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Targeted next-generation sequencing in steroid-resistant nephrotic syndrome: mutations in multiple glomerular genes may influence disease severity.

Bullich G, Trujillano D, Santín S, Ossowski S, Mendizábal S, Fraga G, Madrid Á, Ariceta G, Ballarín J, Torra R, Estivill X, Ars E - Eur. J. Hum. Genet. (2014)

Bottom Line: In the validation cohort, we identified the 42 previously known pathogenic mutations across NPHS1, NPHS2, WT1, TRPC6, and INF2 genes.Two of them were familial cases and presented a more severe phenotype than family members with mutation in only one gene.In conclusion, our results show that massive parallel sequencing is feasible and robust for genetic diagnosis of SRNS/FSGS.

View Article: PubMed Central - PubMed

Affiliation: 1] Molecular Biology Laboratory, Fundació Puigvert, Instituto de Investigaciones Biomédicas Sant Pau (IIB-Sant Pau), Universitat Autònoma de Barcelona, REDinREN, Instituto de Investigación Carlos III, Barcelona, Catalonia, Spain [2] Nephrology Department, Fundació Puigvert, Instituto de Investigaciones Biomédicas Sant Pau (IIB-Sant Pau), Universitat Autònoma de Barcelona, REDinREN, Instituto de Investigación Carlos III, Barcelona, Catalonia, Spain.

ABSTRACT
Genetic diagnosis of steroid-resistant nephrotic syndrome (SRNS) using Sanger sequencing is complicated by the high genetic heterogeneity and phenotypic variability of this disease. We aimed to improve the genetic diagnosis of SRNS by simultaneously sequencing 26 glomerular genes using massive parallel sequencing and to study whether mutations in multiple genes increase disease severity. High-throughput mutation analysis was performed in 50 SRNS and/or focal segmental glomerulosclerosis (FSGS) patients, a validation cohort of 25 patients with known pathogenic mutations, and a discovery cohort of 25 uncharacterized patients with probable genetic etiology. In the validation cohort, we identified the 42 previously known pathogenic mutations across NPHS1, NPHS2, WT1, TRPC6, and INF2 genes. In the discovery cohort, disease-causing mutations in SRNS/FSGS genes were found in nine patients. We detected three patients with mutations in an SRNS/FSGS gene and COL4A3. Two of them were familial cases and presented a more severe phenotype than family members with mutation in only one gene. In conclusion, our results show that massive parallel sequencing is feasible and robust for genetic diagnosis of SRNS/FSGS. Our results indicate that patients carrying mutations in an SRNS/FSGS gene and also in COL4A3 gene have increased disease severity.

No MeSH data available.


Related in: MedlinePlus

Pedigrees of two families with mutations in an SRNS/FSGS gene and COL4A3. (a) In family 10, both siblings had compound heterozygous pathogenic mutations in NPHS2 gene and the more severely affected individual (10-1) carried an additional likely pathogenic variant in COL4A3 gene. (b) In family 253, individuals 253-1 to -4 carried a pathogenic mutation in COL4A3 gene demonstrated to produce exon 46 skipping by reverse transcriptase-PCR and Sanger sequencing and predicted to result in a protein lacking 42 amino acids. Patient 253-1 carried an additional variant in INF2 gene inherited from her mother and developed a more aggressive phenotype than the other affected family members. Cr, creatinine; wt, wild type. The arrows indicate probands. Squares denote males, circles denote females. Filled symbols indicate affected status. Quarter solid symbols indicate microhematuria.
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fig1: Pedigrees of two families with mutations in an SRNS/FSGS gene and COL4A3. (a) In family 10, both siblings had compound heterozygous pathogenic mutations in NPHS2 gene and the more severely affected individual (10-1) carried an additional likely pathogenic variant in COL4A3 gene. (b) In family 253, individuals 253-1 to -4 carried a pathogenic mutation in COL4A3 gene demonstrated to produce exon 46 skipping by reverse transcriptase-PCR and Sanger sequencing and predicted to result in a protein lacking 42 amino acids. Patient 253-1 carried an additional variant in INF2 gene inherited from her mother and developed a more aggressive phenotype than the other affected family members. Cr, creatinine; wt, wild type. The arrows indicate probands. Squares denote males, circles denote females. Filled symbols indicate affected status. Quarter solid symbols indicate microhematuria.

