Original Paper 1609801

European Journal Eur J Hum Genet 1996;4:199-204 of Human Genetics

Markus Kostrzewaa Angelika Köhlera Assignment of Encoding Kerstin Eppelta Lyndsey Hellamb GABAa Receptor Subunits a1r a6, ß2, Nicholas D. Fairweatherb 2 Elaine R. Levyb and Y to a YAC Contig of 5q33 Anthony P. Monacob Ulrich Müllera a Institut für Humangenetik, Justus-Liebig-Universität Giessen, Germany, and b Wellcome Trust Centre for Human Genetics, Headington, Oxford, UK

Keywords Abstract GABAa receptor subunit genes A cluster consisting of the four y-aminobutyric acidA (GABAa) receptor subunit genes GABRA1, GABRA6, GABRB2, and GABRG2 was assigned to 5 a yeast artificial chromosome (YAC) contig of 5q33. Two of the 26 YACs of YAC contig the contig are positive for all four subunit genes. The order of the GABR sub­ unit genes with respect to known anonymous gene loci is cen - D5S380 - D5S403 - D5S529 - GABRB2 - GABRA1/A6 - GABRG2 - D5S422 - tel. This novel YAC contig lies between known YAC contigs of 5q34/q35 and 5q31-q33.

Introduction coded by the genes GABRA1-6, GABRB1-4, GABRG1- 4, GABRD and GABRR1-2 and can be alternatively y-Aminobutyric acid (GABA) is a major inhibitory spliced [3-7], in the mammalian brain. GABA me­ Most of the GABR subunit genes have been mapped to diates its function via specific receptors, mainly of the specific human . The genes coding for the type GABAa. GABAa receptors are ligand-gated ion two subunits pi_2, GABRR1 and GABRR2, are on chro­ channels and permeable to Cl-. Biochemical analyses sug­ mosome 6ql4-q21 [8], GABRD is located in the short gest that the receptors are pentameres of various subunit arm of [9] and GABRA3 has been mapped isoforms [for reviews see ref. 1,2], Presently, 17 different to Xq28 [10, 11]. Several additional subunit genes appear GABAa receptor subunit isoforms are known. Based on to be parts of gene clusters. One cluster consisting of their degree of homology, the subunits are assigned to GABRA5, GABRB3, and GABRG3 has been assigned to families and classes (a, ß, y, 5, and p). Homology within 15qll-ql3 [12—14], Evidence of another cluster compris­ families is 60-80% at the amino acid level and 20-40% ing GABRA2, GABRB1, GABRA4, and GABRG1 comes between classes. Family a comprises six members (ai_é), from both in situ hybridization and deletion mapping families ß and y four each (ßi_4, and yi-4), ô one, and p two experiments [11, 15-18]. These experiments have located (pi_2) members. The GABR receptor subunits are en- the four genes to 4pl4-q21.1. Four additional subunit

© 1996 S. Karger AG, Basel Ulrich Muller Received: KARGER 1018-4813/96/0044-0199$ 10 00/0 Institut fur Humangenetik January 30, 1996 E-Mail [email protected] Justus-Liebig-Universität Revision received: Fax + 41 61 306 12 34 Schlangenzahl 14 March 27, 1996 http://www. karger, ch D-35392 Giessen (Germany) Accepted: April 9, 1996 NOSi CD O< CM CM o CO O co oz oc 0 Cd co Cd oc i oc Cd O tn CÛs CÛ w co co LI ÌLI IOcCOn EIOrt IOLO 3 « co < < CO I».CM Q Û Q O- 0 0- 0 «?! 0 00 00 Q 00 793A6 764D6

72QA1] 744F3 8Û6D6 781A8 727C9 817B2

73ÓG12 B12E11 825B12 775G11 924H9 827E4 786E3 843G6 926D8 795F8 860A12 940G7 91083 860B11 98»: 12 944E2 865F5

910G4 Fig. 1. YAC contig of 5q33. The newly isolated markers are shaded. GABR genes are typed in bold. The present YAC contig is located in the region of the doubly-linked ◄-cen tel contig WC-1432 of the Whitehead Institute for Biomedical Research.

