Clinical and molecular diagnosis of a Costa Rican family with autosomal recessive myotonia congenita (Becker disease) carrying a new mutation in the CLCN1 gene

Myotonia congenita is a muscular disease characterized by myotonia, hypertrophy, and stiffness. It is inherited as either autosomal dominant or recessive known as Thomsen and Becker diseases, respectively. Here we confirm the clinical diagnosis of a family diagnosed with a myotonic condition many years ago and report a new mutation in the CLCN1 gene. The clinical diagnosis was established using ocular, cardiac, neurological and electrophysiological tests and the molecular diagnosis was done by PCR, SSCP and sequencing of the CLCN1 gene. The proband and the other affected individuals exhibited proximal and distal muscle weakness but no hypertrophy or muscular pain was found. The myotatic reflexes were lessened and sensibility was normal. Electrical and clinical myotonia was found only in the sufferers. Slit lamp and electrocardiogram tests were normal. Two affected probands presented diminution of the sensitive conduction velocities and prolonged sensory distal latencies. The clinical spectrum for this family is in agreement with a clinical diagnosis of Becker myotonia. This was confirmed by molecular diagnosis where a new disease-causing mutation (Q412P) was found in the family and absent in 200 unaffected chromosomes. No latent myotonia was found in this family; therefore the ability to cause this subclinical sign might be intrinsic to each mutation. Implications of the structure-function-genotype relationship for this and other mutations are discussed. Adequate clinical diagnosis of a neuromuscular disorder would allow focusing the molecular studies toward the confirmation of the initial diagnosis, leading to a proper clinical management, genetic counseling and improving in the quality of life of the patients and relatives.

La miotonía congénita es una enfermedad muscular caracterizada por miotonía, hipertrofia y rigidez. Se presenta con dos patrones de herencia, autosómica dominante en cuyo caso recibe el nombre de miotonía de Thomsen, o autosómica recesiva conocida como miotonía de Becker. En este trabajo se confirmó el diagnóstico clínico presuntivo hecho hace algunos años en una familia con una condición miotónica y se reporta una nueva mutación en el gen CLCN1. El diagnóstico clínico se estableció después de estudios oculares, cardíacos, neurológicos y electrofisiológicos. El diagnóstico molecular fue hecho mediante la PCR, SSCP y secuenciación del gen CLCN1. El caso índice y los otros individuos afectados exhibieron debilidad muscular proximal y distal, pero no se encontró hipertrofia ni dolor muscular. Los reflejos miotáticos estuvieron disminuidos y la sensibilidad fue normal. Se encontró miotonía clínica y eléctrica solo en los individuos afectados. Las pruebas de lámpara de hendidura y electrocardiograma resultaron normales. Dos individuos afectados presentaron disminución de las velocidades de conducción sensitiva y latencias distales sensoriales prolongadas. El cuadro clínico concuerda con la miotonía de Becker, lo cual se confirmó con el hallazgo de una mutación responsable de la enfermedad en el gen CLCN1 (Q412P), la cual se encontró en la familia y estuvo ausente en 200 cromosomas provenientes de la población general. No se encontró miotonía latente, por lo que probablemente la habilidad de causar este signo subclínico es intrínsica de cada mutación. Afinar el diagnóstico clínico diferencial de las enfermedades neuromusculares permitiría enfocar los estudios moleculares hacia la confirmación del diagnóstico inicial en forma eficiente, lo cual permitiría un manejo clínico y asesoramiento genético más adecuados y una mejora en la calidad de vida de los pacientes y sus familias.

Asociación del polimorfismo del codon 72 del gen p53 con el riesgo de cáncer gástrico en una población de alto riesgo de Costa Rica / Association of the p53 codon 72 polymorphism to gastric cancer risk in a hight risk population of Costa Rica

El cáncer gástrico es la segunda causa de muerte por cáncer en el mundo. Varios factores han sido asociados con el riesgo de llegar a desarrollarlo, entre ellos la predisposición genética. El gen p53 presenta un polimorfismo en el codón 72, el cual ha sido asociado con un mayor riesgo de desarrollar varios tipos de cáncer entre ellos el gástrico. El objetivo de este estudio fue determinar la asociación del polimorfismo localizado en el codón 72 del gen p53 con el riesgo de cáncer gástrico y lesiones gástricas leves en una población de alto riesgo de Costa Rica. El análisis del polimorfismo se llevó a cabo mediante PCR-RFLP, en una muestra de 58 pacientes de cáncer gástrico, 99 personas controles y 41 individuos clasificados como grupos I y II de acuerdo con la clasificación histológica japonesa. No se determinó asociación del polimorfismo del codón 72 de p53 con el riesgo de cáncer gástrico, ni de lesiones gástricas leves en la muestra estudiada. Con base en este estudio y otros que han investigado el polimorfismo del codón 72 del gen p53, no está claro el papel que podría estar jugando dicho polimorfismo en el desarrollo de cáncer gástrico. Mutaciones de novo en el gen p53 producidas durante el desarrollo neoplásico de la enfermedad podrían tener un mayor efecto que polimorfismos de línea germinal de este mismo gen. Existen otros genes polimórficos que también se han asociado con el riesgo de desarrollar cáncer gástrico

