Download Exclusion of the neuronal nicotinic acetylcholine receptor 7 subunit

Molecular Psychiatry (2002) 7, 220–223
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ORIGINAL RESEARCH ARTICLE
Exclusion of the neuronal nicotinic acetylcholine
receptor ␣7 subunit gene as a candidate for catatonic
schizophrenia in a large family supporting the
chromosome 15q13–22 locus
J Meyer1, G Ortega1, K Schraut1, G Nürnberg2,3, F Rüschendorf2,3, K Saar2, R Mössner1,
TF Wienker2,3, A Reis2, G Stöber1 and KP Lesch1
1
Department of Psychiatry and Psychotherapy, University of Wuerzburg, Wuerzburg, Germany; 2Max-Delbrück-Center for
Molecular Medicine, Gene Mapping Center, Berlin, Germany; 3Institute of Medical Biometry, Informatics and Epidemiology,
University of Bonn, Bonn, Germany
Keywords: linkage analysis; chromosome 15; catatonic
schizophrenia; nicotinic acetylcholine receptor
The gene encoding the neuronal nicotinic acetylcholine
receptor ␣7 subunit (CHRNA7) is located on chromosome 15q13.2. This region was suggested to be
involved in the etiopathogenesis of: (a) schizophrenia
combined with a neurophysiological deficit; (b) lithiumresponsive bipolar disorder; and (c) familial catatonic
schizophrenia (periodic catatonia). Therefore, members
of a large family with periodic catatonia strongly supporting the chromosome 15q13–22 region were genotyped with polymorphic markers localized around the
CHRNA7 locus. A recombination event distally of
marker D15S144 leading to the exclusion of the CHRNA7
locus from this candidate region was detected in one
branch of the pedigree. This result provides strong evidence that a gene located telomeric to CHRNA7 is
causative for the pathogenesis of catatonic schizophrenia in this family.
Molecular Psychiatry (2002) 7, 220–223. DOI: 10.1038/
sj/mp/4000970
The gene encoding the neuronal nicotinic acetylcholine receptor ␣7 subunit gene (CHRNA7), located on
chromosome 15q13.2 according to the map provided
by the International Human Genome Sequencing Consortium,1 has been discussed as a candidate for schizophrenia and bipolar disorder. High levels of CHRNA7
gene expression were found in nuclei that receive sensory information, like the thalamic nuclei, the pontine
nuclei, and the superior olive complex.2 Reports on
heavy smoking behavior among schizophrenics further
supported the hypothesis that CHRNA7 might be
involved in the etiopathogenesis of schizophrenia.3
Freedman and associates reported a maximum lod
score (Z = 5.3, ␪ = 0.0, P ⬍ 0.001) for polymorphic
marker D15S1360 mapping in close vicinity to the
CHRNA7 locus for 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 after the stimulus (P50) in schizophrenics.4 In a recent report, this finding was sup-
ported by the same group employing a transmission
disequilibrium approach in 62 parent-child trios.5 Furthermore, they found distinct promoter variants of
CHRNA7 being highly associated with schizophrenia
(␹2 = 12.06, P = 0.0005) by investigating more than 500
schizophrenic and control subjects.6 Turecki and
coworkers7,8 demonstrated that bipolar disorder
(manic-depressive illness) combined with positive
response to lithium treatment maps also to this region
(lod score 3.46, P = 0.000014, for marker ACTC). Riley
et al9 found suggestive evidence for linkage of the locus
with schizophrenia in Southern African Bantu families; Neves-Pereira et al10 and Curtis et al11 failed to
detect linkage of CHRNA7 with schizophrenia. Lai and
colleagues12 found no association of an allelic 2-bp
deletion variant of CHRNA7 with schizophrenia in a
population-based association study.
Periodic catatonia13 (SCZD10, MIM 605419), a familial subtype of catatonic schizophrenia, is a genetically
heterogenous disorder characterized by psychomotoric
disturbances with akinetic and hyperkinetic episodes,
frequently accompanied by hallucinations and
delusions. Successive episodes may lead to an increasingly severe residual catatonic state. Our group has
recently reported confirmed linkage based on a dominant model of inheritance (maximum GENEHUNTERPLUS lod score 3.57, P = 0.000026, at coordinate 35.3
cM) of polymorphic markers between D15S165 and
D15S153 with periodic catatonia (Stöber et al,14 and
unpublished data) by investigating 12 extended pedigrees. Of these, a large family (Figure 2) was found to
support strongly the chromosome 15 locus (maximum
multipoint lod score 2.89, ␪ = 0.029, at D15S1042
(VITESSE)). The aim of present study was to narrow
down this region by genotyping additional markers
around the CHRNA7 locus to determine whether the
gene should be considered as a candidate for
mutational analysis.