genes, GABRAI, GABRA6, GABRB2, and GABRG2, YAC end fragments and inter-Alu-sequences were isolated as pre­ have been mapped to the distal long arm of chromosome viously decribed [23], PCR products were blunt-end-cloned into the EcoRV site of pBluescript II SK + and sequenced. Sequences were 5 (5q31.1-q35) and GABRAI and GABRG2 have been used to generate new primer pairs for STSs. The STSs assigned to a yeast artificial chromosome (YAC) of about were tested for chromosome 5 specificity using human/hamster 450 kb [11, 17, 19-22], These findings suggest the exis­ somatic cell hybrid GM10114 that contains chromosome 5 as the tence of yet another cluster of GABR subunit genes. only human chromosome. Here we demonstrate by YAC mapping and fluores­ Published primer sequences were used for the identification of GABAa receptor subunit genes (GABRA1 [19], GABRA6 [22], cence in situ hybridization (FISH) that subunits GA­ GABRB2 [21], GABRG2 [20]). Anonymous D5S markers were BRAI, GABRA6, GABRB2, and GABRG2 are indeed obtained from the Data Base (D5S380, GDB ID: GOO-186- clustered in the long arm of chromosome 5 (5q33). 594; D5S403, GDB ID: G00-188-048; D5S422, GDB ID: GOO-188- 376; D5S529, GDB ID: G00-195-023). FISH was done according to standard procedures. YAC DNA was either used directly after digoxigenin-11-dUTP or biotin labeling Materials and Methods or after amplification using various Alu primers [24, 25]. The CEPH Mega YAC library pools were screened by PCR fol­ lowing the instructions of CEPH (Centre d’Etude du Polymorphisme Humain). Pulsed-field gel electrophoresis (PFGE) was carried out Results using a CHEF system (Bio Rad). Electrophoresis was performed at 14°C for 25 h (0.5 x TBE, 6 V/cm, 120°, ramp 50-100 s). Sizes of YACs were determined by comparison with the known size of chro­ YACs from the distal long arm of chromosome 5 were mosomes of Saccharomyces cerevisiae strain YPH49. screened to localize GABAa receptor subunit genes GA­ BRAI, GABRA6, GABRB2 and GABRG2 that had pre­ viously been assigned to 5q31.1-q35. The genes were

200 Eur J Hum Genet 1996;4:199-204 Kostrzewa/Köhler/Eppelt/Hellam/ Fairweather/Levy/Monaco/Müller Table 1. Newly developed markers in 5q33

Marker D number Accession Primer sequence Product Annealing name number size, bp temp.,°C

812E11R D5S2513 X94235 CAACACATCACAAATAGAAT 135 52 T AATT GATTT GTC AG AGTT G 825B12R D5S2514 X94236 GAAAACATAACATCATCGCC 154 60 AATT GG AG AAAT AAT G AAAC AT G 910B3R D5S2515 X94237 T GCCC ATCCTT AC AGAAT C A 151 62 GCTT CTTCCCTTTCTT ATTT CA 825B12AR D5S2516 X94238 AGGG AAT AGAAT G AC ACTCT GT 149 59 T AT AC AT GT GATT GGCCT G A 827E4ARL D5S2517 X94239 TTTT CCGATT CT GGTT ACT G 126 60 AAAG AAAAT ATTC AAT GCCT GT 860A12AL D5S2518 X94240 CT GAT G AC AAT AT ACCT GGGT G 128 62 TTCGAGGGAATAATTGAGGA 910B3AR D5S2519 X94241 CT GGGC A AAT G AC AAGT AGG 105 62 GCT AT C AAAAAC AGGT GGC A

