Petr Pohlreich

The role of ATM in breast cancer development.

Complete or partial inability to sense and repair DNA damage increases the risk of developing cancer. The ataxia telangiectasia mutated (ATM) protein kinase has a crucial role in response to DNA double-strand breaks. Hereditary mutations in the ATM gene are the cause of a rare genomic instability syndrome ataxia telangiectasia (AT) characterized, among others, by elevated cancer risk. Although clear in homozygotes, numerous studies have failed to find a link between heterozygotes and cancer. However, there is increasing evidence that ATM heterozygotes have an increased risk of developing breast cancer. First, epidemiological studies conferred an increased risk of breast cancer among AT relatives. Second, in vitro studies of heterozygous cells provide strong evidence of hyperradiosensitivity. Third, some clinical studies found an increased frequency of ATM mutations among high-risk breast cancer families.

The CHEK2 gene I157T mutation and other alterations in its proximity increase the risk of sporadic colorectal cancer in the Czech population.

Checkpoint kinase 2 (CHEK2) gene codes for an important mediator of DNA damage response pathway. Its mutations increase risk of several types of cancer. We analysed selected CHEK2 mutations in 631 Czech colorectal cancer (CRC) patients. The increased risk of CRC was associated with mutations in CHEK2 gene region involving fork head-associated domain [39/631 (6.2%) cases versus 19/683 (2.8%) controls; odds ratio (OR)=2.3; 95% confidence interval (CI)=1.3-4.0; p=0.003], and with the most frequent I157T mutation [30/631 (4.8%) cases versus 17/683 (2.5%) controls; OR=2.0; 95% CI=1.1-3.6; p=0.03]. Prevalence of 1100delC mutation in CRC patients (4/631) did not differ from that in the control population (2/730; p=0.4). The deletion of 5395 bp was not found in any of the successfully analysed CRC cases. We observed no association of analysed mutations with CRC family history. We conclude that the I157T and other alterations in its proximity predispose to sporadic but not to familial CRC in the Czech population.

Screening for genomic rearrangements in BRCA1 and BRCA2 genes in Czech high-risk breast/ovarian cancer patients: high proportion of population specific alterations in BRCA1 gene

Large genomic rearrangements (LGR) represent substantial proportion of pathogenic mutations in the BRCA1 gene, whereas the frequency of rearrangements in the BRCA2 gene is low in many populations. We screened for LGRs in BRCA1 and BRCA2 genes by multiplex ligation-dependent probe amplification (MLPA) in 521 unrelated patients negative for BRCA1/2 point mutations selected from 655 Czech high-risk breast and/or ovarian cancer patients. Besides long range PCR, a chromosome 17-specific oligonucleotide-based array comparative genomic hybridization (aCGH) was used for accurate location of deletions. We identified 14 patients carrying 8 different LGRs in BRCA1 that accounted for 12.3% of all pathogenic BRCA1 mutations. No LGRs were detected in the BRCA2 gene. In a subgroup of 239 patients from high-risk families, we found 12 LGRs (5.0%), whereas two LGRs were revealed in a subgroup of 282 non-familial cancer cases (0.7%). Five LGRs (deletion of exons 1–17, 5–10, 13–19, 18–22 and 21–24) were novel; two LGRs (deletion of exons 5–14 and 21–22) belong to the already described Czech-specific mutations; one LGR (deletion of exons 1–2) was reported from several countries. The deletions of exons 1–17 and 5–14, identified each in four families, represented Czech founder mutations. The present study indicates that screening for LGRs in BRCA1 should include patients from breast or ovarian cancer families as well as high-risk patients with non-familial cancer, in particular cases with early-onset breast or ovarian cancer. On the contrary, our analyses do not support the need to screen for LGRs in the BRCA2 gene. Implementation of chromosome-specific aCGH could markedly facilitate the design of primers for amplification and sequence analysis of junction fragments, especially in deletions overlapping gene boundaries.

