Analysis of CHRNA7 rare variants in aut

RESEARCH ARTICLE

Analysis of CHRNA7 Rare Variants in
Autism Spectrum Disorder Susceptibility
Elena Bacchelli,1 Agatino Battaglia,2 Cinzia Cameli,1 Silvia Lomartire,1 Raffaella Tancredi,2
Susanne Thomson,3 James S Sutcliffe,3 and Elena Maestrini1*
1

Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy

2

Stella Maris Clinical Research Institute for Child and Adolescent Neuropsychiatry, Calambrone (Pisa), Italy
Department of Molecular Physiology & Biophysics and Psychiatry and Vanderbilt Brain Institute, Vanderbilt University,
Nashville, Tennessee

3

Manuscript Received: 16 May 2014; Manuscript Accepted: 30 September 2014

Chromosome 15q13.3 recurrent microdeletions are causally

associated with a wide range of phenotypes, including autism
spectrum disorder (ASD), seizures, intellectual disability, and
other psychiatric conditions. Whether the reciprocal microduplication is pathogenic is less certain. CHRNA7, encoding for the
alpha7 subunit of the neuronal nicotinic acetylcholine receptor,
is considered the likely culprit gene in mediating neurological
phenotypes in 15q13.3 deletion cases. To assess if CHRNA7 rare
variants confer risk to ASD, we performed copy number variant
analysis and Sanger sequencing of the CHRNA7 coding sequence
in a sample of 135 ASD cases. Sequence variation in this
gene remains largely unexplored, given the existence of a fusion
gene, CHRFAM7A, which includes a nearly identical partial
duplication of CHRNA7. Hence, attempts to sequence coding
exons must distinguish between CHRNA7 and CHRFAM7A,
making next-generation sequencing approaches unreliable for
this purpose. A CHRNA7 microduplication was detected in a
patient with autism and moderate cognitive impairment; while
no rare damaging variants were identified in the coding region,
we detected rare variants in the promoter region, previously
described to functionally reduce transcription. This study represents the first sequence variant analysis of CHRNA7 in a sample
of idiopathic autism. Ó 2015 Wiley Periodicals, Inc.

Key words: 15q13.3; neurodevelopmental disorders; copy
number variants; sequence variants

INTRODUCTION
Autism spectrum disorder (ASD) comprises a group of neurodevelopmental traits characterized by repetitive and stereotypic
behaviors and impairments in communication and social interactions, with an onset within the first three years of age. Family
studies indicate a significant genetic basis for ASD susceptibility,
but the underlying genetic architecture is highly complex and
heterogeneous. Recent genome-wide studies have documented
that common variants exert only small individual main effects
on risk, although when common variation (CV) across the genome

Ó 2015 Wiley Periodicals, Inc.

How to Cite this Article:
Bacchelli E, Battaglia A, Cameli C,
Lomartire S, Tancredi R, Thomson S,
Sutcliffe JS, Maestrini E. 2015. Analysis of
CHRNA7 rare variants in Autism spectrum

disorder susceptibility.
Am J Med Genet Part A 9999:1–9.

is considered in aggregate, CV is found to contribute measurably to
ASD risk [Klei et al., 2012]. In addition, rare inherited and de novo
copy number variants (CNVs) and single nucleotide variants
(SNVs) of large effect size have a major role in the etiology of
ASD, contributing in as many as 5–10% of idiopathic cases examined [Devlin and Scherer, 2012].
In spite of the overall relevance of CNVs in autism, each
individual risk CNVs is very rare. Recurrent CNVs, still infrequent or rare, are typically flanked by segmental duplications and
are often implicated in multiple developmental and/or neurological disorders, perhaps not surprisingly given the overlap in
phenotype across conditions. Among recurrent CNVs, the
15q13.3 microdeletion is highly but not always fully penetrant,
and it is significantly enriched in cases of intellectual disability,
autism, epilepsy, schizophrenia, and bipolar disorder [Sharp
et al., 2008; Stefansson et al., 2008; Ben-Shachar et al., 2009
Dibbens et al., 2009; Helbig et al., 2009; Miller et al., 2009;
Pagnamenta et al., 2009; van Bon et al., 2009; Cooper et al., 2011].
This 15q13.3 recurrent microdeletion, resulting in the loss of a
1.5 Mb region between low-copy repeat (LCR) sequences

Conflict of interest: The authors declare no conflict of interest.

