Association between the 2-bp deletion polymorphism in the duplicated version of the alpha7 nicotinic receptor gene and P50 sensory gating.

Abstract

There is considerable evidence implicating the 15q13.3 region in neuropsychiatric disorders, with the α7 nicotinic receptor gene CHRNA7 the most plausible candidate. This region has multiple duplications and many copy number variants (CNVs). A common CNV involves a partial duplication of CHRNA7 (CHRFAM7A), which occurs in either orientation. We examined the distribution of these alternative genomic arrangements in a large cohort of psychiatric patients, their relatives and controls using the 2-bp deletion polymorphism as a marker for the orientation of CHRFAM7A. We investigated three common alleles for association with psychosis and with the P50 sensory gating deficit, which is strongly associated with psychosis and strongly linked to 15q13.3. We found significant within-family association with P50 (empirical P=0.004), which is robust to population stratification. Most of the effect came from the 2-bp deletion allele, which tags the variant of CHRFAM7A in the same orientation as CHRNA7. This allele is associated with the presence of the P50 sensory gating deficit (empirical P=0.0006). Tests comparing within-family and between-family components of association suggest considerable population stratification in the sample. We found no evidence for association with psychosis, but this may reflect lower power using this phenotype. Four out of six previous association studies found association of different psychiatric phenotypes with the same 2-bp deletion allele.European Journal of Human Genetics advance online publication, 16 May 2012; doi:10.1038/ejhg.2012.81.

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ARTICLE
Association between the 2-bp deletion polymorphism
in the duplicated version of the alpha7 nicotinic
receptor gene and P50 sensory gating
Rachel H Flomen1,6,8, Madiha Shaikh2,6, Muriel Walshe2, Katja Schulze2, Mei-Hua Hall3, Marco Picchioni2,4,
Fruhling Rijsdijk5, Timothea Toulopoulou2, Eugenia Kravariti2, Robin M Murray2, Philip Asherson5,
Andrew J Makoff*,1,7and Elvira Bramon2,7
There is considerable evidence implicating the 15q13.3 region in neuropsychiatric disorders, with the a7 nicotinic receptor
gene CHRNA7 the most plausible candidate. This region has multiple duplications and many copy number variants (CNVs).
A common CNV involves a partial duplication of CHRNA7 (CHRFAM7A), which occurs in either orientation. We examined the
distribution of these alternative genomic arrangements in a large cohort of psychiatric patients, their relatives and controls
using the 2-bp deletion polymorphism as a marker for the orientation of CHRFAM7A. We investigated three common alleles
for association with psychosis and with the P50 sensory gating deficit, which is strongly associated with psychosis and
strongly linked to 15q13.3. We found significant within-family association with P50 (empirical P¼0.004), which is robust
to population stratification. Most of the effect came from the 2-bp deletion allele, which tags the variant of CHRFAM7A in
the same orientation as CHRNA7. This allele is associated with the presence of the P50 sensory gating deficit (empirical
P¼0.0006). Tests comparing within-family and between-family components of association suggest considerable population
stratification in the sample. We found no evidence for association with psychosis, but this may reflect lower power using this
phenotype. Four out of six previous association studies found association of different psychiatric phenotypes with the same
2-bp deletion allele.
European Journal of Human Genetics (2013) 21, 76–81; doi:10.1038/ejhg.2012.81; published online 16 May 2012
Keywords: CHRFAM7A; schizophrenia; bipolar disorder
INTRODUCTION
The 15q13.3 region has been implicated in several neurological and
psychiatric disorders. The P50 sensory gating deficit, one of the best
supported endophenotypes of schizophrenia and bipolar disorder,
is strongly linked to this region,1as are two idiopathic epilepsies.2,3Of
the many attempts to demonstrate linkage of schizophrenia and
bipolar disorder to this region, two studies showed linkage to bipolar
disorder,4,5but several found either weak6–10or no linkage to
schizophrenia.11–13Both schizophrenia and bipolar disorder have
also shown association with 15q13.3 markers.14The most plausible
candidate gene in 15q13.3 for the major psychoses and epilepsies is
CHRNA7, the a7 nicotinic acetylcholine receptor gene.15,16However,
15q13.3 is a complex region in the midst of many large segmental
duplications.17,18These are highly variable, resulting in several
different copy number variants (CNVs).
