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

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.

Molecular characterization of five Portuguese patients with pyrimidine 5'-nucleotidase deficient hemolytic anemia showing three new P5'N-I mutations.

Four different gene mutations were identified in five unrelated Portuguese patients with pyrimidine 5'-nucleotidase type I (P5'N-I) deficient chronic hemolytic anemia. Mutations 502G-->C (168Gly-->Arg), 773T-->C (258Ile-->Thr) and the insertion of an Alu element in exon 9, leading to skipping of this exon in the mRNA transcript, are newly described mutations whereas mutation 425T-->C (142Leu-->Pro) has been previously reported.