Correspondence:
Elena Maestrini, Dept. of Pharmacy and Biotechnology, University of
Bologna, via Selmi 3, Bologna 40126, Italy.
E-mail: elena.maestrini@unibo.it
Article first published online in Wiley Online Library
(wileyonlinelibrary.com): 00 Month 2015
DOI 10.1002/ajmg.a.36847

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AMERICAN JOURNAL OF MEDICAL GENETICS PART A

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designated as breakpoints 4 and 5 (BP4 and BP5), contains six
genes (MTMR15, MTMR10, TRPM1, KLF13, OTUD7A, and
CHRNA7) and an miRNA gene (hsa-mir-211).
Strong evidence supporting CHRNA7 as responsible for the
majority of neurodevelopmental phenotypes resulting from deletion, comes from the identification of individuals carrying smaller

deletions which encompass the entire CHRNA7 gene and the first
exon of OTUD7A [Shinawi et al., 2009] or even smaller deletions,
including only CHRNA7 [Masurel-Paulet et al., 2010; Mikhail et al.,
2011; Hoppman-Chaney et al., 2013], and conveying most or all of
the phenotypic abnormalities associated with the larger 15q13.3
recurrent deletions.
Genomic imbalance at chromosome 15q13.3 also includes the
reciprocal microduplication, which has a less certain clinical significance in comparison with the deletions. Nevertheless, de novo
and inherited 15q13.3 duplications are associated with a wide
spectrum of neuropsychiatric disorders, including ASD. The clinical uncertainty of the 15q13.3 duplications could be due to the fact
that a larger sample size is necessary to detect a low penetrant effect
[Szafranski et al., 2010; Moreno-De-Luca et al., 2013].
CHRNA7 encodes the a7 subunit of the neuronal nicotinic
acetylcholine receptor, which is the only subunit able to form a
homopentameric chloride channel receptor, and is highly expressed
in the brain. Receptors containing a7 are localized both pre- and
post-synaptically and regulate the release of both the inhibitory
neurotransmitter GABA and the excitatory neurotransmitter glutamate in the hippocampal formation [Albuquerque et al., 2009].
Alpha-7 nicotinic acetylcholine receptor mediated signaling causes
an influx of Ca2þ into the cell [Vijayaraghavan et al., 1992].

Mobilization of intracellular Ca2þ plays a critical role in synaptic
plasticity and immediate early gene expression associated with
learning and memory [Benfenati, 2007], thus supporting CHRNA7
involvement in the cognitive deficits apparent in neuropsychiatric
disorders. Furthermore, a recent study reported a significantly
reduced CHRNA7 expression in the frontal cortex of individuals
with Rett syndrome or with typical ASD [Yasui et al., 2011]. The
binding of the methyl CpG binding protein 2 (MeCP2), encoded by
MECP2, influences the chromatin loop organization of the much
larger 15q11.2–13.3 region that includes the Prader-Willi/Angelman syndrome region, and is required for optimal expression of AS/

PWS region genes implicated in the ASD phenotype
[Yasui et al., 2011]. Therefore these discoveries suggest that transcription of CHRNA7 is modulated by these regulatory elements
and is involved in ASD-like phenotypes [Yasui et al., 2011].
Based on the evidence that CHRNA7 may be responsible for the
majority of the spectrum of abnormal phenotypic features of
patients with 15q13.3 CNVs, we decided to focus our studies on
this candidate gene. In order to capture the entire spectrum of
genetic variation in CHRNA7 contributing to ASD risk, it is
essential to integrate both CNV and sequence data. However,

