{'fraction_non_alphanumeric': 0.03558419725413281, 'fraction_numerical': 0.02913981507425049, 'mean_word_length': 5.0, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 6, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 2, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Identification of a novel splicing mutation in the fibrinogen Aα chain gene leading to hypofibrinogenaemia in a Chinese pedigree

Fibrinogen is the focal point of the coagulation cascade, which results in the conversion of fibrinogen to fibrin monomer and the eventual covalent crosslinking of the fibrin polymer. Fibrinogen, a 340-kD glycoprotein, is synthesized in the liver as a dimer with each half composed of three different polypeptide chains (Aa, Bb and c). These chains are linked by a network of 29 disulfide bonds to form the circulating protein. Each chain is encoded by a separate gene, FGA, FGB and FGG, which are clustered in a 50-kb region on chromosome 4q32.1. Fibrinogen abnormalities can be classified according to whether there are low or no circulating levels of normal protein (hypofibrinogenaemia or afibrinogenaemia), a mutated species (dysfibrinogenaemia), or a combination (hypodysfibrinogenaemia). Congenital hypofibrinogenaemia is characterized by functional and immunoreactive fibrinogen levels lower than 1.5 g L [1]. Here, we studied a Chinese patient with hypofibrinogenaemia. The proband had low plasma levels of functional and immunoreactive fibrinogen. Sequencing of fibrinogen genes, including exon–intron boundaries and promoter regions, allowed us to identify a heterozygous G>C transition at position +1 of intron 2 of the a-chain gene (FGA IVS2+1G>C). Production of the mutant transcript in transfected HEK 293 cells demonstrated the existence of an aberrant splicing of fibrinogen a-mRNA, leading to a major a-chain truncation associated with hypofibrinogenaemia. The 32-year-old proband had four times of ectopic pregnancy; after ectopic pregnancy, she did not bleed abnormally, did not have anaemia or any other haemostatic challenges. The proband had no acute or chronic liver disorder. No history of bleeding disorders and consanguineous marriages were described in her family (Fig. 1). The results of coagulation tests of all the family members are shown in Table 1. As shown in the table, the proband had prolonged TT and low fibrinogen level (both functional and immunoreactive) whereas all the coagulation tests of other family members were in the normal range. FGA IVS2+1G>C was detected in the patient (Fig. 2). No other mutations were identified in the branch sites and the consensus splicing sequences of other introns. The splice site mutation FGA IVS2+1G>C was not found in any of the other family members. To determine the splicing patterns associated with the FGA IVS2+1G>C mutation, we isolated total RNA from peripheral blood leucocytes of the patient and analysed the transcripts by RT-PCR. Only one band was observed on the agarose gel after electrophoretic separation of RT-PCR products and this band was the same as that of normal control (407 bp) (Fig. 3). The RT-PCR products from the patient were then cloned and sequenced. However, no aberrant transcripts were ever found in 30 individual clones. To verify whether the donor splice-site mutation (FGA IVS2+1G>C) determines the retention of intron 2 in mature mRNA, we made FGA-wt and FGA-mut constructs and transfected them into HEK 293 T cells respectively, total mRNA was extracted after 16 h culture. RT-PCR performed on RNA extracted from FGA-wt-transfected HEK 293 cells allowed us to detect the expected 243-bp long fragment (including a 101-bp exon 2 and a 142-bp Correspondence: Hongli Wang, No.197 Ruijin Second Road, Shanghai 200025, China. Tel.: +86 21 64370045*610609; fax: +86 21 64743206; e-mail: wanghongli602@163.com; wwbzxt@hotmail.com