RET gene mutational diagnosis and precision medicine in Mexico.

Precision medicine is a reality in some diseases; it supports the development of accurate and specific diagnostic methods, new drugs and molecules. Our research team in Mexico, made up of clinical and biomedical researchers, has been performing free RET gene mutational diagnosis for medullary thyroid cancer and multiple endocrine neoplasia (MEN) 2 and 3 for 20 years. RET pathogenic variants in the Mexican population are consistent with reported data: most common mutations are 634/NEM2 and 918/NEM3. Currently, new nanobiotechnology methods are being developed for this type of determination in order to obtain faster, simpler, more sensitive and specific results applicable in all types of laboratories.

La medicina de precisión en algunas enfermedades es una realidad; respalda el desarrollo de métodos diagnósticos certeros y específicos, de nuevas drogas y moléculas. Nuestro equipo de investigación en México, conformado por investigadores clínicos y biomédicos, desde hace 20 años realiza de forma gratuita el diagnóstico mutacional del gen RET y su relación con el cáncer medular de tiroides y la neoplasia endocrina múltiple (NEM) 2 y 3. Las variantes patogénicas de RET en la población mexicana coinciden con los datos reportados: la mayoría con 634/NEM2 y 918/NEM3. Actualmente se están desarrollando nuevos métodos de nanobiotecnología para este tipo de determinaciones, de tal forma que puedan obtenerse resultados más rápidos, simples, sensibles y específicos aplicables en todo tipo de laboratorio.

Evidence for the founder effect of RET533 as the common Greek and Brazilian ancestor spreading multiple endocrine neoplasia 2A.

OBJECTIVES: About one-quarter of patients with medullary thyroid cancer (MTC) have inherited disease due to mutations in the RET gene. A rare mutation in exon 8 (G533C) of RET, previously described in a large Brazilian family with MEN2A, also appeared to be clustering in Greece, whereas it was rarely reported in other ethnic groups. The aim of this study was to identify a possible common ancestry between these carriers. PATIENTS AND METHODS: Twelve RET G533C mutation carriers, four randomly selected from the Brazilian cohort and eight from apparently unrelated Greek families, were studied for a possible common ancestral origin. RET flanking microsatellite markers at chromosome 10q (D10S197, D10S196, D10S1652 and D10S537) were used. RESULTS: Genomic DNA analysis using these markers showed that many of these apparently unrelated individuals shared a common haplotype indicating a common ancestral origin. CONCLUSION: Our data suggest that Brazilian and Greek patients with MTC carrying the G533C mutation in exon 8 of RET gene originate from a common ancestor. Due to historical reasons, we speculate that the more plausible explanation for the origin of this mutation is in Greece.

Multiple endocrine neoplasia: the Chilean experience.

Multiple endocrine neoplasia (MEN) types 1 and 2 are genetic diseases that are inherited as autosomal traits. The major clinical manifestations of multiple endocrine neoplasia type 1 include the so-called "3 P's": parathyroid, pituitary, and pancreatic tumors, including gastroenteroneuroendocrine tumors. Genetic testing can be performed on patients and the potential carriers of the menin gene mutation, but the genotype-phenotype correlation in multiple endocrine neoplasia type 1 is less straightforward than multiple endocrine neoplasia type 2. Most likely, the main advantage of genetic testing in MEN1 is to exclude from further studies those who are negative for the genetic mutation if they belong to a family with a known history of MEN1. In Chile, we started with rearranged during transfection proto-oncogene genetic testing (MEN2) 15 years ago. We carried out a prophylactic total thyroidectomy to prevent medullary thyroid carcinoma in a three-year-old girl who presented with microscopic medullary thyroid carcinoma. More than 90% of the individuals who tested positive using a genetic test achieved a biochemical cure compared with only 27% of patients who receive a clinical diagnosis. Mutations are mainly located in exon 11; the most common is C634W, rather than C634R. Hypertensive crisis was the cause of death in three patients, and extensive distant metastases occurred in nine (including two patients with multiple endocrine neoplasia type 2B) of 14 patients. Earlier recognition of medullary thyroid carcinoma and the other features of the disease, especially pheochromocytoma, will improve the survival rate of patients with multiple endocrine neoplasia.

