RESUMEN
As part of an effort to characterize the hemochromatosis gene, we selected three non-chimeric yeast artificial chromosomes (YACs) overlapping with the YAC B30 previously described and forming an 800 kilobase contig covering the HLA-A/HLA-F region. The precise physical map of these YACs and of the corresponding genomic region were established. Nine concentrated sites of CpG cutter elements, potentially HTF islands, were mapped. In addition, several probes have been generated as tools for mapping and examining transcripts produced in the region. This allowed for the characterization and localization of two new coding sequences, provisionally named HCG (for hemochromatosis candidate gene) and numbered VIII and IX.
Asunto(s)
Cromosomas Humanos Par 6 , Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Sistemas de Lectura Abierta , Mapeo Restrictivo , Northern Blotting , Cromosomas Artificiales de Levadura , Clonación Molecular , Dermatoglifia del ADN , ADN Complementario/genética , Electroforesis en Gel de Campo Pulsado , Biblioteca de Genes , Antígenos HLA/genética , Antígenos HLA-A/genética , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
The hemochromatosis gene (HFE) maps to 6p21.3 and is less than 1 cM from the HLA class I genes; however, the precise physical location of the gene has remained elusive and controversial. The unambiguous identification of a crossover event within hemochromatosis families is very difficult; it is particularly hampered by the variability of the phenotypic expression as well as by the sex- and age-related penetrance of the disease. For these practical considerations, traditional linkage analysis could prove of limited value in further refining the extrapolated physical position of HFE. We therefore embarked upon a linkage-disequilibrium analysis of HFE and normal chromosomes from the Brittany population. In the present report, 66 hemochromatosis families yielding 151 hemochromatosis chromosomes and 182 normal chromosomes were RFLP-typed with a battery of probes, including two newly derived polymorphic markers from the 6.7 and HLA-F loci located 150 and 250 kb telomeric to HLA-A, respectively. The results suggest a strong peak of existing linkage disequilibrium focused within the i82-to-6.7 interval (approximately 250 kb). The zone of linkage disequilibrium is flanked by the i97 locus, positioned 30 kb proximal to i82, and the HLA-F gene, found 250 kb distal to HLA-A, markers of which display no significant association with HFE. These data support the possibility that HFE resides within the 400-kb expanse of DNA between i97 and HLA-F. Alternatively, the very tight association of HLA-A3 and allele 1 of the 6.7 locus, both of which are comprised by the major ancestral or founder HFE haplotype in Brittany, supports the possibility that the disease gene may reside immediately telomeric to the 6.7 locus within the linkage-disequilibrium zone. Additionally, hemochromatosis haplotypes possessing HLA-A11 and the low-frequency HLA-F polymorphism (allele 2) are supportive of a separate founder chromosome containing a second, independently arising mutant allele. Overall, the establishment of a likely "hemochromatosis critical region" centromeric boundary and the identification of a linkage-disequilibrium zone both significantly contribute to a reduction in the amount of DNA required to be searched for novel coding sequences constituting the HFE defect.
Asunto(s)
Marcadores Genéticos , Antígenos HLA-A/genética , Haplotipos , Hemocromatosis/genética , Desequilibrio de Ligamiento , Alelos , Mapeo Cromosómico , Cromosomas Humanos Par 6 , Femenino , Humanos , MasculinoAsunto(s)
Cromosomas Humanos Par 6 , Genes MHC Clase I , Antígenos HLA-A/genética , Hemocromatosis/genética , Mapeo Cromosómico , Clonación Molecular , Sondas de ADN , ADN Complementario , Femenino , Expresión Génica , Biblioteca de Genes , Humanos , Mucosa Intestinal/inmunología , Pulmón/inmunología , Músculo Esquelético/metabolismo , Placenta/inmunología , Embarazo , Seudogenes , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
Haemochromatosis is an inherited disorder of iron metabolism characterized by a general iron over loading. Without diagnosis and early treatment, it is a serous and potentially fatal disease by cardiac failure or hepatocellular carcinoma in particular. Gene prevalence was estimated at 0.06 in Brittany, so that haemochromatosis may be the most common genetic disease in this area. The biochemical defect of the disease is unknown; only one fact is well established: the iron absorption through duodenal mucosa is excessive. However, we don't know if it is a primary event. The gene is also unknown but in 1975 it was located on the short arm of chromosome 6, closely linked to the HLA class I region, less than 1 cM from HLA-A. None of the genes coding for the known iron proteins could be the haemochromatosis gene because of their chromosomal localization. In order to locate this gene with precision, we have used a reverse genetic approach now called positional cloning. Characterization of new polymorphic markers and linkage disequilibrium analysis have led us to locate the gene within a 350 kb region around HLA-A. We have then searched for all the structural genes in this region. Seven new genes have been so identified and located with precision. A structural analysis of these genes was undertaken to find an eventual abnormality in patients.
