Contribution of Recessive Ciliopathy Genes in a Highly Consanguineous Adult Cohort with Biopsy-Proven Focal Segmental Glomerulosclerosis

Contribution of Recessive Ciliopathy Genes in a Highly Consanguineous Adult Cohort with Biopsy-Proven Focal Segmental Glomerulosclerosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Contribution of Recessive Ciliopathy Genes in a Highly Consanguineous Adult Cohort with Biopsy-Proven Focal Segmental Glomerulosclerosis Lava Ahmed, Dlnya Mohammed, Dana Ahmed Sharif This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9125192/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Background Focal segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome in adults and frequently progresses to end-stage renal disease. Emerging evidence suggests recessive ciliopathy genes contribute to glomerular disease, particularly in consanguineous populations, but their role in adult biopsy-proven FSGS remains incompletely defined. This study aimed to determine the frequency and clinical impact of pathogenic variants in recessive ciliopathy genes among adults with FSGS from a highly consanguineous Middle Eastern population. Methods We conducted a case-control study including 35 adults with biopsy-proven FSGS and 20 unaffected individuals with normal kidney function from Sulaymaniyah, Iraq. All participants underwent targeted next-generation sequencing of a 98-gene panel on the Ion S5 platform. Variants were classified according to 2015 American College of Medical Genetics and Genomics (ACMG) criteria; only pathogenic (P) and likely pathogenic (LP) variants in recessive ciliopathy genes were analyzed. Formal rare-variant burden analysis was performed. Demographic and clinical data were compared between groups. Results Biallelic ciliopathy variants were identified in 16 of 35 FSGS patients (45.7%) versus 0 of 20 controls (p < 0.001; OR 34.7, 95% CI 1.9-618.7). Seven ciliopathy genes were implicated: INPP5E (10 patients, 28.6%), BBS2 (7 patients, 20.0%), CPLANE1 (6 patients, 17.1%), CEP290 (4 patients, 11.4%), TCTN2 (1 patient), BBS7 (1 patient), and NPHP3 (1 patient). All identified variants were absent or ultra-rare (allele frequency < 0.000007) in gnomAD v4.1, confirming their rarity. Ciliopathy-positive patients demonstrated universal proteinuria, higher rates of hematuria (81.3% vs 21.1%, p < 0.001), more severe renal dysfunction (serum creatinine 3.6 ± 0.9 vs 1.2 ± 0.4 mg/dL, p < 0.001), and increased hypertension prevalence (68.8% vs 31.6%, p = 0.002) compared with ciliopathy-negative FSGS patients. Conclusions In this highly consanguineous Middle Eastern cohort, recessive ciliopathy gene variants, particularly in INPP5E, BBS2, and CPLANE1, were frequent among adults with biopsy-proven FSGS and associated with severe progressive kidney disease. These findings support incorporating ciliopathy gene testing into the diagnostic evaluation of adults with FSGS from consanguineous families or with positive family history of kidney disease. Ciliopathy Focal segmental glomerulosclerosis Consanguinity Genetic testing Next-generation sequencing Burden analysis Figures Figure 1 1. Background Focal segmental glomerulosclerosis (FSGS) represents a clinically and genetically heterogeneous kidney disorder characterized by sclerosis of some glomeruli with involvement of portions of affected glomeruli ( 1 ). FSGS accounts for approximately 40% of cases of adult nephrotic syndrome and leads to end-stage renal disease (ESRD) as a major worldwide cause ( 2 – 4 ). This clinical heterogeneity reflects diverse underlying mechanisms including podocytopathies, secondary forms, and monogenic causes.( 3 , 5 , 6 ). More than 50 monogenic FSGS genes have been identified, including podocyte structural genes (NPHS1, NPHS2, WT1, LAMB2, CD2AP, TRPC6) and collagen IV genes (COL4A3–COL4A5), which typically underlie early-onset or familial disease ( 5 – 10 ). FSGS has not been the subject of detailed genetic analysis because the genes encoding the proteins of the primary cilium and centrosome are not fully understood ( 11 , 12 ). The primary cilium is a signaling organ responsible for maintaining cellular homeostasis. Mutations in cilia function impair numerous critical processes required for normal glomerular development and maintenance of the filtration barrier. The recent identification of mutations in the TTC21B gene leading to late-onset familial FSGS due to intraflagellar transport defects established that ciliopathies are responsible for more than just congenital and developmental kidney diseases( 11 , 13 , 14 ). In subsequent reports, other multiple ciliopathy genes carrying mutations that are also associated with FSGS have been described. CEP290 mutations that cause Joubert syndrome/nephronophthisis have been described in patients with FSGS ( 15 – 17 ). Similarly, CC2D2A mutations are also associated with FSGS with nephronophthisis overlap ( 15 ). The Joubert syndrome/MORM syndrome causing mutation in INPP5E gene a ciliary protein essential for intraflagellar transport has not yet been described in confirmed cases of FSGS ( 18 – 20 ). BBS2 and BBS7 along with other BBS genes necessary for BBSome complex biogenesis involved in cilia formation have also been identified as genes responsible for causing glomerular disease ( 21 – 24 ) Consanguineous marriages result in an increased chance of homozygous mutations and therefore an increased expression of recessive genes responsible for ciliopathies ( 25 , 26 ). Ciliopathies and other recessive genetic disorders are over-represented in Middle Eastern and North African populations where consanguinity rates are estimated to be above 20% ( 27 – 29 ). This study employed the occurrence of mutations in ciliary genes in a cohort of 35 adult FSGS patients and 20 controls from a highly consanguineous population. Using targeted Next Generation Sequencing (NGS) of a 98 gene panel we found pathogenic mutations in 16 cases (45.7%) confirming the relevance of mutations in ciliary genes in the etiology of FSGS in this population. We describe the clinical and genetic heterogeneity of mutations and contrast them with those identified in controls. These data highlight the importance of the sequencing of ciliary genes in adult patients with FSGS, particularly in cases with a positive family history and/or consanguinity. 2. Methods 2.1 Study Population 35 adult patients with FSGS confirmed by kidney biopsy and 20 controls from the healthy population of Sulaymaniyah, Iraq, with normal kidney function. This study population included FSGS patients whose biopsy kidneys were evaluated by light microscopy (LM) and immunofluorescence and electron microscopy. Patients were excluded if their FSGS was associated with lupus or vasculitis or if it was drug-associated, and obesity-associated glomerulosclerosis. Control Individuals: Healthy controls had: (i) normal serum creatinine (ii) no proteinuria (urine protein <0.15 g/24 hours); (iii) normal blood pressure (<130/80 mmHg); (iv) no personal or family history of kidney disease. 2.2 Ethics approval and consent to participate The study was conducted in compliance with the Declaration of Helsinki and Good Clinical Practice. Ethical approval was given by the Research Ethics Committee of University of Sulaymaniyah, Kurdistan Region, Iraq. Written informed consent was obtained from all participants. Written informed consent was obtained from the parents or guardians of children less than 18 years old. 2.3 Clinical and Laboratory Assessments Patient characteristics at the time of biopsy were recorded including age, sex, family history of kidney disease and parental consanguinity. Additional baseline information included serum creatinine and blood urea nitrogen levels (all in mg/dL), protein in urine, dipstick hematuria ≥1+ and blood pressure measured and history of previous kidney transplant. 2.4 Genetic Analysis and Sequencing DNA from peripheral blood leukocytes was extracted from patients using a Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA). A custom panel of 98 genes was designed using the Ion AmpliSeq Designer software (v6.2; Thermo Fisher Scientific) with respect to the hg19/GRCh37 genome assembly. The genes included in this panel NPHP1, NPHP3, CEP290, TMEM67, BBS1, BBS2, BBS7 and INPP5E, TCTN1–3, CC2D2A and many others. DNA libraries were then prepared using the Ion AmpliSeq Library Kit 2.0 and sequenced on an Ion Torrent S5 (Thermo Fisher Scientific) platform with Ion 540 chips as recommended by the manufacturer. The custom panel contained 2,742 amplicons of length between 125 and 275 bp distributed in two primer pools and it provided in silico 99% coverage of coding exons and exon–intron boundaries. The mean depth of coverage for all genes was over 200× and more than 98% of the targeted bases had a coverage of at least 20×. 2.5 Variant Annotation and Classification All sequencing data was analyzed using Torrent Suite Software from Thermo Fisher Scientific. After primer trimming and read alignment to the hg19/GRCh37 reference genome using TMAP, the Torrent Variant Caller with parameters of 5% minimum variant allele fraction (VAF) and 100× minimum read depth was used to call variants. Annotation was performed using the Franklin platform from Genoox, which integrates population data from gnomAD v4.1, 1000 Genomes and ExAC, as well as prediction scores from SIFT, PolyPhen‑2, CADD, FATHMM, MutationTaster scores, PhyloCSF and GERP conservation scores. MAF in any of the gnomAD v4.1 populations was required to be ≤0.0001 and we were cautious when including Middle Eastern and North African populations to avoid including regional polymorphisms. Variants were classified as pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign, or benign according to the 2015 ACMG guidelines (30). All subsequent analyses were performed using only P and LP variants, with VUS, likely benign, and benign variants excluded. The variants identified here have been submitted to ClinVar (NCBI) and are available under accession numbers SCV007520383–SCV007520396 . The transcript and HGVS nomenclature of the P/LP variants, their zygosity, the number of affected patients and the corresponding allele frequencies in the gnomAD v4.1 are shown in Supplementary Table S1. 2.6 Statistical Analysis All values are given as means ± SD. For comparison of continuous variables, independent-samples t-test or one-way ANOVA was used. For categorical variables, the data were presented as the number and percentage and the chi-square or Fisher’s exact test was used. All the tests were two tailed and p < 0.05 was considered to be statistically significant. We identified carriers to be those with ≥1 P or LP variants in any of the ciliopathy genes in a homozygous (biallelic) configuration as predicted by the AR pattern of inheritance for FSGS. We calculated the OR and 95% CI (Haldane correction) to compare the carrier status versus FSGS using Fisher’s exact test. In addition, gene-specific burden tests for INPP5E, BBS2, CPLANE1 and CEP290 were carried out. The frequency of the identified alleles/alternate bases within the genome aggregation database v4.1 (gnomAD v4.1) and their confirmed rarity, particularly in Middle Eastern and North African populations, was verified for pathogenicity of the identified variants. The SPSS v25.0 statistical software (IBM Corp., Armonk, NY, USA) and Python 3.9 (SciPy library) were used for these analyses. The exploratory gene-specific p-values are provided without adjustment. With four gene-specific burden tests being carried out, the Bonferroni adjustment for a threshold of α = 0.0125 is required. 