Human GABA(B) receptor 1 gene: eight novel sequence variants.

GABA (gamma-aminobutyric acid) is the principal inhibitory neurotransmitter in the brain. The human GABA(B) receptor (GABBR1) maps to the human leukocyte antigen (HLA) region of chromosome 6. Its function and location in a susceptibility region for schizophrenia, epilepsy, and dyslexia make GABBR1 a candidate gene for neurobehavioral disorders. We report the characterization of GABBR1 gene mutations in 100 chromosomes from a mixed American population. Eleven distinct mutations were found, including two previously reported missense mutations (A20V and G489S) and a previously reported silent 1977 T>C transition. Here, we report four novel silent substitutions (39C>T, 1473T>C, 1476T>C, 1545T>C) and four novel intron variants. These DNA variants may be useful in association and linkage studies of neurobehavioral disorders, and in pharmacogenetic studies of drugs targeting GABBR1.

Human gamma-aminobutyric acid B receptor gene: complementary DNA cloning, expression, chromosomal location, and genomic organization.

BACKGROUND: The 6p21.3 region of human chromosome 6 is a genetic locus for schizophrenia, juvenile myoclonic epilepsy, and dyslexia. METHODS: Due to our interest in these disorders we performed complementary DNA (cDNA) hybridization selection on genomic DNA clones spanning this region to identify potential positional-candidate genes. RESULTS: We identified a full-length cDNA with an open reading frame of 2883 bp corresponding to a predicted protein of 961 amino acids that shares greater than 95% homology with the rat gamma-aminobutyric acid B (GABAB) receptor. Northern blot hybridization identified a 4.4-kb transcript in human brain. The human gene mapped to two sites on 6p21.3 separated by 2 Mb. Sequence analysis of both sites showed that the centromeric gene is transcribed, whereas the telomeric site is likely a pseudogene. The transcribed gene is distributed over 22 exons spanning 18 kb of genomic DNA. CONCLUSIONS: The genomic location, tissue expression, and function of the human GABAB receptor gene suggest that it is an important positional-candidate for the neurobehavioral disorders with a genetic locus on 6p21.3. In addition, delineation of the genomic organization will now permit it to be integrated as part of pharmacogenetic studies in trials of anxiolytic, narcotic, antiepileptic, and fluoxetine therapies.

A transcription map of the major histocompatibility complex (MHC) class I region.

We have applied cDNA hybridization selection to nine YACs spanning 3 Mb of genomic DNA from a region centromeric to HLA-A to the histone cluster that lies telomeric to the human major histocompatibility complex (MHC). In addition to Class I genes and pseudogenes, we describe over 63 genes and 23 additional expressed sequence tags distributed throughout the region. Many of the full-length genes belong to gene families. Prominent among these are a group of genes encoding proteins showing homology to the carboxyl-terminal sequences of butyrophilin and an additional group of zinc finger genes. We also detected several previously undefined genes that are specifically expressed in cells of the immune system, indicating a more complex role of the MHC in the immune response than has been appreciated.

Cloning of a new "finger" protein gene (ZNF173) within the class I region of the human MHC.

The human major histocompatability complex contains genes of both immune and nonimmune importance. Recently, several genes encoding novel, non-HLA products have been described in this area. We have performed positional cloning of short fragment cDNA sequences from the class I region of the human MHC using a hybridization selection approach. This report describes isolation of full-length cDNA clones and partial genomic clones that encode a protein that contains two domains rich in cysteine and histidine similar to those characteristic of metal-dependent DNA binding proteins (C3HC4). The predicted protein also contains a domain thought to form a coiled-coil that may promote dimerization. A third feature is a polyglutamic acid region near the carboxyl terminus of the conceptual protein. Because of these properties, we have named this gene product acid finger protein (AFP). Although the biological role of AFP is unknown at present, one potential function is binding of nucleic acids. The gene (ZNF173) is expressed in multiple tissues and is conserved among mammals. In particular, the mouse and human coding regions are highly conserved. In addition to AFP, other related sequences have been localized to the MHC, suggesting that multiple AFP-like genes exist in this area.

Isolation of novel non-HLA gene fragments from the hemochromatosis region (6p21.3) by cDNA hybridization selection.

It has previously been shown that cDNA hybridization selection can identify and recover novel genes from large cloned genomic DNA such as cosmids or YACs. In an effort to identify candidate genes for hemochromatosis, this technique was applied to a 320-kb YAC containing the HLA-A gene. A short fragment cDNA library derived from human duodenum was selected with the YAC DNA. Ten novel gene fragments were isolated, characterized, and localized on the physical map of the YAC.

An integrated approach for identifying and mapping human genes.

We have developed a method for generating expressed-sequence maps of human chromosomes. The method involves several steps that begin with libraries of highly representative short cDNAs prepared by using random oligomers as primers. The cDNA inserts are amplified by PCR with flanking vector primers. Chromosomal region-specific cDNA packets are prepared by hybridization of the cDNA inserts to DNA derived from yeast artificial chromosomes (YACs) assigned to defined regions of human chromosomes. The cDNA packets are cloned into yeast chromosome fragmentation vectors and used for transformation of yeast bearing the YAC used for affinity purification. Sequences in the cDNAs undergo homologous recombination with the corresponding exons in the genomic DNA yielding a set of truncated YACs. Each unique truncation specifies the location of an exon in the YAC. Since all of the truncation events end with the same vector sequence, it is possible to rescue and sequence these ends to generate expressed sequence tags. The method couples rapid purification of region-specific cDNAs with precise mapping of their genes on YACs. Appropriately truncated YACs also provide easy access to gene regulatory sequences. We describe the feasibility of individual steps of the method using the factor IX (F9) gene as a model system and we present the mapping of several expressed sequences corresponding to a 330-kb YAC containing DNA from human chromosome 6p21. In addition, we obtained the sequence, including an intron-exon junction, flanking a particular truncation event.

Physical and genetic mapping of the telomeric major histocompatibility complex region in man and relevance to the primary hemochromatosis gene (HFE).

We performed pulsed-field gel electrophoresis (PFGE) on genomic DNA from a radiation hybrid (RH) cell line and constructed a high-resolution physical map of the major histocompatibility complex class I region in 6p21.3, where the gene for primary hemochromatosis (HFE) is believed to be located. Due to the intact microsegment of hemizygous human genomic DNA preserved in the RH cell line, simplified and distinct restriction fragment banding patterns were generated. Using the RH cell line, we were able to extend the physical map of the HLA class I region to about 3000 kb, order the known HLA class I genes from centromere to telomere: HLA-B, -C, -E, (-A, -H, -G), and -F, and orient the HLA-F gene along the chromosome. The proximity of HLA-F to HLA-A was confirmed by linkage and linkage disequilibrium analysis. This study shows that RH cell lines can be useful for constructing long-range physical maps in specific regions of the human genome with PFGE. Physical and genetic mapping studies of this region are consistent with a localization of the HFE gene proximal or distal to HLA-A.