Detección de una mutación no estándar en el proto-oncogen ret por mutagénesis dirigida / Detection of a non-standard mutation in the ret protoncogene by site directed mutagenesis

MEN2A is an autosomic dominant disease, characterized by medullary thyroid cancer, pheochromocytoma and parathyroid hyperplasia. Mutations in the ret proto-oncogene are associated with this disease, with almost 100% of penetrance. The gene, situated on chromosome 10q11.2, codes for a transmembrane protein with a tyrosinkinase-like receptor function. Mutations that affect its extracellular domain, stimulate spontaneous homodimerization and elevate the basal tyrosinkinase activity. The codon 634 of the gene is considered a hot-spot site, since it is mutated in 85% of the MEN2A families. Our group developed in 2002 an indirect and costless strategy to detect alterations in this site. We present a family suspected of having MEN2A. We applied our PCR based indirect strategy on the DNA of the index patient and found that there was no mutation in that site. Posterior sequencing of exon 10 and 11 confirmed that the mutation affecting this family was in codon 611. Thus, we developed a new costless family-specific strategy based on mutagenic PCR and enzymatic cuts to diagnose all the family members. A seven-year old boy with this mutation was preventively thyroidectomized. In this way, combining the indirect methodology for codon 634 previously developed by our group, and a posterior family-specific mutation detection strategy, we were able to diagnose and intervene presymptomatically the family members, avoiding sending all the samples to foreign centers.(AU)

El síndrome de MEN2A es una enfermedad autosómica dominante que se caracteriza por el desarrollode cáncer medular de tiroides, feocromocitoma e hiperplasia de paratiroides. Mutaciones en elret proto-oncogén se asocian con MEN2A, con una penetrancia cercana al 100%. El gen se encuentra en elcromosoma 10q11.2 y codifica para una proteína transmembrana con función de receptor del tipo tirosina quinasa.Mutaciones que afectan el dominio extracelular de la proteína estimulan la dimerización espontánea del receptory un aumento de la actividad de tirosina quinasa basal. El codón 634 codifica para una cisteína, y es consideradoun sitio hot-spot por encontrarse mutado en el 85% de las familias con MEN2A. Para este sitio, nuestro grupo desarrolló en 2002 una metodología de detección indirecta y económica. Ante una familia sospechada de MEN2A, se aplicó esta estrategia, que reveló un codón 634 sano. Por posterior secuenciación se confirmó que el paciente índice portaba una mutación en el codón 611. Se desarrolló una nueva estrategia familiaespecífica por PCR mutagénica, que permitió diagnosticar en nuestro país a todos los integrantes de la familiacon costos accesibles. Un niño en el cual se halló la mutación, fue tiroidectomizado preventivamente, y a lafecha goza de buena salud. De esta manera, combinando la estrategia de detección de mutaciones en el sitiohot-spot y un posterior diseño de otra metodología familia-específica se pudo diagnosticar e intervenir preventivamente a la familia, sin enviar todas las muestras al extranjero.(AU)

Detección de una mutación no estándar en el proto-oncogen ret por mutagénesis dirigida / Detection of a non-standard mutation in the ret protoncogene by site directed mutagenesis

MEN2A is an autosomic dominant disease, characterized by medullary thyroid cancer, pheochromocytoma and parathyroid hyperplasia. Mutations in the ret proto-oncogene are associated with this disease, with almost 100% of penetrance. The gene, situated on chromosome 10q11.2, codes for a transmembrane protein with a tyrosinkinase-like receptor function. Mutations that affect its extracellular domain, stimulate spontaneous homodimerization and elevate the basal tyrosinkinase activity. The codon 634 of the gene is considered a hot-spot site, since it is mutated in 85% of the MEN2A families. Our group developed in 2002 an indirect and costless strategy to detect alterations in this site. We present a family suspected of having MEN2A. We applied our PCR based indirect strategy on the DNA of the index patient and found that there was no mutation in that site. Posterior sequencing of exon 10 and 11 confirmed that the mutation affecting this family was in codon 611. Thus, we developed a new costless family-specific strategy based on mutagenic PCR and enzymatic cuts to diagnose all the family members. A seven-year old boy with this mutation was preventively thyroidectomized. In this way, combining the indirect methodology for codon 634 previously developed by our group, and a posterior family-specific mutation detection strategy, we were able to diagnose and intervene presymptomatically the family members, avoiding sending all the samples to foreign centers.

