OXGR1 is a candidate disease gene for human calcium oxalate nephrolithiasis.

PURPOSE: Nephrolithiasis (NL) affects 1 in 11 individuals worldwide, leading to significant patient morbidity. NL is associated with nephrocalcinosis (NC), a risk factor for chronic kidney disease. Causative genetic variants are detected in 11% to 28% of NL and/or NC, suggesting that additional NL/NC-associated genetic loci await discovery. Therefore, we employed genomic approaches to discover novel genetic forms of NL/NC. METHODS: Exome sequencing and directed sequencing of the OXGR1 locus were performed in a worldwide NL/NC cohort. Putatively deleterious, rare OXGR1 variants were functionally characterized. RESULTS: Exome sequencing revealed a heterozygous OXGR1 missense variant (c.371T>G, p.L124R) cosegregating with calcium oxalate NL and/or NC disease in an autosomal dominant inheritance pattern within a multigenerational family with 5 affected individuals. OXGR1 encodes 2-oxoglutarate (α-ketoglutarate [AKG]) receptor 1 in the distal nephron. In response to its ligand AKG, OXGR1 stimulates the chloride-bicarbonate exchanger, pendrin, which also regulates transepithelial calcium transport in cortical connecting tubules. Strong amino acid conservation in orthologs and paralogs, severe in silico prediction scores, and extreme rarity in exome population databases suggested that the variant was deleterious. Interrogation of the OXGR1 locus in 1107 additional NL/NC families identified 5 additional deleterious dominant variants in 5 families with calcium oxalate NL/NC. Rare, potentially deleterious OXGR1 variants were enriched in patients with NL/NC compared with Exome Aggregation Consortium controls (χ2 = 7.117, P = .0076). Wild-type OXGR1-expressing Xenopus oocytes exhibited AKG-responsive Ca2+ uptake. Of 5 NL/NC-associated missense variants, 5 revealed impaired AKG-dependent Ca2+ uptake, demonstrating loss of function. CONCLUSION: Rare, dominant loss-of-function OXGR1 variants are associated with recurrent calcium oxalate NL/NC disease.

Extracorporeal membrane oxygenation during the first three waves of the coronavirus disease 2019 pandemic: A retrospective single-center registry study.

BACKGROUND: Despite increasing knowledge about the optimal treatment for patients with severe COVID-19, data from different cohorts suggested that survival of patients treated with ECMO seemed to decline over the course of the pandemic. METHODS: In this non-interventional retrospective single-center registry study we analyzed all consecutive patients tested positive for SARS-CoV-2 infection and supported with VV ECMO in our center during the first three waves of the pandemic. From March 2020 through June 2021, 59 patients have been included. RESULTS: Overall 90-day survival was 32%. Besides changes in drug treatment for COVID-19 and a lower PaO2 /FiO2 ratio before ECMO initiation during the third wave, all other patient baseline characteristics were similar during the three waves. Survival rate was highest during the first wave and lowest during the third wave, yet this difference was not statistically significant. CONCLUSIONS: VV ECMO has shown to be a feasible and safe support option for patients with severe respiratory failure due to COVID-19. The results from this single-center study confirm findings from other cohorts showing declining survival rates of patients treated with VV ECMO during the COVID-19 pandemic, however, the specific reasons for this finding remain unclear.

Incidence and predictors of delirium on the intensive care unit in patients with acute kidney injury, insight from a retrospective registry.

Acute kidney injury (AKI) and delirium are common complications on the intensive care unit (ICU). Few is known about the association of AKI and delirium, as well as about incidence and predictors of delirium in patients with AKI. In this retrospective study, all patients with AKI, as defined by the KDIGO (kidney disease improving global outcome) guideline, treated for more than 24 h on the ICU in an university hospital in 2019 were included and analyzed. Delirium was defined by a NuDesc (Nursing Delirium screening scale) ≥ 2, which is evaluated three times a day in every patient on our ICU as part of daily routine. A total of 383/919 (41.7%) patients developed an AKI during the ICU stay. Delirium was detected in 230/383 (60.1%) patients with AKI. Independent predictors of delirium were: age, psychiatric disease, alcohol abuse, mechanical ventilation, severe shock, and AKI stage II/III (all p < 0.05). The primary cause of illness had no influence on the onset of delirium. Among patients with AKI, the duration of the ICU stay correlated with higher stages of AKI and the presence of delirium (stage I/no delirium: median 1.9 (interquartile range (25th-75th) 1.3-2.9) days; stage II/III/no delirium: 2.6 (1.6-5.5) days; stage I/delirium: 4.1 (2.5-14.3) days; stage II/III/delirium: 6.8 (3.5-11.9) days; all p < 0.01). Delirium, defined as NuDesc ≥ 2 is frequent in patients with AKI on an ICU and independently predicted by higher stages of AKI.