One common CNV includes part of CHRNA7 (exons 5–10), which
is duplicated in the hybrid gene CHRFAM7A that occurs in most
individuals. We found weak evidence for association of the absence of
one copy of CHRFAM7A with psychosis in a large Scottish sample.19
When present, CHRFAM7A can occur in either orientation, with one
orientation strongly associated with the common 2-bp deletion
polymorphisminexon 6,20
(Supplementary Figure 1a). We found no association of the 2-bp
deletion with schizophrenia in the above study, but an association
with schizophrenia has since been reported.21Other studies have
found that the 2-bp deletion is associated with bipolar disorder,22P50
sensory gating deficit23and deficits in episodic memory,24another
endophenotype proposed for schizophrenia. Recent genome-wide
scans for CNVs showed that much rarer CNVs at 15q13.3, usually
involving deletion of B2Mb from CHRFAM7A to CHRNA7, are
overrepresented in many neurological and psychiatric disorders. These
deletions are very rare in the general population (B0.02%), but more
common in schizophrenia, autism/developmental disorders and
some forms of intellectual impairment (B0.2–0.3%) and even
more common in idiopathic generalized epilepsy (B1.0%).25–28
These B2-Mb microdeletions lead to the loss of CHRNA7 and five
other genes (Supplementary Figure 1a), but smaller variants that show
a similar range of phenotypes remove only CHRNA7 and one other
foundonlyinCHRFAM7A
1Department of Clinical Neuroscience, Institute of Psychiatry, King’s College London, London, UK;2NIHR Biomedical Research Centre for Mental Health at the South London
and Maudsley NHS Foundation Trust and Institute of Psychiatry, King’s College London, London, UK;3Psychology Research Laboratory, Harvard Medical School, McLean
Hospital, Belmont, MA, USA;4St Andrews Academic Centre, Institute of Psychiatry, Northampton, UK;5Social, Genetic Developmental Psychiatry Research Centre, Institute of
Psychiatry, King’s College London, London, UK
*Correspondence: Dr A Makoff, Department of Clinical Neuroscience, Institute of Psychiatry, King’s College London, 1, Windsor Walk, Denmark Hill, London, SE5 8AF, UK.
Tel: þ44 207 848 0638; Fax: þ44 207 848 0051; E-mail: andrew.makoff@kcl.ac.uk
6These authors contributed equally to this work.
7These authors jointly directed this work.
8Present address: Department of Medical & Molecular Genetics, King’s College London School of Medicine, Tower Wing, Guy’s Hospital, London, UK.
Received 14 September 2011; revised 16 March 2012; accepted 27 March 2012; published online 16 May 2012
European Journal of Human Genetics (2013) 21, 76–81
& 2013 Macmillan Publishers Limited All rights reserved 1018-4813/13
www.nature.com/ejhg

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gene.29These observations strongly implicate the CHRNA7 region in
schizophrenia and other neuropsychiatric disorders.
P50 sensory gating is an EEG signal that is believed to reflect the
ability to filter out repetitive stimuli and prevent information
overload.30–32The P50 wave is generated in a conditioning-testing
paradigm where the first stimulus activates or conditions the
inhibition phenomenon, whereas the second tests its strength.
Normally, individuals reduce the second (test) response relative to
the first (condition) waves. The diminished test P50 wave is thought
to be due to activation of inhibitory neural circuitry by the
conditional P50 stimuli.1,33–35Compared with controls, patients
with schizophrenia and psychotic bipolar disorder have a relatively
larger P50 response to the second stimulus, from only 20–50%
suppression.31,36–47
Clinically unaffected
of patients with schizophrenia and psychotic bipolar disorder also
have poor P50 suppression, suggesting that this might act as a marker
of genetic risk for these disorders,46–53as it is heritable.54–57
We have examined the effects of CHRFAM7A CNV/2bp deletion
variants on the major psychoses and the P50 sensory gating deficit.
We utilized a large group of families, twin pairs and controls
including several family members diagnosed with schizophrenia or
psychotic bipolar disorder, where P50 measurements were made.
first-degreerelatives
METHODS
The sample
The sample included 871 Caucasian individuals from the national Maudsley
Family and Twin Psychosis Studies. A subgroup of these (N¼445) underwent
EEG recordings from which were derived the P50 suppression ratios,
computed as the ratio of testing to conditioning wave amplitudes (T/C)
expressed as a percentage. Patients satisfied DSM-IV criteria for schizophrenia,
schizoaffective disorder or psychotic bipolar disorder. Their unaffected first-
degree relatives were free of any psychotic illness whereas the controls had no
personal or family history of psychosis. Subjects were excluded if they had a
neurological disorder, head injury with loss of consciousness exceeding 10min
or diagnosis of alcohol or substance dependence in the previous 12 months.
The studies were approved by the Joint South London and Maudsley and the
Institute of Psychiatry NHS Research Ethical Committee or the South East
Research Ethics Committee. All participants gave written informed consent.
Clinical assessments and P50 measurements
Clinical assessment, P50 measurements and analysis were performed as
described in detail elsewhere.31
Genotyping
Genotyping for the CHRFAM7A CNV/2bp deletion polymorphisms required
two sequential assays.19Presence of the 2-bp deletion was determined by limited
cycle PCR using ABI3130 and Genemapper v3.0 software (Life Technologies
Limited, Paisley, UK). Each sample was assayed at least twice to identify three
genotypes (13, 23 and 33) where the 2-bp deletion is present, with alleles defined
as 1¼null CHRFAM7A, 2¼wild-type CHRFAM7A and 3¼CHRFAM7A with
2bp deletion (Supplementary Figures 1a, b). Samples without a 2-bp deletion
were assayed by a Taqman assay (Life Technologies Limited) to determine the
copy number of CHRFAM7A (at least three determinations per sample) to
identify the remaining three genotypes (11, 12 and 22).
Genotyping was performed and analyzed ‘blind’ to patient identity. Several
patients had more than one DNA sample. These and monozygous (MZ) pairs
accounted for 606 genotyped samples, which were compared to assess
genotyping reproducibility, giving an error rate of 6%. Duplicate or MZ pairs
discordant for genotype were excluded.
