A novel WT1 gene mutation in a chinese girl with denys‐drash syndrome

Abstract Objective Denys‐Drash syndrome (DDS) is defined by the triad of Wilms tumor, nephrotic syndrome, and/or ambiguous genitalia. Genetic testing may help identify new gene mutation sites and play an important role in clinical decision‐making. Methods We present a patient with an XY karyotype and female appearance, nephropathy, and Wilms tumor in the right kidney. Genomic DNA was extracted from peripheral blood cells according to standard protocols. “Next‐generation” sequencing (NGS) was performed to identify novel variants. The variant was analyzed with Mutation Taster, and its function was explored by a cell growth inhibition assay. Results We found the first case of Denys‐Drash syndrome with the uncommon missense mutation (c.1420C>T, p.His474 Tyr) in the WT1 gene. In silico analysis, the variant was predicted “disease‐causing” by Mutation Taster. The mutated variant showed a weaker effect in inhibiting tumor cells than wild‐type WT1. Conclusions The uncommon missense mutation (c.1420C>T, p.His474 Tyr) in the WT1 gene may be a crucial marker in DDS.


| Clinical assessment
A comprehensive clinical examination of the abdominal mass was performed. We conducted an ordinary physical examination, laboratory tests [routine blood, urine and fecal, liver and kidney function, electrolyte, tumor makers, and sex-determining region on the Y chromosome (SRY)], and imageological examinations [electrocardiogram, abdominal B-ultrasonography examination, and contrast-enhanced computed tomography (CT)]. After that, a puncture biopsy was performed.

| Clinical treatment
Based on the diagnosis of nephroblastoma (mesenchymal type), the patient was treated with chemotherapy, arterial interventional therapy, and nephroblastoma radical resection. Detailed information is summarized in Table 1.

| Genetic testing
The identification process is a two-step procedure: High-throughput sequencing technologies and validation of suspected diseasecausing mutations (Sanger sequencing).

| High-throughput sequencing
To make a precise diagnosis, we performed targeted "Nextgeneration" sequencing (NGS) of 175 disease-related genes. Written informed consent of the whole family was obtained prior to the collection of 5 mL of their peripheral blood for the following experiment. The study was approved by the ethics committee of the Beijing Genomics Institute (BGI). As for quality control, the data were processed as follows: a.

F I G U R E 1
Discard paired reads if either one read contains adapter contamination (>10 nucleotides aligned to the adapter, allowing ≤10% mismatches); b. Discard paired reads if more than 10% of bases are uncertain in either read; c. Discard paired reads if the proportion of low-quality bases (Phred quality <5) is over 50% in either read.
All downstream bioinformatics analyses were based on high-quality clean data.
The sequencing reads were aligned to the reference human genome (UCSC hg19) using BWA (Burrows-Wheeler Aligner) Multi-Vision software package. 9 After alignment, the output files were used to perform sequencing coverage and depth analysis of the target region, single-nucleotide variants (SNVs), and INDEL calling.
We used SOAPsnp software 10 and Samtools 11 to detect SNVs and indels. All SNVs and indels were filtered and estimated via multiple databases, including NCBI dbSNP, HapMap, 1000 human genome dataset, and database of 100 Chinese healthy adults. 12 Pathogenic variants were assessed under the protocol issued by the American College of Medical Genetics and Genomics (ACMG). 13 The Human Gene Mutation Database (HGMD) was used to screen mutations reported in published studies.

| Verification of the variant
All mutations and potential pathogenic variants were validated by Sanger sequencing on an ABI3730 sequencer. The reference sequence of the WT1 gene is NM_024424.5.

| Functional analysis of the novel variant
To detect the function of the novel WT1 variant, we cloned the wild type and mutated coding DNA sequence (CDS) (NP_077742.3) of the WT1 gene to pcDNA3.1 and compared their function by a cell growth inhibition assay.

| Plasmid construction
Total RNA was extracted from the tumor tissue using TRIzol® Reagent (Thermo Fisher Scientific). We used a QuantiTect reverse

| Cell growth inhibition assay
The cell growth inhibition assay was performed using the MTS method. We seeded 3000 transfected G401 cells from each group in 96-well plates per well and cultured them for 7 days. Cell viability was tested using the MTS Cell Proliferation Assay kit (Promega) according to the standard protocol.

| Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad, Inc.

| Imageological examinations
Abdominal contrast-enhanced CT revealed an approximately 7.1 × 8.0 × 7.6 cm heterogeneous enhancing mass in the upper region of the right kidney ( Figure 3). Ultrasound imaging revealed that the vagina, uterus, and right ovary were almost normal. However, the left ovary was not clearly delineated.

| Laboratory tests and laparoscopic examination
The patient had proteinuria without microscopic hematuria. She un- To determine whether the gonads were normal, laparoscopy was performed when the patient was 29 months old. A small uterus and fallopian tube were observed during the laparoscopic examination.
In addition, both gonads were located at the lateral side of the fallopian tube, so they were partly resected for biopsies. The pathology showed that both gonads were dysgenetic testicular tissue.

