Improved diagnostic testing for ataxia–telangiectasia by immunoblotting of nuclear lysates for ATM protein expression

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Abstract

The laboratory diagnosis of ataxia–telangiectasia (A–T) currently relies upon measurement of serum alphafetoprotein (AFP) and cellular sensitivity to ionizing radiation. A previous report suggests that immunoblotting of whole cell lysates from lymphoblastoid cell lines (LCLs) might be informative for diagnosis. To further evaluate this possibility, and improve sensitivity, we performed immunoblotting for ATM protein on nuclear lysates of 71 consecutive radiosensitive LCLs that were established from patients with clinical features suggestive of A–T. Fifty-two LCLs (73%) contained no detectable ATM protein, with a representative sample (N=25) testing negative for ATM kinase activity, having at least one ATM mutation, and having elevated AFP levels; these results confirmed the diagnosis. Seventeen LCLs (24%) expressed intermediate or normal levels of ATM protein and exhibited normal ATM kinase activity; follow-up studies failed to detect ATM mutations and AFP levels were normal in all but three. Of the remaining two radiosensitive LCLs, one had 35% of normal protein with normal kinase activity and no ATM mutations. The other LCL had 9% of normal protein, with intermediate levels of kinase activity, a homozygous missense ATM mutation, and elevated AFP. Our data suggest that it is very uncommon to encounter bonafide A–T patients with more than trace amounts of ATM protein. We conclude that immunoblotting for ATM protein is of higher specificity for diagnosing A–T than radiosensitivity testing. In addition, we have documented in vitro radiosensitivity in other patients who share some clinical features with A–T.

Introduction

Presently, measurement of serum alphafetoprotein (AFP) and colony survival assay (CSA) are the only clinically validated tests for diagnosing ataxia–telangiectasia (A–T) [1], [2]. AFP is reported to be elevated in about 90% of A–T patients (reviewed in [3]). CSA measures the colony forming ability of lymphoblastoid cell lines (LCLs) after exposure to ionizing radiation (IR). It is abnormal in >99% of A–T LCLs, with a CSA score of <21% as the upper limit for radiosensitivity. On the other hand, we have also observed similar CSA levels of radiosensitivity in LCLs from other patients, including those with other DNA repair disorders (e.g., Nijmegen breakage syndrome), and with primary immunodeficiencies (e.g., X-linked agammaglobulinemia), as well as in patients with neurological findings similar to A–T but of unknown diagnosis [2], [4].

We reported previously that approximately 15% of patients suspected of having A–T had intermediate levels of ATM protein in whole cell lysates [5]. In the current study, we have refined this assay, with a more sensitive measurement of nuclear ATM protein, and added the assessment of ex vivo ATM kinase function. To evaluate the sensitivity and specificity of this new protocol, we characterized 71 consecutive radiosensitive LCLs. We observed that very few (<5%) bonafide A–T patients have more than trace amounts of ATM nuclear protein, and ATM kinase activity was absent in all of these. This study also further documents a subset of radiosensitive (in vitro) patients with as yet undefined disorders.

Section snippets

Radiosensitivity testing

Five to ten milliliters of heparinized blood was referred for testing from patients suspected of having A–T, mostly children under 4 years of age. Peripheral blood lymphocytes (PBL) were isolated on a Ficoll-Hypaque gradient (Amersham Pharmacia, Piscataway, NJ) and transformed with the Epstein–Barr virus. The transformed LCLs were maintained in 15% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA) at 37 °C and 5% CO2. To test for radiosensitivity,

Sensitivity of ATM immunoblotting using nuclear lysates

Immunoblotting of normal whole cell and nuclear lysates were compared in varying concentrations. When 7.5 μg protein was loaded, ATM protein was easily detected in nuclear lysates but was only barely detectable in whole cell lysates (Fig. 1A). At 1 μg, ATM protein was detectable only in trace amounts in nuclear lysates. An antibody to hMre11 protein was used as a control for the amount of protein loaded into each well; at 1 μg, protein was detectable only in nuclear lysates.

To assess the amount of

Discussion

Because the specificity of CSA testing for ataxia–telangiectasia (A–T) was not adequate by itself, we further evaluated the sensitivity and specificity of adjunctive immunoblotting to improve the laboratory diagnosis of A–T [1], [2], [5], [13]. Becker-Catania et al. [5] studied 123 unrelated A–T patients and found that 105 patients (85%) had no ATM protein in immunoblots of whole cell lysates. However, at the time of that report, mutation testing was still unavailable for many of those patients

Acknowledgements

This work was supported by grants from the US National Institutes of Health (NS36323, CA57569), the Ataxia–Telangiectasia Medical Research Foundation (Los Angeles), and the Joseph Drown Foundation. We acknowledge the technical assistance of Eric Olson and Kia Kham-Lee, who assisted in the dHPLC mutation screening.

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