mutation screening of CHRNA7 is complicated by the existence
of a 300 kb duplication, which contains exons 5–10 and the 30 end of
CHRNA7; the duplicated portion of CHRNA7 is fused to exons A–E
of FAM7A resulting in a hybrid gene known as CHRFAM7A [Gault
et al., 1998]. Hence, any attempts at sequencing to detect mutations
must distinguish the nearly identical sequence for the interval
containing exons 5–10 from CHRNA7 and CHRFAM7A. This
renders next-generation sequencing approaches unreliable for
this purpose.
In this study, we have performed CNV analysis and sequence
mutation screening of the coding sequence of the CHRNA7 gene
in a sample of 135 Italian ASD probands, with the aim of
investigating if rare variants in CHRNA7 could play an important
role in ASD.

METHODS
Patients
A total of 135 Italian individuals with ASD from 133 families, were
recruited at the Stella Maris Clinical Research Institute for Child
and Adolescent Neuropsychiatry (Calambrone, Pisa, Italy). ASD

diagnosis was based on the Autism Diagnostic Interview-Revised
(ADI-R) and the Autism Diagnostic Observation Schedule
(ADOS); and a clinical evaluation was undertaken in order to
exclude known syndromes associated with autism. Standard karyotyping, fragile-X testing, EEG, and array-based comparative
genomic hybridization (aCGH) were obtained for all probands.
The main clinical features of the 135 Italian ASD cases included in
this study are reported in Table I. The control sample consists of 174
unrelated Italian individuals with no psychiatric disorders.

TABLE I. Description of the Italian Cohort of ASD Patients Included in CHRNA7 Mutation Screening
Sex

Autism
Atypical
autism
Asperger
syndrome
Total

Intellectual disability

Female
22
6

Epilepsy
2
0

Severe
(20T
c.27C>T
(rs149637464)
Exon 10

c.1–70_1–69delG
g.32322616 delG
Promoter ( 182)

c.1–79G>A

AA ¼ 172;
AG ¼ 2 (0.006)
GG ¼ 174;
GA ¼ 0 (0)
GG ¼ 174;
G/delG ¼ 0 (0)
GG ¼ 122;
GA ¼ 3 (0.012)
AA ¼ 132;
AG ¼ 3 (0.011)
GG ¼ 134;
GA ¼ 1 (0.004)
GG ¼ 134;
G/delG ¼ 1 (0.004)
GG ¼ 134;
GA ¼ 1 (0.004)
c.1–129A>G

EUR*, Europeans include Utah Residents (CEPH) with Northern and Western European ancestry (CEU); TSI, Toscani in Italia; FIN, Finnish in Finland; GBR, British in England and Scotland; IBS, Iberian population in Spain.

—
—

CC ¼ 336;
CT ¼ 43 (0.057)

—
—

—

—

—

AA ¼ 85 (0)

AA ¼ 96;
AG ¼ 2 (0.010)
—

AA ¼ 374;
AG ¼ 5 (0.007)
—

85 CEU
genotype
count (MAF)
98 TSI
genotype
count (MAF)
Italian
controls genotype
count (MAF)
135 ASD
cases genotype
count (MAF)
Protein
NP_000737.1
cDNA

Region
hg19 position
(bp distance from ATG)
(SNP ID)
a: Rare putative functional variants
Promoter ( 241)
g.32322557A>G
(rs188889623)
Promoter ( 191)
g.32322607G>A

TABLE II. Rare Variants Identified in CHRNA7

Controls (1000 genomes)

379 EUR*
genotype
count (MAF)

6

two variants have been previously reported to have a functional
effect on CHRNA7 gene transcription, being strongly associated
with a significant decrease of promoter activity (P < 0,0001; Leonard et al., 2002). While these two variants have been individually
found in a sample of 174 Italian unaffected individuals and are
separately reported in the 1000 Genome project control individuals
(Table II), none of the 174 Italian control individuals or the 379
EUR patients carry both variants.
Segregation analysis of the -86/-241 variants in the Italian family,
showed that the two variants are not on the same chromosome, as
the -86 bp variant was inherited from the mother while the -241 bp
variant was inherited from the father. It is thus plausible that
CHRNA7 expression is significantly decreased in proband
3377_3. Unfortunately, we were not able to test this hypothesis,
since CHRNA7 mRNA is not detectable in blood.

