Sara Frías

A Boolean network model of the FA/BRCA pathway

MOTIVATION Fanconi anemia (FA) is a chromosomal instability syndrome originated by inherited mutations that impair the Fanconi Anemia/Breast Cancer (FA/BRCA) pathway, which is committed to the repair of DNA interstrand cross-links (ICLs). The disease displays increased spontaneous chromosomal aberrations and hypersensitivity to agents that create DNA interstrand cross-links. In spite of DNA damage, FA/BRCA-deficient cells are able to progress throughout the cell cycle, probably due to the activity of alternative DNA repair pathways, or due to defects in the checkpoints that monitor DNA integrity. RESULTS We propose a Boolean network model of the FA/BRCA pathway, Checkpoint proteins and some alternative DNA repair pathways. To our knowledge, this is the largest network model incorporating a DNA repair pathway. Our model is able to simulate the ICL repair process mediated by the FA/BRCA pathway, the activation of Checkpoint proteins observed by recurrent DNA damage, as well as the repair of DNA double-strand breaks and DNA adducts. We generated a series of simulations for mutants, some of which have never been reported and thus constitute predictions about the function of the FA/BRCA pathway. Finally, our model suggests alternative DNA repair pathways that become active whenever the FA/BRCA pathway is defective.

DEB test for Fanconi anemia detection in patients with atypical phenotypes

Pancytopenia, hyperpigmentation, small stature, congenital abnormalities, and predisposition to neoplasia characterize Fanconi anemia (FA). The clinical phenotype is extremely variable, therefore the diagnosis is frequently delayed until the pancytopenia appears, making diagnosis difficult on the basis of clinical manifestations alone. Hypersensitivity of FA cells to the clastogenic effect of diepoxybutane (DEB) provides a unique marker for the diagnosis before the beginning of hematological manifestations. Our aim in this study was to detect FA in children with atypical manifestations to define which conditions should be routinely included in the DEB test screening. We performed the chromosomal breakage test in 34 patients with probable FA and 83 patients with clinical conditions that could suggest FA, but are not usually screened by the DEB test: 20 patients with aplastic anemia, 20 patients with VACTERL association, 20 with radial ray abnormalities, 7 with tracheo‐esophageal fistulae, 12 with anal atresia, and 4 with myelodysplastic syndrome. We found 18 DEB‐positive patients: 12 were in the group of probable FA and 6 in the other groups. Among the last ones: three were included because of aplastic anemia, without any other sign of FA, however when re‐examined, other anomalies were detected. The third patient had anal atresia, renal hypoplasia, pre‐axial polydactyly, and normal blood cell counts and was diagnosed as having VACTERL association. The other two patients lacking physical or hematological signs were identified among the group of radial ray abnormalities. Thus, our results highlight the need to increase the number of abnormalities indicating need for a DEB test. Delay in the diagnosis of FA may have serious consequences for the patients and their family members.

Partial trisomy of the short arm of chromosome 7 due to a familial translocation rcp(7;14) (p11;p11)

A boy with trisomy of the short arm of chromosome 7 is reported. He presented several congenital malformations, such as: dolicocephaly; wide fontanelle; hypertelorism; antimongoloid slanting of the palpebral fissures; small, carp‐shaped mouth; thin neck; extrinsic compression of the oesophagus; congenital dislocation of the shoulders and hips; and a possible cardiac defect. The probands mother had a balanced translocation involving the short arm of chromosomes 7 and 14. In this family the translocation has been transmitted through three generations.

Cytogenetics in Acute Lymphoblastic Leukemia in Mexican Children: An Institutional Experience

BACKGROUND Cytogenetic studies in acute lymphoblastic leukemia (ALL) have identified numerical and structural chromosomal abnormalities related to the diseases pathophysiologic characteristics. These findings correlate with prognosis and response to treatment in ALL patients. The purpose of this study was to define the frequency of chromosomal abnormalities in a group of Mexican children with ALL and to compare these data with those reported in the literature. METHODS Bone marrow chromosome studies with GTG bands were performed in 150 pediatric patients with ALL who were naive to antileukemic treatment and aged from 5 months to 16 years; the majority was diagnosed as L1. RESULTS Among 131 patients, 30 (22.9%) karyotypes were normal and the remaining 101 (77.1%) had abnormal karyotypes with numerical and/or structural abnormalities. Among patients with numerical abnormalities, the most frequent karyotypes were hyperdiploidy with 51-65 chromosomes (30 patients) and hyperdiploidy with 47-50 chromosomes (18 patients). Among recurrent, non-random, and primary structural abnormalities, the most frequent was t(9;22), followed by t(1;19). Aberrations involving band 11q23 were not detected, and only one of two patients with L3 had the t(8;14). Of the secondary non-random abnormalities, dup(1q), del(6q), and i(7)(q10) were found. CONCLUSIONS The frequency and type of chromosomal abnormalities found was comparable to those reported in the literature with similar methodology and pediatric populations; however, the number of cases analyzed should be increased to create a database of Mexican children with ALL, and several patients require molecular analysis to identify chromosomal abnormalities not detected through conventional cytogenetic studies.

