Molecular analysis of the prothrombin gene in two patients

• Main Author: Duke, Leslea Marie
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/4983

A deficiency of prothrombin (Fil) is an extremely rare bleeding disorder. Two patients were studied with a severe form of this disorder which is known as Hypoprothrombinemia. Each patient was identified as being the product of a consanguineous union, and therefore a single homozygous mutation would be expected. To characterize the deficiency, a clinical analysis was performed. Fil antigen and activity levels were provided for each patient. Fli-Vancouver was assessed at 3% Fil activity and 3% Fil antigen levels. Fll-Utrecht exhibited similar levels of 3% for both activity and antigen. The objective of this study was to analyze the HI gene in each patient, and to identify the molecular basis for the disease. Genomic DNA was provided for each patient and the exons of the prothrombin gene were amplified specifically by using the polymerase chain reaction. Primers were designed to include the intron/exon splice junctions and approximately 100 bp 5’ to exon 1 in the amplification reactions. To facilitate directional cloning, restriction sites were incorporated into the primers. In this way, each region could then be analyzed by DNA sequencing. The sequence analyzed in each patient was then compared to the reported sequence of wild-type Fil gene (Degan et al., 1987). Several base changes were observed in both patients, and all were found to be homozygous. A single inserted ‘A’ was found in the region 5’ to exon 1, at nucleotide position -54 , in both Fil-Vancouver and Fll-Utrecht (numbering is according to the sequence of Degen et al., 1987). This insertion was later found in the wild-type sequence reported by Bancroft et al., (1990). It is evident therefore that this alteration is not the cause of the deficiency; however, it does provide the opportunity to focus on the promoter region as a potential region for mutation. At nucleotide position 461, a single ‘ T’ was deleted from the splice region 5’ to exon 2. This change was not expected to interfere with the proper splicing of FIT, as the deleted base occurs in a series of 3 ‘ T’ residues. The function of this base could be easily adapted by either of the flanking bases. This change was observed in both patients. A silent polymorphism resulting from the point mutation of the codon CTA -> CTG was found in exon 2 of Fil-Vancouver and FII-Utrecht. The leucine residue at codon 56 would be unaffected by this mutation. A polymorphism was found in the splice region 3’ to exon 6, which results from the point mutation of G -> A, at nucleotide position 4272. This mutation occurs in both patients, and was also reported by Iwahana et al., (1992). The most significant mutation found was the result of a three bp deletion of the in-frame codon AAG, at nucleotide position 7485-7487. This mutation deletes a single lysine residue at the codon position 301. This mutation was found in Fil-Vancouver, and was found to be homozygous. This deletion occurs in the activation region of FIT, in what would eventually be the A-chain of thrombin (Fila). This mutation is expected to have an effect on the 3-dimensional structure of the polypeptide. A silent polymorphism was identified in exon 10 of both patients. This point mutation resulted from the substitution of ACA -> ACC, at nucleotide position 8903. This threonine at position 388 was unaffected by this change. The molecular basis of the deficiency in FIT-Vancouver appears to be the result of a single deleted lysine residue. This mutation is expected to have global effects on the proper folding of the protein during synthesis, resulting in its degradation. A mutation resulting in the synthesis of an aberrantly folded protein is quite common among disorders where there is little or no generation of protein. The basis of the deficiency in FTT-Utrecht remains elusive. The observed changes observed in the FIT gene were not expected to have a significant effect on the generation of the protein. The intron/exon junctions and splice branch points all remain intact, therefore, the disorder is not expected to be due to an error in splicing. It is possible that the deficiency is in fact, due to a defect in transcription. It is common to find mutations in the upstream promoter and enhancer regions which affect transcription such that minimal amounts of protein are generated. Further studies would be necessary to test the fidelity of the Fil promoter in this patient.