Statistical analyses
Significance of association of the CNV/2bp deletion polymorphisms with
clinical groups was performed by w2-tests, including only one twin from each
MZ pair concordant for psychosis but excluding discordant MZ pairs. In initial
analyses, these tests were also applied to P50, as two categorical groups: P50 T/C
ratios 460 versus o40%, and including one MZ twin per pair using each
average T/C ratio. Family-based association analyses were performed on
categorical phenotypes by the transmission disequilibrium test (TDT) using
Transmit (https://www-gene.cimr.cam.ac.uk/staff/clayton/software/).58Transmit
uses parental genotypes, when present, but can use siblings to derive possible
parental genotypes when absent. As Transmit will not accept twinships, dizygous
(DZ) twins were entered as siblings while one twin per MZ pair were entered as
above. To perform tests for association on continuous phenotype scores
we used QTDT (http://www.sph.umich.edu/csg/abecasis/QTDT/),59,60which
incorporates a modified form of TDT. QTDT will construct possible parental
genotypes where absent and accepts all types of sibships. It can utilize
components variance modeling, which we used as recommended for samples
including families with multiple offspring.60QTDTassesses both between-family
and within-family components of association to provide three tests of
association: ‘total’ combines both components but is invalid in the presence
of population stratification, ‘within’ gives the within-family component and is
robust to stratification and ‘stratification’ compares the components to test for
stratification.
RESULTS
The sample
The sample comprises patients diagnosed with a major psychosis
(mainly schizophrenia or bipolar disorder), their families (including
co-twins), controls and control twin pairs, all of whom were white
Caucasians. Table 1 shows the demographic matching between
patients, relatives and controls. As is often seen with studies
of psychosis, the patient group had significantly more males than
the control group. The P50 subset illustrates the well-established
strength of association between P50 and psychosis,46,61,62with very
strong associations between patients and controls (P¼5?10?9), and
between relatives and controls (P¼6?10?6).
Association study by patient group
Comparison of the distribution of the three alleles among the three
patient groups showed no significant association (Table 2). The
distributions of the six genotype frequencies (see Supplementary
Table 1) were all in Hardy–Weinberg equilibrium (HWE), and there
was also no significant association. As case–control association studies
are vulnerable to population stratification, we investigated possible
family-based association. This was performed by TDT, which
compares the transmission of alleles from heterozygous parents to
affected offspring. As shown in Table 3 (top), there was no evidence
for family-based association with psychosis.
Association study by P50
Allele frequencies for individuals with P50 data are shown in Table 4.
In a preliminary analysis, we compared the allele distributions
between individuals with a T/C ratio 460% (generally regarded as
abnormal) with those o40% (within the normal range). As shown in
Table 4, there was no significant difference between allele frequencies.
Thedistributions ofgenotype
Supplementary Table 2, with all genotypes in HWE.
As with comparisons between patient groups, this type of associa-
tion analysis can be compromised by population stratification.
Furthermore, it does not utilize all available power of the sample as
(a) it reduces the full range of T/C ratios to two discrete groups and
(b) it does not utilize the family relationships. To overcome these
limitations, we analysed the data by QTDT, which compares
transmission of each allele from heterozygous parents with offspring
scored with the full range of T/C ratios.
frequenciesare shownin
Association of CHRFAM7A and P50 sensory gating
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QTDT has three tests of association: ‘total’, ‘within’ and ‘stratifica-
tion’ (see Methods). The tests of total association were not significant
(Table 5). By contrast, the tests of within-family association, which are
robust to population stratification, were clearly significant. The
empirical measurement of significance for global association
(P¼0.004, based on 10000 permutations) is much stronger than
the calculated value (P¼0.02), and is regarded as more reliable60as it
makes no assumptions regarding normality of distribution or degrees
of freedom (Table 5). When each allele was considered alone, allele 3
(empirical P¼0.0006) and, to a much lesser extent, allele 1 (empirical
P¼0.03) were both significant. The tests for stratification were also
significant for global association (empirical P¼0.04) and allele 3
alone (empirical P¼0.004). We also modeled the effects of psychosis
diagnosis, sex or age as covariates, which made very little difference to
the tests of association (Supplementary Table 3).
To provide model-free evidence for the within-family association
found for P50 and to identify its direction, we dichotomized the P50
scores as for the initial analysis and performed TDT using Transmit.
Unsurprisingly, the loss of twin data (used in QTDT) and the loss of
the full range of P50 scores caused considerable loss of power.
Table 1 Details of sample, all of whom were white Caucasians
Affected patients Unaffected relatives Unaffected controlsTotals
Complete sample
aMZ twins
aDZ twins
DSM-IV diagnosis
248
87 (29)
16 (1)
169 schizophrenia
66 bipolar
10 schizoaffective
3 other psychosis
39.0±1.5
17?74
154:94
258
29 (0)
14 (0)
365
b175 (87)
102 (51)
871
b291 (145)
132 (66)
Mean age (±SEM)
Age range
Males: females
47.8±1.9
16?83
113:145
39.9±1.3
19?72
116:249
42.0±0.9
16?83
383:488
P50 sample
aMZ twins
aDZ twins
DSM-IV diagnosis
141
56 (19)
7 (0)
85 schizophrenia
49 bipolar
6 schizoaffective
1 other psychosis
40.2±2.1
21?65
90:51
65.4±8.2
108
18 (0)
5 (0)
196
b78 (38)
67 (32)
445
b152 (74)
79 (37)
Mean age (±SEM)
Age range
Males: females
cP50 T/C ratio
43.0±2.5
19?63
50:58
57.6±7.1
38.1±1.8
19?64
65:131
38.6±4.7
39.9±1.2
19?65
205:240
51.7±3.9
Abbreviations: DZ, dizygous; MZ, monozygous; T/C, ratio of testing to conditioning wave amplitudes.
aNumber of twins for each group with number of twin pairs shown in brackets.
bIncludes one triplet.
cSignificance by t-tests: patients versus controls: P¼5?10?9; patients versus relatives: P¼0.2; relatives versus controls: P¼6?10?6.
