INF2 Mutations Research Articles

Establishing a human-induced pluripotent stem cell line (SMUSHi003-A) from a patient with Charcot-Marie-Tooth disease and focal segmental glomerulosclerosis

INF2 mutations cause Charcot-Marie-Tooth disease (CMT), and /or focal segmental glomerulosclerosis (FSGS) in an autosomal dominant inheritance mode, whose underlying mechanism remainsunclear. Here, we report the generation of an iPSC line from a female patient with CMT and FSGS. The iPSC line from the patient's PBMCscarried aheterozygous INF2 deletion mutation (c.315_323delGCGCGCCGT) within the conserved E2. This line exhibited a normal karyotype, high expression of pluripotency markers, and trilineage differentiation potential. This line can be used to dissect the complex pathomechanism through further induction of differentiation into related cells and as a drug screening tool for INF2-associated diseases.

INF2 and ROBO2 gene mutation in an Indian family with end stage renal failure and follow-up of renal transplantation.

Accurate genetic diagnosis of end-stage renal disease patients with a family history of renal dysfunction is very essential. It not only helps in proper prognosis, but becomes crucial in designating donor for live related renal transplant. We here present a case of family with deleterious mutations in INF2 and ROBO2 and its importance of genetic testing before preparing for kidney transplantation. We report the case of a 29-year-female with end-stage renal disease and rapidly progressive renal failure. Mutational analysis revealed an Autosomal Dominant inheritance pattern and mutation in exon 4 of the INF2 gene (p. Thr215Ser) and exon 26 of the ROBO2 gene (p. Arg1371Cys). Her mother was diagnosed for CKD stage 4 with creatinine level of 4.3 mg/dL. Genetic variants (INF2 and ROBO2) identified in proband were tested in her sisters and mother. Her elder sister was positive for both heterozygous variants (INF2 and ROBO2). Her mother was positive for mutation in INF2 gene, and her donor elder sister did not showed mutation in INF2 gene and had mutation in ROBO2 gene without any clinical symptoms. This case report emphasize that familial genetic screening has allowed us in allocating the donor selection in family where family member had history of genetic defect of Chronic Kidney Disease. Information of the causative renal disorder is extremely valuable for risk-assessment and planning of kidney transplantation.

