Subsequent malignant neoplasms in the pediatric age in retinoblastoma survivors in Argentina.

BACKGROUND: Retinoblastoma survivors in low- and middle-income countries are exposed to high-intensity treatments that potentially place them at higher risk of early subsequent malignant neoplasms (SMNs). METHODS: We followed 714 (403 [56.4%] nonhereditary and 311 [43.5%] hereditary) retinoblastoma survivors diagnosed from August 1987 to December 2016, up to the age of 16 years. We quantified risk of SMNs with cumulative incidence (CI) and standardized incidence ratios (SIR) analysis. Multivariate regression Cox model was used to determine the association of treatments and risk of SMNs. RESULTS: Median follow-up was of 9 years (range: 0.18-16.9) and 24 survivors (3.36%) developed 25 SMNs (n = 22 hereditary, n = 2 nonhereditary). SMNs included sarcomas (osteosarcomas, Ewing sarcomas, rhabdomyosarcomas; n = 12), leukemias (n = 5), and central nervous system tumors (CNS; n = 3). All cases of acute myeloid leukemia (AML) and most of Ewing sarcomas occurred within 5 years of retinoblastoma diagnosis. The type of SMN was the main indicator of mortality (five of five patients with leukemias, six of 12 with sarcomas, and zero of three with CNS tumors died). Compared to the general population, radiation increased the risk of Ewing sarcoma in hereditary survivors by 700-fold (95% CI = 252-2422.6) and chemotherapy increased the risk of AML by 140-fold (95% CI = 45.3-436). The CI of SMNs for hereditary survivors was 13.7% (95% CI = 8.4-22.1) at 15 years. CONCLUSION: Retinoblastoma survivors from Argentina are at higher risk of developing SMNs early in life compared to the general Argentinean population, especially those treated with radiation plus chemotherapy. AML and Ewing sarcoma presented within 5 years of retinoblastoma diagnosis are associated with chemotherapy and radiation exposure.

Prognostic value of mutations in isocitrate dehydrogenase 1 (IDH1) and reverse telomerase transcriptase (TERT ) in Argentine patients` gliomas / Valor pronóstico de mutaciones en Isocitrato dehidrogenasa1 (IDH1) y Telomerasa transcriptasa reversa (TERT ) en gliomas de pacientes argentinos

Background and aim: Gliomas are the most common primary brain tumors, classified according to their histopathological and genetic features. Tumorigenesis depends on alterations in different genes. The aim of this study was the identification of mutations in IDH1 and TERT genes in gliomas of Argentine patients and to correlate them with clinical features and prognosis. Methods: DNA was isolated from 19 biopsies with different glioma grades matched with blood samples. IDH1 and TERT mutations were studied by PCR amplifica-tion and sequencing. Results: Six out of seven patients with low-grade glioma (grade II) harbor IDH1 mutations, mainly without tumor growth and overall survival of more than 12 months. Eleven out of twelve patients with high-grade gliomas (grade III/IV) showed wild type IDH1, mainly with tumor growth and shorter survival than low-grade gliomas. Mutated TERT promoter was present in 5 out of 11 high-grade gliomas, showing the prevalence of polymorphic C allele. In 1 out of 5 low-grade gliomas with a predominance of T allele. TERT and IDH1 mutations were mutually exclusive in most gliomas. Conclusions: Our results show that genetic tests provided a more accurate prognosis than histopathological analysis. The evolution of gliomas can be predicted primarily by the mutational status of IDH1 and secondarily by other markers, such as TERT mutational status

