Genetic Contributions to Lower Urinary Tract Dysfunction.

Lower urinary tract dysfunction (LUTD) can manifest as a spectrum of voiding symptoms in childhood, including urinary urgency, frequency, hesitancy, and incontinence. In severe cases, it can lead to frequent urinary tract infections, hydronephrosis, kidney scarring, and chronic kidney disease. Non-neurogenic neurogenic bladder (NNNB) is a diagnosis of exclusion in which children develop discoordination between the detrusor smooth muscle and external urethral sphincter in the absence of neurological or obstructive lesions, resulting in severe LUTD. Historically, such disorders of voiding were thought to result from behavioral maladaptation. However, it is now increasingly recognized that some individuals may have an underlying genetic etiology for their symptoms. Here, we performed exome sequencing for five probands with NNNB or other forms of severe LUTD, and we identified two individuals with monogenic etiologies for their symptoms. One individual had a homozygous exon 9 deletion in HPSE2 and another had a homozygous single amino acid deletion (p.Gly167del) in ARL6. We performed PCR experiments to identify the breakpoints of the HPSE2 exon 9 deletion and implicate microhomology-mediated end joining as a potential mechanism by which the deletion arose. These findings suggest that genetic testing should be considered for children with severe LUTD.

Safety outcomes of direct oral anticoagulants in older adults with atrial fibrillation: a systematic review and meta-analysis of (subgroup analyses from) randomized controlled trials.

Balancing stroke prevention and risk of bleeding in patients with atrial fibrillation (AF) is challenging. Direct oral anticoagulants (DOACs) are by now considered standard of care for treating patients with AF in international guidelines. Our objective was to assess the safety of long-term intake of DOACs in older adults with AF. We included RCTs in elderly (≥ 65 years) patients with AF. A systematic search in MEDLINE and EMBASE was performed on 19 April 2022. For determination of risk of bias, the RoB 2 tool was applied. We pooled outcomes using random-effects meta-analyses. The quality of evidence was assessed using GRADE. Eleven RCTs with a total of 63,374 patients were identified. Two RCTs compared apixaban with either warfarin or aspirin, four edoxaban with either placebo, aspirin, or vitamin K antagonists (VKAs), two dabigatran with warfarin and three rivaroxaban with warfarin. DOACs probably reduce mortality in elderly patients with AF (HR 0.89 95%CI 0.77 to 1.02). Low-dose DOACs likely reduce bleeding compared to VKAs (HR ranged from 0.47 to 1.01). For high-dose DOACS the risk of bleeding varied widely (HR ranged from 0.80 to 1.40). We found that low-dose DOACs probably decrease mortality in AF patients. Moreover, apixaban and probably edoxaban are associated with fewer major or clinically relevant bleeding (MCRB) events compared to VKAs. For dabigatran and rivaroxaban, the risk of MCRB varies depending on dose. Moreover, subgroup analyses indicate that in the very old (≥ 85) the risk for MCRB events might be increased when using DOACs.Registration: PROSPERO: CRD42020187876.

Categorization of a Universal Coding System to Distinguish Use of Durable Medical Equipment and Supplies in Pediatric Patients.

