Genetic defect responsible for the dysfunctional protein: factor IXLong Beach

DNA sequence analysis of the gene coding for the variant protein, factor IXLong Beach (FIXLB), has identified a transition mutation in an otherwise normal factor IX (FIX) gene. Genomic DNA clones spanning 35 kilobase (kb) pairs of the FIXLB gene were isolated. A gene analysis strategy that specifically characterized exons and their flanking intron sequences predicted the entire amino acid sequence of FIXLB. A thymine to cytosine transition causes the substitution of a threonine codon (ACA) for an isoleucine codon (ATA) in exon VIII of the FIXLB gene. This mutation results in an amino acid substitution at residue 397 of the FIX zymogen and the phenotypic display of hemophilia-B. Previous studies revealed that activated purified FIXLB (FIXaLB) had normal Ca2+, phospholipid, and factor VIIIa binding characteristics. However, FIXaLB activated factor X or factor VII (with their cofactors Ca2+ and phospholipid) at significantly reduced rates, suggesting that the defect in FIXaLB lies near or within the catalytic triad of the FIX heavy chain. Identification of an amino acid substitution near the carboxy-terminus of the FIXaLB heavy chain supports the earlier characterization of this variant protein. Moreover, our data identify a residue in the catalytic domain of FIXa essential for normal function.     

The role of amino-terminal residues of the heavy chain of factor IXa in the binding of its cofactor, factor VIIIa

The purpose of this study is to determine which residues of the factor IXa heavy chain are important for interaction with the cofactor of factor IXa, factor VIIIa. Because the monoclonal antibody (MoAb) FXC008 inhibits interaction between factors IXa and VIIIa, and because it also reacts with residues 181-310 of the factor IXa heavy chain, we used the computer-modelled structure of the factor IXa heavy chain to select charged surface residues likely to interact with FXC008 and/or factor VIIIa. We made mutations in the region of residues 181-310 of the heavy chain of factor IX, and replaced these amino acids individually with those located at the same position in factor X. The mutated factor IX retained complete clotting activity and thus interacted normally with factor VIIIa. Five mutant proteins (factor IXK214F, factor IXK228R, factor IXE240Q, factor IXK247V, and factor IXN260K) reacted with heavy chain-specific MoAbs FXC008 and A-5. Neither factor IXD276K nor factor IXR248H bound to FXC008. Factor IXR252V had reduced affinity to FXC008. Our results suggest the following: (1) factor IXa residues 214, 228, 240, 247, 248, 252, 260, and 276 are not involved in specific interaction with factor VIIIa; and (2) the FXC008 and factor VIIIa binding sites may not share critical residues.     

Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and beta thalassemia

Increasing the expression of the gamma globin genes is considered a useful therapeutic approach to the beta globin diseases. Because butyrate and alpha-amino-n-butyric acid (ABA) augment gamma globin expression in normal neonatal and adult erythroid progenitors, we investigated the effects of sodium butyrate and ABA on erythroid progenitors of patients with beta thalassemia and sickle cell anemia who might benefit from such an effect. Both substances increased fetal hemoglobin (Hb F) expression in Bfu-e from 7% to 30% above levels found in control cultures from the same subjects with sickle cell anemia. The fraction of cultured erythroblasts producing Hb F increased more than 20% with sodium butyrate treatment in 70% of cultures. In most cultures, this produced greater than 20% total Hb F and greater than 70% F cells, levels which have been considered beneficial in ameliorating clinical symptoms. Alpha: non-alpha (alpha-non-alpha) imbalance was decreased by 36% in erythroid progenitors of patients with beta thalassemia cultured in the presence of butyrate compared with control cultures from the same subjects. These data suggest that sodium butyrate may have therapeutic potential for increasing gamma globin expression in the beta globin diseases.     

