Rationale & Objective: Molecular diagnosis of autosomal dominant tubulointerstitial kidney disease (ADTKD) due to variants in the MUC1 gene has long been challenging because variants lie in a large variable number of tandem repeat (VNTR) region, making identification impossible using standard short-read techniques. Previously, we addressed this diagnostic limitation by developing a computational pipeline named VNtyper for easier reliable detection of MUC1 VNTR pathogenic variants from short-read sequences. This led to unexpected diagnoses of ADTKD-MUC1 among patients with kidney disease referred for genetic testing, which we report here. Study Design: Cross-sectional observational study. Setting & Participants: 4,040 patients referred to Necker Enfants-Malades Hospital from 2017 to 2023 for genetic testing for (1) glomerular disease, (2) ciliopathy, (3) congenital anomalies of the kidneys and urinary tracts (CAKUT), (4) ADTKD, or (5) chronic kidney disease (CKD) of unknown origin, in whom MUC1 had not been previously tested by SNaPshot minisequencing. Exposure: Clinical suspicion of ADTKD. Outcome: ADTKD-MUC1 diagnosed using VNtyper. Analytical Approach: Data were collected from patients in whom ADTKD-MUC1 was newly diagnosed and patients in whom ADTKD was clinically suspected were compared with those in whom ADTKD was not. Results: We identified 40 patients with MUC1 variants by VNtyper, including 33 new index patients and 7 relatives. Of the 33 index cases, 20 had been suspected of having ADTKD based on clinical features, and in the other 13 ADTKD had not been considered. In patients in whom ADTKD had not been considered clinically, the detection rate was 0.05% (1 of 1,895) among patients with glomerular disease, 1.2% (4 of 329) among patients with ciliopathy, 0.09% (1 of 1,099) among patients with CAKUT and 2.5% (7 of 285) among patients with CKD of unknown origin. In 6 patients there was no family history of kidney disease, and we confirmed de novo presentation in 2 patients by segregation studies. Limitations: Observational study and selected referral population (may not represent the prevalence or phenotypes in the general kidney disease population). Conclusions: With VNtyper, we were able to diagnose new cases of ADTKD-MUC1 in a large cohort of patients with various phenotypes. Some patients had atypical phenotypes due to a variant in another gene, and some had no family history of kidney disease, suggesting de novo disease, which was confirmed in 2 patients.

Kidney organoids are an emerging tool for disease modeling, especially genetic diseases. Among them, X-linked Alport syndrome (XLAS) is a hematuric nephropathy affecting the glomerular basement membrane (GBM) secondary to pathogenic variations in the COL4A5 gene encoding the α5 subunit of type IV collagen [α5(IV)]. In patients carrying pathogenic variations affecting splicing, the use of antisense oligonucleotides (ASOs) offers immense therapeutic hope. In this study, we develop a framework combining the use of patient-derived cells and kidney organoids to provide evidence of the therapeutic efficacy of ASOs in XLAS patients. Using multiomics analysis, we describe the development of GBM in wild-type and mutated human kidney organoids. We show that GBM maturation is a dynamic process, which requires long organoid culture. Then, using semi-automated quantification of α5(IV) at basement membranes in organoids carrying the splicing variants identified in patients, we demonstrate the efficacy of ASO treatment for α5(IV) restoration. These data contribute to our understanding of the development of GBM and pave the way for a therapeutic screening platform for patients. ### Competing Interest Statement The authors have declared no competing interest. Orphan Kidney Diseases (ORKiD) Agence Nationale de la Recherche, ANR-10-IAHU-01 France 2030 program, DOS0212694