Multiple copies of RUNX1: description of 14 new patients, follow-up, and a review of the literature

RUNX1 over-representation is present in children with acute lymphoblastic leukemia. Although these cases have been related with poor outcome, not all reports describe patient follow-up. To understand its associated clinical features and prognosis, we report on 14 children with ALL and RUNX1 over-representation with laboratory data and outcomes compared to previous reports. Eighty-six children with RUNX1 over-representation have been described, including the 14 patients of this study. Most of them are between 6 and 15 years of age, have low leukocyte counts, pre-B immunophenotype, and three to eight RUNX1 copies. Of the 69 patients with follow-up data, 21 of them relapsed or died, suggesting that RUNX1 over-representation is associated to a poor outcome.

Detection of mosaicism in lymphocytes of parents of free trisomy 21 offspring

Down syndrome (DS) resulting from free trisomy 21 (FT21) has been largely associated with advanced maternal age. However, approximately 60% of FT21 cases are born to young couples. Thus, the etiological factors responsible for these FT21 children must differ from those proposed for maternal age-related FT21. These factors have not been defined. In this study, we analyzed the chromosomes of peripheral blood lymphocytes from three groups of couples aged < or =35 years, to identify chromosomal trisomies: Group I included 5 couples with normal offspring; Group II included 22 couples with one FT21 child; and Group III consisted of 3 couples with recurrent FT21. A total of 13,809 metaphases were analyzed with G-banding and 60,205 metaphases were analyzed with FISH using a 13/21 centromeric probe. Aneuploidy was significantly more frequent in Groups II and III. The frequencies of hyperdiploid cells were 0.19, 0.49 and 0.96% in Groups I-III, respectively. FISH analysis showed that trisomy 21 cell percentages were 0.08, 0.21 and 0.76 for Groups I-III, respectively, and were very similar to those obtained with G-banding. Trisomy 21 mosaicism was found in 2/22 couples with one FT21 offspring, and in 2/3 couples with recurrent FT21. Our data suggest that mosaicism is an important cause of FT21 offspring in young couples, and that aneuploidy is more frequent among couples with FT21 offspring. This may be related with age and other undetermined intrinsic and extrinsic factors.

Analysis of gene rearrangements using a fluorescence in situ hybridization method in Mexican patients with acute lymphoblastic leukemia: experience at a single institution

We evaluated the prevalence of BCR/ABL, MLL, and ETV6/RUNX1 rearrangements as well as CDKN2A (alias p16) deletion in a group of Mexican children with acute lymphoblastic leukemia (ALL) to determine whether the changes coexist, and to compare the incidences found with other reports in the literature. To increase the detection of these abnormalities, we combined conventional cytogenetics and fluorescence in situ hybridization (FISH) analysis. Bone marrow samples were obtained from 59 consecutive children with ALL. FISH detected a total of 63 abnormalities with the selected probes, 34 of which were related to the conventional cytogenetic results. The most common abnormality was the p16 deletion (22.8%), followed by MLL and ETV6/RUNX1 rearrangements (8.7%), and the BCR/ABL fusion was the least frequent (2.7%). The coexistence of two recurrent abnormalities with specific prognostic significance in the same patient was not found. A lesser proportion of the p16 deletion in T-ALL patients was observed, probably related to the low prevalence of this subtype in our population. In addition, we confirmed the low frequency of the ETV6/RUNX1 fusion observed in Hispanics. Due to the different prevalence of these abnormalities in the Mexican population, similar studies should be conducted analyzing new rearrangements, to improve the adequate classification of the abnormalities and the stratification in prognostic groups.

Effect of hydroxyurea and normal plasma on DNA synthesis in lymphocytes from Fanconi anemia patients.