#3760 CYTOSKELETON-ORGANELLE INTERACTION IN LIVING CELL EXPRESSING INF2 VARIANTS CAUSING FSGS AND PERIPHERAL NEUROPATHY

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a common glomerular injury and represents a leading cause of ESRD. Studies with genetic FSGS show that a majority of genes are associated with regulation of podocyte cytoskeletons. INF2 is an actin assembly factor that mediates both polymerization and depolymerization and is the most frequent cause of dominant inherited FSGS. INF2 mutations located in the proximal half of DID (residues Leu57 to Glu184) typically cause CMT with early-onset FSGS, whereas that situating in the distal half (residues Glu184 to Leu245) cause FSGS alone. Previous studies disclosed that INF2 mutations primarily disorganize actin network and alter mitochondria shapes and vesicle motility. However, it remains elusive how the cytoskeletal network is concerted with the organelle morphology and functions in the disease cells. This study aims to characterize the interaction between cytoskeleton and organelle in cells expressing INF2 FSGS or CMT/FSGS variants. Method We characterized the cytoskeletal-organelle interaction in live HeLa or COS-7 cells expressing pathogenic INF2 variants in our cohort of FSGS. We performed a triple labeling by co-transfection of GFP-INF2, cytoskeletal markers (Lifeact-mCherry or EMTB-mScarletI), and by the uptake of organelle markers (Lysotracker or Mitotracker). To clarify the detailed pattern and dynamics of cytoskeletons and organelles, we employed the Spinning Disk Confocal Microscope DragonFly, which allows real-time super-resolution imaging with 70nm resolution. Results Wild-type-INF2 (WT-INF2) cells showed an ER pattern with perinuclear, Golgi accumulation. Mild INF2 variants causing FSGS alone (T161N, N202S) preserved the subcellular distribution essential similar to WT-INF2, while showing focal irregularity of ER networks. In contrast, severe INF2 variants leading to CMT/FSGS (G73D) exhibited a diffuse coarse ER pattern with few Golgi clusters and occasional peripheral accumulation in both cell poles. In live imaging of cells coexpressing GFP-INF2 and Lifeact-mCherry, actin stress fibers were pronouncedly reduced in CMT/FSGS variant (G73D) cells, but were mildly decreased FSGS variant (T161N and N202S) cells, compared with WT-INF2 cells. The filopodia in cells expressing T161N variant were less mobile (n = 10, P< 0.01) but were longer (n = 10, P< 0.0001) than those in cells expressing WT-INF2. T161N variant reduced expression of cortactin that stabilizes actin-branching and narrowed area of cortical actin meshwork compared to the WT-INF2 (n = 11, P< 0.01), suggesting that INF2 could serve the cortical actin formation. HeLa cells coexpressing T161N variant and EMTB showed a bipolar cell elongation along the longitudinal axis (n≥30, P< 0.0001) and disarranged microtubule bundles aligned in parallel, compared with those expressing WT-INF2. A time-lapse imaging of living COS-7 cells revealed that T161N variant increases mitochondrial fragmentation as well as mitochondria-ER interfaces more prominent than WT-INF2. Moreover, T161N variant induced the mitochondria misdistribution at the cell periphery (filopodia and cell edge) in both cell poles. Such aberrant patterns frequently coexisted with disarranged microtubules. Moreover, in lysotracker-labeled living COS-7, T161N variant reduced the motility of the lysosomal vesicles in cortical actin meshwork area, compared with WT-INF2 (n = 100, P< 0.0001). These observations suggest that INF2 variants perturbate the trafficking of organelles through disorganization of actin-microtubule network. Conclusion Our results indicate that INF2 CMT/FSGS variants cause more global and severe organelle dysfunction by disrupting the organelle-cytoskeletal interaction, than INF2 FSGS variants. The microtubule disarrangement might be due to an insufficient capping mechanism by which the microtubules are stably tethered to the cortical actin meshwork. Elucidation of cell-specific factors will help better understanding why podocyte and Schwann cell are susceptible to the cytoskeletal disarrangement.

Podocyte Infolding Glomerulopathy: A Case Series Report and Literature Review.

Podocyte infolding glomerulopathy (PIG) is a peculiar and very rare manifestation in renal pathology. Its underlying pathogenesis mechanism and clinical characteristics remain unclear due to sparse reports. To further elucidate the clinical profile of PIG by carefully reporting our four cases and a comprehensive review of cases in the literature. This study retrospectively reviewed four cases of PIG from 2010 to 2022 in our centre. Clinical and pathological profiles were reported. PIG cases in the literature were searched in the MEDLINE database and analysed together with our cases. Four cases of PIG identified from our centre and 40 cases from the current literature were reported. The pooled analysis of these 44 cases indicated 79.5% (35/44) were females, 93.2% (41/44) were East Asians, and 63.6% (28/44) were reported in Japan. The average age was 42.0 ± 12.5 years old. The average amount of proteinuria at the time of renal biopsy was 3.06 ± 3.2 g/day. The most reported comorbidities were connective tissue diseases, mainly systemic lupus erythematosus, and 20.5% (9/44) of the cases did not have any contaminant disease. Most of the cases (81.8%, 36/44) had been treated with immunosuppressants, of which a combination of corticosteroids and one other type of immunosuppressant was most commonly reported. In addition, 45.4% (20/44) and 34.1% (15/44) of the cases had achieved complete response and partial response, respectively, after treatment. Whole exosome sequencing indicated mutations in the INF2 gene. PIG is a rare condition and seen in relatively younger populations, often associated with connective tissue diseases clinically and one or two other glomerulopathies histologically. The outcomes following immunosuppressive treatment are relatively good. Mutations in INF2 might be involved in the development of PIG; however, the implications of these results need to be investigated.

