RESUMEN
Interleukin-12 (IL-12) is a potent inducer of interferon-gamma production by T cells and is a major factor for the development of T-helper 1 (Th1) cells. It exerts its biological effects through binding to the IL-12 receptor (IL-12R), a heterodimer composed of a 1 and a beta2 subunits. The signaling beta2 chain is expressed on Th1 cells and to a lesser extent on Th0 cells, but not on Th2 cells, rendering these latter cells unresponsive to IL-12. Polymorphisms in the coding region of the IL-12Rbeta2 gene were shown to be associated with atopic disease. Here, we analyzed the 5'-regulatory region of the human IL-12Rbeta2 gene by denaturing high-performance liquid chromatography (Transgenomic WAVE system, San Jose, CA). We found five novel single-nucleotide polymorphisms (SNPs) in the proximal 1.2 kb IL-12Rbeta2 promoter region, i.e. -237C/T, -465A/G, -1023A/G, -1033T/C, and -1035A/G. SNP -465A/G is of particular interest as it determines the integrity of a GATA consensus site. By functional comparison of both -465 alleles in transient transfection assays, we show that promoter activity is increased in case of the -465G allele, disrupting the intact GATA site. Comparison of the prevalence of -465A/G SNP alleles in small cohorts of allergic asthmatic and healthy control individuals provided no evidence for an altered distribution in the asthmatic population. In conclusion, we have identified a novel polymorphic GATA site that may affect transciptional activity of the human IL-12Rbeta2 gene under GATA3-mediated, Th2-polarizing conditions.
Asunto(s)
Regiones Promotoras Genéticas , Receptores de Interleucina/genética , Región de Flanqueo 5' , Asma/genética , Secuencia de Bases , Secuencia de Consenso , Humanos , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Receptores de Interleucina-12RESUMEN
In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (mtDNA) mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of denaturing high performance liquid chromatography (DHPLC). The complete mitochondrial genome was amplified in 13 fragments of 1-2 kb, digested in fragments of 90-600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNA(Leu(UUR)) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNA(Leu(UUR)) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes.
Asunto(s)
Cromatografía Líquida de Alta Presión , ADN Mitocondrial/genética , Pruebas Genéticas/métodos , Genoma , Análisis Heterodúplex/métodos , Mutación/genética , Adenosina Trifosfatasas/genética , Secuencia de Bases , Cardiomiopatías/genética , Cardiomiopatías/patología , Núcleo Celular/genética , Análisis Mutacional de ADN/métodos , Cartilla de ADN/genética , Enzimas de Restricción del ADN/metabolismo , Humanos , Enfermedad de Leigh/genética , Enfermedad de Leigh/patología , Mitocondrias Musculares/genética , Mitocondrias Musculares/patología , Miopatías Mitocondriales/genética , Miopatías Mitocondriales/patología , ATPasas de Translocación de Protón Mitocondriales , Distrofias Musculares/genética , Distrofias Musculares/patología , Desnaturalización de Ácido Nucleico , Polimorfismo Genético/genética , Aminoacil-ARN de Transferencia/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Tamaño de la Muestra , Sensibilidad y Especificidad , Temperatura , Factores de TiempoRESUMEN
Properdin type I deficiency is characterised by complete absence of extracellular properdin, a positive regulator of the alternative pathway of complement activation. Properdin deficiency is associated with increased susceptibility to severe meningococcal disease. We have identified the genetic defect in 10 Dutch families. Six different mutations and one sequence polymorphism in the properdin gene were found. All amino acid substitutions were limited to conserved amino acids in exons 7 and 8 in contrast to the premature stops that were found in other exons. The missense mutations may alter the protein conformation in such a way that properdin will not be secreted and therefore catabolised intracellularly. The decreased properdin levels found in some healthy females carrying one mutated properdin gene were studied for X-inactivation. Most carriers with extreme low or high properdin levels showed preferential X-inactivation for the normal or mutated X chromosome, respectively. We observed some exceptions, suggesting additional regulation of properdin excretion apart from X-inactivation.