Gastric cancer is the second most common cancer associated death cause worldwide. Several factors have been associated with higher risk to develop gastric cancer, among them genetic predisposition. The p53 gene has a polymorphism located at codon 72, which has been associated with higher risk of several types of cancer, including gastric cancer. The aim of this study was to determine the association of p53, codon 72 polymorphism, with the risk of gastric cancer and pre-malignant lesions in a high-risk population from Costa Rica. The genotyping was carried out by PCR-RFLP in 58 gastric cancer patients, 99 controls and 41 individuals classified as group I or II, according to the Japanese histological classification. No association was found for p53, codon 72 polymorphism with neither the risk of gastric cancer nor the risk of less severe gastric lesions in the studied population. Based on this study and taking into account other studies carried out with p53, codon 72 polymorphism, the role of this polymorphism in the development of gastric cancer remains unclear. De novo mutations on p53 gene produced during neoplasic development of this disease might play a greater role than germinal polymorphisms of the gene. Other polymorphic genes have been associated with higher risk to develop gastric cancer

Polimorfismos en los genes de desintoxicación CYP1A1, CYP2E1, GSTT1 y GSTM1 en la susceptibilidad al cáncer gástrico / Polymorphisms in detoxification genes CYP1A1, CYP2E1, GSTT1 and GSTM1 in gastric cancer susceptibility

Cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes are involved in activation and detoxification of many potential carcinogens. Genetic polymorphisms in those enzymes have been found to influence the interindividual susceptibility to cancer. Some polymorphisms of those enzymes have been associated specifically with susceptibility to gastric cancer. We conducted a study in a Costa Rican population, where gastric cancer incidence and mortality rates are among the highest in the world. We investigated whether such variations affected the risk of developing gastric cancer. Subjects included 31 with gastric cancer, 58 controls with gastric injures others than cancer and 51 normal controls confirmed by X-rays (double-contrast) or endoscopic diagnostic. DNA from peripheral white blood cell was obtained from all subjects. Deletion of GSTT1 and GSTM1 was assessed by multiplex PCR and genotyping of CYP2E1 was performed using a PCR-based restriction fragment length polymorphism assay with the restriction enzyme PstI and the gene CYP1A1 using the restriction enzyme MspI The prevalence of CYP1A1 Msp1 polymorphism, GSTT1 and GSTM1 null genotype was similar in the three groups of individuals (p = 0.73, p = 0.88 y p = 0.89 respectively). Our findings suggest that the polymorphism CYP2E1 PstI could be associated with a reduced risk of having gastric cancer (OR = 0.09, IC95%:0.01 - 0.83).

Mutagénesis ambiental y el uso de biomarcadores para predecir el riesgo de cáncer: [revisión] / Environmental mutagenesis and use of biomarkers in cancer risk prediction: [review]

The field of environmental mutagenesis or toxicology genetics aims to study the genetic damage that leads to mutations produced by physical, chemical and biological agents, to identify these agents and analyze their interactions and ways of action. There are enough experimental and epidemiological evidences implicating mutations in oncogenes, tumor suppressor genes and DNA repair genes as determinants in the onset and progression of the neoplastic process. A valuable tool in public and occupational health is the monitoring of populations exposed to potentially hazardous agents. The objective is to protect the health and quality of life of high risk groups on account of the nature of the agents of exposure. Monitoring of genotoxic effects in exposed populations as well as the analysis of susceptibility polymorphism are visualized as key tools in the realm of future public and occupational health in order to prevent the occurrence of environmental and specially occupational origin of tumors. This paper reviews the main concepts concerning this issue and refers to studies on the subject in Costa Rica.

Genotoxicidad de tres plaguicidas utilizados en la actividad bananera de Costa Rica / Genotoxicity of three pesticides used in Costa Rican banana plantations

The in vitro genotoxicity of imazalil and thiabendazole fungicides and the insecticide chlorpyrifos, compounds used in Costa Rican banana plantations, was evaluated with the single-cell gel electrophoresis technique (comet assay). The comet assay is a simple, rapid and low cost technique for quantification of DNA damage. This assay detects DNA single-strand breaks and alkali-labile sites in individual cells. The effects were analyzed by using human lymphocytes exposed to doses of 0, 25, 50, 75 and 100 microg/ml of each pesticide for 30 min at 37 degrees C. The cells were embedded in agarose, lysed, subjected to alkaline electrophoresis (pH >13) for 20 min at 25V, neutralized and dehydrated to be stained with a fluorescent dye and later comets visualization with the epifluorescence microscope. Chlorpyrifos and imazalil induced significant DNA damage in a dose-dependent manner. Chlorpyrifos was the major inductor of DNA breaks. These results indicate that both are genotoxic compounds in vitro. Thiabendazole fungicide did not induced DNA damage using the comet assay for all concentrations tested.