excluded from known YAC contigsof 5q34-q35 [23, 26] Table 2. YAC sizes and FISH results and of 5q31-q33 [27] (not shown). YACs located between YAC Size, kb Chimerism these contigs [28] were positive for GABAa receptor sub­ unit genes. Primers for GABRB2 amplified DNA of 720Al 1 400; 1,300 + YAC 910B3 that contains D5S403. Seven addi­ 727C9 700; 945 + tional D5S403 positive YACs (720A11, 727C9, 736G12, 736G12 360 + 775G11, 786F3, 795F8, 944E2) were negative for all 744F3 850 + GABA receptor subunit genes. Six additional YACs 764D6 615; >1,500 + 775G11 >1,500 + (744F3, 812E11, 843G6, 860A12, 860B11, 865F5) all of 781A8 500 which include D5S422 types positive for GABRG2. Four 786F3 400 D5S422-positive YACs (764D6, 781A8, 827E4, 910G4) 793A6 850 did not contain any GABR subunit gene. Since GABRA1 795F8 945; 1,125; 1,500 and GABRA6 could not be localized on any known YAC 806D6 960 812E11 1,050 from the region, we screened the CEPH library. A total of 817B2 870 8 YACs was isolated. One, 982C12, was positive for both 825B12 1,500 GABRA1 and GABRA6, 5 YACs (793A6, 817B12, 827E4 1,300 924H9, 926D8, 940G7) included GABRG2 in addition 843G6 1,050 860A12 1,050 to GABRA1 and GABRA6 and all four GABRa subunit 860B11 1,050 genes (GABRA 1, GABRA6, GABRB2, and GABRG2) 865F5 1,300 n.d. were detected in two YACs (806D6 and 825B12). In order 910B3 1,500 to refine the resulting YAC contig, several known markers 910G4 >1,500 + (D5S380, D5S403, D5S422, D5S529) were assigned map 924H9 820 positions. In addition, three YAC insert endpieces 926D8 1,300; 1,600 + 940G7 800 n.d. (812E11R, 825B12R, 910B3R) and four inter-Alu se­ 944E2 630 quences (825B12AR, 827E4ARL, 860A12AL, 910B3AR) 982C12 730 + were located on the contig (fig. 1). Sequences of newly developed STSs are given in table 1. Different sizes of the same YACs are The sizes of the 26 YACs of the contig were deter­ probably due to in vivo truncation of an ancestral YAC. mined by PFGE and are listed in table 2. All YACs were

Chromosome 5 GABR Gene Cluster Eur J Hum Genet 1996;4:199-204 201 A B è&

N* ï

%

1* -7

V Fig. 2. A GTG-banded metaphase. * À V B Same metaphase exhibiting the FISH sig­ nal of YAC 806D6 in 5q33. Relevant regions are indicated by arrow heads.

tested for chimerism by FISH. Fourteen YACs appeared be determined, however, whether these genes are indeed to be nonchimeric, and 10 YACs were chimeric (table 2). clustered or whether they are dispersed over a relatively No conclusive results were obtained for YACs 944E2 and large region of . 865F5. The YACs containing the GABR subunit genes The origin of the GABR gene clusters is presently were assigned to 5q33 (fig. 2). unknown. One cluster may have arisen by duplication and subsequent mutation events on one chromosome and then was transposed to other regions of the genome. Discussion Accordingly, the original cluster consisted of one a-, one ß-and one y-subunit as on . This unit was We have demonstrated that genes coding for GABAa dispersed by transposition and underwent further muta­ receptor subunit isoforms cq, a6, ß2, and y2 are clustered tions including a duplication of the a-subunit gene. within 5q33. All four subunits are contained within a It is not known whether clustering of GABR genes has YAC (806D6) of less than 1 Mb. The chromosomal order any functional implications. One could speculate that of the subunit genes is cen - GABRB2 - GABRA1/ clustering facilitates coordination of . GABRA6 - GABRG2 - tel. The two GABRA subunit Support for this notion comes from head-to-head arrange­ genes cannot be separated on this YAC contig indicating ment of GABRB3 and GABRA5 on chromosome 15 close proximity of both genes. Clearly, a high-resolution within less than lOOkb [14] that would allow for simple map is required for their eventual separation. This order coordination of expression of these two genes. In contrast, of GABR subunit genes is in contrast to that of Warring­ GABRA6 of the cluster on chromosome 5 is expressed ton and Bengtsson [29], Their method combining radia­ almost exclusively in the [31, 32] while the tion hybrid mapping, interphase FISH and PFGE indi­ remaining subunit genes are more widely expressed [33], cated the order cen - GABRG2 - GABRA 1 - tel. This finding argues against simultaneous expression of Another cluster of GABR genes, GABRB3, GABRA5, genes within a GABR cluster. and GABRG3, was demonstrated on a YAC contig of A potential function of GABA and its receptors in the 900 kb of proximal 15q [30], The order of the GABR gene origin of human disease has not been proven. A gene locus cluster in 5ql 1-ql 3 is comparable to that of the cluster possibly involved in manic depression has been assigned described here. In both cases, genes coding for subunits ß to distal 5q [34] and one may speculate that GABR genes and y flank the a-subunit gene(s) and the GABRB genes are involved in this disorder. However, there is presently are most centromeric. A third cluster of GABR genes may no direct supporting evidence of this hypothesis. Another be located on chromosome 4 and four subunit genes, study speculates that GABAa receptors might be involved GABRA2, GABRA4, GABRB 1, and GABRG1, have in the pathogenesis of spinal myoclonus [35]. Again, there been assigned to 4p 14-q21.1 [6,11, 15, 17,18]. It needs to is no convincing data in favour of this assumption. Final-