KEY POINTS:Exome sequencing solved 26% of nephronophthisis cases, identifying nephropathy and extrarenal disease genes beyond classic ciliopathy panels. Exome sequencing uncovered GN and tubular nephropathy genes misdiagnosed as ciliopathy-associated nephropathy, underscoring diagnostic overlap in kidney diseases. Patients with nonciliary genetic variants may present with ciliopathy-like extrarenal symptoms, showing phenocopies in kidney ciliopathy diagnostics. BACKGROUND:Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial kidney disease and a leading genetic cause of chronic kidney failure in children and young adults. As a ciliopathy, NPH is caused by biallelic variants in genes encoding proteins involved in the structure and function of primary cilia. The broad clinical spectrum of NPH results in a clinically and genetically heterogeneous disease, posing diagnostic challenges and leaving approximately 30% of cases unresolved with current gene panels. METHODS:After targeted gene panel for ciliopathy-associated genes failed to identify diagnostic variants, exome sequencing (ES) was conducted on 42 unrelated index patients with a clinical diagnosis of NPH, defined as cystic nephropathy progressing to kidney failure within the first two decades of life, or by unspecific CKD accompanied by extrarenal features indicative of a ciliopathy. RESULTS:Pathogenic or likely pathogenic variants were identified in 11 of the 42 patients (26%). Variants were detected in known nephropathy genes ( LAMB2 , COQ8B , COL4A3 , MUC1 ) and a multisystem disease gene with secondary kidney involvement ( AGXT ). In addition, ES elucidated deleterious variants explaining extrarenal phenotypes without corresponding kidney disease in six patients ( APTX , TUBB3 , DHX38 , IQCE, CRX , RPGR ). Variants of unknown significance were identified in three patients, while heterozygous variants in genes associated with recessive disease were observed in three others. A potential candidate gene for syndromic tubulointerstitial nephropathy, SSBP1 , was also identified, suggesting a novel pathway involving mitochondrial dysfunction. CONCLUSIONS:ES enabled the identification of pathogenic variants in known genes associated with kidney diseases, nonkidney conditions, and multisystem disorders with secondary kidney involvement, thereby improving diagnosis accuracy, even in incomplete or atypical cases, and guiding specific diagnostic and therapeutic approaches. The identification of SSBP1 in association with tubulointerstitial nephropathy may provide new insights into the pathogenesis of ciliopathies.

Kidney organoids are an emerging tool for disease modeling, especially genetic diseases. Among these diseases, X-linked Alport syndrome (XLAS) is a hematuric nephropathy affecting the glomerular basement membrane (GBM) secondary to pathogenic variations in the COL4A5 gene encoding the α5 subunit of type IV collagen [α5(IV)]. In patients carrying pathogenic variations affecting splicing, the use of antisense oligonucleotides (ASOs) offers immense therapeutic hope. In this study, we develop a framework combining the use of patient-derived cells and kidney organoids to provide evidence of the therapeutic efficacy of ASOs in XLAS patients. Using multiomics analysis, we describe the development of GBM in WT and mutated human kidney organoids. We show that GBM maturation is a dynamic process, which requires long organoid culture. Then, using semi-automated quantification of α5(IV) at basement membranes in organoids carrying the splicing variants identified in patients, we demonstrate the efficacy of ASO treatment for α5(IV) restoration. These data contribute to our understanding of the development of GBM in kidney organoids and pave the way for a therapeutic screening platform for patients.

Background:The origin of chronic kidney disease (CKD) remains unknown in ≈16% of patients at the time of renal replacement therapy. The aim of this study was to assess the proportion of monogenic kidney diseases in kidney transplant candidates with kidney disease of unknown cause. Methods:Transplant candidates, referred to a nephrogenetic outpatient clinic, had a molecular investigation and were included if they met the following inclusion criteria: absence of diagnosis (including presumed hypertensive nephropathy or vascular or focal segmental glomerulosclerosis lesions) and a glomerular filtration rate <30 ml/min/1.73 m2 before 50 years of age and/or renal morphology abnormality (including renal hypotrophy, cysts and congenital anomalies of the kidney and urinary tract) and/or extrarenal involvement and/or family history of CKD. Results:Eighty-nine patients were evaluated at the nephrogenetic consultation and 84 patients met the inclusion criteria and were tested and included. Half had a family history of CKD. Almost half of the patients (46.4%) had a morphological abnormality of the kidney. Twenty-eight (33.3%) had been biopsied: 21% had focal and segmental hyalinosis lesions and 25% had chronic interstitial nephropathy. Thirty patients (36%) had a positive genetic diagnosis. Of these, 9/30 (30%) had APOL1 high-risk alleles and 21/30 (70%) had monogenic nephropathy. Patients with a positive genetic diagnosis were significantly more likely to have a family history of kidney disease (70% versus 37%; P = .004). Conclusions:Genetic testing enables a diagnosis to be established in 36% of patients, allowing genetic counselling and may help potential living donor evaluations.