Importance: Although durable medical equipment and supplies (DMES) are commonly used to optimize the health and function in pediatric patients, little is known about the prevalence of use and spending on DMES. Objective: To categorize the Healthcare Common Procedure Coding System (HCPCS) for distinguishing DMES types, and to measure the prevalence and related spending of DMES in pediatric patients using Medicaid. Design, Setting, and Participants: This study is a cross-sectional analysis of the 2018 Merative Medicaid Database and included 4 569 473 pediatric patients aged 0 to 21 years enrolled in Medicaid in 12 US states from January 1 to December 31, 2018. Data were analyzed from February 2019 to April 2023. Exposure: DMES exposure was identified with the Centers for Medicare & Medicaid Services HCPCS codes. Three pediatricians categorized HCPCS DMES codes submitted by vendors for reimbursement of dispensed DMES into DMES types and end-organ systems; 15 expert reviewers refined the categorization (2576 DMES codes, 164 DMES types, 14 organ systems). Main Outcomes and Measures: The main outcome was DMES prevalence & Medicaid spending. The χ2 test was used to compare DMES prevalence and Wilcoxon rank sum tests were used to compare per-member-per-year (PMPY) spending by complex chronic conditions (CCC). Results: Of the 4 569 473 patients in the study cohort, 49.3% were female and 56.1% were aged 5 to 15 years. Patients used 133 of 164 (81.1%) DMES types. The DMES prevalence was 17.1% (95% CI, 17.0%-17.2%) ranging from 10.1% (95% CI, 10.0%-10.2%) in patients with no chronic condition to 60.9% (95% CI, 60.8%-61.0%) for patients with 2 or more CCCs. The PMPY DMES spending was $593, ranging from $349 for no chronic condition to $4253 for 2 or more CCCs. Lens (7.9%), vision frames (6.2%), and orthotics for orthopedic injury (0.8%) were the most common DME in patients with no chronic condition. Enteral tube / feeding supplies (19.8%), diapers (19.2%), lower extremity orthotics (12.3%), wheelchair (9.6%), oxygen (9.0%), and urinary catheter equipment (4.2%) were among the most common DMES in children with 2 or more CCCs. Conclusions and Relevance: In this cross-sectional study, HCPCS distinguished a variety of DME types and use across pediatric populations. Further investigation should assess the utility of the HCPCS DMES categorization with efforts to optimize the quality and safety of DMES use.

Prioritization of Monogenic Congenital Anomalies of the Kidney and Urinary Tract Candidate Genes with Existing Single-Cell Transcriptomics Data of the Human Fetal Kidney.

INTRODUCTION: Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first 3 decades of life. Over 40 genes have been identified as causative for isolated human CAKUT. However, many genes remain unknown, and the prioritization of potential CAKUT candidate genes is challenging. To develop an independent approach to prioritize CAKUT candidate genes, we hypothesized that monogenic CAKUT genes are most likely co-expressed along a temporal axis during kidney development and that genes with coinciding high expression may represent strong novel CAKUT candidate genes. METHODS: We analyzed single-cell mRNA (sc-mRNA) transcriptomics data of human fetal kidney for temporal sc-mRNA co-expression of 40 known CAKUT genes. A maximum of high expression in consecutive timepoints of kidney development was found for four of the 40 genes (EYA1, SIX1, SIX2, and ITGA8) in nephron progenitor cells a, b, c, d (NPCa-d). We concluded that NPCa-d are relevant for CAKUT pathogenesis and intersected two lists of CAKUT candidate genes resulting from unbiased whole-exome sequencing (WES) with the 100 highest expressed genes in NPCa-d. RESULTS: Intersection of the 100 highest expressed genes in NPCa-d with WES-derived CAKUT candidate genes identified an overlap with the candidate genes KIF19, TRIM36, USP35, CHTF18, in each of which a biallelic variant was detected in different families with CAKUT. CONCLUSION: Sc-mRNA expression data of human fetal kidney can be utilized to prioritize WES-derived CAKUT candidate genes. KIF19, TRIM36, USP35, and CHTF18 may represent strong novel candidate genes for CAKUT.

Recessive CHRM5 variant as a potential cause of neurogenic bladder.

