A wave of deep intronic mutations in X-linked Alport syndrome.

X-linked Alport syndrome (XLAS) is an inherited kidney disease caused exclusively by pathogenic variants in the COL4A5 gene. In 10-20% of cases, DNA sequencing of COL4A5 exons or flanking regions cannot identify molecular causes. Here, our objective was to use a transcriptomic approach to identify causative events in a group of 19 patients with XLAS without identified mutation by Alport gene panel sequencing. Bulk RNAseq and/or targeted RNAseq using a capture panel of kidney genes was performed. Alternative splicing events were compared to those of 15 controls by a developed bioinformatic score. When using targeted RNAseq, COL4A5 coverage was found to be 23-fold higher than with bulk RNASeq and revealed 30 significant alternative splicing events in 17 of the 19 patients. After computational scoring, a pathogenic transcript was found in all patients. A causative variant affecting COL4A5 splicing and absent in the general population was identified in all cases. Altogether, we developed a simple and robust method for identification of aberrant transcripts due to pathogenic deep-intronic COL4A5 variants. Thus, these variants, potentially targetable by specific antisense oligonucleotide therapies, were found in a high percentage of patients with XLAS in whom pathogenic variants were missed by conventional DNA sequencing.

Overcoming the challenges associated with identification of deep intronic variants by whole genome sequencing.

Whole-genome sequencing (WGS) now allows identification of multiple variants in non-coding regions. The large number of variants identified by WGS however complicates their interpretation. Through identification of the first deep intronic variant in NPHS2, which encodes podocin, a protein implicated in autosomal recessive steroid resistant nephrotic syndrome (SRNS), we compare herein three different tools including a newly developed targeted NGS-based RNA-sequencing to explore the splicing effect of intronic variations. WGS identified two different variants in NPHS2 eventually involved in the disease. Through RT-PCR, exon-trapping Minigene assay and targeted RNA sequencing, we were able to identify the splicing defect in NPHS2 mRNA from patient kidney tissue. Only targeted RNA-seq simultaneously analyzed the effect of multiple variants and offered the opportunity to quantify consequences on splicing. Identifying deep intronic variants and their role in disease is of utmost importance. Alternative splicing can be predicted by in silico tools but always requires confirmation through functional testing with RNA analysis from the implicated tissue remaining the gold standard. When several variants with potential effects on splicing are identified by WGS, a targeted RNA sequencing panel could be of great value.

Investigating genotype-to-phenotype correlation in CHARGE syndrome by deep phenotyping and multiparametric clustering.

CHARGE syndrome, due to CHD7 pathogenic variations, is an autosomal dominant disorder characterized by a large spectrum of severity. Despite the great number of variations reported, no clear genotype-to-phenotype correlation has been reported. Unsupervised machine learning and clustering was undertaken using a retrospective cohort of 42 patients, after deep radiologic and clinical phenotyping, to establish genotype-phenotype correlation for CHD7-related CHARGE syndrome. It resulted in three clusters showing phenotypes of different severities. While no clear genotype-phenotype correlation appeared within the first two clusters, a single patient was outlying the cohort data (cluster 3) with the most atypical phenotype and the most distal frameshift variant in the gene. We added two other patients with similar distal pathogenic variants and observed a tendency toward mild and/or atypical phenotypes. We hypothesized that this finding could potentially be related to escaping nonsense mediated RNA decay, but found no evidence of such decay in vivo for any of the CHD7 pathogenic variation tested. This indicates that this milder phenotype may rather result from the production of a protein retaining all functional domains.

Pseudouridylation defect due to DKC1 and NOP10 mutations causes nephrotic syndrome with cataracts, hearing impairment, and enterocolitis.

