Biochemistry of Factor IX and Molecular Biology of Hemophilia B, страница 12

Of greater value has been the detection of several restriction fragment length polymorphisms (RFLPs) within the factor IX gene that can be used for carrier detection (Fig. 107-10). A single polymorphism within the factor IX coding sequence occurs at residue 148, such that 65% of the white population has a threonine at that locus, and 35% has an alanine. MLID85190593  174 This polymorphism can be detected both immunochemically MLID85216587  175,176 and through oligonucleotide probes. MLID85190593  174 In addition, five intragenic polymorphisms have been described within the introns of the factor IX gene. A TaqI polymorphism at nucleotide 11,111 is found in 35% of the white population. MLID84119514  177 An XmnI polymorphism (G B C) at nucleotide 7,076 is present on 29% of X chromosomes, while a DdeI/HinfI polymorphism resulting from a deletion of nucleotides 5,505 to 5,554 is found at a 24% frequency. MLID85087905  178

Camerino et al. MLID85287199  179 described an MspI polymorphism near nucleotide 16,000 occurring with a 20% frequency, while Hay et al. 180 described a BamHI polymorphism at nucleotide -587, with a 6% frequency. In addition, four polymorphisms have been found to be closely linked to the factor IX gene. Mulligan et al. MLID87192722  181 described an SstI RFLP in the q26-q27 region of the X chromosome with a frequency of approximately 50%. Two closely linked TaqI polymorphisms have also been described. MLID84194100 MLID88131058  182,183 Winship et al. MLID89180371  184 used the polymerase chain reaction (PCR) to unmask an HhaI RFLP 8 kb 3' to the factor IX gene, occurring in 48% of the population studied.

To determine whether a person is a hemophilia B carrier, genomic DNA is isolated from the potential carrier, the patient with hemophilia B, and both parents of the potential carrier. That DNA is digested with the appropriate restriction enzymes, and the digested DNA is run on an appropriate gel matrix. The DNA is transferred to nitrocellulose, and a Southern blot is performed, using labeled factor IX cDNA as the probe. The restriction maps are then compared, with the goal of looking for any polymorphisms found in the patient with hemophilia but not in the normal parent. The same procedure can be used on DNA obtained by amniocentesis to determine whether the fetus of a hemophilia B carrier will be a normal or an affected male, and whether a female fetus will be normal or will carry the hemophilia gene.

With the combination of these polymorphisms, it is predicted that the carrier status of 89% of the potential carrier population (among whites) can be determined with 99.9% certainty. 163 It should be emphasized that these figures apply only to the white population, in whom the most extensive study of RFLPs has taken place. It is clear that the frequency of each polymorphism varies with the ethnic group being studied, such that the polymorphisms currently employed for diagnosis within the white population are inadequate for determining carrier status in either the Japanese MLID87076978  185 or black population. MLID89277343  186

The determination of carrier status using RFLPs can be useful in whites and some other populations that have been well studied. However, even in the white population, some patients cannot be diagnosed with these techniques. The application of PCR to the detection of hemophilia carriers has changed the evaluation of patients with hemophilia B. With PCR, it is now possible to amplify specific exons and then to sequence the DNA to determine the abnormality that led to hemophilia B. With this information, it is then possible to determine whether that mutation is present in the genome of either potential carriers or the fetuses (or both) of those carriers and patients. This obviates the problem of genetic recombination that clouds the use of closely linked restriction polymorphisms to determine the presence or absence of a mutant gene. Moreover, with the use of PCR, it now becomes possible to perform antenatal testing either on cells obtained by amniocentesis or on chorionic villus samples without having to wait the several weeks required to obtain enough fetal cells to do restriction digests.