Genotype-phenotype relationships in ataxia-telangiectasia and variants.

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity, and cancer predisposition. A-T cells are sensitive to ionizing radiation and radiomimetic chemicals and fail to activate cell-cycle checkpoints after treatment with these agents. The responsible gene, ATM, encodes a large protein kinase with a phosphatidylinositol 3-kinase-like domain. The typical A-T phenotype is caused, in most cases, by null ATM alleles that truncate or severely destabilize the ATM protein. Rare patients with milder manifestations of the clinical or cellular characteristics of the disease have been reported and have been designated "A-T variants." A special variant form of A-T is A-TFresno, which combines a typical A-T phenotype with microcephaly and mental retardation. The possible association of these syndromes with ATM is both important for understanding their molecular basis and essential for counseling and diagnostic purposes. We quantified ATM-protein levels in six A-T variants, and we searched their ATM genes for mutations. Cell lines from these patients exhibited considerable variability in radiosensitivity while showing the typical radioresistant DNA synthesis of A-T cells. Unlike classical A-T patients, these patients exhibited 1%-17% of the normal level of ATM. The underlying ATM genotypes were either homozygous for mutations expected to produce mild phenotypes or compound heterozygotes for a mild and a severe mutation. An A-TFresno cell line was found devoid of the ATM protein and homozygous for a severe ATM mutation. We conclude that certain "A-T variant" phenotypes represent ATM mutations, including some of those without telangiectasia. Our findings extend the range of phenotypes associated with ATM mutations.     

An essential function for NBS1 in the prevention of ataxia and cerebellar defects

Nijmegen breakage syndrome (NBS), ataxia telangiectasia and ataxia telangiectasia-like disorder (ATLD) show overlapping phenotypes such as growth retardation, microcephaly, cerebellar developmental defects and ataxia. However, the molecular pathogenesis of these neurological defects remains elusive. Here we show that inactivation of the Nbn gene (also known as Nbs1) in mouse neural tissues results in a combination of the neurological anomalies characteristic of NBS, ataxia telangiectasia and ATLD, including microcephaly, growth retardation, cerebellar defects and ataxia. Loss of Nbn causes proliferation arrest of granule cell progenitors and apoptosis of postmitotic neurons in the cerebellum. Furthermore, Nbn-deficient neuroprogenitors show proliferation defects (but not increased apoptosis) and contain more chromosomal breaks, which are accompanied by ataxia telangiectasia mutated protein (ATM)-mediated p53 activation. Notably, depletion of p53 substantially rescues the neurological defects of Nbn mutant mice. This study gives insight into the physiological function of NBS1 (the Nbn gene product) and the function of the DNA damage response in the neurological anomalies of NBS, ataxia telangiectasia and ATLD.     

Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.     

Investigation of the functional link between ATM and NBS1 in the DNA damage response in the mouse cerebellum

Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are related genomic instability syndromes characterized by neurological deficits. The NBS1 protein that is defective in NBS is a component of the Mre11/RAD50/NBS1 (MRN) complex, which plays a major role in the early phase of the complex cellular response to double strand breaks (DSBs) in the DNA. Among others, Mre11/RAD50/NBS1 is required for timely activation of the protein kinase ATM (A-T, mutated), which is missing or inactivated in patients with A-T. Understanding the molecular pathology of A-T, primarily its cardinal symptom, cerebellar degeneration, requires investigation of the DSB response in cerebellar neurons, particularly Purkinje cells, which are the first to be lost in A-T patients. Cerebellar cultures derived from mice with different mutations in DNA damage response genes is a useful experimental system to study malfunctioning of the damage response in the nervous system. To clarify the interrelations between murine Nbs1 and Atm, we generated a mouse strain with specific disruption of the Nbs1 gene in the central nervous system on the background of general Atm deficiency (Nbs1-CNS-Δ//Atm(-/-)). This genotype exacerbated several features of both conditions and led to a markedly reduced life span, dramatic decline in the number of cerebellar granule neurons with considerable cerebellar disorganization, abolishment of the white matter, severe reduction in glial cell proliferation, and delayed DSB repair in cerebellar tissue. Combined loss of Nbs1 and Atm in the CNS significantly abrogated the DSB response compared with the single mutation genotypes. Importantly, the data indicate that Atm has cellular roles not regulated by Nbs1 in the murine cerebellum.     

