Download Mutational analysis of the connexin 36 gene (CX36)

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. Mutation screening of the
KCNN3 gene reveals a rare frameshift mutation. Mol. Psychiatry
6, 259 – 260.
Browne, D., 1994. Episodic ataxia/myokymia is associated with
point mutations in the human potassium gene KCNA1. Nat.
Genet. 8, 136 – 140.
Browne, D.L., 1995. Identification of two new KCNA1 mutations
in episodic ataxia/myokymia families. Hum. Mol. Genet. 4,
1671 – 1672.
Casaubon, L.K., Melanson, M., Lopes-Cendes, I., Marineau, C.,
Andermann, E., Andermann, F., Weissenbach, J., Prevost, C.,
Bouchard, J.P., Mathieu, J., Rouleau, G.A., 1996. The gene
responsible for a severe form of peripheral neuropathy and agenesis of the corpus callosum maps to chromosome 15q. Am. J.
Hum. Genet. 58, 28 – 34.
Deans, M.R., Gibson, J.R., Sellitto, C., Connors, B.W., Paul, D.L.,
2001. Synchronous activity of inhibitory networks in neocortex
requires electrical synapses containing connexin36. Neuron 31,
477 – 485.
Feigenspan, A., Teubner, B., Willecke, K., Weiler, R., 2001. Expression of neuronal connexin36 in AII amacrine cells of the
mammalian retina. J. Neurosci. 21, 230 – 239.
Freedman, R., Coon, H., Myles-Worsley, M., Orr-Urtreger, A.,
Olincy, A., Davis, A., Polymeropoulos, M., Holik, J., Hopkins,
J., Hoff, M., Rosenthal, J., Waldo, M.C., Reimherr, F., Wender,
P., Yaw, J., Young, D.A., Breese, C.R., Adams, C., Patterson,
L.E., Adler, L.E., Kruglyak, L., Leonard, S., Byerley, W., 1997.
Linkage of a neurophysiological deficit in schizophrenia to a
chromosome 15 locus. Proc. Natl. Acad. Sci. U. S. A. 94, 587 –
592.
Freedman, R., Leonard, S., Gault, J.M., Hopkins, J., Cloninger, C.R.,
Kaufman, C.A., Tsuang, M.T., Farone, S.V., Malaspina, D.,
Svarkic, D.M., Sanders, A., Gejman, P., 2001. Linkage disequilibrium for schizophrenia at the chromosome 15q13 – 14 locus of
the a7-nicotinic acetylcholine receptor subunit gene (CHRNA7).
Am. J. Med. Genet. 105, 20 – 22.
Güldenagel, M., Ammermüller, J., Feigenspan, A., Teubner, B.,
Degen, J., Söhl, G., Willecke, K., Weiler, R., 2001. Visual transmission deficits in mice with targeted disruption of the gap
junction gene connexin 36. J. Neurosci. 21, 6036 – 6044.
Hakak, Y., Walker, J.R., Li, C., Wong, W.H., Davis, K.L., Buxbaum, J.D., Haroutunian, V., Fienberg, A.A., 2001. Genomewide expression analysis reveals dysregulation of myelinationrelated genes in chronic schizophrenia. Proc. Natl. Acad. Sci.
U. S. A. 98, 4746 – 4751.
International Human Genome Sequencing Consortium, 2001. Initial
sequencing of the human genome. Nature 409, 860 – 921.
Leegwater, P.A., Yuan, B.Q., van der Stehen, J., Mulders, J., Konst,
A.A., Boor, P.K., Mejaski-Bosnjak, V., van der Maarel, S.M.,
Frants, R.R., Oudejans, C.B., Schutgens, R.B., Pronk, J.C., van
der Knaap, M.S., 2001. Mutations of MLC1 (KIAA0027), encoding a putative membrane protein, cause megalencephalic
leukoencephalopathy with subcortical cysts. Am. J. Hum.
