The Human Glycine Receptor  Subunit Gene (GLRB ): Structure, Refined Chromosomal Localization, and Population Polymorphism Nicoletta Milani,* Cornel Mu ¨lhardt,* Ruthild G. Weber,² Peter Lichter,² Petra Kioschis,‡ Annemarie Poustka,‡ and Cord-Michael Becker* ,1 * Institut fu ¨r Biochemie, Universita ¨t Erlangen-Nu ¨rnberg, Fahrstrasse 17, D-91054 Erlangen, Germany; and ² Abteilung Organisation Komplexer Genome and ‡Abteilung Molekulare Genomanalyse, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Received January 13, 1998; accepted March 24, 1998 The glycine receptor of the human CNS comprises li- gand-binding 1 and structural  subunits encoded by the GLRA1 and GLRB genes, respectively. Screening of a human hippocampal cDNA library resulted in the iden- tification of the novel subunit transcript  B , differing in the 5-UTR. Analysis of the genomic organization of GLRB showed that the coding region is distributed over nine exons, highly homologous to the GLRA1 gene. By in situ hybridization, the chromosomal localization of GLRB was refined to band 4q31.3. Based on the identical phenotypes of mouse lines carrying mutant alleles of the 1 and  subunit genes, GLRB was assumed to be a candidate gene for those cases of hyperekplexia that cannot be associated with mutations of GLRA1. There- fore, flanking intronic sequences were determined, and DNA samples from more than 30 index patients were subjected to SSCP screening of the entire GLRB coding region. A polymorphism in exon 8 was found both in the normal population and in families affected by hyperek- plexia, although no coding mutation was detectable. © 1998 Academic Press INTRODUCTION The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel mediating synaptic inhibi- tion in spinal cord, brain stem, and other CNS regions. It exists in developmentally regulated, heteromeric iso- forms composed of variants of the ligand-binding polypeptide (1–4) and structural () subunits (Becker et al., 1988; Betz, 1992; Becker, 1995). These subunits share a similar membrane topology in which a large extracellular N-terminal domain is followed by four membrane-spanning segments, TM1–TM4. Structural determinants of ligand binding have been identified within the N-terminal domain of the  polypeptides, while TM2 is thought to line the anion pore of the recep- tor channel (Betz, 1992; Becker, 1995). While the  sub- unit variants possess highly homologous primary struc- tures displaying 80 –90% amino acid identities, the  subunit exhibits a diverging amino acid sequence ac- counting for approximately 47% identity compared to the 1 subunit (Grenningloh et al., 1990; Kingsmore et al., 1994; Matzenbach et al., 1994; Mu ¨ lhardt et al., 1994; Handford et al., 1996). Human and murine glycine recep- tor genes represent a family of genes that share a similar exon–intron organization, suggesting a phylogenetic gene duplication (Shiang et al., 1995). The coding regions of the human GLRA1 gene and the murine Glra1, Glra2, and Glrb genes are spread over nine exons (Matzenbach et al., 1994; Mu ¨ lhardt et al., 1994). The GLRA1 and GLRB genes have been localized to human chromosomal regions 5q32 and 4q32, respectively (Baker et al., 1994; Handford et al., 1996). By synteny homology, these loca- tions are linked to the murine loci on chromosome 11 (29 cM) and chromosome 3 (36 cM) of the Glra1 and Glrb genes, respectively. Interestingly, each of these loci is part of a cluster of amino acid receptor subunit genes also comprising glutamate and GABA A receptor subunits. In contrast, human and murine 2 genes as well as the Glra4 gene are located on the X chromosome (Grennin- gloh et al., 1990; Derry and Barnard, 1991). Hereditary hyperekplexia or startle disease (STHE; MIM 149400) is a rare neurological disorder character- ized by infantile generalized muscular stiffness and excessive startle responses (Andermann et al., 1980). Mutations in the human gene encoding the 1 subunit (GLRA1) are causal to both dominant (Shiang et al., 1993; Shiang et al., 1995; Elmslie et al., 1996; Milani et al., 1996) and recessive (Rees et al., 1994; Brune et al., 1996) forms of hyperekplexia. The missense mutations found give rise to substitutions of amino acid residues located from the TM1 segment to the extracellular loop following segment TM2. Neurological phenotypes rem- iniscent of human hyperekplexia are encountered in three strains of mutant recessive mice. Spasmodic and oscillator mice carry mutant alleles of Glra1 (Ryan et al., 1994; Saul et al., 1994; Kling et al., 1997). In the 1 To whom correspondence should be addressed. Telephone: (49-9131) 85-4190. Fax: (49-9131) 85-2485. E-mail: C.-M.Becker@biochem. uni-erlangen.de. GENOMICS 50, 341–345 (1998) ARTICLE NO. GE985324 341 0888-7543/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.