Lack of large intragenic rearrangements in dihydropyrimidine dehydrogenase (DPYD) gene in fluoropyrimidine-treated patients with high-grade toxicity

PurposeDeficiency of dihydropyrimidine dehydrogenase (DPD) has been associated with severe fluoropyrimidines (FP) toxicity. Mutations in DPD-coding gene (DPYD) were shown to increase the risk of severe toxicity in FP-treated cancer patients. However, the majority of DPYD alterations characterized in these patients has been considered as polymorphisms and known deleterious mutations are rare and present in only limited subgroup of patients with high toxicity. Recently, the common fragile site FRA1E was mapped within DPYD locus but intragenic rearrangements in DPYD gene were not studied so far.MethodsWe performed the analysis of intragenic rearrangements of DPYD using multiplex ligation-dependent probe amplification in 68 patients with high-grade gastrointestinal and/or hematological toxicity developed at the beginning of FP treatment.ResultsWe did not detect any deletion/duplication of one or more DPYD exons in analyzed patients.ConclusionsWe assume that rearrangements in DPYD gene play insignificant role in the development of serious FP-related toxicity.

Mutational analysis of the BRCA1 gene in 30 Czech ovarian cancer patients.

Ovarian cancer is one of the most severe of oncological diseases. Inherited mutations in cancer susceptibility genes play a causal role in 5–10% of newly diagnosed tumours.BRCA1 andBRCA2 gene alterations are found in the majority of these cases. The aim of this study was to analyse theBRCA1 gene in the ovarian cancer risk group to characterize the spectrum of its mutations in the Czech Republic. Five overlapping fragments amplified on both genomic DNA and cDNA were used to screen for the whole proteincoding sequence of theBRCA1 gene. These fragments were analysed by the protein truncation test (PTT) and direct sequencing. Three inactivating mutations were identified in the group of 30 Czech ovarian cancer patients: the 5382insC mutation in two unrelated patients and a deletion of exons 21 and 22 in another patient. In addition, we have found an alternatively spliced product lacking exon 5 in two other unrelated patients. The 5382insC is the most frequent alteration of theBRCA1 gene in Central and Eastern Europe. The deletion of exons 21 and 22 affects the BRCT functional domain of the BRCA1 protein. Although large genomic rearragements are known to be relatively frequent in Western European populations, no analyses have been performed in our region yet.

CHEK2 gene alterations in the forkhead-associated domain, 1100delC and del5395 do not modify the risk of sporadic pancreatic cancer.

Checkpoint kinase 2 gene (CHEK2) alterations increase risk of several cancer types. We analyzed selected CHEK2 alterations in 270 Czech pancreatic cancer patients and in 683 healthy controls. The pancreatic cancer risk was higher in individuals who inherited rare alterations in CHEK2 region involving forkhead-associated domain other than I157T (OR=5.14; 95% CI=0.94-28.23) but the observed association was non-significant (p=0.057). The most frequent I157T mutation did not alter the pancreatic cancer risk and neither the followed deletion of 5395bp nor c.1100delC were found in any of pancreatic cases. We conclude that the I157T, other alterations in its proximity, del5395 and c.1100delC in CHEK2 do not predispose to pancreatic cancer risk in the Czech population.

High frequency of BRCA1/2 and p53 somatic inactivation in sporadic ovarian cancer

Faculty of Medicine, Charles University, Prague, and theGeneral Teaching Hospital in Prague. All analysed tumourswere sporadic i.e. no other tumours were referred to in thefamily history and all the patients were more than 40 yearsold at the time of diagnosis. All participants gave their writ-ten informed consent.All freshly frozen samples were examined by a pathol-ogist to evaluate the proportion of tumourous and nontu-mourous tissue. Only samples with over 50% tumouroustissue were used for our study. The histopathological char-acteristics of analysed tumours are given in table 1. Majorityof patients (21/30 : 70%) were diagnosed in stage III Inter-national Federation of Gynocology and Obstetrics (FIGO).Stages I and IV were similarly represented (4/30 and 5/30:13.3% and 16.7%, respectively), and none of the analysedtumours belong to stage II patient.