Ataxia telangiectasia: new neurons and ATM

Despite the rarity of the human autosomal recessive disease ataxia telangiectasia (A-T) (affecting approximately 1/40000-1/100000), interest in the function of the mutated gene product (ATM) in this syndrome is intense. Mutation of this single gene can lead to a diverse array of features, including cancer, immune defects, infertility and radiosensitivity. However, it is the pronounced and debilitating neurodegeneration that is the hallmark of this disease. Thus, from a clinical perspective, it is ATM function in the nervous system that, arguably, is the most important to understand. Although the case for DNA damage as a causative factor for neurodegeneration in A-T is compelling, new data point to a possible link to defects in neurogenesis. Thus, whereas ATM is important for nervous system development, it could also be important for adult neurogenesis.     

Autosomal recessive cerebellar ataxias

Autosomal recessive cerebellar ataxias (ARCA) are a heterogeneous group of rare neurological disorders involving both central and peripheral nervous system, and in some case other systems and organs, and characterized by degeneration or abnormal development of cerebellum and spinal cord, autosomal recessive inheritance and, in most cases, early onset occurring before the age of 20 years. This group encompasses a large number of rare diseases, the most frequent in Caucasian population being Friedreich ataxia (estimated prevalence 2–4/100,000), ataxia-telangiectasia (1–2.5/100,000) and early onset cerebellar ataxia with retained tendon reflexes (1/100,000). Other forms ARCA are much less common. Based on clinicogenetic criteria, five main types ARCA can be distinguished: congenital ataxias (developmental disorder), ataxias associated with metabolic disorders, ataxias with a DNA repair defect, degenerative ataxias, and ataxia associated with other features. These diseases are due to mutations in specific genes, some of which have been identified, such as frataxin in Friedreich ataxia, α-tocopherol transfer protein in ataxia with vitamin E deficiency (AVED), aprataxin in ataxia with oculomotor apraxia (AOA1), and senataxin in ataxia with oculomotor apraxia (AOA2). Clinical diagnosis is confirmed by ancillary tests such as neuroimaging (magnetic resonance imaging, scanning), electrophysiological examination, and mutation analysis when the causative gene is identified. Correct clinical and genetic diagnosis is important for appropriate genetic counseling and prognosis and, in some instances, pharmacological treatment. Due to autosomal recessive inheritance, previous familial history of affected individuals is unlikely. For most ARCA there is no specific drug treatment except for coenzyme Q10 deficiency and abetalipoproteinemia.     

A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci

The hereditary disorder ataxia telangiectasia (A-T) is associated with striking cellular radiosensitivity that cannot be attributed to the characterized cell cycle checkpoint defects. By epistasis analysis, we show that ataxia telangiectasia mutated protein (ATM) and Artemis, the protein defective in patients with RS-SCID, function in a common double-strand break (DSB) repair pathway that also requires H2AX, 53BP1, Nbs1, Mre11, and DNA-PK. We show that radiation-induced Artemis hyperphosphorylation is ATM dependent. The DSB repair process requires Artemis nuclease activity and rejoins approximately 10% of radiation-induced DSBs. Our findings are consistent with a model in which ATM is required for Artemis-dependent processing of double-stranded ends with damaged termini. We demonstrate that Artemis is a downstream component of the ATM signaling pathway required uniquely for the DSB repair function but dispensable for ATM-dependent cell cycle checkpoint arrest. The significant radiosensitivity of Artemis-deficient cells demonstrates the importance of this component of DSB repair to survival.     

The controlling role of ATM in homologous recombinational repair of DNA damage

The human genetic disorder ataxia telangiectasia (A-T), caused by mutation in the ATM gene, is characterized by chromosomal instability, radiosensitivity and defective cell cycle checkpoint activation. DNA double-strand breaks (dsbs) persist in A-T cells after irradiation, but the underlying defect is unclear. To investigate ATM's interactions with dsb repair pathways, we disrupted ATM along with other genes involved in the principal, complementary dsb repair pathways of homologous recombination (HR) or non-homologous end-joining (NHEJ) in chicken DT40 cells. ATM(-/-) cells show altered kinetics of radiation-induced Rad51 and Rad54 focus formation. Ku70-deficient (NHEJ(-)) ATM(-/-) chicken DT40 cells show radiosensitivity and high radiation-induced chromosomal aberration frequencies, while Rad54-defective (HR(-)) ATM(-/-) cells show only slightly elevated aberration levels after irradiation, placing ATM and HR on the same pathway. These results reveal that ATM defects impair HR-mediated dsb repair and may link cell cycle checkpoints to HR activation.     