| Final Diagnosis and post-treatment follow-up
All clinical manifestations and molecular analyses demonstrated that the patient had Denys-Drash syndrome. After 46 months of followup, the patient was in good condition and showed no signs of tumor recurrence. However, the severity of the nephropathy increased and decreased. Albumin levels (1,030-3,226 mg/L; normal children <100 mg/L) were much higher than normal levels in the first two months, but her microalbuminuria was controlled well thereafter.

| Identification of the novel mutation
Whole exon sequencing revealed that the WT1 mutation was positive. The patient was found to have a heterozygous missense mutation, converting 474 His to 474 Tyr (c.1420C>T, Figure 1) in exon 9 of WT1, encoding a portion of the WT1 zinc-finger 3 domain.

| In silico analysis
The mutant variant of WT1 (c.1420C>T, p. His474 Tyr) was predicted to "disease-causing" by Mutation Taster. The peptide alignment revealed that the histidine in WT1 was a conserved residue ( Figure 6).

| Functional assays of the novel variant
In the cell growth inhibition assay, we found that G401 cells with pcDNA3.1-WT1 (C1420 T) grew faster than those with pcDNA3.1-WT1 (wild type) but slower than the control group. Significant differences were observed between the groups (Figure 7). The result indicates that the mutant variant of WT1 (c.1420C>T, p. His474 Tyr) cannot function as well as the wild-type WT1 as a tumor suppressor gene.

| DISCUSS ION
We present a case of a patient with Wilms tumor, nephropathy, hermaphroditism, and a novel mutation in the WT1 gene. All the pathological features that this patient had met the typical characteristics were described in the cases of Denys-Drash syndrome. 17 In DDS, mutations in the ZF region can abolish the DNA-binding capacity and lead to sex ambiguity because of dysgenetic testis, diffuse mesangial sclerosis with chronic renal disease, and a high incidence of Wilms tumor. 24 In this study, we found that the histidine in Studies have shown that WT1 activates the transcription of the podocalyxin gene. 25,26 The WT1 variant with a mutation in ZF9 may disrupt podocyte development and differentiation, leading to renal insufficiency. In mice with WT1 knockout in podocytes, the podocyte foot processes disappeared and, consequently, glomerulosclerosis and proteinuria developed in mice. The results confirmed the essential role of WT1 in podocyte maturation and maintenance. [27][28][29] In addition, a study of histological specimens from children with DDS found that WT1 expression in podocytes was abnormal in 80% of patients. 30 For this patient, the mutation may play a crucial role in the process of nephrotic syndrome. Fortunately, the nephrotic syndrome was well controlled at the end of the follow-up period.
We will continue to monitor the development of nephropathy in the child closely.
In addition, WT1 also plays a key role in the regulation of SRY expression and sex determination. WT1 and steroidogenic factor 1 (SF-1) are expressed in the urogenital ridge, which develops into the kidneys and gonads. 31 WT1 associates with SF-1 and achieves a synergistic effect. The upregulation of the SRY gene and the promotion of Müllerian inhibiting substance (MIS) both rely on the synergism of WT1 and SF-1 expressed in the bipotential gonad. [32][33][34] The mutant WT1 may disrupt the synergy between WT1 and SF-1, which leads to gonadal abnormalities in 46, XY individuals. 35,36 This may explain the correlation between gonadal abnormalities and mutations in the WT1 gene in this patient.

| CON CLUS ION
We report a novel mutation in WT1 associated with DDS in a child.
The uncommon missense mutation (c.1420C>T, p. His474 Tyr) in the WT1 gene may be a crucial marker in DDS. WT1 sequencing should be considered early in children with sexual dysplasia, especially in patients with a 46, XY karyotype.

ACK N OWLED G EM ENTS
We thank Dr. Zhongwei Gu and Minju Li for kindly providing clinical data and material. Dr. Minju Li gave us valuable advices, and Dr.
Zhongwei Gu provided excellent technical assistance. In addition, we thank the parents of the patient for the support in the study. This study was supported by National Natural Foundation of China (81801939) and Health Commission of Zhejiang Province (2019KY093).

CO N FLI C T O F I NTE R E S T S
There is no conflict of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data used for the analysis in this study are available in China National GeneBank DataBase (URL: https://db.cngb.org/cnsa/, Accession Number: CNP0001670).

F I G U R E 7
The function of WT1 (wild type and mutant variant) in G401 cells. A, The expression level of pcDNA3.1-WT1 (wild type) and pcDNA3.1-WT1 (C1420 T) was examined in a western blot analysis. B, Cell relative growth analysis. 3000 cells from each group were seeded in 96-well plate per well and cultured for 7 days. Thereafter, we detected the cell relative growth rate by the MTS assay. All data are presented as the mean ±standard deviation from three independent experiments. Nonpaired t-test was performed between the groups (**p < 0.05, ***p < 0.005, ns: non-significance)