Background: Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below 30 years of age. Many monogenic forms have been discovered mainly due to comprehensive genetic testing like exome sequencing (ES). However, disease-causing variants in known disease-associated genes still only explain a proportion of cases. Aim of this study was to unravel the underlying molecular mechanism of syndromic CAKUT in a multiplex Arab and a Turkish family with presumed autosomal recessive inheritance. Methods and Results: ES in the index individuals revealed two different rare homozygous variants in FOXD2, a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arab and a missense variant in the Turkish family with family segregation patterns consistent with autosomal-recessive inheritance. CRISPR/Cas9-derived Foxd2 knock-out (KO) mice presented with bilateral dilated renal pelvis accompanied by renal papilla atrophy while extrarenal features included mandibular, ophthalmologic, and behavioral anomalies, recapitulating the phenotype of humans with FOXD2 dysfunction. To study the pathomechanism of FOXD2-dysfunction-mediated developmental renal defects, in a complementary approach, we generated CRISPR/Cas9-mediated KO of Foxd2 in ureteric-bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important in renal/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a cell identity shift towards a stromal cell identity. Histology of Foxd2 KO mouse kidneys confirmed increased fibrosis. Further, GWAS data (genome-wide association studies) suggests that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Conclusions: In summary, our data implicate that FOXD2 dysfunction is a very rare cause of autosomal recessive syndromic CAKUT and suggest disturbances of the PAX2-WNT4 cell signaling axis contribute to this phenotype.

Background and Aims In approximately 10% of adults with chronic kidney disease, a hereditary cause can be identified. Important representatives are Alport syndrome and inherited podocytopathies, which often show the histological picture of focal segmental glomerulosclerosis (FSGS). FSGS is a histological finding of various etiologies (primary, hereditary, secondary). Especially in suspected glomerular kidney disease, kidney biopsy is the diagnostic gold standard. The aim of this study was to evaluate a cohort of individuals with genetically confirmed inherited nephropathy and previous kidney biopsy to determine whether the histological examination can provide a clue to the underlying inherited kidney disease. Biopsies were further investigated by proteomics via liquid-chromatography-mass spectrometry (LC-MS) to potentially elucidate the underlying protein defect. Method The cohort for this retrospective study consisted of 23 individuals with a genetically confirmed inherited nephropathy and a previously performed kidney biopsy. A systematic pathological secondary review of the 23 biopsies was carried out (genetic diagnosis unknown at secondary review). The findings of the biopsies were compared with the molecular genetic results. 9 proband and 9 control biopsies were additionally evaluated through LC-MS. Laser capture microdissection was used to extract glomeruli from the tissue samples, which were then further analyzed on alterations in protein expression secondary to the respective disease-causing variants. Results In the cohort, disease-causing variants were identified in the following genes: COL4A3 (n = 3), COL4A5 (n = 4), WT1 (n = 3), UMOD (n = 3), and each n = 1 for the genes INF2, DAAM2, MUC1, COQ8B, NPHP4, TRIM8, CD2AP, NPHS2, CLCN5, and PAX2. The biopsies showed predominantly segmental glomerulosclerosis and parenchymal scarring, as well as podocyte damage. Four individuals with the histological diagnosis of Alport syndrome were genetically confirmed as having X-chromosomal (n = 2; including one female carrier) and autosomal-recessive (n = 2) Alport syndrome. Proteomics showed heterogeneous results. Proband samples carried variants in COL4A3 (n = 3), COL4A5 (n = 3), ADCK4, NPHP4, and WT1 (the last three each n = 1). COL4A3 was detected in 6/9 of control samples and in 0/9 of proband samples; COL4A5 was detected in 5/9 of control samples and in 0/9 of proband samples. ADCK4, NPHP4, and WT1 could not be detected in this analysis, neither in control, nor in proband samples. Conclusion In this study, molecular genetic diagnostics allowed a more precise disease assignment and thus provided information on therapy, prognosis, recurrence in the transplant, possible extrarenal phenotypes, and inheritance. Histological findings can indicate an inherited disease and help to trigger genetic testing (e.g., Alport syndrome). However, genetic diagnostics can also classify cases for which there are no typical morphological criteria described or if severe scarring impairs morphological diagnosis. Numerous cases of a respective monogenic disease would have to be analyzed in order to establish common histopathological criteria, if present. This is a challenge due to the rapid discovery of new disease-associated genes and the rarity of the respective diseases. LC-MS-based proteomics from kidney biopsy samples showed to be of limited value in further characterizing changes associated with specific variants. Unlike the genome, which is consistent due to the stability of DNA, the proteome is influenced by various effects: Different stages of fibrosis depending on the time of biopsy and other factors like coexistent disease lead to varying protein intensities even in two separate samples that present identical genetic variants. The detected protein intensity patterns could not be sufficiently correlated with the genetic findings. Despite the detection of certain proteins of interest like type IV collagens, their intensity variation due to advanced tissue damage did not allow reliable conclusions on the underlying cause. Alternatively, molecular methods such as MALDI imaging could further visualize these changes.

Disease-causing variants in COL4A3-5 are associated with type-IV-collagen-related nephropathy, a genetically and phenotypically multifaceted disorder comprising Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) and autosomal, X-linked and a proposed digenic inheritance. Initial symptoms of individuals with AS are microscopic hematuria followed by proteinuria leading to kidney failure (90% on dialysis < age 40 years). In contrast, individuals with TBMN, an outdated histology-derived term, present with microscopic hematuria, only some of them develop kidney failure (>50 years of age). An early diagnosis of type-IV-collagen-related nephropathy is essential for optimized therapy and slowing of the disease. Sixty index cases, in whom exome sequencing had been performed and with disease-causing variant(s) in COL4A3-5, were evaluated concerning their clinical tentative diagnosis and their genotype. Of 60 reevaluated individuals with type-IV-collagen-related nephropathy, 72% had AS, 23% TBMN and 5% focal segmental glomerulosclerosis (FSGS) as clinical tentative diagnosis. The FSGS cases had to be re-classified as having type-IV-collagen-related nephropathy. Twelve percent of cases had AS as clinical tentative diagnosis and a monoallelic disease-causing variant in COL4A3/4 but could not be classified as autosomal dominant AS because of limited or conflicting clinical data. This study illustrates the complex clinical and genetic picture of individuals with a type IV-collagen-related nephropathy indicating the need of a refined nomenclature and the more interdisciplinary teamwork of clinicians and geneticists as the key to optimized patient care.