Antecedentes y objetivo: los gliomas son los tumores cerebrales primarios más comunes y se clasifican según sus características histopatológicas y genéticas. La tumorigénesis depende de alteraciones en diferentes genes. El objetivo de este estudio fue identificar mutaciones en los genes IDH1 y TERT en gliomas de pacientes argentinos y correlacionarlos con la evolución clínica. Métodos: se obtu-vieron 19 muestras pareadas de ADN de gliomas y de la sangre. Las mutaciones en IDH1 y TERT se analizaron por PCR y secuenciación. Resultados: la IDH1 mutada se encontró en 6 de los 7 gliomas de bajo grado (grado II), mayormente sin crecimiento tumoral y una sobrevida mayor de 12 meses. La IDH1 salvaje estaba presente en 11 de los 12 gliomas de alto grado (grado III y IV) mayormente con crecimiento tumoral y menor sobrevida que los tumores de bajo grado. Las mutaciones en el promotor del gen TERT se observaron en 5 de los 11 gliomas de alto grado, con la prevalencia de alelo polimórfico C, en cambio, en gliomas de bajo grado TERT mutado estaba presente en 1 de los 5 gliomas con predominio del alelo T. Las mutaciones en IDH1 y TERT fueron mutuamente excluyentes en la mayoría de los gliomas. Conclusiones: el análisis genético provee un pronóstico más certero que el análisis histopatológico. Nuestros resulta-dos muestran que la evolución de gliomas puede predecirse primariamente por el estado mutacional de IDH1 y secundariamente por mutaciones en otros marcadores tales como el TERT

Recurrent Somatic Chromosomal Abnormalities in Relapsed Extraocular Retinoblastoma.

Most reports about copy number alterations (CNA) in retinoblastoma relate to patients with intraocular disease and features of children with extraocular relapse remain unknown, so we aimed to describe the CNA in this population. We evaluated 23 patients and 27 specimens from 4 centers. Seventeen cases had extraocular relapse after initial enucleation and six cases after an initial preservation attempt. We performed an analysis of CNA and BCOR gene alteration by SNP array (Single Nucleotide Polymorfism array), whole-exome sequencing, IMPACT panel and CGH array (Array-based comparative genomic hybridization). All cases presented CNA at a higher prevalence than those reported in previously published studies for intraocular cases. CNA previously reported for intraocular retinoblastoma were found at a high frequency in our cohort: gains in 1q (69.5%), 2p (60.9%) and 6p (86.9%), and 16q loss (78.2%). Other, previously less-recognized, CNA were found including loss of 11q (34.8%), gain of 17q (56.5%), loss of 19q (30.4%) and BCOR alterations were present in 72.7% of our cases. A high number of CNA including 11q deletions, 17q gains, 19q loss, and BCOR alterations, are more common in extraocular retinoblastoma. Identification of these features may be correlated with a more aggressive tumor warranting consideration for patient management.

Analysis of complex structural variants in the DMD gene in one family.

This work describes a family with Duchenne muscular dystrophy (DMD) with a rare case of a symptomatic pregnant woman. The main aim was to perform prenatal molecular diagnosis to provide genetic counseling. The secondary aim was to suggest the molecular mechanisms causing the complex structural variant (cxSV) identified. To accomplish this, we used a multi-technique algorithm including segregation analysis, Multiplex Ligation-dependent Probe Amplification, PCR, X-chromosome inactivation studies, microarrays, whole genome sequencing and bioinformatics. We identified a duplication of exons 38-43 in the DMD gene in all affected and obligate carrier members, proving that this was the DMD-causing mutation. We also observed a skewed X-chromosome inactivation in the symptomatic woman that explained her symptomatology. In addition, we identified a cxSV (duplication of exons 38-43 and deletion of exons 45-54) in the affected boy. The molecular characterization and bioinformatic analyses of the breakpoint junctions allowed us to identify Double Strand Breaks stimulator motifs and suggested the replication-dependent Fork Stalling and Template Switching as the most probable mechanisms leading to the duplication. In addition, the de novo deletion might have been the result of a germline inter-chromosome non-allelic recombination involving the Non-Homologous End Joining mechanism. In conclusion, the diagnostic strategy used allowed us to provide accurate molecular diagnosis and genetic counseling. In addition, the familial molecular diagnosis together with the in-depth characterization of the cxSV helped to determine the chronology of the molecular events, and propose and understand the molecular mechanisms involved in the generation of this complex rearrangement.

