Identification of factor VIII gene mutations in 101 patients with haemophilia A: mutation analysis by inversion screening and multiplex PCR and CSGE and molecular modelling of 10 novel missense substitutions

Abstract

Haemophilia A (HA) is an X-linked bleeding disorder caused by diverse mutations in the human coagulation factor VIII (FVIII) gene. We have analysed DNA from 109 unrelated Indian patients with HA for their FVIII gene defects. Among these patients 89 (82%) had severe (FVIII:C <1%) HA, 11 (10%) had moderate (FVIII:C 1-5%) HA and nine (8%) had mild (FVIII:C 5-30%) HA. These patients were first screened for the common intron 22 and intron 1 inversions. Inversion negative samples were screened for point mutations by a multiplex PCR and conformation sensitive gel electrophoresis strategy. Mutations were identified in 101 of the 109 patients. These included two (2%) intron 1 and 51 (51%) intron 22 inversions, four (4%) gross deletions and 44 (43%) point mutations. Twenty-nine novel causative mutations, including 11 missense, seven frameshift, five nonsense mutations, three splice site defects and three gross deletions were detected. Ten of the novel missense mutations were studied by molecular modelling. Two different (Thr2253Pro and Pro1392fs) mutations were seen in four unrelated families and FVIII gene haplotyping suggested a common founder effect. Seven of these 109 patients had inhibitors. Among them, four had intron 22 inversions, one had a novel gross deletion (delexon 2-9) and one a nonsense mutation (Trp1535Stop). In one of these patients, no mutation could be identified in the FVIII gene. A Thr2253Pro novel mutation and an intron 22 inversion were identified in two female haemophiliacs. The data from this study suggests that the spectrum of gene defects in Indian patients with HA is as heterogeneous as reported in other populations.

Identification of 217 unreported mutations in the F8 gene in a group of 1,410 unselected Italian patients with hemophilia A

“…Our study confirms the well-known correlation between the type of mutation and the severity of HA. The type of mutations found was in agreement with results reported in other settings (Oldenburg and Pavlova 2006;Fernández-López et al 2005;Jayandharan et al 2005;Deszo et al 2006). As would be expected, most severe HA patients carry a molecular defect leading to a null allele (large deletion, inversion, nonsense, and insertion/deletion mutations), whereas missense mutations have been found in the majority of moderate (68%) and mild HA patients (81%).…”

The Italian AICE-Genetics hemophilia A database: results and correlation with clinical phenotype

“…The types of mutations found were in agreement with results reported in other settings (Table 2). [15][16][17][18][19][20][21][22][23][24] As expected, most of patients with severe HA carry a molecular defect predicting a null allele (large deletion, inversion, nonsense, and insertion/deletion mutations), whereas missense mutations have been found in the majority of patients with moderate (68%) and mild HA (80%).…”

Factor VIII genetic mutations and protein alterations in hemophilia A: A review

“…For example, codon with three different amino acid changes are the arginine codon 531in exon 11 , codon 1781 in exon 16 (Faridi et al, 2011) and codon 2150 in exon 23 (Liu et al, 2002;Cutler et al, 2002;Habart et al, 2002;Fernandez-Lopez et al, 2005), which has been reported 61 times (Table 1), since arginine is involved in 25% of all missense mutations found so far and play a crucial role in protein function. Ahmed et al (2005) carried out mutation studies in inversion negative hemophilia A patients by the high performance liquid chromatography (dHPLC) method and found 11 missense mutations, and some other mutations (Ahmed et al, 2005), whereas Jayandharan et al (2005) detected 101 mutations by using multiplex polymerase chain reactions (PCRs) and the Cap analysis gene expression (CAGE) technique, of which 21 were missense mutations (Jayandharan., 2005). There is a report of missense mutation being pre-dominantly found in the A1 and A2 domains (Bogdanova et al, 2005).…”