202 Eur J Hum Genet 1996;4:199-204 Kostrzewa/Köhler/Eppelt/Hellam/ Fairweather/Levy/Monaco/Müller ly, GABRB3 has been implicated in normal facial devel­ Acknowledgements opment. The cleft palate of mice carrying a deletion in the We gratefully acknowledge Dr. D. LePaslier (Centre d’Etude du cpl (cleft palate) locus is rescued by the introduction of Polymorphisme Humain, Paris) for the isolation of YACs. We thank transgenic Gabrb3 [36], Oliver Buckolt for his skillful generation of the artwork and Karen Davis for help in FISH analysis. This work was supported by the European Community [EC GENE-CT93-0050 (DG 12 SSMA)]. APM is a Wellcome Trust Principal Research Fellow.

References

1 Olsen RW, Tobin AJ: Molecular biology of 11 Buckle VJ, Fujita N, Ryder-Cook AS, Derry 18 McLean PJ, Färb DH, Russek SJ: Mapping of GABAa receptors. FASEB J 1990;4:1469— JMJ, Barnard PJ, Lebo RV, Schofield PR, See- the

Chromosome 5 GABR Gene Cluster Eur J Hum Genet 1996;4:199-204 203 27 Li X, Wise CA, Le Pasher D, Hawkins AL, 30 Phillips RL, Rogan PK, Culiat CT, Stubbs L, 33 Wisden W, Laurie DJ, Monyer H, Seeburg PH: Griffin CA, Pittier SJ, Lovett M, Jabs EW: A Rinchik EM, Gottlieb W, Nicholls RD: A YAC The distribution of 13 GABAa receptor sub­ YAC Contig of approximately 3 Mb from hu­ contig spanning 4 genes in distal human chro­ unit mRNAs in the rat brain. I. Telencepha­ man chromosome 5q31-»q33. Genomics mosome 15qll-ql3, mapping of the human lon, diencephalon, mesencephalon. J Neuro- 1992;19:470-477. GABRG3 gene, and effect of homozygous dele­ sci 1992;12:1040-1062. 28 Chumakov IM, Rigault P, Le Gall I, Bellanné- tion of three GABAa receptor genes in mouse. 34 Coon H, Jensen S, Hoff M, Holik J, Plaetke R, Chantelot C, Billault A, Guillou S, Soularue, Am J Hum Genet 1993;53(suppl):A1345. Reimherr F, Wender P, Leppert M, Byerley W: Guasconi A, Poullier E, Gros I et al: A YAC 31 Kato K: Novel GABAa receptor a subunit is A genome-wide search for genes predisposing contig map of the . Nature expressed only in cerebellar granule cells. J Mol to manic-depression, assuming autosomal 1995;377(Suppl): 175-297. Biol 1990;214:619-624. dominant inheritance. Am J Hum Genet 1993; 29 Warrington JA, Bengtsson U: High-resolution 32 Lüddens H, Pritchett DB, Köhler M, Killisch I, 52:1234-1249. physical mapping of human 5q31-q33 using Keinänen K, Monyer H, Sprengel R, Seeburg 35 Simon ES: Involvement of glycine and GABAa three methods: Radiation hybrid mapping, in­ PH: Cerebellar GABAa receptor selective for a receptors in the pathogenesis of spinal myoclo­ terphase fluorescence in situ hybridization, and behavioural antagonist. Nature 1990; nus. Neurology 1995;45:1883-1892. pulsed-field gel electrophoresis. Genomics 346:648-651. 36 Culiat CT, Stubbs LJ, Woychik RP, Russel LB, 1994;24:395-398. Johnson DK, Rinchik EM: Deficiency of the ß3-subunit of the type A y-aminobutyric acid receptor causes cleft palate in mice. Nat Genet 1995;11:344-346.

204 Eur J Hum Genet 1996;4:199-204 Kostrzewa/Köhler/Eppelt/Hcllam/ Fairweather/Levy/Monaco/Müller