Small mutation screening in the DMD gene by whole exome sequencing of an argentine Duchenne/Becker muscular dystrophies cohort.

Dystrophinopathies are neuromuscular X-linked recessive diseases caused by mutations in the DMD gene. This study aimed to identify DMD gene small mutations by Whole Exome Sequencing (WES), in order to confirm clinical diagnosis, identify candidates for Ataluren treatment and perform carrier status testing. Furthermore, was our goal to characterize the DMD sequence variants and identify ancestral haplotypes. We analyzed 40 non-related individuals (38 affected boys with dystrophinopathy presumptive clinical diagnosis and 2 at-risk women) with negative MLPA results. Pathogenic DMD variants were found in 32 boys. Surprisingly, in another 4 patients with absence/deficiency of dystrophin in muscle biopsy, pathogenic variants were found in Limb-girdle muscular dystrophy genes. Therefore, the WES detection rate resulted ∼94% (36/38). We could identify 15 Ataluren candidates and exclude 2 at-risk women. The characterization of the occurrence and diversity of DMD sequence variants from our cohort and from LOVD database, revealed no hotspots but showed exons/introns unlikely to carry small molecular alterations and exons presenting a greater mutagenic abundance than others. Also, we have detected the existence of 2 co-segregating haplotypes blocks. Finally, this work represents the first DMD gene small mutations screening applying WES in an argentine cohort, contributes with the characterization of our population and collaborates with the DMD small mutation's knowledge.

Minimal disseminated disease evaluation and outcome in trilateral retinoblastoma.

Trilateral retinoblastoma (TRb) presents a management challenge, since intracranial tumours are seldom times resectable and quickly disseminate. However, there are no risk factors to predict the final outcome in each patient. OBJECTIVE: To evaluate minimal disseminated disease (MDD) in the bone marrow (BM) and the cerebrospinal fluid (CSF) at diagnosis and during follow-up and reviewing its potential impact in the outcome of patients with TRb. METHODS AND ANALYSIS: We evaluated MDD in five patients with TRb, detecting the mRNA of CRX and/or GD2, in samples from BM and CSF, obtained at diagnosis, follow-up and relapse. RESULTS: Treatment involved intensive systemic chemotherapy in four patients, one did not receive this treatment and died of progression of the disease. Two patients underwent stem cell rescue. Three patients had leptomeningeal relapse and died. One patient remains disease-free for 84 months. RB1 mutations were identified in the five patients, all of them were null mutations. At diagnosis, one patient had tumour cells in the CSF, and none had the BM involved. Only one case of four presented MDD during follow-up in the CSF, without concomitant detection in the BM. On leptomeningeal relapse, no case had MDD in the BM. In all these cases, cells in the CSF were positive for GD2 and/or CRX. CONCLUSION: CSF dissemination always concluded in the death of the patient, without concomitant systemic dissemination denoting the importance of increasing treatment directed to the CSF compartment. The MDD presence could indicate a forthcoming relapse.

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.

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.

Uncommon RB1 somatic mutations in a unilateral retinoblastoma patient.

Retinoblastoma (RB) is the most common primary intraocular malignancy in children. Somatic inactivation of both alleles of the RB1 tumor suppressor gene in a developing retina is a crucial event in the initiation of tumorigenesis in most cases of isolated unilateral retinoblastoma. We analyzed the DNA from tumor tissue and peripheral blood of a unilateral retinoblastoma patient to determine the RB1 mutation status and to provide an accurate genetic counseling. A comprehensive approach, based on our previous experience, was used to identify the causative RB1 mutations. Screening for RB1 mutations was performed by PCR direct sequencing, multiplex ligation-dependent probe amplification (MLPA) and Real Time-PCR analyses. Three different mutations were identified in the tumor DNA, which were absent in blood DNA. The somatic origin of these mutations was vital to rule out the heritable condition in this patient.

