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We studied 10 Mennonite patients who carry the c.6200C>A missense mutation (p.A2067D) in the ATM gene, all of whom exhibited a phenotypic variant of ataxia‐telangiectasia (A‐T) that is characterized by early‐onset dystonia and late‐onset mild ataxia, as previously described. This report provides the pathogenetic evidence for this mutation on cellular functions. Several patients have developed cancer and subsequently experienced life‐threatening adverse reactions to radiation (radiotoxicity) and/or chemotherapy. As the c.6200C>A mutation is, thus far, unique to the Mennonite population and is always associated with the same haplotype or haplovariant, it was important to rule out any possible confounding DNA variant on the same haplotype. Lymphoblastoid cells derived from Mennonite patients expressed small amounts of ATM protein, which had no autophosphorylation activity at ATM Ser1981, and trace‐to‐absent transphosphorylation of downstream ATM targets. A‐T lymphoblastoid cells stably transfected with ATM cDNA which had been mutated for c.6200C>A did not show a detectable amount of ATM protein. The same stable cell line with mutated ATM cDNA also showed a trace‐to‐absent transphosphorylation of downstream ATM targets SMC1pSer966 and KAP1pSer824. From these results, we conclude that c.6200A is the disease‐causing ATM mutation on this haplotype. The presence of at least trace amounts of ATM kinase activity on some immunoblots may account for the late‐onset, mild ataxia of these patients. The cause of the dystonia remains unclear. Because this dystonia‐ataxia phenotype is often encountered in the Mennonite population in association with cancer and adverse reactions to chemotherapy, an early diagnosis is important. We describe and characterize a specific missense change in the ATM gene that associates with a very atypical A‐T phenotype of early‐onset dystonia, mild ataxia, as well as a high cancer risk and hypersensitivity to radiation therapy and chemotherapy. We identify a unique mutation in the ATM gene, c.6200C>A, and a unique associated STR (short tandem repeat) haplotype, and demonstrate with stable transfectants that, although the wild‐type ATM transcript (6200C variant) abrogates the cellular phenotype of A‐T cells, an otherwise identical transcript containing the 6200A variant does not. We show that this mutation is unique, to date, in Mennonite Anabaptists.

Chemical-induced read through of premature stop codons might be exploited as a potential treatment strategy for genetic disorders caused by nonsense mutations. Despite the promise of this approach, only a few read-through compounds (RTCs) have been discovered to date. These include aminoglycosides (e.g., gentamicin and G418) and nonaminoglycosides (e.g., PTC124 and RTC13). The therapeutic benefits of these RTCs remain to be determined. In an effort to find new RTCs, we screened an additional ∼36,000 small molecular weight compounds using a high-throughput screening (HTS) assay that we had previously developed and identified two novel RTCs, GJ071, and GJ072. The activity of these two compounds was confirmed in cells derived from ataxia telangiectasia (A-T) patients with three different types of nonsense mutation in the ATM gene. Both compounds showed activity comparable to stop codons (TGA, TAG, and TAA) PTC124 and RTC13. Early structure-activity relationship studies generated eight active analogs of GJ072. Most of those analogs were effective on all three stop codons. GJ071 and GJ072, and some of the GJ072 analogs, appeared to be well tolerated by A-T cells. We also identified another two active RTCs in the primary screen, RTC204 and RTC219, which share a key structural feature with GJ072 and its analogs.

In an effort to explore the possible causes of human radiosensitivity and identify more rapid assays for cellular radiosensitivity, we interrogated a set of assays that evaluate cellular functions involved in recognition and repair of DNA double-strand breaks: (1) neutral comet assay, (2) radiation-induced γ-H2AX focus formation, (3) the temporal kinetics of structural maintenance of chromosomes 1 phosphorylation, (4) intra-S-phase checkpoint integrity, and (5) mitochondrial respiration. We characterized a unique panel of 19 “radiosensitive” human lymphoblastoid cell lines from individuals with undiagnosed diseases suggestive of a DNA repair disorder. Radiosensitivity was defined by reduced cellular survival using a clonogenic survival assay. Each assay identified cell lines with defects in DNA damage response functions. The highest concordance rate observed, 89% (17/19), was between an abnormal neutral comet assay and reduced survival by the colony survival assay. Our data also suggested that the neutral comet assay would be a more rapid surrogate for analyzing DNA repair/processing disorders.

