title: Rapid detection of monogenic causes of childhood-onset steroid-resistant nephrotic syndrome.
abstract: OBJECTIVE
  In steroid-resistant nephrotic syndrome (SRNS), >21 single-gene causes are known. However, mutation analysis of all known SRNS genes is time and cost intensive. This report describes a new high-throughput method of mutation analysis using a PCR-based microfluidic technology that allows rapid simultaneous mutation analysis of 21 single-gene causes of SRNS in a large number of individuals.
  
  
  METHODS
  This study screened individuals with SRNS; samples were submitted for mutation analysis from international sources between 1996 and 2012. For proof of principle, a pilot cohort of 48 individuals who harbored known mutations in known SRNS genes was evaluated. After improvements to the method, 48 individuals with an unknown cause of SRNS were then examined in a subsequent diagnostic study. The analysis included 16 recessive SRNS genes and 5 dominant SRNS genes. A 10-fold primer multiplexing was applied, allowing PCR-based amplification of 474 amplicons in 21 genes for 48 DNA samples simultaneously. Forty-eight individuals were indexed in a barcode PCR, and high-throughput sequencing was performed. All disease-causing variants were confirmed via Sanger sequencing.
  
  
  RESULTS
  The pilot study identified the genetic cause of disease in 42 of 48 (87.5%) of the affected individuals. The diagnostic study detected the genetic cause of disease in 16 of 48 (33%) of the affected individuals with a previously unknown cause of SRNS. Seven novel disease-causing mutations in PLCE1 (n=5), NPHS1 (n=1), and LAMB2 (n=1) were identified in <3 weeks. Use of this method could reduce costs to 1/29th of the cost of Sanger sequencing.
  
  
  CONCLUSIONS
  This highly parallel approach allows rapid (<3 weeks) mutation analysis of 21 genes known to cause SRNS at a greatly reduced cost (1/29th) compared with traditional mutation analysis techniques. It detects mutations in about 33% of childhood-onset SRNS cases.
authors.affiliation: Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts;
authors.forename: Svjetlana
authors.initials: S
authors.lastname: Lovric
authors.affiliation: Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts;
authors.forename: Humphrey
authors.initials: H
authors.lastname: Fang
authors.affiliation: Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan;
authors.forename: Virginia
authors.initials: V
authors.lastname: Vega-Warner
authors.affiliation: Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts;
authors.forename: Carolin E
authors.initials: CE
authors.lastname: Sadowski
authors.affiliation: Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts;
authors.forename: Heon Yung
authors.initials: HY
authors.lastname: Gee
authors.affiliation: Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts;
authors.forename: Jan
authors.initials: J
authors.lastname: Halbritter
authors.affiliation: Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts;
authors.forename: Shazia
authors.initials: S
authors.lastname: Ashraf
authors.affiliation: Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan;
authors.forename: Pawaree
authors.initials: P
authors.lastname: Saisawat
authors.affiliation: Department of Pediatrics, Center of Pediatric Nephrology & Transplantation, Kasr Al Ainy School of Medicine, Cairo University and Egyptian Group of Orphan Diseases, Cairo, Egypt;
authors.forename: Neveen A
authors.initials: NA
authors.lastname: Soliman
authors.affiliation: Department of Pediatrics, King Abdulaziz University Hospital, Jeddah, Saudi Arabia; and.
authors.forename: Jameela A
authors.initials: JA
authors.lastname: Kari
authors.affiliation: Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan;
authors.forename: Edgar A
authors.initials: EA
authors.lastname: Otto
authors.affiliation: Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts; Howard Hughes Medical Institute, Chevy Chase, Maryland friedhelm.hildebrandt@childrens.harvard.edu.
authors.forename: Friedhelm
authors.initials: F
authors.lastname: Hildebrandt
mesh_terms: D000328:Adult; D017668:Age of Onset; D002675:Child, Preschool; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods*; D004351:Drug Resistance / Q000235:genetics*; D005260:Female; D005820:Genetic Testing / Q000191:economics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007797:Laminin / Q000235:genetics; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D009404:Nephrotic Syndrome / Q000188:drug therapy / Q000453:epidemiology / Q000235:genetics*; D054801:Phosphoinositide Phospholipase C / Q000235:genetics; D013256:Steroids / Q000627:therapeutic use
chemical_list: D007797:Laminin; D008565:Membrane Proteins; D013256:Steroids; C113267:nephrin; C058783:laminin beta2; D054801:Phosphoinositide Phospholipase C; C423320:phospholipase C epsilon
keywords: focal segmental glomerulosclerosis; genetic renal disease; human genetics; molecular genetics; nephrotic syndrome
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