Immunoglobulin heavy chain variable region gene usage in bone marrow transplant recipients: lack of somatic mutation indicates a maturational arrest [see comments]

Many recipients of bone marrow transplant (BMT) make normal amounts of serum immunoglobulin but are deficient in generating specific antibody responses to exogenous stimuli. To determine if abnormal usage of VH genes contributes to this immunodeficiency, the usage of VH genes was determined in peripheral blood B cells of four BMT recipients, two of whom had developed chronic graft versus host disease. The pattern of usage of VH3 or VH4 genes assessed at either 90 days or approximately 1 year after transplant was similar to that observed in healthy subjects and was marked by the over utilization of two elements, one VH3 and one VH4. However, the repertoires of each of the four BMT recipients appeared to be less complex than the repertoires of healthy subjects. The differences were a consequence of the accumulation of somatic mutations among rearrangements in the controls but not in the BMT recipients. The failure to accumulate somatic mutations in rearranged VH genes is consistent with a defect in antigen driven B-cell responses. These results indicate the although the VH gene content of the repertoire has normalized by 90 days posttransplant, a maturational arrest in B-cell differentiation associated with antigen activation persists for at least 1 year after BMT.     

A simple approach to prenatal diagnosis of beta-thalassemia in a geographic area where multiple mutations occur

We describe a simple approach for detecting beta-thalassemia mutations in geographic areas such as southern China where multiple mutations are known to occur. Segments of the beta-globin gene were amplified in vitro by using the polymerase chain reaction. Dot blot hybridization of the amplified DNA with oligonucleotide probes corresponding to the six mutations found in southern China could directly identify the mutations causing beta-thalassemia in the affected families. The increased number of target sequences after amplification allows the use of 35S-labeled probes, which are reusable for up to 3 months. The mutations can be determined in two days.     

Heterogeneous mechanisms of imparied lymphocyte responses in non- Hodgkin's lymphoma

Peripheral blood mononuclear cells (PBMC) from 18 untreated patients with non-Hodgkin's lymphoma (NHL) were studied to characterize the cellular mechanisms contributing to impaired in vitro lymphocyte responses after stimulation by the mitogen conconavalin A (Con-A). In vitro reactivity was quantitated by the 3H-thymidine incorporation in response to an optimal dose of Con-A. All patients demonstrated impaired in vitro reactivities compared to normal controls. These in vitro impairments were partially reversible since patient's cells precultured in media alone for 3 days demonstrated enhanced Con-A responses. In greater than half of the patients, the hyporeactive PBMC suppressed the enhanced reactivities of autologous precultured PBMC when assayed in cocultures. Suppressor activity was detected mainly in those untreated patients presenting with either constitutional symptoms or diffuse histology and in general was not marked compared to the severity of impairments. Adherent monocytes were shown to participate in the suppression of autologous lymphocyte reactivity but only appeared partially responsible for the in vitro impairments. In those patients lacking detectable suppressive activity, preculturing also enhanced Con-A reactivities and was compatible with the presence of a reversible, inhibitory mechanism differing from active suppression. Many patients' hyporeactive PBMC, however, failed to demonstrate normal responses after preculturing. This failure could not be directly attributed to aberrant regulatory populations, but rather appeared to possibly represent an additional intrinsic impairment of potentially reactive populations.     

The reliability and construct validity of the RAQoL: a rheumatoid arthritis-specific quality of life instrument

The present study was designed to test the psychometric properties of the RAQoL, a rheumatoid arthritis (RA)-specific quality of life (QoL) instrument. All stages of the development were conducted simultaneously in The Netherlands and the UK. The content of the draft measure was derived from qualitative interviews with RA patients in both countries. The final version of the RAQoL has 30 items with a 'yes'/'no' response format and takes approximately 6 min to complete. Both language versions have high internal consistency and test-retest reliability (> 0.9), and good sensitivity to discriminate between groups with various disease activity and severity. Given the excellent psychometric properties of the new instrument, it will prove to be a valuable tool for assessing quality of life in clinical trials and for monitoring patients in routine clinical practice.