Using Southern blotting, molecular diagnosis of Inv22 has been available in Argentina since 1995. Shortly after the second recurrent inversion affecting F8, intron 1 (Inv1), was described, our series was reported along with a review of the literature BYL719 nmr estimating that Inv1 causes <3% of severe-HA in Argentina [1]. Inv22 originates from homologous recombination between a 9.5 kb sequence located within F8 intron 22 (int22h-1) and one of two oppositely oriented extragenic copies of int22h (int22h-2 and int22h-3) located by the Xq-telomere. Similarly, Inv1 originates from homologous recombination

between intra- and extragenic 900 bp homologs. Inv22 and Inv1 are occasionally associated with DNA gain/loss or altered DNA sequence, making their genotyping challenging. Liu et al. developed a rapid analysis of Inv22 based on long-distance PCR (LD-PCR) [2]. Our variant of inverse-PCR (inverse shifting-PCR, IS-PCR) that avoids PCR amplification through the int22h region was devised in 2004. In

this technique, BAY 80-6946 genomic DNA is digested with BclI restriction enzyme, and self-ligated producing BclI-DNA circles that provide the template sequence for conventional PCR analysis [3]. The finished sequence of the human X-chromosome indicated that int22h-2 and int22h-3 are inversely oriented to one another and it became clear that only one of these sequences generates inversions through head-to-head pairing with int22h-1. The other copy may generate deletions (Del22) or duplications (Dup22) but not inversions by recombining with equally oriented int22h-1. To support experimental evidence that Inv22 type I results from recombination between int22h-1 and int22h-3 and type II between int22h-1 and int22h-2,

Bagnall et al. hypothesized a non-deleterious 68 kb inversion mediated by large inverted repeats (50 kb) exchanging int22h-2/int22h-3 locations [4]. To distinguish these genomic variants, selleck chemicals llc including haemophilia-causing Inv22 and Del22 and non-causing Dup22, Bagnall et al. developed a LD-PCR-based approach [5]. Our laboratory modified the previous IS-PCR-based approach, which now enables genotyping of both Inv1 and Inv22 from the same template [6] and is applicable to chorionic villus extracted-DNA for prenatal diagnosis [7]. El-Hattab et al. found that hemizygous Dup22 and Del22 associate with intellectual disability and in utero male lethality respectively [8]. The extreme severity of Del22 in males resulting from loss of several genes suggests that reliable Del22 genotyping should be supported by detecting each of the specific juxtaposed sequences of Del22, and the precise DNA loss associated with the ~0.5 Mb deletion [9]. Non-inversion HA- and HB-causative mutations include large deletions of an exon or more that are detected by a consistent absence of contiguous exon-specific PCR products.