INTRODUCTION:Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a common monogenic kidney disease leading to kidney failure usually during mid adulthood. It is due to pathogenic variants in at least five genes. However, despite thorough screening of UMOD, MUC1, REN, HNF1B and SEC61A1, 25 to 50% of families remain without molecular diagnosis. METHODS:Here, we investigated a cohort of 203 families with ADTK, as well as sporadic cases of kidney disease of unknown etiology and cases of chronic kidney disease stage 5 from the Genomics England 100,000 Genomes Project. Expression of JAG1 in kidney and/or urinary epithelial cell (UREC) lines from patients carrying a pathogenic JAG1 variant associated with isolated ADTKD was studied using immunolabelling, Western blotting, targeted RNA-seq and quantitative RT-PCR. Endoplasmic reticulum (ER) stress was tested by analyzing ER protein BiP expression levels in URECs. RESULTS:A pathogenic or likely pathogenic variant in JAG1, the gene associated with Alagille syndrome, was identified in three large families with unsolved ADTKD, and additional rare variants were identified in sporadic cases. In two of the families, the diagnosis of Alagille syndrome was further established in one infant in the fourth or fifth generation; however, none of the 23 adult patients affected with isolated kidney failure (and tubulointerstitial nephritis in individuals with available kidney biopsy) had overt sign of liver, bile duct, heart, eye, or skeletal defect. JAG1 expression studies as well as ER stress analysis suggests that, despite a noteworthy expression of the JAG1-mutated RNAs, the tubulointerstitial renal disease was not due to cell toxicity of an abnormal protein, but rather to haploinsufficiency and loss of function. CONCLUSIONS:JAG1 pathogenic variants can be associated with isolated tubulointerstitial nephropathy which, according to the KDIGO guidelines, should be classified as ADTKD-JAG1 when JAG1 variants lead to isolated chronic kidney disease that fulfills the criteria for ADTKD.

Abstract Background and Aims The human genome includes tandem repeats with variable length (VNTR) and a subset of these repeats have been associated with rare human diseases. Specific frameshift variants in the coding-VNTR of the MUC1 gene cause autosomal dominant tubulointerstitial kidney disease—MUC1 (ADTKD-MUC1). Calling variants from VNTR using short-read sequencing (SRS) is challenging due to poor read mappability, motif complexity (34*60-mer motifs are known up to now), variable repetition, and enormous motif sequence similarity. We have recently developed a computational pipeline called VNtyper, tailored for the precise detection of disease-causing variants within the MUC1 VNTR region using short-read sequencing data. This advancement allowed us to identify overlooked cases in a hereditary renal disease registry, leading to the diagnosis of at least 40 patients. Given that regular exome sequencing with low VNTR coverage proved inefficient for VNtyper, our focus shifted to boost the capture of MUC1 VNTR in exome. This improvement aims to enable the application of VNtyper in ADTKD-MUC1 diagnosis through exome sequencing. Method VNtyper utilizes two independent genotyping algorithms (Kestrel and code-adVNTR) along with MUC1 VNTR-specific reference sequence for the variant detection and is sensitive to the VNTR coverage. We employ Twist custom panels with v1 protocol for target enrichment in both our panel and exome sequencing. This procedure allows for the inclusion of spike-in probes without interfering with other targets. We designed an NTI panel that specifically targets 6 genes associated with ADTKD, including UMOD, MUC1, HNF1B, REN, SEC61A1, and DNAJB11. These genes were captured using a 1x tiling approach, whereas the MUC1 VNTR region was captured with 4x tiling approach. During the exome target enrichment process, we included the NTI probes as a spike-in to boost VNTR coverage. We conducted a routine exome sequencing on a total of 6 samples, consisting of 3 positive controls and 3 negative controls. Additionally, we conducted 16 boosted exome, using 3 negative controls and 13 MUC1 positive samples. Prior to using VNtyper on both exomes, we conducted initial quality controls to verify that there were no alterations in the coverages of other genes. Results The mean coverage of MUC1 VNTR in the regular exome was 72.1x, while in the boosted exome, it exhibited a significant increase to 144x. Adding spike in probes did not have significant effect on the coverage of the contig and there was no alteration in ratio of heterozygosity. As anticipated, in the regular exome, VNtyper was unsuccessful in identifying pathogenic variation in 2 out of 3 true positives. However, all negative controls tested negative. When we used the enhanced exome, we accurately identified all 13 true positive cases, while the negative control cases stayed negative. Through downsampling experiments (reducing read depth from 50% to 5% of the total) on panel data with a mean coverage of 700x, we determined that coverage below 100 should be considered as low coverage for VNTR genotyping using our pipeline. This underscores the critical importance of maintaining adequate coverage for accurate and reliable VNTR genotyping. Conclusion We have achieved successful enhancements in the accuracy and sensitivity of ADTKD diagnosis through the application of VNtyper on clinically boosted exome data. While VNtyper could identify MUC1-positive patients in regular exome data, the effectiveness is influenced by the number of repeats in both alleles and the specific motif in which the variation is present, which significantly decreases the sensitivity. In summary, the VNtyper pipeline demonstrates its ability to detect pathogenic variations in ADTKD-MUC1 on panel data (100% sensitivity), and the improvement in VNTR capture in exome significantly enhances the sensitivity of the ADTKD diagnosis.

