Classification of biopsy findings in individuals with nephropathies usingmolecular genetic testing and proteomics

Abstract 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.