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Schizophrenia Research 58 (2002) 87 – 91 www.elsevier.com/locate/schres Mutational analysis of the connexin 36 gene (CX36) and exclusion of the coding sequence as a candidate region for catatonic schizophrenia in a large pedigree Jobst Meyer *, Marion Mai, Gabriela Ortega, Rainald Mössner, Klaus-Peter Lesch Department of Psychiatry and Psychotherapy, University of Wuerzburg, Fuechsleinstr. 15, D-97080 Würzburg, Germany Received 2 November 2001; accepted 13 January 2002 Abstract The murine connexin 36 gene (Cx36) encodes a gap-junction channel protein which is preferentially expressed in brain and retina. The human orthologue CX36 is located on chromosome 15q14, a region recently shown to contain a susceptibility gene for hereditary catatonic schizophrenia. Therefore, CX36 was considered as a positional candidate for mutational analysis. Three polymorphic sites within CX36 were found by sequencing the two exons, the intron – exon boundaries and the putative promoter region of the gene derived from patients and control subjects. No variant exclusively cosegregates with the disease in a large pedigree that mainly supports the chromosome 15q14 locus, providing evidence that CX36 is not causative for the pathogenesis of catatonic schizophrenia in this family. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Connexin; Catatonic schizophrenia; Mutation analysis 1. Introduction The murine connexin 36 gene (Cx36) is predominantly expressed in neurons of the olfactory bulb, hippocampus, inferior olive and cerebellum (Rash et al., 2000; Teubner et al., 2000). Furthermore, the protein was found to connect pancreatic islet cells with beta cells (Serre-Beinier et al., 2000). Feigenspan et al. (2001) demonstrated that Cx36 is also expressed by AII amacrine cells in homologous and heterologous gap-junctions between AII amacrine and cone * Corresponding author. E-mail address: [email protected] (J. Meyer). bipolar cells in the mouse and rat retina, respectively. The same group concluded that Cx36 is a channelforming gap-junction protein involved in transmission of electrical and metabolic signals between neurons in the central nervous system (Teubner et al., 2000; Güldenagel et al., 2001). Synapses of interneurons containing Cx36 seem to be critical for the generation of widespread, synchronous inhibitory activity (Deans et al., 2001). The human orthologue gene CX36 has been mapped to chromosome 15q14 by Belluardo et al. (1999). It is localized in close vicinity to marker D15S971, which shows a linkage peak (maximum two-point lod score of 11.1 at a recombination fraction of zero; Casaubon et al., 1996) with the recessively inherited Andermann syndrome (periph- 0920-9964/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 0 - 9 9 6 4 ( 0 2 ) 0 0 2 0 6 - 2 88 J. Meyer et al. / Schizophrenia Research 58 (2002) 87–91 eral neuropathy with or without agenesis of the corpus callosum, ACCPN; MIM 218000). Mutations within another member of the connexin family, CX32, lead to X-linked Charcot – Marie –Tooth syndrome, a peripheral demyelinating disorder (Omori et al., 1996). Interestingly, dysregulation of several myelination-related genes has been found in postmortem brains derived from schizophrenic patients, which were assayed by DNA microarray analysis (Hakak et al., 2001). Our group recently found confirmed linkage based on a dominant model of inheritance (maximum GENEHUNTER-PLUS lod score 3.57, p = 0.000026, at coordinate 35.3 cM) of polymorphic markers between D15S144 and D15S1028 with hereditary catatonic schizophrenia (SCZD10, periodic catatonia, MIM 605419) by investigating 12 extended pedigrees. A large family with seven affected members strongly supports the chromosome 15q14– 15 locus (maximum multipoint lod score 2.89, h = 0.029, at D15S1042 (VITESSE); Stöber et al., 2000; Meyer et al., 2002). Meanwhile, further genotyping of this family did lead to the definition of the lower limit at marker D15S132 (Schraut et al., unpublished results), reducing the region of interest to 20.3 cM (nucleotide positions f 28,600,000 –45,420,000 of the draft of chromosome 15; UCSC Genome Browser on April 1, 2001, Freeze: http://www.genome.cse.ucsc.edu/cgi-bin/ hgTracks?position = chr15). This locus has also been described by Freedman et al. (1997, 2001), who reported a maximum lod score (Z = 5.3, h = 0.0, p < 0.001) for polymorphic marker D15S1360 linked to a decrease in the normal inhibition of the response to the