RESUMO
Imprinting defects in 15q11-q13 are a rare but significant cause of Prader-Willi syndrome (PWS) and Angelman syndrome (AS). Patients with an imprinting defect have apparently normal chromosomes 15 of biparental origin, but are recognised by @parental DNA methylation at D15S63 (PW71) or SNURF-SNRPN exon 1. We have investigated the methylation status of five additional loci in 12 such patients with or without a deletion in the imprinting centre. In each patient, the imprinting defect affected all loci tested. During routine diagnostic testing we identified four patients who had a normal methylation pattern at SNURF-SNRPN exon 1, but an abnormal pattern at D15S63. In two of these patients, who were suspected of having PWS, this change was restricted to D15S63. In two patients suspected of having AS, several but not all loci were affected. Using a newly developed methylation-specific PCR test for D15S63 we found that these methylation changes are rare in patients suspected of having AS. Although we can not prove that the methylation changes in the four patients are causally related to their disease, our findings demonstrate that spatially restricted changes in methylation can occur. In some cases, these changes may reflect incomplete imprint spreading.
Assuntos
Síndrome de Angelman/genética , Cromossomos Humanos Par 15/genética , Metilação de DNA , Síndrome de Prader-Willi/genética , Southern Blotting , DNA/genética , DNA/metabolismo , Saúde da Família , Feminino , Impressão Genômica , Humanos , Masculino , Repetições de MicrossatélitesRESUMO
Generalised progressive retinal atrophy (gPRA) is a heterogeneous group of hereditary diseases causing degeneration of the retina in dogs and cats. As a combination of mutations in the RDS/Peripherin and the ROM1 genes leads to the phenotype of retinitis pigmentosa in man we first performed mutation analysis to screen these genes for disease causing mutations followed by the investigation of a digenic inheritance in dogs. We cloned the RDS/Peripherin gene and investigated the RDS/Peripherin and ROM1 genes for disease causing mutations in 13 gPRA-affected dog breeds including healthy animals, obligate gPRA carriers and gPRA-affected dogs. We screened for mutations using single strand conformation polymorphism (SSCP) analysis. Sequence analysis revealed several sequence variations. In the coding region of the RDS/Peripherin gene three nucleotide exchanges were identified (A277C; C316T; G1255A), one of which leads to an amino acid substitution (Ala339Thr). Various silent sequence variations were found in the coding region of the ROM1 gene (A536G, G1006A, T1018C, T1111C, C1150T, C1195T), as well as an amino acid substitution (G252T; Ala54Ser). By excluding the respective gene as a cause for gPRA several sequence variations in the intronic regions were investigated. None of these sequence variations cosegregated with autosomal recessively (ar) transmitted gPRA in 11 breeds. The candidate gene RDS/Peripherin obviously does not harbour the critical mutation causing the autosomal recessive form of gPRA because diseased individuals show heterozygous genotypes for sequence variations in the Miniature Poodle, Dachshund, Australian Cattle Dog, Cocker Spaniel, Chesapeake Bay Retriever, Entlebucher Sennenhund, Sloughi, Yorkshire Terrier, Tibet Mastiff, Tibet Terrier and Labrador Retriever breeds. In the following breeds the ROM1 gene was also excluded indirectly for gPRA: Miniature Poodle, Dachshund, Australian Cattle Dog, Sloughi, Collie, Tibet Terrier, Labrador Retriever and Saarloos/Wolfhound. Digenic inheritance for gPRA is practically excluded for both these genes in four breeds: Miniature Poodle, Dachshund, Labrador Retriever and Saarloos/Wolfhound.