El síndrome de MEN2A es una enfermedad autosómica dominante que se caracteriza por el desarrollode cáncer medular de tiroides, feocromocitoma e hiperplasia de paratiroides. Mutaciones en elret proto-oncogén se asocian con MEN2A, con una penetrancia cercana al 100%. El gen se encuentra en elcromosoma 10q11.2 y codifica para una proteína transmembrana con función de receptor del tipo tirosina quinasa.Mutaciones que afectan el dominio extracelular de la proteína estimulan la dimerización espontánea del receptory un aumento de la actividad de tirosina quinasa basal. El codón 634 codifica para una cisteína, y es consideradoun sitio hot-spot por encontrarse mutado en el 85% de las familias con MEN2A. Para este sitio, nuestro grupo desarrolló en 2002 una metodología de detección indirecta y económica. Ante una familia sospechada de MEN2A, se aplicó esta estrategia, que reveló un codón 634 sano. Por posterior secuenciación se confirmó que el paciente índice portaba una mutación en el codón 611. Se desarrolló una nueva estrategia familiaespecífica por PCR mutagénica, que permitió diagnosticar en nuestro país a todos los integrantes de la familiacon costos accesibles. Un niño en el cual se halló la mutación, fue tiroidectomizado preventivamente, y a lafecha goza de buena salud. De esta manera, combinando la estrategia de detección de mutaciones en el sitiohot-spot y un posterior diseño de otra metodología familia-específica se pudo diagnosticar e intervenir preventivamente a la familia, sin enviar todas las muestras al extranjero.

[Diagnosis by directed mutagenesis of a mutation at the hMSH2 gene associated with hereditary nonpolyposis colorectal cancer]. / Diagnóstico por mutagénesis dirigida de una mutación en el gen hMSH2 vinculada a cáncer colorrectal hereditario no poliposo.

Hereditary nonpolyposis colorectal cancer (NHPCC) is the most common form of inherited colon cancer and one of the most frequent autosomal dominant disorders. HNPCC presents an early onset of colorectal cancer (< 50 years), proximal localization of the colonic tumors, and high risk of developing multiple primary colorectal tumors as well as extracolonic tumors. This disease is caused by mutations in at least four DNA mismatch repair genes, (hMSH2, hMLH1, hPMS1 and hPMS2) and estimations indicate that it affects 1:200-1:2,000 people in the Western populations. The identification of the genes responsible for HNPCC has prompted the search for mutations in affected individuals. DNA from an affected member of a family was sent to a Dutch HNPCC Diagnosis Centre. This Centre reported a germinal mutation, which introduces a premature stopcodon and causes the production of a truncated protein. This particular mutation has not been previously registered in the database of mutations related to this disease. After the identification of the mutation in the index patient, we have developed a quick and efficient procedure for detecting mutations in the rest of the family. The methodology is based on the amplification of the exon 13 in the hMSH2 gene using a forward primer that abuts the mutation site and introduces the cutting sequence of the enzyme Haelll++ only in the wild type allele. At present, seventeen members of the family have been diagnosed and nine have been found to be affected. The methodology is simple, specific, sensitive, inexpensive and applicable in low complexity laboratories.

