Prevalence, Genetics, and Clinical Features of Patients Carrying Podocin Mutations in Steroid-Resistant Nonfamilial Focal Segmental Glomerulosclerosis

Patients

The study included 44 subjects who had received a clinical and pathologic diagnosis of FSGS. Clinical criteria included the presence of steroid-resistant nephrotic syndrome, the exclusion of congenital nephrotic syndrome of the Finnish and the Denys-Drash or Frasier types, and the exclusion of multiplex families that presented with a clear familial inheritance for proteinuria. For first-degree relatives of the patients included in the study, we were able to document normal urinalysis; for all the other relatives, the absence of proteinuria was inferred from an interview. Consanguinity was in all cases excluded with the exception of the parents of one patient (patient 9, Table 1), who were second order cousins. Relevant clinical parameters, including urinalysis and renal function, were normal in all relatives. The histologic hallmarks of FSGS were the presence of at least one area of segmental or global sclerosis with or without Ig deposits (IgM, IgG) in the native glomeruli. Mesangial expansion of both the cellular component and of extracellular matrix was a frequent but not ubiquitous finding. Mean patient age was 22 yr (range, 3 to 32 yr). Ten patients had normal renal function (creatinine, <1.3 mg/dl), 2 had chronic renal failure (creatinine, >1.3 mg/dl), 1 had terminal renal failure in dialysis, and 31 had received a renal allograft. The therapy for primary nephrotic syndrome consisted of long courses of prednisone according to standard protocols (7); all patients were also treated with cyclophosphamide for 2 mo at 2 mg/kg and, in a few cases, with cyclosporin A (CsA) at an initial dose of 4 to 5 mg/kg. Immunosuppressive therapy after renal transplantation followed standard protocols as described previously (8). After transplantation, the urinary parameters were carefully followed every day for 2 mo and then weekly for another 10 mo. Two patients who presented proteinuria greater than 1 g/L posttransplantation were treated following the same protocol used in recurrence of idiopathic FSGS, consisting in plasmapheresis plus cyclophosphamide. A renal biopsy was not performed after recurrence of proteinuria in these cases. Plasmapheresis was performed by removing 1 to 1.5 plasma volumes each cycle according to the patient’s weight for either six cycles on alternate days (patient 6) or for six cycles and then an additional four cycles performed weekly (patient 7). Fluid replacement consisted of fresh plasma or saline solution supplemented with 4% albumin. In those patients treated with cyclophosphamide (2 mg/kg for 2 mo), azathioprine, or mycophenolate mofetil were transiently discontinued until the suspension of cyclophosphamide.

Molecular Analysis of Podocin

DNA was isolated from fresh peripheral blood samples. Molecular analysis of podocin was performed by direct sequencing as described previously by Boute et al. (1). Exons were amplified by PCR using flanking intron primers and subjected to automated sequence analysis by dye-terminator reactions (Automated Sequencer ABI 377; Applied-Biosystem, Milan, Italy).

Generation of New Markers to the NPHS2 Gene

Novel polymorphic DNA markers were found by using a search strategy for microsatellite markers in electronic databases (9). Specifically, a text file containing 16 repeats of dinucleotides AC, AG, AT, and CG was used as a search query for the sequence comparison program BLAST (www3.ncbi.nlm.nih.gov). The queries were searched against sequences of the recently released Human Genome Sequence (www3.ncbi.nlm.nih.gov), with reference to the chromosome 1 working draft sequence segment NT_004470 containing the whole NPHS2 gene.

Primer pairs were designed to the sequence flanking the repeats. One primer (forward) for pairs was labeled with a fluorescent dye to use Automated Sequencer ABI 377. These repeat markers were analyzed for polymorphisms by using DNA samples from 50 to 70 unrelated control individuals and in a three-generation family to confirm the allelic segregation.