second of two identical auditory stimuli, detected by measuring an evoked potential occurring 50 ms (P50) after the stimulus in schizophrenics. Freedman et al. (2001) favour the neuronal nicotinic acetylcholine receptor a7 subunit gene (CHRNA7) as a candidate gene, however, we were able to exclude CHRNA7 from the region of interest in our family by fine-mapping (Meyer et al., 2002). A total of 131 transcripts have been annotated to this part of chromosome 15 according to the draft sequence published by the International Human Genome Sequencing Consortium (2001). Of these genes, 29 are expressed predominantly or exclusively in the brain. We have selected 14 strong candidate genes for a first wave of investigation. Work to identify SNPs and to sequence these genes from patients and controls in comparison is in progress. In order to find the mutation responsible for catatonic schizophrenia in the large family, we have conducted a mutational analysis of the coding sequence, exon – intron boundaries and 5V-regulatory region of CX36, one of the candidate genes mentioned above, which has been selected due to its function and chromosomal localization. 2. Methods 2.1. Clinical The two members of the large family (three generations with 10 healthy and 7 affected members) investigated here were ascertained as previously described in detail by Stöber et al. (2000). All affected family members fulfil the criteria for periodic catatonia, a subtype of catatonic schizophrenia, as described by Leonhard (1999). Periodic catatonia is a genetically heterogeneous disorder characterized by psychosis and psychomotor disturbances. Patients with periodic catatonia express a variable phenotype combining akinetic negativism, hyperkinesia with stereotypies and parakinetic movements as well as increased anxiety, impulsivity and aggressiveness. Acute psychotic episodes may be accompanied by hallucinations and delusions, while successive episodes lead to an increasingly severe catatonic residual state. Based on clinical evidence for anticipation and a cumulative morbidity risk of f 27% in first-degree relatives of patients, a major gene effect was predicted for some forms of periodic catatonia (Lesch et al., 1994; Stöber et al., 1995). Consistent diagnostic classification within the family was achieved by extensive clinical evaluation, and additional information was collected from different sources, including case history, medical records and/or family informants. The study was approved by the Ethics Committee of the University of Wuerzburg and all individuals participated after giving written consent. 2.2. Mutation analysis DNA isolated from blood samples of two patients and three controls was amplified in a T-GRADIENT J. Meyer et al. / Schizophrenia Research 58 (2002) 87–91 Thermocycler (Biometra, Göttingen, Germany). Annotation of the gene and primer selection was based on the sequence provided by the International Human Genome Sequencing Consortium (2001). For these PCRs, we have used eight primer pairs; a complete list is made available on request. PCR was performed in a final volume of 25 Al containing 40 – 60 ng of genomic DNA, 10 pmol of each primer, 200 AM of each NTP, 0.75 or 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris – HCL (pH 8.3 at 25 jC), 0.025 mg ml 1 BSA, 0.025% Tween 20 and 0.5 U of Taq DNA Polymerase (Eurogentec, Seraing, Belgium). Resulting PCR products were sequenced using an ABI 310 automated sequencer (Applied Biosystems, Foster City, USA). 3. Results The putative promotor and both exons of CX36 derived from two affected members of the large family (0732 and 0737; Meyer et al., 2002) that strongly supports the chromosome 15q14– 15 region (Stöber et al., 2000) and three unrelated, healthy controls were investigated. A single putative promoter region of CX36 was predicted by PROSCAN software version 1.7 (http://www.bimas.dcrt.nih.gov/molbio/ proscan/) beginning 60 bp upstream of the translation start. We have investigated about 350 bp of the putative 5V-regulatory region including the entire predicted promoter. Two promoter variants were detected by sequencing of PCR products amplified from the 5Vregulatory region derived from ten chromosomes (Table 1). This A/T promoter-polymorphism is located at nucleotide position-127 relative to the ATG start codon. The coding region and intron –exon junctions of CX36 were also entirely sequenced. The sequencingstrategy included 33 bp of the splice donor and 62 bp 89 of the splice-acceptor sites of the single intron and was extended to the first putative poly(A) signal (ATAAAA) of the 3V-untranslated region (3V-UTR). Two polymorphic sites were detected within the coding region. Both nucleotide-transversions do not alter the amino acid composition. The variants are summarized in Table 1. None of these variants was found to cosegregate exclusively with the disease in the large pedigree described recently by our group (Meyer et al., 2002), providing strong evidence that CX36 does not cause catatonic schizophrenia in this family. 