Late-Onset Schwannomatosis in Two Unrelated Patients With Peripheral Neuropathy and Focal Segmental Glomerulosclerosis due to INF2 Mutations

Late-Onset Schwannomatosis in Two Unrelated Patients With Peripheral Neuropathy and Focal Segmental Glomerulosclerosis due to INF2 Mutations

The Frequency of Genetic Mutations in Pediatric Patients Diagnosed with Nephrotic Syndrome: A Single-Center Retrospective Study in Saudi Arabia.

Limited data are available on the common genetic mutations causing nephrotic syndrome (NS) in the Saudi pediatric population. We therefore conducted this study to estimate the frequency of genetic mutations in pediatric patients diagnosed with NS. We conducted this retrospective cross-sectional study at a single center in Riyadh, Saudi Arabia. The data of pediatric patients diagnosed with NS from 2015 to 2019 were reviewed. Percentages were calculated for categorical variables such as gender and mutant gene. We identified a total of 206 patients diagnosed with NS during the study. Molecular genetic profiling was performed only for 35 patients who met the inclusion criteria. Female patients represented 42.8% of all cases (n = 15). The median age of the patients at diagnosis was 36 months (interquartile range 12-72). Associated anomalies were recognized in 37.14% of the patients (n = 13). Out of the 35 patients, 19 had positive molecular genetic results. Consanguinity was present in 18 (51.42%) of these patients. The most common homozygous mutation detected was PLCE1 (42.1%; n = 8), followed by NPHS1 (26.32%; n = 5). Heterozygous mutations were detected in three children (15.8%). Complement factor B and WT1 mutations accounted for one patient each and both COL4A5 and INF2 mutations were reported in a single child. Two mutant genes of unknown zygosity - CD151 and COL4A3 were also identified. PLCE1 is a major underlying cause of NS. PLCE1 may cause diffuse mesangial sclerosis in the kidney with early-onset NS and a poor prognosis.

Dysregulated Dynein-Mediated Trafficking of Nephrin Causes INF2-related Podocytopathy.

FSGS caused by mutations in INF2 is characterized by a podocytopathy with mistrafficked nephrin, an essential component of the slit diaphragm. Because INF2 is a formin-type actin nucleator, research has focused on its actin-regulating function, providing an important but incomplete insight into how these mutations lead to podocytopathy. A yeast two-hybridization screen identified the interaction between INF2 and the dynein transport complex, suggesting a newly recognized role of INF2 in regulating dynein-mediated vesicular trafficking in podocytes. Live cell and quantitative imaging, fluorescent and surface biotinylation-based trafficking assays in cultured podocytes, and a new puromycin aminoglycoside nephropathy model of INF2 transgenic mice were used to demonstrate altered dynein-mediated trafficking of nephrin in INF2 associated podocytopathy. Pathogenic INF2 mutations disrupt an interaction of INF2 with dynein light chain 1, a key dynein component. The best-studied mutation, R218Q, diverts dynein-mediated postendocytic sorting of nephrin from recycling endosomes to lysosomes for degradation. Antagonizing dynein-mediated transport can rescue this effect. Augmented dynein-mediated trafficking and degradation of nephrin underlies puromycin aminoglycoside-induced podocytopathy and FSGS in vivo. INF2 mutations enhance dynein-mediated trafficking of nephrin to proteolytic pathways, diminishing its recycling required for maintaining slit diaphragm integrity. The recognition that dysregulated dynein-mediated transport of nephrin in R218Q knockin podocytes opens an avenue for developing targeted therapy for INF2-mediated FSGS.

Scrub typhus leading to focal segmental glomerulosclerosis in a child due to genetic predisposition

Scrub typhus is a multisystem disease, caused by genera orientia tsutsugamushi and is currently endemic in India. In children, the disease may vary from a mild to a severe form. Complications include encephalitis, myocarditis, disseminated intravascular coagulation, acute kidney injury, atypical pneumonia, etc. The pathophysiologic mechanisms of renal involvement in scrub typhus include prerenal failure, septic shock, vasculitis, acute tubular injury and direct renal invasion by rickettsia. Here, authors present the case of a previously well 5-year old female child who was admitted to our hospital with a history of high-grade fever and pain abdomen. IgM scrub typhus turned out to be positive and she was adequately treated with doxycycline. She turned afebrile but then gradually developed anasarca, hematuria, proteinuria and persistent stage 2 hypertension. Kidney biopsy was done which revealed focal segmental glomerulosclerosis (FSGS). Further workup of the patient by whole exome sequencing revealed missense mutations in TBX18, INF2 and NPHS1 genes. Mutations in INF2 gene is a recently discovered cause of autosomal dominant FSGS. In our case, the scrub typhus mediated kidney injury probably acted as a trigger in unmasking FSGS in the already genetically susceptible child.

