Human C-terminal CUBN variants associate with chronic proteinuria and normal renal function.

BACKGROUNDProteinuria is considered an unfavorable clinical condition that accelerates renal and cardiovascular disease. However, it is not clear whether all forms of proteinuria are damaging. Mutations in CUBN cause Imerslund-Gräsbeck syndrome (IGS), which is characterized by intestinal malabsorption of vitamin B12 and in some cases proteinuria. CUBN encodes for cubilin, an intestinal and proximal tubular uptake receptor containing 27 CUB domains for ligand binding.METHODSWe used next-generation sequencing for renal disease genes to genotype cohorts of patients with suspected hereditary renal disease and chronic proteinuria. CUBN variants were analyzed using bioinformatics, structural modeling, and epidemiological methods.RESULTSWe identified 39 patients, in whom biallelic pathogenic variants in the CUBN gene were associated with chronic isolated proteinuria and early childhood onset. Since the proteinuria in these patients had a high proportion of albuminuria, glomerular diseases such as steroid-resistant nephrotic syndrome or Alport syndrome were often the primary clinical diagnosis, motivating renal biopsies and the use of proteinuria-lowering treatments. However, renal function was normal in all cases. By contrast, we did not found any biallelic CUBN variants in proteinuric patients with reduced renal function or focal segmental glomerulosclerosis. Unlike the more N-terminal IGS mutations, 37 of the 41 proteinuria-associated CUBN variants led to modifications or truncations after the vitamin B12-binding domain. Finally, we show that 4 C-terminal CUBN variants are associated with albuminuria and slightly increased GFR in meta-analyses of large population-based cohorts.CONCLUSIONCollectively, our data suggest an important role for the C-terminal half of cubilin in renal albumin reabsorption. Albuminuria due to reduced cubilin function could be an unexpectedly common benign condition in humans that may not require any proteinuria-lowering treatment or renal biopsy.FUNDINGATIP-Avenir program, Fondation Bettencourt-Schueller (Liliane Bettencourt Chair of Developmental Biology), Agence Nationale de la Recherche (ANR) Investissements d'avenir program (ANR-10-IAHU-01) and NEPHROFLY (ANR-14-ACHN-0013, to MS), Steno Collaborative Grant 2018 (NNF18OC0052457, to TSA and MS), Heisenberg Professorship of the German Research Foundation (KO 3598/5-1, to AK), Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Centre (SFB) KIDGEM 1140 (project 246781735, to CB), and Federal Ministry of Education and Research (BMB) (01GM1515C, to CB).

APOL1 risk genotype in European steroid-resistant nephrotic syndrome and/or focal segmental glomerulosclerosis patients of different African ancestries.

BACKGROUND: Apolipoprotein L1 (APOL1) risk variants are strongly associated with sporadic focal segmental glomerulosclerosis (FSGS) in populations with African ancestry. We determined the frequency of G1/G2 variants in steroid-resistant nephrotic syndrome (SRNS)/FSGS patients with African or French West Indies ancestry in France and its relationships with other SRNS genes. METHODS: In a cohort of 152 patients (139 families), the APOL1 risk variants were genotyped by direct Sanger sequencing and pathogenic mutations were screened by next-generation sequencing with a panel including 35 SRNS genes. RESULTS: The two risk allele [high-risk (HR)] genotypes were found in 43.1% (66/152) of subjects compared with 18.9% (106/562) in a control population (P < 0.0001): 33 patients homozygous for APOL1 G1 alleles, 4 homozygous for G2 and 29 compound heterozygous for G1 and G2. Compared with patients in the low-risk (LR) group, patients in the HR group were more likely to originate from the French West Indies than from Africa [45/66 (68.2%) versus 30/86 (34.9%); P < 0.0001]. There were more familial cases in the HR group [27 (41.5%) versus 8 (11.4%); P < 0.0001]. However, causative mutations in monogenic SRNS genes were found in only 1 patient in the HR group compared with 16 patients (14 families) in the LR group (P = 0.0006). At diagnosis, patients in the HR group without other mutations were more often adults [35 (53.8%) versus 19 (27.1%); P = 0.003] and had a lower estimated glomerular filtration rate (78.9 versus 98.8 mL/min/1.73 m2; P = 0.02). CONCLUSIONS: The HR genotype is frequent in FSGS patients with African ancestry in our cohort, especially in those originating from the West Indies, and confer a poor renal prognosis. It is usually not associated with other causative mutations in monogenic SRNS genes.

