Evidence of pathogenicity for the leaky splice variant c.1066-6T>G in ATM.

Mild clinical phenotypes of ataxia-telangiectasia (variant A-T) are associated with biallelic ATM variants resulting in residual function of the ATM kinase. At least one regulatory, missense, or leaky splice site mutation resulting in expression of ATM with low level kinase activity was identified in subjects with variant A-T. Studies on the pathogenicity of the germline splicing ATM variant c.1066-6T>G have provided conflicting results. Using whole-exome sequencing, we identified two splice site ATM variants, c.1066-6T>G; [p.?], and c.2250G>A, [p.Ile709_Lys750del], in a compound heterozygous state in a 27-year-old woman who had been diagnosed as having congenital ocular motor apraxia type Cogan in her childhood. Reappraisal of her clinical phenotype revealed consistency with variant A-T. Functional analyses showed reduced expression of ATM protein and residual activity of the ATM kinase at a level consistent with variant A-T. Our results provide evidence for pathogenicity of the leaky ATM splice site variant c.1066-6T>G.

Clinical and Biological Manifestation of RNF168 Deficiency in Two Polish Siblings.

Germline mutations in the RING finger protein gene RNF168 have been identified in a combined immunodeficiency disorder called RIDDLE syndrome. Since only two patients have been described with somewhat different phenotypes, there is need to identify further patients. Here, we report on two Polish siblings with RNF168 deficiency due to homozygosity for a novel frameshift mutation, c.295delG, that was identified through exome sequencing. Both patients presented with immunoglobulin deficiency, telangiectasia, cellular radiosensitivity, and increased alpha-fetoprotein (AFP) levels. The younger sibling had a more pronounced neurological and morphological phenotype, and she also carried an ATM gene mutation in the heterozygous state. Immunoblot analyses showed absence of RNF168 protein, whereas ATM levels and function were proficient in lymphoblastoid cells from both patients. Consistent with the absence of RNF168 protein, 53BP1 recruitment to DNA double-strand breaks (DSBs) after irradiation was undetectable in lymphoblasts or primary fibroblasts from either of the two patients. γH2AX foci accumulated normally but they disappeared with significant delay, indicating a severe defect in DSB repair. A comparison with the two previously identified patients indicates immunoglobulin deficiency, cellular radiosensitivity, and increased AFP levels as hallmarks of RNF168 deficiency. The variability in its clinical expression despite similar cellular phenotypes suggests that some manifestations of RNF168 deficiency may be modified by additional genetic or epidemiological factors.

Functional deficiency of NBN, the Nijmegen breakage syndrome protein, in a p.R215W mutant breast cancer cell line.

BACKGROUND: Mutations in NBN, the gene for Nijmegen Breakage Syndrome (NBS), are thought to predispose women to developing breast cancer, but a breast cancer cell line containing mutations in NBN has not yet been described. The p.R215W missense mutation occurs at sub-polymorphic frequencies in several populations. We aimed to investigate its functional impact in breast cancer cells from a carrier of this NBN mutation. METHODS: Breast cancer cell lines were screened by immunoblotting for NBN protein levels, and the NBN coding region was sequenced for mutation analysis. Radiosensitivity assays and functional studies were performed through immunocytochemistry and immunoblotting, and flow cytometry was employed to assess cell cycle progression. Impedance measurements were used to study the consequences of PARP1 inhibition. Statistical comparisons between cell lines were performed using t-tests. RESULTS: HCC1395 breast cancer cells exhibited reduced NBN protein levels. Direct sequencing identified the NBN p.R215W mutation in the hemizygous state, in addition to a truncation in BRCA1. Mutations in both genes were already present in the heterozygous state in the patient's germline. HCC1395 cells were highly radiosensitive, susceptible to apoptosis and were deficient in the formation of NBN foci. There was also evidence for some impairment in the formation of γH2AX, MDC1, and 53BP1 foci after irradiation; these foci appeared smaller and irregular compared with repair foci in wild-type cells, although ATM signalling was largely unaffected. In line with their deficiency in NBN and BRCA1, HCC1395 cells were particularly sensitive to PARP1 inhibition. CONCLUSION: Our results indicate that the p.R215W mutation in the HCC1395 breast cancer cell line impairs NBN function, making this cell line a potentially useful cellular model for studying defective NBN protein within a mutant BRCA1 background.

Protective role of RAD50 on chromatin bridges during abnormal cytokinesis.

Faithful chromosome segregation is required for preserving genomic integrity. Failure of this process may entail chromatin bridges preventing normal cytokinesis. To test whether RAD50, a protein normally involved in DNA double-strand break repair, is involved in abnormal cytokinesis and formation of chromatin bridges, we used immunocytochemical and protein interaction assays. RAD50 localizes to chromatin bridges during aberrant cytokinesis and subsequent stages of the cell cycle, either decorating the entire bridge or focally accumulating at the midbody zone. Ionizing radiation led to an ∼4-fold increase in the rate of chromatin bridges in an ataxia telangiectatica mutated (ATM)-dependent manner in human RAD50-proficient fibroblasts but not in RAD50-deficient cells. Cells with a RAD50-positive chromatin bridge were able to continue cell cycling and to progress through S phase (44%), whereas RAD50 knockdown caused a deficiency in chromatin bridges as well as an ∼4-fold prolonged duration of mitosis. RAD50 colocalized and directly interacted with Aurora B kinase and phospho-histone H3, and Aurora B kinase inhibition led to a deficiency in RAD50-positive bridges. Based on these observations, we propose that RAD50 is a crucial factor for the stabilization and shielding of chromatin bridges. Our study provides evidence for a hitherto unknown role of RAD50 in abnormal cytokinesis.

