Abstract
Background
Steroid-resistant nephrotic syndrome (SRNS) is a severe cause of progressive renal disease. Genetic forms of SRNS can present with autosomal recessive or autosomal dominant inheritance. Recent studies have identified mutations in multiple podocyte genes responsible for SRNS. Improved sequencing methods (next-generation sequencing, NGS) now promise rapid mutational testing of SRNS genes.
Methods
In the present study, a simultaneous screening of ten SRNS genes in 37 SRNS patients was performed by NGS.
Results
In 38 % of the patients, causative mutations in one SRNS gene were found. In 22 % of the patients, in addition to these mutations, a secondary variant in a different gene was identified.
Conclusions
This high incidence of accumulating sequence variants was unexpected but, although they might have modifier effects, the pathogenic potential of these additional sequence variants seems unclear so far. The example of molecular diagnostics by NGS in SRNS patients shows that these new sequencing technologies might provide further insight into molecular pathogenicity in genetic disorders but will also generate results, which will be difficult to interpret and complicate genetic counseling. Although NGS promises more frequent identification of disease-causing mutations, the identification of causative mutations, the interpretation of incidental findings and possible pitfalls might pose problems, which hopefully will decrease by further experience and elucidation of molecular interactions.
Similar content being viewed by others
References
Vogl I, Eck SH, Benet-Pagès A, Greif PA, Hirv K, Kotschote S, Kuhn M, Gehring A, Bergmann C, Bolz HJ, Stuhrmann M, Biskup S, Metzeler KH, Klein H-G (2012) Diagnostic applications of next-generation sequencing: working towards quality standards/Diagnostische Anwendung von Next-Generation Sequencing: Auf dem Weg zu Qualitätsstandards. Lab Med 36:227–239
A report of the International Study of Kidney Disease in Children (ISKDC) (1978) Nephrotic syndrome in children: prediction of histopathology from clinical and laboratory characteristics at time of diagnosis. A report of ISKDC. Kidney Int 13:159–165
McKinney PA, Feltbower RG, Brocklebank JT, Fitzpatrick MM (2001) Time trends and ethnic patterns of childhood nephrotic syndrome in Yorkshire, UK. Pediatr Nephrol 16:1040–1044
Santin S, Bullich G, Tazon-Vega B, Garcia-Maset R, Gimenez I, Silva I, Ruiz P, Ballarin J, Torra R, Ars E (2011) Clinical utility of genetic testing in children and adults with steroid-resistant nephrotic syndrome. Clin J Am Soc Nephrol 6:1139–1148
Buscher AK, Kranz B, Buscher R, Hildebrandt F, Dworniczak B, Pennekamp P, Kuwertz-Broking E, Wingen AM, John U, Kemper M, Monnens L, Hoyer PF, Weber S, Konrad M (2010) Immunosuppression and renal outcome in congenital and pediatric steroid-resistant nephrotic syndrome. Clin J Am Soc Nephrol 5:2075–2084
Kestila M, Lenkkeri U, Mannikko M, Lamerdin J, McCready P, Putaala H, Ruotsalainen V, Morita T, Nissinen M, Herva R, Kashtan CE, Peltonen L, Holmberg C, Olsen A, Tryggvason K (1998) Positionally cloned gene for a novel glomerular protein nephrin is mutated in congenital nephrotic syndrome. Mol Cell 1:575–582