Diagnóstico por mutagénesis dirigida de una mutación en el gen hMSH2 vinculada a cáncer colorrectal hereditario no poliposo. / [Diagnosis by directed mutagenesis of a mutation at the hMSH2 gene associated with hereditary nonpolyposis colorectal cancer]

Hereditary nonpolyposis colorectal cancer (NHPCC) is the most common form of inherited colon cancer and one of the most frequent autosomal dominant disorders. HNPCC presents an early onset of colorectal cancer (< 50 years), proximal localization of the colonic tumors, and high risk of developing multiple primary colorectal tumors as well as extracolonic tumors. This disease is caused by mutations in at least four DNA mismatch repair genes, (hMSH2, hMLH1, hPMS1 and hPMS2) and estimations indicate that it affects 1:200-1:2,000 people in the Western populations. The identification of the genes responsible for HNPCC has prompted the search for mutations in affected individuals. DNA from an affected member of a family was sent to a Dutch HNPCC Diagnosis Centre. This Centre reported a germinal mutation, which introduces a premature stopcodon and causes the production of a truncated protein. This particular mutation has not been previously registered in the database of mutations related to this disease. After the identification of the mutation in the index patient, we have developed a quick and efficient procedure for detecting mutations in the rest of the family. The methodology is based on the amplification of the exon 13 in the hMSH2 gene using a forward primer that abuts the mutation site and introduces the cutting sequence of the enzyme Haelll++ only in the wild type allele. At present, seventeen members of the family have been diagnosed and nine have been found to be affected. The methodology is simple, specific, sensitive, inexpensive and applicable in low complexity laboratories.

Differential levels of thyroid peroxidase and thyroglobulin messenger ribonucleic acids in congenital goiter with defective thyroglobulin synthesis.

The biosynthesis of thyroid hormones requires iodide, thyroid peroxidase (TPO), thyroglobulin (Tg) and H2O2. We have studied two sisters with congenital large goiters and hypothyroidism. Perchlorate tests were negative. Serum T3 and T4 were decreased, TSH was increased and Tg was within the lower limit of normal. Biochemical and molecular studies were performed on goiter samples obtained after surgery. Tg content in both tissues was negligible. Paper chromatography of labeled iodocompounds showed a decrease in T4, and the presence of a pronase/pancreatin-resistant iodoprotein. TPO activity was normal in the tissues. Sephacryl S-300 gel filtration demonstrated labeled iodoalbumin-like protein and the absence of a Tg peak. Salting out studies of soluble protein fraction gave an abnormal pattern. Agarose gel electrophoresis showed the presence of an iodoalbumin-like protein and the absence of Tg in the tissues. This last finding was confirmed by immunoelectrophoresis. The Tg and TPO mRNAs levels were also analyzed. Dot-blot hybridization studies with pM5 (TPO cDNA) and phTgM2 (Tg cDNA) probes showed increased and decreased signals, respectively. The increase in TPO mRNA can be explained as a compensatory mechanism vis a vis an increase in serum TSH caused by decreased serum T3 and T4 due to the impairment in Tg mRNA. The Tg mRNA of both patients was further studied with four different probes covering 5' and 3' regions (phTgM1, phTgB1, phTgB2 and phTgB3). Hybridization was observed with all four probes, thus excluding a dramatic deletion defect. Northern transfer showed a clear signal of hybridization with the phTgB1 probe in the 8-9 Kb range. We may conclude that the biochemical and molecular abnormality of these patients is characterized by a decrease of Tg mRNA and of Tg translation.

Resultados a 15 años del tratamiento del carcinoma diferenciado de tiroides en una region de bocio endemico / 15 years results in the treatment of differentiated thyroid carcinoma in a region of endemic goiter

Se estudia la sobrevida a 5, 10 y 15 años de 120 carcinomas papilares y 144 foliculares con el método de tabla de vida y chi cuadrado. Se evalúan: a) Factores pronóstico: a) Factores pronóstico: tipo histológico, estadio (I intraglandular, II metástasis en ganglios regionales, III prepagación a estructuras vecinas y IV metástasis a distancia); edad (mayores o mentoes de 40 años) y sexo; b) Tratamiento: cirugía, I131 y hormona tiroidea. Del análisis global a 15 años surge: mayor sobrevida en papilar (84%) que en folicular (66%), p=0,01. Mayor en estadio I (90%) que en II (53%), p < 0,03 y mayor en éste que en IV (15%), pero p = NS; mayor en menores (85%) que en mayores (59%), p < 0;01; mayor en mujeres (75%) que en varones (52%), p = 0,01; a 5 años, pero NS a 10 y 15. Por estadios surge: en I, las diferencias son NS rspecto de hitología, edad y sexo: en II, la diferencia entre papilar (79%) y folicular (15%) es p < 0,001; en menores con papilar, la sobrevida es significativamente mejor que en mayores, pero es NS respecto de sexo. En el folicular II, las diferencias por edad y sexo son NS. En los estadios III y IV, las diferencias son NS en cuanto a histología, edad y sexo, quizás por escaso. La cirugía efectuada en el estadio I fue tiroidectomía subtotal bilateral, casi total y total, en los otros estadios casi total o total, no observándose ninguna diferencia a 15 años. La recidiva por muñón restante en el I fue de 2,5% siempre en mayores de 38 años. La diferencia entre "extirpación simple" y "vaciamientos completos" es p < 0,01 en cuanto a recidivas, pero NS respecto de sobrevida. Se comprueba mayor frecuencia de tetanias definitivas con TTp < 0,01. Los que no recibieron 131I terapéutico quizás presentem mayor mortalidad. Quienes no tomaron hormona tiroidea presentaron mayor mortalidad, p < 0,01. Esto indicaría que los factores pronóstico en orden de importancia son: estadio, edad, tipo histológico y sexo. En el estadio II, el segundo factor sería el tipo histológico. Según sobrevida se identifican tres grupos: 1) bajo riesgo (sobrevida mayor del 90%): estadio I papilar y folicular y II papilar, menores de 40 años; 2) moderado riesgo: (sobrevida del 60 al 90%):...(AU)

Resultados a 15 años del tratamiento del carcinoma diferenciado de tiroides en una region de bocio endemico / 15 year's results in the treatment of differentiated thyroid carcinoma in a region of endemic goiter

Se estudia la sobrevida a 5, 10 y 15 años de 120 carcinomas papilares y 144 foliculares con el método de tabla de vida y chi cuadrado. Se evalúan: a) Factores pronóstico: a) Factores pronóstico: tipo histológico, estadio (I intraglandular, II metástasis en ganglios regionales, III prepagación a estructuras vecinas y IV metástasis a distancia); edad (mayores o mentoes de 40 años) y sexo; b) Tratamiento: cirugía, I131 y hormona tiroidea. Del análisis global a 15 años surge: mayor sobrevida en papilar (84%) que en folicular (66%), p=0,01. Mayor en estadio I (90%) que en II (53%), p < 0,03 y mayor en éste que en IV (15%), pero p = NS; mayor en menores (85%) que en mayores (59%), p < 0;01; mayor en mujeres (75%) que en varones (52%), p = 0,01; a 5 años, pero NS a 10 y 15. Por estadios surge: en I, las diferencias son NS rspecto de hitología, edad y sexo: en II, la diferencia entre papilar (79%) y folicular (15%) es p < 0,001; en menores con papilar, la sobrevida es significativamente mejor que en mayores, pero es NS respecto de sexo. En el folicular II, las diferencias por edad y sexo son NS. En los estadios III y IV, las diferencias son NS en cuanto a histología, edad y sexo, quizás por escaso. La cirugía efectuada en el estadio I fue tiroidectomía subtotal bilateral, casi total y total, en los otros estadios casi total o total, no observándose ninguna diferencia a 15 años. La recidiva por muñón restante en el I fue de 2,5% siempre en mayores de 38 años. La diferencia entre "extirpación simple" y "vaciamientos completos" es p < 0,01 en cuanto a recidivas, pero NS respecto de sobrevida. Se comprueba mayor frecuencia de tetanias definitivas con TTp < 0,01. Los que no recibieron 131I terapéutico quizás presentem mayor mortalidad. Quienes no tomaron hormona tiroidea presentaron mayor mortalidad, p < 0,01. Esto indicaría que los factores pronóstico en orden de importancia son: estadio, edad, tipo histológico y sexo. En el estadio II, el segundo factor sería el tipo histológico. Según sobrevida se identifican tres grupos: 1) bajo riesgo (sobrevida mayor del 90%): estadio I papilar y folicular y II papilar, menores de 40 años; 2) moderado riesgo: (sobrevida del 60 al 90%):...

