Description of a New Simple and Cost-Effective Molecular Testing That Could Simplify MUC1 Variant Detection.

Introduction: Patients with autosomal dominant tubulointerstitial kidney disease (ADTKD) usually present with nonspecific progressive chronic kidney disease (CKD) with mild to negative proteinuria and a family history. ADTKD-MUC1 leads to the formation of a frameshift protein that accumulates in the cytoplasm, leading to tubulointerstitial damage. ADTKD-MUC1 prevalence remains unclear because MUC1 variants are not routinely detected by standard next-generation sequencing (NGS) techniques. Methods: We developed a bioinformatic counting script that can detect specific genetic sequences and count the number of occurrences. We used DNA samples from 27 patients for validation, 11 of them were patients from the Lille University Hospital in France and 16 were from the Wake Forest Hospital, NC. All patients from Lille were tested with an NGS gene panel with our script and all patients from Wake Forest Hospital were tested with the snapshot reference technique. Between January 2018 and February 2023, we collected data on all patients diagnosed with MUC1 variants with this script. Results: A total of 27 samples were tested anonymously by the BROAD Institute reference technique for confirmation and we were able to get a 100% concordance for MUC1 diagnosis. Clinico-biologic characteristics in our cohort were similar to those previously described in ADTKD-MUC1. Conclusion: We describe a new simple and cost-effective method for molecular testing of ADTKD-MUC1. Genetic analyses in our cohort suggest that MUC1 might be the first cause of ADTKD. Increasing the availability of MUC1 diagnosis tools will contribute to a better understanding of the disease and to the development of specific treatments.

Increased burden of rare protein-truncating variants in constrained, brain-specific and synaptic genes in extremely impulsively violent males with antisocial personality disorder.

The genetic correlates of extreme impulsive violence are poorly understood, and there have been few studies that have characterized a large group of affected individuals both clinically and genetically. We performed whole exome sequencing (WES) in 290 males with the life-course-persistent, extremely impulsively violent form of antisocial personality disorder (APD) and analyzed the spectrum of rare protein-truncating variants (rPTVs). Comparisons were made with 314 male controls and publicly available genotype data. Functional annotation tools were used for biological interpretation. Participants were significantly more likely to harbor rPTVs in genes that are intolerant to loss-of-function variants (odds ratio [OR] 2.06; p < 0.001), specifically expressed in brain (OR 2.80; p = 0.036) and enriched for those involved in neurotransmitter transport and synaptic processes. In 60 individuals (20%), we identified rPTVs that we classified as clinically relevant based on their clinical associations, biological function and gene expression patterns. Of these, 37 individuals harbored rPTVs in 23 genes that are associated with a monogenic neurological disorder, and 23 individuals harbored rPTVs in 20 genes reportedly intolerant to loss-of-function variants. The analysis presents evidence in support of a model where presence of either one or several private, functionally relevant mutations contribute significantly to individual risk of life-course-persistent APD and reveals multiple individuals who could be affected by clinically unrecognized neuropsychiatric Mendelian disease. Thus, Mendelian diseases and increased rPTV burden may represent important factors for the development of extremely impulsive violent life-course-persistent forms of APD irrespective of their clinical presentation.

Autosomal dominant ApoA4 mutations present as tubulointerstitial kidney disease with medullary amyloidosis.

Sporadic cases of apolipoprotein A-IV medullary amyloidosis have been reported. Here we describe five families found to have autosomal dominant medullary amyloidosis due to two different pathogenic APOA4 variants. A large family with autosomal dominant chronic kidney disease (CKD) and bland urinary sediment underwent whole genome sequencing with identification of a chr11:116692578 G>C (hg19) variant encoding the missense mutation p.L66V of the ApoA4 protein. We identified two other distantly related families from our registry with the same variant and two other distantly related families with a chr11:116693454 C>T (hg19) variant encoding the missense mutation p.D33N. Both mutations are unique to affected families, evolutionarily conserved and predicted to expand the amyloidogenic hotspot in the ApoA4 structure. Clinically affected individuals suffered from CKD with a bland urinary sediment and a mean age for kidney failure of 64.5 years. Genotyping identified 48 genetically affected individuals; 44 individuals had an estimated glomerular filtration rate (eGFR) under 60 ml/min/1.73 m2, including all 25 individuals with kidney failure. Significantly, 11 of 14 genetically unaffected individuals had an eGFR over 60 ml/min/1.73 m2. Fifteen genetically affected individuals presented with higher plasma ApoA4 concentrations. Kidney pathologic specimens from four individuals revealed amyloid deposits limited to the medulla, with the mutated ApoA4 identified by mass-spectrometry as the predominant amyloid constituent in all three available biopsies. Thus, ApoA4 mutations can cause autosomal dominant medullary amyloidosis, with marked amyloid deposition limited to the kidney medulla and presenting with autosomal dominant CKD with a bland urinary sediment. Diagnosis relies on a careful family history, APOA4 sequencing and pathologic studies.