Mentions: We identified three patients carrying mutations in an SRNS/FSGS gene and also in COL4A3 (Table 3). Patient 266 carried two NPHS1 pathogenic mutations, an in-frame deletion and a frameshift, together with a heterozygous missense mutation in COL4A3, previously reported by Heidet et al.36 She had a congenital NS presenting with microhematuria and no family history of NS. Patient 10-1 and his affected sister (10-2) both carried compound heterozygous missense pathogenic mutations in NPHS2 gene, but only the proband 10-1 harbored a heterozygous missense variant in COL4A3 predicted to be likely pathogenic. Both siblings had early childhood onset of SRNS. Patient 10-1 presented with nephrotic range proteinuria and microhematuria. His renal biopsy revealed FSGS, and he developed ESRD at 12 years. His sibling 10-2 presented with borderline nephrotic range proteinuria but no evidence of microhematuria, renal biopsy showed MCD and she presented normal renal function by the age of 18 years (Figure 1a). Patient 253-1 carried a heterozygous splicing mutation in COL4A3, demonstrated to produce exon 46 skipping by RNA analysis and predicted to result in a protein lacking 42 amino acids, in combination with a missense variant in the exon 12 of INF2. This novel non-conservative substitution, p.R689W, is located at a highly conservative domain (FH2) in the INF2 protein and scored as highly likely pathogenic, using mutation prediction programs. The arginine in the position 689 is totally conserved in mammals and a basic amino acid in all the species. She presented with SRNS and microhematuria at 32 years, and her renal biopsy showed mesangioproliferative lesions with FSGS. Her renal function rapidly deteriorated, reaching ESRD at 33 years. The COL4A3 mutation was inherited from her affected father (253-2) who presented with non-nephrotic range proteinuria and hematuria at 39 years. His renal biopsy showed FSGS, and he reached ESRD at 51 years. The INF2 variant was inherited from her asymptomatic mother (253-5). Two of the proband's uncles carried the COL4A3 mutation, but they only presented microhematuria at 61 (253-3) and 56 years (253-4) (Figure 1b).


Targeted next-generation sequencing in steroid-resistant nephrotic syndrome: mutations in multiple glomerular genes may influence disease severity.

Bullich G, Trujillano D, Santín S, Ossowski S, Mendizábal S, Fraga G, Madrid Á, Ariceta G, Ballarín J, Torra R, Estivill X, Ars E - Eur. J. Hum. Genet. (2014)

Pedigrees of two families with mutations in an SRNS/FSGS gene and COL4A3. (a) In family 10, both siblings had compound heterozygous pathogenic mutations in NPHS2 gene and the more severely affected individual (10-1) carried an additional likely pathogenic variant in COL4A3 gene. (b) In family 253, individuals 253-1 to -4 carried a pathogenic mutation in COL4A3 gene demonstrated to produce exon 46 skipping by reverse transcriptase-PCR and Sanger sequencing and predicted to result in a protein lacking 42 amino acids. Patient 253-1 carried an additional variant in INF2 gene inherited from her mother and developed a more aggressive phenotype than the other affected family members. Cr, creatinine; wt, wild type. The arrows indicate probands. Squares denote males, circles denote females. Filled symbols indicate affected status. Quarter solid symbols indicate microhematuria.
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Related In: Results  -  Collection

