To screen for mutations within the factor VIII gene of 101 patients (85 unrelated), we used denaturing gradient gel electrophoresis (DGGE) after DNA amplification of target regions, including all coding regions except for the middle part (amino acid 757 to amino acid 1649) of the B domain. With this method, missense mutations were identified in 86% of unrelated patients. 41 different mutations were identified: 25 of them have not been described previously. Five of the genotypes are associated with CRM+ and 26 with CRMred status. Patients who are definitely related to each other showed no differences in DNA sequence. One patient showed two different base pair alterations, the first at amino acid 469 [ala(GCA-->gly(GGA)] and the second at position 473 [tyr(TAT)-->cys(TGT)]. One patient with an amino acid change at position 1689 [arg(CGC)-->his(CAC)] has developed an inhibitor against factor VIII.
Screening of the factor VIII (FVIII) gene which spans 186 kb and codes for 26 exons, was originally hampered by its size but is now feasible because rapid DNA scanning methodologies have been developed. The present study for the first time directly compares the three most widely applied screening methods, denaturing gradient gel electrophoresis (DGGE), single-stranded conformational polymorphism (SSCP) and chemical mismatch cleavage (CMC) for their sensitivity of mutation detection in a selected group of ten haemophilia A patients. Nine of these patients are known to be cross-reacting material positive and eight exhibited a mild to moderate phenotype. Of the ten patients screened, we identified mutations in nine by all three screening methods. Of the mutations characterised, two are previously unpublished. T to C (S373P) and G to A (D525N). In one mildly affected haemophiliac, we identified a second T to C sequence change in the 5' untranslated region at -601 bp, probably having no effect on FVIII gene expression. Modelling studies were performed on those mutations lying within the A domains of FVIII (D525N, R527W, I566T) to study the possible effect of these mutations on structure and/or function. When the three methods are performing optimally and have been standardised, our experience is that CMC and DGGE are equally efficient at sequence variation detection while SSCP is slightly less sensitive.
Expression of the 1.9 kb cDNA of murine Calmbp1 has been shown to interfere with the mitotic S-M checkpoint in yeast (J. Cell Sci. 111 (1998) 3609). The physiological function and expression pattern of Calmbp1 in mice, however, are unknown. We have investigated the expression of Calmbp1 in mid-gestation and late-gestation fetuses and in adult organs of the mouse. In Northern blot analyses, using a Calmbp1-specific probe, a single mRNA of more than 7.4 kb was found that showed a progressive decline in total RNA preparations of fetal heads during the period from day E12 to E16. In the adult, this Calmbp1 transcript was detectable by Northern blot analysis exclusively in testis, ovary and spleen of all organs examined. In situ hybridizations revealed that Calmbp1 is expressed (a) in the differentiating central and peripheral nervous system, (b) in the epithelial cells lining the crypts of the small intestine in late gestation and adult mice, (c) in the fetal, but not the adult liver, (d) in both the fetal and adult spleen, where the signal colocalized with hematopoetic cells in the red pulp, (e) in late gestation embryos in the thymus, S-shaped tubules in the kidney, epidermis, and (f) in leptotene, zygotene and pachytene spermatocytes of the adult testis and the follicle epithelium of the activated follicles in the adult ovary.
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