Idiopathic collapsing glomerulopathy is associated with APOL1 high-risk genotypes or Mendelian variants in most affected individuals in a highly admixed population
Authors: Precil D. Neves, Andreia Watanabe, Elieser H. Watanabe, Amanda M. Narcizo, Kelly Nunes, Antonio M. Lerario, Frederico M. Ferreira, Lívia B. Cavalcante, Janewit Wongboonsin, Denise M. Malheiros, Lectícia B. Jorge, Matthew G. Sampson, Irene L. Noronha, Luiz F. Onuchic
Journal: Kidney International (2023)

Collapsing glomerulopathy (CG) is most often associated with fast progression to kidney failure with an incidence apparently higher in Brazil than in other countries. However, the reason for this occurrence is unknown. To better understand this, we performed an integrated analysis of clinical, histological, therapeutic, causative genetic and genetic ancestry data in a highly genetically admixed cohort of 70 children and adult patients with idiopathic CG (ICG). The disease onset occurred at 23 (interquartile range: 17-31) years and approximately half of patients progressed to chronic kidney disease requiring kidney replacement therapy (CKD-KRT) 36 months after diagnosis. Causative genetic bases, assessed by targeted-gene panel or whole-exome sequencing, were identified in 58.6% of patients. Among these cases, 80.5% harbored APOL1 high-risk genotypes (HRG) and 19.5% causative Mendelian variants (MV). Self-reported non-White patients more frequently had HRG. MV was an independent risk factor for progression to CKD-KRT by 36 months and the end of follow-up, while remission was an independent protective factor. All patients with HRG manifested CG at 9-44 years of age, whereas in those with APOL1 low-risk genotype, the disease arose throughout life. HRGs were associated with higher proportion of African genetic ancestry. Novel causative MVs were identified in COL4A5, COQ2 and PLCE1 and previously described causative MVs were identified in MYH9, TRPC6, COQ2, COL4A3 and TTC21B. Three patients displayed HRG combined with a variant of uncertain significance (ITGB4, LAMA5 or PTPRO). MVs were associated with worse kidney prognosis. Thus, our data reveal that the genetic status plays a major role in ICG pathogenesis, accounting for more than half of cases in a highly admixed Brazilian population. Collapsing glomerulopathy (CG) is most often associated with fast progression to kidney failure with an incidence apparently higher in Brazil than in other countries. However, the reason for this occurrence is unknown. To better understand this, we performed an integrated analysis of clinical, histological, therapeutic, causative genetic and genetic ancestry data in a highly genetically admixed cohort of 70 children and adult patients with idiopathic CG (ICG). The disease onset occurred at 23 (interquartile range: 17-31) years and approximately half of patients progressed to chronic kidney disease requiring kidney replacement therapy (CKD-KRT) 36 months after diagnosis. Causative genetic bases, assessed by targeted-gene panel or whole-exome sequencing, were identified in 58.6% of patients. Among these cases, 80.5% harbored APOL1 high-risk genotypes (HRG) and 19.5% causative Mendelian variants (MV). Self-reported non-White patients more frequently had HRG. MV was an independent risk factor for progression to CKD-KRT by 36 months and the end of follow-up, while remission was an independent protective factor. All patients with HRG manifested CG at 9-44 years of age, whereas in those with APOL1 low-risk genotype, the disease arose throughout life. HRGs were associated with higher proportion of African genetic ancestry. Novel causative MVs were identified in COL4A5, COQ2 and PLCE1 and previously described causative MVs were identified in MYH9, TRPC6, COQ2, COL4A3 and TTC21B. Three patients displayed HRG combined with a variant of uncertain significance (ITGB4, LAMA5 or PTPRO). MVs were associated with worse kidney prognosis. Thus, our data reveal that the genetic status plays a major role in ICG pathogenesis, accounting for more than half of cases in a highly admixed Brazilian population. Lay SummaryCollapsing glomerulopathy is a severe kidney disease that often leads to chronic dialysis or kidney transplantation. To expand its understanding, we performed a broad clinical and genetic analysis of a highly admixed patient population with idiopathic collapsing glomerulopathy. Interestingly, we identified a genetic cause in more than half of the 70 evaluated patients. Moreover, in a significant majority of the cases with a genetic form, the disease was associated with the presence of risk variants in both alleles of the APOL1 gene, which are genetic variants associated with African ethnicity. These patients, in fact, displayed a higher proportion of African genetic ancestry. The other genetic cases occurred due to causative variants in other genes, being classified as Mendelian forms. Mendelian cases had a higher risk of progression to kidney failure, and all patients with 2 APOL1 risk alleles manifested collapsing glomerulopathy between 9 and 44 years of age. Collapsing glomerulopathy is a severe kidney disease that often leads to chronic dialysis or kidney transplantation. To expand its understanding, we performed a broad clinical and genetic analysis of a highly admixed patient population with idiopathic collapsing glomerulopathy. Interestingly, we identified a genetic cause in more than half