Mobile ECMO retrieval of patients during the COVID-19 pandemic.

ECMO support is particularly resource-intensive and should be provided in highly specialized centers. Occasionally, ECMO needs to be initiated in non-ECMO centers by mobile ECMO retrieval teams. Subsequently, patients must be transferred on ECMO to the ECMO center. We report single-center data from out-of-center initiations of ECMO during the COVID-19 pandemic. From March 2020 through February 2021, nine patients were connected to ECMO before transfer to our center. Median travel distance (IQR) from the referring hospital to our center was 66 km (20-92), median land travel time (IQR) was 51 minutes (26-92). Personal protective equipment was available for all team members and used throughout the missions. No infections of team members with SARS-CoV-2 occurred. Three patients survived until hospital discharge. Median duration of ECMO (IQR) was 18 days (2-78) in survivors and 19 days (9-42) in non-survivors, respectively. Out-of-center initiation of ECMO during the COVID-19 pandemic was feasible and safe for patients and staff.

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.

Generation of Monogenic Candidate Genes for Human Nephrotic Syndrome Using 3 Independent Approaches.

INTRODUCTION: Steroid-resistant nephrotic syndrome (SRNS) is the second most common cause of chronic kidney disease during childhood. Identification of 63 monogenic human genes has delineated 12 distinct pathogenic pathways. METHODS: Here, we generated 2 independent sets of nephrotic syndrome (NS) candidate genes to augment the discovery of additional monogenic causes based on whole-exome sequencing (WES) data from 1382 families with NS. RESULTS: We first identified 63 known monogenic causes of NS in mice from public databases and scientific publications, and 12 of these genes overlapped with the 63 known human monogenic SRNS genes. Second, we used a set of 64 genes that are regulated by the transcription factor Wilms tumor 1 (WT1), which causes SRNS if mutated. Thirteen of these WT1-regulated genes overlapped with human or murine NS genes. Finally, we overlapped these lists of murine and WT1 candidate genes with our list of 120 candidate genes generated from WES in 1382 NS families, to identify novel candidate genes for monogenic human SRNS. Using this approach, we identified 7 overlapping genes, of which 3 genes were shared by all datasets, including SYNPO. We show that loss-of-function of SYNPO leads to decreased CDC42 activity and reduced podocyte migration rate, both of which are rescued by overexpression of wild-type complementary DNA (cDNA), but not by cDNA representing the patient mutation. CONCLUSION: Thus, we identified 3 novel candidate genes for human SRNS using 3 independent, nonoverlapping hypotheses, and generated functional evidence for SYNPO as a novel potential monogenic cause of NS.

DLG5 variants are associated with multiple congenital anomalies including ciliopathy phenotypes.

BACKGROUND: Cilia are dynamic cellular extensions that generate and sense signals to orchestrate proper development and tissue homeostasis. They rely on the underlying polarisation of cells to participate in signalling. Cilia dysfunction is a well-known cause of several diseases that affect multiple organ systems including the kidneys, brain, heart, respiratory tract, skeleton and retina. METHODS: Among individuals from four unrelated families, we identified variants in discs large 5 (DLG5) that manifested in a variety of pathologies. In our proband, we also examined patient tissues. We depleted dlg5 in Xenopus tropicalis frog embryos to generate a loss-of-function model. Finally, we tested the pathogenicity of DLG5 patient variants through rescue experiments in the frog model. RESULTS: Patients with variants of DLG5 were found to have a variety of phenotypes including cystic kidneys, nephrotic syndrome, hydrocephalus, limb abnormalities, congenital heart disease and craniofacial malformations. We also observed a loss of cilia in cystic kidney tissue of our proband. Knockdown of dlg5 in Xenopus embryos recapitulated many of these phenotypes and resulted in a loss of cilia in multiple tissues. Unlike introduction of wildtype DLG5 in frog embryos depleted of dlg5, introduction of DLG5 patient variants was largely ineffective in restoring proper ciliation and tissue morphology in the kidney and brain suggesting that the variants were indeed detrimental to function. CONCLUSION: These findings in both patient tissues and Xenopus shed light on how mutations in DLG5 may lead to tissue-specific manifestations of disease. DLG5 is essential for cilia and many of the patient phenotypes are in the ciliopathy spectrum.

