Structure, Biology, and Genetics of Factor VIII. Factor VIII Function, страница 9

Before the introduction of modern treatment, severe hemophilia A was a genetically lethal disease in which affected men produced few offspring. Therefore, nearly one-third of the mutant alleles would be lost in each generation. In 1935 Haldane predicted that in order to maintain a constant frequency in the population, about one-third of cases would be the result of novel mutations. The prediction was proved correct, since a large number of different mutations have been found in the factor VIII gene, and many patients have been identified who carry a de novo mutation not present in the X chromosome of their mothers.

Factor VIII Gene Structure and Location

The factor VIII gene is 186 kb long (approximately 0.1% of the DNA of the X chromosome) and contains 26 exons and 25 introns. The nucleotide sequence of the exons, intron-exon boundaries, and 5' and 3' untranslated regions has been determined. MLID85061548 MLID85061550 MLID85061547  17,18,162 The exon length varies from 69 to 262 nucleotides except for exon 14, which is 3,106 nucleotides, and the last exon 26, which has 1,958 nucleotides (Fig. 105-4). There are some large intervening sequences such as IVS22, which is 32 kb and IVS1, IVS6, IVS13, IVS14, and IVS25, which are 14–23 kb long.

The normal factor VIII mRNA is approximately 9 kb, of which the coding sequence is 7,053 nucleotides. There is a CpG island within IVS22 that is associated with two additional transcripts. One transcript of 1.8 kb is produced abundantly in a wide variety of cells. The orientation of this transcript is opposite to that of factor VIII and contains no intervening sequence. MLID90243242  163 This 1,739-nt-long cDNA has been termed factor VIII-associated gene A (F8A) and is conserved in the mouse. MLID92347894  164 The second transcript of 2.5 kb is transcribed in the same direction as factor VIII; after a short exon that may encode for eight amino acids, it utilizes exons 23–26 of the factor VIII gene. MLID93052386  165 This gene has been termed factor VIII-associated gene B (F8B). The two transcripts (F8A and F8B) originate within 122 bases from each other. The sequences of F8A and F8B along with few kilobases of surrounding DNA are also present in two other areas of the X chromosome approximately 400 kb telomeric to factor VIII gene. MLID90243242  163,166 The function of these transcripts and their potential protein products are unknown.

The factor VIII gene maps on the long arm of the X chromosome, in the most distal band Xq28. Haldane and Smith 167 reported linkage of hemophilia A with color blindness, and Boyer and Graham 168 demonstrated close linkage of hemophilia A with polymorphisms at the glucose-6-phosphate-dehydrogenase (G6PD) locus. Additional studies confirmed the close linkage of factor VIII with G6PD. MLID84125351  169 Patterson et al. MLID88212436  170 showed that G6PD and factor VIII genes lie within 500 kb of each other. Pulsed field gel electrophoresis and physical mapping of Xq28 using yeast artificial chromosomes suggested that the factor VIII gene mapped distal to G6PD. MLID92020841  166,171 The order of these loci and the direction of transcription is Xcen-G6PD-3'F8-5'F8-Xqter. MLID93252379  166,172 The distance from factor VIII gene to the Xq telomere is approximately 1 Mb.

Factor VIII Gene Defects

Since the cloning of the factor VIII gene, the DNA of >1,000 hemophilia A patients has been examined for molecular defects. Initially, restriction endonuclease analysis and Southern blot cloning and sequencing were the methods used. The introduction of polymerase chain reaction (PCR) amplification from genomic DNA or from RNA (RT-PCR) revolutionized the mutation detection protocols. Several screening methods for recognition of mutations have been employed, namely, denaturing gradient gel electrophoresis, single-stranded conformational analysis, RNA cleavage analysis, and subsequently direct sequencing of PCR products.