Precision nephrotoxicity testing using 3D in vitro models.

Nephrotoxicity is a significant concern during the development of new drugs or when assessing the safety of chemicals in consumer products. Traditional methods for testing nephrotoxicity involve animal models or 2D in vitro cell cultures, the latter of which lack the complexity and functionality of the human kidney. 3D in vitro models are created by culturing human primary kidney cells derived from urine in a 3D microenvironment that mimics the fluid shear stresses of the kidney. Thus, 3D in vitro models provide more accurate and reliable predictions of human nephrotoxicity compared to existing 2D models. In this review, we focus on precision nephrotoxicity testing using 3D in vitro models with human autologous urine-derived kidney cells as a promising approach for evaluating drug safety.

MANF stimulates autophagy and restores mitochondrial homeostasis to treat autosomal dominant tubulointerstitial kidney disease in mice.

Misfolded protein aggregates may cause toxic proteinopathy, including autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD-UMOD), a leading hereditary kidney disease. There are no targeted therapies. In our generated mouse model recapitulating human ADTKD-UMOD carrying a leading UMOD mutation, we show that autophagy/mitophagy and mitochondrial biogenesis are impaired, leading to cGAS-STING activation and tubular injury. Moreover, we demonstrate that inducible tubular overexpression of mesencephalic astrocyte-derived neurotrophic factor (MANF), a secreted endoplasmic reticulum protein, after the onset of disease stimulates autophagy/mitophagy, clears mutant UMOD, and promotes mitochondrial biogenesis through p-AMPK enhancement, thus protecting kidney function in our ADTKD mouse model. Conversely, genetic ablation of MANF in the mutant thick ascending limb tubular cells worsens autophagy suppression and kidney fibrosis. Together, we have discovered MANF as a biotherapeutic protein and elucidated previously unknown mechanisms of MANF in the regulation of organelle homeostasis, which may have broad therapeutic applications to treat various proteinopathies.

Maternal health and pregnancy outcomes in autosomal dominant tubulointerstitial kidney disease.

Introduction: Autosomal dominant tubulointerstitial kidney disease (ADTKD) is an increasingly recognized cause of chronic kidney disease. ADTKD pregnancy outcomes have not previously been described. Methods: A cross-sectional survey was sent to women from ADTKD families. Results: Information was obtained from 85 afffected women (164 term pregnancies) and 23 controls (50 pregnancies). Only 16.5% of genetically affected women knew they had ADTKD during pregnancy. Eighteen percent of ADTKD mothers had hypertension during pregnancy versus 12% in controls (p = 0.54) and >40% in comparative studies of chronic kidney disease in pregnancy. Eleven percent of births of ADTKD mothers were <37 weeks versus 0 in controls (p < 0.0001). Cesarean section occurred in 19% of pregnancies in affected women versus 38% of unaffected individuals (p = 0.06). Only 12% of babies required a neonatal intensive care unit stay. Conclusions: ADTKD pregnancies had lower rates of hypertension during pregnancy versus other forms of chronic kidney disease, which may have contributed to good maternal and fetal outcomes.

Mitochondrial ROS Triggers KIN Pathogenesis in FAN1-Deficient Kidneys.

Karyomegalic interstitial nephritis (KIN) is a genetic adult-onset chronic kidney disease (CKD) characterized by genomic instability and mitotic abnormalities in the tubular epithelial cells. KIN is caused by recessive mutations in the FAN1 DNA repair enzyme. However, the endogenous source of DNA damage in FAN1/KIN kidneys has not been identified. Here we show, using FAN1-deficient human renal tubular epithelial cells (hRTECs) and FAN1-null mice as a model of KIN, that FAN1 kidney pathophysiology is triggered by hypersensitivity to endogenous reactive oxygen species (ROS), which cause chronic oxidative and double-strand DNA damage in the kidney tubular epithelial cells, accompanied by an intrinsic failure to repair DNA damage. Furthermore, persistent oxidative stress in FAN1-deficient RTECs and FAN1 kidneys caused mitochondrial deficiencies in oxidative phosphorylation and fatty acid oxidation. The administration of subclinical, low-dose cisplatin increased oxidative stress and aggravated mitochondrial dysfunction in FAN1-deficient kidneys, thereby exacerbating KIN pathophysiology. In contrast, treatment of FAN1 mice with a mitochondria-targeted ROS scavenger, JP4-039, attenuated oxidative stress and accumulation of DNA damage, mitigated tubular injury, and preserved kidney function in cisplatin-treated FAN1-null mice, demonstrating that endogenous oxygen stress is an important source of DNA damage in FAN1-deficient kidneys and a driver of KIN pathogenesis. Our findings indicate that therapeutic modulation of kidney oxidative stress may be a promising avenue to mitigate FAN1/KIN kidney pathophysiology and disease progression in patients.