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fig1: Pedigrees of two families with mutations in an SRNS/FSGS gene and COL4A3. (a) In family 10, both siblings had compound heterozygous pathogenic mutations in NPHS2 gene and the more severely affected individual (10-1) carried an additional likely pathogenic variant in COL4A3 gene. (b) In family 253, individuals 253-1 to -4 carried a pathogenic mutation in COL4A3 gene demonstrated to produce exon 46 skipping by reverse transcriptase-PCR and Sanger sequencing and predicted to result in a protein lacking 42 amino acids. Patient 253-1 carried an additional variant in INF2 gene inherited from her mother and developed a more aggressive phenotype than the other affected family members. Cr, creatinine; wt, wild type. The arrows indicate probands. Squares denote males, circles denote females. Filled symbols indicate affected status. Quarter solid symbols indicate microhematuria.
Mentions: We identified three patients carrying mutations in an SRNS/FSGS gene and also in COL4A3 (Table 3). Patient 266 carried two NPHS1 pathogenic mutations, an in-frame deletion and a frameshift, together with a heterozygous missense mutation in COL4A3, previously reported by Heidet et al.36 She had a congenital NS presenting with microhematuria and no family history of NS. Patient 10-1 and his affected sister (10-2) both carried compound heterozygous missense pathogenic mutations in NPHS2 gene, but only the proband 10-1 harbored a heterozygous missense variant in COL4A3 predicted to be likely pathogenic. Both siblings had early childhood onset of SRNS. Patient 10-1 presented with nephrotic range proteinuria and microhematuria. His renal biopsy revealed FSGS, and he developed ESRD at 12 years. His sibling 10-2 presented with borderline nephrotic range proteinuria but no evidence of microhematuria, renal biopsy showed MCD and she presented normal renal function by the age of 18 years (Figure 1a). Patient 253-1 carried a heterozygous splicing mutation in COL4A3, demonstrated to produce exon 46 skipping by RNA analysis and predicted to result in a protein lacking 42 amino acids, in combination with a missense variant in the exon 12 of INF2. This novel non-conservative substitution, p.R689W, is located at a highly conservative domain (FH2) in the INF2 protein and scored as highly likely pathogenic, using mutation prediction programs. The arginine in the position 689 is totally conserved in mammals and a basic amino acid in all the species. She presented with SRNS and microhematuria at 32 years, and her renal biopsy showed mesangioproliferative lesions with FSGS. Her renal function rapidly deteriorated, reaching ESRD at 33 years. The COL4A3 mutation was inherited from her affected father (253-2) who presented with non-nephrotic range proteinuria and hematuria at 39 years. His renal biopsy showed FSGS, and he reached ESRD at 51 years. The INF2 variant was inherited from her asymptomatic mother (253-5). Two of the proband's uncles carried the COL4A3 mutation, but they only presented microhematuria at 61 (253-3) and 56 years (253-4) (Figure 1b).

Bottom Line: In the validation cohort, we identified the 42 previously known pathogenic mutations across NPHS1, NPHS2, WT1, TRPC6, and INF2 genes.Two of them were familial cases and presented a more severe phenotype than family members with mutation in only one gene.In conclusion, our results show that massive parallel sequencing is feasible and robust for genetic diagnosis of SRNS/FSGS.

View Article: PubMed Central - PubMed

Affiliation: 1] Molecular Biology Laboratory, Fundació Puigvert, Instituto de Investigaciones Biomédicas Sant Pau (IIB-Sant Pau), Universitat Autònoma de Barcelona, REDinREN, Instituto de Investigación Carlos III, Barcelona, Catalonia, Spain [2] Nephrology Department, Fundació Puigvert, Instituto de Investigaciones Biomédicas Sant Pau (IIB-Sant Pau), Universitat Autònoma de Barcelona, REDinREN, Instituto de Investigación Carlos III, Barcelona, Catalonia, Spain.

ABSTRACT
Genetic diagnosis of steroid-resistant nephrotic syndrome (SRNS) using Sanger sequencing is complicated by the high genetic heterogeneity and phenotypic variability of this disease. We aimed to improve the genetic diagnosis of SRNS by simultaneously sequencing 26 glomerular genes using massive parallel sequencing and to study whether mutations in multiple genes increase disease severity. High-throughput mutation analysis was performed in 50 SRNS and/or focal segmental glomerulosclerosis (FSGS) patients, a validation cohort of 25 patients with known pathogenic mutations, and a discovery cohort of 25 uncharacterized patients with probable genetic etiology. In the validation cohort, we identified the 42 previously known pathogenic mutations across NPHS1, NPHS2, WT1, TRPC6, and INF2 genes. In the discovery cohort, disease-causing mutations in SRNS/FSGS genes were found in nine patients. We detected three patients with mutations in an SRNS/FSGS gene and COL4A3. Two of them were familial cases and presented a more severe phenotype than family members with mutation in only one gene. In conclusion, our results show that massive parallel sequencing is feasible and robust for genetic diagnosis of SRNS/FSGS. Our results indicate that patients carrying mutations in an SRNS/FSGS gene and also in COL4A3 gene have increased disease severity.

No MeSH data available.


Related in: MedlinePlus