Abstract Background and Aims Autosomal dominant tubulointerstitial kidney disease (ADTKD) due to pathogenic variants in the MUC1 gene is difficult to diagnose since these variants lie in a large variable number tandem repeats (VNTR) and require specialized genetic testing, such as SNaPshot minisequencing. We recently developed a computational pipeline, VNtyper, for easier reliable detection of MUC1 VNTR pathogenic variants and have applied this tool to a large cohort of patients with various phenotypes of kidney disease. The aim of this study is to clinically describe patients in whom MUC1 pathogenic variants were unexpectedly detected with the help of VNtyper. Method We applied a computational pipeline, VNtyper, to a large cohort of patients with suspected hereditary kidney disease referred for genetic testing, regardless of their phenotype. This cohort included patients evaluated from 2017 to 2023 in the Molecular Genetics Department of Necker-Enfants Malades Hospital (Paris, France). Clinical characteristics were collected from patients in whom we detected new MUC1 VNTR pathogenic variants. Results 44 out of 3735 patients tested (1.2%) were newly diagnosed with a MUC1 pathogenic variant. Of these patients, ADTKD was clinically suspected in only 31 out of 44 patients (70%). In 2 patients, there was a pathogenic variant in another gene that could explain the phenotype, in addition to the MUC1 pathogenic variant. In fact, one 52-year-old patient had polycystic kidneys, nephrolithiasis, hematuria and normal kidney function. Her mother also had kidney cysts with absence of chronic kidney disease (CKD). This patient had a pathogenic variant in IFT140 gene (c.2399+1G>T) which could explain her autosomal dominant polycystic kidney disease (ADPKD) phenotype. Whether the MUC1 variant contributes to her disease remains unclear, especially seeing as her kidney function is normal at the age of 52 years. On the other hand, one patient had kidney cysts discovered at 3 days of age. A heterozygous HNF1B deletion was detected which could explain the phenotype. The following detection of a pathogenic MUC1 variant was thus an incidental finding and seemed de novo, although parents declined to get tested. These incidental findings can be challenging, especially when it comes to counselling and follow-up. There was no family history of kidney disease in 7 out of 44 patients (16%), and in one patient, we were able to confirm de novo disease since her parents were tested and were negative. It was unfortunately not possible to get DNA from the parents of the other patients. This is the first case of proven de novo ADTKD-MUC1 described to our knowledge. De novo variants in ADTKD-MUC1 are not surprising since the 7C stretch found in VNTR is a hotspot for mutagenesis. Interestingly, these patients seemed to have a more severe clinical presentation with an early onset of disease (median age at diagnosis 28 years [18, 32]). However, this could be due to a selection bias. In fact, young age of onset of CKD may have prompted clinicians to refer these patients for genetic testing despite negative family history, whereas older patients with possible ADTKD-MUC1 and no family history of kidney disease might be presumed to have another etiology of CKD. One way to confirm this would be to test a large cohort of patients with CKD from unknown etiology with no family history of CKD. Considering the nonspecific clinical manifestations of ADTKD-MUC1 and the phenotypic variability, it has previously been suggested that the most important clue to diagnosis is the presence of CKD in first-degree family. The possibility of de novo cases makes the clinical diagnosis of ADKTD-MUC1 even more challenging. Conclusion With VNtyper, we were able to diagnose new cases of ADTKD-MUC1 in a large cohort of patients with various phenotypes, with a significant prevalence of 1.2%. ADTKD-MUC1 was not suspected in 30% of these patients who would have maybe never been diagnosed otherwise. This is also the first study in which we describe a proven de novo case of ADTKD-MUC1. Pathogenic variants in the MUC1 gene should thus be in the differential diagnosis of all unspecified CKD, even if there is no family history since de novo variants are also possible.

