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

Abstract

Background It is currently not possible to perform single-cell preimplantation genetic diagnosis (PGD) to directly detect the common inversion mutations of the factor VIII (F8) gene responsible for severe hemophilia A (HEMA). As such, PGD for such inversion carriers relies on indirect analysis of linked polymorphic markers. Objectives To simplify linkage-based PGD of HEMA, we aimed to develop a panel of highly polymorphic microsatellite markers located near the F8 gene that could be simultaneously genotyped in a multiplex-PCR reaction. Methods We assessed the polymorphism of various microsatellite markers located ≤ 1 Mb from F8 in 177 female subjects. Highly polymorphic markers were selected for co-amplification with the AMELX/Y indel dimorphism in a single-tube reaction. Results Thirteen microsatellite markers located within 0.6 Mb of F8 were successfully co-amplified with AMELX/Y in a single-tube reaction. Observed heterozygosities of component markers ranged from 0.43 to 0.84, and ∼70-80% of individuals were heterozygous for ≥ 5 markers. The tetradecaplex panel successfully identified fully informative markers in a couple interested in PGD for HEMA because of an intragenic F8 point mutation, with haplotype phasing established through a carrier daughter. In-vitro fertilization (IVF)-PGD involved single-tube co-amplification of fully informative markers with AMELX/Y and the mutation-containing F8 amplicon, followed by microsatellite analysis and amplicon mutation-site minisequencing analysis. Conclusions The single-tube multiplex-PCR format of this highly polymorphic microsatellite marker panel simplifies identification and selection of informative markers for linkage-based PGD of HEMA. Informative markers can also be easily co-amplified with mutation-containing F8 amplicons for combined mutation detection and linkage analysis.

Inverse PCR to perform long-distance haplotyping: main applications to improve preimplantation genetic diagnosis in hemophilia

“…We note that in PGD [18], the F8-Inv22 cannot be diagnosed in single or few cells given its structural complexity neither by Southern blot analysis [10], long-range PCR [19], nor by iPCR [16]. We thus included four F8Int21 [CA]n STR-informative Inv22 carriers without affected relatives (i.e., obligate carriers or sisters of deceased probands) requesting genetic counseling to perform PGD and assessed them using the PI approach (Table 3).…”

Identification of Novel Microsatellite Markers Flanking the SMN1 and SMN2 Duplicated Region and Inclusion Into a Single-Tube Tridecaplex Panel for Haplotype-Based Preimplantation Genetic Testing of Spinal Muscular Atrophy

“…Approximately 0.5 Mb of DNA sequences upstream and downstream of the chromosome 5q13.2 duplicated region were downloaded from the UCSC genome browser (GRCh37/hg19 assembly). The strategy for identification and selection of markers and for primer design have been described elsewhere (Chen et al, 2015; Zhao et al, 2017). Briefly, di-, tri-, tetra-, and penta- nucleotide repeats with >80% sequence match and with alignment scores of >54, 80, 66, and 52, respectively, were selected.…”

Four Decades of Carrier Detection and Prenatal Diagnosis in Hemophilia A: Historical Overview, State of the Art and Future Directions

“…Single cells biopsied from cultured embryos at the stage of 6 to 8 cells are examined for the hemophilic haplotype using multi-allelic STR markers within and in close proximity to the F8 gene. Predetermined informative STR markers are co-amplified with AMELX/Y indel polymorphism for sex determination in a single-tube PCR reaction [32][33][34][35]. At least two informative STR markers, preferably located upstream and downstream of the F8 gene, must be used to avoid the risk of misdiagnosis due to recombination between the marker and the F8 mutation.…”