3. Results 3.1. Study Cohort Characteristics 35 adults with biopsy‑proven FSGS and 20 healthy controls were studied in this cohort (Table 1). The mean age at diagnosis of FSGS was 25.1 ± 7.9 years (range 14–52). Eighty percent of FSGS patients (28/35) were from consanguineous families, which is higher than none of the controls (0/20; p<0.001). Pathogenic or likely pathogenic variants in genes encoding subunits of the cilia were identified in 16 of 35 FSGS patients (45.7%) and in 0 of 20 controls (p<0.001). Clinical characteristics of Ciliopathy positive FSGS patients had universal proteinuria (16/16, 100%) and frequent hematuria (13/16, 81.3%). They had a significantly worse renal function (higher serum creatinine levels, 3.6 ± 0.9 vs 1.2 ± 0.4 mg/dL; BUN, 76.4 ± 31.2 vs 29.7 ± 10.5 mg/dL) and higher ESRD performed transplantation rates (31.3% vs 10.5%) than the ciliopathy negative patients with FSGS. Table 1. Demographic, Clinical, and Laboratory Characteristics of Study Participants Characteristic FSGS Ciliopathy-Positive (n=16) FSGS Ciliopathy-Negative (n=19) Healthy Controls (n=20) P-value Age, years (mean ± SD, range) 25.1 ± 7.9 (14–52) 27.4 ± 8.1 (16–48) 26.8 ± 7.5 (18–45) NS Male/Female, n 10/6 11/8 12/8 NS Clinical Features Consanguinity, n (%) 13 (81.3) 3 (15.8) 0 (0) <0.001 Family history of kidney disease, n (%) 16 (100) 5 (26.3) 0 (0) <0.001 Laboratory Parameters at Baseline Serum creatinine, mg/dL (mean ± SD) 3.6 ± 0.9 1.2 ± 0.4 0.8 ± 0.2 <0.001 Blood urea nitrogen, mg/dL (mean ± SD) 76.4 ± 31.2 28.3 ± 12.1 16.2 ± 5.8 <0.001 Proteinuria ≥0.5 g/24 hr, n (%) 16 (100) 5 (26.3) 0 (0) <0.001 Hematuria (dipstick ≥1+), n (%) 13 (81.3) 4 (21.1) 0 (0) <0.001 Clinical Outcomes Hypertension, n (%) 11 (68.8) 6 (31.6) 0 (0) 0.002 ESRD/Kidney transplant, n (%) 5 (31.3) 2 (10.5) 0 (0) 0.053 NS, not significant; ESRD, end-stage renal disease; eGFR, estimated glomerular filtration rate. 3.2. Rare-Variant Burden Analysis To assess the degree of enrichment of recessive ciliopathy variants we performed gene set burden testing on the biallelic pathogenic/likely pathogenic carrier status. Of the 35 FSGS patients, 16 (45.7%) were carriers, compared to 0 of the 20 controls (Fisher’s exact p<0.001) giving an odds ratio of 34.7 (95% CI 1.9–618.7) after Haldane correction. The distribution of these recessive ciliopathy genes among FSGS patients is shown in Figure 1. Gene specific burden tests for INPP5E, BBS2, CPLANE1 and CEP290 are shown in (Supplementary Table S2). There is enrichment for INPP5E (10/35 cases vs 0/20; OR 17.0, 95% CI 1.0–294.0; p=0.011) and BBS2 (7/35 vs 0/20; OR 10.7, 95% CI 0.6–199.5; p=0.053). There is weaker evidence for carrier status for CPLANE1 (6/35; p=0.076) and CEP290 (4/35; p=0.285). The wide CI are due to the small number of cases in this analysis and the fact that we have not identified any carriers in the control cohort, so we simply treat the gene level p values as hypotheses to be tested in larger data sets. Further studies with larger multi-center cohorts will be needed to confirm our observation of an enrichment of ciliopathy variants in adult FSGS. Sequencing data were re analyzed from over 730,000 individuals in gnomAD v4.1 and examined the prevalence of the common frameshift mutations in INPP5E and BBS2 as well as all reported truncating mutations in CPLANE1. The INPP5E c.367_368delGC (p. Ala123ProfsTer27) mutation that results in a premature stop codon at c.385 (p. Ala123ProfsTer27)) was present in homozygote form in all 10 FSGS individuals carrying an INPP5E mutation, was not present in gnomAD or in the exomes of any of the individuals in the Middle Eastern populations. The 3 BBS2 frameshift mutations c.1254delT, c.1725delT, c.106_107delCA as well as 5 of the reported CPLANE1 frameshift mutations were not present or were found to be ultra rare (AF < 0.000007) as were variants in CEP290, TCTN2, BBS7 and NPHP3.The universal absence or extreme rarity of these truncating mutations, and the fact that they are present in homozygote form only in individuals with FSGS confirms their role as pathogenic mutations in this disease. 3.3. Genetic Findings: Spectrum of Ciliopathy Mutations Targeted panel sequencing identified 16 of 35 FSGS patients (45.7%) with pathogenic or likely pathogenic ciliopathy variants. The 35 pathogenic/likely pathogenic variant alleles were detected in 16 individuals; 15 frameshift mutations were identified, and all were truncating (100%). Using the ACMG 2015 standards, 6 variants (40.0%) were classified as pathogenic, and 9 (60.0%) as likely pathogenic. There were no VUS, likely benign, or benign variants that were retained in these patients who had variants in the ciliopathy genes. 3.3.1 Ciliopathy Gene Variant Frequency and Spectrum The 16 ciliopathy‑positive patients carried variants in seven genes: INPP5E, BBS2, CPLANE1, CEP290, TCTN2, BBS7, and NPHP3 (Table 2). Mutations in INPP5E were identified in 10 of 16 ciliopathy positive patients (62.5%) and in 5 of 18 (28.6%) of the full FSGS cohort. All carried the same homozygous frameshift mutation c.367_368delGC (p.Ala123ProfsTer27) which is a population specific founder mutation. Mutations in BBS2 were present in 7 of 16 (43.8%; 20.0% of the full FSGS cohort) ciliopathy positive patients and were identified as three frameshift mutations; c.1254delT, c.1725delT and c.106_107delCA which were all present in biallelic combinations. CPLANE1 variants occurred in 6 of 16 ciliopathy‑positive patients (37.5%; 17.1% of the cohort) across five frameshift alleles (c.1819delT, c.1868_1869insT, c.8263delA, c.6599delT, c.3912delA). CEP290 variants were detected in 4 patients (11.4% of the cohort), TCTN2 in 1 patient, and BBS7 and NPHP3 in 1 patient each. Complete variant information, including ACMG classification, number of patients carrying each allele, and gnomAD v4.1 allele counts and frequencies, is presented in Table 3 and Supplementary Table S1. 3.4 Association between ciliopathy genes and genotype–phenotype correlations 3.4.1 Age at FSGS diagnosis and clinical presentation There was no statistically significant difference in age at diagnosis between genes (p > 0.05, Table 2). INPP5E, BBS2, CEP290 and CPLANE1 mutations typically presented in early to mid-twenties, while BBS7 and NPHP3 cases presented in their thirties, more in keeping with adult onset FSGS than childhood podocytopathy. 3.4.2 Biochemical parameters: serum creatinine and urea Baseline renal function was associated with the expression of the gene encoding creatinine and BUN (p < 0.001 and p < 0.05, respectively, (Tables 2). The highest levels of BUN were recorded for the INPP5E, CPLANE1 and BBS2 genes, while the lowest BUN values were recorded for the CEP290, NPHP3 and BBS7 genes. These values corresponded to those recorded for creatinine, indicating that the extent of renal impairment was gene-specific. 3.4.3 Proteinuria and hematuria Proteinuria was present in all 16 ciliopathy positive patients, compared to 26.3% of ciliopathy negative FSGS patients and none of the controls (both p<0.001). Hematuria was also more common in ciliopathy positive patients (81.3% vs 21.1% and 0%; p<0.001), particularly in CEP290, TCTN2, BBS7 and INPP5E mutation carriers. 3.4.4 Hypertension and transplant outcomes Hypertension was more common in ciliopathy positive than ciliopathy negative individuals (68.8% vs 31.6%; p=0.051) and was not detected in controls (p<0.001). Kidney transplantation for ESRD had occurred in 31.3% of ciliopathy positive individuals and 10.5% of ciliopathy negative individuals, with no further transplants recorded. Table 2. Genotype-Phenotype Correlations by Ciliopathy Gene Gene Patients (n) Variants (n) Unique Mutations Age at Dx (yr) Creatinine (mg/dL) BUN (mg/dL) Consanguinity (%) Family Hx (%) Hematuria (%) Hypertension (%) INPP5E 10 10 c.367_368delGC 23.2±8.4 4.0±1.3 88.6±25.3 33.3 77.8 88.9 22.2 BBS2 7 9 c.1254delT c.1725delT c.106_107delCA 27.3±4.1 3.8±0.4 80.3±12.8 28.6 75.0 71.4 33.3 CPLANE1 6 9 c.8263delA c.1868_1869insT c.1819delT c.6599delT c.3912delA 29.3±10.8 3.7±2.3 88.5±38.9 66.7 100 33.3 50.0 CEP290 4 4 c.2462delT c.6416_6417delAA c.2523delA 24.8±6.9 1.8±0.8 45.6±20.1 50.0 75.0 100 0 TCTN2 1 1 c.1206delT 23.0 2.5 68.5 100 100 100 0 BBS7 1 1 c.1759_1760delAG 30.0 4.0 52.0 0 100 100 0 NPHP3 1 1 c.2161delA 30.0 4.0 44.0 100 100 33.3 0 Dx, diagnosis; BUN, blood urea nitrogen; Hx, history 3.5. Inheritance Patterns and Consanguinity All 16 patients with FSGS that were found to have mutations in the genes associated with ciliopathy had biallelic mutations (Table 3). Consanguinity was reported in 81.3% of the ciliopathy positive patient’s vs 15.8% of the ciliopathy negative FSGS patients (p<0.001) a 5.3-fold enrichment. Consanguinity rates for each gene were as follows: all carriers in the TCTN2 and NPHP3 cohorts were consanguineous and although the consanguinity rate for CPLANE1 carriers was not as high at 66.7%, it was still clearly present. For INPP5E, CEP290 and BBS2 carriers, consanguinity rates were present but were lower. There was only one patient with a BBS7 mutation and they had no reported family relationships but because of the small number of carriers this was not felt to be statistically significant. Table 3. Detailed Variant Classification and ACMG Annotation Gene Transcript cDNA Change Protein Change Mutation Type ACMG Class Number of Patients Inheritance INPP5E NM_019892.6 c.367_368delGC p.Ala123ProfsTer27 Frameshift deletion LP 10 AR BBS2 NM_031885.5 c.1254delT p.Phe418LeufsTer40 Frameshift deletion LP 6 AR BBS2 NM_031885.5 c.1725delT p.Phe575LeufsTer8 Frameshift deletion P 2 AR BBS2 NM_031885.5 c.106_107delCA p.Gln36AsnfsTer8 Frameshift deletion LP 1 AR CPLANE1 NM_023073.3 c.8263delA p.Thr2755HisfsTer2 Frameshift deletion P 1 AR CPLANE1 NM_023073.3 c.1819delT p.Tyr607ThrfsTer6 Frameshift deletion P 3 AR CPLANE1 NM_023073.3 c.1868_1869insT p.Leu623PhefsTer8 Frameshift insertion LP 3 AR CPLANE1 NM_023073.3 c.6599delT p.Leu2200CysfsTer85 Frameshift deletion LP 1 AR CPLANE1 NM_023073.3 c.3912delA p.Gly1305GlufsTer13 Frameshift deletion LP 1 AR CEP290 NM_025114.4 c.2462delT p.Leu821CysfsTer7 Frameshift deletion LP 2 AR CEP290 NM_025114.4 c.6416_6417delAA p.Lys2139SerfsTer3 Frameshift deletion LP 1 AR CEP290 NM_025114.4 c.2523delA p.Glu842ArgfsTer15 Frameshift deletion LP 1 AR TCTN2 NM_024809.5 c.1206delT p.Phe402LeufsTer12 Frameshift deletion LP 1 AR BBS7 NM_176824.3 c.1759_1760delAG p.Arg587GlufsTer3 Frameshift deletion LP 1 AR NPHP3 NM_153240.5 c.2161delA p.Met721Ter Frameshift deletion LP 1 AR ACMG, American College of Medical Genetics and Genomics; P, pathogenic; LP, likely pathogenic; AR, autosomal recessive. Summary: Total distinct variants identified: 15 (all frameshift mutations). Pathogenic (P): 6 (40.0%). Likely pathogenic (LP): 9 (60.0%). 4. Discussion 4.1. Overview and key messages This study indicates that recessive ciliopathy gene variants are important contributors to adult FSGS in this highly consanguineous cohort. Mutations in genes encoding ciliopathy proteins were identified in 45.7% of FSGS patients but none of the controls, and all 35 alleles contained truncating pathogenic/likely pathogenic mutations according to ACMG guidelines. Ciliopathy positive patients had more severe disease: universal nephrotic range proteinuria, hematuria, worse renal function, more consanguinity and family history suggestive of recessive inheritance and thus a significant association between mutations in ciliopathy genes and adult FSGS in this patient group.( 31, 32). Research on FSGS sequencing has shown that monogenic causes exist in 10-40% of cases which primarily affect patients with steroid-resistant disease, those who develop the condition at young ages and have family history of the disease(33, 34). The studies focused on two gene groups which include podocyte structural genes (NPHS1, NPHS2, ACTN4, TRPC6) and collagen IV genes(5, 32, 35). Research on ciliopathy genes has focused primarily on their role in developmental renal disease and cystic nephronophthisis instead of their role as the main factor in adult FSGS (36, 37). Our results extend this paradigm by showing that, at least in a consanguineous Middle Eastern cohort, recessive ciliopathy genes form one of the largest identifiable genetic categories of FSGS and can present with a “pure” glomerular phenotype. We recently demonstrated by rare variant burden analysis an enrichment of bi-allelic recessive ciliopathy variants in our FSGS cases compared to controls (OR 34.7, 95% CI 1.9–618.7, p<0.001), which suggests a major genetic role for ciliopathy in our data set. Of the 34 bi-allelic recessive ciliopathy variants detected, the 3 genes with the highest enrichment scores were INPP5E, BBS2 and CPLANE1, with INPP5E and BBS2 having p values of less than 0.005. All variants were absent or ultra rare in the gnomAD v4.1 exome data set (MAF <0.000007) including in Middle Eastern populations, thus common polymorphisms can be ruled out. 4.2. Gene-specific patterns: INPP5E, BBS2, CPLANE1 and others Mutations in the INPP5E gene were the most common mutations identified in this group of patients. All 10 INPP5E mutant carriers had the homozygous c.367_368delGC (p.Ala123ProfsTer27) mutation and this allele was not detected in the control population. INPP5E carriers had the highest average serum creatinine and urea levels and also the highest rate of hematuria (88.9%). The age of onset of the kidney disease was variable and ranged from the second to the third decade of life. INPP5E mutations are associated with Joubert syndrome, MORM (mental retardation, ocular malformations, microcephaly, skeletal abnormalities) syndrome and kidney disease. Our data suggests that mutations in INPP5E can also cause adult onset FSGS with significant renal injury in a consanguineous family . (38-40). BBS2 mutations were found in 7 patients (3 different frameshift mutations: c.1254delT, c.1725delT and c.106_107delCA) with ciliopathy. Patients with BBS2 mutations had heavy proteinuria, elevated serum creatinine, more frequently hematuria and hypertension compared to INPP5E patients. Mutations in BBS2 encode a subunit of the BBSome complex. BBSome mutations were identified in patients with Bardet–Biedl syndrome (BBS), our adult patients with biallelic mutations in BBS2 did not show any clinical features suggestive of the BBS syndrome suggesting that the renal phenotype in adult patients with mutations in the BBSome may be more pronounced and dominant over any possible limited or non-obvious extra-renal manifestations.