Case Report: a novel variant in WT1 leads to focal segmental glomerulosclerosis and uterovaginal anomalies through exon skipping

Abstract

Background: Podocytopathies are a varied set of renal diseases in which podocytes are unable to perform their typical filtration function within the glomerulus. This typically leads to edema, proteinuria, and hypoalbuminemia early in life. Among podocytopathies, focal segmental glomerulosclerosis (FSGS) is characterized by histology demonstrating segmental and focal sclerosis of the glomerular tuft. FSGS affects an estimated 1-20 per one million individuals and leads to significant morbidity and mortality related to renal failure. While FSGS can be attributed to many causes, such as drug reactions and infections, underlying pathogenic genetic variants play an increasingly well-recognized role in this disease.

Case: A 38-year-old 46,XX female patient of self-reported Cambodian ancestry was evaluated due to her history of atypical uterovaginal morphology. She had a history of hypertension and nephrotic range proteinuria that was diagnosed early in adulthood. A kidney biopsy at that time revealed FSGS. Following worsening renal function and subsequent end-stage renal disease (ESRD), she underwent a kidney transplant at 33 years of age. After kidney transplant, she presented with hematocolpos and was found to have distal vaginal atresia and an arcuate uterus. She underwent vaginoplasty and then had regular menses. She was noted to have persistently elevated follicle stimulating hormone levels, consistent with primary ovarian insufficiency, but with normal anti-Müllerian hormone levels. Assessment of her family history was suggestive of other individuals in her family with similar renal disease and uterine differences. Genetic analysis identified a WT1 variant (c.1338A>C; p. =) of uncertain significance that is also present in her similarly affected mother. To help clarify the potential impact of this variant, we completed a mini-gene assay to detect in vitro splicing changes in the presence of the WT1 variant sequence uncovered in this individual. This demonstrated resultant aberrant splicing that further supports the pathogenicity of the uncovered variant for this individual.

Conclusions: To our knowledge, this represents the first case of a podocytopathy with co-occurring uterovaginal anomalies due to exon skipping in WT1. The patient exhibited a severe course of chronic kidney dysfunction requiring a kidney transplant. Clinical RNA sequencing to clarify variants impacting splicing remains challenging due to tissue- specific gene expression for genes such as WT1, thus, research-based assays may be beneficial to understand the consequence of rare or previously uncharacterized variants.

Keywords: WT1; kidney transplant; nephrotic syndrome; podocytopathy; renal genetics; splicing.

Figure 1

Timeline of the clinical course and evaluation.

Figure 2

Arcuate uterus in an individual harboring WT1 c.1338A>C. Axial (left), coronal (middle), and sagittal (right) MRI images demonstrating atypical uterine morphology. The arrow indicates arcuate uterine morphology. The arrowhead indicates a lack of clear uterine septation. The bracket highlights atypical vaginal morphology without a clear vaginal septum.

Figure 3

Pedigree of the family with multiple individuals affected with renal disease and uterovaginal differences. The proband (III-1) as well as one full sister (III-2) and their mother (II-2) are affected with renal disease and variable uterine differences (shaded shapes). Asterisks designate individuals that underwent sequencing and were found to harbor the WT1 c.1338A>C variant (II-2 and III-3). Individuals in generation I are deceased, although their causes of death are not due to renal disease.

Figure 4

Mini-gene assay demonstrates exon 9 skipping due to WT1 c.1338A>C. (A) Schematic of mini-gene sequence encompassing the 3,772 base pairs that constitute exon 8 through exon 10 of WT1. This was present within a pCDNA3.1 vector downstream of a T7 promotor for the expression in HEK293-T cells. Following transfection of either a control pCDNA3.1 vector containing the wild-type mini-gene sequence of exon 8–exon 10 or a site-directed variant with the base change corresponding to c.1338A>C, RNA was collected and reverse transcriptase PCR amplified to detect changes in splicing compared to control. (B) Smaller bands present for three biological replicates of the c.1338A>C vector-transfected cells that were not present in controls indicating likely exon 9 skipping due to this variant. (C) Quantified relative proportion of exon 9 skipping over the three replicates. Error bar indicates standard deviation.