Congenital fibrinogen deficiency in Pakistan and identification of five novel mutations

Objectives: The aim of the study is to identify the underlying mutations of afibrinogenemia and possibly to correlate bleeding symptoms with the identified genotype. Methods: Subjects with inherited bleeding tendency in the absence of any acquired cause of hemostatic defect were included in the study. Afibrinogenemia was diagnosed after analysis of PT, APTT, and measurements of functional and antigen fibrinogen levels. Genomic DNA was extracted from whole blood by the QiAamp DNA Blood mini kit (Qiagen).Genomic DNA was PCR amplified by using sense and antisense primers designed on the basis of known sequences of the three fibrinogen genes and intergenic regions (GenBank accession numbers M64982, M64983, M10014, U36478, and AF229198). Molecular analysis was performed by DNA sequencing on both strands, by using the Big Dye Terminator Cycle Sequencing kit and an ABI-3130-XL automated DNA sequencer (Applied Biosystems). The Variant Reporter software (Applied Biosystems) was used for mutation detection. Results: A total of 21 afibrinogenemic patients (10 males and 11 females) came to our attention: their mean age was 11.54 +/- 11.2 years (range 1–40) and the mean bleeding score was 13.3 +/- 6.7.The most common bleeding manifestations were bruises, umbilical cord bleeding, gum bleeding, hematomas, circumcision bleeding, epistaxis, and menorrhagia. Genetic analysis was completed in nine cases, revealing a total of six different mutations: four in FGA (p.Arg 129 X, p.Cys8 X, p.Gln 200X,p. Gln162 X, the last three being novel) and two in FGB (p.Arg 47 X, p.Thr 407 Lys, both being novel). All afibrinogenemic patients resulted homozygous for the identified mutation. Conclusion: We have reported five novel mutations leading to congenital afibrinogenemia in a cohort of 21 patients.