(41, 42). Ciliopathy genes in our cohort, including CPLANE1, CEP290, TCTN2, BBS7 and NPHP3, demonstrated consistent associations with reduced renal function and FSGS biopsy proven disease. The classical nephronophthisis related ciliopathy syndromes caused by mutations in STK11/LKB1, CTNS and TTC21B genes are characterized by tubulointerstitial fibrosis, cysts and bland urinalysis with minimal proteinuria and hematuria in late-stage kidney disease. Patients carrying mutations in STK11 and TTC21L in the CEP290 locus uniformly developed proteinuria, and also hematuria in some cases within the CEP290 positive subgroup, indicating early onset kidney disease characterized by glomerular dysfunction with increased permeability to proteins and cells. Our findings, together with earlier reports of FSGS linked to ciliary genes, such as TTC21B, NPHP4, CRB2 and CC2D2A, suggest that disrupted function of distinct ciliary modules may give rise to a unifying glomerular disease process that manifests as FSGS and podocyte damage.(13, 37, 43-45). 4.3. Pathophysiologic implications Primary cilia have been implicated in signaling in podocytes and also in cytoskeletal organization(46, 47). These common features of the 35-truncating LOF mutations and the uniform FSGS histology and severe proteinuria suggest that mutations in these genes destabilize the podocyte and lead to disruption of the filtration barrier. Gene-specific functions may also contribute to the observed phenotypic heterogeneity of mutations within a gene. For example, mutations in genes encoding ciliary proteins involved in signaling (such as INPP5E and CEP290) were associated with more hematuria suggesting more extensive damage to the endothelium, GBM and/or podocytes. In contrast, mutations in CPLANE1, a component of the transition zone complex, were most commonly found in patients with universal proteinuria but relatively little hematuria, suggesting an important role of this protein in the maintenance of the structure and function of the foot processes and slit diaphragm of the podocyte. The mutations in BBS2/BBS7 may contribute to the relative resistance of the podocyte to signals from vasopressin and other molecules, blood pressure and thereby to the development of hypertension and progression of the renal disease. ( 36, 37, 48). 4.4. Clinical implications and genetic testing strategy Genetic testing for an expanded panel of ciliopathy genes in adult patients with FSGS revealed disease causing mutations in a subset of consanguineous families. Single gene panel testing for NPHS and COL4A genes may not detect mutations present in certain populations, indicating that the use of broad panels of genes is required and that single gene sequencing may not be sufficient in some cases (35). Genetic testing can help identify biallelic mutations in ciliopathy genes and thus lead to a definitive diagnosis for affected individuals and their families. This test is not immunology dependent and is autosomal-recessive as each of the children of an affected parent will have a 25% chance of developing the disease. This test may be also helpful to determine if an individual is a carrier and to facilitate family planning especially in large families. The identification of mutations in a gene that leads to the diagnosis of the renal disease associated with ciliopathy genes will also allow complete cascade testing for possible extra renal features of the disease including neurological, ocular, skeletal and endocrine (49). Early genetic testing can be used in patients with suspected hereditary FSGS for diagnosis and clinical management (50, 51). 4.5. Limitations and future directions While the cohort is small, it is the first study to our knowledge to examine the role of recessive ciliopathy genes in adult FSGS in a highly consanguineous cohort from a Middle Eastern population. The strong enrichment of biallelic truncating mutations in affected individuals relative to controls supports an association. However, there are limitations to this study. The cohort was small (35 cases and 20 controls) and examination of some of the genes (BBS7, NPHP3 and TCTN2) had only a single affected individuals per gene which may not provide sufficient power to make any gene-specific genotype-phenotype association. Genotype-phenotype analyses were performed in without multivariable adjustment because of insufficient power for multiple regression analyses to account for any potential confounders. Rare variant burden analyses were performed in an unadjusted fashion and only Haldane corrected gene level p values were reported because of multiple testing. The overall burden analysis between cases and controls seemed robust, but should be interpreted with caution at the gene level, particularly when the p values are close to the multiple testing threshold such as BBS2 and CPLANE1. A major limitation is that this is a cross-sectional study, and we do not capture the natural history of the disease such as time to ESRD and to kidney transplantation as well as post-transplant outcomes. We did not also perform a detailed evaluation of extra-renal manifestations (neuroimaging, ophthalmology, audiology and endocrinology). Thus, it is possible that subtle manifestations of ciliopathy have been overlooked. Lack of consanguinity between affected individuals and their extended family members meant that we were not always able to perform segregation analysis and Sanger confirmation, which would have allowed us to further demonstrate co-segregation. However, the presence of biallelic truncating mutations in affected individuals with complete absence or near absence of the variants in gnomAD in the context of consanguinity provides compelling evidence of their pathogenicity. A multi-center study that includes affected individuals with different ages at diagnosis, in addition to a larger control cohort, will be required to assess gene-specific risks of disease, as well as including more comprehensive extra-renal evaluation and functional in vitro or in vivo assays to better understand the disease-causing pathways at the level of cilia biology, e.g. roles of INPP5E, CPLANE1 and CEP290 in FSGS. 5. Conclusions Mutations in ciliopathy genes appear to be important genetic contributor for adult FSGS in highly consanguineous populations. In this study, biallelic pathogenic or likely pathogenic variants were identified in 45.7% of patients and mutations in INPP5E, BBS2 and CPLANE1 were identified accounting for most genetically explained cases. In conclusion, mutations in ciliopathy genes should be considered in the genetic testing panel for adult FSGS, particularly in consanguineous families and those with strong family history of kidney diseases. Abbreviations ACMG: American College of Medical Genetics and Genomics BBS: Bardet-Biedl syndrome BUN: Blood urea nitrogen CI: Confidence interval eGFR: Estimated glomerular filtration rate ESRD: End-stage renal disease FSGS: Focal segmental glomerulosclerosis gnomAD: Genome Aggregation Database LP: Likely pathogenic MAF: Minor allele frequency NGS: Next-generation sequencing OR: Odds ratio P: Pathogenic SD: Standard deviation VUS: Variant of uncertain significance Declarations Ethics approval and consent to participate The research team executed their study based on Declaration of Helsinki guidelines and Good Clinical Practice standards. Ethical approval was obtained from the Research Ethics Committee of the University of Sulaymaniyah, Kurdistan Region, Iraq. All study participants received written informed consent before starting the research but participants under 18 required their parent or legal guardian to give consent. Consent for publication Not applicable. The manuscript does not contain any identifiable individual data or images. Availability of data and materials The pathogenic and likely pathogenic variants identified in this study have been deposited in ClinVar, a publicly accessible database of human genetic variation and its relationship to health (NCBI ClinVar, https://www.ncbi.nlm.nih.gov/clinvar/). The variants are available under accession numbers SCV007520383–SCV007520396 and can be accessed via our submitter page, Molecular Lab (submitter ID 510454), at: https://www.ncbi.nlm.nih.gov/clinvar/submitters/510454/. Competing interests The authors declare that they have no competing interests. Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Authors' contributions Lava I. Ahmed conceived and designed the study, obtained ethical approval, supervised the research, performed DNA extraction, library preparation and next-generation sequencing, conducted statistical analysis, and drafted the manuscript. Dlnya A. Mohammed, MD, PhD contributed to study design, supervised laboratory work, assisted in data interpretation, and critically reviewed and revised the manuscript. Dana Ahmed Sharif, MD recruited patients, collected and managed clinical data, reviewed kidney biopsy reports, and contributed to clinical interpretation of genotype–phenotype correlations. All authors reviewed and approved the final manuscript and agree to be accountable for all aspects of the work. Acknowledgements The authors thank all participating patients and their families for their valuable contribution to this study. We thank the laboratory and technical staff and all collaborators who supported DNA extraction, library preparation, and sequencing. We are grateful to the University of Sulaimani for providing institutional and infrastructural support that made this research possible. References de Cos M, Meliambro K, Campbell KN. Novel treatment paradigms: focal segmental glomerulosclerosis. Kidney Int Rep. 2023;8(1):30–5. Rout P, Hashmi MF, Baradhi KM. Focal Segmental Glomerulosclerosis. StatPearls. Treasure Island (FL): StatPearls Publishing Copyright © 2025. StatPearls Publishing LLC.; 2025. Ni Cathain D, Reidy D, Bagnasco S, Kant S. Focal and Segmental Glomerulosclerosis: A Comprehensive State-of-the-Art Review. Sclerosis. 2025;3(3):24. Gbadegesin R, Lavin P, Foreman J, Winn M. Pathogenesis and therapy of focal segmental glomerulosclerosis: an update. Pediatr Nephrol. 2011;26(7):1001–15. Chen YM, Liapis H. Focal segmental glomerulosclerosis: molecular genetics and targeted therapies. BMC Nephrol. 2015;16:101. Lepori N, Zand L, Sethi S, Fernandez-Juarez G, Fervenza FC. 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DNA replication stress underlies renal phenotypes in CEP290-associated Joubert syndrome. J Clin Invest. 2015;125(9):3657–66. Gupta M, Lewis TR, Stuck MW, Spencer WJ, Klementieva NV, Arshavsky VY, Pazour GJ. Inpp5e Is Critical for Photoreceptor Outer Segment Maintenance. bioRxiv. 2024. Drole Torkar A, Avbelj Stefanija M, Bertok S, Trebušak Podkrajšek K, Debeljak M, Stirn Kranjc B, et al. Novel insights into monogenic obesity syndrome due to INPP5E gene variant: a case report of a female patient. Front Endocrinol. 2021;12:581134. Travaglini L, Brancati F, Silhavy J, Iannicelli M, Nickerson E, Elkhartoufi N, et al. Phenotypic spectrum and prevalence of INPP5E mutations in Joubert syndrome and related disorders. Eur J Hum Genet. 2013;21(10):1074–8. Zhao Y, Rahmouni K. BBSome: a New Player in Hypertension and Other Cardiovascular Risks. Hypertension. 2022;79(2):303–13. Meyer JR, Krentz AD, Berg RL, Richardson JG, Pomeroy J, Hebbring SJ, Haws RM. Kidney failure in Bardet–Biedl syndrome. Clin Genet. 2022;101(4):429–41. Min J, Xiao R, Fu Q, Huang Y, Wang H. Compound heterozygous mutations in BBS7 cause kidney abnormalities in Bardet-Biedl syndrome. Genes Dis. 2026;13(3):101792. Singh KK, Kumar R, Prakash J, Krishna A. Bardet-Biedl syndrome presenting with steroid sensitive nephrotic syndrome. Indian J Nephrol. 2015;25(5):300–2. Howie AJ. Genetic studies of focal segmental glomerulosclerosis: a waste of scientific time? Pediatr Nephrol. 2020;35(1):9–16. Pollak MR, Familial FSGS. Adv Chronic Kidney Dis. 2014;21(5):422–5. Al-Mousa H, Al-Saud B. Primary Immunodeficiency Diseases in Highly Consanguineous Populations from Middle East and North Africa: Epidemiology, Diagnosis, and Care. Front Immunol. 2017;8:678. Anwar WA, Khyatti M, Hemminki K. Consanguinity and genetic diseases in North Africa and immigrants to Europe. Eur J Pub Health. 