Micronúcleos y otras anormalidades nucleares en el epitelio oral de mujeres expuestas ocupacionalmente a plaguicidas / Micronuclei and other nuclear abnormalities in the oral epithelium of female workers exposed to pesticides

In order to study if banana fields labour exposure to pesticides produces some kind of DNA damage, we determine the presence of micronuclei in epithelial oral cells in working women in Guapiles and Siquirres, Costa Rica, as an effect biomarker. We also analyzed other abnormalities in the nucleus of those cells such as broken-egg, karyolysis or kariorrhexis, to see if there was some kind of genotoxicity or citotoxicity. The women group exposed to pesticides worked in packing bananas plant from different independent farms. The control group of women had never done any farming tasks; they did not live in the banana fields, neither their husband. We got information about the life style, medical and familial history of the participants through an interview. We did not found any significant increment in the frequency of micronuclei form the exposed group compared with the controls. The other nuclei abnormalities showed signs of citotoxicity or genotoxicity in the controls, associated with the intake of coffee and dental x-rays. These results do not rule out at that pesticides used in packing bananas are agents capable of producing damage to the DNA, but it seems that micronuclei from the oral epithelium is not the most adequate marker to measure it.

Aberraciones cromosómicas en trabajadoras expuestas a plaguicidas / Chromosomic aberrations in female workers exposed to pesticides

The purpose of this work was to determine if the occupational exposure to those pesticides used at banana plantations' packaging plants produces genetic damage to somatic cells of female workers. Chromosomal aberrations were scored in lymphocytes of 20 women, 10 female exposed workers and 10 female controls. Workers were recruited from independent farms from two locations in Costa Rica, during January through June in 1996 and 1997. These females had a minimum of three months of work, had never received chemotherapy or radiotherapy and did some of these labors: sealing, spraying or weighting of bananas. Control unexposed females lived in the same area, were of similar age and neither them nor their husbands/mates had ever worked in pesticide related labors. For each female, 100 mitotic figures were scored. The kind of aberrations detected were acentric fragments, dicentric chromosomes, rings, gaps and breaks. Among workers, 16% of cells (n=1000) had one or more abnormalities, whereas control unexposed females had 6% of cells (n=1000) with comparable anomalies (p < 0.05). In conclusion, the pesticide exposure is a risk factor for chromosome aberrations in female somatic cells.

Disabilities caused by unstable mutations in Costa Rica

Myotonic dystrophy and fragile X syndrome are two genetically determined relatively common disabilities. Both are examples of a new type of mutation mechanism called unstable or dynamic mutations, triple repeats expansions or DNA amplification. Fragile X syndrome is recognized as the main cause of hereditary mental retardation and myotonic dystrophy is considered the most common muscular dystrophy of adults. This is a prospective non randomized study of clinically affected people, in order to confirm the diagnosis with molecular techniques (Southern blot and PCR) and to perform cascade screening of the rest of the family to offer them adequate genetic counseling. We were able to corroborate the initial diagnosis in most clinical cases of myotonic dystrophy, but in the cases of mental retardation more than half studies were negative for fragile X syndrome, stressing the difficulties encountered by medical practitioners to diagnose this syndrome. The reasons for this are several; probable the main culprit is the subtle and unspecific clinical picture affected individuals exhibit, particularly children before puberty. Cascade screening, genetic counseling and selective abortion are the only tools available to prevent these disabling diseases for the moment.

Las mutaciones inestables, nuevo reto para el consejo genético de enfermedades hereditarias: [revisión] / Unstable mutations, new challenges for genetic counseling of inherited disorders: [review]

Unstable mutations or amplification of DNA tandem repeats sequences constitute a new kind of genetic alteration discovered in the 90's that cause hereditary diseases. This mutation has been found inside or near important genes involved in the normal neurological function in human beings. In some cases, the presence of the amplification causes altered expression of the genes, their inactivation or the synthesis of a protein with new functions. Some common characteristics of these diseases are that they affect the central nervous system and are degenerative in nature. Most of them show genetic anticipation meaning that the severity of the manifestations increases in each generation and appear at an earlier age. In most cases, the severity of the symptoms is positively correlated with the size of the amplification. Twenty illnesses caused by this kind of mutations have been identified so far. Briefly, this work reviews the current knowledge about this topic.