Neurogenic bladder is caused by disruption of neuronal pathways regulating bladder relaxation and contraction. In severe cases, neurogenic bladder can lead to vesicoureteral reflux, hydroureter, and chronic kidney disease. These complications overlap with manifestations of congenital anomalies of the kidney and urinary tract (CAKUT). To identify novel monogenic causes of neurogenic bladder, we applied exome sequencing (ES) to our cohort of families with CAKUT. By ES, we have identified a homozygous missense variant (p.Gln184Arg) in CHRM5 (cholinergic receptor, muscarinic, 5) in a patient with neurogenic bladder and secondary complications of CAKUT. CHRM5 codes for a seven transmembrane-spanning G-protein-coupled muscarinic acetylcholine receptor. CHRM5 is shown to be expressed in murine and human bladder walls and is reported to cause bladder overactivity in Chrm5 knockout mice. We investigated CHRM5 as a potential novel candidate gene for neurogenic bladder with secondary complications of CAKUT. CHRM5 is similar to the cholinergic bladder neuron receptor CHRNA3, which Mann et al. published as the first monogenic cause of neurogenic bladder. However, functional in vitro studies did not reveal evidence to strengthen the status as a candidate gene. Discovering additional families with CHRM5 variants could help to further assess the genes' candidate status.

Genetic Variants in ARHGEF6 Cause Congenital Anomalies of the Kidneys and Urinary Tract in Humans, Mice, and Frogs.

BACKGROUND: About 40 disease genes have been described to date for isolated CAKUT, the most common cause of childhood CKD. However, these genes account for only 20% of cases. ARHGEF6, a guanine nucleotide exchange factor that is implicated in biologic processes such as cell migration and focal adhesion, acts downstream of integrin-linked kinase (ILK) and parvin proteins. A genetic variant of ILK that causes murine renal agenesis abrogates the interaction of ILK with a murine focal adhesion protein encoded by Parva , leading to CAKUT in mice with this variant. METHODS: To identify novel genes that, when mutated, result in CAKUT, we performed exome sequencing in an international cohort of 1265 families with CAKUT. We also assessed the effects in vitro of wild-type and mutant ARHGEF6 proteins, and the effects of Arhgef6 deficiency in mouse and frog models. RESULTS: We detected six different hemizygous variants in the gene ARHGEF6 (which is located on the X chromosome in humans) in eight individuals from six families with CAKUT. In kidney cells, overexpression of wild-type ARHGEF6 -but not proband-derived mutant ARHGEF6 -increased active levels of CDC42/RAC1, induced lamellipodia formation, and stimulated PARVA-dependent cell spreading. ARHGEF6-mutant proteins showed loss of interaction with PARVA. Three-dimensional Madin-Darby canine kidney cell cultures expressing ARHGEF6-mutant proteins exhibited reduced lumen formation and polarity defects. Arhgef6 deficiency in mouse and frog models recapitulated features of human CAKUT. CONCLUSIONS: Deleterious variants in ARHGEF6 may cause dysregulation of integrin-parvin-RAC1/CDC42 signaling, thereby leading to X-linked CAKUT.

Potentially Inadequate Medications in the Elderly: PRISCUS 2.0.

BACKGROUND: The term potentially inadequate medication (PIM) is used to describe substances that may be unsuitable for use inthe elderly and should be avoided. The PRISCUS list, published in 2010, was the first catalog of PIM designed for the Germandrug market to become adopted in practice. While 24% of German patients aged ≥ 65 years were prescribed at least one PIMper year in 2009, the proportion in 2019 was only 14.5%. METHODS: In a three-round Delphi process, experts from clinical practice and research evaluated whether selected substancesare PIM for the elderly. The participants were provided with dedicated literature including systematic reviews carried out for theparticular purposes of this project. RESULTS: Fifty-nine persons took part in the Delphi process and, in addition, contributed comments and therapeutic alternatives.Altogether, 187 substances were classed as PIM. One hundred thirty-three of the substances now listed were not in the originalPRISCUS list: these include some oral antidiabetics, all of the selective COX-2 inhibitors, and moderately long acting benzodiazepinessuch as oxazepam. For some other substances, e.g., proton pump inhibitors (PPI), the advisability of treatment formore than 8 weeks was considered as potentially inappropriate, as was the use of ibuprofen in doses >1200 mg/day and formore than 1 week without PPI. Risperidone for more than 6 weeks is also PIM. CONCLUSION: The new, greatly extended PRISCUS list must now be validated in epidemiological and prospective studies and itspracticability in routine daily use must be verified.