The formin INF2 in disease: progress from 10years of research.

Formins are a conserved family of proteins that primarily act to form linear polymers of actin. Despite their importance to the normal functioning of the cytoskeleton, for a long time, the only two formin genes known to be a genetic cause of human disorders were DIAPH1 and DIAPH3, whose mutation causes two distinct forms of hereditary deafness. In the last 10years, however, the formin INF2 has emerged as an important target of mutations responsible for the appearance of focal segmental glomerulosclerosis, which are histological lesions associated with glomerulus degeneration that often leads to end-stage renal disease. In some rare cases, focal segmental glomerulosclerosis concurs with Charcot-Marie-Tooth disease, which is a degenerative neurological disorder affecting peripheral nerves. All known INF2 gene mutations causing disease map to the exons encoding the amino-terminal domain. In this review, we summarize the structure, biochemical features and functions of INF2, conduct a systematic and comprehensive analysis of the pathogenic INF2 mutations, including a detailed study exon-by-exon of patient cases and mutations, address the impact of the pathogenic mutations on the structure, regulation and known functions of INF2, draw a series of conclusions that could be useful for INF2-related disease diagnosis, and suggest lines of research for future work on the molecular mechanisms by which INF2 causes disease.

Regulation of INF2-mediated actin polymerization through site-specific lysine acetylation of actin itself

INF2 is a formin protein that accelerates actin polymerization. A common mechanism for formin regulation is autoinhibition, through interaction between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD). We recently showed that INF2 uses a variant of this mechanism that we term "facilitated autoinhibition," whereby a complex consisting of cyclase-associated protein (CAP) bound to lysine-acetylated actin (KAc-actin) is required for INF2 inhibition, in a manner requiring INF2-DID. Deacetylation of actin in the CAP/KAc-actin complex activates INF2. Here we use lysine-to-glutamine mutations as acetylmimetics to map the relevant lysines on actin for INF2 regulation, focusing on K50, K61, and K328. Biochemically, K50Q- and K61Q-actin, when bound to CAP2, inhibit full-length INF2 but not INF2 lacking DID. When not bound to CAP, these mutant actins polymerize similarly to WT-actin in the presence or absence of INF2, suggesting that the effect of the mutation is directly on INF2 regulation. In U2OS cells, K50Q- and K61Q-actin inhibit INF2-mediated actin polymerization when expressed at low levels. Direct-binding studies show that the CAP WH2 domain binds INF2-DID with submicromolar affinity but has weak affinity for actin monomers, while INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WT-actin. Actin in complex with full-length CAP2 is predominately ATP-bound. These interactions suggest an inhibition model whereby CAP/KAc-actin serves as a bridge between INF2 DID and DAD. In U2OS cells, INF2 is 90-fold and 5-fold less abundant than CAP1 and CAP2, respectively, suggesting that there is sufficient CAP for full INF2 inhibition.

Mutations in INF2 may be associated with renal histology other than focal segmental glomerulosclerosis

