In vitro transcription of compound heterozygous hypofibrinogenemia Matsumoto IX; first identification of FGB IVS6 deletion of 4 nucleotides and FGG IVS3-2A>G causing abnormal RNA splicing

Genetic analyses of novel compound heterozygous hypodysfibrinogenemia, Tsukuba I: FGG c.1129+62_65 del AATA and FGG c.1299+4 del A

“…In order to analyze all exons and exon-intron boundaries in the A-, B-, and -chain genes, PCR and direct sequencing were performed as described elsewhere [12]. Furthermore, PCR products were subcloned into pCR2.1 plasmid vectors (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions [13].…”

“…Reverse-transcriptase (RT) reactions were performed using the oligo dT primer and RT from Moloney murine leukemia virus [13], followed by PCR amplification with two pairs of primers: the sense primer located in FGG-exon 8 (5'-CATGTTCAAGGTGGGACCTG-3') and antisense primer located in FGG-exon 9 (5'-TTCATGGAATACCACCGGGT-3') for minigene I, or the sense primer located in FGG-exon 9 (5'-GTGGCACTTACTCAAAAGCATC-3') and antisense primer located in FGG-3' UTR (5'-CTCTCTGTTCAGATAAAGTCC-3') for minigene II.…”

Molecular analysis of afibrinogenemic mutations caused by a homozygous FGA1238 bp deletion, and a compound heterozygous FGA1238 bp deletion and novel FGA c.54+3A>C substitution

“…Nine volumes of blood were collected into plastic tubes containing 1 volume of 3.2% trisodium citrate. Separated plasma was used for coagulation tests and buffy coat cells were extracted to prepare genomic DNA [9]. This analysis was approved by the Ethics Committee for Genetic Analysis of Shinshu University School of Medicine.…”

“…Blood coagulation screening tests; prothrombin time (PT), activated partial thromboplastine time (APTT), the fibrinogen concentration, which was determined by the thrombin time method, and the immunologic fibrinogen concentration were measured as described elsewhere [9].…”

“…To analyze all exons and exon-intron boundaries in the Aα-, Bβ-, and γ-chain genes, PCR and direct sequencing were performed as described elsewhere [9]. To examine the large deletion of the genes, long-range PCR for the entire Aα-, Bβ-, and γ-chain genes was performed using the TaKaRa LA Taq (Takara Bio Inc., Otsu, Japan) and the pair of primers for Aα-chain: sense primer: 5'-CAGCTAGCTTACCTAAGCACC-3' and antisense primer: 5'-GTTAAGGAAGAAATGCAAGGG-3', Bβ-chain: sense primer:…”