2014;24(suppl1):57–63. Al-Kandari Y, Bahzad S, Ramadan D, Alsharhan H, Hussain M, Al-Herz W. Attitudes of parents with a child with autosomal recessive disease toward consanguinity. INQUIRY: J Health Care Organ Provis Financing. 2025;62:00469580251366872. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, 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(5):405–24. Sambharia M, Rastogi P, Thomas CP, editors. Monogenic focal segmental glomerulosclerosis: a conceptual framework for identification and management of a heterogeneous disease. American Journal of Medical Genetics Part C: Seminars in Medical Genetics; 2022: Wiley Online Library. Lepori N, Zand L, Sethi S, Fernandez-Juarez G, Fervenza FC. Clinical and pathological phenotype of genetic causes of focal segmental glomerulosclerosis in adults. Clin Kidney J. 2018;11(2):179–90. Braunisch MC, Riedhammer KM, Herr P-M, Draut S, Günthner R, Wagner M, et al. Identification of disease-causing variants by comprehensive genetic testing with exome sequencing in adults with suspicion of hereditary FSGS. Eur J Hum Genet. 2021;29(2):262–70. Miao J, e Vairo FP, Hogan MC, Erickson SB, El Ters M, Bentall AJ, et al. editors. Identification of genetic causes of focal segmental glomerulosclerosis increases with proper patient selection. Mayo Clinic Proceedings; 2021: Elsevier. Sambharia M, Rastogi P, Thomas CP. Monogenic focal segmental glomerulosclerosis: A conceptual framework for identification and management of a heterogeneous disease. Am J Med Genet C Semin Med Genet. 2022;190(3):377–98. Stokman MF, Saunier S, Benmerah A. Renal Ciliopathies: Sorting Out Therapeutic Approaches for Nephronophthisis. Front Cell Dev Biology. 2021;Volume 9–2021. Srivastava S, Molinari E, Raman S, Sayer JA. Many Genes-One Disease? Genetics of Nephronophthisis (NPHP) and NPHP-Associated Disorders. Front Pediatr. 2017;5:287. Dillard KJ, Hytönen MK, Fischer D, Tanhuanpää K, Lehti MS, Vainio-Siukola K, et al. A splice site variant in INPP5E causes diffuse cystic renal dysplasia and hepatic fibrosis in dogs. PLoS ONE. 2018;13(9):e0204073. Hakim S, Dyson JM, Feeney SJ, Davies EM, Sriratana A, Koenig MN, et al. Inpp5e suppresses polycystic kidney disease via inhibition of PI3K/Akt-dependent mTORC1 signaling. Hum Mol Genet. 2016;25(11):2295–313. Xu W, Jin M, Hu R, Wang H, Zhang F, Yuan S, Cao Y. The Joubert Syndrome Protein Inpp5e Controls Ciliogenesis by Regulating Phosphoinositides at the Apical Membrane. J Am Soc Nephrol. 2017;28(1):118–29. Forsythe E, Sparks K, Best S, Borrows S, Hoskins B, Sabir A, et al. Risk Factors for Severe Renal Disease in Bardet-Biedl Syndrome. J Am Soc Nephrol. 2017;28(3):963–70. Gupta N, D'Acierno M, Zona E, Capasso G, Zacchia M, editors. Bardet–Biedl syndrome: The pleiotropic role of the chaperonin-like BBS6, 10, and 12 proteins. American Journal of Medical Genetics Part C: Seminars in Medical Genetics. Wiley Online Library; 2022. Hildebrandt F, Attanasio M, Otto E. Nephronophthisis: disease mechanisms of a ciliopathy. J Am Soc Nephrol. 2009;20(1):23–35. Stokman M, Lilien M, Knoers N. Nephronophthisis-related ciliopathies. GeneReviews®[Internet]. 2023. Simms RJ, Eley L, Sayer JA, Nephronophthisis. Eur J Hum Genet. 2009;17(4):406–16. Corbeil D, Thamm K, Karbanová J, Fargeas CA, Jászai J. The primary cilium as a multifunctional organelle: emerging roles and unanswered questions. Cell Commun Signal. 2025;23(1):406. Pala R, Alomari N, Nauli SM. Primary Cilium-Dependent Signaling Mechanisms. Int J Mol Sci. 2017;18(11). Hardee I, Soldatos A, Davids M, Vilboux T, Toro C, David KL, et al. Defective ciliogenesis in INPP5E-related Joubert syndrome. Am J Med Genet A. 2017;173(12):3231–7. Simms RJ, Hynes AM, Eley L, Sayer JA. Nephronophthisis: a genetically diverse ciliopathy. Int J Nephrol. 2011;2011:527137. Snoek R, Nguyen TQ, van der Zwaag B, van Zuilen AD, Kruis HME, van Gils-Verrij LA, et al. Importance of Genetic Diagnostics in Adult-Onset Focal Segmental Glomerulosclerosis. Nephron. 2019;142(4):351–8. Yao T, Udwan K, John R, Rana A, Haghighi A, Xu L, et al. Integration of Genetic Testing and Pathology for the Diagnosis of Adults with FSGS. Clin J Am Soc Nephrol. 2019;14(2):213–23. Additional Declarations No competing interests reported. Supplementary Files SupplementarytableS1.docx Supplementary Table S1. Mutation carrying pathogenic and likely pathogenic recessive ciliopathy gene variants were identified in adult individuals with confirmed FSGS by biopsy. The genes, transcripts, HGVS cDNA and protein change, zygosity, number of affected individuals, ACMG classification and gnomAD v4.1 allele counts and frequencies are provided in the table below. SupplementarytableS2.docx Supplementary Table S2. Rare variant burden analysis: comparison of carrier frequencies of biallelic ciliopathy genes between FSGS cases and controls. This table shows the number of carriers, prevalence, odds ratio (OR) with 95% confidence interval (CI) and the p-value from the Fisher’s exact test with Haldane correction for each gene. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 18 May, 2026 Reviews received at journal 15 May, 2026 Reviews received at journal 02 May, 2026 Reviewers agreed at journal 24 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers invited by journal 22 Apr, 2026 Editor assigned by journal 30 Mar, 2026 Submission checks completed at journal 29 Mar, 2026 First submitted to journal 29 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9125192","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":631013821,"identity":"25dc9475-60d9-4e7c-8a04-76544e406c8a","order_by":0,"name":"Lava Ahmed","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYBACPgh1AIiTgYQBkGYHYh4wwg7YEFrSEiBamInXkmMAYUO14ARs7GcMPzDU3InmZ8/5Jl1QcNje4DAD44O3bQwyfAdwaOHJMZZgOPYsd2bP223SMwwOJ244zMBsOLeNgUcSlxagFyQY2A7nbriRu02axyAtAWgLmzQvUIsBLi38z5J/MPw7nLv/Rs4zkBaQw9h/49UikXxMgrENaItEDhtQiw0j0GFszPi1PD5mkdh3OHfGmWfG1kAtiTMPMzZLzjkngdMv/PyJzTc+fDuc29+e/PA2zx8Je77jzQc/vCmzsccVYmCQgMplbAASEpDIIhGQoWUUjIJRMAqGJQAASWxWXU3WT0QAAAAASUVORK5CYII=","orcid":"","institution":"University of Sulaimani","correspondingAuthor":true,"prefix":"","firstName":"Lava","middleName":"","lastName":"Ahmed","suffix":""},{"id":631013822,"identity":"c925afde-29e0-4967-a909-2056e7dcc3da","order_by":1,"name":"Dlnya Mohammed","email":"","orcid":"","institution":"University of Sulaimani","correspondingAuthor":false,"prefix":"","firstName":"Dlnya","middleName":"","lastName":"Mohammed","suffix":""},{"id":631013823,"identity":"602a08bd-6967-4289-bd12-f076cec084b8","order_by":2,"name":"Dana Ahmed Sharif","email":"","orcid":"","institution":"University of Sulaimani","correspondingAuthor":false,"prefix":"","firstName":"Dana","middleName":"Ahmed","lastName":"Sharif","suffix":""}],"badges":[],"createdAt":"2026-03-14 22:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9125192/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9125192/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108406629,"identity":"13549936-2131-4d52-8c0a-b6ad492f3541","added_by":"auto","created_at":"2026-05-04 09:44:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16427,"visible":true,"origin":"","legend":"\u003cp\u003eThe number of adults with FSGS confirmed by kidney biopsy and with confirmed biallelic pathogenic or likely pathogenic variants in the listed recessive ciliopathy genes.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9125192/v1/fee3734873494c98b766603b.png"},{"id":108804889,"identity":"21ac1f98-c2d5-435f-8b94-dbf1db33b68d","added_by":"auto","created_at":"2026-05-08 15:24:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":389690,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9125192/v1/7292bfdd-4acc-4f1a-b4da-27a9911b3748.pdf"},{"id":108406631,"identity":"02b92e8d-ee28-4fa7-96e7-65c7991f297e","added_by":"auto","created_at":"2026-05-04 09:44:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20602,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table S1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMutation carrying pathogenic and likely pathogenic recessive ciliopathy gene variants were identified in adult individuals with confirmed FSGS by biopsy. The genes, transcripts, HGVS cDNA and protein change, zygosity, number of affected individuals, ACMG classification and gnomAD v4.1 allele counts and frequencies are provided in the table below.\u003c/p\u003e","description":"","filename":"SupplementarytableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9125192/v1/81d4f0e8504c78984dce1bde.docx"},{"id":108493687,"identity":"176958c2-3cea-4637-9e3e-ea1a16ef1752","added_by":"auto","created_at":"2026-05-05 10:01:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table S2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRare variant burden analysis: comparison of carrier frequencies of biallelic ciliopathy genes between FSGS cases and controls. This table shows the number of carriers, prevalence, odds ratio (OR) with 95% confidence interval (CI) and the p-value from the Fisher’s exact test with Haldane correction for each gene.\u003c/p\u003e","description":"","filename":"SupplementarytableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9125192/v1/5fdefe8786403dc645aa5041.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Contribution of Recessive Ciliopathy Genes in a Highly Consanguineous Adult Cohort with Biopsy-Proven Focal Segmental Glomerulosclerosis","fulltext":[{"header":"1. Background","content":"\u003cp\u003eFocal segmental glomerulosclerosis (FSGS) represents a clinically and genetically heterogeneous kidney disorder characterized by sclerosis of some glomeruli with involvement of portions of affected glomeruli (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). FSGS accounts for approximately 40% of cases of adult nephrotic syndrome and leads to end-stage renal disease (ESRD) as a major worldwide cause (\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). This clinical heterogeneity reflects diverse underlying mechanisms including podocytopathies, secondary forms, and monogenic causes.(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMore than 50 monogenic FSGS genes have been identified, including podocyte structural genes (NPHS1, NPHS2, WT1, LAMB2, CD2AP, TRPC6) and collagen IV genes (COL4A3\u0026ndash;COL4A5), which typically underlie early-onset or familial disease (\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFSGS has not been the subject of detailed genetic analysis because the genes encoding the proteins of the primary cilium and centrosome are not fully understood (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The primary cilium is a signaling organ responsible for maintaining cellular homeostasis. Mutations in cilia function impair numerous critical processes required for normal glomerular development and maintenance of the filtration barrier. The recent identification of mutations in the TTC21B gene leading to late-onset familial FSGS due to intraflagellar transport defects established that ciliopathies are responsible for more than just congenital and developmental kidney diseases(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn subsequent reports, other multiple ciliopathy genes carrying mutations that are also associated with FSGS have been described. CEP290 mutations that cause Joubert syndrome/nephronophthisis have been described in patients with FSGS (\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Similarly, CC2D2A mutations are also associated with FSGS with nephronophthisis overlap (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The Joubert syndrome/MORM syndrome causing mutation in INPP5E gene a ciliary protein essential for intraflagellar transport has not yet been described in confirmed cases of FSGS (\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). BBS2 and BBS7 along with other BBS genes necessary for BBSome complex biogenesis involved in cilia formation have also been identified as genes responsible for causing glomerular disease (\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eConsanguineous marriages result in an increased chance of homozygous mutations and therefore an increased expression of recessive genes responsible for ciliopathies (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Ciliopathies and other recessive genetic disorders are over-represented in Middle Eastern and North African populations where consanguinity rates are estimated to be above 20% (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study employed the occurrence of mutations in ciliary genes in a cohort of 35 adult FSGS patients and 20 controls from a highly consanguineous population. Using targeted Next Generation Sequencing (NGS) of a 98 gene panel we found pathogenic mutations in 16 cases (45.7%) confirming the relevance of mutations in ciliary genes in the etiology of FSGS in this population. We describe the clinical and genetic heterogeneity of mutations and contrast them with those identified in controls. These data highlight the importance of the sequencing of ciliary genes in adult patients with FSGS, particularly in cases with a positive family history and/or consanguinity.