Diagnóstico citogenético-molecular del síndrome Xq frágil / Diagnostic testing in fragile X syndrome

El síndrome Xq frágil (SXF) es una causa frecuente de retraso mental (RM); se estima que uno de cada 4.000 varones y una década 6.000 mujeres lo presentan. Clínicamente los individuos afectados se caracterizan por presentar déficit intelectual y cognitivo, déficit de lenguaje, macroorquidismo, fascie alargada y orejas prominentes, entre otras dismorfias faciales. A nivel molecular es posible distinguir fundamentalmente dos tipos de alelos mutados: premutacion y mutación completa, las cuales corresponden a amplificación del trinucleótido CGG localizado en el primer exón del gen FMR1; las premutaciones presentan entre 52 y 200 repetidos y las mutaciones completas sobre 200 CGG, con hipermetilación de la región promotora del gen FMR1 e inhibición de la expresión de la proteína FMRP, causante del RM y dismorfias características de este síndrome. Desde que se identifico la mutación en 1991, la pesquisa de pacientes afectados se inicia por el examen clínico y luego el análisis citogenetico clásico y el test de screening basado en PCR para individuos varones y análisis molecular directo del gen FMR 1 por Southern Blot con la sonda Stb 12.3 para pacientes mujeres; los varones que presentan un PCR alterado deben ser confirmados por Southern Blot. El PCR debe ser usado como método de screening solo en varones con RM, sin historia familiar; es un sensible, rápido, de bajo costo y permite determinar el numero de repetidos CGG. Proponemos el uso conjunto de estos métodos para optimizar el estudio molecular directo del gen FMR1 y establecer un protocolo mas eficiente en la pesquisa de afectados, el estudio de familiares a riesgo y el consejo genético adecuado.

Tamizaje clínico y análisis de mutaciones en el gen FMR1 en 99 varones con características clínicas del síndrome de X-frágil / Clinical screening and FMR1 gene mutation analysis in male patients with Fragile X syndrome

Introducción: El síndrome de X frágil (SXF) es una causa frecuente de retraso mental (RM), se presenta en 1 de 4 000 hombres y en 1 de 8 000 mujeres. A nivel molecular existen principalmente tres tipos de alteraciones: premutación, mutación completa y mosaicos, todas las cuales corresponden a amplificación del trinucleótido CGG localizado en el primer exón del gen FMR1: las premutaciones presentan entre 52 y 200 repetidos; las mutaciones completas, sobre 200 CGG, presentan hipermetilación de la región promotora del gen FMR1 e inhibición de la expresión de la proteína FMRP, causante del RM y dismorfias características de este síndrome. Los mosaicos presentan mutación completa y premutación o metilación parcial del gen FMR1. Los pacientes con SXF son diagnosticados clínicamente según un protocolo de tamizaje que considera 15 características clínicas que entrega un puntaje máximo de 30 puntos en individuos afectados. Objetivo: Definir criterios clínicos específicos para población chilena que ayuden a identificar a los individuos que deban ser sometidos a estudios moleculares confirmatorios de SXF. Pacientes y Método: Se consideraron 99 pacientes varones referidos al INTA por presentar retraso mental y características clínicas sugerentes del SXF; a todos se les realizó evaluación clínica utilizando el protocolo descrito por Buttler y estudio molecular con análisis directo del gen FMR1 por Southern blot. Resultados: 23 de los 99 pacientes estudiados presentaron una mutación en FMR1 y puntaje clínico entre 16 y 27 puntos; los 76 casos restantes con puntajes clínicos entre 10 y 26 puntos, no presentaron mutación en el gen FMR1. Se evaluaron las características clínicas en ambos grupos y se observó que 4 de ellas se asocian significativamente a la mutación, siendo tres de ellas independientes de la edad de los pacientes. Conclusiones: Con estos resultados y a fin de optimizar el estudio molecular directo del gen FMR1, proponemos que el criterio de selección de pacientes sea a través del examen clínico y que todo individuo con puntaje ³ 15 puntos debe ser sometido al estudio molecular.

[Clinical, genetic and molecular features in 45 patients with Prader-Willi syndrome]. / Caracterización clínico-genético-molecular de 45 pacientes chilenos con Síndrome de Prader Willi.

BACKGROUND: Prader-Willi syndrome (PWS) is a neurogenetic disease characterized by neonatal hypotonia, retarded mental and motor development, hypogonadism, hyperphagia, morbid obesity and dysmorphic facial features. It has an incidence of 1:12.000-15.000 newborns and is caused by abnormalities in genes located in 15q11q13. PWS is one of the most frequent genetic disorders and microdeletion syndromes. It is also the most common cause of obesity from genetic origin and it was the first disease in which imprinting and uniparental disomy were recognized as cause of genetic disorders. Seventy to seventy five percent of PWS cases are due to 15q11q13 deletions, 20-25% to uniparental disomy and 1% to mutations in the imprinting center. AIM: To analyze the clinical, genetic and molecular features of patients with PWS, seen at one institution. PATIENTS AND METHODS: Retrospective review of 45 patients (27 males) with PWS seen at the Genetics Outpatient Clinic at INTA. RESULTS: Twenty three (51.1%) patients had a deletion, 13 (28.9%) patients did not have a deletion. In nine patients, fluorescence in situ hybridization (FISH) study was not performed, therefore the presence of deletion was unknown. The clinical score was 8 points for patients younger than 3 years (n=11) and 11.5 points for patients older than 3 years (n=34); for patients aged 12 months or less, the clinical score was 7 points. Mean clinical score was 11 points for patients with deletion and 10 points for patients without deletion. CONCLUSIONS: Most patients with PWS have a deletion; the phenotype depends on age and the clinical score is useful for Chilean patients with PWS.

