RESUMO
Many bipolar affective disorder (BD) susceptibility loci have been identified but the molecular mechanisms responsible for the disease remain to be elucidated. In the locus 4p16, several candidate genes were identified but none of them was definitively shown to be associated with BD. In this region, the PPP2R2C gene encodes the Bgamma-regulatory subunit of the protein phosphatase 2A (PP2A-Bgamma). First, we identified, in two different populations, single nucleotide polymorphisms and risk haplotypes for this gene that are associated to BD. Then, we used the Bgamma subunit as bait to screen a human brain cDNA library with the yeast two-hybrid technique. This led us to two new splice variants of KCNQ2 channels and to the KCNQ2 channel itself. This unusual K+ channel has particularly interesting functional properties and belongs to a channel family that is already known to be implicated in several other monogenic diseases. In one of the BD populations, we also found a genetic association between the KCNQ2 gene and BD. We show that KCNQ2 splice variants differ from native channels by their shortened C-terminal sequences and are unique as they are active and exert a dominant-negative effect on KCNQ2 wild-type (wt) channel activity. We also show that the PP2A-Bgamma subunit significantly increases the current generated by KCNQ2wt, a channel normally inhibited by phosphorylation. The kinase glycogen synthase kinase 3 beta (GSK3beta) is considered as an interesting target of lithium, the classical drug used in BD. GSK3beta phosphorylates the KCNQ2 channel and this phosphorylation is decreased by Li+.
Assuntos
Transtorno Bipolar/genética , Transtorno Bipolar/metabolismo , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ2/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Tálamo/metabolismo , Animais , Antimaníacos/farmacologia , Argentina , Células COS , Estudos de Casos e Controles , Chlorocebus aethiops , Inibidores Enzimáticos/farmacologia , Frequência do Gene , Predisposição Genética para Doença , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Haplótipos , Humanos , Desequilíbrio de Ligação , Cloreto de Lítio/farmacologia , Potenciais da Membrana , Razão de Chances , Fosforilação , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Fosfatase 2 , Medição de Risco , Fatores de Risco , Tálamo/efeitos dos fármacos , Transfecção , Reino UnidoRESUMO
To clone the human major histocompatibility complex (MHC), 53 YACs, with an average size of 490 kb, were isolated and characterized from the CEPH YAC library. These YACs were organized in a single large contig covering more than 4000 kb. Furthermore, a complete physical map of the previously uncloned HLA class I region was established from partial and/or total digestions of 15 YACs spanning 2000 kb. This resulted in the establishment of the first YAC contig that spans the entire MHC region and constitutes an essential step in the isolation of all of the genes present in the region.
Assuntos
Genes MHC Classe I , Hominidae/genética , Complexo Principal de Histocompatibilidade , Animais , Sequência de Bases , Linhagem Celular , Cromossomos Artificiais de Levedura , Clonagem Molecular , Consanguinidade , Primers do DNA , Biblioteca Gênica , Antígenos HLA-D/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Mapeamento por RestriçãoRESUMO
Because of their large size, YACs provide is a powerful tool for physical mapping studies of complex genomes. As it will be advantageous to have genomic libraries of clones with large inserts for analyzing megabase sized regions of the human genome, we have investigated a number of parameters in order to increase the insert size of the YACs. We constructed a genomic library currently containing more than 85,000 YAC clones. Mean sizes of YACs produced at several stages of construction of the library range from 430 kb to 1,200 kb, representing 13 haploid equivalents of the human genome. This library was organized in order to allow rapid screen of YACs for large scale physical mapping of the human genome.