The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.      

Near-haploid cell line in megakaryoblastic transformation of Philadelphia-positive chronic myeloid leukemia

A case of near haploidy in a patient with an acute megakaryoblastic transformation of Philadelphia (Ph)-positive chronic myeloid leukemia (CML) was studied. Cytogenetic studies revealed persistence of a Ph-positive pseudodiploid cell line and the emergence of a Ph-positive near-haploid cell line, i.e., 46,XX,t(9;22)(q34;q11)/28,XX,t(9;22),+8,+14,+18,+29. The near-haploid cell line is a rare cytogenetic finding. The patient rapidly deteriorated and died.     

Mutation screening analysis of the retinoblastoma related gene RB2/p130 in sporadic ovarian cancer and head and neck squamous cell cancer.

AIMS: To investigate the involvement of the RB2/p130 gene in the pathogenesis of sporadic ovarian cancer in addition to head and neck squamous cell carcinoma (HNSCC). METHODS: Paired tumour and patient matched normal DNA samples from 43 sporadic ovarian tumours and 39 normal/tumour HNSCC DNA samples were screened. The mutation screen used polymerase chain reaction (PCR) amplification followed by single strand conformation polymorphism analysis and direct sequencing of the PCR products. Exons 19 and 20 (B domain) and exons 21 and 22 (C-terminus) were analysed for mutations. These exons were chosen because most of the point mutations in RB2/p130 are located in the C-terminal region and mutations in these exons have been identified previously in nasopharyngeal carcinomas and primary lung tumours. RESULTS: No abnormal band shifts were seen in the samples analysed, and no bands directly sequenced revealed the presence of mutations. CONCLUSIONS: Genetic alterations in the RB2/p130 gene (exons 19-22) are unlikely to be involved directly in the pathogenesis of sporadic ovarian cancer or HNSCC.