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

Edgar A. Otto, Juliana Helou, Susan J. Allen, John F. O'Toole, Eric L. Wise, Shazia Ashraf, Massimo Attanasio, Weibin Zhou, Matthias T F Wolf, Friedhelm Hildebrandt

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)418-426
Number of pages9
JournalHuman Mutation
Volume29
Issue number3
DOIs
StatePublished - Mar 2008

Fingerprint

Deoxyribonuclease I
Mutation
Nonsense Codon
Apium graveolens
Frameshift Mutation
Retinitis Pigmentosa
Multiplex Polymerase Chain Reaction
Kidney Diseases
Complex Mixtures
Microsatellite Repeats
Sepharose
Chronic Kidney Failure
Gels
Costs and Cost Analysis

Keywords

  • CEL I endonuclease
  • Homozygosity mapping
  • Mutation detection
  • Nephronophthisis
  • NPHP1
  • NPHP2/INVS
  • NPHP3
  • NPHP4
  • NPHP5/IQCB1
  • Senior-Løken syndrome

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

Cite this

Otto, E. A., Helou, J., Allen, S. J., O'Toole, J. F., Wise, E. L., Ashraf, S., ... Hildebrandt, F. (2008). Mutation analysis in nephronophthisis using a combined approach of homozygosity mapping, CEL I endonuclease cleavage, and direct sequencing. Human Mutation, 29(3), 418-426. https://doi.org/10.1002/humu.20669

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

In: Human Mutation, Vol. 29, No. 3, 03.2008, p. 418-426.

Research output: Contribution to journalArticle

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. / Mutation analysis in nephronophthisis using a combined approach of homozygosity mapping, CEL I endonuclease cleavage, and direct sequencing. In: Human Mutation. 2008 ; Vol. 29, No. 3. pp. 418-426.
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AU - Wise, Eric L.

AU - Ashraf, Shazia

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