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

Abstract

The extraordinary heterogeneous nature of the deleterious mutations in the F8 gene that lead to functional deficiency of clotting factor VIII in haemophilia A makes routine direct mutation profiling difficult. When direct mutation analysis cannot be performed or a causative/candidate mutation is not found, a second-line approach to track the defective F8 gene within at-risk families is linkage genetic analysis with, tried-and-tested, F8-intragenic and/or extragenic non-recombining multiallelic short tandem repeats (STR). Although several typing STR loci within and around F8 have been described, there is need for improving assessment, because the combined informativeness of available assays rarely reaches 100%. Here, we characterized a newly identified 0.28 cM-resolution marker-set, consisting of a dinucleotide STR located on F8 intron 21 (F8Int21; [AC](n)) and three extragenic tetranucleotide STR located on GAB3 intron 1 (GAB3Int1; [TAAA](n)) and TMLHE intron 1 (TMLHEInt1.1; [GAAA](n) and TMLHEInt1.3; [ATTC](n)). Heterozygosity rates determined in 100 unrelated females ranged from 0.25 (GAB3Int1) to 0.63 (F8Int21). The set rendered a combined informativeness of 0.91 for at least one marker and 0.60 for a minimum of two loci, with at least one F8-intragenic. Multiallelic interlocus non-random association analysis revealed that GAB3Int1 is not in significant gametic disequilibrium (GD) with F8Int21, F8Int9.2, TMLHEInt1.3 or TMLHEInt1.1. Gametic disequilibrium breakdown attests historical recombination between GAB3Int1 and the F8 gene. Through computational analysis of reference assembly sequence data, we note in the GD breakdown region and in the F8 gene a higher than average density of the 13-mer CCNCCNTNNCCNC consensus motif, commonly associated with recombination hotspots.

Single‐tube tetradecaplex panel of highly polymorphic microsatellite markers < 1 Mb from F8 for simplified preimplantation genetic diagnosis of hemophilia A

“…The panel also includes the AMELX/Y dimorphism for gender determination. Of the 13 STR markers, 11 have been studied previously as smaller panels in several populations [9,11,13,14,[16][17][18][19]27]. The remaining two markers, TMLHEInt2 and HEMA154498.9, are being investigated for the first time and they are highly polymorphic.…”

“…Microsatellite markers, also referred to as short tandem repeats (STRs), have been widely used in linkage analysis as they are highly polymorphic and widespread within the human genome. A list of STRs located in and around the F8 gene has been reported previously [10], some of which have been multiplexed in small panels [9,[11][12][13][14][15][16][17][18][19][20][21][22]. However, a majority of these STRs have not been tested experimentally and their heterozygosity and polymorphism indices are unknown.…”

5meCpG Epigenetic Marks Neighboring a Primate-Conserved Core Promoter Short Tandem Repeat Indicate X-Chromosome Inactivation

“…Human genomic DNA was extracted from either peripheral blood or mouth epithelial cells (swabs) utilizing a commercial Illustra blood genomic Prep Mini Spin kit (GE Healthcare, Little Chalfont, UK) [21]. Genomic DNA from blood samples from female carriers of the F8 defect, the Dutch population subset and the marmoset necropsy tissues (blood, muscle, liver, brain and skin) was extracted via phenol-chloroform and ethanol precipitation [22].…”

Somatic/Germinal Mosaicism of a F8 Promoter Deletion Confounds Clinical Predictions in a Family with Haemophilia A: Key Role of Genotype Quantitation

“…Se realizaron PCR simples o en multiplex, usando 10-50 ng de ADNg (o 100-150 ng en multiplex), 0,5 U de Taq ADN Polimerasa (Promega, USA), 0,5 μM de cada primer (Tabla M&M.4), 200 μM de dNTPs, 1,5 mM de MgCl2 y buffer Taq polimerasa provisto por el fabricante, en un volumen final de 20 μl. (Edelmann et al, 2002); * (Fimiani et al, 2006); # (Machado-Medina-Acosta, 2009); δ (Liang et al, 2009); ‡ (Machado et al, 2011).…”