Introduction: Unlike idiopathic nephrotic syndrome (NS), hereditary podocytopathies are not expected to recur after kidney transplantation. However, some reports of posttransplant recurrence of NS in patients carrying variants in the NPHS2 gene have been described, notably with the p.Arg138Gln variant, which is more prevalent in Europe. The objective of this study was to assess the risk of recurrence after kidney transplantation in a large cohort of patients with biallelic NPHS2 pathogenic variants. Methods: Since January 2010, 61 patients identified at Necker-Enfants Malades Hospital and 56 enrolled in the PodoNet Registry with biallelic variants in the NPHS2 gene were transplanted and were compared with 44 transplanted children with steroid-resistant NS (SRNS) without any identified pathogenic variant. Results: Of the 117 patients, 23 carried the p.Arg138Gln variant in the homozygous state and 16 in the compound heterozygous state. The other 78 patients carried different variants in the homozygous (n = 44) or compound heterozygous state. Only 1 patient with NPHS2-related SRNS experienced posttransplant recurrence (median follow-up of cohort 8.5 years [2.5–15]). Conversely, 7 of 44 patients (16%) without any identified pathogenic variant recurred within a maximum of 7 days after transplantation (median follow-up 8.9 years [0.6–13.9]). Conclusion: In this large cohort, the risk of patients with causative variants in the NPHS2 gene to develop NS recurrence after kidney transplantation was extremely low. This is coherent with the pathophysiology of intrinsic slit-diaphragm disease. These data are reassuring and should be considered when counselling patients, making living kidney donation, whether related or not, a safe choice.

Introduction: Unlike idiopathic nephrotic syndrome (NS), hereditary podocytopathies are not expected to recur after kidney transplantation. However, some reports of posttransplant recurrence of NS in patients carrying variants in the NPHS2 gene have been described, notably with the p.Arg138Gln variant, which is more prevalent in Europe. The objective of this study was to assess the risk of recurrence after kidney transplantation in a large cohort of patients with biallelic NPHS2 pathogenic variants. Methods: Since January 2010, 61 patients identified at Necker -Enfants Malades Hospital and 56 enrolled in the PodoNet Registry with biallelic variants in the NPHS2 gene were transplanted and were compared with 44 transplanted children with steroid -resistant NS (SRNS) without any identified pathogenic variant. Results: Of the 117 patients, 23 carried the p.Arg138Gln variant in the homozygous state and 16 in the compound heterozygous state. The other 78 patients carried different variants in the homozygous ( n 1 / 4 44) or compound heterozygous state. Only 1 patient with NPHS2 -related SRNS experienced posttransplant recurrence (median follow-up of cohort 8.5 years [2.5-15]). Conversely, 7 of 44 patients (16%) without any identified pathogenic variant recurred within a maximum of 7 days after transplantation (median follow-up 8.9 years [0.6-13.9]). Conclusion: In this large cohort, the risk of patients with causative variants in the NPHS2 gene to develop NS recurrence after kidney transplantation was extremely low. This is coherent with the pathophysiology of intrinsic slit -diaphragm disease. These data are reassuring and should be considered when counselling patients, making living kidney donation, whether related or not, a safe choice.

The human genome includes tandem repeats with variable length (VNTR) and a subset of these repeats have been associated with rare human diseases. Specific frameshift variants in the coding-VNTR of the MUC1 gene cause autosomal dominant tubulointerstitial kidney disease – MUC1 (ADTKD-MUC1). Calling variants from VNTR using short-read sequencing (SRS) is challenging due to poor read mappability, motif complexity (34*60-mer motifs are known up to now), variable repetition, and enormous motif sequence similarity.