Informativeness of a novel multiallelic marker-set comprising an F8 intron 21 and three tightly linked loci for haemophilia A carriership analysis

FILIPE BRUM MACHADO ; ANTONIO FRANCISCO ALVES DA SILVA; LILIANA CARMEN ROSSETTI; CARLOS DANIEL DE BRASI; ENRIQUE MEDINA-ACOSTA

HAEMOPHILIA

WILEY-BLACKWELL PUBLISHING, INC

Lugar: Londres; Año: 2011 vol. 17 p. 257 - 266

1351-8216

About 40-50% of severe haemophilia A (HEMA) cases are caused by inversions involving F8-intron 22 (Inv22) and F8-intron 1 inversions (Inv1). These can be directly assessed through Southern blotting, long-range PCR, or inverse shifting-PCR and/or sequencing. The extraordinary heterogeneous nature of the remaining half of deleterious mutations makes routine direct mutant profiling difficult, even when sequencing is available. Linkage genetic analysis using F8 intragenic and/or closely linked extragenic short tandem repeats (STR) allows carrier-tracking the defective F8 gene within at-risk families. Although several typing STR loci around and within F8 have been described, there is a need to further improving assessment, because the cumulative informativeness of available assays rarely reaches 100%. We have characterized the newly identified dinucleotide STRs located F8 intron 21 (F8Int21; [1]n) and the extragenic tetranucleotide STRs located on GAB3 intron 1 (GAB3Int1; [TAAA]n) and TMLHE intron 1 (TMLHEInt1.1; [GAAA]n and TMLHEInt1.3; [ATTC]n), and developed them as DNA markers suitable for polymorphism segregation analysis, using multiplex quantitative fluorescence polymerase chain reaction. We determined heterozygosity and allele frequencies in a 101 unrelated females. Heterozigosity rates ranged from 0.26 (GAB3Int1) to 0.58 (TMLHEInt1.1 and TMLHEInt1.3). The assay rendered a cumulative heterozygosity of 0.71 for a minimum of two informative markers, with at least one F8 intragenic. The utility of the multiplex assay was exemplified by haplotyping 20 males screened positive for Inv22-1 and Inv22-2 type mutations and by segregation analysis of haplotypes from a kindred with two severe HEMA-affected full siblings and their non-affected maternal half sibling.