Copy Number Variation Analysis Facilitates Identification of Genetic Causation in Patients with Congenital Anomalies of the Kidney and Urinary Tract.

Background: Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease among children and adults younger than 30 yr. In our previous study, whole-exome sequencing (WES) identified a known monogenic cause of isolated or syndromic CAKUT in 13% of families with CAKUT. However, WES has limitations and detection of copy number variations (CNV) is technically challenging, and CNVs causative of CAKUT have previously been detected in up to 16% of cases. Objective: To detect CNVs causing CAKUT in this WES cohort and increase the diagnostic yield. Design setting and participants: We performed a genome-wide single nucleotide polymorphism (SNP)-based CNV analysis on the same CAKUT cohort for whom WES was previously conducted. Outcome measurements and statistical analysis: We evaluated and classified the CNVs using previously published predefined criteria. Results and limitations: In a cohort of 170 CAKUT families, we detected a pathogenic CNV known to cause CAKUT in nine families (5.29%, 9/170). There were no competing variants on genome-wide CNV analysis or WES analysis. In addition, we identified novel likely pathogenic CNVs that may cause a CAKUT phenotype in three of the 170 families (1.76%). Conclusions: CNV analysis in this cohort of 170 CAKUT families previously examined via WES increased the rate of diagnosis of genetic causes of CAKUT from 13% on WES to 18% on WES + CNV analysis combined. We also identified three candidate loci that may potentially cause CAKUT. Patient summary: We conducted a genetics study on families with congenital anomalies of the kidney and urinary tract (CAKUT). We identified gene mutations that can explain CAKUT symptoms in 5.29% of the families, which increased the percentage of genetic causes of CAKUT to 18% from a previous study, so roughly one in five of our patients with CAKUT had a genetic cause. These analyses can help patients with CAKUT and their families in identifying a possible genetic cause.

Development of a deprescribing manual for frail older people for use in the COFRAIL study and in primary care.

Introduction: Many older adults are affected by multimorbidity and subsequent polypharmacy which is associated with adverse outcomes. This is especially relevant for frail older patients. Polypharmacy may be reduced via deprescribing. As part of the complex intervention in the COFRAIL study, we developed a deprescribing manual to be used by general practitioners (GPs) in family conferences, in which GPs, patients and caregivers jointly discuss treatments. Methods: We selected indications with a high prevalence in older adults in primary care (e.g. diabetes mellitus, hypertension) and conducted a literature search to identify deprescribing criteria for these indications. We additionally reviewed clinical practice guidelines. Based on the extracted information, we created a deprescribing manual which was then piloted in an expert workshop and in family conferences with volunteer patients according to the inclusion and exclusion criteria of the study protocol. Results: Initially, 13 indications/topics were selected. The literature search identified deprescribing guides, reviews and clinical trials as well as lists of potentially inappropriate medication and systematic reviews on the risk and benefits of specific drugs and drug classes in older patients. After piloting and revisions, the deprescribing manual now covers 11 indications/topics. In each chapter, patient- and medication-related deprescribing criteria, monitoring and communication strategies, and information about concerns related to the use of specific drugs in older patients are provided. Discussion: We found varying deprescribing strategies in the literature, which we consolidated in our deprescribing manual. Whether this approach leads to successful deprescribing in family conferences is being investigated in the cluster-randomised controlled COFRAIL study. Plain Language Summary: Development of a manual to help doctors to identify which medications can be withdrawn Many older adults suffer from chronic diseases and take multiple medications concurrently. This can lead to side effects and other undesired events. We developed a manual to help doctors identify which medications can be withdrawn, so that they can discuss this with their patients. This manual was used in the COFRAIL study where doctors, patients and caregivers met in family conferences to discuss their preferences and decide together how future treatments should be handled. The manual contains information on common medications, symptoms and diseases in older patients such as diabetes and high blood pressure. Before the manual was used in the study, it was tested by volunteer patients and their doctors and caregivers to make sure that it is user-friendly.