Primary hyperparathyroidism as the first clinical manifestation of multiple endocrine neoplasia type 2A in a 5-year-old child.

BACKGROUND: Primary hyperparathyroidism occurs in only 10%-30% of patients with multiple endocrine neoplasia type 2A (MEN2A), rarely as the sole clinical manifestation, and is usually diagnosed after the third decade of life. SUMMARY: A 5-year-old girl was referred for prophylactic thyroidectomy as she carried the p.C634R RET mutation. She was clinically asymptomatic, with a normally palpable thyroid and with the cervical region free of lymphadenopathy or other nodules. Preoperative tests revealed hypercalcemia associated with elevation of parathyroid hormone (PTH) (calcium = 11.2 mg/dL, calcium ion = 1.48 mmol/L, phosphorus = 4.0 mg/dL, alkaline phosphatase = 625 U/L, parathyroid hormone (PTH) PTH = 998 pg/mL). A thyroid ultrasound was normal and parathyroid scintigraphy with (99m)Tc-Sestamibi revealed an area of radioconcentration in the upper half of the left thyroid lobe suggesting hyperfunctioning parathyroid tissue. She underwent total thyroidectomy and parathyroidectomy and developed hypocalcemia. The anatomopathological examination showed no histopathological changes in the thyroid tissue and an adenoma of the parathyroid gland, confirming the diagnosis of hyperparathyroidism. CONCLUSIONS: Primary hyperparathyroidism can be a precocious manifestation of MEN2A. This case report highlights that asymptomatic hypercalcemia should be scrutinized in children related to patients with MEN2A who carry a mutation in the RET proto-oncogene, especially mutations in the codon 634, before the currently recommended age of 8 years.

[Familial isolated primary hyperparathyroidism--description and analyses of six cases]. / Hiperparatireoidismo primário familiar isolado--análise e descrição de uma família com seis casos índices.

Our objective is to evaluate and describe one family with six cases of familial isolated primary hyperparathyroidism (HFI), a rare hereditable disorder with an autossomal dominant mode of inheritance. It is characterized by a primary hyperparathyroidism without association with other endocrine tumors or diseases. The HFI diagnosis relied on the demonstration of hypercalcemia, inappropriately high levels of parathyroid hormone, and parathyroid adenomas, plus exclusion of NEM 1/2a and HPT/TM syndrome in this family. We analyzed the description of the first diagnosis, surgical approach, postoperative histopathological results and their development process. The first patient, treated in our institute twenty years ago, has recidivated eleven years after the treatment. Her sister had had the same diagnosis, which motivated us to investigate theirs relatives. The analysis of the characteristics that run in these patients' family has contributed to facilitate their diagnosis and therapeutic treatment, including clinical and genetic orientation of this family.

[Genetic screening of multiple endocrine neoplasia type 2: experience of the USP Endocrine Genetics Unit]. / Rastreamento gênico da neoplasia endócrina múltipla tipo 2: experiência da Unidade de Endocrinologia Genética da USP.

Multiple endocrine neoplasia type 2 (MEN-2) is an inherited tumor syndrome that includes medullary thyroid carcinoma (MTC), primary hyperparathyroidism, pheochromocytoma and other non-endocrine diseases. Since the first RET missense mutations in association with MEN-2 were identified, RET mutation analysis had a great impact in the clinical management of MEN-2, such as in early diagnosis and treatment of MTC. Presently, early total thyroidectomy provides real cure of MTC for cases in which molecular diagnosis has been performed at early ages. After RET mutation identification, family members should be screened for this mutation by using methods as DGGE, SSCP, restriction enzyme, genetic sequencing or mini-sequencing. In this paper, we briefly review our experience with the direct RET gene sequencing and DGGE approaches. In 50 typical MEN-2 patients analyzed using both methods, we found no false results suggesting that DGGE is a reliable screening method for RET proto-oncogene mutation analysis.

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

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.

[Multiple endocrine neoplasia type 2]. / Neoplasia endócrina múltipla tipo 2.

