Nephrotic Syndrome Gene TBC1D8B Is Required for Endosomal Maturation and Nephrin Endocytosis in Drosophila.

BACKGROUND: Variants in TBC1D8B cause nephrotic syndrome. TBC1D8B is a GTPase-activating protein for Rab11 (RAB11-GAP) that interacts with nephrin, but how it controls nephrin trafficking or other podocyte functions remains unclear. METHODS: We generated a stable deletion in Tbc1d8b and used microhomology-mediated end-joining for genome editing. Ex vivo functional assays utilized slit diaphragms in podocyte-like Drosophila nephrocytes. Manipulation of endocytic regulators and transgenesis of murine Tbc1d8b provided a comprehensive functional analysis of Tbc1d8b. RESULTS: A null allele of Drosophila TBC1D8B exhibited a nephrocyte-restricted phenotype of nephrin mislocalization, similar to patients with isolated nephrotic syndrome who have variants in the gene. The protein was required for rapid nephrin turnover in nephrocytes and for endocytosis of nephrin induced by excessive Rab5 activity. The protein expressed from the Tbc1d8b locus bearing the edited tag predominantly localized to mature early and late endosomes. Tbc1d8b was required for endocytic cargo processing and degradation. Silencing Hrs, a regulator of endosomal maturation, phenocopied loss of Tbc1d8b. Low-level expression of murine TBC1D8B rescued loss of the Drosophila gene, indicating evolutionary conservation. Excessive murine TBC1D8B selectively disturbed nephrin dynamics. Finally, we discovered four novel TBC1D8B variants within a cohort of 363 patients with FSGS and validated a functional effect of two variants in Drosophila, suggesting a personalized platform for TBC1D8B-associated FSGS. CONCLUSIONS: Variants in TBC1D8B are not infrequent among patients with FSGS. TBC1D8B, functioning in endosomal maturation and degradation, is essential for nephrin trafficking.

Contributions of Rare Gene Variants to Familial and Sporadic FSGS.

BACKGROUND: Over the past two decades, the importance of genetic factors in the development of FSGS has become increasingly clear. However, despite many known monogenic causes of FSGS, single gene defects explain only 30% of cases. METHODS: To investigate mutations underlying FSGS, we sequenced 662 whole exomes from individuals with sporadic or familial FSGS. After quality control, we analyzed the exome data from 363 unrelated family units with sporadic or familial FSGS and compared this to data from 363 ancestry-matched controls. We used rare variant burden tests to evaluate known disease-associated genes and potential new genes. RESULTS: We validated several FSGS-associated genes that show a marked enrichment of deleterious rare variants among the cases. However, for some genes previously reported as FSGS related, we identified rare variants at similar or higher frequencies in controls. After excluding such genes, 122 of 363 cases (33.6%) had rare variants in known disease-associated genes, but 30 of 363 controls (8.3%) also harbored rare variants that would be classified as "causal" if detected in cases; applying American College of Medical Genetics filtering guidelines (to reduce the rate of false-positive claims that a variant is disease related) yielded rates of 24.2% in cases and 5.5% in controls. Highly ranked new genes include SCAF1, SETD2, and LY9. Network analysis showed that top-ranked new genes were located closer than a random set of genes to known FSGS genes. CONCLUSIONS: Although our analysis validated many known FSGS-causing genes, we detected a nontrivial number of purported "disease-causing" variants in controls, implying that filtering is inadequate to allow clinical diagnosis and decision making. Genetic diagnosis in patients with FSGS is complicated by the nontrivial rate of variants in known FSGS genes among people without kidney disease.

A risk allele for focal segmental glomerulosclerosis in African Americans is located within a region containing APOL1 and MYH9.

Genetic variation at the MYH9 locus is linked to the high incidence of focal segmental glomerulosclerosis (FSGS) and non-diabetic end-stage renal disease among African Americans. To further define risk alleles with FSGS we performed a genome-wide association analysis using more than one million single-nucleotide polymorphisms in 56 African-American and 61 European-American patients with biopsy-confirmed FSGS. Results were compared to 1641 European Americans and 1800 African Americans as unselected controls. While no association was observed in the cohort of European Americans, the case-control comparison of African Americans found variants within a 60 kb region of chromosome 22 containing part of the APOL1 and MYH9 genes associated with increased risk of FSGS. This region spans different linkage disequilibrium blocks, and variants associating with disease within this region are in linkage disequilibrium with variants which have shown signals of natural selection. APOL1 is a strong candidate for a gene that has undergone recent natural selection and is known to be involved in the infection by Trypanosoma brucei, a parasite common in Africa that has recently adapted to infect human hosts. Further studies will be required to establish which variants are causally related to kidney disease, what mutations caused the selective sweep, and to ultimately determine if these are the same.

Autosomal Dominant Tubulointerstitial Kidney Disease-Uromodulin Misclassified as Focal Segmental Glomerulosclerosis or Hereditary Glomerular Disease.

INTRODUCTION: Focal segmental glomerulosclerosis (FSGS) is a histopathologically defined kidney lesion. FSGS can be observed with various underlying causes, including highly penetrant monogenic renal disease. We recently identified pathogenic variants of UMOD, a gene encoding the tubular protein uromodulin, in 8 families with suspected glomerular disease. METHODS: To validate pathogenic variants of UMOD, we reviewed the clinical and pathology reports of members of 8 families identified to have variants of UMOD. Clinical, laboratory, and pathologic data were collected, and genetic confirmation for UMOD was performed by Sanger sequencing. RESULTS: Biopsy-proven cases of FSGS were verified in 21% (7 of 34) of patients with UMOD variants. The UMOD variants seen in 7 families were mutations previously reported in autosomal dominant tubulointerstitial kidney disease-uromodulin (ADTKD-UMOD). For one family with 3 generations affected, we identified p.R79G in a noncanonical transcript variant of UMOD co-segregating with disease. Consistent with ADTKD, most patients in our study presented with autosomal dominant inheritance, subnephrotic range proteinuria, minimal hematuria, and renal impairment. Kidney biopsies showed histologic features of glomerular injury consistent with secondary FSGS, including focal sclerosis and partial podocyte foot process effacement. CONCLUSION: Our study demonstrates that with the use of standard clinical testing and kidney biopsy, clinicians were unable to make the diagnosis of ADTKD-UMOD; patients were often labeled with a clinical diagnosis of FSGS. We show that genetic testing can establish the diagnosis of ADTKD-UMOD with secondary FSGS. Genetic testing in individuals with FSGS histology should not be limited to genes that directly impair podocyte function.