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Study Population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e35 adult patients with FSGS confirmed by kidney biopsy and 20 controls from the healthy population of Sulaymaniyah, Iraq, with normal kidney function. This study population included FSGS patients whose biopsy kidneys were evaluated by light microscopy (LM) and immunofluorescence and electron microscopy. Patients were excluded if their FSGS was associated with lupus or vasculitis or if it was drug-associated, and obesity-associated glomerulosclerosis. Control Individuals: Healthy controls had: (i) normal serum creatinine (ii) no proteinuria (urine protein \u0026lt;0.15 g/24 hours); (iii) normal blood pressure (\u0026lt;130/80 mmHg); (iv) no personal or family history of kidney disease.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Ethics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in compliance with the Declaration of Helsinki and Good Clinical Practice. Ethical approval was given by the Research Ethics Committee of University of Sulaymaniyah, Kurdistan Region, Iraq. Written informed consent was obtained from all participants. Written informed consent was obtained from the parents or guardians of children less than 18 years old.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Clinical and Laboratory Assessments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatient characteristics at the time of biopsy were recorded including age, sex, family history of kidney disease and parental consanguinity. Additional baseline information included serum creatinine and blood urea nitrogen levels (all in mg/dL), protein in urine, dipstick hematuria \u0026ge;1+ and blood pressure measured and history of previous kidney transplant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Genetic Analysis and Sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA from peripheral blood leukocytes was extracted from patients using a Wizard\u0026reg; Genomic DNA Purification Kit (Promega, Madison, WI, USA). A custom panel of 98 genes was designed using the Ion AmpliSeq Designer software (v6.2; Thermo Fisher Scientific) with respect to the hg19/GRCh37 genome assembly. The genes included in this panel NPHP1, NPHP3, CEP290, TMEM67, BBS1, BBS2, BBS7 and INPP5E, TCTN1\u0026ndash;3, CC2D2A and many others. DNA libraries were then prepared using the Ion AmpliSeq Library Kit 2.0 and sequenced on an Ion Torrent S5 (Thermo Fisher Scientific) platform with Ion 540 chips as recommended by the manufacturer. The custom panel contained 2,742 amplicons of length between 125 and 275 bp distributed in two primer pools and it provided in silico 99% coverage of coding exons and exon\u0026ndash;intron boundaries. The mean depth of coverage for all genes was over 200\u0026times; and more than 98% of the targeted bases had a coverage of at least 20\u0026times;.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Variant Annotation and Classification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll sequencing data was analyzed using Torrent Suite Software from Thermo Fisher Scientific. After primer trimming and read alignment to the hg19/GRCh37 reference genome using TMAP, the Torrent Variant Caller with parameters of 5% minimum variant allele fraction (VAF) and 100\u0026times; minimum read depth was used to call variants. Annotation was performed using the Franklin platform from Genoox, which integrates population data from gnomAD v4.1, 1000 Genomes and ExAC, as well as prediction scores from SIFT, PolyPhen‑2, CADD, FATHMM, MutationTaster scores, PhyloCSF and GERP conservation scores. MAF in any of the gnomAD v4.1 populations was required to be \u0026le;0.0001 and we were cautious when including Middle Eastern and North African populations to avoid including regional polymorphisms. Variants were classified as pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign, or benign according to the 2015 ACMG guidelines (30). All subsequent analyses were performed using only P and LP variants, with VUS, likely benign, and benign variants excluded. The variants identified here have been submitted to ClinVar (NCBI) and are available under accession numbers \u003cstrong\u003eSCV007520383\u0026ndash;SCV007520396\u003c/strong\u003e. The transcript and HGVS nomenclature of the P/LP variants, their zygosity, the number of affected patients and the corresponding allele frequencies in the gnomAD v4.1 are shown in Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll values are given as means \u0026plusmn; SD. For comparison of continuous variables, independent-samples t-test or one-way ANOVA was used. For categorical variables, the data were presented as the number and percentage and the chi-square or Fisher\u0026rsquo;s exact test was used. All the tests were two tailed and p \u0026lt; 0.05 was considered to be statistically significant. We identified carriers to be those with \u0026ge;1 P or LP variants in any of the ciliopathy genes in a homozygous (biallelic) configuration as predicted by the AR pattern of inheritance for FSGS. We calculated the OR and 95% CI (Haldane correction) to compare the carrier status versus FSGS using Fisher\u0026rsquo;s exact test. In addition, gene-specific burden tests for INPP5E, BBS2, CPLANE1 and CEP290 were carried out. The frequency of the identified alleles/alternate bases within the genome aggregation database v4.1 (gnomAD v4.1) and their confirmed rarity, particularly in Middle Eastern and North African populations, was verified for pathogenicity of the identified variants. The SPSS v25.0 statistical software (IBM Corp., Armonk, NY, USA) and Python 3.9 (SciPy library) were used for these analyses. The exploratory gene-specific p-values are provided without adjustment. With four gene-specific burden tests being carried out, the Bonferroni adjustment for a threshold of \u0026alpha; = 0.0125 is required.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Study Cohort Characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e35 adults with biopsy‑proven FSGS and 20 healthy controls were studied in this cohort (Table 1). The mean age at diagnosis of FSGS was 25.1 \u0026plusmn; 7.9 years (range 14\u0026ndash;52). Eighty percent of FSGS patients (28/35) were from consanguineous families, which is higher than none of the controls (0/20; p\u0026lt;0.001). Pathogenic or likely pathogenic variants in genes encoding subunits of the cilia were identified in 16 of 35 FSGS patients (45.7%) and in 0 of 20 controls (p\u0026lt;0.001). Clinical characteristics of Ciliopathy positive FSGS patients had universal proteinuria (16/16, 100%) and frequent hematuria (13/16, 81.3%). They had a significantly worse renal function (higher serum creatinine levels, 3.6 \u0026plusmn; 0.9 vs 1.2 \u0026plusmn; 0.4 mg/dL; BUN, 76.4 \u0026plusmn; 31.2 vs 29.7 \u0026plusmn; 10.5 mg/dL) and higher ESRD performed transplantation rates (31.3% vs 10.5%) than the ciliopathy negative patients with FSGS.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Demographic, Clinical, and Laboratory Characteristics of Study Participants\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"3\" cellpadding=\"0\" width=\"685\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFSGS Ciliopathy-Positive (n=16)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFSGS Ciliopathy-Negative (n=19)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHealthy Controls (n=20)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eP-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAge, years (mean \u0026plusmn; SD, range)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e25.1 \u0026plusmn; 7.9 (14\u0026ndash;52)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e27.4 \u0026plusmn; 8.1 (16\u0026ndash;48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e26.8 \u0026plusmn; 7.5 (18\u0026ndash;45)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eMale/Female, n\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10/6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e11/8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12/8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eClinical Features\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eConsanguinity, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e13 (81.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e3 (15.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFamily history of kidney disease, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e16 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e5 (26.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLaboratory Parameters at Baseline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eSerum creatinine, mg/dL (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e3.6 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1.2 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0.8 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBlood urea nitrogen, mg/dL (mean \u0026plusmn; SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e76.4 \u0026plusmn; 31.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e28.3 \u0026plusmn; 12.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e16.2 \u0026plusmn; 5.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eProteinuria \u0026ge;0.5 g/24 hr, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e16 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e5 (26.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eHematuria (dipstick \u0026ge;1+), n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e13 (81.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e4 (21.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eClinical Outcomes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eHypertension, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e11 (68.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e6 (31.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eESRD/Kidney transplant, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e5 (31.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2 (10.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0.053\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNS, not significant; ESRD, end-stage renal disease; eGFR, estimated glomerular filtration rate.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRare-Variant Burden Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the degree of enrichment of recessive ciliopathy variants we performed gene set burden testing on the biallelic pathogenic/likely pathogenic carrier status. Of the 35 FSGS patients, 16 (45.7%) were carriers, compared to 0 of the 20 controls (Fisher\u0026rsquo;s exact p\u0026lt;0.001) giving an odds ratio of 34.7 (95% CI 1.9\u0026ndash;618.7) after Haldane correction. The distribution of these recessive ciliopathy genes among FSGS patients is shown in Figure 1. Gene specific burden tests for INPP5E, BBS2, CPLANE1 and CEP290 are shown in (Supplementary Table S2). There is enrichment for INPP5E (10/35 cases vs 0/20; OR 17.0, 95% CI 1.0\u0026ndash;294.0; p=0.011) and BBS2 (7/35 vs 0/20; OR 10.7, 95% CI 0.6\u0026ndash;199.5; p=0.053). There is weaker evidence for carrier status for CPLANE1 (6/35; p=0.076) and CEP290 (4/35; p=0.285). The wide CI are due to the small number of cases in this analysis and the fact that we have not identified any carriers in the control cohort, so we simply treat the gene level p values as hypotheses to be tested in larger data sets. Further studies with larger multi-center cohorts will be needed to confirm our observation of an enrichment of ciliopathy variants in adult FSGS.\u003c/p\u003e\n\u003cp\u003eSequencing data were re analyzed from over 730,000 individuals in gnomAD v4.1 and examined the prevalence of the common frameshift mutations in INPP5E and BBS2 as well as all reported truncating mutations in CPLANE1. The INPP5E c.367_368delGC (p. Ala123ProfsTer27) mutation that results in a premature stop codon at c.385 (p. Ala123ProfsTer27)) was present in homozygote form in all 10 FSGS individuals carrying an INPP5E mutation, was not present in gnomAD or in the exomes of any of the individuals in the Middle Eastern populations. The 3 BBS2 frameshift mutations c.1254delT, c.1725delT, c.106_107delCA as well as 5 of the reported CPLANE1 frameshift mutations were not present or were found to be ultra rare (AF \u0026lt; 0.000007) as were variants in CEP290, TCTN2, BBS7 and NPHP3.The universal absence or extreme rarity of these truncating mutations, and the fact that they are present in homozygote form only in individuals with FSGS confirms their role as pathogenic mutations in this disease.