410Arg, p.Gln142*, p.Glu437Lys, p.Arg458*, and p.Arg458Gln) in WT1 were associated with gonadoblastoma in a total of 15 cases (including our two cases). CONCLUSIONS: WT1 alterations account for 3% of 46,XY DSD patients in our cohort. 46,XY DSD patients harboring exonic WT1 pathogenic variants carry a small but definitive risk of gonadoblastoma; hence, these patients require a gonadoblastoma surveillance with a more stringent surveillance in those harboring a gonadoblastoma-associated variant.

Intra-Abdominal Desmoplastic Small Round Cell Tumor: Current Treatment Options and Perspectives.

Intra-abdominal desmoplastic small round cell tumor (IDSRCT) is a rare and highly malignant soft tissue neoplasm, which is characterized by rapid progression and poor prognosis. The mechanism underlying the development of this neoplasm remains elusive, but all cases are characterized by the chromosomal translocation t (11;22) (p13; q12), which results in a formation of EWSR1-WT1 gene fusion. The diagnosis of IDSRCT is often made with core-needle tissue biopsy specimens or laparoscopy or laparotomy. Immunohistochemical analyses have shown the co-expression of epithelial, neuronal, myogenic, and mesenchymal differentiation markers. FISH or reverse transcription polymerase chain reaction detecting EWS-WT1 fusion can be performed to assist in molecular confirmation. There is no standard of care for patients with IDSRCT currently, and majority of newly diagnosed patients received the aggressive therapy, which includes >90% resection of surgical debulking, high-dose alkylator-based chemotherapy, and radiotherapy. More recently, targeted therapy has been increasingly administered to recurrent IDSRCT patients and has been associated with improved survival in clinical conditions. Immunotherapy as a possible therapeutic strategy is being explored in patients with IDSRCT. In this review, we summarize currently available knowledge regarding the epidemiology, potential mechanisms, clinical manifestations, diagnosis, treatment, and prognosis of IDSRCT to assist oncologists in comprehensively recognizing and accurately treating this malignancy.