Complete remission of Cdx-2 positive primary testicular carcinoid tumor: 10-years follow-up and literature review.

BACKGROUND: The neuroendocrine cells can cause a variety of malignancies throughout the human body known as the neuroendocrine tumors (NETs) or carcinoid tumors. The primary testicular carcinoid tumor (PTCT) accounts for less than 1% of the testicular neoplasms and for only 0.2% of all carcinoid tumors representing already a very rare neoplastic entity. Here, we present a patient with a history of an exceptionally rare primary testicular carcinoid tumor, staining positive for Cdx-2 along with a literature review. CASE PRESENTATION: A 44-year old patient without significant past medical history was diagnosed in September 2009 with primary testicular carcinoid tumor, which was surprisingly staining positively for Cdx-2, too. At the time of the initial diagnosis the tumor was already showing histopathological infiltration of veins. DOTA-TATE-PET/CT imaging and endoscopy studies did not show any signs of distant metastases and in particular no gastrointestinal manifestation following no further medical indication for systemic chemotherapy. The continuous and close follow-up of the patient has reached a total of over 10 years at the time of publication remaining in complete remission. CONCLUSION: The diagnosis of primary testicular carcinoid is based on histopathology. The detailed histopathologic assessment of biomarkers based on immunohistochemistry is very important for the classification and the prognosis of the primary testicular carcinoid tumor. Primary testicular carcinoid tumor with Cdx-2 positive stain outlines an exceptionally rare neoplastic entity without a consensus about general follow-up guidelines, requiring close clinical and imaging aftercare and consideration in Cdx-2 positive metastatic tumor of unknown origin.

Loss of Anks6 leads to YAP deficiency and liver abnormalities.

ANKS6 is a ciliary protein that localizes to the proximal compartment of the primary cilium, where it regulates signaling. Mutations in the ANKS6 gene cause multiorgan ciliopathies in humans, which include laterality defects of the visceral organs, renal cysts as part of nephronophthisis and congenital hepatic fibrosis (CHF) in the liver. Although CHF together with liver ductal plate malformations are common features of several human ciliopathy syndromes, including nephronophthisis-related ciliopathies, the mechanism by which mutations in ciliary genes lead to bile duct developmental abnormalities is not understood. Here, we generated a knockout mouse model of Anks6 and show that ANKS6 function is required for bile duct morphogenesis and cholangiocyte differentiation. The loss of Anks6 causes ciliary abnormalities, ductal plate remodeling defects and periportal fibrosis in the liver. Our expression studies and biochemical analyses show that biliary abnormalities in Anks6-deficient livers result from the dysregulation of YAP transcriptional activity in the bile duct-lining epithelial cells. Mechanistically, our studies suggest, that ANKS6 antagonizes Hippo signaling in the liver during bile duct development by binding to Hippo pathway effector proteins YAP1, TAZ and TEAD4 and promoting their transcriptional activity. Together, this study reveals a novel function for ANKS6 in regulating Hippo signaling during organogenesis and provides mechanistic insights into the regulatory network controlling bile duct differentiation and morphogenesis during liver development.

Subcutaneous Enoxaparin Safely Facilitates Bedside Sustained Low-Efficiency Hemodialysis in Hypercoagulopathic Coronavirus Disease 2019 Patients-A Proof-of-Principle Trial.