Fanconi anemia (FA) is characterized at the cellular level by a high frequency of spontaneous chromosomal aberrations; crosslinking agents cause an abnormal increase in the frequency of chromosomal damage, and semiconservative DNA synthesis is severely inhibited. Deoxyribonucleotides are needed in both semiconservative and repair DNA synthesis. To investigate the involvement of deoxyribonucleotide pools in the inhibition of DNA synthesis in FA, we evaluated the effect on FA lymphocytes of hydroxyurea (HU), an inhibitor of ribonucleotide reductase which is known to alter the intracellular levels of deoxyribonucleotides. To achieve this goal, lymphocyte cultures of 4 FA patients and 4 normal individuals were used. Cultures were treated with HU and/or mitomycin C and normal human plasma. All cultures were processed to detect the number of DNA synthesizing nuclei by autoradiography. Scoring of 2000 nuclei for each kind of culture every 6 h in the last 24 h of incubation showed that, in long incubation periods, DNA synthesis in FA is largely inhibited by HU and this hypersensitivity may be partially decreased by addition of normal human plasma. It is known that recovery from damage induced by HU involves several enzymes such as flavin oxido-reductase, superoxide dismutase and catalase which are involved in the production or scavenging of O2 radicals; FA cells are deficient in the detoxification of oxygen and this could explain the response of FA cells to HU.

Chromosome instability with bleomycin and x‐ray hypersensitivity in a boy with Nijmegen breakage syndrome

We report on a Mexican boy with microcephaly, short stature, and a high frequency of chromosome aberrations with rearrangements involving chromosomes 7 and 14, typical of ataxia telangiectasia (AT) patients. He had neither ataxia nor telangiectasia, and his immunological status and serum alpha feto protein (AFP) level were normal. Bleomycin hypersensitivity, which has been demon-strated in AT patients, was tested in the patient using AT and normal subjects for comparison. The frequency of spontaneously occurring chromosome aberrations in lymphocyte cultures was significantly higher in the patient and the AT patient than in the normal subject. Four cells from the patient showed structural rearrangements involving chromosomes 7 or 14, with breakpoints typical for AT. When exposed to 5.0 micrograms bleomycin, the lymphocytes from the AT patient showed the highest sensitivity to this agent; our patient had an intermediate sensitivity. In both patients several rearrangements involving chromosomes 7 and 14 were scored, while none were observed in the normal subject. A colony survival assay (CSA) [Huo et al., 1994: Cancer Res 54:2544-2547], using a lymphoblastoid cell line (LCL) derived from our patient, showed a survival fraction (SF) of 7%, which is in the same range as in AT patients. The clinical picture, together with the cytogenetic and radiosensitivity results, suggests that our patient fits the variable spectrum of Nijmegen breakage syndrome.

Differential expression of TP53 associated genes in Fanconi anemia cells after mitomycin C and hydroxyurea treatment

Fanconi anemia (FA) is a rare, heritable chromosomal instability disease characterized by several congenital defects and cancer predisposition. Functional interactions between specific FA proteins and DNA damage response and repair activities have been reported, but the interplay between these mechanisms for maintaining genomic stability are not well understood. Many DNA damage response proteins are transcriptionally regulated by the tumor suppressor protein p53 (TP53), suggesting an important regulatory role for the DNA damage and stress response pathway. To better understand the association between FA and the DNA damage stress response we analyzed the levels of chromosomal damage and damage mediated gene transcription responses in lymphoblastoid cells derived from normal individuals and patients carrying the most common FA complementation group (FA-A). Chromosomal aberrations were first measured after exposure to mitomicyn C (MMC) or hydroxyurea (HU). Aliquots of the same cell were than assayed for the transcriptional response of 21 DNA damage and stress response genes using quantitative real-time PCR. The FA-A lymphoblastoid cells showed significant increases in the frequency of chromosome aberrations relative to non-FA-A lymphoblastoid lines after MMC treatment. The MMC induced damage was correlated with a general increase in expression of TP53-modulated DNA damage stress response genes involved in processes such as DNA repair, cell cycle progression, and apoptosis. Following HU treatment FA cells showed a decreased induction of CAs with much less transcriptional differences between targeted genes. Overall, the differences between the normal and FA-A cells after genotoxic treatments imply an increased activation and reliance of FA cells on the down-stream activities of TP53 for prevention of cell killing and chromosome damage from interstrand crosslinks but not for general replication arrest and double strand breaks. Furthermore, these results imply a regulatory connection between the FA pathway and activation of TP53 for responding to DNA damage. Alterations in the regulation of the DNA damage response may be related to the complex phenotypes seen in FA patients.