Detection of new mutations and molecular pathology of mild and moderate haemophilia A patients from southern Brazil

Abstract

A total of 76 unrelated male patients with mild (n = 55) or moderate (n = 21) haemophilia A living in the southern Brazilian state of Rio Grande do Sul were studied by direct sequencing of all F8 26 exons, the 5' UTR and 3' UTR, intron-exon junctions and the promoter region. When no mutation was found, a multiplex ligation-dependent probe amplification analysis was performed. We identified the disease-causing mutations in 69 patients, who showed 33 different mutations: 27 missense, one small deletion, two small duplications and three splice site mutations. Seven missense and two splice site mutations were not previously reported in HAMSTeRS and were not identified in any current literature search. Nine recurrent mutations were found, one of them never described before (p.Tyr1786Phe). Haplotype analysis indicated that this mutation had originated in the Brazilian population as a single event in a common ancestor. The possible influence of these mutations in the determination of the disease was carefully considered, including bioinformatic tools. These data add to the general knowledge of the disease and can also be useful for HA diagnosis and detection of carriers in the southern Brazilian population.

A germline variant affects putative miRNA‐binding sites at the F8 3′UTR and acts as a potential haemophilia A phenotype modifier in Southern Brazilian patients

“…Recently, an alternative that has been explored involves the regulation by microRNAs (miRNAs), that negatively regulate gene expression by binding to the 3 0 UTRs of target mRNAs [3]. The aim of this study was to investigate, for the first time, a SNP previously found in the 3 0 UTR of F8 in HA patients (c.8728 A>G rs1050705, position 1672 of the mRNA) [4] and evaluate whether this SNP may alter FVIII expression.…”

“…We identified the diseasecausing mutations in 69 (91%) of the 76 screened patients, which showed 33 different mutations: 27 (82%) missense, one (3%) small deletion, two (6%) small duplications and three (9%) splice site mutations. To verify the disease-causing nature of the alterations found, we examined 100 normal chromosomes from blood bank donors of the same ethnic groups [4]. For the current in silico analysis, prediction tools were used to investigate whether the SNP altered any miRNA-binding sites (miRBS) in the F8 3 0 UTR.…”

“…Recurrent mutations, which probably should have a common origin, would be an especially favourable test for our results. Sixteen mild cases with a p.Ile1679Thr recurrent mutation [4] were included in this analysis. We selected miRNA-binding sites having DDG values below À10 and sites that had been previously predicted by miRanda and RNAhybrid (miR-26a-5p, miR-26b-5p and miR-1297) as having DDG values above À10.…”

“…They all have the G allele and their mean FVIII:C level was 11.9 U dL À1 (6-17 U dL À1 ). Seven patients with another recurrent mutation (p.Arg1749Cys) [4] were also included in the analysis and they all have the A allele. The mean FVIII:C level for these patients was 31.8 U dL À1 (21-40 U dL À1 ).…”

Factor VIII mutations and inhibitor formation in a southern Brazilian population

“…They had their F8 gene and inhibitor status investigated [3,4], but no formal statistical analysis between the two sets of characteristics had been made. The present study describes such an analysis.…”