Table 2 Comparison of allele frequencies by patient group
AlleleAffected patientsUnaffected relativesUnaffected controls
1
2
3
Totals
44 (0.13)
141 (0.42)
151 (0.45)
336
55 (0.14)
157 (0.41)
170 (0.45)
382
54 (0.11)
226 (0.47)
206 (0.42)
486
Alleles defined as 1 (null CHRFAM7A), 2 (wt CHRFAM7A), 3 (CHRFAM7A with 2bp deletion).
Global significance for overall allele frequency comparisons: patients versus controls: w2¼1.87,
2 degree of freedom (df), P¼0.4; relatives versus controls: w2¼3.48, 2df, P¼0.18.
Table 3 Comparison of transmissions to offspring affected with
psychosis or T/C ratio 460%
AllelePhenotype
Observed
transmissions
Expected
transmissions
aSignificance
(w2, df, P)
1
2
3
Totals
Psychosis 42 (0.13)
136 (0.43)
142 (0.44)
320
42 (0.13)
136 (0.43)
142 (0.44)
320
w2¼0.01, 1df, P¼0.9
w2¼0.00, 1df, P¼1.0
w2¼0.00, 1df, P¼1.0
1
2
3
Totals
P50 T/C460% 22 (0.11)
82 (0.40)
100 (0.49)
204
26 (0.13)
85 (0.42)
93 (0.46)
204
w2¼2.31, 1df, P¼0.13
w2¼0.74, 1df, P¼0.4
w2¼3.76, 1df, P¼0.05
Abbreviations: df, degree of freedom; T/C, ratio of testing to conditioning wave amplitudes.
For psychosis, global significance: w2¼0.01, 2df, P¼1.0.
For P50, global significance: w2¼4.41, 2df, P¼0.11.
aSignificance determined by transmission disequilibrium test.
Nominally significant P-value shown in bold.
Table 4 Comparison of allele frequencies by P50 T/C ratios
Allele
All T/C
ratios
T/C ratios
460%
T/C ratios
o40%
aSignificance
1
2
84 (0.12)
298 (0.44)
28 (0.12)
95 (0.40)
38 (0.13)
132 (0.46)
w2¼0.27, 1df, P¼0.6
w2¼2.06, 1df,
P¼0.15
w2¼3.17, 1df,
P¼0.07
3296 (0.44) 115 (0.48)116 (0.41)
Totals678238 286
Abbreviations: df, degree of freedom; T/C, ratio of testing to conditioning wave amplitudes.
aGlobal significance for allele frequency comparisons T/C ratios 460% versus o40%:
w2¼3.18, 2df, P¼0.2.
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Nonetheless, nominally significant association with allele 3 was
detected (w2¼3.76, 1 degree of freedom (df), P¼0.05; Table 3,
bottom). These results support the QTDT within-family tests of
association and show that the direction is of elevated P50 scores
associated with increased transmission of allele 3.
DISCUSSION
Within-family
CHRFAM7Avariants with P50 T/C ratio, with most of the association
between elevated ratios and raised allele 3 (2bp deletion) frequencies.
However, w2-tests and tests combining between- and within-family
associations, that do not control for stratification, were not signifi-
cant, although such tests considering allele 3 alone almost reached
nominal significance. This pattern of findings could indicate a type I
error in the within-family tests of association. However, they may also
be explained by population stratification for the genetic variants
investigated here, which would invalidate between-family tests,
whereas within-family tests remain robust. Supporting this, we found
that tests of population stratification for these genetic variants were
significant, indicating that only the within-family association test is
valid and suggesting that significant between-family association may
have been masked by population stratification.59Similar results were
found for other associations.63,64
Although our cohort were all white Caucasians, some genetic
heterogeneity within this ethnic group would not be surprising.
Investigating the sample with a panel of ethnically specific SNPs
might clarify this issue, although CHRFAM7A variants investigated
here show a wide allele frequency variation between ethnicities,21
suggesting that stratification effects may be particularly pronounced
for these variants. Although population stratification is a plausible
explanation for the difference between within- and between-family
tests of association, other factors may be involved.
One potential source of error could be incorrect genotype calls,
which are known to increase type I errors in family-based association
studies.65,66Therefore, the observed within-family association might
be because of genotype errors that we estimated at 6%. Using
simulated error rates up to 10% in SNPs, type I errors were found
to be highest where both alleles had very different frequencies and
minimal with alleles of equal frequency,65with similar observations
for multiallelic polymorphisms.66Our strongest association was due
to an excess of allele 3 transmissions from 13 and 23 heterozygotes.
testsprovideevidenceforassociationofthe
Allele 3 frequency is close to the combined frequencies of the other
two alleles, where the error-induced type I error rate is expected to be
low. This suggests that genotype errors are unlikely to account for the
significant within-family association.
