De Novo CUL3 Variant in a Child Presenting With Hypertension and Kidney Failure

Introduction

Monogenic diseases are an underappreciated but important cause of chronic kidney disease.¹ They are estimated to be responsible for 70% of kidney failure cases in children and 10% to 15% in adults. Genetic testing plays a crucial role in the management of kidney diseases by identifying the molecular etiology, providing more accurate prognostic insights, and guiding management and therapy.S1–S3 Nevertheless, the interpretation of genetic variants may prove challenging, requiring collaboration between (pediatric) nephrologists and clinical and molecular geneticists. We present a case of thrombotic microangiopathy (TMA), malignant hypertension, and kidney failure in which a de novo CUL3 variant was identified and highlight the importance of genetic workup and a transdisciplinary approach.

Case Presentation

An 11-year-old Caucasian girl with an antenatally diagnosed congenital cystic adenomatoid malformation in the left chest, developmental coordination disorder and short stature (−2.0 SD), presented with kidney failure, oliguria, and early-onset malignant hypertension (Supplementary Tables S1 and S2). Initial blood tests (Supplementary Figure S1) indicated chronic kidney failure, as suggested by elevated parathormone and anemia with low reticulocyte counts, and signs of TMA. Renal ultrasound showed normal-sized kidneys with bilateral hyperechogenic cortex. Kidney biopsy (Supplementary Figure S2) showed severe glomerular damage with pseudocrescent formation, without immune complex deposition (total renal chronicity score of 9).S4 Gene panel sequencing for atypical hemolytic uremic syndrome yielded normal results (Supplementary Methods), and TMA resolved after blood pressure correction. Subsequent more extensive genetic testing revealed a de novo variant in CUL3.

Despite optimized peritoneal dialysis, dietary restrictions, and triple antihypertensive therapy, blood pressure control remained suboptimal (Supplementary Figure S3; Supplementary Table S1). Conventional hemodiafiltration (HDF) failed, prompting a switch to in-center nocturnal HDF (3 × 9 h/wk) with low-sodium dialysate and strict dry weight monitoring. Given the emerging efficacy of hydrochlorothiazide in adults with therapy-resistant hypertension and advanced chronic kidney disease, a trial with hydrochlorothiazide was implemented.² This approach enabled blood pressure control, tapering of antihypertensive therapy, and nephrectomy deferral.

After 20 months of dialysis, she underwent a living-related donor kidney transplant. Because steroids and tacrolimus pose a risk for malignant hypertension and TMA in the early posttransplant period, we adhered to a low-steroid immunosuppressive strategy.³ The peri-transplant course was uneventful with adequate blood pressure control, and she was discharged 10 days posttransplant. Currently, at 22 months posttransplant, she remains clinically stable, with blood pressure controlled on amlodipine monotherapy.

Results

The unexplained kidney failure in this girl prompted exome sequencing with virtual panel analysis of known nephropathy genes (Supplementary Methods). This revealed a de novo heterozygous frameshift variant in exon 9 of CUL3 (c.1349del, p.[Ser450LeufsTer5], NM_003590.4) (Figure 1a; Supplementary Methods). This variant was classified as a variant of uncertain significance (ClinGen Bayesian P = 0.812, corresponding to a “hot” class 3 variant) according to the Bayesian revision of the guidelines of the American College of Medical Genetics and Genomics, using the Sherloc (semiquantitative, hierarchical evidence-based rules for locus interpretation) rulesS5^,S6 (Supplementary Table S3). Nonsense variants in CUL3 have been linked to impaired neurodevelopment (OMIM 619239), whereas variants that trigger exon 9 skipping cause AD pseudohypoaldosteronism type 2E (OMIM 614496) and early-onset arterial hypertension.4, 5, 6 However, in silico splicing analysis and RNA-sequencing (Figure 1c; Supplementary Figure S4; Supplementary Table S4; Supplementary Methods) supported the hypothesis that the c.1349del variant can induce exon 9 skipping.S7^,S8 Based on these results the CUL3 variant was reclassified as likely pathogenic (class 4, P = 0.949, Supplementary Table S4 and S5).

Figure 1.

