Int22h-related inversions causing hemophilia A: A novel insight into their origin and a new more discriminant PCR test for their detection

Abstract

Background: Intrachromosomal, homologousrecombination of the duplicon int22h-1 with int22h-2 orint22h-3 causes inversions accounting for 45% of severe hemophilia A, hence the belief that int22h-2 and int22h-3 are in opposite orientation to int22h-1. However, inversions involving int22h-2 are five times rarer than those involving its virtually identical copy: int22h-3. Recent sequencing has indicated that int22h-2 and int22h-3 form the internal part of the arms of an imperfect palindrome so that int22h-2, in the centromeric arm, has the same orientation as int22h-1 and, upon recombination with int22h-1, should produce deletions and duplications but not inversions. Aim: This work aims to provide rapid tests for all the mutations that can result from recombinations between the int22h sequences and to investigate whether int22h-2-related inversions causing hemophilia A arise in chromosomes, where the arms of the palindrome have recombined so that int22h-2 and int22h-3 swap places and orientation. Patients/methods: Twenty patients with int22h-related inversions were examined together with a control and inversion carriers using reverse transcription-polymerase chain reaction (RTPCR), long-range PCR and sequencing. Results and conclusions: Analysis of mRNA in patients and a control provided evidence confirming the palindromic arrangement of int22h-2 and int22h-3 and the proposed inversion polymorphism that allows int22h-2 to be in the telomeric arm of the palindrome and in opposite orientation to int22h-1. New long-range PCR reactions were used to develop a single tube test that detects and discriminates inversions involving int22h-2 or int22h-3 and a two-tube test that can distinguish imversion, deletions, and duplication due to recombination between int22h sequences. © 2006 International Society on Thrombosis and Haemostasis.