Assuntos
Doenças do Cão/genética , Proteínas do Olho/genética , Proteínas de Filamentos Intermediários/genética , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Degeneração Retiniana/veterinária , Substituição de Aminoácidos , Animais , Sequência de Bases , Gatos , Bovinos , Clonagem Molecular , Primers do DNA/genética , Cães , Variação Genética , Dados de Sequência Molecular , Periferinas , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Degeneração Retiniana/genética , Especificidade da EspécieRESUMO
We investigated the gene encoding the beta subunit of cGMP phosphodiesterase (PDE6B) as a candidate for generalized progressive retinal atrophy (gPRA), an autosomal recessively transmitted eye disease in dogs. The PDE6B gene was isolated from a genomic library. Single-strand conformation polymorphism analysis revealed eight intronic variations in different subsets of the 14 dog breeds investigated. In addition, we identified an 8-bp insertion after codon 816 in certain Sloughi dogs. Analysis of PRA-affected and obligatory carrier Sloughis showed that this mutation cosegregates with disease status in a large pedigree. All other exchanges identified were not located in functionally relevant parts of the gene (e.g., in the splice signal consensus sites). In most dog breeds (Labrador retriever, Tibetan mastiff, dachshund, Tibetan terrier, miniature poodle, Australian cattle dog, cocker spaniel, collie, Saarloos wolfhound, Chesapeake Bay retriever, and Yorkshire terrier), PDE6B was excluded as a candidate gene for gPRA because heterozygous allele constellations were detected in diseased animals. Therefore, the PDE6B sequence variations did not segregate together with the mutation(s) causing gPRA. Direct and indirect DNA tests concerning gPRA can be offered now for a variety of different dog breeds.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , Doenças do Cão/genética , Éxons/genética , Mutagênese Insercional/genética , Degeneração Retiniana/genética , Degeneração Retiniana/veterinária , 3',5'-GMP Cíclico Fosfodiesterases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Cruzamento , Clonagem Molecular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Análise Mutacional de DNA , Doenças do Cão/enzimologia , Cães , Feminino , Variação Genética/genética , Genótipo , Humanos , Íntrons/genética , Masculino , Dados de Sequência Molecular , Linhagem , Polimorfismo Conformacional de Fita Simples , Regiões Promotoras Genéticas/genética , Subunidades Proteicas , Degeneração Retiniana/enzimologiaRESUMO
The imprinted domain on human chromosome 15 consists of two oppositely imprinted gene clusters, which are under the coordinated control of an imprinting center (IC) at the 5' end of the SNURF-SNRPN gene. One gene cluster spans the centromeric part of this domain and contains several genes that are transcribed from the paternal chromosome only (MKRN3, MAGEL2, NDN, SNURF-SNRPN, HBII-13, HBII-85 and HBII-52). Apart from the HBII small nucleolar RNA (snoRNA) genes, each of these genes is associated with a 5' differentially methylated region (DMR). The second gene cluster maps to the telomeric part of the imprinted domain and contains two genes (UBE3A and ATP10C), which in some tissues are preferentially expressed from the maternal chromosome. So far, no DMR has been identified at these loci. Instead, maternal-only expression of UBE3A may be regulated indirectly through a paternally expressed antisense transcript. We report here that a processed antisense transcript of UBE3A starts at the IC. The SNURF-SNRPN sense/UBE3A antisense transcription unit spans more than 460 kb and contains at least 148 exons, including the previously identified IPW exons. It serves as the host for the previously identified HBII-13, HBII-85 and HBII-52 snoRNAs as well as for four additional snoRNAs (HBII-436, HBII-437, HBII-438A and HBII-438B), newly identified in this study. Almost all of those snoRNAs are encoded within introns of this large transcript. Northern blot analysis indicates that most if not all of these snoRNAs are indeed expressed by processing from these introns. As we have not obtained any evidence for other genes in this region, which, from the mouse data appears to be critical for the neonatal Prader-Willi syndrome phenotype, a lack of these snoRNAs may be causally involved in this disease.
Assuntos
Autoantígenos/genética , Impressão Genômica , Ligases/genética , Proteínas Nucleares/genética , RNA Nucleolar Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas , Adulto , Síndrome de Angelman/genética , Sequência de Bases , Northern Blotting , Cromossomos Humanos Par 15/genética , DNA Complementar/química , DNA Complementar/genética , Éxons , Dosagem de Genes , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes/genética , Humanos , Íntrons , Dados de Sequência Molecular , Síndrome de Prader-Willi/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Distribuição Tecidual , Transcrição Gênica/genética , Ubiquitina-Proteína Ligases , Proteínas Centrais de snRNPRESUMO
Although genetic predisposition for inflammatory bowel disease (IBD) is well established, little is known about the accountable genes. The pathogenesis of IBD is characterized by an imbalanced activation of Th1- and Th2-lymphocytes. IL-10 represents an anti-inflammatory cytokine which downregulates the production of Th1-derived cytokines. To evaluate the role of the IL-10 gene in IBD, two polymorphisms in the promoter region (G/A at position -1082 and C/A at position -592) were genotyped in 142 patients with Crohn's disease (CD), 104 patients with ulcerative colitis (UC), and 400 healthy controls. Significant differences were not apparent, neither in the allele frequencies of either polymorphism, nor in the haplotype frequencies. Screening of the coding region of the IL-10 gene by polymerase chain reaction--single strand conformation polymorphism (PCR-SSCP) analysis revealed a rare sequence variation in exon 1 leading to an amino acid exchange (G-->A; G15R) in two patients with CD and five healthy controls. Therefore, polymorphisms of the IL-10 gene are not demonstrably involved in the predisposition of IBD in our cohorts of patients.