To cite this article: Rossetti LC, Radic CP, Larripa IB, De Brasi CD. Developing a new generation of tests for genotyping hemophilia-causative rearrangements involving int22h and int1h hotspots in the factor VIII gene. J Thromb Haemost 2008; 6: 830-6.Summary. Background: Inversions of F8-intron 22 (Inv22) and F8-intron 1 (Inv1) are responsible for 45-50% of severe hemophilia A cases. Objective: In order to improve the molecular diagnosis of Inv22 and Inv1, and to enable rapid discrimination of Inv22-type 1 and Inv22-type 2 patterns, int22h-mediated deletions (Del22) and duplications (Dup22), we developed a genotyping system based on a novel inverse shifting-polymerase chain reaction (IS-PCR) approach. Methods: IS-PCR involved BclI restriction, followed by self-ligation to create ÔBclI circlesÕ, and finally PCR analysis. Three PCR analysis tests were developed: (i) Inv22-diagnostic for a pattern-sensitive detection of deleterious mutations (Inv22 and Del22) from non-deleterious variants (Dup22 and normal); (ii) Inv1-diagnostic; and (iii) Inv22-complementary for discrimination between Inv22 and Del22, and between Dup22 and normal. For rapid molecular analysis of F8, the Inv22 and Inv1 diagnostic tests can be performed simultaneously. The optional Inv22-complementary test need only be used for specific purposes. Results and Conclusions: Diagnostic tests were validated using previously studied samples. IS-PCR evaluated carrier mosaicisms and performed robustly over wide ranges of DNA qualities and procedural conditions. IS-PCR improved the molecular diagnosis of hemophilia A. This genotyping strategy may potentially be adapted to virtually all known rearrangements in the human genome.

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Although large deletions from the coagulation factor VIII gene, F8, are responsible for 5% of severe hemophilia A (seHA), few of them have been fully characterised. A detailed description of a large partial deletion of the F8 caused by unequal recombination between homeologous AluSx-derived sequences is presented. The proband, a case of isolated hemophilia A with a high inhibitor titre (5700 BU), showed a consistent absence of PCR-amplification of exons 4 to 10, EX4_EX10del. Two approaches were used to narrow down the deletion breakpoints: a direct physical analysis based on PCR (that additionally permits carrier detection in the family); and, under the hypothesis that the mutation resulted from homologous recombination, sequence alignments of F8 intron 3 and 10. Both approaches indicate an unequal crossing over (CO) between two Alu-related sequences. Both elements involved were derived from the AluSx-subfamily consensus and demonstrate 86% sequence identity (with only single-base mismatches), with three gaps (of 2, 3 and 14-bases) and two main tracts of perfectly homologous sequence (28 and 24-bp). The short stretch of intron 10 embedded into intron 3 sequence, linked to the CO, represents a typical hallmark of homologous recombination (double-strand break repair model). A detailed description of EX4_EX10del mutation is c.[338+3485delins1687+2223_1687+2225; 338+3551_1687+2291 del]. The common involvement of unequal homologous recombination mediated by repetitive elements allowed us to suggest that our experimental design (based on intron sequence alignments) may be successfully applied to rearrangements involved in other X-linked inherited diseases. Like other Alu-rich genes throughout the human genome, Alu-mediated homologous recombination in F8 may be an important cause of hemophilia by promoting large DNA deletions.