Trends in SARS-CoV-2 cycle threshold values in the Czech Republic from April 2020 to April 2022.

The inability to predict the evolution of the COVID-19 epidemic hampered abilities to respond to the crisis effectively. The cycle threshold (Ct) from the standard SARS-CoV-2 quantitative reverse transcription-PCR (RT-qPCR) clinical assay is inversely proportional to the amount of SARS-CoV-2 RNA in the sample. We were interested to see if population Ct values could predict future increases in COVID-19 cases as well as subgroups that would be more likely to be affected. This information would have been extremely helpful early in the COVID-19 epidemic. We therefore conducted a retrospective analysis of demographic data and Ct values from 2,076,887 nasopharyngeal swab RT-qPCR tests that were performed at a single diagnostic laboratory in the Czech Republic from April 2020 to April 2022 and from 221,671 tests that were performed as a part of a mandatory school surveillance testing program from March 2021 to March 2022. We found that Ct values could be helpful predictive tools in the real-time management of viral epidemics. First, early measurement of Ct values would have indicated the low viral load in children, equivalent viral load in males and females, and higher viral load in older individuals. Second, rising or falling median Ct values and differences in Ct distribution indicated changes in the transmission in the population. Third, monitoring Ct values and positivity rates would have provided early evidence as to whether prevention measures are effective. Health system authorities should thus consider collecting weekly median Ct values of positively tested samples from major diagnostic laboratories for regional epidemic surveillance.

MANF stimulates autophagy and restores mitochondrial homeostasis to treat toxic proteinopathy.

Misfolded protein aggregates may cause toxic proteinopathy, including autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD- UMOD ), one of the leading hereditary kidney diseases, and Alzheimer’s disease etc. There are no targeted therapies. ADTKD is also a genetic form of renal fibrosis and chronic kidney disease, which affects 500 million people worldwide. For the first time, in our newly generated mouse model recapitulating human ADTKD- UMOD carrying a leading UMOD deletion mutation, we show that autophagy/mitophagy and mitochondrial biogenesis are severely impaired, leading to cGAS- STING activation and tubular injury. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel endoplasmic reticulum stress-regulated secreted protein. We provide the first study that inducible tubular overexpression of MANF after the onset of disease stimulates autophagy/mitophagy and clearance of the misfolded UMOD, and promotes mitochondrial biogenesis through p-AMPK enhancement, resulting in protection of kidney function. Conversely, genetic ablation of endogenous MANF upregulated in the mutant mouse and human tubular cells worsens autophagy suppression and kidney fibrosis. Together, we discover MANF as a novel biotherapeutic protein and elucidate previously unknown mechanisms of MANF in regulating organelle homeostasis to treat ADTKD, which may have broad therapeutic application to treat various proteinopathies.

Influence of glycoprotein MUC1 on trafficking of the Ca2+-selective ion channels, TRPV5 and TRPV6, and on in vivo calcium homeostasis.

Polymorphism of the gene encoding mucin 1 (MUC1) is associated with skeletal and dental phenotypes in human genomic studies. Animals lacking MUC1 exhibit mild reduction in bone density. These phenotypes could be a consequence of modulation of bodily Ca homeostasis by MUC1, as suggested by the previous observation that MUC1 enhances cell surface expression of the Ca2+-selective channel, TRPV5, in cultured unpolarized cells. Using biotinylation of cell surface proteins, we asked whether MUC1 influences endocytosis of TRPV5 and another Ca2+-selective TRP channel, TRPV6, in cultured polarized epithelial cells. Our results indicate that MUC1 reduces endocytosis of both channels, enhancing cell surface expression. Further, we found that mice lacking MUC1 lose apical localization of TRPV5 and TRPV6 in the renal tubular and duodenal epithelium. Females, but not males, lacking MUC1 exhibit reduced blood Ca2+. However, mice lacking MUC1 exhibited no differences in basal urinary Ca excretion or Ca retention in response to PTH receptor signaling, suggesting compensation by transport mechanisms independent of TRPV5 and TRPV6. Finally, humans with autosomal dominant tubulointerstitial kidney disease due to frame-shift mutation of MUC1 (ADTKD-MUC1) exhibit reduced plasma Ca concentrations compared to control individuals with mutations in the gene encoding uromodulin (ADTKD-UMOD), consistent with MUC1 haploinsufficiency causing reduced bodily Ca2+. In summary, our results provide further insight into the role of MUC1 in Ca2+-selective TRP channel endocytosis and the overall effects on Ca concentrations.

