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

Abstract

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.

Figure 1

Immunoblots showing ATM expression in AT7LA cells transfected with normal pMAT1 or variants. Constructs were induced with CdCl₂. Mre11 was used as a protein loading control. NAT2 is a normal LCL.

Figure 2

A. AT7LA cells transfected with pMAT1 were induced for 2, 4, 8, and 17h with CdCl₂. Significant levels of ATM mRNA expression were detected at 8 and 17h post-induction. B. ATM mRNA levels at 8h post-induction, detected by quantitative real time PCR. First bar on the left represents endogenous mRNA, without induction by CdCl₂. Data were normalized to pMAT1-induced transcription levels (second bar).

Figure 2

A. AT7LA cells transfected with pMAT1 were induced for 2, 4, 8, and 17h with CdCl₂. Significant levels of ATM mRNA expression were detected at 8 and 17h post-induction. B. ATM mRNA levels at 8h post-induction, detected by quantitative real time PCR. First bar on the left represents endogenous mRNA, without induction by CdCl₂. Data were normalized to pMAT1-induced transcription levels (second bar).

Figure 3

Immunoblot showing radiation-induced phosphorylation. A. ATM-S1981. Stably transfected cells were induced with CdCl₂ and irradiated with 2 Gy. β-actin was used as a protein loading control. B. SMC1-S966. Stably transfected cells were induced with CdCl₂ and irradiated with 10 Gy. Total SMC1 levels are shown in bottom panel, using an antibody that does not crossreact with phosphorylated SMC1.

Figure 3

Immunoblot showing radiation-induced phosphorylation. A. ATM-S1981. Stably transfected cells were induced with CdCl₂ and irradiated with 2 Gy. β-actin was used as a protein loading control. B. SMC1-S966. Stably transfected cells were induced with CdCl₂ and irradiated with 10 Gy. Total SMC1 levels are shown in bottom panel, using an antibody that does not crossreact with phosphorylated SMC1.

Figure 4

A. The radiosensitivity of AT7LA cells was abrogated by transfection with wild type construct, pMAT1. NAT9: normal; AT7LA: AT cell. Bar: speckled area denotes radiosensitivity range; diagonal-hatching denotes normal range. B. The radiosensitivity of each transfected LCL. Normal, intermediate and A-T ranges are indicated by dotted lines. NAT2 is a control LCL. C, control; M, mutation; ON, ‘operationally neutral’ variant; P, benign polymorphism.

Figure 4

A. The radiosensitivity of AT7LA cells was abrogated by transfection with wild type construct, pMAT1. NAT9: normal; AT7LA: AT cell. Bar: speckled area denotes radiosensitivity range; diagonal-hatching denotes normal range. B. The radiosensitivity of each transfected LCL. Normal, intermediate and A-T ranges are indicated by dotted lines. NAT2 is a control LCL. C, control; M, mutation; ON, ‘operationally neutral’ variant; P, benign polymorphism.