RNA modifications play a fundamental role in cellular function. Pseudouridylation, the most abundant RNA modification, is catalyzed by the H/ACA small ribonucleoprotein (snoRNP) complex that shares four core proteins, dyskerin (DKC1), NOP10, NHP2, and GAR1. Mutations in DKC1, NOP10, or NHP2 cause dyskeratosis congenita (DC), a disorder characterized by telomere attrition. Here, we report a phenotype comprising nephrotic syndrome, cataracts, sensorineural deafness, enterocolitis, and early lethality in two pedigrees: males with DKC1 p.Glu206Lys and two children with homozygous NOP10 p.Thr16Met. Females with heterozygous DKC1 p.Glu206Lys developed cataracts and sensorineural deafness, but nephrotic syndrome in only one case of skewed X-inactivation. We found telomere attrition in both pedigrees, but no mucocutaneous abnormalities suggestive of DC. Both mutations fall at the dyskerin-NOP10 binding interface in a region distinct from those implicated in DC, impair the dyskerin-NOP10 interaction, and disrupt the catalytic pseudouridylation site. Accordingly, we found reduced pseudouridine levels in the ribosomal RNA (rRNA) of the patients. Zebrafish dkc1 mutants recapitulate the human phenotype and show reduced 18S pseudouridylation, ribosomal dysregulation, and a cell-cycle defect in the absence of telomere attrition. We therefore propose that this human disorder is the consequence of defective snoRNP pseudouridylation and ribosomal dysfunction.

Long-Term Efficacy and Safety of Repeated Rituximab to Maintain Remission in Idiopathic Childhood Nephrotic Syndrome: An International Study.

BACKGROUND: Long-term outcomes after multiple courses of rituximab among children with frequently relapsing, steroid-dependent nephrotic syndrome (FRSDNS) are unknown. METHODS: A retrospective cohort study at 16 pediatric nephrology centers from ten countries in Asia, Europe, and North America included children with FRSDNS who received two or more courses of rituximab. Primary outcomes were relapse-free survival and adverse events. RESULTS: A total of 346 children (age, 9.8 years; IQR, 6.6-13.5 years; 73% boys) received 1149 courses of rituximab. A total of 145, 83, 50, 28, 22, and 18 children received two, three, four, five, six, and seven or more courses, respectively. Median (IQR) follow-up was 5.9 (4.3-7.7) years. Relapse-free survival differed by treatment courses (clustered log-rank test P<0.001). Compared with the first course (10.0 months; 95% CI, 9.0 to 10.7 months), relapse-free period and relapse risk progressively improved after subsequent courses (12.0-16.0 months; HRadj, 0.03-0.13; 95% CI, 0.01 to 0.18; P<0.001). The duration of B-cell depletion remained similar with repeated treatments (6.1 months; 95% CI, 6.0 to 6.3 months). Adverse events were mostly mild; the most common adverse events were hypogammaglobulinemia (50.9%), infection (4.5%), and neutropenia (3.7%). Side effects did not increase with more treatment courses nor a higher cumulative dose. Only 78 of the 353 episodes of hypogammaglobulinemia were clinically significant. Younger age at presentation (2.8 versus 3.3 years; P=0.05), age at first rituximab treatment (8.0 versus 10.0 years; P=0.01), and history of steroid resistance (28% versus 18%; P=0.01) were associated with significant hypogammaglobulinemia. All 53 infective episodes resolved, except for one patient with hepatitis B infection and another with EBV infection. There were 42 episodes of neutropenia, associated with history of steroid resistance (30% versus 20%; P=0.04). Upon last follow-up, 332 children (96%) had normal kidney function. CONCLUSIONS: Children receiving repeated courses of rituximab for FRSDNS experience an improving clinical response. Side effects appear acceptable, but significant complications can occur. These findings support repeated rituximab use in FRSDNS.

Targeted next-generation sequencing in a large series of fetuses with severe renal diseases.

We report the screening of a large panel of genes in a series of 100 fetuses (98 families) affected with severe renal defects. Causative variants were identified in 22% of cases, greatly improving genetic counseling. The percentage of variants explaining the phenotype was different according to the type of phenotype. The highest diagnostic yield was found in cases affected with the ciliopathy-like phenotype (11/15 families and, in addition, a single heterozygous or a homozygous Class 3 variant in PKHD1 in three unrelated cases with autosomal recessive polycystic kidney disease). The lowest diagnostic yield was observed in cases with congenital anomalies of the kidney and urinary tract (9/78 families and, in addition, Class 3 variants in GREB1L in three unrelated cases with bilateral renal agenesis). Inheritance was autosomal recessive in nine genes (PKHD1, NPHP3, CEP290, TMEM67, DNAJB11, FRAS1, ACE, AGT, and AGTR1), and autosomal dominant in six genes (PKD1, PKD2, PAX2, EYA1, BICC1, and MYOCD). Finally, we developed an original approach of next-generation sequencing targeted RNA sequencing using the custom capture panel used for the sequencing of DNA, to validate one MYOCD heterozygous splicing variant identified in two male siblings with megabladder and inherited from their healthy mother.

