Abdellatif Errami

Brca2 (XRCC11) Deficiency Results in Radioresistant DNA Synthesis and a Higher Frequency of Spontaneous Deletions

ABSTRACT We show here that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11 is defective in the breast cancer susceptibility gene Brca2. The very complex phenotype of V-C8 cells is complemented by a single human chromosome 13 providing the BRCA2 gene, as well as by the murine Brca2 gene. The Brca2 deficiency in V-C8 cells causes hypersensitivity to various DNA-damaging agents with an extreme sensitivity toward interstrand DNA cross-linking agents. Furthermore, V-C8 cells show radioresistant DNA synthesis after ionizing radiation, suggesting that Brca2 deficiency affects cell cycle checkpoint regulation. In addition, V-C8 cells display tremendous chromosomal instability and a high frequency of abnormal centrosomes. The mutation spectrum at the hprt locus showed that the majority of spontaneous mutations in V-C8 cells are deletions, in contrast to wild-type V79 cells. A mechanistic explanation for the genome instability phenotype of Brca2-deficient cells is provided by the observation that the nuclear localization of the central DNA repair protein in homologous recombination, Rad51, is reduced in V-C8 cells.

XR-C1, a new CHO cell mutant which is defective in DNA-PKcs, is impaired in both V(D)J coding and signal joint formation

DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (DSB) repair and V(D)J recombination. We have isolated a new X-ray-sensitive CHO cell line, XR-C1, which is impaired in DSB repair and which was assigned to complementation group 7, the group that is defective in the XRCC7 / SCID ( Prkdc ) gene encoding the catalytic subunit of DNA-PK (DNA-PKcs). Consistent with this complementation analysis, XR-C1 cells lackeddetectable DNA-PKcs protein, did not display DNA-PK catalytic activity and were complemented by the introduction of a single human chromosome 8 (providing the Prkdc gene). The impact of the XR-C1 mutation on V(D)J recombination was quite different from that found in most rodent cells defective in DNA-PKcs, which are preferentially blocked in coding joint formation, whereas XR-C1 cells were defective in forming both coding and signal joints. These results suggest that DNA-PKcs is required for both coding and signal joint formation during V(D)J recombination and that the XR-C1 mutant cell line may prove to be a useful tool in understanding this pathway.

Concurrent methylation of promoters from tumor associated genes predicts outcome in acute myeloid leukemia.

By assessment of the methylation status of 25 candidate tumor suppressor genes (TSGs) in 119 acute myeloid leukemia (AML) patients and 5 controls, we aimed to determine whether simultaneous methylation of multiple TSGs exerts prognostic impact. Methylation-specific multiplex ligation probe amplification (MS-MLPA) revealed methylation of at least one TSG in 59/119 patients, while no methylation was found in controls. Methylation of different TSGs within patients was substantially correlated (intra-class correlation; 0.38). ESR1 methylation (34/119) strongly predicted concurrent methylation of other genes, OR 7.33 (95%CI 4.13–12.99). A Cox regression model that included the three most frequently methylated TSGs ESR1, CDKN2B/p15 and IGSF4, showed ESR1 to have opposite effects on overall survival (OS) compared with the other two, HR 0.22 (95% CI 0.09–0.53) and HR 1.66 (95% CI 0.73–3.79), HR 1.61 (95%CI 0.66–3.93). By assessment of CDKN2B/p15 and IGSF4 methylation, patients with methylation at multiple loci can be identified. Accumulation of methylation aberrancies is much more pronounced in ESR1 methylated patients. When combined, the methylation status of ESR1, CDKN2B/p15 and IGSF4 enable identification of patient subgroups with large differences in OS (p <0.0001). This study shows that methylation profiling allows risk stratification in AML. In addition, ESR1 methylation may reflect a biological pathway that leads to hypermethylation of multiple genes, which is reflected by methylation of IGSF4 and/or CDKN2B/p15.

A new X-ray sensitive CHO cell mutant of ionizing radiation group 7,XR-C2, that is defective in DSB repair but has only a mild defect in V(D)J recombination.

The DNA-dependent protein kinase (DNA-PK) complex plays a key role in DNA double-strand break (DSB) repair and V(D)J recombination. Using a genetic approach we have isolated cell mutants sensitive to ionizing radiation (IR) in the hope of elucidating the mechanism and components required for these pathways. We describe here, an X-ray-sensitive and DSB repair defective Chinese hamster ovary (CHO) cell line, XR-C2, which was assigned to the X-Ray Cross Complementation (XRCC) group 7. This group of mutants is defective in the XRCC7/SCID/Prkdc gene, which encodes the catalytic subunit of DNA-PK (DNA-PKcs). Despite the fact that XR-C2 cells expressed normal levels of DNA-PKcs protein, no DNA-PK catalytic activity could be observed in XR-C2, confirming the genetic analyses that these cells harbor a dysfunctional gene for DNA-PKcs. In contrast to other IR group 7 mutants, which contain undetectable or low levels of DNA-PKcs protein and which show a severe defect in V(D)J recombination, XR-C2 cells manifested only a mild defect in both coding and signal junction formation. The unique phenotype of the XR-C2 mutant suggests that a normal level of kinase activity is critical for radiation resistance but not for V(D)J recombination, whereas the overall structure of the DNA-PKcs protein appears to be of great importance for this process.

