Dezsö David

Analysis of the consequences of premature termination codons within factor VIII coding sequences.

Summary.  Inhibitor antibody formation is a complication of factor VIII (FVIII) replacement therapy due to a failure to synthesize sufficient FVIII protein to induce immune tolerance. The incidence of nonsense mutations in inhibitor patients is high, however, this association is variable according to the position of the mutation. We have studied the effect of nonsense mutations on accumulation of FVIII mRNA, protein translation and secretion. Appropriately processed mRNA was detected in cells transfected with wild‐type R1966X and R2116X expression constructs and no evidence of nonsense‐mediated decay was observed. All constructs directed the translation of detectable intracellular FVIII antigen, however, secreted FVIII was detected only in conditioned media of cells transfected with wild‐type cDNA. We have also analyzed ectopic FVIII mRNA transcripts in the lymphocytes of six hemophilia A patients with nonsense mutations (Q139X, R583X, R1941X, R1966X and two unrelated patients with R2116X). FVIII mRNA was detectable in every case. In R1941X and R1966X only normally spliced transcripts were present. In Q139X, R583X and R2116X aberrantly spliced transcripts were observed with two distinct patterns in two individuals with the R2116X mutation. No correlation between mutation, transcript pattern and incidence of inhibitor development was apparent.

Female haemophiliac homozygous for the factor VIII intron 22 inversion mutation, with transcriptional inactivation of one of the factor VIII alleles.

Summary.  Phenotypic expression of X‐linked recessive disorders, including haemophilia A, is rare in females. This report describes a female with sporadic severe haemophilia A. The female patient and her family members were evaluated by coagulation assays. Visible detectable disturbance of X chromosome structure or number, as well as 2N von Willebrand disease, were excluded as possible explanations of the haemophilia A phenotype. Molecular studies, factor VIII (FVIII) intron 22 inversion mutation analysis showed that the severe haemophilia A phenotype is the result of a maternally inherited, distal, FVIII gene inversion and a paternally inherited de novo, also distal, FVIII gene inversion. Furthermore, comparative single‐stranded conformation polymorphism analysis revealed the absence of detectable maternally inherited abnormal FVIII gene transcript in the patients peripheral blood lymphocytes. X chromosome methylation analysis indicates that this could be explained by preferential inactivation of the maternally inherited X chromosome carrying the distal FVIII gene inversion.

Molecular characterization of a 7p15–21 homozygous deletion in a Wilms tumor

Recent molecular studies have shown a relatively high rate of loss of heterozygosity (LOH) at band 7p15–21 in Wilms tumor. We previously reported that the minimal common region of LOH was located between markers D7S517 and D7S503 in bands 7p15–21. We also reported the identification of one Wilms tumor (GOS44) bearing a homozygous, interstitial deletion at a locus within this region. Homogeneous primary cell cultures have been derived from this tumor and have been used for all the subsequent analyses. Using PCR and a panel of STS markers mapping between D7S517 and D7S503, the physical boundaries of the homozygous deletion were determined to be between D7S638 and D7S644. The deleted region spans approximately 3 Mbp of genomic sequence and includes seven known genes (KIAA0744, KIAA0713, AHR, AGR2, NET6, HSPC028, and DGKB.) as well as five predicted genes with similarities to genes of known function (LOC‐91802, ‐116364, ‐96009, ‐92511, and ‐92512). The proximal breakpoint was found to lie between exon 6 and exon 7 of KIAA0744, and the distal breakpoint lay between exon 17 and exon 18 of DGKB. It is unlikely that a functional fusion gene product was generated as a consequence of the fusion between these two genes, because they are oriented in opposite directions on the chromosome. This is the only reported homozygous deletion recorded so far in Wilms tumor, and it provides the means to identify the tumor‐suppressor gene located in this deletion.

Characterization of a splicing mutation in the factor VIII gene at the RNA level

Haemophilia A is an X-linked bleeding disorder caused by mutations in the coagulation factor VIII (FVIII) gene. The identification and characterization of naturally occurring disease-producing mutations allows the recognition of new mechanisms of pathogenesis in haemophilia A. Analysis of the illegitimately transcribed FVIII mRNA in a severely affected patient has revealed that the A→G transition at position −2 of the acceptor splice site of intron 4 results in the skipping of exon 5 in 90% of the processed pre-mRNA. Another minor mRNA species arising from the skipping of exons 4 and 5 has also been observed. The skipping of exon 5 predicts the removal of the corresponding 13 amino acids from the A1 domain of FVIII. A novel missense mutation, C329S, in exon 8 of FVIII gene has been identified in another patient.

Phenotypes of Carboxyl-Terminal Rhodopsin Mutations in Autosomal Dominant Retinitis Pigmentosa

We compared the ocular findings in 13 patients from 4 families with autosomal dominant retinitis pigmentosa and 4 different mutations in the carboxyl-terminal sequence of rhodopsin. Phenotypic similarities were found among patients with point mutations predicting the amino acid changes valine-345-methionine, proline-347-serine or proline-347-leucine in the rhodopin molecule. These patients had no measurable rod function and an early impairment of cone function, which was most profound in the proline-347-serine genotype. One patient with a valine-345-methionine mutation showed a regional predilection of fundus abnormalities and cone sensitivity loss. A different phenotype with relatively mild disease expression could be observed in a family with a deletion of 8 base pairs (codons 341–343). One 34 year-old member showed regionally varying rod sensitivity loss, which was less severe in the peripheral visual field, and well maintained cone function, as measured by electroretinography and psychophysical tests.