According to Freedman et al5 and the results of the
4th chromosome 15 workshop (http: //www.medgen.
ubc.ca/medgen/scw15.html), polymorphic markers
Exclusion of the nicotinic acetylcholine receptor
J Meyer et al
around CHRNA7 and the gene itself are assembled in
the following order (coordinates in centiMorgans (cM)
according to the Marshfield map are given in brackets):
centromer — D15S165 (20.24) — D15S1043 (20.24) —
duplication of exons 5–10 of CHRNA7 — D15S1031
(21.58) — CHRNA7 — D15S1010 (23.89) — D15S144
(25.30) — D15S1040 (28.35) — ACTC (31.46) —
D15S1042 (32.58) — D15S659 (43.47) — D15S1028
(45.62) — D15S1033 (52.33) — D15S987 (59.05) —
D15S153 (62.40) — telomer (Figure 1). We have
recently restricted the chromosomal candidate region
of the large family (Figure 2) strongly supporting this
locus to approximately 42 cM between D15S165 and
D15S153 by a genome-wide linkage scan (Stöber et al,14
and unpublished data). In order to decide whether
CHRNA7 should be considered as a candidate for
mutational analysis, we have genotyped the members
of this family, many of whom were affected with catatonic schizophrenia, with additional markers. Of these
were informative D15S1010, D15S144, D15S1040,
ACTC, D15S968, D15S659, D15S1028, and D15S987,
respectively. Genotyping led to the exclusion of
CHRNA7 together with the duplication of exons 5–10
of this gene15 from the region of interest due to a recombination event between D15S144 and D15S1040 in one
branch of the pedigree (Figure 2), narrowing further
down the region to approximately 20.3 cM between
markers D15S144 and D15S1028. A recombination
between markers D15S659 (43.47 cM; not shown) and
D15S1028 (45.62 cM; Figure 2) in proband 0935 defines
the distal limit of the disease region.
Several positive linkage findings for psychiatric disorders have been reported for the chromosome 15q13–
22 region.4,5,7,8,14 Of these, manic-depressive illness
and periodic catatonia share bipolarity as a common
feature, thus providing evidence that pleiotrophic
effects of different mutations within one gene may be
responsible for different clinical phenotypes. However,
a potential clustering of more than one disease gene in
this region cannot be ruled out. In order to find the
gene(s) responsible for the diseases, narrowing down
the chromosomal region and exclusion of candidate
genes should be of high priority.
The neuronal nicotinic acetylcholine receptor ␣7
subunit gene (CHRNA7) has been suggested as a candidate gene for schizophrenia,4,5 although, mutational
analysis of the CHRNA7 coding region revealed no
variant uniquely found in a significant proportion of
schizophrenic patients so far.16 Exclusion of CHRNA7
as a candidate for periodic catatonia in a large family
by haplotype mapping with densely spaced markers
strongly supports the notion that a gene located telomeric to CHRNA7 within a 20.3-cM region is causative
for the disease in this family.
221
Methods
Clinical
The multiplex family was ascertained as previously
described in detail.14 Consistent diagnostic classification within the family segregating periodic catatonia described here 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.
Figure 1 Schematic map of polymorphic markers and genes
around the CHRNA7 locus on chromosome 15q13.2. Coordinates in centiMorgans (cM) according to the Marshfield map
are given in brackets.
Genotyping
Polymorphic markers around CHRNA7 were selected
from the Marshfield map. Fragments were amplified in
a T-GRADIENT Thermocycler (Biometra, Göttingen,
Germany) in the presence of ␣-32P-dCTP and separated
Molecular Psychiatry
Exclusion of the nicotinic acetylcholine receptor
J Meyer et al
222
Figure 2 Segregating of polymorphic markers around CHRNA7 with periodic catatonia. Affected family members are symbolized by black diamonds. Six polymorphic markers out of 24 totally tested within this region are shown. A recombination
event distally from D15S144 in proband 0730 and descendants of 0730 led to exclusion of CHRNA7 from the chromosomal
disease region (boxed). Haplotypes from deceased persons or persons not available for genotyping were deduced from the data
of the previously reported linkage scan,14 and given in brackets. The double-crossover at marker ACTC in proband 0825
(haplotypes 1, 3) is confirmed by the results of the linkage scan (Stöber et al14 and data not shown).
on 6% polyacrylamide gels. PCR was performed in a
final volume of 25 ␮l containing 60 ng of genomic
DNA, 10 pmol of each primer, 200 ␮M of each dNTP,
1.0 or 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH
8.3 at 25°C), 0.025 mg ml−1 BSA, 0.025% Tween 20,
and 0.5 U of Taq DNA polymerase (Eurogentec, Seraing, Belgium). Gels were dried and exposed to Kodak
X-OMAT AR films. Resulting alleles were integrated
into the haplotype data set derived from the genome
wide linkage scan described previously (Stöber et al,14
and unpublished data).
4
5
6
7
Acknowledgements
The authors would like to express their gratitude to the
study participants and their family. 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).
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Correspondence: J Meyer, PhD, Dept of Psychiatry and Psychotherapy, University of Wuerzburg, 97080 Wuerzburg, Germany.
E-mail: jobst.meyer얀mail.uni-wuerzburg.de
Received 30 March 2001; revised 27 June 2001; accepted 10 July 2001
Molecular Psychiatry