Biochemistry of Factor IX and Molecular Biology of Hemophilia B, страница 11

Approximately one-third of the cases of hemophilia B fall within the group termed cross-reactive material positive. Such patients have normal levels of factor IX antigen and variable levels of factor IX activity, due to the presence of a dysfunctional factor IX molecule. Mutations have been described that affect post-translational protein processing, g-carboxylation, lipid binding, EGF domain function, zymogen activation, and substrate recognition and/or enzymatic activity. In addition, a series of patients has been reported to display the hemophilia BM phenotype. This has proved to be a heterogeneous set of mutations, as noted below.

A developing theme in understanding the molecular basis of hemophilia B is the discovery of duplicate mutations in apparently unrelated patients. To date, 378 unique point mutations (both missense and nonsense) have been reported. MLID93324412  69 Several groups have postulated that the CpG dinucleotide sequence is a mutational hot spot. MLID86235434 MLID87065092 MLID78246993  167–169 The CpG sequence is a component of four of the six arginine codons; as a mutational hot spot, it may represent an important cause for the development of spontaneous point mutations. Furthermore, using a mathematical model, the 20 CpG dinucleotides in the factor IX coding sequence showed an observed mutation rate 150 times the rate that would be expected for the predicted transition rate. MLID90280441  170 Of 51 single base pair substitutions found in one study, 27 were at CpG dinucleotides, which represents a 38-fold excess for mutations at these sites. MLID90287697  171 In another study, point mutations at CpG dinucleotides were estimated to be increased by 77-fold over the expected rate. MLID89371752  172

In patients with mutant plasma proteins, the mutation of arginine to another amino acid also appears to be a developing theme. Since internal point mutations are likely to destabilize protein structure and lead to markedly diminished circulating protein levels, patients preselected for circulating mutant protein antigen are likely to have amino acid substitutions on the protein surface. Arginines, located on the protein surface, are used by proteases of the trypsin family to hydrolyze adjacent bonds during protein processing and zymogen activation. It would thus appear, given the functional importance of arginines, that their mutation leads to phenotypically obvious defects in protein function.

Molecular Diagnosis

Hemophilia B is diagnosed by the finding of isolated, hereditary factor IX deficiency. Factor IX deficiency may be observed by prolongation of the activated partial thromboplastin time and by failure of plasma from these patients to correct the clotting time of known factor IX-deficient plasma. These findings, combined with a lifelong history of bleeding or a family history of bleeding disorders, secure the diagnosis. The level of factor IX antigen present in the plasma of each patient permits the hemophilia to be characterized as antigen negative or antigen positive. Within a particular cohort, the factor IX activity level is usually constant, reflecting that each family displays the same genetic defect and that each defect is associated with a particular phenotype.

Because of the bleeding disorder associated with moderate or severe hemophilia B, and because of the sex-linked nature of the disorder, genetic counseling for affected persons and potential carriers becomes a major issue. Carrier analysis has been done by differentiating between factor IX activity and factor IX antigen levels. However, this method has been at best only 80% effective, partially because of the preponderance of mutations yielding markedly diminished factor IX antigen and partially because of "lyonization," which causes differential levels of inactivation of the X chromosome carrying the mutant gene. MLID77222501  173

With knowledge of the sequence of the factor IX gene, it is possible to probe for the precise genetic defect among potential carriers. If the genetic defect for a particular cohort is known, the DNA of potential carriers can be screened using stringent hybridization to oligonucleotide probes to detect whether they carry that genetic defect. This method is handicapped, since as many as one-third of hemophilia cases in any generation arise from new mutations. It would likely be difficult to detect these using probes for specific mutations.