Uncommon RB1 somatic mutations in a unilateral retinoblastoma patient

Retinoblastoma (RB) is the most common primary intraocular malignancy in children. Somatic inactivation of both alleles of the RB1 tumor suppressor gene in a developing retina is a crucial event in the initiation of tumorigenesis in most cases of isolated unilateral retinoblastoma. We analyzed the DNA from tumor tissue and peripheral blood of a unilateral retinoblastoma patient to determine the RB1 mutation status and to provide an accurate genetic counseling. A comprehensive approach, based on our previous experience, was used to identify the causative RB1 mutations. Screening for RB1 mutations was performed by PCR direct sequencing, multiplex ligation-dependent probe amplification (MLPA) and Real Time-PCR analyses. Three different mutations were identified in the tumor DNA, which were absent in blood DNA. The somatic origin of these mutations was vital to rule out the heritable condition in this patient.

El retinoblastoma (RB) es el cáncer ocular más común de la niñez. La inactivación somática de ambos alelos del gen supresor de tumores RB1 en la retina en desarrollo es un evento crucial en la iniciación de la tumorigénesis en la mayoría de los casos de retinoblastoma unilateral. Nosotros analizamos el ADN de tumor y de sangre periférica de un paciente con retinoblastoma unilateral para identificar las mutaciones y así proveer un asesoramiento genético a la familia. Para ello utilizamos un protocolo basado en nuestra previa experiencia para identificar todas las mutaciones en el gen RB1 que causaron el RB. El rastreo de mutaciones se realizó por medio de los siguientes análisis: PCR-secuenciación, amplificación multiplex de sondas ligadas (MLPA) y PCR-Tiempo Real. Se encontraron tres mutaciones diferentes en el ADN del tumor, las cuales estaban ausentes en el ADN de la sangre. El origen somático de estas mutaciones es importante para indicar que la enfermedad no es hereditaria.

Molecular diagnosis of dystrophinopathies using a multi-technique analysis algorithm.

INTRODUCTION: Dystrophinopathies are X-linked recessive neuromuscular diseases caused by mutations in the dystrophin gene. In this study we aimed to detect mutations within the dystrophin gene in DMD patients, to determine the carrier status of women, and to perform a prenatal diagnosis. METHODS: We analyzed 17 individuals from 2 unrelated families with a history of DMD. We used multiplex PCR, multiplex ligation-dependent probe amplification (MLPA), and short tandem-repeat (STR) segregation analysis to accurately detect and characterize the mutations and to identify the at-risk haplotype. RESULTS: The selected methodology allowed for characterization of 2 single-exon out-of-frame deletions in affected patients. Nine of 13 women and a fetus were excluded from being carriers. Three recombination events were found and suggested that germline mosaicism had occurred in both families. CONCLUSIONS: This methodology proved to be efficient for characterizing the disease-causing mutation in affected individuals and for assessing the carrier status in healthy relatives. These findings helped inform precise genetic counseling and contributed to characterization of the disease in the Argentine population.

Symptomatic female carriers of Duchenne muscular dystrophy (DMD): genetic and clinical characterization.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the dystrophin gene and is characterized by muscle degeneration and death. DMD affects males; females being asymptomatic carriers of mutations. However, some of them manifest symptoms due to a translocation between X chromosome and an autosome or to a heterozygous mutation leading to inactivation of most of their normal X chromosome. Six symptomatic female carriers and two asymptomatic were analyzed by: I) Segregation of STRs-(CA)n and MLPA assays to detect a hemizygous alteration, and II) X chromosome inactivation pattern to uncover the reason for symptoms in these females. The symptomatic females shared mild but progressive muscular weakness and increased serum creatin kinase (CK) levels. Levels of dystrophin protein were below normal or absent in many fibers. Segregation of STRs-(CA)n revealed hemizygous patterns in three patients, which were confirmed by MLPA. In addition, this analysis showed a duplication in another patient. X chromosome inactivation assay revealed a skewed X inactivation pattern in the symptomatic females and a random inactivation pattern in the asymptomatic ones. Our results support the hypothesis that the DMD phenotype in female carriers of a dystrophin mutation has a direct correlation with a skewed X-chromosome inactivation pattern.