Unlike P50, the psychosis phenotype showed no evidence for
association with the CHRFAM7A alleles investigated. This is con-
sistent with the hypothesis that endophenotypes may confer greater
power to detect association than the disease phenotype, where the
variance is spread over a wider range of genetic and environmental
factors. Furthermore, the psychosis phenotype is not amenable to
quantitative family-based studies such as QTDT, which under certain
circumstances may also have more power than categorical association
tests.
Of the six previous studies involving CHRFAM7A variants and
either a psychosis phenotype or endophenotype,19,21–24,67four found
a significant association with the 2-bp deletion. However, there is no
consistency with the phenotype involved. Raux et al23found a higher
frequency of the 2-bp deletion allele in individuals with a high P50
T/C ratio, but no association with schizophrenia. Our data support
these results. By contrast, Sinkus et al21found no association with
P50, but significant association with schizophrenia in both Caucasians
and African-Americans. Dempster et al24found an association
between the same allele and episodic memory deficits, a possible
endophenotype of schizophrenia. Finally, Hong et al22found an
association between the 2-bp deletion and bipolar disorder. It is
possible that at least some of these four studies may be detecting the
same genetic effect as in the present study.
The biological effect of the 2-bp deletion in CHRFAM7A is difficult
to infer as the role of CHRFAM7A, found uniquely in humans, is
poorly understood. The gene is transcribed, as mRNA sequences
have been detected.17,68Two recent studies demonstrated that
co-expression of CHRFAM7A with CHRNA7 significantly reduced
acetylcholine currents.69,70This appeared to be a posttranslational
effect, and the presence of the 2-bp deletion in exon 6 of CHRFAM7A
further reduced these currents.70The CHRFAM7A sequence predicts
two different potential proteins that contain some a7 nicotinic
receptor amino-acid sequence, one being prevented by the 2-bp
deletion, whereas the other is only possible in its presence. However,
there is no evidence for either protein.
Another possibility is that the 2-bp deletion is linked to a
functional polymorphism. Previously, we found that CHRFAM7A
commonly exists in either orientation (Supplementary Figure 1a) and
that the 2-bp deletion is strongly associated with CHRFAM7A in the
same orientation as CHRNA7.20This suggests that the 2-bp deletion
tags the orientation of CHRFAM7A, although this does depend on a
single study. It is possible that the orientation of the gene might affect
its expression, as the DNA environment upstream of the likely
location of the CHRFAM7A promoter would be altered by the
inversion. However, there is an alternative way in which the
orientation of CHRFAM7A might exert a biological effect. The rare
15q13.3 microdeletion is strongly associated with many neuro-
psychiatric conditions, including schizophrenia.25–27This deletion is
recurrentand probably arises
recombination when two direct repeats misalign during meiosis.
Two large direct repeats likely to be responsible include CHRNA7 and
CHRFAM7A71(see Supplementary Figure 1a). The occurrence of
these two genes in the same orientation therefore predisposes, in a
very small minority of cases, to the 15q13.3 microdeletion in the next
generation. A similar misalignment of these direct repeats might
occur during DNA replication. An accumulation of such 15q13.3
microdeletions in key neural cells might mimic some of the
from non-allelic homologous
Table 5 Quantitative transmission disequibrium test results for
P50 T/C ratio
Allele
Association
test
aCalculated significance
bEmpirical significance
All
1
2
3
total
w2¼4.29, 2df, P¼0.12
w2¼2.44, 1df, P¼0.12
w2¼0.74, 1df, P¼0.4
w2¼3.44, 1df, P¼0.06
All
1
2
3
within
w2¼7.62, 2df, P¼0.02
w2¼4.35, 1df, P¼0.04
w2¼0.62, 1df, P¼0.4
w2¼7.07, 1df, P¼0.008
P¼0.004 (10,000 permutations)
P¼0.03 (10,000 permutations)
P¼0.0006 (100,000 permutations)
All
1
2
3
stratification
w2¼4.06, 2df, P¼0.13
w2¼2.09, 1df, P¼0.15
w2¼0.24, 1df, P¼0.6
w2¼4.34, 1df, P¼0.04
P¼0.04 (1,000 permutations)
P¼0.13 (1,000 permutations)
P¼0.004 (10,000 permutations)
Abbreviation: T/C, ratio of testing to conditioning wave amplitudes.
aCalculated significance was derived from the differences in log likelihood scores (w2) and
degrees of freedom (df) between the null and alternative models.
bEmpirical significance was derived from multiple permutation tests, number of tests as shown.
Nominally significant P-values shown in bold.
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phenotypes associated with patients who have inherited this deletion.
If it occurs, it would be limited to individuals with at least one copy of
CHRFAM7A in the CHRNA7 orientation. Our finding of an
association between the P50 sensory gating deficit with the 2-bp
deletion allele provides an indirect evidence for an association with
this orientation of CHRFAM7A, which is consistent with this
intriguing possible mechanism.
CONFLICT OF INTEREST
The authors declare no conflict of interest
ACKNOWLEDGEMENTS
We thank all study participants. This work was supported by NARSAD awards
to AM and EB and Wellcome Trust Research Training Fellowships to MP and
EB, with further support provided by the Wellcome Trust and NIHR. EB holds
a MRC new investigator award. We also thank R Pinto for her advice and
assistance with QTDT.