Caracterización clínico-genético-molecular de 45 pacientes chilenos con Síndrome de Prader Willi / Clinical, genetic and molecular features in 45 patients with Prader-Willi Syndrome

Background: Prader-Willi syndrome (PWS) is a neurogenetic disease characterized by neonatal hypotonia, retarded mental and motor development, hypogonadism, hyperphagia, morbid obesity and dysmorphic facial features. It has an incidence of 1:12.000-15.000 newborns and is caused by abnormalities in genes located in 15q11q13. PWS is one of the most frequent genetic disorders and microdeletion syndromes. It is also the most common cause of obesity from genetic origin and it was the first disease in which imprinting and uniparental disomy were recognized as cause of genetic disorders. Seventy to seventy five percent of PWS cases are due to 15q11q13 deletions, 20-25percent to uniparental disomy and 1percent to mutations in the imprinting center. Aim: To analyze the clinical, genetic and molecular features of patients with PWS, seen at one institution. Patients and methods: Retrospective review of 45 patients (27 males) with PWS seen at the Genetics Outpatient Clinic at INTA. Results: Twenty three (51.1percent) patients had a delection, 13 (28.9percent) patients did not have a deletion. In nine patients, fluorescence in situ hybridization (FISH) study was not performed, therefore the presence of deletion was unknown. The clinical score was 8 points for patients younger than 3 years (n=11) and 11.5 points for patients older than 3 years (n=34); for patients aged 12 months or less, the clinical score was 7 points. Mean clinical score was 11 points for patients with deletion and 10 points for patients without deletion. Conclusions: Most patients with PWS have a deletion; the phenotype depends on age and the clinical score is useful for Chilean patients with PWS .

Diagnóstico molecular de los síndromes de Prader-Willi y de Angelman: análisis de metilación, citogenética y FISH / Methylation, citogenetic and FISH tests in the molecular diagnosis of Prader-Willi and Angelman syndromes

Background: The diagnosis of Prader-Willi and Angelman syndromes is difficult, since their phenotypic manifestations are variable and unspecific. The study of the methylation state of DNA in l5(q11-q13) using polymerase chain reaction, called methylation test, allows the diagnosis of most patients with Prader-Willi and Angelman syndromes, irrespective if the underlying molecular alteration is a deletion, uniparental disomy or a punctual imprinting mutation. Aim: To assess the effectiveness of methylation test in the diagnosis of Prader-Willi and Angelman syndromes. Patients and methods : Thirty seven cases with a presumptive diagnosis of Prader-Willi syndrome and 25 with the presumptive diagnosis of Angelman syndrome were studied. Methylation test was done in genomic DNA obtained from peripheral Iymphocytes. Results: Methylation test confirmed the clinical diagnosis in 11 of 37 patients with PraderWilli (30 percent) and 6 of 25 patients with Angelman syndrome (24 percent). Conclusions: Clinical criteria overestimate the diagnosis of Prader-Willi and Angelman syndromes. The initial diagnosis should be confirmed with the methylation test and, if necessary, with FISH that will detect most deletions in the region

Análisis molecular directo de mutaciones en el gen FMR-1 en pacientes con síndrome de Xq frágil y sus familias / Direct molecular analysis of FMR-1 gene mutation in patients with fragile Xq syndrome and their families

Background: The unequivocal diagnosis of fragile Xq syndrome is based in the direct analysis of the underlying FMR-1 gene mutation, that consists in an increased number of trinucleotide CGG repetitions. Aim: To study families with fragile Xq syndrome, using the Southern technique for the analysis of the mutation. Subjects and methods: Fifteen individuals, pertaining to 6 families with fragile Xq syndrome, were studied. Clinical, cytogenetic and molecular analysis using Southern technique, were done. Results: Five male individuals had a clinically evident syndrome, confirmed by cytogenetic analysis that showed fragility in 10 to 29 percent of studied cells. One subject with a clinical picture suggesting fragile Xq had a normal cytogenetic study. The other studied subjects were the mothers of the five subjects with the syndrome, that must be carriers, and four brothers. Molecular analysis showed that seven subjects (5 males) had a complete mutation, five (4 females) were carriers of a pre mutation and three (2 males) did not have the mutation. Conclusions: The southern technique allows to verify the normal condition of FRAXA locus, identify carriers and to detect complete mutations in fragile Xq syndrome

Hallazgos citogenéticos en pacientes con síndrome de Down / Cytogenetic findings in patients with Down syndrome

Con el objeto de establecer la frecuencia de trisomia 21 no clásicas se analizaron los estudios cromosómicos de 201 pacientes, derivados para estudio citogenético en linfocitos de sangre periférica con el diagnóstico clínico de síndrome de Down. De éstos, 161 (80%) resultaron trisomías 21 clásicas, 7(3,5%) trisomías 21 por translocación, 5(2,5%) trisomías 21 en mosaico, 6(3%) trisomías 21 más otra alteración cromosómica y 22(11%) tenía estudios citogenéticos normales. En 26,8% de los pacientes el diagnóstico fue hecho en el período de recién nacido y la proporción de hombres y mujeres fue de (1,18:1)