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

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.

Prenatal diagnosis of Duchenne/Becker muscular dystrophy by short tandem repeat segregation analysis in Argentine families.

INTRODUCTION: Duchenne/Becker muscular dystrophies (DMD/BMD) are X-linked recessive diseases caused by mutations in the dystrophin gene. METHODS: We used multiplex polymerase chain reaction (PCR) and short tandem repeat (STR) segregation analysis for DMD/BMD-carrier detection and prenatal diagnosis. RESULTS: Twenty-four at-risk pregnancies were evaluated: 17 were excluded from carrying dystrophin gene mutations with 95-100% certainty. Of the remaining cases, 2 were determined to carry a dystrophin gene mutation with 95-100% certainty. Three cases had a 67% probability of carrying the mutation, and 2 others were not informative. The certainty of the test increased to ~100% in some cases due to the identification of several genetic events: 4 recombinations; 4 de novo mutations; and 8 deletions encompassing some of the STRs evaluated. DISCUSSION: Overall, 19 of 24 (79%) molecular prenatal diagnoses were informative, indicating that multiplex PCR/STR segregation analysis is a reliable method for carrier detection and prenatal diagnosis when other more sophisticated techniques are unavailable.

Neurofibromatosis type 2: molecular and clinical analyses in Argentine sporadic and familial cases.

Neurofibromatosis 2 is a familial syndrome characterized by the development of schwannomas, meningiomas and ependymomas. Most of them are benign however, their location in the nervous system has harmful effects on important cranial and spinal structures. These tumors are developed as the outcome of NF2 gene (22q12) inactivation. The NF2 protein, merlin or schwannomin belongs to the Ezrin, Radixin, Moesin (ERM) family involved in the cytoskeletal network and has a tumor suppressor function. Inactivating mutations occur as "de novo" (more frequently) or as inherited, and most of them are frameshift or nonsense. Our aim is to study NF2 gene alterations in Argentine patients and relate them to clinical features. 10 families and 29 single patients were analyzed for: 1) at-risk haplotype by STR-segregation analysis and 2) NF2 gene mutations by SSCP/heteroduplex/sequencing. The at-risk haplotype was uncovered in 8 families and mutations were identified in 5 patients. The molecular data are in full agreement with the clinical features supporting previous reports. The obtained results were important for the detection of mutation-carrying relatives and exclusion of other individuals from risk.

Gene expression profiling of the hedgehog signaling pathway in human meningiomas.

The Hedgehog (Hh) signaling pathway has an important role during embryogenesis and in adult life, regulating proliferation, angiogenesis, matrix remodeling and stem-cell renewal. Deregulation of the Hh pathway is involved in tumor development, since mutations in several components of this pathway were found in patients with basal cell carcinoma, medulloblastoma and other tumors; however, the role of Hh in meningiomas has not been studied yet. Meningiomas represent 30% of primary cranial tumors, are mostly benign and prevail in the second half of life. Novel therapies for meningiomas such as targeted molecular agents could use Hh pathway components. To provide information concerning molecular alterations, by use of real-time RT-PCR, we studied expression at the mRNA level of 32 Hh pathway and target genes in 36 meningioma specimens of different grades. mRNA levels of 16 genes, involved mainly in Hh pathway activation and cell proliferation, increased in meningiomas in comparison with normal tissue, whereas those of 7 genes, mainly related to Hh pathway repression, decreased. The most significant changes occurred in signal transduction (SMO) and GLI-transcription factor genes, and the target FOXM1 mRNA attained the highest values; their over-expression was found in aggressive and in benign tumors. Some proliferation-related genes (SPP1, IGF2) were overexpressed in higher meningioma grades. A correlation in expression between genes with a similar function was also found. Our results show a marked activation of the Hh pathway in meningiomas, which may be important for their biological and clinical characterization and would be useful for gene therapy.

Gene expression profiling of ErbB receptors and ligands in human meningiomas.