Mele C, Iatropoulos P, Donadelli R, Calabria A, Maranta R, Cassis P, Buelli S, Tomasoni S, Piras R, Krendel M, Bettoni S, Morigi M, Delledonne M, Pecoraro C, Abbate I, Capobianchi MR, Hildebrandt F, Otto E, Schaefer F, Macciardi F, Ozaltin F, Emre S, Ibsirlioglu T, Benigni A, Remuzzi G, Noris M (2011) MYO1E mutations and childhood familial focal segmental glomerulosclerosis. N Engl J Med 365:295–306
Hinkes BG, Mucha B, Vlangos CN, Gbadegesin R, Liu J, Hasselbacher K, Hangan D, Ozaltin F, Zenker M, Hildebrandt F (2007) Nephrotic syndrome in the first year of life: two-thirds of cases are caused by mutations in 4 genes (NPHS1, NPHS2, WT1, and LAMB2). Pediatrics 119:e907–e919
Hinkes B, Wiggins RC, Gbadegesin R, Vlangos CN, Seelow D, Nurnberg G, Garg P, Verma R, Chaib H, Hoskins BE, Ashraf S, Becker C, Hennies HC, Goyal M, Wharram BL, Schachter AD, Mudumana S, Drummond I, Kerjaschki D, Waldherr R, Dietrich A, Ozaltin F, Bakkaloglu A, Cleper R, Basel-Vanagaite L, Pohl M, Griebel M, Tsygin AN, Soylu A, Muller D, Sorli CS, Bunney TD, Katan M, Liu J, Attanasio M, O’Toole JF, Hasselbacher K, Mucha B, Otto EA, Airik R, Kispert A, Kelley GG, Smrcka AV, Gudermann T, Holzman LB, Nurnberg P, Hildebrandt F (2006) Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible. Nat Genet 38:1397–1405
Kim JM, Wu H, Green G, Winkler CA, Kopp JB, Miner JH, Unanue ER, Shaw AS (2003) CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility. Science 300:1298–1300
Reiser J, Polu KR, Moller CC, Kenlan P, Altintas MM, Wei C, Faul C, Herbert S, Villegas I, Avila-Casado C, McGee M, Sugimoto H, Brown D, Kalluri R, Mundel P, Smith PL, Clapham DE, Pollak MR (2005) TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function. Nat Genet 37:739–744
Weins A, Kenlan P, Herbert S, Le TC, Villegas I, Kaplan BS, Appel GB, Pollak MR (2005) Mutational and biological analysis of alpha-actinin-4 in focal segmental glomerulosclerosis. J Am Soc Nephrol 16:3694–3701
Brown EJ, Schlondorff JS, Becker DJ, Tsukaguchi H, Tonna SJ, Uscinski AL, Higgs HN, Henderson JM, Pollak MR (2010) Mutations in the formin gene INF2 cause focal segmental glomerulosclerosis. Nat Genet 42:72–76
Ozaltin F, Ibsirlioglu T, Taskiran EZ, Baydar DE, Kaymaz F, Buyukcelik M, Kilic BD, Balat A, Iatropoulos P, Asan E, Akarsu NA, Schaefer F, Yilmaz E, Bakkaloglu A (2011) Disruption of PTPRO causes childhood-onset nephrotic syndrome. Am J Hum Genet 89:139–147
Gupta IR, Baldwin C, Auguste D, Ha KC, El Andalousi J, Fahiminiya S, Bitzan M, Bernard C, Akbari MR, Narod SA, Rosenblatt DS, Majewski J, Takano T (2013) ARHGDIA: a novel gene implicated in nephrotic syndrome. J Med Genet 50:330–338
Akilesh S, Suleiman H, Yu H, Stander MC, Lavin P, Gbadegesin R, Antignac C, Pollak M, Kopp JB, Winn MP, Shaw AS (2011) Arhgap24 inactivates Rac1 in mouse podocytes, and a mutant form is associated with familial focal segmental glomerulosclerosis. J Clin Invest 121:4127–4137