The term multiple endocrine neoplasia (MEN) was introduced by Steiner et al. in 1968 to describe disorders that include a combination of endocrine tumors. The Wermer syndrome was designed as MEN 1 and the Sipple syndrome as MEN 2. Sizemore et al. (1974) completed that the MEN 2 category included 2 subgroups: patients with medullary thyroid carcinoma (MTC), pheochromocytoma, and parathyroid disease and a normal appearance (MEN 2A) and other without parathyroid disease but with mucosal neuromas and mesodermal abnormalities (MEN 2B). MTC is usually the first tumor diagnosed. The diagnosis of MTC has several implications: disease extent should be evaluated, pheochromocytoma and hyperparathyroidism should be screened and whether the MTC is sporadic or hereditary should be determined by a direct analysis of the RET proto-oncogene. Here, the pathological characteristics, genetic abnormalities, and clinical features of MEN 2 are discussed. The diagnostic and therapeutic approaches used to patients with clinical disease and carriers identified through familiar screening are also described. Progresses related especially to genetic screening and earlier intervention have permitted an improvement in the long-term outcome. However, treatment for disseminated disease is still ineffective.

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)

Molecular and biochemical screening for the diagnosis and management of medullary thyroid carcinoma in multiple endocrine neoplasia type 2A.

Patients with Multiple Endocrine Neoplasia (MEN) type 2A are at risk for early medullary thyroid carcinoma (MTC). We performed different screening tests for MTC--a recently reported biochemical screening test using omeprazole-induced calcitonin (CT) stimulation and DNA analysis--in fifteen members of two non-consanguineous Brazilian families with MEN 2A. RET proto-oncogene analysis was carried out by direct DNA sequencing of PCR-amplified products for exons 10 and 11. Family 1 showed a germline mutation (C634Y) in three individuals; a sister and a brother with symptomatic MTC; the former also presented with pheochromocytoma and hyperparathyroidism, and her son was a nine-year-old boy of previously unknown status. Family 2 showed the C634R mutation only in the index case, who presented with cutaneous lichen amyloidosis in addition to MTC, pheochromocytoma and hyperparathyroidism. Neither her parents nor her four brothers showed this genetic abnormality, suggesting a de novo RET proto-oncogene mutation in this patient. The controls and patients presented normal basal gastrin levels and a significant increase after omeprazole. Basal CT levels were elevated in patients with MTC and undetectable in control and asymptomatic family members. No subject showed any increase in CT levels after omeprazole treatment. In conclusion, the two most frequent RET proto-oncogene mutations in MEN 2A are present in Brazilian families. In addition, the specificity of basal and omeprazole-stimulated calcitonin is rather limited, and the efficacy of the omeprazole test still needs to be systematically examined. Therefore, RET proto-oncogene analysis must be the first choice for a screening procedure to identify gene carriers in MEN 2A family members and to permit early prophylactic treatment of MTC.

[Multiple endocrine neoplasia: a clinical model for applying molecular genetic techniques]. / Neoplasias endocrinas múltiples: un modelo clínico para aplicar técnicas de genética molecular.

Multiple endocrine neoplasias (MEN) are syndromes inherited as autosomal dominant. The application of the techniques of molecular biology has made possible the identification of the genes causing MEN 1 and 2. The gene responsible for MEN 1 belongs to the family of tumor suppressor genes and encodes for a protein named MENIN whose function remains to be elucidated. The identification of mutant MEN 1 gene carriers who are at risk of developing this syndrome requires frequent biochemical screening for the development of endocrine tumors. MEN 2 is a consequence of mutations in the Ret proto-oncogene (c-Ret). This gene encodes for a tyrosine kinase receptor thought to play a role in the development of neural crest-derived tissue. Members of kindred with either MEN 2A or MEN 2B should be screened by direct DNA testing early in life for mutations in c-Ret. Those with the mutation should be advised to have thyroidectomy at five years of age in children with MEN 2A and earlier in children with MEN 2B. Some cases of sporadic MTC are actually MEN 2A or Familial MTC after c-Ret testing is done, therefore routine application of this test is recommended in all cases of apparent sporadic MTC.