Metabolites of De Novo Purine Synthesis: Metabolic Regulators and Cytotoxic Compounds.

Cytotoxicity of de novo purine synthesis (DNPS) metabolites is critical to the pathogenesis of three known and one putative autosomal recessive disorder affecting DNPS. These rare disorders are caused by biallelic mutations in the DNPS genes phosphoribosylformylglycineamidine synthase (PFAS), phosphoribosylaminoimidazolecarboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS), adenylosuccinate lyase (ADSL), and aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) and are clinically characterized by developmental abnormalities, psychomotor retardation, and nonspecific neurological impairment. At a biochemical level, loss of function of specific mutated enzymes results in elevated levels of DNPS ribosides in body fluids. The main pathogenic effect is attributed to the accumulation of DNPS ribosides, which are postulated to be toxic to the organism. Therefore, we decided to characterize the uptake and flux of several DNPS metabolites in HeLa cells and the impact of DNPS metabolites to viability of cancer cell lines and primary skin fibroblasts. We treated cells with DNPS metabolites and followed their flux in purine synthesis and degradation. In this study, we show for the first time the transport of formylglycinamide ribotide (FGAR), aminoimidazole ribotide (AIR), succinylaminoimidazolecarboxamide ribotide (SAICAR), and aminoimidazolecarboxamide ribotide (AICAR) into cells and their flux in DNPS and the degradation pathway. We found diminished cell viability mostly in the presence of FGAR and AIR. Our results suggest that direct cellular toxicity of DNPS metabolites may not be the primary pathogenetic mechanism in these disorders.

Autosomal dominant tubulointerstitial kidney disease: A review.

The clinical characteristics of autosomal dominant tubulointerstitial kidney disease (ADTKD) include bland urinary sediment, slowly progressive chronic kidney disease (CKD) with many patients reaching end stage renal disease (ESRD) between age 20 and 70 years, and autosomal dominant inheritance. Due to advances in genetic diagnosis, ADTKD is becoming increasingly recognized as a cause of CKD. Pathogenic variants in UMOD, MUC1, and REN are the most common causes of ADTKD. ADTKD-UMOD is also associated with hyperuricemia and gout. ADTKD-REN often presents in childhood with mild hypotension, CKD, hyperkalemia, acidosis, and anemia. ADTKD-MUC1 patients present only with CKD. This review describes the pathophysiology, genetics, clinical manifestation, and diagnosis for ADTKD, with an emphasis on genetic testing and genetic counseling suggestions for patients.

An intermediate-effect size variant in UMOD confers risk for chronic kidney disease.

The kidney-specific gene UMOD encodes for uromodulin, the most abundant protein excreted in normal urine. Rare large-effect variants in UMOD cause autosomal dominant tubulointerstitial kidney disease (ADTKD), while common low-impact variants strongly associate with kidney function and the risk of chronic kidney disease (CKD) in the general population. It is unknown whether intermediate-effect variants in UMOD contribute to CKD. Here, candidate intermediate-effect UMOD variants were identified using large-population and ADTKD cohorts. Biological and phenotypical effects were investigated using cell models, in silico simulations, patient samples, and international databases and biobanks. Eight UMOD missense variants reported in ADTKD are present in the Genome Aggregation Database (gnomAD), with minor allele frequency (MAF) ranging from 10-5 to 10-3. Among them, the missense variant p.Thr62Pro is detected in ∼1/1,000 individuals of European ancestry, shows incomplete penetrance but a high genetic load in familial clusters of CKD, and is associated with kidney failure in the 100,000 Genomes Project (odds ratio [OR] = 3.99 [1.84 to 8.98]) and the UK Biobank (OR = 4.12 [1.32 to 12.85). Compared with canonical ADTKD mutations, the p.Thr62Pro carriers displayed reduced disease severity, with slower progression of CKD and an intermediate reduction of urinary uromodulin levels, in line with an intermediate trafficking defect in vitro and modest induction of endoplasmic reticulum (ER) stress. Identification of an intermediate-effect UMOD variant completes the spectrum of UMOD-associated kidney diseases and provides insights into the mechanisms of ADTKD and the genetic architecture of CKD.

Vignette-Based Reflections to Inform Genetic Testing Policies in Living Kidney Donors.