Ashraf S, Gee HY, Woerner S, Xie LX, Vega-Warner V, Lovric S, Fang H, Song X, Cattran DC, Avila-Casado C, Paterson AD, Nitschke P, Bole-Feysot C, Cochat P, Esteve-Rudd J, Haberberger B, Allen SJ, Zhou W, Airik R, Otto EA, Barua M, Al-Hamed MH, Kari JA, Evans J, Bierzynska A, Saleem MA, Bockenhauer D, Kleta R, El Desoky S, Hacihamdioglu DO, Gok F, Washburn J, Wiggins RC, Choi M, Lifton RP, Levy S, Han Z, Salviati L, Prokisch H, Williams DS, Pollak M, Clarke CF, Pei Y, Antignac C, Hildebrandt F (2013) ADCK4 mutations promote steroid-resistant nephrotic syndrome through CoQ10 biosynthesis disruption. J Clin Invest 123:5179–5189
Heeringa SF, Chernin G, Chaki M, Zhou W, Sloan AJ, Ji Z, Xie LX, Salviati L, Hurd TW, Vega-Warner V, Killen PD, Raphael Y, Ashraf S, Ovunc B, Schoeb DS, McLaughlin HM, Airik R, Vlangos CN, Gbadegesin R, Hinkes B, Saisawat P, Trevisson E, Doimo M, Casarin A, Pertegato V, Giorgi G, Prokisch H, Rotig A, Nurnberg G, Becker C, Wang S, Ozaltin F, Topaloglu R, Bakkaloglu A, Bakkaloglu SA, Muller D, Beissert A, Mir S, Berdeli A, Varpizen S, Zenker M, Matejas V, Santos-Ocana C, Navas P, Kusakabe T, Kispert A, Akman S, Soliman NA, Krick S, Mundel P, Reiser J, Nurnberg P, Clarke CF, Wiggins RC, Faul C, Hildebrandt F (2011) COQ6 mutations in human patients produce nephrotic syndrome with sensorineural deafness. J Clin Invest 121:2013–2024
Srichai MB, Konieczkowski M, Padiyar A, Konieczkowski DJ, Mukherjee A, Hayden PS, Kamat S, El-Meanawy MA, Khan S, Mundel P, Lee SB, Bruggeman LA, Schelling JR, Sedor JR (2004) A WT1 co-regulator controls podocyte phenotype by shuttling between adhesion structures and nucleus. J Biol Chem 279:14398–14408
Barbaux S, Niaudet P, Gubler MC, Grunfeld JP, Jaubert F, Kuttenn F, Fekete CN, Souleyreau-Therville N, Thibaud E, Fellous M, McElreavey K (1997) Donor splice-site mutations in WT1 are responsible for Frasier syndrome. Nat Genet 17:467–470
Hasselbacher K, Wiggins RC, Matejas V, Hinkes BG, Mucha B, Hoskins BE, Ozaltin F, Nurnberg G, Becker C, Hangan D, Pohl M, Kuwertz-Broking E, Griebel M, Schumacher V, Royer-Pokora B, Bakkaloglu A, Nurnberg P, Zenker M, Hildebrandt F (2006) Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders. Kidney Int 70:1008–1012
McCarthy HJ, Bierzynska A, Wherlock M, Ognjanovic M, Kerecuk L, Hegde S, Feather S, Gilbert RD, Krischock L, Jones C, Sinha MD, Webb NJ, Christian M, Williams MM, Marks S, Koziell A, Welsh GI, Saleem MA, Group RtUSS (2013) Simultaneous sequencing of 24 genes associated with steroid-resistant nephrotic syndrome. Clin J Am Soc Nephrol 8:637–648
A report of the International Study of Kidney Disease in Children (ISKDC) (1981) The primary nephrotic syndrome in children. Identification of patients with minimal change nephrotic syndrome from initial response to prednisone. A report of ISKDC. J Pediatr 98:561–564
Liu Z, Blattner SM, Tu Y, Tisherman R, Wang JH, Rastaldi MP, Kretzler M, Wu C (2011) Alpha-actinin-4 and CLP36 protein deficiencies contribute to podocyte defects in multiple human glomerulopathies. J Biol Chem 286:30795–30805
Abid A, Khaliq S, Shahid S, Lanewala A, Mubarak M, Hashmi S, Kazi J, Masood T, Hafeez F, Naqvi SA, Rizvi SA, Mehdi SQ (2012) A spectrum of novel NPHS1 and NPHS2 gene mutations in pediatric nephrotic syndrome patients from Pakistan. Gene 502:133–137