In 2010, INF2 mutations were associated with autosomal-dominant focal segmental glomerulosclerosis (FSGS), clinically presenting with moderate proteinuria in adolescence. However, in the meantime, cases with more severe clinical courses have been described, including progression to end-stage renal disease (ESRD) during childhood. INF2 mutations in patients with isolated FSGS are clustered in exons 2 to 4, encoding the diaphanous inhibitory domain, involved in the regulation of the podocyte actin cytoskeleton. We report a family with 14 affected individuals (autosomal-dominant mode of inheritance), most of whom presented with nephrotic-range proteinuria, hypertension, and progressive renal failure. Four members received a kidney transplant without disease recurrence. Two patients underwent renal biopsy with the result of minimal-change glomerulopathy and IgA nephropathy respectively. We performed mutational analysis of ACTN4, CD2AP, COQ6, INF2, LAMB2, NPHS1, NPHS2, PLCE1, TRPC6, and WT1 in the index patient by next-generation sequencing. Additionally, in 6 affected and 2 unaffected family members target diagnostics were performed. We identified a novel heterozygous mutation c.490G>C (p.(Ala164Pro) in exon 3 of the INF2 gene in the index patient and 6 additionally examined affected family members. In silico analysis predicted it as "probably damaging". Additionally, three patients and 2 unaffected relatives harbored a novel heterozygous variant in ACTN4 (c.1149C>G, p.(Ile383Met)) with uncertain pathogenicity. Mutations in INF2 are associated with familial proteinuric diseases - irrespective of the presence of FSGS and in the case of rapid disease progression. Therefore, mutational analysis should be considered in patients with renal histology other than FSGS and severe renal phenotype.

Thrombotic Microangiopathy in Inverted Formin 2-Mediated Renal Disease.

The demonstration of impaired C regulation in the thrombotic microangiopathy (TMA) atypical hemolytic uremic syndrome (aHUS) resulted in the successful introduction of the C inhibitor eculizumab into clinical practice. C abnormalities account for approximately 50% of aHUS cases; however, mutations in the non-C gene diacylglycerol kinase-ε have been described recently in individuals not responsive to eculizumab. We report here a family in which the proposita presented with aHUS but did not respond to eculizumab. Her mother had previously presented with a post-renal transplant TMA. Both the proposita and her mother also had Charcot-Marie-Tooth disease. Using whole-exome sequencing, we identified a mutation in the inverted formin 2 gene (INF2) in the mutational hotspot for FSGS. Subsequent analysis of the Newcastle aHUS cohort identified another family with a functionally-significant mutation in INF2 In this family, renal transplantation was associated with post-transplant TMA. All individuals with INF2 mutations presenting with a TMA also had aHUS risk haplotypes, potentially accounting for the genetic pleiotropy. Identifying individuals with TMAs who may not respond to eculizumab will avoid prolonged exposure of such individuals to the infectious complications of terminal pathway C blockade.

INF2 mutations and FSGS in Chinese families

INF2 mutations and FSGS in Chinese families

The Disease-Associated Formin INF2/EXC-6 Organizes Lumen and Cell Outgrowth during Tubulogenesis by Regulating F-Actin and Microtubule Cytoskeletons

We investigate how outgrowth at the basolateral cell membrane is coordinated with apical lumen formation in the development of a biological tube by characterizing exc-6, a gene required for C. elegans excretory cell (EC) tubulogenesis. We show that EXC-6 is orthologous to the human formin INF2, which polymerizes filamentous actin (F-actin) and binds microtubules (MTs) in vitro. Dominant INF2 mutations cause focal segmental glomerulosclerosis (FSGS), a kidney disease, and FSGS+Charcot-Marie-Tooth neuropathy. We show that activated INF2 can substitute for EXC-6 in C. elegans and that disease-associated mutations cause constitutive activity. Using genetic analysis and live imaging, we show that exc-6 regulates MT and F-actin accumulation at EC tips and dynamics of basolateral-localized MTs, indicating that EXC-6 organizes F-actin and MT cytoskeletons during tubulogenesis. The pathology associated with INF2 mutations is believed to reflect misregulation of F-actin, but our results suggest alternative or additional mechanisms via effects on MT dynamics.

Novel INF2 mutations in an Italian cohort of patients with focal segmental glomerulosclerosis, renal failure and Charcot-Marie-Tooth neuropathy.

