Int22h-related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their detection.
BACKGROUND
Intrachromosomal, homologous recombination of the duplicon int22h-1 with int22h-2 or int22h-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.


OBJECTIVE
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.


METHODS
Twenty patients with int22h-related inversions were examined together with a control and inversion carriers using reverse transcription-polymerase chain reaction (RT-PCR), long-range PCR and sequencing.


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 inversions, deletions, and duplications due to recombination between int22h sequences.
Bagnall
R D
RD
Department of Medical and Molecular Genetics, King's College School of Medicine, London, UK. richard.bagnall@genetics.kcl.ac.uk
Giannelli
F
F
Green
P M
PM
D001483:Base Sequence; D007446:Chromosome Inversion; D041321:Chromosomes, Human, X / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D005169:Factor VIII / Q000235:genetics; D005260:Female; D006467:Hemophilia A / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D005169:Factor VIII