Reliable and cost-effective approach for diagnosis of heterozygous F8/F9 large deletions by quantitative real-time PCR.

ABELLEYRO MM; RADIC CP; TETZLAFF T; MARCHIONE V; FUNDIA AF; LARRIPA IB; ROSSETTI LC; DE BRASI CD

HAEMOPHILIA

WILEY-BLACKWELL PUBLISHING, INC

Lugar: Londres; Año: 2015 vol. 21 p. 247 - 251

1351-8216

To improve cost-effectiveness of carrier diagnosis of X-linked large deletions, hemophilia genes (F8 and F9) were used to adjust a qPCR-based strategy to assess carrier probability by use of healthy male and female samples to represent carriers and non-carriers, respectively.Quantitative real-time-PCR amplification protocols on four X-linked targets (F8 promoter,exon 6 and exon 26; and F9 exon 8) and two autosomal references (CTLA4) were adjusted to obtain T/R (target/reference) ratios from 14 females at-risk of being carriers of large deletions and control groups of 15 males (46;XY) and 15females (46,XX). T/R ratio ranges from the male and female control groups did not overlap indicating the reliability of our measuring approach. Normal controls?T/R data were represented by Normal probability curves using their respective means and standard deviations. Case T/R values were interpolated into male (Px) and female (Pxx) probability to calculate carrier probability (Pc) as Px-/(Px-+Pxx). Nine females-at-risk were diagnosed as carriers of gross deletions, (Pc=100%), and five, as non-carriers (Pc ranged 0-1%). Our carrier diagnosis approach was validated on all four X-linked targets by comparing the diagnosis of six females with heterozygous large deletions perfectly matching those obtained by other strategies (e.g., gap-PCR). The presented qPCR-based measuring system using normal controls has proven robust and accurate for carrier diagnosis of hemophilia-causative large deletions.These results prompt us to include this approach into the routine protocol forF8 and F9 mutational analysis and anticipate reliable results also when applied to other X-linked disorders.