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eGenetic Findings: Spectrum of Ciliopathy Mutations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTargeted panel sequencing identified 16 of 35 FSGS patients (45.7%) with pathogenic or likely pathogenic ciliopathy variants. The 35 pathogenic/likely pathogenic variant alleles were detected in 16 individuals; 15 frameshift mutations were identified, and all were truncating (100%). Using the ACMG 2015 standards, 6 variants (40.0%) were classified as pathogenic, and 9 (60.0%) as likely pathogenic. There were no VUS, likely benign, or benign variants that were retained in these patients who had variants in the ciliopathy genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.1 Ciliopathy Gene Variant Frequency and Spectrum\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 16 ciliopathy‑positive patients carried variants in seven genes: INPP5E, BBS2, CPLANE1, CEP290, TCTN2, BBS7, and NPHP3 (Table 2).\u0026nbsp;Mutations in INPP5E were identified in 10 of 16 ciliopathy positive patients (62.5%) and in 5 of 18 (28.6%) of the full FSGS cohort. All carried the same homozygous frameshift mutation c.367_368delGC (p.Ala123ProfsTer27) which is a population specific founder mutation. Mutations in BBS2 were present in 7 of 16 (43.8%; 20.0% of the full FSGS cohort) ciliopathy positive patients and were identified as three frameshift mutations; c.1254delT, c.1725delT and c.106_107delCA which were all present in biallelic combinations. CPLANE1 variants occurred in 6 of 16 ciliopathy‑positive patients (37.5%; 17.1% of the cohort) across five frameshift alleles (c.1819delT, c.1868_1869insT, c.8263delA, c.6599delT, c.3912delA). CEP290 variants were detected in 4 patients (11.4% of the cohort), TCTN2 in 1 patient, and BBS7 and NPHP3 in 1 patient each.\u0026nbsp;Complete variant information, including ACMG classification, number of patients carrying each allele, and gnomAD v4.1 allele counts and frequencies, is presented in Table 3 and Supplementary Table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Association between ciliopathy genes and genotype\u0026ndash;phenotype correlations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.1 Age at FSGS diagnosis and clinical presentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no statistically significant difference in age at diagnosis between genes (p \u0026gt; 0.05, Table 2). INPP5E, BBS2, CEP290 and CPLANE1 mutations typically presented in early to mid-twenties, while BBS7 and NPHP3 cases presented in their thirties, more in keeping with adult onset FSGS than childhood podocytopathy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.2 Biochemical parameters: serum creatinine and urea\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBaseline renal function was associated with the expression of the gene encoding creatinine and BUN (p \u0026lt; 0.001 and p \u0026lt; 0.05, respectively, (Tables 2). The highest levels of BUN were recorded for the INPP5E, CPLANE1 and BBS2 genes, while the lowest BUN values were recorded for the CEP290, NPHP3 and BBS7 genes. These values corresponded to those recorded for creatinine, indicating that the extent of renal impairment was gene-specific.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.3 Proteinuria and hematuria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteinuria was present in all 16 ciliopathy positive patients, compared to 26.3% of ciliopathy negative FSGS patients and none of the controls (both p\u0026lt;0.001). Hematuria was also more common in ciliopathy positive patients (81.3% vs 21.1% and 0%; p\u0026lt;0.001), particularly in CEP290, TCTN2, BBS7 and INPP5E mutation carriers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.4 Hypertension and transplant outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHypertension was more common in ciliopathy positive than ciliopathy negative individuals (68.8% vs 31.6%; p=0.051) and was not detected in controls (p\u0026lt;0.001). Kidney transplantation for ESRD had occurred in 31.3% of ciliopathy positive individuals and 10.5% of ciliopathy negative individuals, with no further transplants recorded.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Genotype-Phenotype Correlations by Ciliopathy Gene\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"3\" cellpadding=\"0\" width=\"1048\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePatients (n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariants (n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnique Mutations\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge at Dx (yr)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCreatinine (mg/dL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eBUN (mg/dL)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eConsanguinity (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFamily Hx (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHematuria (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eHypertension (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eINPP5E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.367_368delGC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e23.2\u0026plusmn;8.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e4.0\u0026plusmn;1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e88.6\u0026plusmn;25.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e33.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e77.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e88.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e22.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBBS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.1254delT\u003c/p\u003e\n \u003cp\u003ec.1725delT\u003c/p\u003e\n \u003cp\u003ec.106_107delCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e27.3\u0026plusmn;4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e3.8\u0026plusmn;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e80.3\u0026plusmn;12.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e28.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e75.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e71.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e33.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCPLANE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.8263delA\u003c/p\u003e\n \u003cp\u003ec.1868_1869insT\u003c/p\u003e\n \u003cp\u003ec.1819delT\u003c/p\u003e\n \u003cp\u003ec.6599delT\u003c/p\u003e\n \u003cp\u003ec.3912delA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e29.3\u0026plusmn;10.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e3.7\u0026plusmn;2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e88.5\u0026plusmn;38.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e66.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e33.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e50.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCEP290\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.2462delT\u003c/p\u003e\n \u003cp\u003ec.6416_6417delAA\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;c.2523delA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e24.8\u0026plusmn;6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1.8\u0026plusmn;0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e45.6\u0026plusmn;20.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e50.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e75.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTCTN2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.1206delT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e23.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e68.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBBS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.1759_1760delAG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e30.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e52.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNPHP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.2161delA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e30.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e44.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e33.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eDx, diagnosis; BUN, blood urea nitrogen; Hx, history\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Inheritance Patterns and Consanguinity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 16 patients with FSGS that were found to have mutations in the genes associated with ciliopathy had biallelic mutations (Table 3). Consanguinity was reported in 81.3% of the ciliopathy positive patient\u0026rsquo;s vs 15.8% of the ciliopathy negative FSGS patients (p\u0026lt;0.001) a 5.3-fold enrichment. Consanguinity rates for each gene were as follows: all carriers in the TCTN2 and NPHP3 cohorts were consanguineous and although the consanguinity rate for CPLANE1 carriers was not as high at 66.7%, it was still clearly present. For INPP5E, CEP290 and BBS2 carriers, consanguinity rates were present but were lower. There was only one patient with a BBS7 mutation and they had no reported family relationships but because of the small number of carriers this was not felt to be statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u003c/strong\u003e Detailed Variant Classification and ACMG Annotation\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"3\" cellpadding=\"0\" width=\"779\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTranscript\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ecDNA Change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein Change\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMutation Type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eACMG Class\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of Patients\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eInheritance\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eINPP5E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_019892.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.367_368delGC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Ala123ProfsTer27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBBS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_031885.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.1254delT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Phe418LeufsTer40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBBS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_031885.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.1725delT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Phe575LeufsTer8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBBS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_031885.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.106_107delCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Gln36AsnfsTer8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCPLANE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_023073.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.8263delA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Thr2755HisfsTer2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCPLANE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_023073.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.1819delT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Tyr607ThrfsTer6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCPLANE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_023073.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.1868_1869insT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Leu623PhefsTer8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift insertion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCPLANE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_023073.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.6599delT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Leu2200CysfsTer85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCPLANE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_023073.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.3912delA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Gly1305GlufsTer13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCEP290\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_025114.