Genetic Defect Analysis Of A Chinese Family With Congenital Afibrinogenemia

Objectives To determain the causative gene mutation of fibrinogen genes in a proband with congenital afibrinogenemia and her family members by genetic analysis and explore the possible molecular pathogenesis from the level of molecular genetics.Methods 1 APTT, PT, TT of the proband and her family members were assessed by conventional laboratory methods; Plasma fibrinogen was detected by both immuneturbidity and clauss method. 2 Western-blot was performed on examining expression of plasma Fg α, β and γ peptide chain between family members and healthy control. 3 Genetic DNA from peripheral blood was extracted between family members and healthy control by Genome Extraction Kit, and all the exons and exon- intronic boundaries and promoter region of the three fibrinogen genes(FGA, FGB, FGG)were detected by direct sequencing after amplifying. The whole genome sequencing on the proband and her mother was performed by next generation sequencing to screen genetic mutations associated with disease. Further, gene polymorphism need to be excluded by comparing with NCBI. 4 The discoverable gene mutation was copied into the website of neural network to predict the gene mutation which can cause the change of the splice site. Peripheral blood DNA of prohand and healthy control was used as template respectively. primers were designed to amplify the mutation sequence and normal sequence. The DNA fragment was cloned into pc DNA3.1(-) vector to construct small gene plasmid FGG(Wt-FGG) containing mutation sequence and small gene plasmid FGG(Nt-FGG) containing normal sequence and the 2 plasmid were transfected into COS-7 cells respectively. RNA which extracted from cells were reverse transcripted into c DNA by RT-PCR. Target fragment were sequenced for generation after verifying by agarose gel electrophoresis. The results of sequencing were compared with normal m RNA sequence in NCBI. DNASIS software was used to analyse the changes of amino acid after gene mutation.Results 1 The coagulation function of APTT>200s、PT and TT>100s in proband and her brother, wheares all the values in her parents were normal. Plasma Fg was 0 in proband and her brother according to immune turbidity method and cluss method, wheares her parents, values were slightly lower than the normal healthy control. 2 Western-blot showed no fibrinogen expression in prohand and her brother. Although the α, β and γ fibrinogen chains(65KD, 52 KD, 46 KD, respectively) were found in proband’s parents, the value were lower than the normal control. 3 There was no gene mutation was found in exon-intronic boundaries and promoter region, whereas, a homozygous A to G mutation was found at nucleotide 4266 in exon 5 of FGA gene, P 331 Thr>Ala in proband and her brother and the parents were heterozygous mutation in the same position. This mutation had been reported as a polymorphism site. The next generation sequencing of whole genome revealed that a homozygous G to A mutation was found in the intronic3 of FGG gene, FGG IVS3+5G>A, together with a heterozygous found in her mother for the same mutation position. Generation sequencing was used to verify the same location of other family members. The results showed that her little brother was homozygous mutation as same as proband and her father was heterozygous as same as mother. Meanwhile, detection of 30 normal healthy controls could not be found in the same gene mutation. Gene polymorphism of SNP was excluded using NCBI database. 4 The splice site prediction software showed the disappearance of donor splice sites, not activated cryptic splice site and produced new splice site. Based on amplified fragment(766bp) from the peripheral blood DNA of prohand and healthy control, Agarose Gel Electrophoresis revealed that the c DNA was approximately 280 bp and 460 bp between Wt-FGG and Nt-FGG, respectively. Sequencing results suggested that m RNA of WtFGG was lack of exon 3 compare with Nt-FGG(184bp). Compared with normal in NCBI, abnormal m RNA resulting from gene mutation missed amino acids from 16 th to 76 th which located in exon 3. DNASIS showed that stop codons formed in the second amino acids of exon 4, truncated gamma chain only contained 16 amino acids according to vitro construction of cell model after transcription.Conclusions 1 Congenital afibrinogenemia is caused by the mutation G-A located in the fibrinogen FGA gene intron 3 of the fifth nucleotide in these family, which has not been described in our country. 2 After the splice site mutations lead to abnormal m RNA splicing, the skipping of exon 3 formed truncated γ chain. It is the possible pathogenesis of this family which fibrinogen can not be synthesize.