OBJECTIVES: Renal replacement therapy in coronavirus disease 2019 patients is complicated by increased activation of the coagulation system. This may worsen the quality of hemodialysis and contribute to a shortage of dialysis machines as well as plastic disposables during the pandemic. This study describes a simple and safe protocol of anticoagulation with low-molecular-weight heparin in combination with bedside sustained low-efficiency hemodialysis in coronavirus disease 2019 patients. DESIGN: Monocentric observational cross-over trial investigating sustained low-efficiency hemodialysis with unfractionated heparin following sustained low-efficiency hemodialysis with low-molecular-weight heparin. SETTING: Coronavirus disease 2019-ICU in a German Tertiary Care University Hospital. PATIENTS: Three consecutive severe coronavirus disease 2019 patients receiving nine sustained low-efficiency hemodialysis therapies with unfractionated heparin followed by 18 sustained low-efficiency hemodialysis therapies with low-molecular-weight heparin. INTERVENTIONS: Switch from IV unfractionated heparin to subcutaneous low-molecular-weight heparin enoxaparin in therapeutic doses for patients receiving bedside sustained low-efficiency hemodialysis. MEASUREMENTS AND MAIN RESULTS: Nine renal replacement therapy sessions in patients anticoagulated with high doses of unfractionated heparin had to be discontinuated prematurely because of clotting of tubes or membrane and poor quality of hemodialysis. In the same patients, the switch to anticoagulation with therapeutic doses of the low-molecular-weight heparin enoxaparin allowed undisturbed bedside sustained low-efficiency hemodialysis for at least 12 hours. Quality of hemodialysis was excellent, no bleeding event was observed. CONCLUSIONS: Systemic anticoagulation with subcutaneous enoxaparin provides an effective and safe renal replacement procedure in critically ill patients with coronavirus disease 2019 and hypercoagulability. The protocol reduces the risk of filter clotting, blood loss, and poor dialysis quality and may also prevent systemic thromboembolism.

ADCK4 Deficiency Destabilizes the Coenzyme Q Complex, Which Is Rescued by 2,4-Dihydroxybenzoic Acid Treatment.

BACKGROUND: Mutations in ADCK4 (aarF domain containing kinase 4) generally manifest as steroid-resistant nephrotic syndrome and induce coenzyme Q10 (CoQ10) deficiency. However, the molecular mechanisms underlying steroid-resistant nephrotic syndrome resulting from ADCK4 mutations are not well understood, largely because the function of ADCK4 remains unknown. METHODS: To elucidate the ADCK4's function in podocytes, we generated a podocyte-specific, Adck4-knockout mouse model and a human podocyte cell line featuring knockout of ADCK4. These knockout mice and podocytes were then treated with 2,4-dihydroxybenzoic acid (2,4-diHB), a CoQ10 precursor analogue, or with a vehicle only. We also performed proteomic mass spectrometry analysis to further elucidate ADCK4's function. RESULTS: Absence of Adck4 in mouse podocytes caused FSGS and albuminuria, recapitulating features of nephrotic syndrome caused by ADCK4 mutations. In vitro studies revealed that ADCK4-knockout podocytes had significantly reduced CoQ10 concentration, respiratory chain activity, and mitochondrial potential, and subsequently displayed an increase in the number of dysmorphic mitochondria. However, treatment of 3-month-old knockout mice or ADCK4-knockout cells with 2,4-diHB prevented the development of renal dysfunction and reversed mitochondrial dysfunction in podocytes. Moreover, ADCK4 interacted with mitochondrial proteins such as COQ5, as well as cytoplasmic proteins such as myosin and heat shock proteins. Thus, ADCK4 knockout decreased the COQ complex level, but overexpression of ADCK4 in ADCK4-knockout podocytes transfected with wild-type ADCK4 rescued the COQ5 level. CONCLUSIONS: Our study shows that ADCK4 is required for CoQ10 biosynthesis and mitochondrial function in podocytes, and suggests that ADCK4 in podocytes stabilizes proteins in complex Q in podocytes. Our study also suggests a potential treatment strategy for nephrotic syndrome resulting from ADCK4 mutations.

Recuperation of severe tumoral calcinosis in a dialysis patient: A case report.