Assessing the quality of evidence on safety: specifications for application and suggestions for adaptions of the GRADE-criteria in the context of preparing a list of potentially inappropriate medications for older adults.

BACKGROUND: Systematic reviews that synthesize safety outcomes pose challenges (e.g. rare events), which raise questions for grading the strength of the body of evidence. This is maybe one reason why in many potentially inappropriate medication (PIM) lists the recommendations are not based on formalized systems for assessing the quality of the body of evidence such as GRADE. In this contribution, we describe specifications and suggest adaptions of the GRADE system for grading the quality of evidence on safety outcomes, which were developed in the context of preparing a PIM-list, namely PRISCUS. METHODS: We systematically assessed each of the five GRADE domains for rating-down (study limitations, imprecision, inconsistency, indirectness, publication bias) and the criteria for rating-up, considering if special considerations or revisions of the original approach were indicated. The result was gathered in a written document and discussed in a group-meeting of five members with various background until consensus. Subsequently, we performed a proof-of-concept application using a convenience sample of systematic reviews and applied the approach to systematic reviews on 19 different clinical questions. RESULTS: We describe specifications and suggest adaptions for the criteria "study limitations", imprecision, "publication bias" and "rating-up for large effect". In addition, we suggest a new criterion to account for data from subgroup-analyses. The proof-of-concept application did not reveal a need for further revision and thus we used the approach for the systematic reviews that were prepared for the PRISCUS-list. We assessed 51 outcomes. Each of the proposed adaptions was applied. There were neither an excessive number of low and very low ratings, nor an excessive number of high ratings, but the different methodological quality of the safety outcomes appeared to be well reflected. CONCLUSION: The suggestions appear to have the potential to overcome some of the challenges when grading the methodological quality of harms and thus may be helpful for producers of evidence syntheses considering safety.

Safety of dipeptidyl peptidase-4 inhibitors in older adults with type 2 diabetes: a systematic review and meta-analysis of randomized controlled trials.

