Mutation analysis in nephronophthisis using a combined approach of homozygosity mapping, CEL I endonuclease cleavage, and direct sequencing.
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.
Otto
Edgar A
EA
Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA.
Helou
Juliana
J
Allen
Susan J
SJ
O'Toole
John F
JF
Wise
Eric L
EL
Ashraf
Shazia
S
Attanasio
Massimo
M
Zhou
Weibin
W
Wolf
Matthias T F
MT
Hildebrandt
Friedhelm
F
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
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