ErbB family receptors mediate major cellular functions implied in tumorigenesis, though their role in meningiomas was not thoroughly studied. Meningiomas represent 30% of primary cranial tumors, are mostly benign, and prevail in the second half of life. Tumor therapy requires information about molecular alterations, thus we studied expression of ErbB receptor and ligand genes by real-time RT-PCR in different meningioma grades. Receptors were overexpressed (ErbB1, ErbB2) or underexpressed (ErbB3, ErbB4). Ligands EGF, TGFA, AREG, DTR, BTD were underexpressed and the neuregulins were overexpressed or underexpressed. A strong ErbB1-ErbB2 correlation was found. These data might be useful for gene therapy.

T3 receptors in human pituitary tumors.

OBJECTIVE: The purpose of this work was to investigate the synthesis of T3 receptors in human tumors of the anterior pituitary gland, its relationship with the hormone synthesized and/or secreted by the tumor and the post-surgical evolution of the patient. METHODS: Patients were evaluated clinically and by magnetic nuclear resonance to classify the adenoma according to their size. Hormonal concentrations in sera were determined by radioimmunoassay. Immunohistochemistry of the pituitary hormones was performed in the tumors. Tumors were obtained at surgery and immediately frozen in ice, transported to the laboratory and stored at -70 degrees C. Reverse transcription was performed with purified RNA from the tumors. RESULTS: Out of 33 pituitary tumors, 29 had RNA for T3 receptors synthesis (88%). They were present in different histological specimens, the tumors were grades 1-4 according to their size, and there was no relationship between the size of the tumor and the presence of T3 receptor RNAs. The post-surgical evolution of the patient was mostly dependent on the size and not on the presence of T3 receptors. DISCUSSION: The presence of thyroid hormone receptors in pituitary tumors is in line with two important characteristics of these tumors: they are histologically benign and well differentiated.

Asymptomatic Becker muscular dystrophy in a family with a multiexon deletion.

We report a Becker muscular dystrophy (BMD) family with one 5-year-old affected patient and a 69-year-old asymptomatic grandfather. Dystrophin gene multiplex polymerase chain reaction and multiplex ligation-dependant probe amplification analysis showed that both males carried an in-frame deletion of exons 45-55. Segregation analysis revealed two additional asymptomatic boys in this family. Our finding supports previous predictions that exons 45-55 are the optimal multiexon skipping target in antisense gene therapy to transform the severe Duchenne muscular dystrophy into the milder BMD, or even asymptomatic, phenotype.

Expression of p16(INK4A) gene in human pituitary tumours.

Pituitary adenomas comprise 10-15% of primary intracranial tumours but the mechanisms leading to tumour development are yet to be clearly established. The retinoblastoma pathway, which regulates the progression through the cell cycle, is often deregulated in different types of tumours. We studied the cyclin-dependent kinase inhibitor p16(INK4A) gene expression at mRNA level in human pituitary adenomas. Forty-six tumour specimens of different subtypes, 21 clinically non-functioning, 12 growth hormone-secreting, 6 prolactin-secreting, 6 adrenocorticotropin-secreting, and 1 thyrotropin-secreting tumours were studied. All clinically non-functioning and most of the hormone-secreting tumours were macroadenomas (38/46). The RT-PCR assay and electrophoresis of the PCR-products showed that p16(INK4A) mRNA was undetectable in: 62% of non-functioning, 8% of growth hormone-secreting, 17% of prolactin-secreting and 17% of adrenocorticotropin-secreting adenomas. Forty percent of all macroadenomas and 25% of microadenomas had negative p16(INK4A) mRNA, the latter results suggest that the absence of p16(INK4A) product might be an early event in tumours with no expression of this suppressor gene. Within the non-functioning adenomas 63% were "null cell" and 37% were positive for some hormone, both subgroups showed similar percentage of cases with absence of p16(INK4A) mRNA. Our results show that clinically non-functioning macroadenomas have impaired p16(INK4A) expression in a clearly higher proportion than any other pituitary tumour subtype investigated. Other regulatory pathways may be implicated in the development of tumours with positive p16(INK4A) expression.