Genotyping the Hemophilia Inversion Hotspot by Use of Inverse PCR

2005/04/29 by Liliana Carmen Rossetti, Claudia Pamela Radic, Irene Beatriz Larripa +2 · 20 citations
Medicine · Biochemistry, Genetics and Molecular Biology · #Hemophilia Treatment and Research #Virus-based gene therapy research #Blood Coagulation and Thrombosis Mechanisms

paper · pdf · doi:10.1373/clinchem.2004.046490

Abstract

BACKGROUND: Factor VIII intron 22 inversions (Inv22) cause 40%-45% of severe cases of hemophilia A in all human populations. Currently, Inv22 can be analyzed either by Southern blotting or by rapid long-distance-PCR-based approaches. We describe an alternative method using inverse-PCR (I-PCR). METHODS: I-PCR involved 3 steps: (a) BclI restriction; (b) self-ligation of restriction fragments, providing BclI rings; and (c) standard multiplex-PCR analysis. PCR was achieved by use of a set of 3 primers that yielded a 487-bp amplicon for the nonrearranged intragenic allele and a 559-bp amplicon for the Inv22 allele. Specific primer sites were targeted by masking relevant regions for human repeats and low-complexity DNA. Inv22 I-PCR was applied to samples from 16 individuals (8 women and 8 men) representing 24 X chromosomes previously genotyped by Southern blotting. Additionally, we evaluated the sensitivity and the ability to assess eventual Inv22 carrier mosaicisms by experiments using artificial DNA mixtures (Inv22 + no-Inv22 male samples). RESULTS: Results for previously genotyped samples agreed with results of Southern blot analyses. As expected, cell composition of the artificial mosaic was linearly reflected by the relative intensities of Inv22 signals. I-PCR was estimated to detect Inv22-positive cells at concentrations as low as approximately 5%. CONCLUSION: The proposed technique provides a rapid tool for Inv22 genotyping.

Citations

• Basic local alignment search tool

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• Burden of rare genetic disorders in India: twenty-two years' experience of a tertiary centre. [europepmc]
• Inverse Shifting-PCR Modified by Capillary Electrophoresis for Detecting F8 int22h and int1h Inversions in Severe Hemophilia A Patients and Probable Carriers. [europepmc]
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