In-depth study of the c.1349del CUL3 variant. (a) Schematic representation of the normal splicing of CUL3, and c.1349del CUL3–induced nonsense-mediated mRNA decay (NMD) and nonsense-associated altered splicing (NAS). NMD and NAS are competitive RNA quality control mechanisms, and the NMD inhibitor cycloheximide (CHX) blocks the NMD pathway in favor of the NAS pathway.S16 After inhibition of the NMD pathway, the c.1349del allele frequency increased from 8% in the untreated patient sample to 34% in the sample treated with CHX. NMD leads to haploinsufficiency; however, exon 9 skipping via NAS is predicted to produce an abnormal Cullin 3 protein missing part of the 4-helix-bundle domain (CUL3-Δ9). Cullin 3 protein structure was taken from the AlphaFold database.S17^,S18 The AlphaFold 3 server was used to predict the CUL3-Δ9 protein structure.S19 (b) Sashimi plots of exons 7 to 10 of CUL3. RNA-sequencing (RNA-Seq) on cultured peripheral blood mononuclear cells showed exon 9 skipping in only 3 of 230 (1.3%) splice junction reads in the patient sample; however, this increased to 14 of 111 (12.6%) after treatment of the cell cultures with CHX. RNA-Seq was performed on untreated and treated (+ CHX) cultured peripheral blood mononuclear cells of the patient and 2 controls. (c) In silico splicing analysis. The Human Splicing Finder (GENOMNIS) and EX-SKIP (not shown) predict that the c.1349del variant significantly alters the ratio between exonic splicing enhancer (ESE) motifs and exonic splicing silencer (ESS) motifs.S7^,S8 The red line represents the exonic splicing ratio (ESR) profile for the CUL3 c.1349del allele. The blue line represents the wild type allele. An ESR value > 0 signifies that there are more exonic splicing silencers than enhancers; a value > 0 signifies the opposite. (d) Pathophysiological effects of CUL3-Δ9. The CUL3-Δ9 protein disrupts the interaction with the other constituents of the Cullin-RING ubiquitin ligase, KLHL3 (Kelch-like 3), and RING (really interesting new gene).⁶ This interferes with ubiquitination and proteasomal degradation of With no lysine (WNK) kinases and results in increased activation of the Na⁺-Cl⁻ cotransporter (NCC) in the distal convoluted tubule (DCT), and electrolyte abnormalities and hypertension.⁸ CUL3-Δ9 also causes hypertension via vascular smooth muscle and endothelial mechanisms, possibly explaining the more severe and early-onset hypertension seen in this type of PHA.⁸ VAF, variant allele frequency.

Discussion

We present a child with kidney failure and TMA secondary to hypertension, in whom we identified a de novo heterozygous frameshift variant in CUL3. This frameshift variant, which affects the exonic splicing motifs in exon 9 of CUL3, may explain the early-onset hypertension in our patient, as is typically seen in CUL3-associated PHA2.

Until now, only variants causing a skip of exon 9 and an in-frame deletion of 4 amino acids in exon 10 (c.1420_1431del12, p. Phe474_Met477del) were linked to CUL3-associated PHA2.^6,7 Exon 9 encodes 57 amino acids in the 4-helix bundle domain of Cullin 3 (CUL3-Δ9), and mounting evidence suggests that CUL3-Δ9 causes electrolyte abnormalities and hypertension via different pathways⁸ (Figure 1c). In contrast, nonsense and frameshift variants, like ours, are associated with neurodevelopmental disorders, presenting with global developmental delay, mild to severe intellectual disability, seizures, and autism spectrum disorder, but typically without kidney disease. De novo variants in the human genome are rare, which supports the pathogenicity of the identified CUL3 variant.S9–S11 However, this apparent phenotype-genotype paradox was puzzling. Therefore, we hypothesized that the c.1349del variant in CUL3 might induce exon skipping, produce CUL3-Δ9 protein, and cause early-onset hypertension in our patient through so-called nonsense-associated altered splicing. Nonsense-associated altered splicing is an RNA quality control mechanism that upregulates transcripts that skip a disturbing premature truncation codon–containing exon via alternative splicing. It is hypothesized to be a rescue mechanism that competes with nonsense-mediated mRNA decay and allows the production of an alternative (partially) functional protein instead of a completely inactive one (Figure 1a).S12^,S13 The mechanisms underlying nonsense-associated altered splicing are incompletely understood; however, one model proposes a disruption of exonic splicing enhancer and exonic splicing silencer motifs and their ratio.S14 Notably, intron 8 of the CUL3 gene features a weak acceptor site with a Berkeley Drosophila Genome Project Splice Site Prediction score of 0.51, making it susceptible to subtle changes in cis-regulatory splicing motifs.S15 Our in silico and RNA-sequencing data support the hypothesis of nonsense-associated altered splicing through a change in the exonic splicing enhancer / exonic splicing silencer ratio, although exon skipping was only observed in a minority of splice junction reads (Figure 1b and c). Further research is needed to assess its effect on RNA splicing, protein synthesis and function.