Asunto(s)
Compensación de Dosificación (Genética) , Meningitis Meningocócica/genética , Mutación Missense , Properdina/deficiencia , Properdina/genética , ADN/análisis , ADN/sangre , Análisis Mutacional de ADN , Exones , Femenino , Humanos , Linfocitos/fisiología , Masculino , Países Bajos , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Polimorfismo Conformacional Retorcido-Simple , Properdina/metabolismo , Cromosoma X/genéticaRESUMEN
We report on a case of generalized mosaicism for trisomy 22. At chorionic villus sampling (CVS) in the 37th week of pregnancy, a 47,XX,+22 karyotype was detected in all cells. The indication for CVS was severe unexplained symmetrical intrauterine growth retardation (IUGR) and a ventricular septal defect (VSD) was noted. In cultured cells from amniotic fluid taken simultaneously, only two out of ten clones were trisomic. At term, a growth-retarded girl with mild dysmorphic features was born. Lymphocytes showed a normal 46,XX[50] karyotype; both chromosomes 22 were maternal in origin (maternal uniparental disomy). Investigation of the placenta post-delivery using fluorescence in situ hybridization showed a low presence of trisomy 22 cells in only one out of 14 biopsies. In cultured fibroblasts of skin tissue, a mosaic 47,XX,+22[7]/46,XX[25] was observed. Clinical follow-up is given up to 19 months.
Asunto(s)
Cromosomas Humanos Par 22/genética , Retardo del Crecimiento Fetal/genética , Repeticiones de Microsatélite/genética , Mosaicismo/genética , Trisomía/genética , Anomalías Múltiples/genética , Adulto , Alelos , Biopsia , Muestra de la Vellosidad Coriónica , Femenino , Retardo del Crecimiento Fetal/complicaciones , Retardo del Crecimiento Fetal/fisiopatología , Estudios de Seguimiento , Defectos del Tabique Interventricular/complicaciones , Defectos del Tabique Interventricular/embriología , Defectos del Tabique Interventricular/genética , Humanos , Recién Nacido , Cariotipificación , Padres , Placenta/patología , Embarazo , Resultado del Embarazo , Tercer Trimestre del Embarazo , Diagnóstico Prenatal/métodos , Trisomía/diagnósticoRESUMEN
Properdin deficiency carrier identification is relevant, because properdin-deficient persons have an increased risk of contracting meningococcal disease. Vaccination against meningococcal disease at a young age may provide protection. Accurate detection of this deficiency is needed. Microsatellite haplotyping with the PFCI and PFC2 markers closely linked to the properdin gene locus at Xp11.3-Xp11.23 may offer an easy and accurate identification of carriers of the properdin deficiency gene. The chance to study 91 relatives belonging to 10 families with complete (type 1) properdin deficiency offered a unique opportunity to assess whether properdin type 1 deficiency is associated with a distinct microsatellite haplotype. Haplotyping with the closely linked PFC1 and 2 markers yielded five different haplotypes, which did not support the concept of a founder effect. Among the 28 women carriers, two had normal properdin levels and in five the PFC1,2 polymorphism was not informative owing to homozygosity. Extending the microsatellite haplotyping with three additional markers (DXS1126, DXS426 and DXS7) yielded informative haplotypes in all meioses. We concluded that microsatellite haplo-typing using five markers in close proximity to the properdin gene locus is an accurate method of detecting carriers of the properdin deficiency gene and of properdin-deficient persons within a family at a young age.
Asunto(s)
Haplotipos , Heterocigoto , Repeticiones de Microsatélite , Properdina/deficiencia , Femenino , Humanos , Masculino , Cromosoma XRESUMEN
Using a panel of patient cell lines with chromosomal breakpoints, we constructed a physical map for the short arm of human chromosome 11. We focused on 11p15, a chromosome band harboring at least 25 known genes and associated with the Beckwith-Wiedemann syndrome, several childhood tumors, and genomic imprinting. This underlines the need for a physical map for this region. We divided the short arm of chromosome 11 into 18 breakpoint regions, and a large series of new and previously described genes and markers was mapped within these intervals using fluorescence in situ hybridization. Cosmid fingerprint analysis showed that 19 of these markers were included in cosmid contigs. A detailed 10-Mb pulsed-field physical map of the region 11p15.3-pter was constructed. These three different approaches enabled the high-resolution mapping of 210 markers, including 22 known genes.