Lack of large genomic deletions in BRIP1, PALB2, and FANCD2 genes in BRCA1/2 negative familial breast cancer.

16% of the familial BC cases [2]. Other BC sus-ceptibility genes include TP53 [16], PTEN [15], ATM [24],LKB1/STK11 [8], CHEK2 [28], BRIP1/FANCJ [27], andPALB2/FANCN [22]. However to date, the majority offamilial BC cases can not be attributed to mutations in oneof the known susceptibility genes.The discovery of the breast cancer susceptibility geneBRCA2asthegenedefectiveintheFanconianemia(FA)-D1complementation group [11], the identification of BRIP1(BRCA1 InteractingProtein)[3, 12,13] andPALB2 (PartnerAnd Localizer of BRCA2) [23, 32] as the genes responsiblefor the FA-J and FA-N complementation groups, respec-tively, established a clear link between breast cancer sus-ceptibility and FA. Fanconi anemia (FA) is a recessivelyinherited chromosomal instability syndrome with autosomalor X-linked mode of inheritance, and is characterized by anincreased susceptibility to several forms of malignancies [1,17].Thediseaseiscausedbymutationsinoneofthe13genesso far identified [18]. The FA gene products interact in acommon pathway whereby most of the proteins (FANCA, -B, -C, -E, -F, -G, -L, and -M) form a multiprotein complexthat is required for the monoubiquitination of FANCD2 andFANCI.However,thismodificationstepisnotinfluencedbyFANCD1(BRCA2),FANCJ(BRIP1),orFANCN(PALB2),and hence these proteins seem to act downstream of thisprocess. FANCD2 and FANCI are thought to form a proteincomplex (ID complex), which translocates to DNA damagesites where it co-localizes with the downstream FA proteins,BRCA2/FANCD1, BRIP1/FANCJ, and PALB2/FANCNand other proteins that are involved in the recognition andrepair of DNA damage, such as BRCA1, ATM, NBS, andRAD51 [31].Several studies explored whether heterozygous femalecarriers for a mutation in one of the FA genes are atincreased risk for breast cancer. To date, only mutations inthe ‘‘downstream’’ FA genes have been found to signifi-cantly elevate the risk of developing breast cancer. Het-erozygous mutations in BRIP1/FANCJ and PALB2/FANCNappear to increase the risk 2- and 2.3-fold, respectively [22,27]. Risk assessment has been based on screening fortruncating mutations in these genes in familial breastcancer (FBC) patients lacking mutations in BRCA1/2.Furthermore, FANCD2 mutations, have been suggested toplay a role in the development of breast cancer based onobservations of Fancd2 knockout mice, which demon-strated a high incidence of epithelial tumors, includingmammary and ovarian tumors [10]. However, in humans asignificant contribution of FANCD2 mutations to FBCcould not be established [14, 26].

Deletions within COL11A1 in Type 2 stickler syndrome detected by multiplex ligation- dependent probe amplification (MLPA)

BackgroundCOL11A1 is a large complex gene around 250 kb in length and consisting of 68 exons. Pathogenic mutations in the gene can result in Stickler syndrome, Marshall syndrome or Fibrochondrogenesis. Many of the mutations resulting in either Stickler or Marshall syndrome alter splice sites and result in exon skipping, which because of the exon structure of collagen genes usually leaves the message in-frame. The mutant protein then exerts a dominant negative effect as it co-assembles with other collagen gene products. To date only one large deletion of 40 kb in the COL11A1, which was detected by RT-PCR, has been characterized. However, commonly used screening protocols, utilizing genomic amplification and exon sequencing, are unlikely to detect such large deletions. Consequently the frequency of this type of mutation is unknown.Case presentationsWe have used Multiplex Ligation-Dependent Probe Amplification (MLPA) in conjunction with exon amplification and sequencing, to analyze patients with clinical features of Stickler syndrome, and have detected six novel deletions that were not found by exon sequencing alone.ConclusionExon deletions appear to represent a significant proportion of type 2 Stickler syndrome. This observation was previously unknown and so diagnostic screening of COL11A1 should include assays capable of detecting both large and small deletions, in addition to exon sequencing.