Targeted disruption of the cell-cycle checkpoint gene ATR leads to early embryonic lethality in mice.

Checkpoints of DNA integrity are conserved throughout evolution, as are the kinases ATM (Ataxia Telangiectasia mutated) and ATR (Ataxia- and Rad-related), which are related to phosphatidylinositol (PI) 3-kinase [1] [2] [3]. The ATM gene is not essential, but mutations lead to ataxia telangiectasia (AT), a pleiotropic disorder characterised by radiation sensitivity and cellular checkpoint defects in response to ionising radiation [4] [5] [6]. The ATR gene has not been associated with human syndromes and, structurally, is more closely related to the canonical yeast checkpoint genes rad3(Sp) and MEC1(Sc) [7] [8]. ATR has been implicated in the response to ultraviolet (UV) radiation and blocks to DNA synthesis [8] [9] [10] [11], and may phosphorylate p53 [12] [13], suggesting that ATM and ATR may have similar and, perhaps, complementary roles in cell-cycle control after DNA damage. Here, we report that targeted inactivation of ATR in mice by disruption of the kinase domain leads to early embryonic lethality before embryonic day 8.5 (E8.5). Heterozygous mice were fertile and had no aberrant phenotype, despite a lower ATR mRNA level. No increase was observed in the sensitivity of ATR(+/-) embryonic stem (ES) cells to a variety of DNA-damaging agents. Attempts to target the remaining wild-type ATR allele in heterozygous ATR(+/-) ES cells failed, supporting the idea that loss of both alleles of the ATR gene, even at the ES-cell level, is lethal. Thus, in contrast to the closely related checkpoint gene ATM, ATR has an essential function in early mammalian development.

ATM germline mutations in classical ataxia-telangiectasia patients in the Dutch population.

Germline mutations in the ATM gene are responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T). In our study, we have determined the ATM mutation spectrum in 19 classical A-T patients, including some immigrant populations, as well as 12 of Dutch ethnic origin. Both the protein truncation test (PTT) and the restriction endonuclease fingerprinting (REF) method were used and compared for their detection efficiency, identifying 76% and 60% of the mutations, respectively. Most patients were found to be compound heterozygote. Seventeen mutations were distinct, of which 10 were not reported previously. Mutations are small deletions or point mutations frequently affecting splice sites. Moreover, a 16.7-kb genomic deletion of the 3' end of the gene, most likely a result of recombination between two LINE elements, was identified. The most frequently found mutation, identified in three unrelated Turkish A-T individuals, was previously described to be a Turkish A-T founder mutation. The presence of a founder mutation among relatively small ethnic population groups in Western Europe could indicate a high carrier frequency in such communities. In patients of Dutch ethnic origin, however, no significant founder effect could be identified. The observed genetic heterogeneity including the relative high percentage of splice-site mutations had no reflection on the phenotype. All patients manifested classical A-T and increased cellular radioresistant DNA synthesis.

A G-->A transition creates a branch point sequence and activation of a cryptic exon, resulting in the hereditary disorder neurofibromatosis 2.

We describe a G-->A transition within intron 5 of the NF2 gene. This mutation creates a consensus splice branch point sequence. To our knowledge this is the first report of a mutation that creates a functional branch point sequence in a human hereditary disorder. The new branch point sequence is located 18 bp upstream of a consensus splice acceptor site. A consensus splice donor site is found 106 bp 3' of the acceptor site. Asa consequence the G-->A transition results in an alternatively spliced mRNA containing an additional exon 5a of 106 bp derived from intron sequences. We cloned the mutant cDNA and show that due to an in-frame stop codon the cDNA codes for a truncated NF2 protein. The mutation was observed in three affected members of an NF2 family. In a tumour of one of the family members both alternatively spliced and wild-type mRNA were found, although the wild-type allele of the gene is absent due to an interstitial deletion on chromosome 22. We also show that immunoprecipitations reveal the presence of full-length wild-type NF2 protein in the tumour lysate. These data support the hypothesis that some degree of normal splicing of the mutant precursor RNA is taking place. It is therefore likely that this residual activity of the mutant allele explains the relatively mild phenotype in the family. These data also indicate that complete inactivation of the gene is not required for tumour formation.