1 Freedman R, Coon H, Myles-Worsley M et al: Linkage of a neurophysiological deficit
in schizophrenia to a chromosome 15 locus. Proc Natl Acad Sci USA 1997; 94:
587–592.
2 Elmslie FV, Rees M, Williamson MP et al: Genetic mapping of a major susceptibility
locus for juvenile myoclonic epilepsy on chromosome 15q. Hum Mol Genet 1997; 6:
1329–1334.
3 Neubauer BA, Fiedler B, Himmelein B et al: Centrotemporal spikes in families with
rolandic epilepsy: linkage to chromosome 15q14. Neurology 1998; 51: 1608–1612.
4 Edenberg HJ, Foroud T, Conneally PM et al: Initial genomic scan of the NIMH genetics
initiative bipolar pedigrees: Chromosomes 3, 5, 15, 16, 17, and 22. Am J Med Genet
1997; 74: 238–246.
5 Turecki G, Grof P, Grof E et al: Mapping susceptibility genes for bipolar disorder: a
pharmacogenetic approach based on excellent response to lithium. Mol Psychiatry
2001; 6: 570–578.
6 Kaufmann CA, Suarez B, Malaspina D et al: NIMH Genetics Initiative Millenium
Schizophrenia Consortium: linkage analysis of African-American pedigrees. Am J Med
Genet 1998; 81: 282–289.
7 Leonard S, Gault J, Moore T et al: Further investigation of a chromosome 15 locus in
schizophrenia: analysis of affected sibpairs from the NIMH Genetics Initiative.
Am J Med Genet 1998; 81: 308–312.
8 Riley BP, Makoff A, Mogudi-Carter M et al: Haplotype transmission disequilibrium and
evidence for linkage of the CHRNA7 gene region to schizophrenia in Southern African
Bantu families. Am J Med Genet 2000; 96: 196–201.
9 Gejman PV, Sanders AR, Badner JA, Cao Q, Zhang J.: Linkage analysis of schizophrenia
to chromosome 15. Am J Med Genet 2001; 105: 789–793.
10 De Luca V, Wong AHC, Muller D, Wong GWH, Tyndale R, Kennedy JL: Evidence
of association between smoking and alpha7 nicotinic receptor subunit gene in
schizophrenia patients. Neuropsychopharmacology 2004; 29: 1522–1526.
11 Neves-Pereira M, Bassett AS, Honer WG, Lang D, King NA, Kennedy JL: No evidence
for linkage of the CHRNA7 gene region in Canadian schizophrenia families. Am J Med
Genet 1998; 81: 361–363.
12 Curtis L, Blouin JL, Radhakrishna U et al: No evidence for linkage between
schizophrenia and markers at chromosome 15q13-14. Am J Med Genet 1999; 88:
109–112.
13 Meyer J, Ortega G, Schraut K et al: Exclusion of the neuronal nicotinic acetylcholine
receptor alpha7 subunit gene as a candidate for catatonic schizophrenia in a
large family supporting the chromosome 15q13-22 locus. Mol Psychiatry 2002; 7:
220–223.
14 Stassen HH, Bridler R, Hagele S et al: Schizophrenia and smoking: evidence for a
common neurobiological basis? Am J Med Genet 2000; 96: 173–177.
15 Leonard S, Freedman R: Genetics of chromosome 15q13-q14 in schizophrenia.
Biol Psychiatry 2006; 60: 115–122.
16 Mulley JC, DibbensLM: Chipping
complex genetics: the 15q13.3 microdeletion shows the way. Genome Med 2009;
1: 33.
17 Riley BP, Williamson M, Collier DA, Wilkie H, Makoff AJ A: 3Mb map of a large
segmental duplication overlapping a7 nicotinic acetylcholine receptor gene (CHRNA7)
at human 15q13– q14. Genomics 2002; 79: 197–209.
18 Makoff A, Flomen RH: Detailed analysis of 15q11-q14 sequence fully reveals large
segmental duplications at breakpoints for Prader –Willi and Angelman syndromes and
other 15q genomic disorders. Genome Biol 2007; 8: R114.1–R114.16.
19 Flomen RH, Collier DA, Osborne S et al: Association study of CHRFAM7A copy number
and 2 bp deletion polymorphisms with schizophrenia and bipolar affective disorder.
Am J Med Genet 2006; 141: 571–575.
away atthecommon epilepsieswith
20 Flomen RH, Davies AF, Di Forti M et al: The copy number variant involving part of
the alpha 7 nicotinic receptor gene (CHRNA7) contains a polymorphic inversion.
Eur J Hum Genet 2008; 16: 1364–1371.
21 Sinkus ML, Lee MJ, Gault J et al: A 2-base pair deletion polymorphism in the partial
duplication of the alpha7 nicotinic acetylcholine gene (CHRFAM7A) on chromosome
15q14 is associated with schizophrenia. Brain Res 2009; 1291: 1–11.
22 Hong CJ, Lai IC, Liou LL, Tsai SJ: Association study of the human partially duplicated
alpha7 nicotinic acetylcholine receptor genetic variant with bipolar disorder. Neurosci
Lett 2004; 355: 69–72.