Genet. 68, 831 – 838.
J. Meyer et al. / Schizophrenia Research 58 (2002) 87–91
Leonhard, K., 1999. Classification of Endogenous Psychoses and
their Differentiated Etiology, 2nd edn. Springer, New York.
Lesch, K.P., Stöber, G., Balling, U., Franzek, E., Li, S.H., Ross,
C.A., Newman, M., Beckmann, H., Riederer, P., 1994. Triplet
repeats in clinical subtypes of schizophrenia: variation at the
DRPLA (B 37 CAG repeat) locus is not associated with periodic
catatonia. J. Neural Transm.: Gen. Sect. 98, 153 – 157.
Meyer, J., Huberth, A., Ortega, G., Syagailo, Y.V., Jatzke, S., Mössner, R., Strom, T.M., Ulzheimer-Teuber, I., Stöber, G., Schmitt,
G., Lesch, K.P., 2001. A missense mutation in a novel gene
encoding a putative cation channel is associated with catatonic
schizophrenia in a large pedigree. Mol. Psychiatry 6, 302 – 306.
Meyer, J., Ortega, G., Schraut, K., Nürnberg, G., Rüschendorf, F.,
Saar, K., Mössner, R., Wienker, T.F., Reis, A., Stöber, G., Lesch,
K.P., 2002. Exclusion of the neuronal nicotinic acetylcholine
receptor a7 subunit gene as a candidate for catatonic schizophrenia in a large family supporting the chromosome 15q13 – 22
locus. Mol. Psychiatry 7, 220 – 223.
Neusch, C., Rozengurt, N., Jacobs, R.E., Lester, H.A., Kofuji, P.,
2001. Kir4.1 potassium channel subunit is crucial for oligodendrocyte development and in vivo myelination. J. Neurosci. 21,
5429 – 5438.
Omori, Y., Mesnil, M., Yamasaki, H., 1996. Connexin 32 mutations
from X-linked Charcot – Marie – Tooth disease patients: functional defects and dominant negative effects. Mol. Biol. Cell
7, 907 – 916.
91
Rash, J.E., Staines, W.A., Yasumura, T., Patel, D., Furman, C.S.,
Stelmack, G.L., Nagy, I.J., 2000. Immunogold evidence that
neuronal gap junctions in adult rat brain and spinal chord contain connexin-36 but not connexin-32 or connexin-43. Proc.
Natl. Acad. Sci. U. S. A. 97, 7573 – 7578.
Serre-Beinier, V., Le Gurun, S., Belluardo, N., Trovato-Salinaro, A.,
Charollais, A., Haefliger, J.A., Condorelli, D.F., Meda, P., 2000.
Cx36 preferentially connects beta-cells within pancreatic islets.
Diabetes 49, 727 – 734.
Stöber, G., Franzek, E., Lesch, K.P., Beckmann, H., 1995. Periodic
catatonia: a schizophrenic subtype with major gene effect and
anticipation. Eur. Arch. Psychiatry Clin. Neurosci. 245, 135 –
141.
Stöber, G., Saar, K., Rüschendorf, F., Meyer, J., Nürnberg, G.,
Jatzke, S., Franzek, E., Reis, A., Lesch, K.P., Wienker, T.F.,
Beckmann, H., 2000. Splitting schizophrenia: periodic catatonia-susceptibility locus on chromosome 15q15. Am. J. Hum.
Genet. 67, 1201 – 1207.
Teubner, B., Degen, J., Söhl, G., Güldenagel, M., Bukauskas, F.F.,
Trexler, E.B., Verselis, V.K., De Zeeuw, C.I., Lee, C.G., Kozak,
C.A., Petrasch-Parwez, E., Dermietzel, R., Willecke, K., 2000.
Functional expression of the murine connexin 36 gene coding
for a neuron-specific gap junctional protein. J. Membr. Biol.
176, 249 – 262.