REGISTRATION: PROSPERO: CRD42020210645. INTRODUCTION: We aimed to assess the safety of dipeptidyl peptidase-4 (DPP-4) inhibitors in older patients with type 2 diabetes with inadequate glycaemic control. METHODS: We included randomized controlled trials (RCTs) in older (⩾65 years) patients with type 2 diabetes. The intervention group was randomized to treatment with any DPP-4 inhibitors. A systematic search in MEDLINE and Embase was performed in December 2020. For assessing the risk of bias, RoB 2 tool was applied. The quality of evidence was assessed using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. We pooled outcomes using random effects meta-analyses. RESULTS: We identified 16 RCTs that included 19,317 patients with a mean age of greater than 70 years. The mean HbA1c level ranged between 7.1 and 10.0 g/dl. Adding DPP-4 inhibitors to standard care alone may increase mortality slightly [risk ratio (RR) 1.04; 95% confidence interval (CI) 0.89-1.21]. Adding DPP-4 inhibitors to standard care increases the risk for hypoglycaemia (RR 1.08; 95% CI 1.01-1.16), but difference in overall adverse events is negligible. DPP-4 inhibitors added to standard care may reduce mortality compared with sulfonylureas (RR 0.88; 95% CI 0.75-1.04). DPP-4 inhibitors probably reduce the risk for hypoglycaemia compared with sulfonylureas (magnitude of effect not quantifiable because of heterogeneity) but difference in overall adverse events is negligible. There is insufficient evidence on hospitalizations, falls, fractures, renal impairment and pancreatitis. CONCLUSION: There is no evidence that DPP-4 inhibitors in addition to standard care decrease mortality but DPP-4 inhibitors increase hypoglycaemia risk. Second-line therapy in older patients should be considered cautiously even in drugs with a good safety profile such as DPP-4 inhibitors. In case second-line treatment is necessary, DPP-4 inhibitors appear to be preferable to sulfonylureas. PLAIN LANGUAGE SUMMARY: Safety of dipeptidyl peptidase-4 inhibitors in older adults with type 2 diabetes: Introduction:: We performed the review to assess the safety of dipeptidyl peptidase-4 (DPP-4) inhibitors in older type 2 diabetes patients with blood sugar outside the normal level.Methods:: To answer the question, we searched various electronic databases. We included studies in older (⩾65 years) patients with type 2 diabetes that assessed the safety of DPP-4 inhibitors. The data from the different studies were quantitatively summarized using statistical methods. We assessed the quality of the data to judge the certainty of the findings.Results:: We identified 16 studies that included 19,317 patients with a mean age greater than 70 years. The average blood sugar level of patients in the included studies was slightly or moderately increased. Adding DPP-4 inhibitors to standard care alone may increase mortality slightly. Adding DPP-4 inhibitors to standard care increases the risk for hypoglycaemia, but difference in overall adverse events is negligible. DPP-4 inhibitors added to standard care may reduce mortality compared with sulfonylureas. DPP-4s probably reduce the risk of hypoglycaemia compared with sulfonylureas (magnitude of effect not quantifiable because of heterogeneity) but difference in overall adverse events is negligible. There is insufficient evidence on hospitalizations, falls, fractures, renal impairment and pancreatitis.Conclusion:: There is no evidence that DPP-4 inhibitors in addition to standard care decrease mortality but DPP-4 inhibitors increase the risk that blood sugar falls below normal. Adding DPP-4 inhibitorss to standard care in older patients should be considered cautiously even in drugs with a good safety profile such as DPP-4 inhibitors. In case additional treatment is necessary, DPP-4 inhibitors appear to be preferable to sulfonylureas.

Whole exome sequencing identifies potential candidate genes for spina bifida derived from mouse models.

Spina bifida (SB) is the second most common nonlethal congenital malformation. The existence of monogenic SB mouse models and human monogenic syndromes with SB features indicate that human SB may be caused by monogenic genes. We hypothesized that whole exome sequencing (WES) allows identification of potential candidate genes by (i) generating a list of 136 candidate genes for SB, and (ii) by unbiased exome-wide analysis. We generated a list of 136 potential candidate genes from three categories and evaluated WES data of 50 unrelated SB cases for likely deleterious variants in 136 potential candidate genes, and for potential SB candidate genes exome-wide. We identified 6 likely deleterious variants in 6 of the 136 potential SB candidate genes in 6 of the 50 SB cases, whereof 4 genes were derived from mouse models, 1 gene was derived from human nonsyndromic SB, and 1 gene was derived from candidate genes known to cause human syndromic SB. In addition, by unbiased exome-wide analysis, we identified 12 genes as potential candidates for SB. Identification of these 18 potential candidate genes in larger SB cohorts will help decide which ones can be considered as novel monogenic causes of human SB.

The effect of gender-affirming hormone treatment on serum creatinine in transgender and gender-diverse youth: implications for estimating GFR.