TBC1D8B Loss-of-Function Mutations Lead to X-Linked Nephrotic Syndrome via Defective Trafficking Pathways.

Steroid-resistant nephrotic syndrome (SRNS) is characterized by high-range proteinuria and most often focal and segmental glomerulosclerosis (FSGS). Identification of mutations in genes causing SRNS has improved our understanding of disease mechanisms and highlighted defects in the podocyte, a highly specialized glomerular epithelial cell, as major factors in disease pathogenesis. By exome sequencing, we identified missense mutations in TBC1D8B in two families with an X-linked early-onset SRNS with FSGS. TBC1D8B is an uncharacterized Rab-GTPase-activating protein likely involved in endocytic and recycling pathways. Immunofluorescence studies revealed TBC1D8B presence in human glomeruli, and affected individual podocytes displayed architectural changes associated with migration defects commonly found in FSGS. In zebrafish we demonstrated that both knockdown and knockout of the unique TBC1D8B ortholog-induced proteinuria and that this phenotype was rescued by human TBC1D8B mRNA injection, but not by either of the two mutated mRNAs. We also showed an interaction between TBC1D8B and Rab11b, a key protein in vesicular recycling in cells. Interestingly, both internalization and recycling processes were dramatically decreased in affected individuals' podocytes and fibroblasts, confirming the crucial role of TBC1D8B in the cellular recycling processes, probably as a Rab11b GTPase-activating protein. Altogether, these results confirmed that pathogenic variations in TBC1D8B are involved in X-linked podocytopathy and points to alterations in recycling processes as a mechanism of SRNS.

Neurological involvement in monogenic podocytopathies.

Genetic studies of hereditary nephrotic syndrome (NS) have identified more than 50 genes that, if mutated, are responsible for monogenic forms of steroid-resistant NS (SRNS), either isolated or syndromic. Most of these genes encode proteins expressed in the podocyte with various functions such as transcription factors, mitochondrial proteins, or enzymes, but mainly structural proteins of the slit diaphragm (SD) as well as cytoskeletal binding and regulator proteins. Syndromic NS is sometimes associated with neurological features. Over recent decades, various studies have established links between the physiology of podocytes and neurons, both morphologically (slit diaphragm and synapse) and functionally (signaling platforms). Variants in genes expressed in different compartments of the podocyte and neurons are responsible for phenotypes associating kidney lesions with proteinuria (mainly Focal and Segmental Glomerulosclerosis (FSGS) or Diffuse Mesangial Sclerosis (DMS)) and central and/or peripheral neurological disorders. The Galloway-Mowat syndrome (GAMOS, OMIM#251300) associates neurological defects, microcephaly, and proteinuria and is caused by variants in genes encoding proteins of various functions (microtubule cytoskeleton regulation (WDR73), regulation of protein synthesis via transfer RNAs (KEOPS and WDR4 complexes)). Pierson syndrome (OMIM#609049) associating congenital nephrotic syndrome and central neurological and ophthalmological anomalies is secondary to variants in LAMB2, involved in glomerular and ocular basement membranes. Finally, Charcot-Marie-Tooth-FSGS (OMIM#614455) combines peripheral sensory-motor neuropathy and proteinuria and arises from INF2 variants, resulting in cytoskeletal polymerization defects. This review focuses on genetic syndromes associating nephrotic range proteinuria and neurological involvement and provides the latest advances in the description of these neuro-renal disorders.

Cystic kidney diseases associated with mutations in phosphomannomutase 2 promotor: a large spectrum of phenotypes.