23 Raux G, Bonnet-Brilhault F, Louchart S et al: The -2 bp deletion in exon 6 of the ‘alpha
7-like’ nicotinic receptor subunit gene is a risk factor for the P50 sensory gating
deficit. Mol Psychiatry 2002; 7: 1006–1011.
24 Dempster EL, Toulopoulou T, McDonald C et al: Episodic memory performance
predicted by the 2 bp deletion in exon 6 of the ‘alpha 7-like’ nicotinic receptor
subunit gene. Am J Psychiatry 2006; 163: 1832–1834.
25 Stefansson H, Rujescu D, Cichon S et al: Large recurrent microdeletions associated
with schizophrenia. Nature 2008; 455: 232–236.
26 International Schizophrenia Consortium. Rare chromosomal deletions and duplications
increase risk of schizophrenia. Nature 2008; 455: 237–241.
27 Helbig I, Mefford HC, Sharp AJ et al: 15q13.3 microdeletions increase risk of
idiopathic generalized epilepsy. Nat Genet 2009; 41: 160–162.
28 van Bon BW, Mefford HC, Menten B et al: Further delineation of the 15q13
microdeletion and duplication syndromes: a clinical spectrum varying from
non-pathogenic to a severe outcome. J Med Genet 2006; 46: 511–523.
29 Shinawi M, Schaaf CP, Bhatt SS et al: A small recurrent deletion within 15q13.3 is
associated with a range of neurodevelopmental phenotypes. Nat Genet 2009; 41:
1269–1271.
30 Freedman R, Adler L, Myles-Worsley M et al: Inhibitory gating of an evoked response to
repeated auditory stimuli in schizophrenic and normal subjects. Archives Gen
Psychiatr 1996; 53: 1114–1121.
31 Shaikh M, Hall MH, Schulze K et al: Do COMT, BDNF and NRG1 polymorphisms
influence P50 sensory gating in psychosis? Psychol Med 2011; 41: 263–276.
32 Bramon E, Rabe-Hesketh S, Sham P, Murray RM, Frangou S: Meta-analysis of the
P300 and P50 waveforms in schizophrenia. Schizophr Res 2004; 70: 315–329.
33 Adler LE, Pachtman E, Franks RD, Pecevich M, Waldo MC, Freedman R: Neurophy-
siological evidence for a defect in neuronal mechanisms involved in sensory gating in
schizophrenia. Biol Psychiatry 1982; 17: 639–654.
34 Hall MH, Rijsdijk F, Kalidindi S et al: Genetic overlap between bipolar illness and
event-related potentials. Psychol Med 2007; 37: 667–678.
35 Hall MH, Rijsdijk F, Picchioni M et al: Substantial shared genetic influences on
schizophrenia and event-related potentials. Am J Psychiatry 2007; 164: 804–812.
36 Franks R, Adler L, Waldo M, Alpert J, Freedman R: Neurophysiological studies of
sensory gating in mania: comparison with schizophrenia. Biol Psychiatry 1983; 18:
989–1005.
37 Freedman R, Adler L, Waldo M, Pachtman E, Franks R: Neurophysiological evidence
for a defect in inhibitory pathways in schizophrenia: comparison of medicated and
drug-free patients. Biol Psychiatry 1983; 18: 537–551.
38 Freedman R, Adler LE, Gerhardt GA et al: Neurobiological studies of sensory gating in
schizophrenia. Schizophr Bull 1987; 13: 669–678.
39 Nagamoto HT, Adler LE, Waldo MC, Freedman R: Sensory gating in schizophrenics and
normal controls: effects of changing stimulation interval. Biol Psychiatry 1989; 25:
549–561.
40 Baker N, Staunton M, Adler L et al: Sensory gating deficits in psychiatric inpatients:
relation to catecholamine metabolites in different diagnostic groups. Biol Psychiatry
1990; 27: 519–528.
41 Judd LL, McAdams L, Budnick B, Braff DL: Sensory gating deficits in schizophrenia:
new results. Am J Psychiatry 1992; 149: 488–493.
42 Ward PB, Hoffer LD, Leibert B, Catts SV, O’Donnell M, Adler LE: Replication of a P50
auditory gating deficit in Australian patients with schizophrenia. Psychiat Res 1996;
64: 121–135.
43 Clementz BA, Geyer MA, Braff DL: P50 suppression among schizophrenia and
normal comparison subjects: a methodological analysis. Biol Psychiatry 1997; 41:
1035–1044.
44 Clementz BA, Geyer MA, Braff DL: Multiple site evaluation of P50 suppression
among schizophrenia and normal comparison subjects. Schizophr Res 1998; 30:
71–80.
45 Olincy A, Martin L: Diminished suppression of the P50 auditory evoked potential
in bipolar disorder subjects with a history of psychosis. Am J Psychiatry 2005; 162:
43–49.
46 Schulze KK, Hall MH, McDonald C et al: P50 auditory evoked potential suppression in
bipolar disorder patients with psychotic features and their unaffected relatives. Biol
Psychiatry 2007; 62: 121–128.
47 Sanchez-Morla EM, Garcia-Jimenez MA, Barabash A et al: P50 sensory gating deficit
is a common marker of vulnerability to bipolar disorder and schizophrenia. Acta
Psychiatr Scand 2008; 117: 313–318.