Identification of genetic causes for sporadic steroid-resistant nephrotic syndrome in adults.

Monogenic forms of Steroid-Resistant Nephrotic Syndrome (SRNS) have been widely characterized, but genetic screening paradigms preferentially address congenital, infantile onset, and familial cases. Our aim was to characterize the distribution of disease-causing gene mutations in adults with sporadic SRNS or focal segmental glomerulosclerosis (FSGS). We selected adult patients with non-syndromic, biopsy-proven FSGS or SRNS in the absence of known family history. Strict clinical criteria included lack of response to glucocorticoids and cyclosporine, and no recurrence after kidney transplantation. Mutations in SRNS genes were detected using a targeted gene panel. Sixteen of 135 tested participants (11.8%) carried pathogenic mutations in monogenic SRNS genes, and 14 others (10.4%) carried two APOL1 high-risk alleles. Autosomal recessive disease was diagnosed in 5 participants, autosomal dominant disease in 9, and X-linked disease in 2. Four participants carried a de novo heterozygous mutation. Among the 16 participants with identified mutations in monogenic SNRS genes, 7 (43.7%) had type IV collagen mutations. Mutations in monogenic SNRS genes were identified primarily in participants with proteinuria onset before 25 years of age, while the age at disease onset was variable in those with APOL1 high-risk genotype. Mean age at diagnosis was lower and renal survival was worse in participants with identified mutations in SNRS genes than in those without mutations. We found a significant rate of pathogenic mutations in adults with SRNS, with Type IV collagen mutations being the most frequent. These findings may have immediate impact on clinical practice.

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.

LMX1B mutations cause hereditary FSGS without extrarenal involvement.

LMX1B encodes a homeodomain-containing transcription factor that is essential during development. Mutations in LMX1B cause nail-patella syndrome, characterized by dysplasia of the patellae, nails, and elbows and FSGS with specific ultrastructural lesions of the glomerular basement membrane (GBM). By linkage analysis and exome sequencing, we unexpectedly identified an LMX1B mutation segregating with disease in a pedigree of five patients with autosomal dominant FSGS but without either extrarenal features or ultrastructural abnormalities of the GBM suggestive of nail-patella-like renal disease. Subsequently, we screened 73 additional unrelated families with FSGS and found mutations involving the same amino acid (R246) in 2 families. An LMX1B in silico homology model suggested that the mutated residue plays an important role in strengthening the interaction between the LMX1B homeodomain and DNA; both identified mutations would be expected to diminish such interactions. In summary, these results suggest that isolated FSGS could result from mutations in genes that are also involved in syndromic forms of FSGS. This highlights the need to include these genes in all diagnostic approaches to FSGS that involve next-generation sequencing.

Cysteamine therapy delays the progression of nephropathic cystinosis in late adolescents and adults.

Nephropathic cystinosis is a multisystem autosomal recessive disease caused by cystine accumulation, which is usually treated by oral cysteamine. In order to determine long-term effects of this therapy, we enrolled 86 adult patients (mean age 26.7 years) diagnosed with nephropathic cystinosis, 75 of whom received cysteamine. Therapy was initiated at a mean age of 9.9 years with a mean duration of 17.4 years. By last follow-up, 78 patients had end-stage renal disease (mean age 11.1 years), 62 had hypothyroidism (mean age 13.4), 48 developed diabetes (mean age 17.1 years), and 32 had neuromuscular disorders (mean age 23.3 years). Initiating cysteamine therapy before 5 years of age significantly decreased the incidence and delayed the onset of end-stage renal disease, and significantly delayed the onset of hypothyroidism, diabetes, and neuromuscular disorders. The development of diabetes and hypothyroidism was still significantly delayed, however, in patients in whom therapy was initiated after 5 years of age, compared with untreated patients. The life expectancy was significantly improved in cysteamine-treated versus untreated patients. Thus, cysteamine decreases and delays the onset of complications and improves life expectancy in cystinosis. Hence, cysteamine therapy should be introduced as early as possible during childhood and maintained lifelong.

INF2 mutations in Charcot-Marie-Tooth disease with glomerulopathy.

