Mutation analysis in nephronophthisis using a combined approach of homozygosity mapping, CEL I endonuclease cleavage, and direct sequencing

被引:69
作者
Otto, Edgar A. [1 ]
Helou, Juliana [1 ]
Allen, Susan J. [1 ]
O'Toole, John E. [1 ]
Wise, Eric L. [1 ]
Ashraf, Shazia [1 ]
Attanasio, Massimo [1 ]
Zhou, Weibin [1 ]
Wolf, Matthias T. E. [1 ]
Hildebrandt, Friedhelm [1 ]
机构
[1] Univ Michigan, Dept Pediat, Ann Arbor, MI 48109 USA
关键词
nephronophthisis; Senior-Loken syndrome; mutation detection; CEL I endonuclease; NPHP1; NPHP2/INVS; NPHP3; NPHP4; NPHP5/IQCB1; homozygosity mapping;
D O I
10.1002/humu.20669
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
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 (NTHP1-8) 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 NTHP1 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-Loken 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.
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收藏
页码:418 / 426
页数:9
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