Statistical Analysis

The differences in clinical parameters between FSGS patient carriers and noncarriers of podocin mutations were evaluated with one-way ANOVA. Data are expressed as mean ± SD.

Podocin Mutations

Among the group of 44 FSGS patients, nine nonfamilial patients carrying podocin mutations (Table 1) were found. Patients 4, 5, and 8 presented composite heterozygous mutations, whereas the remaining six were homozygous. Overall, we found the same mutations described by Boute et al. (1), with the exception of two novel mutations at exon 4 in position 506 resulting in a T to C transition (L169P) and in a T insertion in position 467/8, producing a frameshift (Figure 1). In comparison with 75 normal DNA sequences, we could exclude that L169P was a polymorphism.

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Figure 1. Two novel mutations at exon 4 in the NPHS2 gene detected by direct sequencing in two patients.

Markers to the NPHS2 Gene and Haplotype Analysis

A set of new markers (1 dinucleotide repeat and 4 single nucleotide polymorphisms [SNPs]) was identified in the NPHS2 gene (Table 2) and used to genotype patients carrying the more frequent mutations 419delG, R138Q, and L169P (Table 3). The dinucleotide repeats marker located in intron 1 of NPHS2 gene presented 5 alleles from 301 to 309 bp, with a polymorphism information content value of 0.66. Of the four SNPs identified, the one in exon 2 (c288C>T: S96S) was already reported by Wu et al. (10), and the others, one in intron 7 (EX7+7A>G) and two in exon 8 (c951T>C: A317A; c1038A>G: L346L), are of new description.

Haplotype reconstruction for the nine affected patients was carried out by using all polymorphisms above. Three of the four SNPs (c288C>T, EX7+7A>G, and c1038A>G) showing low heterozygosity were not informative. On the basis of analysis of the two informative polymorphisms (Intron1 and c951T>C), a founder effect was suggested for the 419delG mutation. On the other hand, a common haplotype was identified in three of the six haplotypes carrying the R138Q mutations (Table 3).

Clinical Features and Recurrence in Carriers of the Podocin Mutations

The clinical features of each patient carrying mutations of podocin are reported in Table 1, including the therapy of nephrotic syndrome at onset and the clinical outcome relative to renal function. Of the nine patients, two had normal renal function and massive proteinuria at the ages of 3 and 10 yr. Seven had developed end-stage renal failure and had received a renal allograft. Major clinical characteristics were comparable in FSGS patients with podocin mutations and in those without, including pathologic features and outcome. Accordingly, children with podocin mutations developed nephrotic syndrome at 3.83 ± 4.43 yr, and those with idiopathic FSGS presented proteinuria at 5.25 ± 2.92 yr. In all cases, an oral steroid regimen of 2 mg/kg and in pulse (10 mg/kg), in combination with cyclophosphamide (2 mg/kg for 2 mo) failed to induce remission. Two patients (patients 5 and 9) who still had their native kidneys and normal renal function at the ages of 10 and 4 yr, respectively, had been treated with CsA at an initial dose of 4 to 5 mg/kg and successive reduction to 2 to 3 mg/kg. The response to the drug differed; in patient 5, the treatment failed to induce any change, whereas in patient 9, the only subject with the L169P mutation, we observed a stable reduction of proteinuria below the nephrotic range. In the remaining population of podocin mutation carriers, the mean age at which end-stage renal failure developed was 9.57 ± 4.69 yr, which was comparable to the 10.2 ± 5 yr in those with idiopathic FSGS. After renal transplantation, two patients (patients 6 and 7) presented a mild recurrence of proteinuria (2 to 3 g/d) after at least 10 d of normal urinalysis and normal renal function. In one case (patient 6), proteinuria occurred after 10 d, and in the other (patient 7) after 300 d. The therapy followed established protocols based on plasmapheresis and cyclophosphamide, which induced in both cases prompt remission of proteinuria. Renal function and urinalysis after 6 and 8 mo after the short course of plasmapheresis were normal.