Tsukaguchi H, Sudhakar A, Le TC, Nguyen T, Yao J, Schwimmer JA, Schachter AD, Poch E, Abreu PF, Appel GB, Pereira AB, Kalluri R, Pollak MR (2002) NPHS2 mutations in late-onset focal segmental glomerulosclerosis: R229Q is a common disease-associated allele. J Clin Invest 110:1659–1666
Jeanpierre C, Denamur E, Henry I, Cabanis MO, Luce S, Cecille A, Elion J, Peuchmaur M, Loirat C, Niaudet P, Gubler MC, Junien C (1998) Identification of constitutional WT1 mutations, in patients with isolated diffuse mesangial sclerosis, and analysis of genotype/phenotype correlations by use of a computerized mutation database. Am J Hum Genet 62:824–833
Matejas V, Hinkes B, Alkandari F, Al-Gazali L, Annexstad E, Aytac MB, Barrow M, Blahova K, Bockenhauer D, Cheong HI, Maruniak-Chudek I, Cochat P, Dotsch J, Gajjar P, Hennekam RC, Janssen F, Kagan M, Kariminejad A, Kemper MJ, Koenig J, Kogan J, Kroes HY, Kuwertz-Broking E, Lewanda AF, Medeira A, Muscheites J, Niaudet P, Pierson M, Saggar A, Seaver L, Suri M, Tsygin A, Wuhl E, Zurowska A, Uebe S, Hildebrandt F, Antignac C, Zenker M (2010) Mutations in the human laminin beta2 (LAMB2) gene and the associated phenotypic spectrum. Hum Mutat 31:992–1002
Schultheiss M, Ruf RG, Mucha BE, Wiggins R, Fuchshuber A, Lichtenberger A, Hildebrandt F (2004) No evidence for genotype/phenotype correlation in NPHS1 and NPHS2 mutations. Pediatr Nephrol 19:1340–1348
Beltcheva O, Martin P, Lenkkeri U, Tryggvason K (2001) Mutation spectrum in the nephrin gene (NPHS1) in congenital nephrotic syndrome. Hum Mutat 17:368–373
Ruf RG, Lichtenberger A, Karle SM, Haas JP, Anacleto FE, Schultheiss M, Zalewski I, Imm A, Ruf EM, Mucha B, Bagga A, Neuhaus T, Fuchshuber A, Bakkaloglu A, Hildebrandt F, Arbeitsgemeinschaft Fur Padiatrische Nephrologie Study G (2004) Patients with mutations in NPHS2 (podocin) do not respond to standard steroid treatment of nephrotic syndrome. J Am Soc Nephrol 15:722–732
Koziell A, Grech V, Hussain S, Lee G, Lenkkeri U, Tryggvason K, Scambler P (2002) Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration. Hum Mol Genet 11:379–388
Pelletier J, Bruening W, Kashtan CE, Mauer SM, Manivel JC, Striegel JE, Houghton DC, Junien C, Habib R, Fouser L, Fine RN, Haber DASBL, Housman D (1991) Germline mutations in the Wilms’ tumor suppressor gene are associated with abnormal urogenital development in Denys-Drash syndrome. Cell 67:437–447
Lenkkeri U, Mannikko M, McCready P, Lamerdin J, Gribouval O, Niaudet PM, Antignac CK, Kashtan CE, Homberg C, Olsen A, Kestila M, Tryggvason K (1999) Structure of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1) and characterization of mutations. Am J Hum Genet 64:51–61
Aya K, Tanaka H, Seino Y (2000) Novel mutation in the nephrin gene of a Japanese patient with congenital nephrotic syndrome of the Finnish type. Kidney Int 57:401–404
Lovric S, Fang H, Vega-Warner V, Sadowski CE, Gee HY, Halbritter J, Ashraf S, Saisawat P, Soliman NA, Kari JA, Otto EA, Hildebrandt F, Nephrotic Syndrome Study G (2014) Rapid detection of monogenic causes of childhood-onset steroid-resistant nephrotic syndrome. Clin J Am Soc Nephrol 9:1109–1116
