Diana Parma

Expression of c-myc and c-fos oncogenes in different rat brain regions during postnatal development

We have studied the expression of c‐myc and c‐fos proto‐oncogenes in various areas of the central nervous system during postnatal development, c‐myc mRNA levels increased during the first 5 days and then decreased over the next 15 days in all nervous regions studied, c‐fos mRNA levels changed in a different way in four brain areas. While in cerebral cortex and cerebellum there was a sharp decrease during the first 10 days, in white matter and hypothalamus c‐fos transcript levels remained high during the same period, decreasing at a later stage.

Spectrum of RB1 mutations in argentine patients: 20-years experience in the molecular diagnosis of retinoblastoma.

ABSTRACT Background: Retinoblastoma is a hereditary cancer of childhood caused by mutations in the RB1 tumor suppressor gene. An early diagnosis is critical for survival and eye preservation, thus identification of RB1 mutations is important for unequivocal diagnosis of hereditary retinoblastoma and risk assessment in relatives. Methods: We studied 144 families for 20 years, performing methodological changes to improve detection of mutation. Segregation analysis of polymorphisms, MLPA, FISH and cytogenetic assays were used for detection of “at risk haplotypes” and large deletions. Small mutations were identified by heteroduplex/DNA sequencing. Results: At risk haplotypes were identified in 11 familial and 26 sporadic cases, being useful for detection of asymptomatic carriers, risk exclusion from relatives and uncovering RB1 recombinations. Ten large deletions (eight whole gene deletions) were identified in six bilateral/familial and four unilateral retinoblastoma cases. Small mutations were identified in 29 cases (four unilateral retinoblastoma patients), being the majority nonsense/frameshift mutations. Genotype-phenotype correlations confirm that the retinoblastoma presentation is related to the type of mutation, but some exceptions may occur and it is crucial to be considered for genetic counseling. Three families included second cousins with retinoblastoma carrying different haplotypes, which suggest independent mutation events. Conclusion: This study enabled us to obtain information about molecular and genetic features of patients with retinoblastoma in Argentina and correlate them to their phenotype.

Clinical, cytogenetic, and molecular testing of Argentine patients with retinoblastoma

PURPOSE The purpose of this study is to determine the clinical, chromosomal, and molecular characteristics of Argentine patients with unilateral and bilateral retinoblastoma. STUDY DESIGN Eighty-six patients belonging to 82 families were studied; 59% of them were examined during the first year of life. Leukocoria was the most common reason for consultation. Other presenting signs were strabismus and glaucoma. Enucleation of the affected eye was performed in 85% of the cases and the complication rate was 13%. RESULTS An appropriate therapy allowed the survival of 84 of the 86 patients. Two children with malformations and growth retardation had an abnormal karyotype with a deletion in 13q14. Segregation analysis of polymorphic sites within the retinoblastoma gene and the parental origin of the allele lost in the tumor were analyzed in 30 of the 82 families. Five mutant alleles transmitted through the germline and six de novo germline mutant alleles were identified in 12 patients with hereditary retinoblastoma. Most de novo germline mutant alleles were paternally derived. Molecular analysis of nonhereditary retinoblastoma showed loss of heterozygosity in three of eight cases. From these, two maternal alleles and one paternal allele were lost, thus not indicating a significant difference in the parental origin for the lost allele. CONCLUSIONS These data are useful for deoxyribonucleic acid diagnosis of susceptibility to retinoblastoma in relatives of hereditary patients, even if mutations have not been identified.

Detection of mutations in argentine retinoblastoma patients by segregation of polymorphisms, exon analysis and cytogenetic test.

The aim of this study was to detect chromosomal and molecular abnormalities in 16 Argentine families with retinoblastoma (RB). Chromosomes were analyzed by G-banding, DNA from leukocytes and tumors was studied by segregation of polymorphisms within RB gene (RB1) and the DNA from chorionic villus by sequencing. The karyotype of an Rb236 bilateral patient with dismorphic signs revealed a deletion in 13q13–21. Polymorphism and exon analyses showed a deletion in the 3′ end of RB1 in an Rb72 patient. The mutant RB1 allele, detected by loss of heterozygosity (LOH) in the tumor, was identified in 14 out of 18 tumors. The analysis of chorionic villus revealed a mutation, a C-to-T transition in exon 18. Molecular and cytogenetic analyses in families with RB offer valuable information on how to assess the risk of tumor development.