ATM, the Mre11/Rad50/Nbs1 complex, and topoisomerase I are concentrated in the nucleus of Purkinje neurons in the juvenile human brain

The genetic disease ataxia telangiectasia (AT) results from mutations in the ataxia telangiectasia mutated (ATM) gene. AT patients develop a progressive degeneration of cerebellar Purkinje neurons. Surprisingly, while ATM plays a criticial role in the cellular reponse to DNA damage, previous studies have localized ATM to the cytoplasm of rodent and human Purkinje neurons. Here we show that ATM is primarily localized to the nucleus in cerebellar Purkinje neurons in postmortem human brain tissue samples, although some light cytoplasmic ATM staining was also observed. No ATM staining was observed in brain tissue samples from AT patients, verifying the specificity of the antibody. We also found that antibodies against components of the Mre11/Rad50/Nbs1 (MRN) complex showed strong staining in Purkinje cell nuclei. However, while ATM is present in both the nucleoplasm and nucleolus, MRN proteins are excluded from the nucleolus. We also observed very high levels of topoisomerase 1 (TOP1) in the nucleus, and specifically the nucleolus, of human Purkinje neurons. Our results have direct implications for understanding the mechanisms of neurodegeneration in AT and AT-like disorder.     

Requirement of the MRN complex for ATM activation by DNA damage

The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. Here we show that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD.     

Accumulation of DNA damage and reduced levels of nicotine adenine dinucleotide in the brains of Atm-deficient mice.

Ataxia-telangiectasia (A-T) is a human genetic disorder caused by mutational inactivation of the ATM gene. A-T patients display a pleiotropic phenotype, in which a major neurological feature is progressive ataxia due to degeneration of cerebellar Purkinje and granule neurons. Disruption of the mouse Atm locus creates a murine model of A-T that exhibits most of the clinical and cellular features of the human disease, but the neurological phenotype is barely expressed. We present evidence for the accumulation of DNA strand breaks in the brains of Atm(-/-), supporting the notion that ATM plays a major role in maintaining genomic stability. We also show a perturbation of the steady state levels of pyridine nucleotides. There is a significant decrease in both the reduced and the oxidized forms of NAD and in the total levels of NADP(T) and NADP(+) in the brains of Atm(-/-) mice. The changes in NAD(T), NADH, NAD(+), NADP(T), and NADP(+) were progressive and observed primarily in the cerebellum of 4-month-old Atm(-/-) mice. Higher rates of mitochondrial respiration were also recorded in 4-month-old Atm(-/-) cerebella. Taken together, our findings support the hypothesis that absence of functional ATM results in continuous stress, which may be an important cause of the degeneration of cerebellar neurons in A-T.     

Hypothesis: Ataxia-telangiectasia: Is ATM a sensor of oxidative damage and stress?

Ataxia-telangiectasia (A-T) is a pleiotropic recessive disorder characterized cerebellar ataxia, immunodeficiency, specific developmental defects, profound predisposition to cancer and acute radiosensitivity. Functional inactivation of single gene product, ATM, accounts for this compound phenotype. We suggest that ATM acts as a sensor of reactive oxygen species and/or oxidative damage cellular macromolecules, including DNA. In turn, ATM induces signalling through multiple pathways, thereby coordinating acute phase stress responses with cell cycle checkpoint control and repair of oxidative damage. Absence of ATM is proposed to limit the repair of insidious oxidative damage that can occur under normal physiological conditions, ultimately leading to apoptosis of particularly sensitive cells, such as neurons and thymocytes.     

