The relevance of molecular biology studies in the genetic counselling of argentine retinoblastoma families

Objetivo: Evaluar la importancia de la detección de mutaciones del gen RB1 en el asesoramiento genético de las familias argentinas con retinoblastoma. Métodos: Se incluyeron en este estudio 34 familias argentinas con Retinoblastoma (Rb) bilateral y unilateral. Se analizaron 130 muestras de ADN de leucocitos, tumores y vellosidades coriónicas, por ensayos de Biología Molecular indirectos y directos, como Southern blot, segregación de los polimorfismos BamHI, Rbi4, XbaI y Rb 1.20 (PCR-RFLP, PCR-STR), PCR-heteroduplex y secuenciación del gen RB1. Resultados: El análisis molecular fue informativo en 18 familias de las 34 incluidas en el estudio (53%), el 56% con Rb bilateral y el 44% con Rb unilateral. Se contó con muestras de ADN tumoral de 11 pacientes que se estudiaron para detectar pérdida de heterocigosidad (LOH), que posibilitó identificar el alelo RB1 mutado en 9 pacientes (82%). Cuando no se analizaron las muestras tumorales, los estudios fueron informativos solo en 9 de los 23 pacientes (39%); se utilizó la detección directa en 17 pacientes (41% informativo) e indirecta en 20 (60% informativo). Conclusiones: Los resultados demuestran la necesidad de contar con ADN del tumor, cuando el paciente fue enucleado, y acentúan la importancia de la detección directa de la mutación en familias con Rb esporádico temprano sin muestra tumoral. Los estudios de biología molecular contribuyeron con el adecuado asesoramiento genético de pacientes argentinos y sus familiares y el diseño apropiado de su tratamiento temprano.Objective: Evaluate the relevance of RB1 mutations detection in the genetic counselling of Argentine retinoblastoma families. Methods: We included in this study 34 Argentine families with bilateral and unilateral Retinoblastoma (Rb). 130 DNA samples from leukocytes, tumors and chorionic villus were analyzed by indirect and direct molecular biology assays like Southern blot, segregation of polymorphisms BamHI, Rbi4, XbaI y Rb 1.20 (PCR-RFLP, PCR-STR), PCR-heteroduplex and sequencing of RB1 gene. Results: Molecular biology analysis was informative in 18 out of 34 families studied (53%), 56% with bilateral and 44% with unilateral Rb. DNA tumor samples of 11 patients were available and could be studied by loss of heterozygosity (LOH) detection, that allowed us to identify the mutated RB1 allele in 9 (82%) patients. When tumor samples were not analized, the studies were informative only in 9 out of 23 patients (39%); we used direct mutation detection in 17 (41% informative) and indirect assays in 20 (60% informative) patients. Conclusions: The results prove the necessity to have DNA tumor, when the patient has been enucleated, and emphasize the importance of direct mutation detection in families with early sporadic Rb without tumor sample. The RB1 molecular biology contributed to the adequate genetic counselling of Argentine patients and relatives and their appropriate early treatment planning.Fil: Parma, Diana Lidia. Universidad de Buenos Aires; ArgentinaFil: Dalamon, Viviana Karina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires; ArgentinaFil: Fernández, C. Universidad de Buenos Aires; ArgentinaFil: Szijan, Irena. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Damel, A.. Universidad de Buenos Aires; Argentin

Retinoblastoma gene mutations in primary human bladder cancer.

Inactivation of the retinoblastoma (RB) gene is known to be implicated in the pathogenesis of several types of human cancers. Since structural alterations of the RB gene have not been well examined in human bladder cancer, we looked for mutations in the entire coding region of this gene using polymerase chain reaction (PCR) and single-strand conformational polymorphism analysis of RNA. We also examined allelic loss of the RB gene using PCR-based restriction fragment length polymorphism analysis. Of 30 samples obtained from patients with bladder cancer, eight (27%) were found to have RB gene mutations. DNA sequencing of the PCR products revealed five cases with single point mutations and three cases with small deletions. These mutations included one (10%) of ten low-grade (grade 1) tumours, four (50%) of eight intermediate-grade (grade 2) tumours and three (25%) of 12 high-grade (grade 3) tumours. Likewise, mutations were found in four (21%) of 19 superficial (pTa and pT1) tumours and four (36%) of 11 invasive (pT2 or greater) tumours. In 15 informative cases, loss of heterozygosity at the RB locus was shown in five cases (33%), three cases with RB mutations and two without them. These results suggest that RB gene mutations are involved in low-grade and superficial bladder cancers as well as in high-grade and invasive cancers

