Putative low penetrance cancer susceptibility genes may have under appreciated role in the overall genetic predisposition to breast cancer and other common malignancies. To test their possible functional role, we used a variety of approaches to examine several candidate breast cancer susceptibility genes, including BACH1 (Lei and Vorechovsky, 2003; Luo et al., 2002), CDH (Lei et al., 2002b; Salahshor et al., 2001), ATM (Lei et al., 2002a; Stankovic et al., 2001) and MMP3 (Lei et al., 2002c). The methods employed mutation analysis (Luo et al., 2002; Vorechovsky, 2002), analysis of alternative pre-mRNA splicing (Kralovicova et al., 2004), subcellular localization (Lei and Vorechovsky, 2003), promoter activity assays (Lei et al., 2002b), haplotype analysis (Stankovic et al., 2001) and allelic association (Lei et al., 2002c). Full details are described in references below: - Kralovicova, J., Houngninou-Molango, S., Kramer, A., and Vorechovsky, I. (2004). Branch sites haplotypes that control alternative splicing. Hum Mol Genet 18, in press. - Lei, H., Pospisilova, D., Lindblom, A., and Vorechovsky, I. (2002a). Re: Dominant Negative ATM Mutations in Breast Cancer Families. J Natl Cancer Inst 94, 951-952. - Lei, H., Sjoberg-Margolin, S., Salahshor, S., Werelius, B., Jandakova, E., Hemminki, K., Lindblom, A., and Vorechovsky, I. (2002b). CDH1 mutations are present in both ductal and lobular breast cancer, but promoter allelic variants show no detectable breast cancer risk. Int J Cancer 98, 199-204. - Lei, H., and Vorechovsky, I. (2003). BACH 517C->T transition impairs protein translocation to nucleus: a role in breast cancer susceptibility? Int J Cancer 104, 389-391. - Lei, H., Zaloudik, J., and Vorechovsky, I. (2002c). Lack of association of the -1171 (5A) allele of the MMP3 promoter with breast cancer. Clin Chem 48, 798-799. - Luo, L., Lei, H., Du, Q., von Wachenfeldt, A., Kockum, I., Luthman, H., Vorechovsky, I., and Lindblom, A. (2002). No mutations in the BACH1 gene in BRCA1 and BRCA2 negative breast-cancer families linked to 17q22. Int J Cancer 98, 638-639. - Salahshor, S., Haixin, L., Huo, H., Kristensen, V. N., Loman, N., Sjoberg-Margolin, S., Borg, A., Borresen-Dale, A. L., Vorechovsky, I., and Lindblom, A. (2001). Low frequency of E-cadherin alterations in familial breast cancer. Breast Cancer Res 3, 199-207. - Stankovic, T., Taylor, A. M., Yuille, M. R., and Vorechovsky, I. (2001). Recurrent ATM mutations in T-PLL on diverse haplotypes: no support for their germline origin. Blood 97, 1517-1518. - Vorechovsky, I. (2002). Mutation analysis of large genomic regions in tumor DNA using single-strand conformation polymorphism. Lessons from the ATM gene. Methods Mol Med 68, 115-124.
Mechanism(s) of immunodeficiency consequent to inactivation of the ATM gene which might enhance tumorigenesis in A-T patients
Ataxia telangiectasia (A-T) is an autosomal recessive multisystem disorder characterized by cerebellar ataxia, conjunctival and cutaneous telangiectasias, immune deficiency, chromosome aberrations, radiation hypersensitivity, and a high incidence of tumors, mainly of lymphoid origin. The mechanism(s) of the immunodeficiency consequent to inactivation of the ATM gene, which might underlie both impaired lymphocyte development and enhanced lymphomagenesis, have not yet been fully elucidated. In the course of this project we reported that the T-cell receptor (TCR) variable beta (BV)–chain repertoire of 9 A-T patients was restricted by diffuse expansions of some variable genes prevalently occurring within the CD4 subset and clustering to certain TCRBV genes (eg, 5.1,11, 14, and 23). In addition, the study of the third complementarity-determining region (CDR3) showed, in all patients, significantly altered profiles in most BV genes examined suggesting diffuse oligoclonal expansions. The sequencing of TCR CDR3 regions revealed completely normal V(D)J coding joints and confirmed a reduced diversity of the antigen-receptor repertoire. The B-cell repertoire was similarly restricted and skewed by diffuse oligoclonal expansions with normal V(D)J joints. Thymic output, evaluated by measuring TCR rearrangement excision circles, was extremely low. The majority of peripheral T cells had the phenotype and the function of effector memory cells, indicating that in vivo they are able to respond normally by terminal differentiation to antigenic stimulation. These results indicate that ATM mutation limits the generation of a wide repertoire of normally functioning T and B cells.