Although our patient’s phenotype deviates from typical PHA2, there is clinical evidence to support the CUL3 variant’s pathogenicity. The high-anion gap metabolic acidosis and hyperkalemia at presentation were likely chronic kidney disease–related. Nevertheless, although serum potassium, bicarbonate, and chloride levels deviated from reported means for exon 9-skipping CUL3 variants, they remain within the outlier range in Boyden et al.⁶’s cohort. Furthermore, a comprehensive TMA workup found no alternative explanation other than hypertension (Supplementary Tables S1 and S2). Interestingly, Blackburn et al.⁵ recently reported early-onset hypertension without hyperkalemia in 5 (out of 35) patients with de novo loss-of-function CUL3 variants, further supporting the phenotype’s variability in CUL3 variants. Finally, our patient presented with short stature and developmental coordination disorder, both associated with pathogenic CUL3 variants.4, 5, 6, 7

Ultimately, this case underscores the effectiveness of an extended HDF regimen with low-sodium dialysate for controlling hypertension in patients with severe (monogenic) hypertension and kidney failure, even without nephrectomy. Extended hemodialysis/HDF regimens have shown beneficial effects on blood pressure control and left ventricular hypertrophy and should be considered for dialysis patients with therapy-resistant hypertension.⁹ In addition, we postulate that the initiation of hydrochlorothiazide improved blood pressure control, although its impact relative to the extended HDF regimen is unclear. Previous research highlighted that thiazides reduce ambulatory systolic blood pressure in adults with therapy-resistant hypertension and advanced chronic kidney disease by improving fluid management and reducing peripheral resistance.²

In conclusion, this unique and atypical case of kidney failure in a patient with a de novo heterozygous frameshift variant in exon 9 of CUL3 highlights the importance of genetic testing and its potential impact on clinical decision making; it also emphasizes the challenges in interpreting genetic variants and the crucial role of transdisciplinary care (Table 1).

Table 1.

Teaching points

•We describe a case of early-onset hypertension, TMA, and kidney failure in which a de novo CUL3 variant was identified.

•Genetic testing supplements conventional practice in unexplained cases of kidney failure and TMA.

•Genetic test results can be challenging to interpret and are subject to transdisciplinary discussion.

•Extended HD/HDF regimens as well as addition of hydrochlorothiazide may have beneficial effects in patients with therapy-resistant hypertension and chronic kidney disease.

HD, hemodialysis; HDF, hemodiafiltration; TMA, thrombotic microangiopathy.

Disclosure

All the authors declared no competing interests.

Patient Consent

The authors declare that they have obtained consent from the patient discussed in the report.

Supplementary Material

Supplementary File (PDF)

Supplementary Methods. Supplementary References. Figure S1. Schematic overview of the clinical and biochemical presentation and management. Figure S2. Picture of a hematoxylin-eosin–stained section of the kidney biopsy. Figure S3. Schematic overview of the management of the therapy-resistant hypertension. Figure S4.In silico splicing analysis using the Human Splicing Finder. Table S1. Summary of laboratory results and blood pressure measurements prior to CKD diagnosis and during follow-up. Table S2. Overview of the non-genetic diagnostic work-up for the early-onset hypertension. Table S3. Criteria used for classifying the CUL3 c.1349del variant. Table S4. Human Splicing Finder prediction of broken and created exonic splicing motifs. Table S5. Pathogenic frameshift and nonsense variants in exon 9 of CUL3.