Giglio S, Provenzano A, Mazzinghi B, Becherucci F, Giunti L, Sansavini G, Ravaglia F, Roperto RM, Farsetti S, Benetti E, Rotondi M, Murer L, Lazzeri E, Lasagni L, Materassi M, Romagnani P (2015) Heterogeneous genetic alterations in sporadic nephrotic syndrome associate with resistance to immunosuppression. J Am Soc Nephrol 26:230–236
Mir S, Yavascan O, Berdeli A, Sozeri B (2012) TRPC6 gene variants in Turkish children with steroid-resistant nephrotic syndrome. Nephrol Dial Transplant 27:205–209
Boyer O, Benoit G, Gribouval O, Nevo F, Tete MJ, Dantal J, Gilbert-Dussardier B, Touchard G, Karras A, Presne C, Grunfeld JP, Legendre C, Joly D, Rieu P, Mohsin N, Hannedouche T, Moal V, Gubler MC, Broutin I, Mollet G, Antignac C (2011) Mutations in INF2 are a major cause of autosomal dominant focal segmental glomerulosclerosis. J Am Soc Nephrol 22:239–245
Hoefele J, Wolf MT, O’Toole JF, Otto EA, Schultheiss U, Deschenes G, Attanasio M, Utsch B, Antignac C, Hildebrandt F (2007) Evidence of oligogenic inheritance in nephronophthisis. J Am Soc Nephrol 18:2789–2795
Bergmann C, von Bothmer J, Ortiz Bruchle N, Venghaus A, Frank V, Fehrenbach H, Hampel T, Pape L, Buske A, Jonsson J, Sarioglu N, Santos A, Ferreira JC, Becker JU, Cremer R, Hoefele J, Benz MR, Weber LT, Buettner R, Zerres K (2011) Mutations in multiple PKD genes may explain early and severe polycystic kidney disease. J Am Soc Nephrol 22:2047–2056
Weber S, Gribouval O, Esquivel EL, Moriniere V, Tete MJ, Legendre C, Niaudet P, Antignac C (2004) NPHS2 mutation analysis shows genetic heterogeneity of steroid-resistant nephrotic syndrome and low post-transplant recurrence. Kidney Int 66:571–579
Lowik M, Levtchenko E, Westra D, Groenen P, Steenbergen E, Weening J, Lilien M, Monnens L, van den Heuvel L (2008) Bigenic heterozygosity and the development of steroid-resistant focal segmental glomerulosclerosis. Nephrol Dial Transplant 23:3146–3151
Caridi G, Perfumo F, Ghiggeri GM (2005) NPHS2 (Podocin) mutations in nephrotic syndrome. Clinical spectrum and fine mechanisms. Pediatr Res 57:54R–61R
Voelkerding KV, Dames S, Durtschi JD (2010) Next-generation sequencing for clinical diagnostics-principles and application to targeted resequencing for hypertrophic cardiomyopathy: a paper from the 2009 William Beaumont Hospital Symposium on Molecular Pathology. J Mol Diagn 12:539–551
Behjati S, Tarpey PS (2013) What is next-generation sequencing? Arch Dis Child Educ Pract Ed 98:236–238
Acknowledgments
We thank the patients and their families for participation in this study. JH, AKB, MCL, and SW received financial support by the German Society of Pediatric Nephrology.
Conflict of interest
The author(s) declare that they have no competing interests.
Ethical approval
The study was approved by the ethics committee of the University of Duisburg-Essen and is in adherence to the Declaration of Helsinki.
Informed consent
Informed consent was obtained from the patients or the patients’ guardians.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Weber, S., Büscher, A.K., Hagmann, H. et al. Dealing with the incidental finding of secondary variants by the example of SRNS patients undergoing targeted next-generation sequencing. Pediatr Nephrol 31, 73–81 (2016). https://doi.org/10.1007/s00467-015-3167-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00467-015-3167-6