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X-chromosome inactivation (XCI) is the epigenetic transcriptional silencing of an X-chromosome during the early stages of embryonic development in female eutherian mammals. XCI assures monoallelic expression in each cell and compensation for dosage-sensitive X-linked genes between females (XX) and males (XY). DNA methylation at the carbon-5 position of the cytosine pyrimidine ring in the context of a CpG dinucleotide sequence (5meCpG) in promoter regions is a key epigenetic marker for transcriptional gene silencing. Using computational analysis, we revealed an extragenic tandem GAAA repeat 230-bp from the landmark CpG island of the human X-linked retinitis pigmentosa 2 RP2 promoter whose 5meCpG status correlates with XCI. We used this RP2 onshore tandem GAAA repeat to develop an allele-specific 5meCpG-based PCR assay that is highly concordant with the human androgen receptor (AR) exonic tandem CAG repeat-based standard HUMARA assay in discriminating active (Xa) from inactive (Xi) X-chromosomes. The RP2 onshore tandem GAAA repeat contains neutral features that are lacking in the AR disease-linked tandem CAG repeat, is highly polymorphic (heterozygosity rates approximately 0.8) and shows minimal variation in the Xa/Xi ratio. The combined informativeness of RP2/AR is approximately 0.97, and this assay excels at determining the 5meCpG status of alleles at the Xp (RP2) and Xq (AR) chromosome arms in a single reaction. These findings are relevant and directly translatable to nonhuman primate models of XCI in which the AR CAG-repeat is monomorphic. We conducted the RP2 onshore tandem GAAA repeat assay in the naturally occurring chimeric New World monkey marmoset (Callitrichidae) and found it to be informative. The RP2 onshore tandem GAAA repeat will facilitate studies on the variable phenotypic expression of dominant and recessive X-linked diseases, epigenetic changes in twins, the physiology of aging hematopoiesis, the pathogenesis of age-related hematopoietic malignancies and the clonality of cancers in human and nonhuman primates.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the dystrophin gene and is characterized by muscle degeneration and death. DMD affects males; females being asymptomatic carriers of mutations. However, some of them manifest symptoms due to a translocation between X chromosome and an autosome or to a heterozygous mutation leading to inactivation of most of their normal X chromosome. Six symptomatic female carriers and two asymptomatic were analyzed by: I) Segregation of STRs-(CA)n and MLPA assays to detect a hemizygous alteration, and II) X chromosome inactivation pattern to uncover the reason for symptoms in these females. The symptomatic females shared mild but progressive muscular weakness and increased serum creatin kinase (CK) levels. Levels of dystrophin protein were below normal or absent in many fibers. Segregation of STRs-(CA)n revealed hemizygous patterns in three patients, which were confirmed by MLPA. In addition, this analysis showed a duplication in another patient. X chromosome inactivation assay revealed a skewed X inactivation pattern in the symptomatic females and a random inactivation pattern in the asymptomatic ones. Our results support the hypothesis that the DMD phenotype in female carriers of a dystrophin mutation has a direct correlation with a skewed X-chromosome inactivation pattern.

The factor VIII gene (F8) intron 22 inversion (Inv22) is a paradigmatic duplicon-mediated rearrangement, found in about one half of patients with severe hemophilia A worldwide. The identification of this prevalent cause of hemophilia was delayed for nine years after the F8 characterization in 1984. The aim of this review is to present the wide diversity of practical approaches that have been developed for genotyping the Inv22 (and related int22h rearrangements) since discovery in 1993. The sequence— Southern blot, long distance-PCR and inverse shifting-PCR—for Inv22 genotyping is an interesting example of scientific ingenuity and evolution in order to resolve challenging molecular diagnostic problems.

Hemophilia A (HA) provides excellent models to analyze genotype–phenotype relationships and mutational mechanisms. NhF8ld's breakpoints were characterized using case‐specific DNA‐tags, direct‐ or inverse‐polymerase chain reaction amplification, and Sanger sequencing. DNA‐break's stimulators (n = 46), interspersed repeats, non‐B‐DNA, and secondary structures were analyzed around breakpoints versus null hypotheses (E‐values) based on computer simulations and base‐frequency probabilities. Nine of 18 (50%) severe‐HA patients with nhF8lds developed inhibitors, 1/8 affecting one exon and 8/10 (80%) affecting multi‐exons. NhF8lds range: 2–165 kb. Five (45%) nhF8lds involve F8‐extragenic regions including three affecting vicinal genes (SMIM9 and BRCC3) but none shows an extra‐phenotype not related to severe‐HA. The contingency analysis of recombinogenic motifs at nhF8ld breakpoints indicated a significant involvement of several DNA‐break stimulator elements. Most nhF8ld's breakpoint junctions showed microhomologies (1–7 bp). Three (27%) nhF8lds show complexities at the breakpoints: an 8‐bp inverted‐insertion, and the remnant two, inverted‐ and direct‐insertions (46–68 bp) supporting replicative models microhomology‐mediated break‐induced replication/Fork Stalling and Template Switching. The remnant eight (73%) nhF8lds may support nonhomologous end joining/microhomology‐mediated end joining models. Our study suggests the involvement of the retroposition machinery (e.g., Jurka‐targets, Alu‐elements, long interspersed nuclear elements, long terminal repeats), microhomologies, and secondary structures at breakpoints playing significant roles in the origin of the upmost severe phenotype in HA.