BACKGROUND: Equations for estimated glomerular filtration rate (eGFR) based on serum creatinine include terms for sex/gender. For transgender and gender-diverse (TGD) youth, gender-affirming hormone (GAH) treatment may affect serum creatinine and in turn eGFR. METHODS: TGD youth were recruited for this prospective, longitudinal, observational study prior to starting GAH treatment. Data collected as part of routine clinical care were abstracted from the medical record. RESULTS: For participants designated male at birth (DMAB, N = 92), serum creatinine decreased within 6 months of estradiol treatment (mean ± SD 0.83 ± 0.12 mg/dL to 0.76 ± 0.12 mg/dL, p < 0.001); for participants designated female at birth (DFAB, n = 194), serum creatinine increased within 6 months of testosterone treatment (0.68 ± 0.10 mg/dL to 0.79 ± 0.11 mg/dL, p < 0.001). Participants DFAB treated with testosterone had serum creatinine similar to that of participants DMAB at baseline, whereas even after estradiol treatment, serum creatinine in participants DMAB remained higher than that of participants DFAB at baseline. Compared to reference groups drawn from the National Health and Nutritional Examination Survey, serum creatinine after 12 months of GAH was more similar when compared by gender identity than by designated sex. CONCLUSION: GAH treatment leads to changes in serum creatinine within 6 months of treatment. Clinicians should consider a patient's hormonal exposure when estimating kidney function via eGFR and use other methods to estimate GFR if eGFR based on serum creatinine is concerning.

A truncating NRIP1 variant in an Arabic family with congenital anomalies of the kidneys and urinary tract.

Congenital anomalies of the kidneys and urinary tract (CAKUT) constitute the most common cause of early-onset chronic kidney disease. In a previous study, we identified a heterozygous truncating variant in nuclear receptor-interacting protein 1 (NRIP1) as CAKUT causing via dysregulation of retinoic acid signaling. This large family remains the only family with NRIP1 variant reported so far. Here, we describe one additional CAKUT family with a truncating variant in NRIP1. By whole-exome sequencing, we identified one heterozygous frameshift variant (p.Asn676Lysfs*27) in an isolated CAKUT patient with bilateral hydroureteronephrosis and right grade V vesicoureteral reflux (VUR) and in the affected father with left renal hypoplasia. The variant is present twice in a heterozygous state in the gnomAD database of 125,000 control individuals. We report the second CAKUT family with a truncating variant in NRIP1, confirming that loss-of-function mutations in NRIP1 are a novel monogenic cause of human autosomal dominant CAKUT.

Whole-exome sequencing identifies FOXL2, FOXA2 and FOXA3 as candidate genes for monogenic congenital anomalies of the kidneys and urinary tract.

BACKGROUND: Congenital anomalies of the kidneys and urinary tract (CAKUT) constitute the most common cause of chronic kidney disease in the first three decades of life. Variants in four Forkhead box (FOX) transcription factors have been associated with CAKUT. We hypothesized that other FOX genes, if highly expressed in developing kidneys, may also represent monogenic causes of CAKUT. METHODS: We here performed whole-exome sequencing (WES) in 541 families with CAKUT and generated four lists of CAKUT candidate genes: (A) 36 FOX genes showing high expression during renal development, (B) 4 FOX genes known to cause CAKUT to validate list A, (C) 80 genes that we identified as unique potential novel CAKUT candidate genes when performing WES in 541 CAKUT families and (D) 175 genes identified from WES as multiple potential novel CAKUT candidate genes. RESULTS: To prioritize potential novel CAKUT candidates in the FOX gene family, we overlapped 36 FOX genes (list A) with lists C and D of WES-derived CAKUT candidates. Intersection with list C identified a de novo FOXL2 in-frame deletion in a patient with eyelid abnormalities and ureteropelvic junction obstruction, and a homozygous FOXA2 missense variant in a patient with horseshoe kidney. Intersection with list D identified a heterozygous FOXA3 missense variant in a CAKUT family with multiple affected individuals. CONCLUSIONS: We hereby identified FOXL2, FOXA2 and FOXA3 as novel monogenic candidate genes of CAKUT, supporting the utility of a paralog-based approach to discover mutated genes associated with human disease.

Reverse phenotyping facilitates disease allele calling in exome sequencing of patients with CAKUT.