BACKGROUND: One of the common late sequela in patients with end-stage renal disease (ESRD) is the calcium phosphate disorder leading to chronic hypercalcemia and hyperphosphatemia causing the precipitation of calcium salt in soft tissues. Tumoral calcinosis is an extremely rare clinical manifestation of cyst-like soft tissue deposits in different periarticular regions in patients with ESRD and is characterized by extensive calcium salt containing space-consuming painful lesions. The treatment of ESRD patients with tumoral calcinosis manifestation involves an increase in or switching of renal replacement therapy regimes and the adjustment of oral medication with the goal of improved hypercalcemia and hyperphosphatemia. CASE SUMMARY: We describe a 40-year-old woman with ESRD secondary to IgA-nephritis and severe bilateral manifestation of tumoral calcinosis associated with hypercalcemia, hyperphosphatemia and tertiary hyperparathyroidism. The patient was on continuous ambulatory peritoneal dialysis and treatment with vitamin D analogues. After switching her to a daily hemodialysis schedule and adjusting the medical treatment, the patient experienced a significant dissolution of her soft tissue calcifications within a couple of weeks. Complete remission was achieved 11 mo after the initial diagnosis. CONCLUSION: Reduced patient compliance and subsequent insufficiency of dialysis regime quality contribute to the aggravation of calcium phosphate disorder in a patient with ESRD leading to the manifestation of tumoral calcinosis. However, the improvement of the treatment strategy and reinforcement of patient compliance enabled complete remission of this rare disease entity.

Mutations in KIRREL1, a slit diaphragm component, cause steroid-resistant nephrotic syndrome.

Steroid-resistant nephrotic syndrome is a frequent cause of chronic kidney disease almost inevitably progressing to end-stage renal disease. More than 58 monogenic causes of SRNS have been discovered and majority of known steroid-resistant nephrotic syndrome causing genes are predominantly expressed in glomerular podocytes, placing them at the center of disease pathogenesis. Herein, we describe two unrelated families with steroid-resistant nephrotic syndrome with homozygous mutations in the KIRREL1 gene. One mutation showed high frequency in the European population (minor allele frequency 0.0011) and this patient achieved complete remission following treatment, but later progressed to chronic kidney disease. We found that mutant KIRREL1 proteins failed to localize to the podocyte cell membrane, indicating defective trafficking and impaired podocytes function. Thus, the KIRREL1 gene product has an important role in modulating the integrity of the slit diaphragm and maintaining glomerular filtration function.

Corticosteroid treatment exacerbates nephrotic syndrome in a zebrafish model of magi2a knockout.

Recently, recessive mutations of MAGI2 were identified as a cause of steroid-resistant nephrotic syndrome (SRNS) in humans and mice. To further delineate the pathogenesis of MAGI2 loss of function, we generated stable knockout lines for the two zebrafish orthologues magi2a and magi2b by CRISPR/Cas9. We also developed a novel assay for the direct detection of proteinuria in zebrafish independent of transgenic background. Whereas knockout of magi2b did not yield a nephrotic syndrome phenotype, magi2a-/- larvae developed ascites, periorbital edema, and proteinuria, as indicated by increased excretion of low molecular weight protein. Electron microscopy demonstrated extensive podocyte foot process effacement. As in human SRNS, we observed genotype/phenotype correlation, with edema onset occurring earlier in zebrafish with truncating alleles (5-6 days post fertilization) versus hypomorphic alleles (19-20 days post fertilization). Paradoxically, corticosteroid treatment exacerbated the phenotype, with earlier onset of edema. In contrast, treatment with cyclosporine A or tacrolimus had no significant effect. Although RhoA signaling has been implicated as a downstream mediator of MAGI2 activity, targeting of the RhoA pathway did not modify the nephrotic syndrome phenotype. In the first CRISPR/Cas9 zebrafish knockout model of SRNS, we found that corticosteroids may have a paradoxical effect in the setting of specific genetic mutations.

Treatment with 2,4-Dihydroxybenzoic Acid Prevents FSGS Progression and Renal Fibrosis in Podocyte-Specific Coq6 Knockout Mice.

