ALTERED LOCALIZATION OF THE CUBN–AMN COMPLEX IN RENAL TISSUE OF A PATIENT WITH PROCHOB

Cubilin (CUBN) is expressed at the proximal tubule brush border, where it forms a complex with Amnionless (AMN) at its N-terminus to mediate albumin reabsorption. AMN and CUBN are known to be interdependent for their plasma membrane expression. Because CUBN forms a complex with AMN at its N-terminus, pathogenic variants in the CUBN N-terminus or AMN result in the failure of CUBN to be expressed at the cell membrane. Since CUBN is also involved in vitamin B12 absorption in the small intestine, pathogenic variants in the CUBN N-terminus or AMN have long been known to cause Imerslund-Gräsbeck Syndrome (IGS), characterized by tubular proteinuria and megaloblastic anemia due to vitamin B12 malabsorption. Recently, biallelic variants in the CUBN C-terminus, which is the proposed binding site for albumin, have been reported to cause chronic benign proteinuria (PROCHOB), which presents with tubular proteinuria alone, suggesting an impairment of this binding. PROCHOB is known for clinical features such as persistent proteinuria that does not respond to steroids or ACE inhibitors, yet without progression to renal function deterioration. However, there are no reports analyzing the localization changes of the CUBN–AMN complex in the renal tissue of PROCHOB patients.

We identified a pediatric patient with compound heterozygous variants in the CUBN C-terminus who presented with persistent tubular proteinuria consistent with PROCHOB. Renal biopsy tissue obtained from this patient was examined. After deparaffinization of paraffin-embedded sections, immunofluorescence staining was performed using antibodies against both the N- and C-terminal regions of CUBN and against AMN. Staining was conducted according to a standardized protocol, employing fluorescently labeled secondary IgG antibodies and counterstaining with DAPI. Fluorescence microscopy was used to qualitatively assess expression levels and localization within proximal tubules. As a control, renal tissue from an age-matched patient with nephrotic syndrome was examined under identical conditions. Images were evaluated independently by at least two investigators under blinded conditions, and localization patterns were compared with those of controls.

The subject is a pediatric patient who was noted to have proteinuria of approximately 2 g/gCr during infancy, with high levels of urinary alpha1 microglobulin (a1MG). A renal biopsy at age 1 showed focal glomerular sclerosis. Trio whole-exome sequencing of the patient and parents was performed in 2017 at age 2, but no variants considered causative of the proteinuria were identified. As the patient entered school age, persistent proteinuria of 0.5–1 g/gCr unresponsive to medication continued. Since the clinical course was similar to PROCHOB reported in 2020, we re-evaluated the initial sequencing data and identified compound heterozygous variants in CUBN (maternal: C-terminal splice variant; paternal: C-terminal missense variant). Although the missense variant was classified as a VUS under ACMG/AMP guidelines, it was extremely rare in allele frequency and predicted to be pathogenic in silico. In the renal tissue of this case, expression of CUBN N-terminus, C-terminus, and AMN remained in the proximal tubules compared to the control, but their localization to the brush border was abolished.

The identification of a C-terminal missense variant, together with the absence of anemia, suggests partial retention of function. In renal biopsy, both CUBN and AMN showed markedly reduced apical localization, indicating impaired targeting of the complex to the brush border. C-terminal variants may therefore compromise the structural stability and folding of CUBN, disrupting complex formation and localization. Given that the degree of proteinuria in this patient was comparable to IGS and accompanied by elevated urinary a1MG, PROCHOB may result in urinary loss of not only albumin but also multiple CUBN ligands, similar to IGS.