Diagnostico por mutagenesis dirigida de una mutacion en el gen hMSH2 vinculada a cancer colorrectal hereditario no poliposo / Diagnosis of hereditary nonpolyposis colorectal cancer by directed mutagenesis in a mutation at the hMSH2 gene

El cáncer colorrectal hereditario no poliposo (HNPCC) es la forma más común de cáncer de colon hereditario, y una de las afecciones autosómicas dominantes más frecuentes. Clínicamente se caracteriza por su temprana apacición (< 50 años), la localización proximal de los tumores colónicos y un alto riesgo de desarrollar tumores colorrectales primarios múltiples y extracolónicos. La enfermedad es causada por diferentes mutaciones en alguno de los por lo menos cuatro genes reparadores de discordancias del AND (genes MMR: hMSH2, hHLH1, hPMS1 y hPMS2. Se calcula que afecta a 1:200 1:2000 personas de la población occidental. La identificación de estos genes responsables de HNPCC ha permitido la búsqueda de mutaciones germinales en individuos afectados. En una familia mendocina con cáncer de colon hereditario se realizó la búsqueda del gen afectado a través de un centro holandés de diagnóstico de HNPCC donde detectaron una mutación en el exón 13 del gen hMSH2. La mutación introduce un codón de finalización temprano lo que provoca la expresión de una proteína truncada. Esta mutación en particular no estaba registrada en la base de datos de mutaciones relacionadas con HNPCC. Luego de la detección en el paciente índice, desarrollamos en nuestro laboratorio un procedimiento rápido y eficiente para detectar mutaciones en el resto de los familiares. La metodología consistió en la amplificación del exón 13 del gen hMSH2 mediante un cebador para el extremo 5 que linda con el sitio de la mutación puntual e introduce parte de la secuencia de corte para la enzima Haelll que es completada sólo en el alelo sano. Este análisis genético nos permitió hasta la fecha diagnosticar 17 individuos de los cuales 9 resultaron afectados y están entrando en un programa de seguimiento clínico y consultoría genética. (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.

Management of overt and subclinical hypothyroidism: factors influencing L-thyroxine dosage

With the aim of establishing optimal dosage schedules, 171 women with either orvet (OH, n=80) or subclinical (SCH, n=91) hypothyroidism were assessed before and 6 months after starting L-thyroxine (LT4) replacement therapy. Each group was further classified into four subgroups according to post-therapy serum TSH level, as follows; A) complete suppression; B) partial suppression; C) normal range and D) above normal range (insufficient response). In all subgroups, LT4 doses were higher for OH than for SCH, whether expressed as total daily dose (mug) or as a function of either actual or ideal body weight (mug/kg BW). In OH, LT4 dose was higher for subgroups A or B as compared with either C or D. In SCH, subgroup A received a larger dose than the other subgroups. Post-treatment serum thyroxine levels showed the same pattern for both OH and SCH. Mean LT4 dose was similar in patients with high and normal antithyroid antibodies and in patients with goiter and in those without it. In goitrous patients thyroid volume decreased in subgroup B, particularly in those patients that had elevated antithyroid antibodies, but not in subgroup C. In OH patients a significant negative correlation was found between daily LT4 dose per Kg actual BW and actual BW, especially in subgroup C for patients with a body mass index > 27 kg/cm2 (r = -0.90, p<0.001). In subgroup C of the SCH group, a negative correlation between LT4 dose and age was noticed. Both in OH and in SCH, LT4 dose per kg actual BW required to obtain a serum TSH within the normal range was lower in women with a body mass index (BM) > 27 kg/m2 than in those with a BMI = 27 kg/m2. LT4 doses for subgroup C did not differ from those needed in hypothyroid patients with previous Graves disease, in either OH or SCH patients. (AU)