BACKGROUND: Although studies have identified >55 genes as causing steroid-resistant nephrotic syndrome (SRNS) and localized its pathogenesis to glomerular podocytes, the disease mechanisms of SRNS remain largely enigmatic. We recently reported that individuals with mutations in COQ6, a coenzyme Q (also called CoQ10, CoQ, or ubiquinone) biosynthesis pathway enzyme, develop SRNS with sensorineural deafness, and demonstrated the beneficial effect of CoQ for maintenace of kidney function. METHODS: To study COQ6 function in podocytes, we generated a podocyte-specific Coq6 knockout mouse (Coq6podKO ) model and a transient siRNA-based COQ6 knockdown in a human podocyte cell line. Mice were monitored for development of proteinuria and assessed for development of glomerular sclerosis. Using a podocyte migration assay, we compared motility in COQ6 knockdown podocytes and control podocytes. We also randomly assigned 5-month-old Coq6podKO mice and controls to receive no treatment or 2,4-dihydroxybenzoic acid (2,4-diHB), an analog of a CoQ precursor molecule that is classified as a food additive by health authorities in Europe and the United States. RESULTS: Abrogation of Coq6 in mouse podocytes caused FSGS and proteinuria (>46-fold increases in albuminuria). In vitro studies revealed an impaired podocyte migration rate in COQ6 knockdown human podocytes. Treating Coq6podKO mice or cells with 2,4-diHB prevented renal dysfunction and reversed podocyte migration rate impairment. Survival of Coq6podKO mice given 2,4diHB was comparable to that of control mice and significantly higher than that of untreated Coq6podKO mice, half of which died by 10 months of age. CONCLUSIONS: These findings reveal a potential novel treatment strategy for those cases of human nephrotic syndrome that are caused by a primary dysfunction in the CoQ10 biosynthesis pathway.

Whole-Exome Sequencing Enables a Precision Medicine Approach for Kidney Transplant Recipients.

BACKGROUND: Whole-exome sequencing (WES) finds a CKD-related mutation in approximately 20% of patients presenting with CKD before 25 years of age. Although provision of a molecular diagnosis could have important implications for clinical management, evidence is lacking on the diagnostic yield and clinical utility of WES for pediatric renal transplant recipients. METHODS: To determine the diagnostic yield of WES in pediatric kidney transplant recipients, we recruited 104 patients who had received a transplant at Boston Children's Hospital from 2007 through 2017, performed WES, and analyzed results for likely deleterious variants in approximately 400 genes known to cause CKD. RESULTS: By WES, we identified a genetic cause of CKD in 34 out of 104 (32.7%) transplant recipients. The likelihood of detecting a molecular genetic diagnosis was highest for patients with urinary stone disease (three out of three individuals), followed by renal cystic ciliopathies (seven out of nine individuals), steroid-resistant nephrotic syndrome (nine out of 21 individuals), congenital anomalies of the kidney and urinary tract (ten out of 55 individuals), and chronic glomerulonephritis (one out of seven individuals). WES also yielded a molecular diagnosis for four out of nine individuals with ESRD of unknown etiology. The WES-related molecular genetic diagnosis had implications for clinical care for five patients. CONCLUSIONS: Nearly one third of pediatric renal transplant recipients had a genetic cause of their kidney disease identified by WES. Knowledge of this genetic information can help guide management of both transplant patients and potential living related donors.

Mutations of ADAMTS9 Cause Nephronophthisis-Related Ciliopathy.

Nephronophthisis-related ciliopathies (NPHP-RCs) are a group of inherited diseases that are associated with defects in primary cilium structure and function. To identify genes mutated in NPHP-RC, we performed homozygosity mapping and whole-exome sequencing for >100 individuals, some of whom were single affected individuals born to consanguineous parents and some of whom were siblings of indexes who were also affected by NPHP-RC. We then performed high-throughput exon sequencing in a worldwide cohort of 800 additional families affected by NPHP-RC. We identified two ADAMTS9 mutations (c.4575_4576del [p.Gln1525Hisfs∗60] and c.194C>G [p.Thr65Arg]) that appear to cause NPHP-RC. Although ADAMTS9 is known to be a secreted extracellular metalloproteinase, we found that ADAMTS9 localized near the basal bodies of primary cilia in the cytoplasm. Heterologously expressed wild-type ADAMTS9, in contrast to mutant proteins detected in individuals with NPHP-RC, localized to the vicinity of the basal body. Loss of ADAMTS9 resulted in shortened cilia and defective sonic hedgehog signaling. Knockout of Adamts9 in IMCD3 cells, followed by spheroid induction, resulted in defective lumen formation, which was rescued by an overexpression of wild-type, but not of mutant, ADAMTS9. Knockdown of adamts9 in zebrafish recapitulated NPHP-RC phenotypes, including renal cysts and hydrocephalus. These findings suggest that the identified mutations in ADAMTS9 cause NPHP-RC and that ADAMTS9 is required for the formation and function of primary cilia.