Family history of kidney disease increases risk of end-stage kidney disease (ESKD) in donors. Pre-donation genetic testing is recommended in evaluation guidelines and regulatory policy. Collaborating across several institutions, we describe cases to illustrate the utility as well as practical issues in incorporating genetic testing in transplant protocols. Case 1 is from 2009, before pervasive genetic testing. A healthy 27-year-old Caucasian male had an uneventful donor evaluation for his mother, who had early onset ESKD of unclear cause. He participated in paired-exchange kidney donation, but developed progressive kidney disease and gout over the next 10 years. A uromodulin gene mutation (NM_003361.3(UMOD):c.377 G>A p.C126Y) was detected and kidney biopsy showed tubulointerstitial kidney disease. The patient subsequently required kidney transplantation himself. Case 2 was a 36-year-old African American female who had an uneventful kidney donor evaluation. She underwent gene panel-based testing to rule out ApolipoproteinL1 risk variants, for which was negative. Incidentally, a sickle-cell trait (NM_000518.5(HBB):c.20A>T p.Glu7Val) was noted, and she was declined for kidney donation. This led to significant patient anguish. Case 3 was a 26-year-old Caucasian female who underwent panel-based testing because the potential recipient, her cousin, carried a variant of uncertain significance in the hepatocyte nuclear factor-1-ß (HNF1B) gene. While the potential donor did not harbor this variant, she was found to have a likely pathogenic variant in complement factor I (NM_000204.4(CFI):c.1311dup:p.Asp438Argfs*8), precluding kidney donation. Our cases emphasize that while genetic testing can be invaluable in donor evaluation, transplant centers should utilize detailed informed consent, develop care pathways for secondary genetic findings, and share experience to develop best practices around genetic testing in donors.

Genetic Etiologies for Chronic Kidney Disease Revealed through Next-Generation Renal Gene Panel.

INTRODUCTION: Chronic kidney disease (CKD) is a major public health issue in the USA. Identification of monogenic causes of CKD, which are present in ∼10% of adult cases, can impact prognosis and patient management. Broad gene panels can provide unbiased testing approaches, which are advantageous in phenotypically heterogeneous diseases. However, the use and yield of broad genetic panels by nephrologists in clinical practice is not yet well characterized. METHODS: Renal genetic testing, ordered exclusively for clinical purposes, predominantly by general and transplant nephrologists within the USA, was performed on 1,007 consecutive unique patient samples. Testing was performed using a commercially available next-generation sequencing-based 382 gene kidney disease panel. Pathogenic (P) and likely pathogenic (LP) variants were reported. Positive findings included a monoallelic P/LP variant in an autosomal dominant or X-linked gene and biallelic P/LP variants in autosomal recessive genes. RESULTS: Positive genetic findings were identified in 21.1% (212/1,007) of cases. A total of 220 positive results were identified across 48 genes. Positive results occurred most frequently in the PKD1 (34.1%), COL4A5 (10.9%), PKD2 (10.0%), COL4A4 (6.4%), COL4A3 (5.9%), and TTR (4.1%) genes. Variants identified in the remaining 42 genes comprised 28.6% of the total positive findings, including single positive results in 26 genes. Positive results in >1 gene were identified in 7.5% (16/212) of cases. CONCLUSIONS: Use of broad panel genetic testing by clinical nephrologists had a high success rate, similar to results obtained by academic centers specializing in genetics.

Phenylbutyrate rescues the transport defect of the Sec61α mutations V67G and T185A for renin.

The human Sec61 complex is a widely distributed and abundant molecular machine. It resides in the membrane of the endoplasmic reticulum to channel two types of cargo: protein substrates and calcium ions. The SEC61A1 gene encodes for the pore-forming Sec61α subunit of the Sec61 complex. Despite their ubiquitous expression, the idiopathic SEC61A1 missense mutations p.V67G and p.T185A trigger a localized disease pattern diagnosed as autosomal dominant tubulointerstitial kidney disease (ADTKD-SEC61A1). Using cellular disease models for ADTKD-SEC61A1, we identified an impaired protein transport of the renal secretory protein renin and a reduced abundance of regulatory calcium transporters, including SERCA2. Treatment with the molecular chaperone phenylbutyrate reversed the defective protein transport of renin and the imbalanced calcium homeostasis. Signal peptide substitution experiments pointed at targeting sequences as the cause for the substrate-specific impairment of protein transport in the presence of the V67G or T185A mutations. Similarly, dominant mutations in the signal peptide of renin also cause ADTKD and point to impaired transport of this renal hormone as important pathogenic feature for ADTKD-SEC61A1 patients as well.