Asunto(s)
Hemocromatosis/genética , Inversión Cromosómica , Mapeo Cromosómico , Clonación Molecular/métodos , Frecuencia de los Genes , Tamización de Portadores Genéticos , Antígenos HLA/genética , Antígeno HLA-A1/genética , Hemocromatosis/epidemiología , Hemocromatosis/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Desequilibrio de Ligamiento , Biología Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Antígenos HLA-ERESUMEN
Haemochromatosis is an inherited disorder of iron metabolism characterized by a general iron over loading. Without diagnosis and early treatment, it is a serious and potentially fatal disease by cardiac failure or hepatocellular carcinoma in particular. Gene prevalence was estimated at 0.06 in Brittany, so that haemochromatosis may be the most common genetic disease in this area. The biochemical defect of the disease is unknown; only one fact is well established: the iron absorption through duodenal mucosa is excessive. However we don't know if it is a primary event. The gene is also unknown but in 1975 it was located on the short arm of chromosome 6, closely linked to the HLA class I region, less than 1 cM from HLA-A. None of the genes coding for the known iron proteins could be the haemochromatosis gene because of their chromosomal localization. In order to locate this gene with precision, we have used a reverse genetic approach now called positional cloning. Characterization of new polymorphic markers and linkage disequilibrium analysis, have led us to locate the gene within a 350 kb region around HLA-A. We have then searched for all the structural genes in this region. Seven new genes have been so identified and located with precision. A structural analysis of these genes was undertaken to find an eventual abnormality in patients.
Asunto(s)
Hemocromatosis/genética , HumanosRESUMEN
A yeast artificial chromosome (YAC B30) with a 320 kb insert of genomic DNA which includes the HLA-A gene was used to screen a cDNA library of human duodenal mucosa. Seven cDNA clones were isolated which correspond to seven new non-HLA class I structural genes. These new genes are located within a region that may well contain the gene responsible for hemochromatosis and have therefore been named HCG I-VII (Hemochromatosis Candidate Gene). HCG I, III, V and VI are probably single copy genes, situated at 180, 155, 140 and 230 kb centromeric to HLA-A, respectively. HCG II, IV and VII present several copies: one copy of HCG II, one of HCG IV and one of HCG VII are centromeric to HLA-A (at 30, 70 and 100 kb respectively). Another copy of HCG IV is 20 kb telomeric to HLA-A. Each of the genes localized on the YAC B30 is associated with an CpG/HTF island.
Asunto(s)
ADN/genética , Genes MHC Clase I , Genes , Antígenos HLA-A/genética , Southern Blotting , Línea Celular , Cromosomas Fúngicos , Duodeno , Biblioteca de Genes , Hemocromatosis/genética , Humanos , Mucosa Intestinal/inmunología , Especificidad de Órganos , Mapeo Restrictivo , Saccharomyces cerevisiae/genéticaRESUMEN
Genetic haemochromatosis (HFE) is a frequent and potentially fatal disease. Early phlebotomies may prevent complications. The recessive gene for HFE is unknown but closely linked to the HLA-A locus. No direct test for homozygosity for HFE is currently available, apart from HLA typing within the family of a patient with confirmed HFE. During a reverse genetic approach to identify the gene, we found three anonymous genomic probes (P3, P5, and I.82) derived from previously cloned YACs and physically mapped in the HLA class I region. P3 and P5 probes recognise 3 loci (P3A, P3B, P5) and I.82 one locus about 100 kb from HLA-A. Using five biallelic polymorphisms (I.82/BglII, P3B/EcoRV, P3B/PstI, P5/HindIII, P3A/PstI), we tested 198 HLA typed subjects from the families of 22 haemochromatosis patients. The information from the five polymorphisms was sufficient to identify unequivocally extended restriction haplotypes in all families. The restriction haplotypes cosegregate with the HFE allele and enable identification of genotypically identical sibs in all families studied. The linked DNA markers described in this article avoid the disadvantages of HLA serological typing and can be used in genetic counselling of HFE families.
Asunto(s)
Marcadores Genéticos , Pruebas Genéticas/métodos , Hemocromatosis/diagnóstico , ADN , Sondas de ADN , Femenino , Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Masculino , Linaje , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Two yeast artificial chromosomes of the HLA class I region were subcloned. Four of the subclones studied displayed restriction polymorphisms that corresponded to six bi-allelic series. Allelic distribution of the anonymous markers was then studied by comparing a control population with a group of patients with familial haemochromatosis. Only one marker presents an unequivocal association with the haemochromatosis gene and is 100 kb centromeric to HLA-A. This association however is not as strong as with HLA-A3. The results suggest two possible locations for the haemochromatosis gene: less than 100 kb centromeric to the HLA-A locus, or on the telomeric side.
Asunto(s)
Cromosomas Humanos Par 6 , Genes MHC Clase I , Marcadores Genéticos/genética , Antígenos HLA-A/genética , Hemocromatosis/genética , Alelos , Sondas de ADN/genética , Humanos , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
The class I region of the human major histocompatibility complex contains genes encoding the classical transplantation antigens (HLA-A, B, and C), at least three new class I genes (HLA-E, F, and G) and many class I pseudogenes (including HLA-H). By pulse field gel electrophoresis and using five rare cutter enzymes, we have constructed a precise and continuous map of 1200 kilobases (kb) around HLA-A. The blots were hybridized with HLA-A, E, and F-specific probes and with new probes derived from yeast artificial chromosomes and cosmids of the class I region. We have compared the genomic organization of the same 1200 kb in three homozygous lymphoblastoid cell lines corresponding to three different HLA haplotypes (A3, A24, and A31). The differences in size observed may have been caused by insertions and deletions and may prove valuable in understanding the evolution of the HLA chromosomal region.