Overexpression of E2F1 associated with LOH at RB locus and hyperphosphorylation of RB in non-small cell lung carcinoma

金沢大学大学院医学系研究科保健学専攻Purpose E2F1 plays a critical role in cell proliferation, and its function is controlled by the retinoblastoma (RB) protein. We examined the expression of E2F1 and the aberration of RB gene and protein to elucidate what factors contribute to the overexpression of E2F1 in non-small cell lung carcinomas. Methods The expression level of E2F1 in tissues of non-small cell lung carcinomas was measured by means of quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. For RB, we examined loss of heterozygosity (LOH) by PCR-restriction fragment length polymorphism and a variable number of tandem repeats, and protein expression by immunohistochemistry. Results Fifteen cases of carcinoma (46%) showed high transcription levels of E2F1 gene. Immunohistochemically, almost all (14 of 15) cases overexpressing E2F1 mRNA were positive for E2F1 protein. LOH at the RB locus was found in 13 of 30 informative cases. In 13 cases with LOH, ten showed overexpression of E2F1 mRNA and protein. Immunohistochemical positivity for phosphorylated RB protein was also closely correlated with overexpression of E2F1. Conclusions Our results suggest that overexpression of E2F1, induced both by LOH at the RB locus and anomalous phosphorylation of the RB protein, is involved in the development of non-small cell lung carcinoma

Physical mapping of a chromosomal region deleted in small cell lung cancer

Characteristic deletions have been associated with a number of solid tumours, which represent the sites of tumour suppressor genes. The deletion of 3p21 sequences in small cell lung carcinoma has been demonstrated. To identify and isolate this tumour suppressor gene by positional cloning, a precise knowledge of the boundaries of the deletion is required. A detailed physical map of this region is important in the identification of genes with potential tumour suppressing properties. The thesis describes the physical mapping of a region of the human genome, 3p21, which is consistently deleted in small cell lung carcinoma, SCLC. These deletions are thought to indicate the presence of a tumour suppressor gene. Somatic cell hybrids and pulsed field gel electrophoresis were used to map within the deleted region. Somatic cell hybrids were established to provide a mapping resource for the fine mapping of markers on 3p21. Each panel of hybrids were characterised with available 3p markers and genes. One hybrid containing a portion of 3p21 representing a part of the deletion was used further in physical mapping studies. A gene was isolated from this hybrid and it's genomic structure determined. A cosmid encompassing this gene was used to initiate a chromosomal walk and construction of a contig in this region. Using pulsed field gel electrophoresis, a long range map around this gene was generated and a physical link to another marker in 3p21 shown. This defined region was analysed in SCLC tumours using similar techniques

Linkage studies in a Li-Fraumeni family with increased expression of p53 protein but no germline mutation in p53.

We report a family with the Li-Fraumeni syndrome (LFS) in whom we have been unable to detect a mutation in the coding sequence of the p53 gene. Analysis of linkage to three polymorphic markers within p53 enabled direct involvement of p53 to be excluded. This is the first example of a LFS family in whom exclusion of p53 has been possible. Four affected members of the family with sarcoma or premenopausal breast cancer showed increased expression of p53 protein in their normal tissues as detected by immunohistochemistry. It therefore appears that the LFS phenotype has been conferred by an aberrant gene, showing a dominant pattern of inheritance, which may be acting to compromise normal p53 function rather than by a mutation in p53 itself. In order to try to determine the chromosomal location of this putative gene, we have carried out studies of linkage to candidate loci. By these means we have excluded involvement of Rb1 and BRCA1 on chromosomes 13q and 17q respectively. The MDM2 oncogene on chromosome 12q was considered to be the prime candidate as MDM2 is amplified in sarcomas and the MDM2 product binds to p53. Furthermore, p53 mutation and amplification of MDM2 have been shown to be mutually exclusive events in tumour development. Linkage analysis to two polymorphic markers within MDM2 yielded a three-point LOD score of -5.4 at a recombination fraction theta equal to zero. Therefore MDM2 could be excluded. It is possible that the gene which is responsible for cancer susceptibility in this family, possibly via interaction with p53, will be important in the histogenesis of breast cancer in general. We are now carrying out further studies to locate and identify this gene