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.2462delT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Leu821CysfsTer7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCEP290\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_025114.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.6416_6417delAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Lys2139SerfsTer3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eCEP290\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_025114.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.2523delA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Glu842ArgfsTer15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTCTN2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_024809.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.1206delT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Phe402LeufsTer12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eBBS7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_176824.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.1759_1760delAG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Arg587GlufsTer3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNPHP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNM_153240.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ec.2161delA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003ep.Met721Ter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFrameshift deletion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eLP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eACMG, American College of Medical Genetics and Genomics; P, pathogenic; LP, likely pathogenic; AR, autosomal recessive. Summary: Total distinct variants identified: 15 (all frameshift mutations). Pathogenic (P): 6 (40.0%). Likely pathogenic (LP): 9 (60.0%).\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cstrong\u003e4.1. Overview and key messages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study indicates that recessive ciliopathy gene variants are important contributors to adult FSGS in this highly consanguineous cohort. Mutations in genes encoding ciliopathy proteins were identified in 45.7% of FSGS patients but none of the controls, and all 35 alleles contained truncating pathogenic/likely pathogenic mutations according to ACMG guidelines. Ciliopathy positive patients had more severe disease: universal nephrotic range proteinuria, hematuria, worse renal function, more consanguinity and family history suggestive of recessive inheritance and thus a significant association between mutations in ciliopathy genes and adult FSGS in this patient group.( 31, 32).\u003c/p\u003e\n\u003cp\u003eResearch on FSGS sequencing has shown that monogenic causes exist in 10-40% of cases which primarily affect patients with steroid-resistant disease, those who develop the condition at young ages and have family history of the disease(33, 34). The studies focused on two gene groups which include podocyte structural genes (NPHS1, NPHS2, ACTN4, TRPC6) and collagen IV genes(5, 32, 35). Research on ciliopathy genes has focused primarily on their role in developmental renal disease and cystic nephronophthisis instead of their role as the main factor in adult FSGS (36, 37). Our results extend this paradigm by showing that, at least in a consanguineous Middle Eastern cohort, recessive ciliopathy genes form one of the largest identifiable genetic categories of FSGS and can present with a “pure” glomerular phenotype.\u003c/p\u003e\n\u003cp\u003eWe recently demonstrated by rare variant burden analysis an enrichment of bi-allelic recessive ciliopathy variants in our FSGS cases compared to controls (OR 34.7, 95% CI 1.9–618.7, p\u0026lt;0.001), which suggests a major genetic role for ciliopathy in our data set. Of the 34 bi-allelic recessive ciliopathy variants detected, the 3 genes with the highest enrichment scores were INPP5E, BBS2 and CPLANE1, with INPP5E and BBS2 having p values of less than 0.005. All variants were absent or ultra rare in the gnomAD v4.1 exome data set (MAF \u0026lt;0.000007) including in Middle Eastern populations, thus common polymorphisms can be ruled out.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2. Gene-specific patterns: INPP5E, BBS2, CPLANE1 and others\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMutations in the INPP5E gene were the most common mutations identified in this group of patients. All 10 INPP5E mutant carriers had the homozygous c.367_368delGC (p.Ala123ProfsTer27) mutation and this allele was not detected in the control population. INPP5E carriers had the highest average serum creatinine and urea levels and also the highest rate of hematuria (88.9%). The age of onset of the kidney disease was variable and ranged from the second to the third decade of life. INPP5E mutations are associated with Joubert syndrome, MORM (mental retardation, ocular malformations, microcephaly, skeletal abnormalities) syndrome and kidney disease. Our data suggests that mutations in INPP5E can also cause adult onset FSGS with significant renal injury in a consanguineous family\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e(38-40).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBBS2 mutations were found in 7 patients (3 different frameshift mutations: c.1254delT, c.1725delT and c.106_107delCA) with ciliopathy. Patients with BBS2 mutations had heavy proteinuria, elevated serum creatinine, more frequently hematuria and hypertension compared to INPP5E patients. Mutations in BBS2 encode a subunit of the BBSome complex. BBSome mutations were identified in patients with Bardet–Biedl syndrome (BBS), our adult patients with biallelic mutations in BBS2 did not show any clinical features suggestive of the BBS syndrome suggesting that the renal phenotype in adult patients with mutations in the BBSome may be more pronounced and dominant over any possible limited or non-obvious extra-renal manifestations.(41, 42).\u003c/p\u003e\n\u003cp\u003eCiliopathy genes in our cohort, including CPLANE1, CEP290, TCTN2, BBS7 and NPHP3, demonstrated consistent associations with reduced renal function and FSGS biopsy proven disease. The classical nephronophthisis related ciliopathy syndromes caused by mutations in STK11/LKB1, CTNS and TTC21B genes are characterized by tubulointerstitial fibrosis, cysts and bland urinalysis with minimal proteinuria and hematuria in late-stage kidney disease. Patients carrying mutations in STK11 and TTC21L in the CEP290 locus uniformly developed proteinuria, and also hematuria in some cases within the CEP290 positive subgroup, indicating early onset kidney disease characterized by glomerular dysfunction with increased permeability to proteins and cells. Our findings, together with earlier reports of FSGS linked to ciliary genes, such as TTC21B, NPHP4, CRB2 and CC2D2A, suggest that disrupted function of distinct ciliary modules may give rise to a unifying glomerular disease process that manifests as FSGS and podocyte damage.(13, 37, 43-45).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3. Pathophysiologic implications\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimary cilia have been implicated in signaling in podocytes and also in cytoskeletal organization(46, 47). These common features of the 35-truncating LOF mutations and the uniform FSGS histology and severe proteinuria suggest that mutations in these genes destabilize the podocyte and lead to disruption of the filtration barrier. Gene-specific functions may also contribute to the observed phenotypic heterogeneity of mutations within a gene. For example, mutations in genes encoding ciliary proteins involved in signaling (such as INPP5E and CEP290) were associated with more hematuria suggesting more extensive damage to the endothelium, GBM and/or podocytes. In contrast, mutations in CPLANE1, a component of the transition zone complex, were most commonly found in patients with universal proteinuria but relatively little hematuria, suggesting an important role of this protein in the maintenance of the structure and function of the foot processes and slit diaphragm of the podocyte. The mutations in BBS2/BBS7 may contribute to the relative resistance of the podocyte to signals from vasopressin and other molecules, blood pressure and thereby to the development of hypertension and progression of the renal disease. ( 36, 37, 48).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4. Clinical implications and genetic testing strategy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenetic testing for an expanded panel of ciliopathy genes in adult patients with FSGS revealed disease causing mutations in a subset of consanguineous families. Single gene panel testing for NPHS and COL4A genes may not detect mutations present in certain populations, indicating that the use of broad panels of genes is required and that single gene sequencing may not be sufficient in some cases (35).\u003c/p\u003e\n\u003cp\u003eGenetic testing can help identify biallelic mutations in ciliopathy genes and thus lead to a definitive diagnosis for affected individuals and their families. This test is not immunology dependent and is autosomal-recessive as each of the children of an affected parent will have a 25% chance of developing the disease. This test may be also helpful to determine if an individual is a carrier and to facilitate family planning especially in large families. The identification of mutations in a gene that leads to the diagnosis of the renal disease associated with ciliopathy genes will also allow complete cascade testing for possible extra renal features of the disease including neurological, ocular, skeletal and endocrine (49). Early genetic testing can be used in patients with suspected hereditary FSGS for diagnosis and clinical management (50, 51).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5. Limitations and future directions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile the cohort is small, it is the first study to our knowledge to examine the role of recessive ciliopathy genes in adult FSGS in a highly consanguineous cohort from a Middle Eastern population. The strong enrichment of biallelic truncating mutations in affected individuals relative to controls supports an association. However, there are limitations to this study. The cohort was small (35 cases and 20 controls) and examination of some of the genes (BBS7, NPHP3 and TCTN2) had only a single affected individuals per gene which may not provide sufficient power to make any gene-specific genotype-phenotype association. Genotype-phenotype analyses were performed in without multivariable adjustment because of insufficient power for multiple regression analyses to account for any potential confounders. Rare variant burden analyses were performed in an unadjusted fashion and only Haldane corrected gene level p values were reported because of multiple testing. The overall burden analysis between cases and controls seemed robust, but should be interpreted with caution at the gene level, particularly when the p values are close to the multiple testing threshold such as BBS2 and CPLANE1. A major limitation is that this is a cross-sectional study, and we do not capture the natural history of the disease such as time to ESRD and to kidney transplantation as well as post-transplant outcomes. We did not also perform a detailed evaluation of extra-renal manifestations (neuroimaging, ophthalmology, audiology and endocrinology). Thus, it is possible that subtle manifestations of ciliopathy have been overlooked. Lack of consanguinity between affected individuals and their extended family members meant that we were not always able to perform segregation analysis and Sanger confirmation, which would have allowed us to further demonstrate co-segregation. However, the presence of biallelic truncating mutations in affected individuals with complete absence or near absence of the variants in gnomAD in the context of consanguinity provides compelling evidence of their pathogenicity. A multi-center study that includes affected individuals with different ages at diagnosis, in addition to a larger control cohort, will be required to assess gene-specific risks of disease, as well as including more comprehensive extra-renal evaluation and functional in vitro or in vivo assays to better understand the disease-causing pathways at the level of cilia biology, e.g. roles of INPP5E, CPLANE1 and CEP290 in FSGS.\u003c/p\u003e"},{"header":"5. Conclusions ","content":"\u003cp\u003eMutations in ciliopathy genes appear to be important genetic contributor for adult FSGS in highly consanguineous populations. In this study, biallelic pathogenic or likely pathogenic variants were identified in 45.7% of patients and mutations in INPP5E, BBS2 and CPLANE1 were identified accounting for most genetically explained cases. In conclusion, mutations in ciliopathy genes should be considered in the genetic testing panel for adult FSGS, particularly in consanguineous families and those with strong family history of kidney diseases.