Mutations of INF2 represent the major cause of familial autosomal dominant (AD) focal segmental glomerulosclerosis (FSGS). A few patients present neurological symptoms of Charcot-Marie-Tooth (CMT) disease but the prevalence of the association has not been assessed yet. We screened 28 families with AD FSGS and identified 8 INF2 mutations in 9 families (32 patients overall), 3 of which were new. Mutations were in all cases localized in the diaphanous-inhibitory domain (DID) of the protein. Clinical features associated with INF2 mutations in our patient cohort included mild proteinuria (1.55 g/L; range 1-2.5) and haematuria as a unique symptom that was recognized at a median age of 21.75 years (range 8-30). Eighteen patients developed end-stage renal disease during their third decade of life; 12 patients presented a creatinine range between 1.2 and 1.5 mg/dL and 2 were healthy at 45 and 54 years of age. CMT was diagnosed in four cases (12.5%); one of these patients presented an already known mutation on exon 2 of INF2, whereas the other patients presented the same mutation on exon 4, a region that was not previously associated with CMT. We confirmed the high incidence of INF2 mutations in families with AD FSGS. The clinical phenotype was mild at the onset of the disease, but evolution to ESRD was frequent. The incidence of CMT has, for the first time, been calculated here to be 12.5% of mutation carriers. Our findings support INF2 gene analysis in families in which renal failure and/or neuro-sensorial defects are inherited following an AD model.

INF2-Mediated Severing through Actin Filament Encirclement and Disruption

INF2 is a formin protein with the unique ability to accelerate both actin polymerization and depolymerization, the latter requiring filament severing. Mutations in INF2 lead to the kidney disease focal segmental glomerulosclerosis (FSGS) and the neurological disorder Charcot-Marie Tooth disease (CMTD). Here, we compare the severing mechanism of INF2 with that of the well-studied severing protein cofilin. INF2, like cofilin, binds stoichiometrically to filament sides and severs in a manner that requires phosphate release from the filament. In contrast to cofilin, however, INF2 binds ADP and ADP-Pi filaments equally well. Furthermore, two-color total internal reflection fluorescence (TIRF) microscopy reveals that a low number of INF2 molecules, as few as a single INF2 dimer, are capable of severing, while measurable cofilin-mediated severing requires more extensive binding. Hence, INF2 is a more potent severing protein than cofilin. While a construct containing the FH1 and FH2 domains alone has some severing activity, addition of the C-terminal region increases severing potency by 40-fold, and we show that the WH2-resembling DAD motif is responsible for this increase. Helical 3D reconstruction from electron micrographs at 20 Å resolution provides a structure of filament-bound INF2, showing that the FH2 domain encircles the filament. We propose a severing model in which FH2 binding and phosphate release causes local filament deformation, allowing the DAD to bind adjacent actin protomers, further disrupting filament structure.

The various Charcot–Marie–Tooth diseases

This review focuses on recent advances in the diagnostic approaches and the underlying pathophysiological mechanisms of Charcot-Marie-Tooth (CMT) disease. We also discuss the emerging therapies for this hereditary neuropathy. To date, numerous genes are implicated in CMT, and new genes have recently been found to be associated with this neuropathy (INF2, FBLN5, etc.). Some specific or evocative clinical signs of CMT subtypes (proteinuria with INF2 mutations, etc.) have been identified. Characteristic pathological findings, which may suggest gene mutations, are also recognized by nerve biopsy (mainly ultrastructural lesions). CMT disease is the most common inherited neuromuscular disorder, with a fairly homogeneous clinical phenotype (progressive distal muscle weakness and atrophy, foot deformities, distal sensory loss, and depressed tendon reflexes). With more than 40 genes implicated, an update of the present and rather confusing classification of CMT is needed. Over the last few years, new mutated genes have been discovered. Although nerve biopsy is not routinely carried out in CMT neuropathies, it may show characteristic features, which can orientate the search for the mutated gene. There are currently no effective medications for CMT, but clinical trials are ongoing or planned.