BACKGROUND: Fluorescence in situ hybridization has improved the detection of genomic aberrations in chronic lymphocytic leukemia. We used this method to identify chromosomal abnormalities in patients with chronic lymphocytic leukemia and assessed their prognostic implications. METHODS: Mononuclear cells from the blood of 325 patients with chronic lymphocytic leukemia were analyzed by fluorescence in situ hybridization for deletions in chromosome bands 6q21, 11q22-23, 13q14, and 17p13; trisomy of bands 3q26, 8q24, and 12q13; and translocations involving band 14q32. Molecular cytogenetic data were correlated with clinical findings. RESULTS: Chromosomal aberrations were detected in 268 of 325 cases (82 percent). The most frequent changes were a deletion in 13q (55 percent), a deletion in 11q (18 percent), trisomy of 12q (16 percent), a deletion in 17p (7 percent), and a deletion in 6q (7 percent). Five categories were defined with a statistical model: 17p deletion, 11q deletion, 12q trisomy, normal karyotype, and 13q deletion as the sole abnormality; the median survival times for patients in these groups were 32, 79, 114, 111, and 133 months, respectively. Patients in the 17p- and 11q-deletion groups had more advanced disease than those in the other three groups. Patients with 17p deletions had the shortest median treatment-free interval (9 months), and those with 13q deletions had the longest (92 months). In multivariate analysis, the presence or absence of a 17p deletion, the presence or absence of an 11q deletion, age, Binet stage, the serum lactate dehydrogenase level, and the white-cell count gave significant prognostic information. CONCLUSIONS: Genomic aberrations in chronic lymphocytic leukemia are important independent predictors of disease progression and survival. These findings have implications for the design of risk-adapted treatment strategies.
Discovery of genetic alterations in human neoplasms of the gene encoding for the PPP2R1A and PPP2R1B isoforms of the subunit A of the serine-threonine phosphatase 2A
The phosphatase 2A (PP2A) is one of the major cellular serine-threonine phosphatases. It was recently shown that the gene encoding for the beta isoform of its subunit A, PPP2R1B, is altered in human lung and colorectal carcinomas, suggesting a role in human tumorigenesis. In the course of this project, we reported the detection of mutations in breast, lung carcinomas and melanomas in the genes of both alpha (PPP2R1A) and beta isoforms. Mutations affecting PPP2R1B were found in four breast carcinomas, while mutations in PPP2R1A were found in carcinomas of the breast and of the lung and in one melanoma. Most of the mutations affecting PPP2R1B were exons deletions, suggesting abnormal splicing. These splicing abnormalities were detected in tumor samples in the absence of the normal splicing product, and were not found in several normal controls. In one case, a homozygous deletion present in tumor DNA, and not in the matched normal control was demonstrated. Mutations affecting the PPP2R1A gene were nucleotide substitutions changing highly conserved amino acids and one frame-shift. Although the frequency of alterations is low, the inclusion of both isoforms of subunit A in the genes mutated in human cancer and the addition of breast cancer to the list of neoplasms in which PPP2R1B is altered, strengthen the potential role of PP2A in human tumorigenesis. The result has been reported in a well-recognized international scientific journal. At present, the use of the result and its expected benefits are limited because of the low frequency at which this alteration is detected and the lack of association with clinical parameters.