BACKGROUND: Charcot-Marie-Tooth neuropathy has been reported to be associated with renal diseases, mostly focal segmental glomerulosclerosis (FSGS). However, the common mechanisms underlying the neuropathy and FSGS remain unknown. Mutations in INF2 were recently identified in patients with autosomal dominant FSGS. INF2 encodes a formin protein that interacts with the Rho-GTPase CDC42 and myelin and lymphocyte protein (MAL) that are implicated in essential steps of myelination and myelin maintenance. We therefore hypothesized that INF2 may be responsible for cases of Charcot-Marie-Tooth neuropathy associated with FSGS. METHODS: We performed direct genotyping of INF2 in 16 index patients with Charcot-Marie-Tooth neuropathy and FSGS who did not have a mutation in PMP22 or MPZ, encoding peripheral myelin protein 22 and myelin protein zero, respectively. Histologic and functional studies were also conducted. RESULTS: We identified nine new heterozygous mutations in 12 of the 16 index patients (75%), all located in exons 2 and 3, encoding the diaphanous-inhibitory domain of INF2. Patients presented with an intermediate form of Charcot-Marie-Tooth neuropathy as well as a glomerulopathy with FSGS on kidney biopsy. Immunohistochemical analysis revealed strong INF2 expression in Schwann-cell cytoplasm and podocytes. Moreover, we demonstrated that INF2 colocalizes and interacts with MAL in Schwann cells. The INF2 mutants perturbed the INF2-MAL-CDC42 pathway, resulting in cytoskeleton disorganization, enhanced INF2 binding to CDC42 and mislocalization of INF2, MAL, and CDC42. CONCLUSIONS: INF2 mutations appear to cause many cases of FSGS-associated Charcot-Marie-Tooth neuropathy, showing that INF2 is involved in a disease affecting both the kidney glomerulus and the peripheral nervous system. These findings provide new insights into the pathophysiological mechanisms linking formin proteins to podocyte and Schwann-cell function. (Funded by the Agence Nationale de la Recherche and others.).

Mutations in INF2 are a major cause of autosomal dominant focal segmental glomerulosclerosis.

The recent identification of mutations in the INF2 gene, which encodes a member of the formin family of actin-regulating proteins, in cases of familial FSGS supports the importance of an intact actin cytoskeleton in podocyte function. To determine better the prevalence of INF2 mutations in autosomal dominant FSGS, we screened 54 families (78 patients) and detected mutations in 17% of them. All mutations were missense variants localized to the N-terminal diaphanous inhibitory domain of the protein, a region that interacts with the C-terminal diaphanous autoregulatory domain, thereby competing for actin monomer binding and inhibiting depolymerization. Six of the seven distinct altered residues localized to an INF2 region that corresponded to a subdomain of the mDia1 diaphanous inhibitory domain reported to co-immunoprecipitate with IQ motif-containing GTPase-activating protein 1 (IQGAP1). In addition, we evaluated 84 sporadic cases but detected a mutation in only one patient. In conclusion, mutations in INF2 are a major cause of autosomal dominant FSGS. Because IQGAP1 interacts with crucial podocyte proteins such as nephrin and PLCε1, the identification of mutations that may alter the putative INF2-IQGAP1 interaction provides additional insight into the pathophysiologic mechanisms linking formin proteins to podocyte dysfunction and FSGS.

Genotype-phenotype correlations in non-Finnish congenital nephrotic syndrome.

Mutations in NPHS1, which encodes nephrin, are the main causes of congenital nephrotic syndrome (CNS) in Finnish patients, whereas mutations in NPHS2, which encodes podocin, are typically responsible for childhood-onset steroid-resistant nephrotic syndrome in European populations. Genotype-phenotype correlations are not well understood in non-Finnish patients. We evaluated the clinical presentation, kidney histology, and disease progression in non-Finnish CNS cases by mutational screening in 107 families (117 cases) by sequencing the entire coding regions of NPHS1, NPHS2, PLCE1, WT1, LAMB2, PDSS2, COQ2, and NEPH1. We found that CNS describes a heterogeneous group of disorders in non-Finnish populations. We identified nephrin and podocin mutations in most families and only rarely found mutations in genes implicated in other hereditary forms of NS. In approximately 20% of cases, we could not identify the underlying genetic cause. Consistent with the major role of nephrin at the slit diaphragm, NPHS1 mutations associated with an earlier onset of disease and worse renal outcomes than NPHS2 mutations. Milder cases resulting from mutant NPHS1 had either two mutations in the cytoplasmic tail or two missense mutations in the extracellular domain, including at least one that preserved structure and function. In addition, we extend the spectrum of known NPHS1 mutations by describing long NPHS1 deletions. In summary, these data demonstrate that CNS is not a distinct clinical entity in non-Finnish populations but rather a clinically and genetically heterogeneous group of disorders.

