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Large genomic rearrangements within the PCDH15 gene are a significant cause of USH1F syndrome.

Le Guédard S, Faugère V, Malcolm S, Claustres M, Roux AF - Mol. Vis. (2007)

Bottom Line: Failure to screen for gross genomic rearrangements is likely to significantly lower the mutation detection rate.The intron sizes of PCDH15 are up to 150 kb, and the first three exons of the gene cover 0.42 Mb.The genomic structure of any gene should be taken into consideration when designing a mutation screening strategy.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Génétique Moléculaire du CHU de Montpellier, Institut Universitaire de Recherche Clinique, Montpellier, France.

ABSTRACT

Purpose: Protocadherin-15 (PCDH15) is one of the five genes currently identified as being mutated in Usher 1 syndrome and defines Usher syndrome type 1F (USH1F). When PCDH15 was systematically analyzed for mutations in a cohort of USH1 patients, a number of deletions were found. Here we characterize these deletions as to extent, position, and breakpoints.

Methods: Microsatellite and single nucleotide polymorphism (SNP) analyses, used in a preliminary survey of an Usher cohort of 31 patients, revealed large deletions in three patients. These deletions were further characterized by semiquantitative PCR assays to narrow down the breakpoints.

Results: The analysis of the three large deletions revealed that all six breakpoints are different. The breakpoint junction was identified in one patient and the four other breakpoints were mapped to 4 kb. There were no specific distinguishing features of the isolated breakpoints.

Conclusions: A complete screen of PCDH15 should include a search for large deletions. Failure to screen for gross genomic rearrangements is likely to significantly lower the mutation detection rate. A likely explanation for the high rate of such deletions is the unusual gene structure. PCDH15 gene spans nearly 1 Mb for a corresponding open reading frame (ORF) of 7,021 bp. The intron sizes of PCDH15 are up to 150 kb, and the first three exons of the gene cover 0.42 Mb. The genomic structure of any gene should be taken into consideration when designing a mutation screening strategy.

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Schematic localization of the deletion breakpoints on the PCDH15 gene and their analysis. A: Localization of the six deletion breakpoints on the bacterial artificial chromosome (BAC) clones. B: The breakpoint junction fragment identified in patient U297 is aligned with the wild-type sequences spanning the 5' and 3' breakpoints. The deleted sequences are crossed out.
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f2: Schematic localization of the deletion breakpoints on the PCDH15 gene and their analysis. A: Localization of the six deletion breakpoints on the bacterial artificial chromosome (BAC) clones. B: The breakpoint junction fragment identified in patient U297 is aligned with the wild-type sequences spanning the 5' and 3' breakpoints. The deleted sequences are crossed out.

Mentions: To narrow down the deletion breakpoints, we used PCR walking methods that included laboratory-designed amplicons localized in a first step every 50 kb both upstream and downstream of the identified deletions. The primers were chosen according to the sequence of the bacterial artificial chromosome (BAC) clones (their accession number is given in Figure 1A). Once a breakpoint was localized between two adjacent amplicons, further primers were designed for new amplicons until this initial 50 kb distance was reduced to a maximum 4 kb interval. Each breakpoint interval thus characterized by PCR walking is positioned on the BAC clones (Figure 2A).


Large genomic rearrangements within the PCDH15 gene are a significant cause of USH1F syndrome.

Le Guédard S, Faugère V, Malcolm S, Claustres M, Roux AF - Mol. Vis. (2007)

Schematic localization of the deletion breakpoints on the PCDH15 gene and their analysis. A: Localization of the six deletion breakpoints on the bacterial artificial chromosome (BAC) clones. B: The breakpoint junction fragment identified in patient U297 is aligned with the wild-type sequences spanning the 5' and 3' breakpoints. The deleted sequences are crossed out.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533038&req=5

f2: Schematic localization of the deletion breakpoints on the PCDH15 gene and their analysis. A: Localization of the six deletion breakpoints on the bacterial artificial chromosome (BAC) clones. B: The breakpoint junction fragment identified in patient U297 is aligned with the wild-type sequences spanning the 5' and 3' breakpoints. The deleted sequences are crossed out.
Mentions: To narrow down the deletion breakpoints, we used PCR walking methods that included laboratory-designed amplicons localized in a first step every 50 kb both upstream and downstream of the identified deletions. The primers were chosen according to the sequence of the bacterial artificial chromosome (BAC) clones (their accession number is given in Figure 1A). Once a breakpoint was localized between two adjacent amplicons, further primers were designed for new amplicons until this initial 50 kb distance was reduced to a maximum 4 kb interval. Each breakpoint interval thus characterized by PCR walking is positioned on the BAC clones (Figure 2A).

Bottom Line: Failure to screen for gross genomic rearrangements is likely to significantly lower the mutation detection rate.The intron sizes of PCDH15 are up to 150 kb, and the first three exons of the gene cover 0.42 Mb.The genomic structure of any gene should be taken into consideration when designing a mutation screening strategy.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Génétique Moléculaire du CHU de Montpellier, Institut Universitaire de Recherche Clinique, Montpellier, France.

ABSTRACT

Purpose: Protocadherin-15 (PCDH15) is one of the five genes currently identified as being mutated in Usher 1 syndrome and defines Usher syndrome type 1F (USH1F). When PCDH15 was systematically analyzed for mutations in a cohort of USH1 patients, a number of deletions were found. Here we characterize these deletions as to extent, position, and breakpoints.

Methods: Microsatellite and single nucleotide polymorphism (SNP) analyses, used in a preliminary survey of an Usher cohort of 31 patients, revealed large deletions in three patients. These deletions were further characterized by semiquantitative PCR assays to narrow down the breakpoints.

Results: The analysis of the three large deletions revealed that all six breakpoints are different. The breakpoint junction was identified in one patient and the four other breakpoints were mapped to 4 kb. There were no specific distinguishing features of the isolated breakpoints.

Conclusions: A complete screen of PCDH15 should include a search for large deletions. Failure to screen for gross genomic rearrangements is likely to significantly lower the mutation detection rate. A likely explanation for the high rate of such deletions is the unusual gene structure. PCDH15 gene spans nearly 1 Mb for a corresponding open reading frame (ORF) of 7,021 bp. The intron sizes of PCDH15 are up to 150 kb, and the first three exons of the gene cover 0.42 Mb. The genomic structure of any gene should be taken into consideration when designing a mutation screening strategy.

Show MeSH
Related in: MedlinePlus