Mutation analysis in nephronophthisis using a combined approach of homozygosity mapping, CEL I endonuclease cleavage, and direct sequencing Communicated by Peter Byers
Otto, Edgar A.; Helou, Juliana; Allen, Susan J.; O'Toole, John F.; Wise, Eric L.; Ashraf, Shazia; Attanasio, Massimo; Zhou, Weibin; Wolf, Matthias T. F.; Hildebrandt, Friedhelm
2008-03
Citation
Otto, Edgar A.; Helou, Juliana; Allen, Susan J.; O'Toole, John F.; Wise, Eric L.; Ashraf, Shazia; Attanasio, Massimo; Zhou, Weibin; Wolf, Matthias T.F.; Hildebrandt, Friedhelm (2008). "Mutation analysis in nephronophthisis using a combined approach of homozygosity mapping, CEL I endonuclease cleavage, and direct sequencing Communicated by Peter Byers ." Human Mutation 29(3): 418-426. <http://hdl.handle.net/2027.42/58034>
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. Hum Mutat 29(3), 418–426, 2008. © 2007 Wiley-Liss, Inc.Publisher
Wiley Subscription Services, Inc., A Wiley Company
ISSN
1059-7794 1098-1004
Other DOIs
PMID
18076122
Types
Article
URI
http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=18076122&dopt=citationMetadata
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