We studied 10 Mennonite patients who carry the c.6200C>A missense mutation (p.A2067D) in the ATM gene, all of whom exhibited a phenotypic variant of ataxia‐telangiectasia (A‐T) that is characterized by early‐onset dystonia and late‐onset mild ataxia, as previously described. This report provides the pathogenetic evidence for this mutation on cellular functions. Several patients have developed cancer and subsequently experienced life‐threatening adverse reactions to radiation (radiotoxicity) and/or chemotherapy. As the c.6200C>A mutation is, thus far, unique to the Mennonite population and is always associated with the same haplotype or haplovariant, it was important to rule out any possible confounding DNA variant on the same haplotype. Lymphoblastoid cells derived from Mennonite patients expressed small amounts of ATM protein, which had no autophosphorylation activity at ATM Ser1981, and trace‐to‐absent transphosphorylation of downstream ATM targets. A‐T lymphoblastoid cells stably transfected with ATM cDNA which had been mutated for c.6200C>A did not show a detectable amount of ATM protein. The same stable cell line with mutated ATM cDNA also showed a trace‐to‐absent transphosphorylation of downstream ATM targets SMC1pSer966 and KAP1pSer824. From these results, we conclude that c.6200A is the disease‐causing ATM mutation on this haplotype. The presence of at least trace amounts of ATM kinase activity on some immunoblots may account for the late‐onset, mild ataxia of these patients. The cause of the dystonia remains unclear. Because this dystonia‐ataxia phenotype is often encountered in the Mennonite population in association with cancer and adverse reactions to chemotherapy, an early diagnosis is important.

We studied 10 Mennonite patients who carry the c.6200C>A missense mutation (p.A2067D) in the ATM gene, all of whom exhibited a phenotypic variant of ataxia-telangiectasia (A-T) that is characterized by early-onset dystonia and late-onset mild ataxia, as previously described. This report provides the pathogenetic evidence for this mutation on cellular functions. Several patients have developed cancer and subsequently experienced life-threatening adverse reactions to radiation (radiotoxicity) and/or chemotherapy. As the c.6200C>A mutation is, thus far, unique to the Mennonite population and is always associated with the same haplotype or haplovariant, it was important to rule out any possible confounding DNA variant on the same haplotype. Lymphoblastoid cells derived from Mennonite patients expressed small amounts of ATM protein, which had no autophosphorylation activity at ATM Ser1981, and trace-to-absent transphosphorylation of downstream ATM targets. A-T lymphoblastoid cells stably transfected with ATM cDNA which had been mutated for c.6200C>A did not show a detectable amount of ATM protein. The same stable cell line with mutated ATM cDNA also showed a trace-to-absent transphosphorylation of downstream ATM targets SMC1pSer966 and KAP1pSer824. From these results, we conclude that c.6200A is the disease-causing ATM mutation on this haplotype. The presence of at least trace amounts of ATM kinase activity on some immunoblots may account for the late-onset, mild ataxia of these patients. The cause of the dystonia remains unclear. Because this dystonia-ataxia phenotype is often encountered in the Mennonite population in association with cancer and adverse reactions to chemotherapy, an early diagnosis is important. We describe and characterize a specific missense change in the ATM gene that associates with a very atypical A-T phenotype of early-onset dystonia, mild ataxia, as well as a high cancer risk and hypersensitivity to radiation therapy and chemotherapy. We identify a unique mutation in the ATM gene, c.6200C>A, and a unique associated STR (short tandem repeat) haplotype, and demonstrate with stable transfectants that, although the wild-type ATM transcript (6200C variant) abrogates the cellular phenotype of A-T cells, an otherwise identical transcript containing the 6200A variant does not. We show that this mutation is unique, to date, in Mennonite Anabaptists.