Functional characterization connects individual patient mutations in ataxia telangiectasia mutated (ATM) with dysfunction of specific DNA double-strand break-repair signaling pathways.

Ataxia telangiectasia mutated (ATM) has multiple functions in homologous recombination (HR) and nonhomologous end joining (NHEJ), which lead to conflicting data regarding its DNA double-strand break-repair (DSBR) functions in previous studies. To explore the effect of clinically relevant ATM mutations, we characterized DSBR between mutated EGFP genes and ATM kinase signaling in 9 lymphoblastoid cell lines (LCLs) derived from patients with ataxia telangiectasia (AT) with defined vs. 3 control LCLs without ATM mutations. Our study revealed that the DSBR phenotype in AT cells is not uniform but appears to depend on the mutation, causing up to 32-fold increased or up to 3-fold decreased activities in particular pathways. Comparison with a further 10 LCLs mutated in downstream factors (BRCA1, BRCA2, Nibrin, Rad50, and Chk2) showed that the most diametrically opposed DSBR patterns in AT cells phenocopied NBN/RAD50 or BRCA1 mutations. Notably, reexpressing wild-type ATM reversed these defects by 2.3- to 3.5-fold. Our data suggest that ATM stimulates repair proteins such as Nibrin, which execute HR, single-strand annealing (SSA), and NHEJ. Concomitantly, ATM minimizes error-prone repair (SSA and NHEJ) through activation of surveillance factors such as BRCA1. Since the outcome of the individual defect can be diametrically opposed, distinguishing repair patterns in patients with ATM mutations may also be relevant regarding therapeutic responses.

Human RAD50 deficiency in a Nijmegen breakage syndrome-like disorder.

The MRE11/RAD50/NBN (MRN) complex plays a key role in recognizing and signaling DNA double-strand breaks (DSBs). Hypomorphic mutations in NBN (previously known as NBS1) and MRE11A give rise to the autosomal-recessive diseases Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively. To date, no disease due to RAD50 deficiency has been described. Here, we report on a patient previously diagnosed as probably having NBS, with microcephaly, mental retardation, 'bird-like' face, and short stature. At variance with this diagnosis, she never had severe infections, had normal immunoglobulin levels, and did not develop lymphoid malignancy up to age 23 years. We found that she is compound heterozygous for mutations in the RAD50 gene that give rise to low levels of unstable RAD50 protein. Cells from the patient were characterized by chromosomal instability; radiosensitivity; failure to form DNA damage-induced MRN foci; and impaired radiation-induced activation of and downstream signaling through the ATM protein, which is defective in the human genetic disorder ataxia-telangiectasia. These cells were also impaired in G1/S cell-cycle-checkpoint activation and displayed radioresistant DNA synthesis and G2-phase accumulation. The defective cellular phenotype was rescued by wild-type RAD50. In conclusion, we have identified and characterized a patient with a RAD50 deficiency that results in a clinical phenotype that can be classified as an NBS-like disorder (NBSLD).

Nijmegen Breakage Syndrome mutations and risk of breast cancer.

Mutations in the NBS1 gene have been identified as disease-causing mutations in patients with Nijmegen Breakage Syndrome (NBS), but their clinical impact on breast cancer susceptibility has remained uncertain. We determined the frequency of 2 NBS mutations, 657del5 and R215W, in two large series of breast cancer cases and controls from Northern Germany and from the Republic of Belarus. The 5-bp-deletion 657del5 was identified in 15/1,588 cases (0.9%) from Belarus and in 1/1,076 cases (0.1%) from Germany but in only 1/1,014 population controls from Belarus and 0/1017 German controls (p < 0.01). The missense substitution R215W was observed in 9/1,588 Byelorussian and 9/1,076 German patients (0.6% and 0.8%, respectively) but was also present in 5/1,014 Byelorussian and 2/1,017 German control individuals (adjusted OR = 1.9, 95%CI 0.8-4.6, p = 0.18). Studies of lymphoblastoid cell lines revealed that NBS1/p95 protein levels were reduced to 70% in cells from a heterozygous breast cancer patient carrying R215W and to 15% in cells from a NBS patient compound heterozygous for 657del5/R215W suggesting that the R215W substitution may be associated with protein instability. Levels of radiation-induced phosphorylation of Nbs1/p95(Ser343) were reduced to 60% and 35% of wildtype, respectively. Neither age at diagnosis nor family history of breast cancer differed significantly between carriers and noncarriers of NBS mutations. The combined data are in line with an about 3-fold increase in breast cancer risk for female NBS heterozygotes (OR 3.1; 95%CI 1.4-6.6) and indicate that the 657del5 deletion and perhaps the R215W substitution contribute to inherited breast cancer susceptibility in Central and Eastern Europe.