[Early diagnosis of multiple endocrine neoplasia type 2 (MEN 2) by detection of mutated RET proto-oncogene carriers]. / Diagnóstico precoz de neoplasia endocrina múltiple tipo 2 (MEN 2) por la deteccion de portadores de mutaciones en el proto-oncogen RET]

RET proto-oncogene mutation results in a dominant autosomic inherited syndrome (MEN 2) presenting three distinct subtypes: MEN 2A, MEN 2B, and familial medullary thyroid carcinoma (FMTC). Detection of RET proto-oncogene mutation is a predictor before clinical or biochemical evidence of the disease is present and leads to preventive thyroid removal since there is no effective treatment for metastases. The aim of the present study was to characterize mutations in the RET proto-oncogene in affected patients and to identify potential carriers in their families. Two families with FMTC (5 and 6 members), 4 with MEN 2A (5, 5, 4 and 3 members) and 2 with MEN 2B (5 and 1 members), were studied. DNA was obtained from blood samples in all patients and from thyroid or from pheonochromocytoma tissues in patients submitted to surgery. PCR amplification was performed using specific primers for exons 10, 11 and 16, followed by direct sequencing. Mutations at codon 634 in exon 11 were found in 16 subjects with FMTC and MEN 2A: TGC --> CGC (cysteine to arginine) in 9 cases, TGC --> TAC (cysteine to tyrosine) in 3, and TGC --> TTC (cysteine to phenilalanine) in 4. A unique mutation of codon 918 in exon 16, ATG --> ACG (methionine to threonine), was found in both MEN 2B affected patients. The mutations detected in DNA from peripheral blood were the same as those present in DNA extracted from tumor material. RET mutations were detected in all affected patients, confirming the diagnosis, and in 10 members of their families. In five of the carriers total thyroidectomy was performed. Anatomopathological study showed C-cells hyperplasia or in-situ microcarcinoma in two children (9 and 12 y) with no clinical signs of diseases and medullary thyroid carcinoma in three adults, who were previously unaware of the presence of thyroid nodules. The early detection of RET mutation followed by total thyroidectomy may prevent the development of the disease, specially in affected families, and avoid the fatal outcome of delayed medullary thyroid carcinoma diagnosis.

Diagnostico precoz de neoplasia endocrina multiple tipo 2 (MEN 2) por la deteccion de portadores de mutaciones en el proto-oncogen RET / Early diagnosis of multiple endocrine neoplasia type 2 by detection of matated RET proto-oncogene carriers

El MEN 2 es un síndrome hereditario autosómico dominante, en el cual mutaciones del RET dan origen a tres fenotipos diferentes: carcinoma medular de tiroides familiar (CMTF), MEN 2A y MEN 2B. La identificación de mutaciones en el proto-oncogen RET predice el desarrollo de la enfermedad antes de las evidencias clínicas y bioquímicas. En este trabajo se identificaron portadores del RET por caracterización de mutaciones en pacientes y sus familiares. Se estudiaron 21 familias con CMTF (5 y 6 miembros), 4 con MEN 2A (dos de 5, una de 4 y otra de 3 miembros) y 2 con MEN 2B (5 y 1 miembros). Se obtuvieron muestras de ADN de sangre, en todos los casos y de tejido de feocromocitoma y/o tejido tiroideo en los operados. Se utilizó PCR para amplificar los exones 10, 11 y 16 con oligonucleótidos específicos, realizándose secuenciación directa de los fragmentos. En las familias con CMTF y con MEN 2A se encontraron mutaciones en el codón 634 del exón 11 en 16 sujetos, dectándose 9 casos con la mutación TGC r CGC (cisteína a arginina), 3 con TGC r TAC (cisteína a tirosina) y 4 con TGC r TTC (cisteína a fenilalanina). En los pacientes con MEN 2 B se encontró una mutación en el codón 918 del exón 16 ATG r ACG (meitonina a treonina). En tejido tumoral se detectó la misma mutación que en sangre periférica. El diagnóstico de MEN 2 fue confirmado en los 8 pacientes y detectado en 10 familiares. En los 5 portadores tiroidectomizados se encontró hiperplasia de células C o microcarcinoma in situ en 2 niños (9 y 12 años) y CMT en 3 adultos. La detección temprana de mutaciones del RET, especialmente en familiares seguida por tiroidectomía total, podría prevenir el desarrollo de CMT, modificado el desenlace fatal que ocurre cuando es diagnosticado tardíamente.