Nbs1 is required for ATR-dependent phosphorylation events

Nijmegen breakage syndrome (NBS) is characterised by microcephaly, developmental delay, characteristic facial features, immunodeficiency and radiosensitivity. Nbs1, the protein defective in NBS, functions in ataxia telangiectasia mutated protein (ATM)-dependent signalling likely facilitating ATM phosphorylation events. While NBS shares overlapping characteristics with ataxia telangiectasia, it also has features overlapping with ATR-Seckel (ATR: ataxia-telangiectasia and Rad3-related protein) syndrome, a subclass of Seckel syndrome mutated in ATR. We show that Nbs1 also facilitates ATR-dependent phosphorylation. NBS cell lines show a similar defect in ATR phosphorylation of Chk1, c-jun and p-53 in response to UV irradiation- and hydroxyurea (HU)-induced replication stalling. They are also impaired in ubiquitination of FANCD2 after HU treatment, which is ATR dependent. Following HU-induced replication arrest, NBS and ATR-Seckel cells show similarly impaired G2/M checkpoint arrest and an impaired ability to restart DNA synthesis at stalled replication forks. Moreover, NBS cells fail to retain ATR in the nucleus following HU treatment and extraction. Our findings suggest that Nbs1 functions in both ATR- and ATM-dependent signalling. We propose that the NBS clinical features represent the result of these combined defects.     

Mutations associated with variant phenotypes in ataxia-telangiectasia.

We have identified 14 families with ataxia-telangiectasia (A-T) in which mutation of the ATM gene is associated with a less severe clinical and cellular phenotype (approximately 10%-15% of A-T families identified in the United Kingdom). In 10 of these families, all the homozygotes have a 137-bp insertion in their cDNA caused by a point mutation in a sequence resembling a splice-donor site. The second A-T allele has a different mutation in each patient. We show that the less severe phenotype in these patients is caused by some degree of normal splicing, which occurs as an alternative product from the insertion-containing allele. The level of the 137-bp PCR product containing the insertion was lowest in two patients who showed a later onset of cerebellar ataxia. A further four families who do not have this insertion have been identified. Mutations detected in two of four of these are missense mutations, normally rare in A-T patients. The demonstration of mutations giving rise to a slightly milder phenotype in A-T raises the interesting question of what range of phenotypes might occur in individuals in whom both mutations are milder. One possibility might be that individuals who are compound heterozygotes for ATM mutations are more common than we realize.     

Two unrelated patients with MRE11A mutations and Nijmegen breakage syndrome-like severe microcephaly

MRE11 and NBS1 function together as components of a MRE11/RAD50/NBS1 protein complex, however deficiency of either protein does not result in the same clinical features. Mutations in the NBN gene underlie Nijmegen breakage syndrome (NBS), a chromosomal instability syndrome characterized by microcephaly, bird-like faces, growth and mental retardation, and cellular radiosensitivity. Additionally, mutations in the MRE11A gene are known to lead to an ataxia-telangiectasia-like disorder (ATLD), a late-onset, slowly progressive variant of ataxia-telangiectasia without microcephaly. Here we describe two unrelated patients with NBS-like severe microcephaly (head circumference -10.2 SD and -12.8 SD) and mutations in the MRE11A gene. Both patients were compound heterozygotes for a truncating or missense mutation and carried a translationally silent mutation. The truncating and missense mutations were assumed to be functionally debilitating. The translationally silent mutation common to both patients had an effect on splicing efficiency resulting in reduced but normal MRE11 protein. Their levels of radiation-induced activation of ATM were higher than those in ATLD cells.     

ATM gene expression is associated with differentiation and angiogenesis in infiltrating breast carcinomas

The product of the ATM gene, mutated in the human genetic disorder ataxia-telangiectasia (A-T) plays a key role in the detection and repair of DNA double-strand breaks. A-T is defined by progressive cerebellar ataxia, telangiectasia, sensitivity to ionising radiation and genomic instability with cancer predisposition. On the other hand, increased angiogenesis is essential for tumor growth and metastasis. The aim of this study was to investigate ATM expression in breast carcinomas and its relationship to neoangiogenesis. METHODS AND RESULTS: Fifty-two breast tumors from 51 patients, 38 of them with concomitant in situ component (CIS), were analyzed by immunohistochemistry for the expression of ATM. CD34 expression was used for the morphometric evaluation of vasculature. ATM was positive in 1 to 10% of normal epithelial cells. ATM expression was reduced in 55.8% of infiltrating carcinomas, non-reduced in 34.6%, and increased in 9.6%. Expression of ATM in CIS was similar to the infiltrating component in 71% of cases and reduced in 23.7% of them. High-grade ductal infiltrating carcinomas showed lower ATM expression than low-grade ones. Reduced ATM expression also correlated with increased microvascular area. CONCLUSIONS: Reduced ATM expression in breast carcinomas correlated with tumor differentiation and increased microvascular parameters, supporting its role in neoangiogenesis and tumor progression in breast carcinogenesis.     

