title: Mutation analysis in nephronophthisis using a combined approach of homozygosity mapping, CEL I endonuclease cleavage, and direct sequencing.
abstract: Nephronophthisis (NPHP), an autosomal recessive kidney disease, is the most frequent genetic cause of chronic renal failure in the first three decades of life. Mutations in eight genes (NPHP1-8) have been identified. We here describe a combined approach for mutation screening of NPHP1, NPHP2, NPHP3, NPHP4, and NPHP5 in a worldwide cohort of 470 unrelated patients with NPHP. First, homozygous NPHP1 deletions were detected in 97 patients (21%) by multiplex PCR. Second, 25 patients with infantile NPHP were screened for mutations in inversin (NPHP2/INVS). We detected a novel compound heterozygous frameshift mutation (p.[Q485fs]+[R687fs]), and a homozygous nonsense mutation (p.R899X). Third, 37 patients presenting with NPHP and retinitis pigmentosa (Senior-Løken syndrome [SLS]) were screened for NPHP5/IQCB1 mutations by direct sequencing. We discovered five different (three novel) homozygous premature termination codon (PTC) mutations (p.F142fsX; p.R461X; p.R489X; p.W444X; and c.488-1G>A). The remaining 366 patients were further investigated for mutations in NPHP1, NPHP3, and NPHP4. We applied a "homozygosity only" strategy and typed three highly polymorphic microsatellite markers at the respective loci. A total of 32, eight, and 14 patients showed homozygosity, and were screened by heteroduplex crude celery extract (CEL I) endonuclease digests. The sensitivity of CEL I was established as 92%, as it detected 73 out of 79 different known mutations simply on agarose gels. A total of 10 novel PTC mutations were found in NPHP1 (p.P186fs, p.R347X, p.V492fs, p.Y509X, and c.1884+1G>A), in NPHP3 (c.3812+2T>C and p.R1259X), and in NPHP4 (p.R59X, p.T1004fs, and p.V1091fs). The combined homozygosity mapping and CEL I endonuclease mutation analysis approach allowed us to identify rare mutations in a large cohort of patients at low cost.
authors: Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA.
authors: Edgar A
authors: EA
authors: Otto
authors: Juliana
authors: J
authors: Helou
authors: Susan J
authors: SJ
authors: Allen
authors: John F
authors: JF
authors: O'Toole
authors: Eric L
authors: EL
authors: Wise
authors: Shazia
authors: S
authors: Ashraf
authors: Massimo
authors: M
authors: Attanasio
authors: Weibin
authors: W
authors: Zhou
authors: Matthias T F
authors: MT
authors: Wolf
authors: Friedhelm
authors: F
authors: Hildebrandt
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000951:Antigens, Neoplasm; D001483:Base Sequence; D002148:Calmodulin-Binding Proteins / Q000235:genetics; D018797:Cell Cycle Proteins; D002874:Chromosome Mapping; D003598:Cytoskeletal Proteins; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics; D004720:Endonucleases; D005260:Female; D017353:Gene Deletion; D005808:Genes, Recessive; D006720:Homozygote; D006801:Humans; D007674:Kidney Diseases / Q000150:complications / Q000235:genetics*; D008297:Male; D008565:Membrane Proteins; D018895:Microsatellite Repeats; D009154:Mutation; D009363:Neoplasm Proteins; D017354:Point Mutation; D011506:Proteins / Q000235:genetics*; D012174:Retinitis Pigmentosa / Q000150:complications / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000951:Antigens, Neoplasm; D002148:Calmodulin-Binding Proteins; D018797:Cell Cycle Proteins; C493903:Cep290 protein, human; D003598:Cytoskeletal Proteins; D017931:DNA Primers; C114503:INVS protein, human; C498171:IQCB1 protein, human; D008565:Membrane Proteins; C108725:NPHP1 protein, human; C491578:NPHP4 protein, human; D009363:Neoplasm Proteins; D011506:Proteins; D014157:Transcription Factors; D004720:Endonucleases; C406922:CEL I nuclease