María Rodríguez Roque

A simple PCR-based genotyping method for M105I mutation of alpha-SNAP enhances the study of early pathological changes in hyh phenotype

alpha-SNAP is an essential component of the protein machinery responsible for membrane fusion events in different cell types. The hyh (hydrocephalus with hop gait) mouse carries a missense mutation in Napa gene that results in a point mutation (M105I) in alpha-SNAP protein. Homozygous animals for the mutant allele have been identified by the clinical and/or neuropathological phenotype, or by direct sequencing of PCR products. The aims of the present study were (i) to develop a high-throughput technique to genotype hyh mice, (ii) to correlate genotype-phenotype, and (iii) to analyze the earliest pathological changes of hyh mutant mice. As no restriction sites are affected by the hyh mutation, we resolved this problem by creating a BspHI restriction site with a modified (mismatch) polymerase chain reaction (PCR) primer in wild-type allele. This artificially created restriction site (ACRS)-PCR technique is a simple, rapid and reliable method to genotype hyh mice in a day-work procedure. Biochemical and histological analysis of genotyped hyh embryos at different developmental stages allowed us to identify and characterize the earliest brain pathological changes of the hyh phenotype, including the first signs of neuroepithelial disruption and neuronal ectopia. In addition, genotype-phenotype analysis of 327 animals confirmed that (i) hyh is a single-gene autosomal recessive disorder, and (ii) the disorder has 100% penetrance (i.e., the mutation was only present in affected mice). The genotyping method described here enhances the potentiality of hyh mouse as a unique in vivo model to study the role of membrane trafficking in different developmental and physiological processes.

A PCR-mutagenesis strategy for rapid detection of mutations in codon 634 of the ret proto-oncogene related to MEN 2A.

BackgroundMultiple endocrine neoplasias type 2A (MEN 2A) is a dominantly inherited cancer syndrome. Missence mutations in the codon encoding cysteine 634 of the ret proto-oncogene have been found in 85% of the MEN 2A families. The main tumour type always present in MEN 2A is medullar thyroid carcinoma (MTC). Only 25% of all MTC are hereditary, and generally they are identified by a careful family history. However, some familial MTCs are not easily detected by this means and underdiagnosis of MEN 2A is suspected.MethodsDNA samples from MEN 2A patients were amplified by PCR. The products were incubated with the restriction enzyme Bst ApI or Bgl I.The samples were loaded in non-denaturing 10% Polyacrilamyde Gel and run at 120 volts for 40 min. The gels were stained with 10 μg/ml ethidium bromide, and the bands were visualized under a UV lamp.ResultsWe developed a PCR-mutagenic method to check the integrity of the three bases of the cysteine 634 codon.ConclusionThe method can be used to detect inherited mutations in MTC patients without a clear family history. The method is relatively simple to use as a routine test in these patients to decrease the underdiagnosis of MEN 2A. In addition, the assay can be used to screen affected families with any mutation in cysteine 634.

Uso combinado de calcio y pentagastrina en el diagnóstico y seguimiento del cáncer medular de tiroides

El cancer medular de tiroides (CMT), en sus formas hereditaria y esporadica, es una neoplasia derivada de las celulas C tiroideas. Estas celulas se caracterizan por su capacidad para sintetizar la hormona calcitonina. La determinacion de calcitonina plasmatica tras estimulo combinado con calcio y pentagastrina es desde hace anos un metodo sensible para la deteccion precoz del cancer medular de tiroides. Sin embargo, desde que se demostro que mutaciones en el protooncogen RET son las responsables del CMT hereditario, el diagnostico de los individuos portadores de la enfermedad se comenzo a realizar por tecnicas de biologia molecular. Desde 1988 hemos utilizado la prueba de estimulo combinado con calcio y pentagastrina para el diagnostico y seguimiento del CMT. A partir de 1996 incorporamos el diagnostico molecular en tres familias afectadas por neoplasia endocrina multiple tipo 2A (NEM 2A). La prueba de estimulo se continuo utilizando para el seguimiento de los pacientes tratados por cirugia y para detectar el desarrollo de CMT en dos ninos afectados cuyos padres demoraron la decision de la cirugia profilactica. Nuestros resultados demuestran que la prueba es un excelente marcador de la evolucion de CMT en pacientes afectados y un metodo sensible para detectar CMT en sus estadios iniciales. Si bien la biologia molecular es la herramienta de leccion para diagnosticar el CMT hereditario e incluso para decidir sobre una tiroidectomia profilactica, la prueba de estimulo mantiene una innegable vigencia en el seguimiento de individuos tratados por CMT hereditario y esporadico.