Neuron, Vol. 3. 647-654, November, 1989, Copyright 0 1989 by Cell Press Chromosomal localization of GABA, Receptor Subunit Genes: Relationship to Human Genetic Disease Veronica J. Buckle: Norihisa Fujita,+* Allan S. Ryder-Cook,+ Jonathan M. J. Derry,+ Pene J. Barnard,+ Roger V. lebo,§ Peter R. Schofield,II** Peter H. Seeburg,II Alan N. Bateson,+ Mark C. Darlison,+ and Eric A. Barnard+ * Nuffield Department of Clinical Medicine University of Oxford Oxford OX3 9DU England +MRC Molecular Neurobiology Unit MRC Centre Cambridge CB2 2QH England 5 Howard Hughes Medical Institute University of California San Francisco, California 94143 II Laboratory of Molecular Neuroendocrinology ZMBH University of Heidelberg D6900 Heidelberg Federal Republic of Germany Summary Hybridization of CABAA receptor probes to human chromosomes in situ and to DNA from sorted human chromosomes has localized the genes encoding a b subunit and three isoforms of the a subunit. The a2 and b genes are both located on chromosome 4 in bands p12-p13 and may be adjacent. The al gene is on chromosome 5 (bands q34-q35) and the a3 gene is on the X chromosome. The a3 locus was mapped also on the mouse X chromosome using genetic break-point analysis in an interspecies pedigree. The combined results locate the human a3 gene within band Xq28, in a location that makes it a candidate gene for the X-linked form of manic depression. Introduction The GABA* receptor is the major inhibitory neurotrans- mitter receptor of the vertebrate brain. It is known to contain a and p subunits, and three isoforms of the a subunit and one of the b subunit have been identified by cDNA cloning from the bovine brain (Schofield et al., 1987; Levitan et al., 1988). The cDNA sequences show that the a subunits are homologous but derive from different genes. Furthermore, the corresponding mRNAs have different distributions between brain regions and between cell types in those regions (Wisden et al., 1988, 1989). It can be expected that the loss of any of these *Present address: Ciba-Geigy (Japan) Ltd., Takarazuka 665, Japan. ** Present address: Pacific Biotechnology Ltd., 72-76 McLachlan Avenue, Rushcutters Bay 2011, Australia. subunits as a functional entity due to mutation could be deleterious in some brain locations. Hence, these genes could be candidates for neurologic and psychiatric in- herited disorders in certain cases; possibilities where this should be considered include those conditions benefit- ting from treatment with valproate or benzodiazepines (which potentiate the action of GABA). It is therefore of both clinical and fundamental interest to know on which chromosomes these GABA* receptor genes are located and whether some are associated on a single chromosome. From a clinical point of view it is necessary to make this determination in the human ge- nome, but it is also of value to do so in the mouse, in which genetic mapping and breeding of mutants with genetic defects of the central nervous system are more advanced than in other mammals and the homology of certain chromosomal regions of linked genes with those found in man (linkage analysis) can give further defini- tion to gene locations on human chromosomes. We re- port here on both these species and find a suggestive as- sociation of a GABAA receptor gene with an inherited disease, meriting detailed investigation. Results Either the cloned bovine GABAA receptor subunit cDNAs (Schofield et al., 1987; Levitan et al., 1988) encoding three forms of the a subunit, al, a2, and a3, and the b subunit, or corresponding human brain cDNAs (Schofield et al., 1989) were used to generate probes (see Experi- mental Procedures). The four human brain subunits are almost identical in amino acid sequence to the corre- sponding bovine subunits (approximately 98% identity; Schofield et al., 1989; P R. Schofield and l? H. Seeburg, unpublished data), justifying the cross-hybridization of bovine probes to human chromosomes used in some cases. Similarly, the a3 subunits of bovine and rat are ap- proximately 99% identical in amino acid sequence (I? R. Schofield and P H. Seeburg, unpublished data). so the bovine a3 probe was likewise used directly to hybridize to mouse DNA. Chromosome Assignment Using DNAs from Sorted Human Chromosomes We have used a set of nitrocellulose filters (Lebo et al., 1984) containing dual laser-sorted human chromo- somes (DNA spot-blots) to determine the locations of four GABAA receptor subunit genes. These were sequen- tially hybridized with the corresponding bovine cDNA probes (see Experimental Procedures). An example of these results, for the B subunit gene, is shown in Figure 1. A strong hybridization signal is seen with DNA from chromosome 4 only; all other chromosomes are neg- ative. When hybridized with the al probe, strong positive signals were detected with chromosome 4 and with chromosome 5 (Table 1). The a2 probe hybridized most