Molecular diagnosis of haemophilia A at Centro Hospitalar de Coimbra in Portugal: study of 103 families – 15 new mutations

Abstract

Haemophilia A (HA), the most commonly inherited bleeding disorder, has well known phenotype heterogeneity, influenced by the type of mutation, modulating factors and development of inhibitors. Nowadays, new technologies in association with bioinformatics tools allow a better genotype/phenotype correlation. With the main objective of identifying familial carrier women and to offer prenatal diagnosis, 141 HA patients belonging to 103 families, followed or referred to the Haemophilia Centre of CHC, E.P.E., were studied. Molecular diagnosis strategy was based on HA severity: IVS22 and IVS1 inversions, direct sequencing and MLPA technique. New missense and splicing mutations were further analyzed using molecular modelling. Genotype/phenotype correlation was assessed taking into account the known modulating factors. During this study, mutations were detected in 102/103 families, carrier status was determined in 83 women and 14 prenatal diagnoses were performed. In a total of 46 different mutations identified, 15 have not been reported previously by the HAMSTeRS and HGMD. Genotype/phenotype correlation revealed two cases with a clinical picture less severe than expected by the type of mutation identified. Six patients developed inhibitors: five severe (IVS22, IVS1, large deletion) and one mild (p. Gln2265Lys). The adopted strategy allowed the identification of 99% of the molecular alterations underlying the HA phenotype (98% detection rate for severe and 100% for moderate and mild). Evaluation of genotype-phenotype correlation was complemented with structural protein modelling of newly identified missense mutations, contributing to better understanding of the disease-causing mechanisms and to deepening knowledge on protein structure-function.

Multiplex ligation‐dependent probe amplification as first mutation screening for large deletions and duplications in haemophilia

“…Owing to the low frequency of CNV, in most cases, the analysis should go ahead with the screening, so the additional cost for MLPA might seem inappropriate. The use of the MLPA as first molecular diagnostic has not been suggested by other laboratories . Anyway Rost et al suggest “applying MLPA prior to any screening method for point mutations in the F8 gene” as “MLPA is a reliable, easy‐to‐use and fast method” and the whole F8 or F9 gene can be analyzed in one assay and in our opinion these advantages are rewarding.…”

Gene and Stem Cell Therapies for Fetal Care

“…For individuals with a family history of hemophilia A, which represents about 75% of hemophilia cases, prenatal diagnosis for hemophilia A is available, encouraged, and cost-effective, even in low-resource countries . Hemophilia A is now detectable in utero by performing digital polymerase chain reaction on the small number of fetal cells present within the mother’s peripheral blood .…”

In utero stem cell transplantation and gene therapy: rationale, history, and recent advances toward clinical application

“…For individuals with a family history of HA (~75% of HA cases), prenatal diagnosis for HA is feasible, available, and is both encouraged and cost-effective, even when considering developing third world countries. 235–246 Moreover, another recent study has shown it is now possible to diagnose HA in utero by performing digital PCR on the small number of fetal cells present within the mother’s peripheral blood, making it possible to diagnose HA prenatally with essentially zero risk to the fetus or mother. 245 Despite the availability of prenatal screening, still, ~1 in 5,000 boys born each year worldwide are affected with HA.…”