[15 years' results in the treatment of differentiated thyroid carcinoma in a region of endemic goiter]. / Resultados a 15 años del tratamiento del carcinoma diferenciado de tiroides en una región de bocio endemico.

We have evaluated the results of differentiated thyroid carcinoma treatment after 15 years of follow up. The group consisted of 120 cases of papillary type and 144 cases of follicular type; the mean age of the patients was 38.2 years for papillary and 46.5 for the follicular. The ratio men/women was 1/4 for the papillary and 1/3.2 for the follicular. The prognostic factors evaluated included: a) histologic types according to WHO classification, b) clinical stage, c) age and d) sex. The staging of the disease followed the classification suggested by Smendal, stage I: intraglandular disease; stage II: regional lymph node metastases; stage III: invasion of other tissues in the neck or mediastinum and stage IV: distant metastases. The basic treatment was surgery with the use of 131I and thyroid hormone. The results were statistically analyzed with the conventional life table method or chi square. At 15 years the overall survival was 84 +/- 4% for the papillary type and 66 +/- 9% for the follicular type (p = 0.01) (Fig. 1). The patients treated in stage I had a survival of 90 +/- 5%, significantly higher than those in stage II (53 +/- 8%), p less than 0.03. In stage III and IV the small number of patients made it difficult to interpret the results statistically (Fig. 2). The age of the patients appears to be another prognostic factor. The group under 40 years had 85 +/- 5% of survival compared to 59 +/- 4% over 40 years, p less than 0.01 (Fig. 3).(ABSTRACT TRUNCATED AT 250 WORDS)

Resultados a 15 años del tratamiento del carcinoma diferenciado de tiroides en una región de bocio endemico. / [15 years results in the treatment of differentiated thyroid carcinoma in a region of endemic goiter]

We have evaluated the results of differentiated thyroid carcinoma treatment after 15 years of follow up. The group consisted of 120 cases of papillary type and 144 cases of follicular type; the mean age of the patients was 38.2 years for papillary and 46.5 for the follicular. The ratio men/women was 1/4 for the papillary and 1/3.2 for the follicular. The prognostic factors evaluated included: a) histologic types according to WHO classification, b) clinical stage, c) age and d) sex. The staging of the disease followed the classification suggested by Smendal, stage I: intraglandular disease; stage II: regional lymph node metastases; stage III: invasion of other tissues in the neck or mediastinum and stage IV: distant metastases. The basic treatment was surgery with the use of 131I and thyroid hormone. The results were statistically analyzed with the conventional life table method or chi square. At 15 years the overall survival was 84 +/- 4

for the papillary type and 66 +/- 9

for the follicular type (p = 0.01) (Fig. 1). The patients treated in stage I had a survival of 90 +/- 5

, significantly higher than those in stage II (53 +/- 8

), p less than 0.03. In stage III and IV the small number of patients made it difficult to interpret the results statistically (Fig. 2). The age of the patients appears to be another prognostic factor. The group under 40 years had 85 +/- 5

of survival compared to 59 +/- 4

over 40 years, p less than 0.01 (Fig. 3).(ABSTRACT TRUNCATED AT 250 WORDS)