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACMG: American College of Medical Genetics and Genomics\u003cbr\u003e\u0026nbsp;BBS: Bardet-Biedl syndrome\u003cbr\u003e\u0026nbsp;BUN: Blood urea nitrogen\u003cbr\u003e\u0026nbsp;CI: Confidence interval\u003cbr\u003e\u0026nbsp;eGFR: Estimated glomerular filtration rate\u003cbr\u003e\u0026nbsp;ESRD: End-stage renal disease\u003cbr\u003e\u0026nbsp;FSGS: Focal segmental glomerulosclerosis\u003cbr\u003e\u0026nbsp;gnomAD: Genome Aggregation Database\u003cbr\u003e\u0026nbsp;LP: Likely pathogenic\u003cbr\u003e\u0026nbsp;MAF: Minor allele frequency\u003cbr\u003e\u0026nbsp;NGS: Next-generation sequencing\u003cbr\u003e\u0026nbsp;OR: Odds ratio\u003cbr\u003e\u0026nbsp;P: Pathogenic\u003cbr\u003e\u0026nbsp;SD: Standard deviation\u003cbr\u003e\u0026nbsp;VUS: Variant of uncertain significance\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research team executed their study based on Declaration of Helsinki guidelines and Good Clinical Practice standards. Ethical approval was obtained from the Research Ethics Committee of the University of Sulaymaniyah, Kurdistan Region, Iraq. All study participants received written informed consent before starting the research but participants under 18 required their parent or legal guardian to give consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. The manuscript does not contain any identifiable individual data or images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pathogenic and likely pathogenic variants identified in this study have been deposited in ClinVar, a publicly accessible database of human genetic variation and its relationship to health (NCBI ClinVar, https://www.ncbi.nlm.nih.gov/clinvar/). The variants are available under accession numbers SCV007520383\u0026ndash;SCV007520396 and can be accessed via our submitter page, Molecular Lab (submitter ID 510454), at: https://www.ncbi.nlm.nih.gov/clinvar/submitters/510454/.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLava I. Ahmed conceived and designed the study, obtained ethical approval, supervised the research, performed DNA extraction, library preparation and next-generation sequencing, conducted statistical analysis, and drafted the manuscript.\u003c/p\u003e\n\u003cp\u003eDlnya A. Mohammed, MD, PhD contributed to study design, supervised laboratory work, assisted in data interpretation, and critically reviewed and revised the manuscript.\u003c/p\u003e\n\u003cp\u003eDana Ahmed Sharif, MD recruited patients, collected and managed clinical data, reviewed kidney biopsy reports, and contributed to clinical interpretation of genotype\u0026ndash;phenotype correlations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors reviewed and approved the final manuscript and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank all participating patients and their families for their valuable contribution to this study. We thank the laboratory and technical staff and all collaborators who supported DNA extraction, library preparation, and sequencing. We are grateful to the\u0026nbsp;\u003cstrong\u003eUniversity of Sulaimani\u003c/strong\u003e for providing institutional and infrastructural support that made this research possible.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ede Cos M, Meliambro K, Campbell KN. Novel treatment paradigms: focal segmental glomerulosclerosis. Kidney Int Rep. 2023;8(1):30\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRout P, Hashmi MF, Baradhi KM. Focal Segmental Glomerulosclerosis. StatPearls. Treasure Island (FL): StatPearls Publishing Copyright \u0026copy; 2025. StatPearls Publishing LLC.; 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi Cathain D, Reidy D, Bagnasco S, Kant S. Focal and Segmental Glomerulosclerosis: A Comprehensive State-of-the-Art Review. Sclerosis. 2025;3(3):24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGbadegesin R, Lavin P, Foreman J, Winn M. 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Front Pediatr. 2017;5:287.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDillard KJ, Hyt\u0026ouml;nen MK, Fischer D, Tanhuanp\u0026auml;\u0026auml; K, Lehti MS, Vainio-Siukola K, et al. A splice site variant in INPP5E causes diffuse cystic renal dysplasia and hepatic fibrosis in dogs. PLoS ONE. 2018;13(9):e0204073.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHakim S, Dyson JM, Feeney SJ, Davies EM, Sriratana A, Koenig MN, et al. Inpp5e suppresses polycystic kidney disease via inhibition of PI3K/Akt-dependent mTORC1 signaling. Hum Mol Genet. 2016;25(11):2295\u0026ndash;313.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu W, Jin M, Hu R, Wang H, Zhang F, Yuan S, Cao Y. The Joubert Syndrome Protein Inpp5e Controls Ciliogenesis by Regulating Phosphoinositides at the Apical Membrane. J Am Soc Nephrol. 2017;28(1):118\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForsythe E, Sparks K, Best S, Borrows S, Hoskins B, Sabir A, et al. Risk Factors for Severe Renal Disease in Bardet-Biedl Syndrome. J Am Soc Nephrol. 2017;28(3):963\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta N, D'Acierno M, Zona E, Capasso G, Zacchia M, editors. Bardet\u0026ndash;Biedl syndrome: The pleiotropic role of the chaperonin-like BBS6, 10, and 12 proteins. American Journal of Medical Genetics Part C: Seminars in Medical Genetics. Wiley Online Library; 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHildebrandt F, Attanasio M, Otto E. Nephronophthisis: disease mechanisms of a ciliopathy. J Am Soc Nephrol. 2009;20(1):23\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStokman M, Lilien M, Knoers N. Nephronophthisis-related ciliopathies. GeneReviews\u0026reg;[Internet]. 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimms RJ, Eley L, Sayer JA, Nephronophthisis. Eur J Hum Genet. 2009;17(4):406\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorbeil D, Thamm K, Karbanov\u0026aacute; J, Fargeas CA, J\u0026aacute;szai J. The primary cilium as a multifunctional organelle: emerging roles and unanswered questions. Cell Commun Signal. 2025;23(1):406.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePala R, Alomari N, Nauli SM. Primary Cilium-Dependent Signaling Mechanisms. Int J Mol Sci. 2017;18(11).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHardee I, Soldatos A, Davids M, Vilboux T, Toro C, David KL, et al. Defective ciliogenesis in INPP5E-related Joubert syndrome. Am J Med Genet A. 2017;173(12):3231\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimms RJ, Hynes AM, Eley L, Sayer JA. Nephronophthisis: a genetically diverse ciliopathy. Int J Nephrol. 2011;2011:527137.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSnoek R, Nguyen TQ, van der Zwaag B, van Zuilen AD, Kruis HME, van Gils-Verrij LA, et al. Importance of Genetic Diagnostics in Adult-Onset Focal Segmental Glomerulosclerosis. Nephron. 2019;142(4):351\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao T, Udwan K, John R, Rana A, Haghighi A, Xu L, et al. Integration of Genetic Testing and Pathology for the Diagnosis of Adults with FSGS. Clin J Am Soc Nephrol. 2019;14(2):213\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-nephrology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnep","sideBox":"Learn more about [BMC Nephrology](http://bmcnephrol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bnep/default.aspx","title":"BMC Nephrology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ciliopathy, Focal segmental glomerulosclerosis, Consanguinity, Genetic testing, Next-generation sequencing, Burden analysis","lastPublishedDoi":"10.21203/rs.3.rs-9125192/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9125192/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eBackground\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFocal segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome in adults and frequently progresses to end-stage renal disease. Emerging evidence suggests recessive ciliopathy genes contribute to glomerular disease, particularly in consanguineous populations, but their role in adult biopsy-proven FSGS remains incompletely defined. This study aimed to determine the frequency and clinical impact of pathogenic variants in recessive ciliopathy genes among adults with FSGS from a highly consanguineous Middle Eastern population.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethods\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe conducted a case-control study including 35 adults with biopsy-proven FSGS and 20 unaffected individuals with normal kidney function from Sulaymaniyah, Iraq. All participants underwent targeted next-generation sequencing of a 98-gene panel on the Ion S5 platform. Variants were classified according to 2015 American College of Medical Genetics and Genomics (ACMG) criteria; only pathogenic (P) and likely pathogenic (LP) variants in recessive ciliopathy genes were analyzed. Formal rare-variant burden analysis was performed. Demographic and clinical data were compared between groups.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBiallelic ciliopathy variants were identified in 16 of 35 FSGS patients (45.7%) versus 0 of 20 controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; OR 34.7, 95% CI 1.9-618.7). Seven ciliopathy genes were implicated: INPP5E (10 patients, 28.6%), BBS2 (7 patients, 20.0%), CPLANE1 (6 patients, 17.1%), CEP290 (4 patients, 11.4%), TCTN2 (1 patient), BBS7 (1 patient), and NPHP3 (1 patient). All identified variants were absent or ultra-rare (allele frequency\u0026thinsp;\u0026lt;\u0026thinsp;0.000007) in gnomAD v4.1, confirming their rarity. Ciliopathy-positive patients demonstrated universal proteinuria, higher rates of hematuria (81.3% vs 21.1%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), more severe renal dysfunction (serum creatinine 3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 vs 1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mg/dL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and increased hypertension prevalence (68.8% vs 31.6%, p\u0026thinsp;=\u0026thinsp;0.002) compared with ciliopathy-negative FSGS patients.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusions\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn this highly consanguineous Middle Eastern cohort, recessive ciliopathy gene variants, particularly in INPP5E, BBS2, and CPLANE1, were frequent among adults with biopsy-proven FSGS and associated with severe progressive kidney disease. These findings support incorporating ciliopathy gene testing into the diagnostic evaluation of adults with FSGS from consanguineous families or with positive family history of kidney disease.\u003c/p\u003e","manuscriptTitle":"Contribution of Recessive Ciliopathy Genes in a Highly Consanguineous Adult Cohort with Biopsy-Proven Focal Segmental Glomerulosclerosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 09:44:45","doi":"10.21203/rs.3.rs-9125192/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-18T09:17:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-15T12:38:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-02T17:03:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76716483658221357146102968605381246032","date":"2026-04-24T12:05:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"34374396735028723225541366899593166289","date":"2026-04-23T02:07:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-22T11:51:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-30T08:56:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-29T22:07:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Nephrology","date":"2026-03-29T22:04:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-nephrology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnep","sideBox":"Learn more about [BMC Nephrology](http://bmcnephrol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bnep/default.aspx","title":"BMC Nephrology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9d9ef301-591e-4dda-9c47-0fffe832f89b","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-18T09:17:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-15T12:38:17+00:00","index":36,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-02T17:03:52+00:00","index":35,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T09:24:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 09:44:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9125192","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9125192","identity":"rs-9125192","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}