Genetic variants in the LAMA5 gene in pediatric nephrotic syndrome.

BACKGROUND: Nephrotic syndrome (NS), a chronic kidney disease, is characterized by significant loss of protein in the urine causing hypoalbuminemia and edema. In general, ∼15% of childhood-onset cases do not respond to steroid therapy and are classified as steroid-resistant NS (SRNS). In ∼30% of cases with SRNS, a causative mutation can be detected in one of 44 monogenic SRNS genes. The gene LAMA5 encodes laminin-α5, an essential component of the glomerular basement membrane. Mice with a hypomorphic mutation in the orthologous gene Lama5 develop proteinuria and hematuria. METHODS: To identify additional monogenic causes of NS, we performed whole exome sequencing in 300 families with pediatric NS. In consanguineous families we applied homozygosity mapping to identify genomic candidate loci for the underlying recessive mutation. RESULTS: In three families, in whom mutations in known NS genes were excluded, but in whom a recessive, monogenic cause of NS was strongly suspected based on pedigree information, we identified homozygous variants of unknown significance (VUS) in the gene LAMA5. While all affected individuals had nonsyndromic NS with an early onset of disease, their clinical outcome and response to immunosuppressive therapy differed notably. CONCLUSION: We here identify recessive VUS in the gene LAMA5 in patients with partially treatment-responsive NS. More data will be needed to determine the impact of these VUS in disease management. However, familial occurrence of disease, data from genetic mapping and a mouse model that recapitulates the NS phenotypes suggest that these genetic variants may be inherited factors that contribute to the development of NS in pediatric patients.

Panel sequencing distinguishes monogenic forms of nephritis from nephrosis in children.

BACKGROUND: Alport syndrome (AS) and atypical hemolytic-uremic syndrome (aHUS) are rare forms of chronic kidney disease (CKD) that can lead to a severe decline of renal function. Steroid-resistant nephrotic syndrome (SRNS) is more common than AS and aHUS and causes 10% of childhood-onset CKD. In recent years, multiple monogenic causes of AS, aHUS and SRNS have been identified, but their relative prevalence has yet to be studied together in a typical pediatric cohort of children with proteinuria and hematuria. We hypothesized that identification of causative mutations by whole exome sequencing (WES) in known monogenic nephritis and nephrosis genes would allow distinguishing nephritis from nephrosis in a typical pediatric group of patients with both proteinuria and hematuria at any level. METHODS: We therefore conducted an exon sequencing (WES) analysis for 11 AS, aHUS and thrombotic thrombocytopenic purpura-causing genes in an international cohort of 371 patients from 362 families presenting with both proteinuria and hematuria before age 25 years. In parallel, we conducted either WES or high-throughput exon sequencing for 23 SRNS-causing genes in all patients. RESULTS: We detected pathogenic mutations in 18 of the 34 genes analyzed, leading to a molecular diagnosis in 14.1% of families (51 of 362). Disease-causing mutations were detected in 3 AS-causing genes (4.7%), 3 aHUS-causing genes (1.4%) and 12 NS-causing genes (8.0%). We observed a much higher mutation detection rate for monogenic forms of CKD in consanguineous families (35.7% versus 10.1%). CONCLUSIONS: We present the first estimate of relative frequency of inherited AS, aHUS and NS in a typical pediatric cohort with proteinuria and hematuria. Important therapeutic and preventative measures may result from mutational analysis in individuals with proteinuria and hematuria.