Investigations of the molecular determinants of maize streak virus replication

Includes bibliographical references.Geminiviruses replicate via a rolling circle mechanism, which initiates at the origin of replication located within the long intergenic region (LIR). The viral replication associated-protein (Rep) in conjunction with the host's DNA replication machinery is responsible for the initiation and termination of the replication cycle from a stem-loop structure, located within the LIR and conserved throughout the three genera of Geminiviridae. The specific interaction between the Rep protein with sequences within the intergenic region has been well characterised for the begomoviruses and to some extent the curtoviruses; however, this interaction in the mastreviruses, and in particular maize streak virus (MSV), has yet to be fully explored. A theoretical model has been proposed based on sequence data and informed by the current understanding of replication specificity in begomoviruses. Due to the lack of conservation of the stem sequence of the stem-loop structure amongst mastreviruses, the model implicates a pair of nucleotide sequence repeats called iterons. These are located within the stem structure, and on the complementary sense side of the LIR. The former is the putative site of Rep interaction with the LIR. These iterons would therefore potentially act as the determinants of replication specificity amongst mastreviruses

Analysis of the relationship between genomic instability, heterozygosity levels and phenotype in Saccharomyces cerevisiae

2018 Fall.Includes bibliographical references.Understanding the forces that mediate genome evolution is a central problem in genetics, with implications for diverse processes that range from speciation, to biotechnological applications, to human disease. The central theme of my dissertation was the characterization of two forces, genomic instability and natural selection, that significantly impact genome structure by influencing the levels of genomic heterozygosity. While genomic instability processes can act to erode heterozygosity from the genome, natural selection may favor the maintenance of heterozygous alleles in cases where there is a positive correlation between heterozygosity and higher fitness. In Chapter I, I reviewed different types of mitotic mutations that can result in the appearance of tracts of homozygosity in genomes and recent discoveries about the temporal accumulation of such events. I also introduce the concept of heterosis, a phenomenon characterized by a positive correlation between genomic heterozygosity and phenotype in many species, and its potential role in contributing to the long-term maintenance of genomic heterozygosity. In Chapter II, I describe the characterization of a mechanism of systemic genomic instability in yeast that challenges the conventional model of gradual and independent accumulation of mutations. We showed that a subset of mitotic cells within a population experience bursts of genomic instability, which results in multiple independent events of loss-of-heterozygosity (LOH) accumulating over one or a few generations of mitotic cell division. We named this outcome "systemic genomic instability". The occurrence of this phenomenon was initially identified in the heterozygous yeast strain JAY270, and then validated in a conventional laboratory strain background, whose genome is almost fully homozygous. Elevated rates of coincident LOH was also observed in mutant strains incapable of entering meiosis, indicating cryptic initiation of meiotic recombination followed by return-to-growth in a few cells in the population was not responsible for the higher than expected rates of coincident LOH. This finding brings to light a novel and intriguing mechanism of genomic instability in yeast that has relevant parallels to bursts of accumulation of copy number alterations in the human genome, providing a powerful experimental model system to dissect the fundamental mechanisms responsible for the generation of rapid changes in chromosome structure. In Chapter III, we explored the role that genomic heterozygosity plays on the superior industrial traits of the JAY270 strain. In the previous Chapter we showed that mitotic recombination leading to LOH occurs at a high frequency during JAY270's clonal propagation. These LOH events act against the long-term maintenance of genomic heterozygosity, yet about 60% of JAY270's genome has remained heterozygous over time. We hypothesized that specific heterozygous alleles may have a positive impact on the traits of this strain and therefore were maintained through selection. We generated a collection of inbred strains derived from JAY270, and assessed them phenotypically under different growth conditions. Our results demonstrated that genomic heterozygosity indeed has a substantial impact on two important industrial traits of this strain – heat stress tolerance and growth kinetics. We identified several genomic regions potentially associated with those traits and conducted experiments to investigate the bulk contributions of heterozygosity blocks in three specific chromosomes. This study revealed candidate regions containing loci that potentially underlie important industrial traits of JAY270 and details on the extent to which heterozygosity may impact JAY270's genome evolution and phenotype. The combined results of these research projects provide important insights about the role of genomic instability mechanisms and their phenotypic outcomes in determining genome evolution, contributing discoveries that may have important practical implications for diverse fields, including biotechnology, cancer development and evolution, as well as genome sciences