Actin Monomers Activate Inverted Formin 2 by Competing with Its Autoinhibitory Interaction

INF2 is an unusual formin protein in that it accelerates both actin polymerization and depolymerization, the latter through an actin filament-severing activity. Similar to other formins, INF2 possesses a dimeric formin homology 2 (FH2) domain that binds filament barbed ends and is critical for polymerization and depolymerization activities. In addition, INF2 binds actin monomers through its diaphanous autoregulatory domain (DAD) that resembles a Wiskott-Aldrich syndrome protein homology 2 (WH2) sequence C-terminal to the FH2 that participates in both polymerization and depolymerization. INF2-DAD is also predicted to participate in an autoinhibitory interaction with the N-terminal diaphanous inhibitory domain (DID). In this work, we show that actin monomer binding to the DAD of INF2 competes with the DID/DAD interaction, thereby activating actin polymerization. INF2 is autoinhibited in cells because mutation of a key DID residue results in constitutive INF2 activity. In contrast, purified full-length INF2 is constitutively active in biochemical actin polymerization assays containing only INF2 and actin monomers. Addition of proteins that compete with INF2-DAD for actin binding (profilin or the WH2 from Wiskott-Aldrich syndrome protein) decrease full-length INF2 activity while not significantly decreasing activity of an INF2 construct lacking the DID sequence. Profilin-mediated INF2 inhibition is relieved by an anti-N-terminal antibody for INF2 that blocks the DID/DAD interaction. These results suggest that free actin monomers can serve as INF2 activators by competing with the DID/DAD interaction. We also find that, in contrast to past results, the DID-containing N terminus of INF2 does not directly bind the Rho GTPase Cdc42.

Genetic screening in adolescents with steroid-resistant nephrotic syndrome

Genetic screening paradigms for congenital and infantile nephrotic syndrome are well established; however, screening in adolescents has received only minor attention. To help rectify this, we analyzed an unselected adolescent cohort of the international PodoNet registry to develop a rational screening approach based on 227 patients with nonsyndromic steroid-resistant nephrotic syndrome aged 10-20 years. Of these, 21% had a positive family history. Autosomal dominant cases were screened for WT1, TRPC6, ACTN4, and INF2 mutations. All other patients had the NPHS2 gene screened, and WT1 was tested in sporadic cases. In addition, 40 sporadic cases had the entire coding region of INF2 tested. Of the autosomal recessive and the sporadic cases, 13 and 6%, respectively, were found to have podocin-associated nephrotic syndrome, and 56% of them were compound heterozygous for the nonneutral p.R229Q polymorphism. Four percent of the sporadic and 10% of the autosomal dominant cases had a mutation in WT1. Pathogenic INF2 mutations were found in 20% of the dominant but none of the sporadic cases. In a large cohort of adolescents including both familial and sporadic disease, NPHS2 mutations explained about 7% and WT1 4% of cases, whereas INF2 proved relevant only in autosomal dominant familial disease. Thus, screening of the entire coding sequence of NPHS2 and exons 8-9 of WT1 appears to be the most rational and cost-effective screening approach in sporadic juvenile steroid-resistant nephrotic syndrome.

Mutations in the INF2 gene account for a significant proportion of familial but not sporadic focal and segmental glomerulosclerosis

Mutations in the inverted formin 2 gene (INF2) have recently been identified as the most common cause of autosomal dominant focal and segmental glomerulosclerosis (FSGS). In order to quantify the contribution of various genes contributing to FSGS, we sequenced INF2 where all mutations have previously been described (exons 2 to 5) in a total of 215 probands and 281 sporadic individuals with FSGS, along with other known genes accounting for autosomal dominant FSGS (ACTN4, TRPC6 and CD2AP) in 213 probands. Variants were classified as disease-causing if they altered the amino acid sequence, were not found in control samples, and in families segregated with disease. Mutations in INF2 were found in a total of 20 of the 215 families (including those previously reported) in our cohort of autosomal dominant familial nephrotic syndrome or FSGS, thereby explaining disease in 9 percent. INF2 mutations were found in 2 of 281 individuals with sporadic FSGS. In contrast, ACTN4 and TRPC6-related disease accounted for 3 and 2 percent of our familial cohort, respectively. INF2-related disease showed variable penetrance, with onset of disease ranging widely from childhood to adulthood and commonly leading to ESRD in the third and fourth decade of life. Thus, mutations in INF2 are more common, although still minor, monogenic cause of familial FSGS when compared to other known autosomal dominant genes associated with FSGS.