Radiation, DNA damage and cancer

The characterization of the rare, radiation-sensitive and cancer-prone syndromes, ataxia telangiectasia and Nijmegen breakage syndrome, has demonstrated that genetic predisposition increases the risk of developing cancer after exposure to ionizing radiation (IR). Molecular analyses of these disorders provide valuable insights into the normal function of these two gene products in the cellular response to IR-induced DNA damage. Their contribution to a cellular radiosensitive phenotype and their role in sporadic cancers can now be fully assessed. For example, the gene ataxia telangiectasia mutated (ATM) has recently been shown to be a tumour suppressor gene in T-cell prolymphocytic leukaemia, and there is increasing evidence that individuals with one mutated ATM or Nijmegen breakage syndrome (NBS1) allele have an increased predisposition to cancer.     

Whole-exome sequencing identifies homozygous AFG3L2 mutations in a spastic ataxia-neuropathy syndrome linked to mitochondrial m-AAA proteases

We report an early onset spastic ataxia-neuropathy syndrome in two brothers of a consanguineous family characterized clinically by lower extremity spasticity, peripheral neuropathy, ptosis, oculomotor apraxia, dystonia, cerebellar atrophy, and progressive myoclonic epilepsy. Whole-exome sequencing identified a homozygous missense mutation (c.1847G>A; p.Y616C) in AFG3L2, encoding a subunit of an m-AAA protease. m-AAA proteases reside in the mitochondrial inner membrane and are responsible for removal of damaged or misfolded proteins and proteolytic activation of essential mitochondrial proteins. AFG3L2 forms either a homo-oligomeric isoenzyme or a hetero-oligomeric complex with paraplegin, a homologous protein mutated in hereditary spastic paraplegia type 7 (SPG7). Heterozygous loss-of-function mutations in AFG3L2 cause autosomal-dominant spinocerebellar ataxia type 28 (SCA28), a disorder whose phenotype is strikingly different from that of our patients. As defined in yeast complementation assays, the AFG3L2(Y616C) gene product is a hypomorphic variant that exhibited oligomerization defects in yeast as well as in patient fibroblasts. Specifically, the formation of AFG3L2(Y616C) complexes was impaired, both with itself and to a greater extent with paraplegin. This produced an early-onset clinical syndrome that combines the severe phenotypes of SPG7 and SCA28, in additional to other "mitochondrial" features such as oculomotor apraxia, extrapyramidal dysfunction, and myoclonic epilepsy. These findings expand the phenotype associated with AFG3L2 mutations and suggest that AFG3L2-related disease should be considered in the differential diagnosis of spastic ataxias.     

Altered Telomere Nuclear Matrix Interactions and Nucleosomal Periodicity in Ataxia Telangiectasia Cells before and after Ionizing Radiation Treatment

Cells derived from ataxia telangiectasia (A-T) patients show a prominent defect at chromosome ends in the form of chromosome end-to-end associations, also known as telomeric associations, seen at G(1), G(2), and metaphase. Recently, we have shown that the ATM gene product, which is defective in the cancer-prone disorder A-T, influences chromosome end associations and telomere length. A possible hypothesis explaining these results is that the defective telomere metabolism in A-T cells are due to altered interactions between the telomeres and the nuclear matrix. We examined these interactions in nuclear matrix halos before and after radiation treatment. A difference was observed in the ratio of soluble versus matrix-associated telomeric DNA between cells derived from A-T and normal individuals. Ionizing radiation treatment affected the ratio of soluble versus matrix-associated telomeric DNA only in the A-T cells. To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, we examined such interactions in human cells expressing either a dominant-negative effect or complementation of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix similar to that of A-T cells. A-T fibroblasts transfected with wild-type ATM gene had corrected telomere-nuclear matrix interactions. Further, we found that A-T cells had different micrococcal nuclease digestion patterns compared to normal cells before and after irradiation, indicating differences in nucleosomal periodicity in telomeres. These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix, and alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene.