The diagnosis of a rare genetic disease begins an odyssey toward treatment and cure for patients, clinicians, and researchers. Even though a treatment may not have been identified, there are many ways in which the clinician can help the patient. Information available to patients and their participation in research is dependent on how many individuals have previously been diagnosed with the condition and how much prior research has been conducted. When only a few cases have been reported, information will be limited to case reports, and each newly diagnosed patient is critical to learning about the disease. As more information becomes available, patient foundations form and researchers develop a long-term interest in the condition. In addition to informing the patient about diagnosis, prognosis, and the genetics of the underlying condition, clinicians should also help to guide the patient toward patient foundations and provide information about research participation to help better characterize the condition and find treatments. In rare disease research, each patient is important in providing clinical information as well as genetic and other biological samples for study. Researchers take this information provided by patients, analyze the data, and provide patients information about their condition. As patients and researchers work together, progress in research occurs, eventually leading to a treatment. The clinician, patient, clinical researcher, and basic scientist are all critical to success toward a potential therapy.

INTRODUCTION:Biallelic variants in Fanconi Anemia-associated Nuclease 1 (FAN1) cause karyomegalic tubulointerstitial nephropathy (KIN), a condition poorly characterized in terms of kidney survival, patient survival, and clinical characteristics. Therefore, we undertook a cross-sectional collaborative study to better characterize KIN-FAN1. METHODS:To gather data, we distributed a REDCap survey on clinical characteristics and genetic variants of KIN-FAN1 to colleagues and case report authors. RESULTS:Based on the survey, we identified 86 families affected (122 individuals) from 22 countries. There were 56 families (83 individuals) with a genetic diagnosis of KIN-FAN1, including 38 distinct FAN1 variants, and 30 families (39 individuals) with KIN with no predisposing risk factors and without molecular FAN1 testing. The median age at presentation was 38.5 years (interquartile range: 29-43), 62% male. Of the cohort, 46% had asymptomatic elevation of liver function tests, 39% had pulmonary complications, and 6% developed cancer. The median age of kidney failure was 45 years (95% confidence interval (CI): 38-56). Of the cohort, 27.1% died at a median age of 55 years (95% CI: 43-75). Pulmonary complications was/were the cause of death in 15.4% of patients on dialysis and 23.1% of kidney transplant recipients. Compared to other variants, patients with the p.W707X-FAN1 variant were at a significantly higher risk of pulmonary complications (adjusted odds ratio: 8.26 (95% CI: 1.7-40.1) and had a significantly shorter lifespan (hazard ratio: 3.24 (95% CI: 1.13-9.28). No genetic covariates were statistically associated with the progression to kidney failure. CONCLUSIONS:Patients with KIN-FAN1 develop kidney failure at a median age of 45 years. Survival is compromised with many dying of pulmonary disease.

KEY POINTS:Single-molecule real-time sequencing with the PacMUC1 script resolved exact MUC1 variable tandem repeat structure and full allelic variation. In 300 individuals, the protocol identified 215 distinct MUC1 tandem repeat alleles with 80 repeat units and nine frameshift mutation types. Probe extension assay identified 90% of families with frameshift mutations, detection of frameshifted mucin-1 aided genetically unresolved cases. BACKGROUND:ADTKD- MUC1 is caused by frameshift mutations in the MUC1 gene, producing a frameshifted neoprotein (MUC1fs) toxic to kidney cells. The gene's variable number of tandem repeats (VNTR), with approximately 80% guanine/cytosine content, has made it largely inaccessible to standard short-read sequencing, leaving the reference sequence and natural variation poorly defined and complicating mutation detection. METHODS:Using single-molecule real-time (SMRT) sequencing, we characterized MUC1 VNTR in 300 individuals, including 279 from 143 families suspected of having ADTKD- MUC1 , assessing VNTR length, repeat structure, and frameshift mutations. Results were compared with the Clinical Laboratory Improvement Amendments-approved probe-extension assay, detecting the prevalent 59dupC mutation, and with MUC1fs immunohistochemistry, which detects the pathogenic protein independent of the underlying genomic change. RESULTS:We identified 215 unique VNTR alleles composed of 80 distinct repeat units, 46 (58%) of which were novel, and nine distinct frameshift mutations present on 52 mutated alleles. Overall, MUC1 frameshift mutations were identified in 71 of 143 families (50%) with suspected ADTKD- MUC1 , comprising 135 affected individuals (48%). The SMRT assay outperformed the probe-extension assay by identifying frameshift mutations in two families with previously inconclusive results and in eight additional families whose mutations were undetectable by the probe-extension design. When successful, SMRT assay showed 100% concordance with probe-extension assay at the family level and 98% at the individual level, with discordance attributable to allelic dropout inherent to both long-range PCR amplification and long-read sequencing. Analysis of the mutational spectrum confirmed 59dupC as the most prevalent mutation, affecting approximately 90% of families, while the other eight mutation types occurred at most twice. CONCLUSIONS:The SMRT assay outperformed the Clinical Laboratory Improvement Amendments-approved probe-extension assay by detecting essentially all VNTR-associated frameshift mutations. The probe-extension assay identified approximately 90% of affected families. MUC1fs immunohistochemistry added diagnostic value in genetically unresolved cases by detecting the pathogenic protein independent of the underlying mutation.

Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a genetically heterogeneous disorder characterized by progressive chronic kidney disease, primarily affecting the tubules and interstitium. It results from pathogenic variants in several known genes, including UMOD, mucin-1 (MUC1), REN, APOA4, and others. Despite its clinical significance, ADTKD is often under-recognized due to nonspecific histopathological and clinical features and limited referral for genetic testing, which remains the gold standard for diagnosis. In addition, due to technical difficulties, genetic testing for ADTKD-MUC1 is not currently performed in multigene panels and cannot be obtained through whole exome or genome sequencing. Recent advances in kidney disease research underscore the critical role of comprehensive genetic testing in elucidating the molecular etiology of unsolved cases. However, challenges persist in the diagnosis of specific ADTKD subtypes, particularly those caused by pathogenic MUC1 pathogenic variants. This review provides a detailed analysis of the genetic basis, clinical presentation, and diagnostic approaches for ADTKD, with an emphasis on the limitations in current testing methodologies for ADTKD-MUC1. We further explore recent advancements in our understanding of ADTKD and highlight future directions for improving diagnostic accuracy and patient care through enhanced genetic testing technologies.

Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD) is the third most common inherited monogenic kidney disorder. Mutations in UMOD and MUC1 account for most cases, with the disease characterized by progressive eGFR decline leading to kidney failure. No disease-modifying therapies exist, and transplantation is the only current option. Designing trials for ADTKD is hampered by small patient numbers, variable progression rates, and uncertainty around optimal endpoints. Building on natural history data from Wake Forest School of Medicine, nonlinear mixed-effects models were developed to describe eGFR decline in UMOD and MUC1 variants. These models formed the foundation for a web-based clinical trial simulation (CTS) tool (https://app.cop.ufl.edu/adtkd/) built in R Shiny. The tool allows users to define trial populations, configure design parameters, and estimate statistical power by simulating placebo vs. assumed treatment arms. Drug effects were modeled as percentage changes in key parameters of the developed disease progression models describing individual-level eGFR trajectory over age: DPT50 (age at which eGFR is half of its maximum decline), γ 1 $$ {\gamma}_1 $$ and γ 2 $$ {\gamma}_2 $$ (steepness before and after DPT50). Both slope-based eGFR change and end-of-trial eGFR measures functioned as effective surrogate endpoints. Herein, we present the model-based CTS tool developed for ADTKD and demonstrate its use in designing and evaluating clinical trial scenarios, illustrated by two representative case studies. The CTS tool provides a pragmatic framework for optimizing ADTKD trial design. By enabling scenario testing and highlighting genotype-specific considerations, it supports efficient, cost-effective planning and represents an example of model-informed drug development in rare kidney diseases.

Abstract Background ADTKD- MUC1 is one of the major entities of ADTKD caused by frameshift variants in the MUC1 VNTR that standard short-read sequencing fails to detect. Existing 59dupC-targeted probe-extension assays do not allow for broad screening and cannot detect atypical non-dupC variants. Recently, VNtyper, a Kestrel-based genotyping pipeline with optional code-adVNTR cross-validation for MUC1 VNTR genotyping from short-read sequencing data allowed to circumvent this diagnostic limitation, but needed further development for easy access and rapid sample processing. Methods We developed VNtyper 2, by refactoring VNtyper into a modular, production-grade tool with a companion web platform, VNtyper-Online ( https://vntyper.org ), for freely available browser-based analysis with short turnaround time and without local bioinformatics infrastructure. We validated VNtyper 2 on 400 simulated samples generated with MucOneUp and 142 clinical exomes with independently confirmed genotypes. Results In simulation, VNtyper 2 detected the canonical 59dupC variant with 96% sensitivity and 100% specificity. Reference-standard validation on 142 samples yielded 90.6% sensitivity and 98.2% specificity overall, with cohort-dependent performance across the Twist Exome v2 French-German cohort (98% sensitivity, 87.5% specificity) and the KAPA HyperExome V2 (Roche) Czech-US cohort (79.4% sensitivity, 100% specificity). Screening of 3582 exomes and targeted panels from international CKD referral programmes identified 51 positive individuals, including 9 with atypical non-dupC frameshift variants that would have been missed by 59dupC-targeted probe-extension assays. In unselected CKD cohorts, a descriptive random-effects summary estimated a detection rate of 1.4% (95% CI 0.6 to 3.1%). Conclusions VNtyper 2 and VNtyper-Online are open-source tools for MUC1 VNTR genotyping from short-read data and can support locally validated workflows when VNTR coverage is adequate. By improving accessibility and turnaround time, these tools democratize MUC1 diagnostics at global scale. For its integration into routine diagnostics, we propose an expert-informed two-pathway workflow developed through European ADTKD-Net consortium consensus.

Introduction: Pathogenic (P) variants in COL4A3/A4/A5 genes are known to cause thin glomerular basement membrane (GBM) or Alport-related kidney disease; however, the exact diagnostic yield of genetic testing remains unknown. Methods: In this retrospective genotype-phenotype correlation study, we screened the patient populations of 2 major US medical centers for individuals who underwent kidney biopsy, and who had documented genetic testing results on a large 385-kidney disease gene panel. We correlated GBM thickness, estimated glomerular filtration rate, proteinuria, and hematuria with genotyping results. Results: We identified 115 patients with coexisting histopathology and genetic testing data, of which 49 had ultrastructural abnormalities of the GBM. Among those 49 cases, 9 had a heterozygous pathogenic or likely pathogenic (P/LP) variant in one of the COL4A genes, and 9 additional patients had COL4A variants of uncertain significance (VUS). Thirty-one patients with thin GBM were COL4A3/A4/A5 wildtype. One patient with a P variant in COL4A4 had no GBM abnormalities. Three COL4A VUS were upgraded to P/LP through experimental testing. Presence of P/LP variants in COL4A genes correlated with GBM thickness, but not with other clinical parameters. Among 31 thin GBM cases with no COL4A variant, we found variants in steroid-resistant nephrotic syndrome-, congenital anomalies of the kidneys and urinary tract (CAKUT)-, and autosomal dominant tubulointerstitial kidney disease (ADTKD) genes characterized as P/ LP or as "high-risk VUS." Immune-mediated glomerular injury was as frequent in biopsy specimens with thin GBM as with normal GBM. Conclusion: In our study, almost two-thirds of patients with thin GBM have no variant in COL4A3/A4/A5 genes. Our data suggest that genetic testing may not obviate the need for kidney biopsy.

Introduction:Mitochondrial DNA (mtDNA) is not routinely analyzed in inherited kidney disease. We evaluated mtDNA variation in families who remained genetically unresolved despite extensive testing. Methods:We reviewed pedigrees from the Wake Forest-Charles University Rare Inherited Kidney Disease Registry to identify genetically unresolved families with suspected maternal inheritance, performed mtDNA genotyping, clinically characterized variant carriers, and functionally evaluated disease-associated mitochondrial variants. Results:Among 33 families with evidence of maternal inheritance, 18 (55%) carried one of seven disease-associated mtDNA variant types, including homoplasmic recurrent single-nucleotide insertions in the second light-strand promoter (LSP2; 9 families), novel MT-TW and MT-TL2 variants (2 and 1 families, respectively), and previously reported MT-TF and heteroplasmic MT-ND5 variant (5 and 1 families, respectively). In 16 families, variants occurred on distinct haplotypes, consistent with independent mutational events and rapid enrichment to homoplasmy across generations. Maternal transmission was strongly supported, with below-normal kidney function observed in 54/60 (90%) offspring of affected mothers versus 1/17 (6%) offspring of affected fathers (p = 1.23 × 10 ⁻11 ). Pathogenicity was further supported by predicted deleterious structural effects and functional evidence of impaired mitochondrial transcription and translation, respiratory chain deficiency, and CoQ10 depletion. Affected individuals predominantly presented with chronic tubulointerstitial kidney disease, occasionally accompanied by gout and only sporadically with extrarenal manifestations. The rate of kidney disease progression appeared to vary both between and within families. Overall, 109/119 genetically affected individuals or obligate at-risk carriers were clinically affected; most unaffected carriers were younger than 45 years of age. Clinical status was unavailable for an additional 66 obligate at-risk carriers. Conclusions:These findings establish the physiological relevance of the LSP2 promoter, support routine assessment of the mitochondrial genome in inherited kidney disease, and highlight mtDNA variants as an important cause of familial and sporadic tubulointerstitial kidney disease of previously unexplained etiology. Lay Summary:Many inherited kidney diseases remain unexplained because routine genetic testing focuses on genes in the cell nucleus and does not examine mitochondrial DNA-the small genome in the cell's energy-providing mitochondria, inherited only from the mother. We studied 33 families with chronic kidney disease whose family histories suggested maternal inheritance and identified disease-causing mitochondrial DNA variants in 18 (55%). Nine families carried variants in LSP2, a recently discovered mitochondrial regulatory element, highlighting its importance in normal mitochondrial function and disease. Others carried pathogenic variants in mitochondrial tRNA genes required for mitochondrial protein synthesis. Laboratory studies showed that these variants impair mitochondrial energy conversion. In all families, the predominant manifestation was slowly progressive kidney disease, sometimes leading to dialysis or kidney transplantation. These findings identify pathogenic mitochondrial DNA variants as an underrecognized cause of inherited kidney disease and support the inclusion of mitochondrial DNA analysis in routine genetic testing.

Abstract Introduction Autosomal-dominant tubulointerstitial kidney disease (ADTKD) is characterized by chronic kidney disease (CKD) with an average age of end-stage renal disease (ESRD) of approximately 45 years, bland urinary sediment, the absence of proteinuria and autosomal dominant inheritance. While several causative genes have been found, there remain families in whom no molecular diagnosis has been identified (ADTKD-NMD). Methods We identified BICC1 truncating variants in several families with ADTKD-NMD in the Wake Forest Rare Inherited Kidney Disease Registry and then screened families in our database and other referred families for BICC1 truncating variants. We performed segregation analysis and characterized affected individuals for clinical and histopathologic phenotypes. We analyzed oligomer formation of BICC1 mutants with wild-type BICC1-, ANKS3- and ANKS6 proteins through co-immunoprecipitation and Western blotting, and we tested for posttranscriptional regulation of the BICC1 target mRNA, Dand5 , in a Luciferase reporter assay. Results We found 6 heterozygous truncating mutations in BICC1 segregating with the ADTKD phenotype in 8 independent pedigrees worldwide. Affected individuals developed kidney failure in the 6 th to 7 th decade of life that was characterized pathologically by tubular atrophy and interstitial fibrosis. The truncated gene products localized to cytoplasmic bodies and demonstrated various degrees of self-association or binding to the known interaction partners, ANKS3 and ANKS6. While the wild-type BICC1 gene product acts as a posttranscriptional repressor of target mRNAs, all truncation variants exhibited increased expression of substrate mRNA. Conclusions Truncating variants in BICC1 are a novel cause of ADTKD, segregating with the disease phenotype and upregulating BICC1 target gene expression through a dominant-negative- or a gain-of-function mode of action. Translational Statement Novel disease gene discoveries have a high potential for translational impact. Autosomal-dominant tubulointerstitial kidney disease (ADTKD) is reported to occur in 1-2 individuals per 100,000 but is underrecognized and underdiagnosed. Although several genes have been associated with ADTKD, many cases remain genetically unresolved. Our discovery of BICC1 as a novel gene in ADTKD will lead to improved disease recognition, prognostication and counseling. Through its role in gene regulation, BICC1 is a bona fide target for the molecular study of kidney fibrosis and atrophy, a final common pathway in CKD, as well as for the development of future therapies.

Autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD-UMOD) is one of the leading hereditary kidney diseases. Currently there is no targeted treatment. To illuminate human relevance of mesencephalic astrocyte-derived neurotrophic factor (MANF)-based therapy, we have established patient induced pluripotent stem cell (iPSC)-derived kidney organoid model carrying UMOD p.H177-R185del, the leading mutation causing ADTKD. We have discovered that MANF can directly bind and repress ER calcium release channel IP3R1, thus enhancing AMPK-induced autophagy in a TRIB3-dependent manner. The therapeutic implication of this finding may well be extended to other protein misfolding diseases.