MLPA analysis of an Argentine cohort of patients with dystrophinopathy: Association of intron breakpoints hot spots with STR abundance in DMD gene.

Dystrophinopathies are X-linked recessive diseases caused by mutations in the DMD gene. Our objective was to identify mutations in this gene by Multiplex Ligation Probe Amplification (MLPA), to confirm the clinical diagnosis and determine the carrier status of at-risk relatives. Also, we aimed to characterize the Dystrophinopathies argentine population and the DMD gene. We analyzed a cohort of 121 individuals (70 affected boys, 11 symptomatic women, 37 at-risk women and 3 male villus samples). The MLPA technique identified 56 mutations (45 deletions, 9 duplications and 2 point mutations). These results allowed confirming the clinical diagnosis in 63% (51/81) of patients and symptomatic females. We established the carrier status of 54% (20/37) of females at-risk and 3 male villus samples. We could establish an association between the most frequent deletion intron breakpoints and the abundance of dinucleotide microsatellites loci, despite the underlying mutational molecular mechanism remains to be elucidated. The MLPA demonstrate, again, to be the appropriate first mutation screening methodology for molecular diagnosis of Dystrophinopathies. The reported results permitted to characterize the Dystrophinopathies argentine population and lead to better understanding of the genetic and molecular basis of rearrangements in the DMD gene, useful information for the gene therapies being developed.

Importancia de los estudios de biología molecular en el asesoramiento genético de familias argentinas con retinoblastoma

espanolObjetivo: Evaluar la importancia de la deteccion de mutaciones del gen RB1 en el asesoramiento genetico de las familias argentinas con retinoblastoma. Metodos: 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 corionicas, por ensayos de Biologia Molecular indirectos y directos, como Southern blot, segregacion de los polimorfismos BamHI, Rbi4, XbaI y Rb 1.20 (PCR-RFLP, PCR-STR), PCR-heteroduplex y secuenciacion del gen RB1. Resultados: El analisis 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 conto con muestras de ADN tumoral de 11 pacientes que se estudiaron para detectar perdida de heterocigosidad (LOH), que posibilito 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 utilizo la deteccion 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 acentuan la importancia de la deteccion directa de la mutacion en familias con Rb esporadico temprano sin muestra tumoral. Los estudios de biologia molecular contribuyeron con el adecuado asesoramiento genetico de pacientes argentinos y sus familiares y el diseno apropiado de su tratamiento temprano. EnglishObjective: 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.

RB1 gene mutations in Argentine retinoblastoma patients. Implications for genetic counseling

Retinoblastoma (RB) is an inherited childhood ocular cancer caused by mutations in the tumor suppressor RB1 gene. Identification of RB1 mutations is essential to assess the risk of developing retinoblastoma in the patients´ relatives. Retinoblastoma is a potentially curable cancer and an early diagnosis is critical for survival and eye preservation. Unilateral retinoblastoma is mostly non-heritable and results from two somatic mutations whereas bilateral retinoblastoma is heritable and results from one germline and one somatic mutation, both have high penetrance, 90%. The purpose of this study was to identify causative RB1 mutations in RB patients with different clinical presentations. A comprehensive approach was used to study a cohort of 34 patients with unilateral, bilateral and trilateral retinoblastoma. Blood and tumor DNA was analyzed by sequencing and multiplex ligation-dependent probe amplification (MLPA) assay. Validation of an insertion mutation was performed by cloning the PCR product. Most of the patients in our cohort had unilateral RB, eight patients had bilateral RB and one patient had a trilateral tumor with ocular and suprasellar/sellar locations. Other tumors in addition to retinoblastoma were also found in the affected families. One patient had two syndromes, retinoblastoma and schwannomatosis, and another RB patient had a father with a retinoma. Five out of the 25 unilateral RB patients carried germinal mutations (20%), which were mostly missense mutations. The bilateral and trilateral patients carried splice-site, nonsense and frameshift mutations as well as a whole RB1 gene deletion. Missense mutations were associated with mild phenotype: unilateral retinoblastoma, retinoma or no tumor. In this study we identified causative RB1 mutations in most bilateral RB patients and in some unilateral RB patients, including five novel mutations. These data are crucial for genetic counseling and confirm the need to perform complete genetic screening for RB1 mutations in both constitutional and tumor tissues.