RESUMEN
We describe a young woman with Prader-Willi syndrome (PWS) due to a mosaic imprinting defect. Three independent assays revealed a reduced proportion of nonmethylated SNURF-SNRPN alleles in peripheral blood DNA: methylation-specific PCR followed by denaturing high-performance liquid chromatography (MSP/DHPLC), methylation-sensitive restriction enzyme analysis and methylation-specific real-time PCR analysis. Microsatellite analysis and fluorescence in situ hybridisation revealed apparently normal chromosomes 15 of biparental origin. Based on the MSP/DHPLC and real-time PCR results, we estimate that approximately 50% of the patient's blood cells have an imprinting defect and 50% of the cells are normal. Apart from a rather normal facial appearance, the proband has typical features of PWS.
Asunto(s)
Impresión Genómica , Mosaicismo , Síndrome de Prader-Willi/genética , Adulto , Niño , Cromatografía Líquida de Alta Presión , Metilación de ADN , Femenino , Humanos , Hibridación Fluorescente in Situ , Repeticiones de Microsatélite , Reacción en Cadena de la PolimerasaRESUMEN
The Prader-Willi syndrome (PWS) region contains several genes transcribed from the paternal chromosome only. We have previously identified a testis-specific gene, C15orf2, which maps between NDN and SNURF-SNRPN and is expressed from both alleles. Here we report on two novel genes (prader-willi region non-protein-coding RNA 1 and 2) located between NDN and C15orf2. By database search we found five partially duplicated copies, of which only one of each appears to be active. PWRN2 is expressed only in testis and is biallelic. PWRN1 is biallelically expressed in testis and kidney, but monoallelically in fetal brain. Methylation analysis of a CpG island 15 kb upstream of exon 1 showed absence of methylation in spermatozoa, but methylated and unmethylated alleles in fetal brain. Reinvestigation of C15orf2 revealed that this gene is also expressed in fetal brain and that expression in this tissue is monoallelic. We conclude that PWRN1 and C15orf2 may play a role in PWS.
Asunto(s)
Empalme Alternativo/fisiología , Síndrome de Angelman/metabolismo , Encéfalo/metabolismo , Citocinas/metabolismo , Síndrome de Prader-Willi/metabolismo , Alelos , Síndrome de Angelman/genética , Encéfalo/embriología , Citocinas/genética , Metilación de ADN , Feto/anatomía & histología , Expresión Génica , Impresión Genómica , Humanos , Masculino , Síndrome de Prader-Willi/genética , Proteínas , Transcripción Genética/fisiologíaRESUMEN
Angelman syndrome is a neurogenetic disorder caused by the loss of function of the imprinted UBE3A gene in 15q11-q13. In a small group of patients, the disease is due to an imprinting defect (ID) that silences the maternal UBE3A allele. The presence of a faint maternal band detected by methylation-specific PCR analysis of the SNURF-SNRPN locus in approximately one-third of patients who have an ID but no imprinting center deletion suggested that these patients are mosaics of ID cells and normal cells. In two patients studied, somatic mosaicism was proven by molecular and cellular cloning, respectively. X inactivation studies of cloned fibroblasts from one patient suggest that ID occurred before the blastocyst stage. To quantify the degree of mosaicism, we developed a novel quantitative methylation assay based on real-time PCR. In 24 patients tested, the percentage of normal cells ranged from <1% to 40%. Regression analysis suggests that patients with a higher percentage of normally methylated cells tend to have milder clinical symptoms than patients with a lower percentage. In conclusion, we suggest that the role of mosaic imprinting defects in mental retardation is underestimated.
Asunto(s)
Síndrome de Angelman/genética , Impresión Genómica , Mosaicismo , Síndrome de Angelman/fisiopatología , Células Cultivadas , Clonación Molecular , Islas de CpG , Metilación de ADN , Cartilla de ADN , Compensación de Dosificación (Genética) , Fibroblastos/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Humanos , Repeticiones de Microsatélite , Proteínas del Tejido Nervioso/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas de Unión al ARN/genética , Análisis de Regresión , Análisis de Secuencia de ADN , Piel/citologíaRESUMEN
Although uniparental disomy often results from the postzygotic rescue of a meiotic non-disjunction event, mosaicism is usually confined to the placenta. We describe a girl with Prader-Willi syndrome (PWS) who is mosaic for normal cells and cells with maternal uniparental disomy 15 [upd(15)mat] in blood and skin. Somatic mosaicism was confirmed by cloning and genotyping of skin fibroblasts. X inactivation studies indicated that upd occurred prior to X inactivation. RNA samples from the cloned cells were used in DNA microarray experiments to study the effect of upd(15)mat on the gene expression pattern of fibroblasts. Proof of principle was obtained by detecting several chromosome 15 genes known to be imprinted. We did not obtain any evidence for novel 15q genes showing imprinted expression in fibroblasts. Differentially expressed genes on other chromosomes are candidates for downstream genes regulated by an imprinted gene and may play a role in the pathogenesis of PWS. The finding of strongly reduced mRNA levels in upd(15)mat cells of the gene encoding secretogranin II (SCG2), which is a precursor of the dopamine releasing factor secretoneurin, raises the question whether hyperphagia in patients with PWS might be due to a defect in dopamine-modulated food reward circuits.