Functional and computational assessment of missense variants in the ataxia-telangiectasia mutated (ATM) gene: mutations with increased cancer risk.

The functional consequences of missense variants are often difficult to predict. This becomes especially relevant when DNA sequence changes are used to determine a diagnosis or prognosis. To analyze the consequences of 12 missense variants in patients with mild forms of ataxia-telangiectasia (A-T), we employed site-directed mutagenesis of ataxia-telangiectasia mutated (ATM) cDNA followed by stable transfections into a single A-T cell line to isolate the effects of each allele on the cellular phenotype. After induction of the transfected cells with CdCl2, we monitored for successful ATM transcription and subsequently assessed: 1) intracellular ATM protein levels; 2) ionizing radiation (IR)-induced ATM kinase activity; and 3) cellular radiosensitivity. We then calculated SIFT and PolyPhen scores for the missense changes. Nine variants produced little or no correction of the A-T cellular phenotype and were interpreted to be ATM mutations; SIFT/PolyPhen scores supported this. Three variants corrected the cellular phenotype, suggesting that they represented benign variants or polymorphisms. SIFT and PolyPhen scores supported the functional analyses for one of these variants (c.1709T>C); the other two were predicted to be "not tolerated" (c.6188G>A and c.6325T>G) and were classified as "operationally neutral." Genotype/phenotype relationships were compared: three deleterious missense variants were associated with an increased risk of cancer (c.6679C>T, c.7271T>G, and c.8494C>T). In situ mutagenesis represents an effective experimental approach for distinguishing deleterious missense mutations from benign or operationally neutral missense variants.

ATM gene founder haplotypes and associated mutations in Polish families with ataxia-telangiectasia.

Ataxia-telangiectasia (A-T) is an early onset autosomal recessive ataxia associated with characteristic chromosomal aberrations, cell cycle checkpoint defects, cancer susceptibility, and sensitivity to ionizing radiation. We utilized the protein truncation test (PTT), and single strand conformation polymorphism (SSCP) on cDNA, as well as denaturing high performance liquid chromatography (dHPLC) on genomic DNA (gDNA) to screen for mutations in 24 Polish A-T families. Twenty-six distinct Short Tandem Repeat (STR) haplotypes were identified. Three founder mutations accounted for 58% of the alleles. Three-quarters of the families had at least one recurring (shared) mutation, which was somewhat surprising given the low frequency of consanguinity in Poland. STR haplotyping greatly improved the efficiency of mutation detection. We identified 44 of the expected 48 mutations (92%): sixty-nine percent were nonsense mutations, 23% caused aberrant splicing, and 5% were missense mutations. Four mutations have not been previously described. Two of the Polish mutations have been observed previously in Amish and Mennonite A-T patients; this is compatible with historical records. Shared mutations shared the same Single Nucleotide Polymorphism (SNP) and STR haplotypes, indicating common ancestries. The Mennonite mutation, 5932 G>T, is common in Russian A-T families, and the STR haplovariants are the same in both Poland and Russia. Attempts to correlate phenotypes with genotypes were inconclusive due to the limited numbers of patients with identical mutations.