Characterisation of ATM Mutations in Slavic Ataxia Telangiectasia Patients

Ataxia telangiectasia (AT) is a genomic instability syndrome characterised, among others, by progressive cerebellar degeneration, oculocutaneous telangiectases, immunodeficiency, elevated serum alpha-phetoprotein level, chromosomal breakage, hypersensitivity to ionising radiation and increased cancer risk. This autosomal recessive disorder is caused by mutations in the ataxia telangiectasia mutated (ATM) gene coding for serine/threonine protein kinase with a crucial role in response to DNA double-strand breaks. We characterised genotype and phenotype of 12 Slavic AT patients from 11 families. Mutation analysis included sequencing of the entire coding sequence, adjacent intron regions, 3′UTR and 5′UTR of the ATM gene and multiplex ligation-dependent probe amplification (MLPA) for the detection of large deletions/duplications at the ATM locus. The high incidence of new and individual mutations demonstrates a marked mutational heterogeneity of AT in the Czech Republic. Our data indicate that sequence analysis of the entire coding region of ATM is sufficient for a high detection rate of mutations in ATM and that MLPA analysis for the detection of deletions/duplications seems to be redundant in the Slavic population.

The postribosomal particle of rabbit liver contains protein-synthesis factors and serum albumin mRNA

As demonstrated by indirect immunoprecipitation and polyacrylamide gel electrophoresis, an 85S particle separated by sucrose density-gradient centrifugation from the postribosomal pellet of rabbit liver, is able to synthesize serum albumin if supplemented with both ribosomal subunits and sources of energy. It is retained on heparin bound to Sepharose 4B, contains translatable mRNA and apparently all protein factors required for translation. This particle may represent a highly organized protein synthesizing machinery, the combination of which with ribosomes results in formation of new protein molecules.

Utilization of Initiator Transfer Ribonucleic Acid Modified by Carcinogens Cholesteryl 14-Methylhexadecanoate and N-Methyl-N-Nitrosourea for Cell-Free Protein Synthesis

(3H)Cholesteryl 14-methylehexadecanoate becomes bound to initiator tRNA from rat liver if incubated with it at 37°C. The binding of the ester was competitively inhibited by N-methyl-N-nitrosourea which has been previously demonstrated to alkylate guanine. It thus appears that the lipid reacts with guanosine residues in the molecule of tRNAF. Initiator tRNA preparations pretreated with cholesteryl 14-methylhexadecanoate were charged significantly better with L-methionine in the presence of aminoacyl-tRNA synthetases than comparable control preparations. The effect of the ester was in this respect comparable with that of N-methyl-N-nitrosourea. Incubation of (35S) Met-tRNAF modified by the lipid or N-methyl-N-nitrosourea with partially purified eIF-2 resulted in an enhanced formation of the ternary complex of this protein-synthesis factor. Also the sythesis of the 80S initiation complex was stimulated in the presence of these modified Met-tRNAs. Protein synthesis in a system composed of rabbit reticulocyte polyribosomes and partially purified initiation and elongation factors was stimulated if unfractionated aminoacyl-tRNA from rat liver pretreated with cholesteryl 14-methylhexadecanoate of N-methyl-N-nitrosourea was used. No changes were found in poly(U)-dependent peptide elongation when using aminoacyl-tRNA modified by either of these agents. Evidence was presented previously that cholesteryl 14-methylhexadecanoate is involved in the control of protein synthesis by affecting the binding sites of protein-synthesis factors and ribosomes for aminoacyl-tRNA, and protein phosphorylation. The present results suggest that the modification of initiator tRNA by this lipid may represent another pathway for its modulation of gene translation.