PURPOSE: Congenital anomalies of the kidneys and urinary tract (CAKUT) constitute the leading cause of chronic kidney disease in children. In total, 174 monogenic causes of isolated or syndromic CAKUT are known. However, syndromic features may be overlooked when the initial clinical diagnosis of CAKUT is made. We hypothesized that the yield of a molecular genetic diagnosis by exome sequencing (ES) can be increased by applying reverse phenotyping, by re-examining the case for signs/symptoms of the suspected clinical syndrome that results from the genetic variant detected by ES. METHODS: We conducted ES in an international cohort of 731 unrelated families with CAKUT. We evaluated ES data for variants in 174 genes, in which variants are known to cause isolated or syndromic CAKUT. In cases in which ES suggested a previously unreported syndromic phenotype, we conducted reverse phenotyping. RESULTS: In 83 of 731 (11.4%) families, we detected a likely CAKUT-causing genetic variant consistent with an isolated or syndromic CAKUT phenotype. In 19 of these 83 families (22.9%), reverse phenotyping yielded syndromic clinical findings, thereby strengthening the genotype-phenotype correlation. CONCLUSION: We conclude that employing reverse phenotyping in the evaluation of syndromic CAKUT genes by ES provides an important tool to facilitate molecular genetic diagnostics in CAKUT.

Exome survey of individuals affected by VATER/VACTERL with renal phenotypes identifies phenocopies and novel candidate genes.

The acronym VATER/VACTERL refers to the rare nonrandom association of the following component features (CFs): vertebral defects (V), anorectal malformations (ARM) (A), cardiac anomalies (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb anomalies (L). For the clinical diagnosis, the presence of at least three CFs is required, individuals presenting with only two CFs have been categorized as VATER/VACTERL-like. The majority of VATER/VACTERL individuals displays a renal phenotype. Hitherto, variants in FGF8, FOXF1, HOXD13, LPP, TRAP1, PTEN, and ZIC3 have been associated with the VATER/VACTERL association; however, large-scale re-sequencing could only confirm TRAP1 and ZIC3 as VATER/VACTERL disease genes, both associated with a renal phenotype. In this study, we performed exome sequencing in 21 individuals and their families with a renal VATER/VACTERL or VATER/VACTERL-like phenotype to identify potentially novel genetic causes. Exome analysis identified biallelic and X-chromosomal hemizygous potentially pathogenic variants in six individuals (29%) in B9D1, FREM1, ZNF157, SP8, ACOT9, and TTLL11, respectively. The online tool GeneMatcher revealed another individual with a variant in ZNF157. Our study suggests six biallelic and X-chromosomal hemizygous VATER/VACTERL disease genes implicating all six genes in the expression of human renal malformations.

Recessive NOS1AP variants impair actin remodeling and cause glomerulopathy in humans and mice.

Nephrotic syndrome (NS) is a leading cause of chronic kidney disease. We found recessive NOS1AP variants in two families with early-onset NS by exome sequencing. Overexpression of wild-type (WT) NOS1AP, but not cDNA constructs bearing patient variants, increased active CDC42 and promoted filopodia and podosome formation. Pharmacologic inhibition of CDC42 or its effectors, formin proteins, reduced NOS1AP-induced filopodia formation. NOS1AP knockdown reduced podocyte migration rate (PMR), which was rescued by overexpression of WT Nos1ap but not by constructs bearing patient variants. PMR in NOS1AP knockdown podocytes was also rescued by constitutively active CDC42Q61L or the formin DIAPH3 Modeling a NOS1AP patient variant in knock-in human kidney organoids revealed malformed glomeruli with increased apoptosis. Nos1apEx3-/Ex3- mice recapitulated the human phenotype, exhibiting proteinuria, foot process effacement, and glomerulosclerosis. These findings demonstrate that recessive NOS1AP variants impair CDC42/DIAPH-dependent actin remodeling, cause aberrant organoid glomerulogenesis, and lead to a glomerulopathy in humans and mice.