4. Discussion Myelination-related genes have recently come into focus as candidates for the etiopathogenesis of schizophrenia (Hakak et al., 2001). Additionally, recent work suggests involvement of genes encoding channel-proteins in both myelinopathies and psychoses of the schizophrenic spectrum combined with motor impairment. Bowen et al. (2001) published a frameshift mutation in the calcium-activated potassium channel gene KCNN3 in a schizophrenic patient, and our group has recently reported a missense mutation within KIAA0027 (WKL1, MLC1) suggestively linked with catatonic schizophrenia in a large pedigree (Meyer et al., 2001). Recessive mutations within KIAA0027 cause a severe neurological disorder, megalencephalic leukoencephalopathy with subcortical cysts (MLC; MIM 604004; Leegwater et al., 2001). KIAA0027 is a brain-specific putative nonselective cation channel that shares most identical amino acid positions with the voltage-gated potassium channel KCNA1, which leads to myokymia combined with periodic ataxia if mutated (MIM 160120; Browne, 1994, 1995). Another related protein, the inwardly rectifying potassium channel Kcnj10, causes hypo- Table 1 Variants of the CX36 gene Variant Position Frequency Accession number T: 7/10; A: 3/10 NT_010253.8 C: 7/10; T: 3/10 XM_007605 G: 9/10; A: 1/10 XM_007605 _ Numbering of nucleotide (nt) positions are provided according to GenBank accession number XM 007605 for the cDNA and NT_010253.8 for the genomic sequence. T !A TCC ! TCT GAG ! GAA 5V-regulatory region; nt 135853 codon 196 (serine); nt 588 codon 296 (glutamic acid); nt 888 90 J. Meyer et al. / Schizophrenia Research 58 (2002) 87–91 myelination, vacuolation, axonal swelling, motor impairment, and tremor in Kcnj10 / mice (Neusch et al., 2001). Agartz et al. (2001) reported white matter abnormalities in schizophrenic patients detected by a diffusion tensor imaging study; their results are in good agreement with the hypothesis that some forms of schizophrenia putatively resemble channelopathies. Since also a member of the connexin gene family, CX32, was found to be involved in a demyelination syndrome (Omori et al., 1996), we have considered another member of this family, CX36, as a positional candidate for mutational analysis. CX36 encodes a neuronal gap-junction related channel protein involved in transmission of signals across cell – cell boundaries (Teubner et al., 2000). We did not find mutations in CX36 exclusively inherited together with the disease in the large family supporting the 15q14 – 15 locus by investigating the coding region, predicted promoter sequence, and exon –intron boundaries. We cannot rule out, that mutations in a distant regulatory region not covered by our sequencing strategy may cause the disease. However, our work, together with the findings mentioned above, suggests that the investigation of other channel-encoding genes localized in this chromosomal region should be considered. Acknowledgements The authors would like to express their gratitude to the study participants and their family, and G. Stöber for sharing blood samples of family members. This research was supported in part by grants from the Ministry of Research (BMBF, 01GA9802/5). KPL is supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 581). References Agartz, I., Andersson, J.L., Skare, S., 2001. Abnormal brain white matter in schizophrenia: a diffusion tensor imaging study. NeuroReport 12, 2251 – 2254. Belluardo, N., Trovato-Salinaro, A., Mudo, G., Hurd, Y.L., Condorelli, D.F., 1999. Structure, chromosomal localization, and brain expression of human Cx36 gene. J. Neurosci. Res. 57, 740 – 752. Bowen, T., Williams, N., Norton, N., Spurlock, G., Wittekindt, O.H., Morris-Rosendahl, D.J., Williams, H., Brzustowicz, L., Hoogendoorn, B., Zammit, S., Jones, G., Sanders, R.D., Jones, L.A., McCarthy, G., Jones, S., Bassett, A., Cardno, A.G., Owen, M.J., O’Donovan, M.C., 2001. 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