of the 70 evaluated patients. Moreover, in a significant majority of the cases with a genetic form, the disease was associated with the presence of risk variants in both alleles of the APOL1 gene, which are genetic variants associated with African ethnicity. These patients, in fact, displayed a higher proportion of African genetic ancestry. The other genetic cases occurred due to causative variants in other genes, being classified as Mendelian forms. Mendelian cases had a higher risk of progression to kidney failure, and all patients with 2 APOL1 risk alleles manifested collapsing glomerulopathy between 9 and 44 years of age. Collapsing glomerulopathy (CG) is an entity first described by Weiss et al.1Weiss M.A. Daquioag E. Margolin E.G. Pollak V.E. Nephrotic syndrome, progressive irreversible renal failure, and glomerular "collapse": a new clinicopathologic entity?.Am J Kidney Dis. 1986; 7: 20-28Abstract Full Text PDF PubMed Scopus (161) Google Scholar in 6 patients who presented with nephrotic syndrome (NS) and underwent fast chronic kidney disease progression with requirement of kidney replacement therapy (CKD-KRT). As per the Columbia classification, CG is classified as a histologic subtype of focal segmental glomerulosclerosis (FSGS), whereas other authors recommend it to be classified separately from FSGS, using the term CG.2D'Agati V.D. Fogo A.B. Bruijn J.A. Jennette J.C. Pathologic classification of focal segmental glomerulosclerosis: a working proposal.Am J Kidney Dis. 2004; 43: 368-382Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar,3Schwimmer J.A. Markowitz G.S. Valeri A. Appel G.B. 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Marasa M. et al.Clinical real-time genome sequencing to solve the complex and confounded presentation of a child with focal segmental glomerulosclerosis and multiple malignancies.Kidney Int Rep. 2022; 7: 2312-2316Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar genes have also been associated with CG. The assessment of CG pathogenesis, clinical features, and treatment, however, has currently been limited by the lack of studies including large numbers of CG cases as well as addressing specific CG issues. A number of questions, therefore, remain unclear. It is currently unknown how frequently CG is associated with APOL1 HRG and Mendelian pathogenic variants and how it is distributed throughout life. Along this line, it is unclear how APOL1-associated and Mendelian forms of CG are distributed throughout different age ranges. Moreover, we do not know if and how a non-African genetic background impacts the APOL1 HRG effect. To address these fundamental points, we performed the current study to evaluate molecular-genetic, clinical, and histologic parameters in a large, highly admixed Brazilian population of adult and pediatric patients diagnosed with ICG. Adult and pediatric patients with histologic diagnosis of CG established by kidney biopsy were initially selected and screened for potential secondary causes of this disorder based on clinical evaluation and complementary tests. Those with negative assessment were classified as ICG cases and initially included in this retrospective cohort. These patients were then submitted to comprehensive demographic, clinical, laboratory, and therapeutic responsiveness characterization. Therapeutic response was assessed based on the course of proteinuria and estimated glomerular filtration rate (eGFR). As per the 2021 Kidney Disease: Improving Global Outcomes (KDIGO) on glomerular diseases,42Rovin B.H. Adler S.G. Barratt J. et al.KDIGO 2021 clinical practice guideline for the management of glomerular diseases.Kidney Int. 2021; 100: S1-S276Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar a reduction in proteinuria by 50% or more from baseline, however without reaching normal levels, was considered partial remission, whereas full return to the normal range was deemed as complete remission. Progression to CKD-KRT was the primary analyzed outcome, whereas the secondary outcomes comprised time to reach CKD-KRT, response to therapy—categorized into partial remission, complete remission, or no response—and development of CG in the graft. All kidney biopsies were evaluated by specialized renal pathologists. The study was approved by the ethics committee of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo under the numbers CAAE 17279219.8.0000.0068 for the adult patients and CAAE 70756317.7.0000.0068 for the pediatric patients. DNA was extracted from peripheral blood using the QIAamp DNA Blood Mini Kit (Qiagen). Samples were submitted to target sequencing using 2 customized gene panels or to whole-exome sequencing (WES). The first customized gene panel was assembled to capture 845 target regions including all exons of 42 genes associated with NS (ACTN4, ADCK4, ARHGDIA, NPHS2, CD2AP, COQ2, CRB2, CUBN, DGKE, EMP2, EYA1, COQ6, FAT1, INF2, ITGA3, ITGB4, KANK1, KANK2, KANK4, LAMB2, LMX1B, MYO1E, NPHS1, PAX2, PDSS2, PLCE1, PODXL, PTPRO, SCARB2, SMARCAL1, TRPC6, TTC21B, WDR73, WT1, XPO5, APOL1, ARHGAP24, MEFV, NXF5, NUP107, NUP205, and NUP93), with a mean coverage >99%. DNA libraries were prepared applying the Nextera Rapid Capture enrichment protocol (Illumina) following the manufacturer's instructions. The second customized gene panel was designed to generate 579 target regions of 20 genes related to glomerulopathies and/or NS (NEIL1, COL4A3, COL4A4, COL4A5, CFH, CFI, CD46, C3, CFHR5, ADAMTS13, FN1, MCP1, ANLN, MYH9, MAGI2, TNS2, DLC1, CDK20, ITSN1, and ITSN2), also with a mean coverage >99%. In this case, DNA libraries were prepared using the Ampliseq protocol (Illumina) according to the manufacturer's orientations. The MiSeq platform (Illumina) was used to sequence the targeted regions. This task was carried out using the MiSeq Reagent Kit v2 as dual index and paired-end runs (2 × 150 cycles for Nextera Rapid Capture enrichment and 2 × 250 cycles for Ampliseq). WES was performed using 2 protocols. In the first one, DNA libraries were prepared on the Nextera Exome Rapid Capture enrichment protocol (Illumina). In this case, sequencing was performed in the NextSeq500 platform (Illumina) as dual index and paired-end runs (2 × 150 cycles) using the NextSeq High-Output Kit. In the second protocol, DNA libraries were prepared with a customized Illumina kit (Illumina) developed for the Mendelics Genomic Analysis facility (Mendelics). This approach included approximately 33 MB of targeted regions, coverage of at least 95% of targeted regions with ≥10 reads of horizontal coverage, and 100× mean depth. Each allele of the target regions located in autosomes in all patients and in X chromosomes in women was therefore represented in approximately 50% of the reads. Nova Seq 6000 (Illumina) was used for targeted gene enrichment and sequencing. The generated sequencing reads were aligned to the human reference genome GRCh37/hg19 using the Burrows-Wheeler Aligner (BWA-MEM aligner, GNU General Public License version 0.7.17; MIT License) and screened for duplicate reads with the Biobambam tool. Variant calling was performed with FreeBayes and the resulting variant call formats were annotated with the ANNOVAR software package (http://annovar.openbioinformatics.org).43Wang K. Li M. Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (9312) Google Scholar To warrant similar variant analysis in all cases, the patients submitted to WES were analyzed for the same genes included in the 2 customized gene panels. The resulting data were filtered targeting variants with at least 10 reads and with low frequency (≤0.1% according to the American College of Medical Genetics and Genomics [ACMG] guidelines) in control populations (gnomAD [https://gnomad.broadinstitute.org/], AbraOM [https://abraom.ib.usp.br/], and SELAdb [http://intranet.fm.usp.br/sela/]). It must be noted that AbraOM and SELAdb are Brazilian databases generated for populations of the São Paulo state, Brazil, which represent the background population of the current study. All but 2 patients, in fact, live/lived in the São Paulo state. The variants were ranked thereafter according to their pathogenicity potential. Stop-gain, splice-site disrupting, and frameshift variants were initially considered, since they are related to potential loss of function. Prediction of pathogenicity of missense variants was based on multiple in silico programs, including PolyPhen2, Mutation Assessor, SIFT, and PROVEAN. The assessed variants had their sequencing reads visually inspected using Integrative Genomics Viewer. Sanger sequencing was performed to confirm the potential causative variants, to proceed with segregation analysis when parental DNA was available, and to confirm APOL1 G1 and/or G2 variants. Pathogenicity of potentially causative variants was assessed applying the ACMG criteria.44Richards S. Aziz N. Bale S. et al.Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Genet Med. 2015; 17: 405-424Abstract Full Text Full Text PDF PubMed Scopus (19424) Google Scholar Variants of uncertain significance (VUSs) in COQ2 (coenzyme Q2), associated with high pathogenicity scores predicted by in silico programs and phenotypes strongly correlated with the mutated gene, were submitted to additional molecular mechanics evaluation to significantly strengthen the assessment of pathogenicity. This analysis was included in a recently submitted manuscript from our group, which strongly supported pathogenicity for both VUSs.45Watanabe A. Patogênese Genética-Molecular da Síndrome Nefrótica Córtico-Resistente, Síndrome Nefrótica Congênita e Glomeruloesclerose Segmentar e Focal: Realidade e Contribuições da Análise de Uma População Pediátrica Brasileira.Doctoral thesis. University of São Paulo School of Medicine. 2023Crossref Google Scholar ACMG-based pathogenic and likely pathogenic variants as well as the COQ2 VUSs were considered causative for the disease. Segregation was analyzed in all pedigrees with available structure and information. All monogenic variants and APOL1-HRG genotyping were confirmed by Sanger sequencing. All patients were genotyped with a specifically designed high-density single nucleotide polymorphism (SNP) array (Infinium Global Screening Array version 3.0; Illumina) to ascertain the individual genetic ancestry. The data were cleaned and filtered according to the manufacturer's recommendations using the GWASTools pipeline.46Gogarten S.M. Bhangale T. Conomos M.P. et al.GWASTools: an R/Bioconductor package for quality control and analysis of genome-wide association studies.Bioinformatics. 2012; 28: 3329-3331Crossref PubMed Scopus (138) Google Scholar Relatedness analyses of the genetic data were performed using the methodology contained in the R SNPRelate package.47Zheng X. Levine D. Shen J. et al.A high-performance computing toolset for relatedness and princi