Miotomía congénita: Caracterización clínica de una familia costarricense afectada por la Enfermedad de Thomsen / Myotonia congenital: Clinic characterization of a family affected by Thomsen`s disease

La miotonía congénita es una enfermedad muscular hereditaria caracterizada por excitabilidad incrementada de la fibra muscular, que se refleja en miotonía clínica, además de rigidez e hipertrofia. Su herencia puede ser de forma autosómica dominante o recesiva, designadas como miotonía generalizada de Thomsen y Becker respectivamente. Se establece el diagnóstico clínico correcto de una familia costarricense previamente había sido diagnosticada con distrofia miotónica tipo 1, pero cuyo estudio a nivel molecular y clínico descartó una distrofia miotónica después de realizar exámenes físicos, electromiográficos, oculares y electrocardiograma de varios miembros de la familia. Los individuos poseían la fuerza normal pero presentaban el fenómeno mejor en los brazos, era más evidente en la musculatura proximal que en la distal, además de ser generalizada e iba desapareciendo conforme el paciente repetía varias veces el movimiento. Ahora bien, con los estudios moleculares es necesario confirmar el dignóstico clínico propuesto en este trabajo. Los pacientes presentan un cuadro clínico claramente diferente de la DM1, compatible con miotonía congénita, y que por su forma de herencia; autosómica dominante; debe clasificarse como enfermedad de Thomsen.

Daño del ADN en trabajadoras bananeras expuestas a plaguicidas en Limón, Costa Rica / DNA damage in female workers exposed to pesticides in banana plantations at Limón, Costa Rica

Pesticide use in Costa Rica is very high and all year round. A high percentage of what is sprayed remains in the environment and in the living organisms around. This situation brings contamination and health problems to people in contact with them. The onset of adverse effects may be in the short or the long term, and symptoms vary widely, from headaches to cancer. Much research in this area has been devoted to acute or chronic effects, and not until recently to the genotoxic effect of pesticides. This study evaluated the genotoxic effect of pesticides used in banana packing activities, using the comet assay (single cell electrophoresis) as the biological marker in lymphocytes. This was a case-control double blind study of 30 exposed women from 15 banana farms and 28 women not occupationally exposed to pesticides from the same geographic area. Results show damage to single stranded DNA after working from 5 to 15 years (R2 = 0.12). In Costa Rica we do not have an historical record of the kind of pesticides used in banana farms, the period of time and for how long were they used. This prevented further analysis concerning dose, frequency of exposure and use of new or old kind of pesticides in the farms in relation to DNA damage. The comet assay is of value in the genetic monitoring of pesticide exposed populations.

Micronuclei frequency in lymphocytes of individuals occupationally exposed to pesticides

Pesticides have been widely used in developing countries over the years. A large amount of these remains in the environment and organisms. Pesticide pollution is detrimental to human health. The effects can be seen on a short or a long-term basis and the symptoms can vary from headache to cancer. Only a minority of studies focuses on their genotoxic effect. This study assesses the genotoxic effect of the pesticides used at banana-packaging plants with binucleate micronuclei assay using cultured lymphocytes. The studied population included 32 exposed and 37 unexposed women from Costa Rica. There is no significant difference between the two groups. However, women who worked at the packaging plant and had stillbirths or spontaneous abortions were 1.45 times more (alpha = 0.06) likely to have an increased micronuclei frequency than their coworkers who lacked those disorders; this may indicate genetic susceptibility. In vitro pesticides studies and susceptibility biomarkers are needed to identify subgroups with higher risks.

Tamizaje de sitio frágil en el cromosoma X en una población de retardados mentales: hallazgos preliminares / Screening for the X chromosome fragile site in a mentally retarded population

El sitio frágil del cromosoma X es la anomalía cromosómica más común, después de la trosomía 21, entre varones con retardo mental de etiología genética, y además se asocia con deficiencia mental en más de la mitad de los casos de mujeres portadoras. Este estudio en el primer intento de averiguar sis sucede esto mismo en nuestro país, en una muestra de retardados mentales seleccionados. El tamizaje se está realizado en la Escuela de Eseñanza Especial "Fernando Centeno Güel" mediante análisis cromosómico de un mínimno de 100 figuras mitóticas de cada individuo, obtenidas del cultivo por 92 horas, de sangre periférica en medio 199 suplementado al 5% con suero fetal bovino. Los resultados preliminares obtenidos confirman la presencia de este marcador cromosómico también entre nuestros retardados mentales y con una frecuencia (5%) similar a la informada en la literatura. Las implicaciones concomitantes al diagnóstico del síndrome del X frágil son muy importantes en varios aspectos: la asesoría genética adecuada y oportuna podría prevenir en muchos casos la concepción de individuos afectados, el tratamiento con ácido fólico parece promisorio y por último, existe la opción del diagnóstico fetal y prevención post-concepción