Several genes highly expressed in podocytes (e.g., ACTN4, INF2, and TRPC6) had previously been reported to be a cause of familial and sporadic autosomal dominant forms of FSGS. Herein, we present the molecular genetic analysis of INF2 (using the HRM method and Sanger sequencing) performed on a large cohort of patients with FSGS/MCD (FSGS = 77 patients, MCD = 32 patients), as well as in a group of patients (6) characterized by a positive family history for ESRD in combination with advanced CKD/ESRD at the time of diagnosis. These types of analyses can lead to the identification of new causal mutations; therefore, they are very important in clinical practice for the diagnosis of patients.
The INF2 protein, belonging to the formin family, plays a key role in the influence of the actin cytoskeleton dependent processes in podocytes because of its capability to accelerate both the polymerisation and depolymerisation of actin. The protein consists of three main parts including: the DID domain, two FH domains, and the DAD domain. Previous studies have demonstrated the abnormal distribution of the detected mutations; all of them having been identified in exons encoding the DID domain, suggesting its crucial role for the protein function. The DID domain appears to be important for actin cytoskeleton regulation [7, 8].
All identified substitutions in the exons were determined to be disease causing candidates if they: (a) segregated with the disease (in the case that other relatives were also affected, and we had samples of their DNA); (b) were predicted to be damaging by the Mutational Taster program , PolyPhen-2 , PROVEAN , and SIFT ; (c) were not carried by both healthy family members (whose DNA samples we had) as well as the healthy controls; and (d) were not present in control chromosomes in the db SNP and 1000 Genomes project.
We identified the mutation p.Arg218Gln in INF2 with a damaging effect on the podocyte function in two brothers from a family with the positive history for ESRD. This mutation was first described by Brown et al. in a large family with the familial form of FSGS . Both brothers quickly progressed to ESRD, aged 27 and 31, respectively.
The other detected casual mutation in INF2 was p.Arg214His, also first identified by Brown et al. . The mutation was found in a woman with a perihilar variant of FSGS and simultaneously suffering from diabetes mellitus. Although the family history of the proband was positive for renal diseases (father and uncle), her two healthy daughters had refused the opportunity for genetic testing. The incidences of this variant in our analysis were 0.9% (the whole cohort of patients), 1.3% (only the cohort of FSGS patients), and 5.6% (the cohort of FSGS patients with a positive family history), respectively. Both identified casual mutations are localized in the DID domain of INF2, which is necessary for normal protein function because it is involved in actin cytoskeleton regulation.
The mutational analysis of INF2 also identified other different exonic changes and intronic substitutions that seemed to have no effect on the phenotype of the patients included in this study.
The most frequent substitution in INF2 was p.P35P within exon 2, and with an allele frequency of more than 97%. This incidence corresponds with the results available in the ExAC Browser (http://exac.broadinstitute.org/) and gnomAD Browser (http://gnomad.broadinstitute.org/) databases, confirming the sensitivity of HRM. Other highly frequent changes in INF2 were p.D1022D and p.D880D, with allele frequencies of 68% and 60%, respectively.
The HRM analysis was a very effective method for mutational screening in such a large cohort of patients. It proved to be of sufficient sensitivity in the detection of single nucleotide substitutions.
According to previous studies focused on INF2, it appears that this gene is responsible for a high percentage of patients with familial FSGS [5, 17, 18]. Even though the proportion of mutations in sporadic FSGS is significantly lower [9, 18], we included both familiar and idiopathic patients with FSGS. Although the incidence of pathogenic mutations was lower than our assumptions at the beginning of the study, we established the method for the molecular analysis of INF2 and analyzed a large cohort of patients with FSGS/MCD; confirming that MCD cases are less likely to harbor deleterious genetic variants in those genes implicated in FSGS [4,5,6, 19]. This was the first molecular genetic analysis focused on INF2 in the Czech Republic. A mutational analysis of INF2 should be performed in all patients with a positive family history of FSGS or unknown ESRD with an autosomal dominant inheritance. Proteinuria is frequently mild or moderate, the patients are asymptomatic, and advanced renal insufficiency is already present, so a renal biopsy is not indicated in many cases.