Whole F9 PCR amplification and detection of large deletions in hemizygous Probands

RADIC CP; MARCHIONE VD; ROSSETTI CL

World Federation for Hemophilia Genetics Laboratory Manual.

World Federation of Hemophilia

Año: 2022;

The analysis of all relevant sequences of the F9 (including promoter region, all exonic-coding sequences (8 exons) and intronic sequences associated splicing sites) represented in 12 PCR products, should be done over severe, mild and moderate Haemophilia B (HB) cases. Large deletions can be primary detected by the consistent absence of one or more contiguous PCR amplification products. In cases with large deletion involving all F9 products, multiplex PCR amplification is perform with PCR products of F9 and an uninvolved autosomal region. The small variants associated to hemophilia can be determined by direct sequencing of the 12 PCR products or the selected PCR products if a screening is previously performed. The primers used are designed with molecular sizes (260-583 bp) and characteristics that follow the recommendations of the screening method CSGE (Conformation Sensitive Gel Electrophoresis) (Ganguly, 2002) (See chapter 5, CSGE screening for F8 & F9).Early genotypic characterization constitutes a solid and advantageous tool for a treatment design adjusted to each patient, as well as being the only reliable tool to offer genetic counseling to females at risk of the family and invasive, non-invasive and/or preimplantation prenatal genetic diagnosis.