Diagnostico precoz de neoplasia endocrina multiple tipo 2 (MEN 2) por la deteccion de portadores de mutaciones en el proto-oncogen RET / Early diagnosis of multiple endocrine neoplasia type 2 by detection of matated RET proto-oncogene carriers

El MEN 2 es un síndrome hereditario autosómico dominante, en el cual mutaciones del RET dan origen a tres fenotipos diferentes: carcinoma medular de tiroides familiar (CMTF), MEN 2A y MEN 2B. La identificación de mutaciones en el proto-oncogen RET predice el desarrollo de la enfermedad antes de las evidencias clínicas y bioquímicas. En este trabajo se identificaron portadores del RET por caracterización de mutaciones en pacientes y sus familiares. Se estudiaron 21 familias con CMTF (5 y 6 miembros), 4 con MEN 2A (dos de 5, una de 4 y otra de 3 miembros) y 2 con MEN 2B (5 y 1 miembros). Se obtuvieron muestras de ADN de sangre, en todos los casos y de tejido de feocromocitoma y/o tejido tiroideo en los operados. Se utilizó PCR para amplificar los exones 10, 11 y 16 con oligonucleótidos específicos, realizándose secuenciación directa de los fragmentos. En las familias con CMTF y con MEN 2A se encontraron mutaciones en el codón 634 del exón 11 en 16 sujetos, dectándose 9 casos con la mutación TGC r CGC (cisteína a arginina), 3 con TGC r TAC (cisteína a tirosina) y 4 con TGC r TTC (cisteína a fenilalanina). En los pacientes con MEN 2 B se encontró una mutación en el codón 918 del exón 16 ATG r ACG (meitonina a treonina). En tejido tumoral se detectó la misma mutación que en sangre periférica. El diagnóstico de MEN 2 fue confirmado en los 8 pacientes y detectado en 10 familiares. En los 5 portadores tiroidectomizados se encontró hiperplasia de células C o microcarcinoma in situ en 2 niños (9 y 12 años) y CMT en 3 adultos. La detección temprana de mutaciones del RET, especialmente en familiares seguida por tiroidectomía total, podría prevenir el desarrollo de CMT, modificado el desenlace fatal que ocurre cuando es diagnosticado tardíamente. (AU)

[Multiple endocrine neoplasia type 2A. Report of a case with an unusually aggressive outcome]. / Neoplasia endocrina múltiple tipo 2A. Presentación de un caso de evolución inhabitualmente agresiva.

We report a 28 years old woman who consulted for diarrhea of two years and a thyroid nodule. A medullary thyroid carcinoma was diagnosed and a thyroidectomy performed. There was a local relapse two months later and distant metastases were found five months later. A MIBG-1131 scintigraphic image of the adrenals lead to the suspicion of a bilateral pheochromocytoma. The surgical resection of the adrenals confirmed the diagnosis. There was no response to chemotherapy and the patient continued with severe hypercalcemia, repeated infections, persistent diarrhea and cachexia, dying one year after the diagnosis. There was no family history of the disease. We conclude that this is a particularly aggressive presentation of a multiple endocrine neoplasia type 2A.

Early presentation of metastatic medullary carcinoma in multiple endocrine neoplasia, type IIA: implications for therapy.

A girl 5 years 11 months of age, belonging to an extensive kindred with multiple endocrine neoplasia, type IIA (MEN IIA), was found to have multifocal medullary thyroid carcinoma with metastasis in one paraglandular lymph node after positive findings on a calcium-pentagastrin stimulation test. Her sister, 3 years 8 months of age, also had an elevated calcitonin level, and thyroidectomy revealed C-cell hyperplasia and a focus of medullary thyroid carcinoma. These two cases underscore the need for prophylactic thyroidectomies in MEN IIA patients as young as 5 years of age and strict yearly provocative screening beginning at age 1 year.