48 Siegel C, Waldo M, Mizner G, Adler L, Freedman R: Deficits in sensory gating in
schizophrenic patients and their relatives. Arch Gen Psychiatry 1984; 41: 607–612.
49 Waldo MC, Adler LE, Freedman R: Defects in auditory sensory gating and
their apparent compensation in relatives of schizophrenics. Schizophr Res 1988; 1:
19–24.
50 Waldo M, Myles-Worsley M, Madison A, Byerley W, Freedman R: Sensory gating
deficits in parents of schizophrenics. Am J Med Genet 1995; 60: 506–511.
Association of CHRFAM7A and P50 sensory gating
R Flomen et al
80
European Journal of Human Genetics

Page 6

51 Stevens KE, Freedman R, Collins AC et al: Genetic correlation of inhibitory gating of
hippocampal auditory evoked response and alpha-bungarotoxin-binding nicotinic
cholinergic receptors in inbred mouse strains. Neuropsychopharmacol 1996; 15:
152–162.
52 Clementz B, Geyer M, Braff D: Poor P50 suppression among schizophrenia patients
and their first-degree biological relatives. Am J Psychiatry 1998; 155: 1691–1694.
53 Waldo MC,Adler LE,LeonardS
factors in the first-degree relatives of schizophrenic people. Biol Psychiatry 2000;
47: 231–239.
54 Young D, Waldo M, Rutledge JH, Freedman R: Heritability of inhibitory gating of the
P50 auditory-evoked potential in monozygotic and dizygotic twins. Biol Psychiatry
1996; 33: 113–117.
55 Hall MH, Schulze K, Rijsdijk F et al: Heritability and reliability of P300, P50 and
duration mismatch negativity. Behav Genet 2006; 36: 845–857.
56 Anokhin AP, Vedeniapin AB, Heath AC, Korzyukov O, Boutros NN: Genetic and
environmental influences on sensory gating of mid-latency auditory evoked responses:
a twin study. Schizophr Res 2007; 89: 312–319.
57 Thaker GK: Neurophysiological endophenotypes across bipolar and schizophrenia
psychosis. Schizophr Bull 2008; 34: 760–773.
58 Clayton D: A generalization of the transmission/disequilibrium test for uncertain-
haplotype transmission. Am J Hum Genet 1999; 65: 1170–1177.
59 Abecasis GR, Cardon LR, Cookson WO: A general test of association for quantitative
traits in nuclear families. Am J Hum Genet 2000; 66: 279–292.
60 Abecasis GR, Cookson WO, Cardon LR: Pedigree tests of transmission disequilibrium.
Eur J Hum Genet 2000; 8: 545–551.
61 Bramon E, Rabe-Hesketh S, Sham P, Murray RM, Frangou S: Meta-analysis
of the P300 and P50 waveforms in schizophrenia. Schizophr Res 2004; 70:
315–329.
et al:Familialtransmission ofrisk
62 Hall MH, Schulze K, Sham P et al: Further evidence for shared genetic effects between
psychotic bipolar disorder and P50 suppression: a combined twin and family study.
Am J Med Genet 2008; 147B: 619–627.
63 Illot NE, Saudino KJ, Asherson P: Genetic influences on attention deficit hyperactivity
disorder symptoms from age 2 to 3: a quantitative and molecular genetic investigation.
BMC Psychiatry 2010; 10: 102.
64 Zhang H, He J-W, Gao G et al: Polymorphisms in the HOXD4 gene are not associated
with peak bone mineral density in Chinese nuclear families. Acta Pharmacol Sinica
2010; 31: 977–983.
65 Gordon D, Heath SC, Liu X, Ott J: A transmission/disequilibrium test that allows for
genotyping errors in the analysis of single-nucleotide polymorphism data. Am J Hum
Genet 2001; 69: 371–380.
66 Mitchell AA, Cutler DJ, Chakravarti A: Undetected genotyping errors cause apparent
overtransmission of common alleles in the transmission/disequilibrium test. Am J Hum
Genet 2003; 72: 598–610.
67 Lai IC, Hong CJ, Tsai SJ: Association study of a nicotinic receptor variant with
schizophrenic disorders. Neuropsychobiol 2001; 43: 15–18.
68 Gault J, Robinson M, Berger R et al: Genomic organization and partial duplication of
the human alpha7 neuronal nicotinic acetylcholine receptor gene (CHRNA7).
Genomics 1998; 52: 173–185.
69 de Lucas-Cerrillo AM, Maldifassi MC, Arnalich F et al: Function of partially duplicated
human a7 nicotinic receptor subunit CHRFAM7A gene: potential implications for the
cholinergic anti-inflammatory response. J Biol Chem 2011; 286: 594–606.
70 Araud T, Graw S, Berger R et al: The chimeric gene CHRFAM7A, a partial duplication
of the CHRNA7 gene, is a dominant negative regulator of a7*nAChR function.
Biochem Pharmacol 2011; 82: 904–914.
71 Makoff A, Flomen R: Common inversion polymorphisms and rare microdeletions at
15q13.3. Eur J Hum Genet 2009; 17: 149–150.
Supplementary Information accompanies the paper on European Journal of Human Genetics website (http://www.nature.com/ejhg)
Association of CHRFAM7A and P50 sensory gating
R Flomen et al
81
European Journal of Human Genetics