BACKGROUND: Co-occurrence of polycystic kidney disease and hyperinsulinemic hypoglycemia has been reported in children in a few families associated with a variant in the promotor of the PMM2 gene, at position -167 upstream of the coding sequence. PMM2 encodes phosphomannomutase 2, a key enzyme in N-glycosylation. While biallelic coding PMM2 mutations are involved in congenital disorder of glycosylation CDG1A, that particular variant in the promoter of the gene, either in the homozygous state or associated with a mutation in the coding exons of the gene, is thought to restrict the N-glycosylation defect to the kidney and the pancreas. METHODS: Targeted exome sequencing of a panel of genes involved in monogenic kidney diseases. RESULTS: We identified a PMM2 variant at position -167 associated with a pathogenic PMM2 variant in the coding exons in 3 families, comprising 6 cases affected with a cystic kidney disease. The spectrum of phenotypes was very broad, from extremely enlarged fetal cystic kidneys in the context of a COACH-like syndrome, to isolated cystic kidney disease with small kidneys, slowly progressing toward kidney failure in adulthood. Hypoglycemia was reported only in one case. CONCLUSION: These data show that the PMM2 promotor variation, in trans of a PMM2 coding mutation, is associated with a wide spectrum of kidney phenotypes, and is not always associated with extra-renal symptoms. When present, extra-renal defects may include COACH-like syndrome. These data prompt screening of PMM2 in unresolved cases of fetal hyperechogenic/cystic kidneys as well as in cystic kidney disease in children and adults. Graphical Abstract.

The spectrum of kidney function alterations in adolescents with a solitary functioning kidney.

BACKGROUND: A precise assessment of glomerular filtration rate is key to delineate the care of children with a solitary functioning kidney (SFK). Data regarding measured GFR (mGFR) in this population is restricted to a single study of 77 individuals, which suggested that a GFR estimation (eGFR) method based on creatinine and cystatin C (eGFR-CKiD2) performed better than Schwartz's equation (eGFR-Schwartz). METHODS: We measured GFR in 210 consecutive adolescents (7 to 22 years old) with an SFK referred to our institution between 2014 and 2019 and in 43 young candidates for kidney donation (18 to 25 years old). We compared the distribution of mGFR in both groups and determined the factors associated with reduced mGFR in adolescents with an SFK. We further compared different eGFR formulas with mGFR and assessed the association of mGFR and eGFRs with PTH and FGF23, two early indicators of GFR reduction. RESULTS: While adolescents with an SFK had a similar median mGFR to healthy controls (103 ± 24ml/min/1.73m2 vs. 107 ± 12 ml/min/1.73m2), the fraction of individuals with an mGFR below 90 ml/min/1.73m2 was higher in patients with SFK (23% vs. 5% in controls; P = 0.005). Multiple linear regression identified older age, ipsilateral abnormalities of the urinary tract, lack of compensatory hypertrophy, and treated hypertension as independent factors associated with reduced mGFR. A smaller bias using eGFR-Schwartz (95% confidence interval (95%CI): 3 to 7) was revealed when compared to other eGFR. Compared to eGFR-Schwartz, mGFR showed a stronger correlation with PTH (r = 0.04 vs. r = 0.1) and FGF23 (r = 0.03 vs. r = 0.05). CONCLUSION: SFK is not a benign condition, since 20% of the patients display altered kidney function. Our results raise caution regarding the use of the cystatin-based equation. mGFR shows a better ability than eGFR-Schwartz to differentiate patients showing early homeostatic adaptation to GFR reduction.

Long-term kidney and liver outcome in 50 children with autosomal recessive polycystic kidney disease.