Nephrin mutations can cause childhood-onset steroid-resistant nephrotic syndrome.

Classically, infants with mutations in NPHS1, which encodes nephrin, present with nephrotic syndrome within the first 3 mo of life (congenital nephrotic syndrome of the Finnish-type), and children with mutations in NPHS2, which encodes podocin, present later with steroid-resistant nephrotic syndrome. Recently, however, NPHS2 mutations have been identified in children with congenital nephrotic syndrome. Whether NPHS1 mutations similarly account for some cases of childhood steroid-resistant nephrotic syndrome is unknown. In this study, 160 patients who belonged to 142 unrelated families and presented with nephrotic syndrome at least 3 mo after birth were screened for NPHS1 variants once mutations in NPHS2 had been excluded. Compound heterozygous NPHS1 mutations were identified in one familial case and nine sporadic cases. Mutations included protein-truncating nonsense and frameshift mutations, as well as splice-site and missense variants. Mutations were classified as "severe" or "mild" using prediction algorithms and functional assays. Most missense variants trafficked normally to the plasma membrane and maintained the ability to form nephrin homodimers and to heterodimerize with NEPH1, suggesting retained function. The presence of at least one "mild" mutation in these patients likely explains the later onset and milder course of disease. These results broaden the spectrum of renal disease related to nephrin mutations.

NPHS2 mutation analysis shows genetic heterogeneity of steroid-resistant nephrotic syndrome and low post-transplant recurrence.

BACKGROUND: Mutations of NPHS2 are causative in familial autosomal-recessive (AR) and sporadic steroid-resistant nephrotic syndrome (SRNS). This study aimed to determine the spectrum of NPHS2 mutations and to establish genotype-phenotype correlations. METHODS: NPHS2 mutation analysis was performed in 338 patients from 272 families with SRNS: 81 families with AR SRNS, 172 patients with sporadic SRNS, and 19 patients with diffuse mesangial sclerosis (DMS). RESULTS: Twenty-six different pathogenic NPHS2 mutations were detected, including 13 novel mutations. The mutation detection rate was 43% for familial AR and 10.5% for sporadic SRNS, confirming genetic heterogeneity. No pathogenic NPHS2 mutations were found in DMS patients. Age at onset in patients with two pathogenic mutations was earlier, especially in cases with frameshift, truncating, and the R138Q missense mutations. Patients with only one NPHS2 mutation or variant had late-onset NS. Triallelic inheritance was observed in one patient with a homozygous R138Q mutation and a de novo NPHS1 mutation. Among 32 patients with two NPHS2 mutations who underwent kidney transplantation, only one developed late recurrence of focal segmental glomerulosclerosis (FSGS). Among 25 patients with sporadic SRNS and post-transplantation recurrence, we detected a heterozygous NPHS2 mutation in one case, and heterozygous variants/polymorphisms in 3 cases. CONCLUSION: Patients with two pathogenic NPHS2 mutations present with early-onset SRNS and very low incidence of post-transplantation recurrence. Heterozygous NPHS2 variants may play a role in atypical cases with mild, late-onset course, and recurrence after transplantation.

Long-term social outcome of children after kidney transplantation.

BACKGROUND: There are few data concerning the social outcome at adult age of children who received a kidney transplant. The aim of this study was to collect information on this outcome in a cohort of 366 children who underwent transplantation between 1973 and 1985. METHODS: Information was obtained through a simple questionnaire in 244 patients. The mean age of the patients was 31.7 years, and they had undergone grafting at a mean age of 11.9 years. RESULTS: As of December 2000 or at last visit, 77% had a functioning graft. The mean height was 156.6 cm for male patients and 147.4 cm for female patients. The distribution of educational level was lower than national averages: 27.4% were at the lowest level versus 3% of the general population, 41.4% were at the middle level, 31.2% had reached the baccalaureate level, and 11% had followed a university cursus. Activity was similar to the general population: 73% had paid employment versus 72%, 6.5% were unemployed versus 10.5%, and 18.7% received a disablement pension. Among the 149 male patients, 39 (27%) had a marital life and 12 (8.3%) had children, whereas among the 95 female patients, 48 (50%) had a marital life and 26 (27%) had at least one child. Lodging was the parent's home in 46% and independent in 54%. Multivariate analysis showed a significant correlation between educational level, paid activity, marital life, and independent housing with final height. CONCLUSIONS: The long-term social outcome of patients who underwent grafting in childhood more than 15 years previously is encouraging. The importance of reaching a normal height is stressed.