There are 3 major forms of autosomal dominant tubulointerstitial kidney disease (ADTKD): ADTKD due to UMOD mutations, MUC1 mutations, and mutations in the REN gene encoding renin. Lack of knowledge about these conditions contributes to frequent nondiagnosis, but with even limited knowledge, nephrologists can easily obtain a diagnosis and improve patient care. There are 3 cardinal features of these disorders: (1) the conditions are inherited in an autosomal dominant manner and should be considered whenever both a parent and child suffer from kidney disease; the presence of even more affected family members provides further support. (2) These conditions are associated with a bland urinary sediment, ruling out glomerular disorders. (3) There is a variable rate of decline in kidney function. The mean age of ESRD is approximately 45, but the range is from 17 to >75. ADTKD-UMOD is often but not always associated with gout in the teenage years. ADKTKD-REN is associated with signs of hyporeninemia: mild hypotension, mild hyperkalemia, anemia in childhood, and hyperuricemia and gout in the teenage years. The only clinical manifestation of ADTKD-MUC1 is slowly progressive CKD. Diagnosis should be made by genetic testing, and kidney biopsy should be avoided.

Background:ADTKD-MUC1 is caused by frameshift mutations in MUC1 gene that produce a frameshifted protein (MUC1fs) toxic to kidney cells. The gene's variable number of tandem repeats (VNTR), with high GC content, makes it largely inaccessible to standard sequencing. As a result, both the reference sequence and natural variation in this region remain poorly defined, complicating mutation detection and data interpretation. Standard methods also fail to pinpoint the exact VNTR unit affected, limiting insight into mutation mechanisms and genotype-phenotype correlations. Methods:We employed Single Molecule, Real-Time (SMRT) sequencing and characterized the genomic sequence of MUC1 in 300 individuals including 279 individuals from 143 families suspected of having ADTKD-MUC1. We compared these results to those obtained using the CLIA-approved mass spectrometry-based probe extension (PE) assay, which specifically detect the most prevalent 59dupC mutation. We correlated the structural features of the MUC1 VNTR with the rate of kidney function decline in affected individuals. Results:We identified MUC1 consensus sequences for 205 unique VNTR alleles, with 9 distinct types of frameshift mutations present on 52 distinct mutated VNTR alleles. MUC1 frameshift mutations were identified in 71 of 143 families (50%) with suspected ADTKD, comprising 135 genetically affected individuals (48%). The SMRT assay exhibited complete concordance and revealed that the PE assay is capable of detecting frameshift mutations in approximately 85% of affected families. The constellation of VNTR structures supports a genotype-progression model, in which fast progressors exhibit a significantly lower number of repeat units on the wild-type allele and a higher number of repeats on the mutation-bearing allele, including an increased number of frameshifted repeat units. Conclusions:SMRT sequencing outperforms current diagnostic methods for ADTKD-MUC1 and reveals the prognostic value of VNTR structures. Although their contribution to disease progression is modest (~6% variance explained), it remains biologically and clinically meaningful.

Introduction:Although 10% of adults with chronic kidney disease (CKD) have a monogenic cause, the characteristics of monogenic CKD in older adults (aged ≥ 60 years) are less characterized. We aimed to assess the clinical and genetic spectrum of older adults with CKD and the clinical utility of genetic findings. Methods:The diagnostic yield of clinically validated disease-causing variants and their type ("typical" vs. "later-onset" phenotypes) were analyzed in older patients with suspected monogenic CKD who were referred to an Irish registry according to predetermined criteria. Independent genetic diagnosis and kidney survival time predictors were analyzed using marginal logistic and Cox regression analyses. Results:Two hundred sixty-five adults (from 202 families) were aged ≥ 60 years at the time of genetic testing, of which 74.3% (197/265) progressed to kidney failure. Diagnostic variants were found in 60.4% (122/202) families, including 39% of noncystic kidney disease families. Variants causing "later-onset" phenotypes were more prevalent in patients with disease-onset ≥ 60 years (56% vs. 8.3%; P ≤ 0.001), which include genetic variants in: IFT140, ALG5, ALG9, DNAJB11, COL4A5 in females, monoallelic COL4A3, and the UMOD p.Thr62Pro variant, associated with delayed onset of kidney failure compared with "typical" variants (hazard ratio: 0.52; 95% confidence interval: 0.27-0.98; P = 0.043). A family history of CKD and a priori cystic kidney disease diagnosis independently predicted genetic diagnosis (P ≤ 0.05). In 24% of older adults with positive results, the treatment plan was modified. Conclusion:In older patients with CKD, genetic testing revealed enriched variants associated with less-penetrant phenotypes, often with a family history of CKD, which affects clinical management.