BACKGROUND: Autosomal recessive polycystic kidney disease (ARPKD) is a rare ciliopathy characterized by congenital hepatic fibrosis and cystic kidney disease. Lack of data about long-term follow-up makes it difficult to discuss timing and type of organ transplantation. Our objectives were to evaluate long-term evolution and indications for transplantation, from birth to adulthood. METHODS: Neonatal survivors and patients diagnosed in postnatal period with ARPKD between 1985 January and 2017 December from 3 French pediatric centers were retrospectively enrolled in the study. RESULTS: Fifty patients with mean follow-up 12.5 ± 1 years were enrolled. ARPKD was diagnosed before birth in 24%, and at mean age 1.8 years in others. Thirty-three patients were < 1 year of age at first symptoms, which were mostly kidney-related. These most often presented high blood pressure during follow-up. Portal hypertension was diagnosed in 29 patients (58%), 4 of them with bleeding from esophageal varices. Eight patients presented cholangitis (> 3 episodes in three children). Liver function was normal in all patients. Nine children received a kidney transplant without liver complications. A 20-year-old patient received a combined liver-kidney transplant (CLKT) for recurrent cholangitis, and a 15-year-old boy an isolated liver transplant for uncontrollable variceal bleeding despite portosystemic shunt. CONCLUSIONS: Long-term outcome in patients with ARPKD is heterogeneous, and in this cohort did not depend on age at diagnosis except for blood pressure. Few patients required liver transplantation. Indications for liver or combined liver-kidney transplantation were limited to recurrent cholangitis or uncontrollable portal hypertension. Liver complications after kidney transplantation were not significant.

Association between 25(OH) vitamin D and graft survival in renal transplanted children.

BACKGROUND: In children, vitamin D deficiency is common after renal transplantation. Besides promoting bone and muscle development, vitamin D has immunomodulatory effects, which could protect kidney allografts. The purpose of this study was to assess the association between vitamin D status and the occurrence of renal rejection. METHODS: We studied a retrospective cohort of 123 children, who were transplanted at a single institution between September 2008 and April 2019. Patients did not receive vitamin D supplementation systematically. In addition, factors influencing vitamin D status were analyzed using univariate and multivariate analyses. RESULTS: Median 25-hydroxy-vitamin D (25-OH-D) concentration was close to reference values at the time of transplantation (30 ng/mL (min-max 5-100)), but rapidly decreased within the first 3 months to 19 ng/mL (min-max 3-91) (P < .001). The overall acute rejection rate was 7%. The clinical rejection rate (5% vs 9%), subclinical rejection (12% vs 36%), and borderline changes (21% vs 28%) were not statistically different during the follow-up between the 3-month 25-OH-D < 20 ng/mL and 3-month 25-OH-D > 20 ng/mL groups. There was a correlation between the 25-OH-D levels and PTH concentration at 3 months (r = -.2491, P = .01), but no correlation between the 3-month 25-OH-D and the season of the year (F = 0.19, P = .90; F = 1.34, P = .27, respectively). Multivariate analyses revealed that age and mGFR at 3 months, were independent predictors of mGFR at 12 months. CONCLUSION: Our data show that vitamin D deficiency can develop rapidly after transplantation; vitamin D levels at 3 months are not associated with lower mGFR or a higher rejection rate at 1 year in children as opposed to adult recipients.

Clinical and genetic heterogeneity in familial steroid-sensitive nephrotic syndrome.

BACKGROUND: Familial steroid-sensitive nephrotic syndrome (SSNS) is a rare condition. The disease pathophysiology remains elusive. However, bi-allelic mutations in the EMP2 gene were identified, and specific variations in HLA-DQA1 were linked to a high risk of developing the disease. METHODS: Clinical data were analyzed in 59 SSNS families. EMP2 gene was sequenced in families with a potential autosomal recessive (AR) inheritance. Exome sequencing was performed in a subset of 13 families with potential AR inheritance. Two variations in HLA-DQA1 were genotyped in the whole cohort. RESULTS: Transmission was compatible with an AR (n = 33) or autosomal dominant (AD, n = 26) inheritance, assuming that familial SSNS is a monogenic trait. Clinical features did not differ between AR and AD groups. All patients, including primary (n = 7) and secondary steroid resistant nephrotic syndrone (SRNS), (n = 13) were sensitive to additional immunosuppressive therapy. Both HLA-DQA1 variations were found to be highly linked to the disease (OR = 4.34 and OR = 4.89; p < 0.001). Exome sequencing did not reveal any pathogenic mutation, neither did EMP2 sequencing. CONCLUSIONS: Taken together, these results highlight the clinical and genetic heterogeneity in familial SSNS. Clinical findings sustain an immune origin in all patients, whatever the initial steroid-sensitivity. The absence of a variant shared by two families and the HLA-DQA1 variation enrichments suggest a complex mode of inheritance.