DISCUSSION
Microdeletions of chromosome 15q13.3 have been associated with
multiple neurological and neuropsychiatric phenotypes, with the
strongest enrichment observed in cases of idiopathic generalized
epilepsy (IGE), but also among patients with ID, autism and
schizophrenia [Marshall et al., 2008; Sharp et al., 2008; Stefansson
et al., 2008; Cooper et al., 2011; Kaminsky et al., 2011; Sanders et al.,
2011]. The reciprocal microduplications of 15q13.3 have been more
challenging to interpret, being detected across the same spectrum of
neuropsychiatric disorders of the microdeletions, but with high
variability in expressivity and reduced penetrance and more often
inherited than de novo as compared with deletions.
CHRNA7 is thought to be the causative gene for the neurological
phenotypes in patients with 15q13.3 CNVs, but, given the genomic
complexity and the presence of an almost identical partial duplication, sequence analysis of the coding region has been performed
only in a handful of patients carrying the microdeletion, with
different clinical phenotypes [Masurel-Paulet et al., 2010].Therefore, in order to analyze the contribution of CHRNA7 rare variants
in ASD susceptibility, we screened a well-characterized cohort of
135 Italian ASD individuals for the presence of structural and
sequence variants.
CNV analysis led to identification of a small 15q13.3 microduplication, which involves the entire CHRNA7 gene and the first
exon of OTUD7A longer isoform, in an ASD proband. Even if it has
not been determined where the duplicated genetic material exactly
resides, the microduplication is most likely generated by NAHR
mediated by LCRs in BP4 and BP5, thus lying in tandem with itself,
as previously described [Szafranski et al., 2010]. Interestingly this
proband, beyond a clinical diagnosis of autism and moderate
cognitive impairment, developed complex partial seizures with
secondary generalization, at age of 13 years.
Whereas epilepsy has been strongly associated with microdeletions of CHRNA7 [Helbig et al., 2009; Shinawi et al., 2009], seizures
are not reported as a common features of patients carrying the
microduplication [Szafranski et al., 2010]. Notably, two peaks of
seizure onset have been reported in ASD, one in early childhood
[Volkmar and Nelson, 1990] and the other in adolescence and
continuing through adulthood [Tuchman and Cuccaro, 2011].
Therefore, it is not possible to exclude that some participants of

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BACCHELLI ET AL.
pediatric cohorts may develop epilepsy at a later time. To better
understand the complex genotype-phenotype correlations of the
reciprocal microdeletions and microduplication, a detailed clinical
characterization of the individuals carrying the CNV would be very
useful, especially if followed up over time. Nevertheless, the cooccurrence of ASD and epilepsy in the proband with the CHRNA7
duplication, may suggest that the microduplication involving
CHRNA7 could have the same role of the deletion in ASD/epilepsy
susceptibility although with lower penetrance. In accordance with
this hypothesis, CHRNA7 transcript levels were recently found
reduced in neuronal cells [Meguro-Horike et al., 2011] or brain
samples with maternal 15q duplication [Hogart et al., 2009], in
contrast to what is expected according to the gene copy number.
The observation that deletions and duplications at the same locus
may yield similar phenotypes is quite common and it could be
explained by the sensitivity of certain cellular functions to dosage
imbalance, as described for the 1q21.1 region [Harvard et al., 2011].
Further evidence for dosage sensitivity of CHRNA7 has been
provided by a very recent study that shows the co-segregation of
a CHRNA7 triplication with neuropsychiatric and cognitive phenotypes in a three generations family [Soler-Alfonso et al., 2014].
The phenotypic variability at locus 15q13.3 may also be controlled by second-site CNVs, in line with the recently proposed
“two-hit model” for severe developmental delay [Girirajan et al.,
2010]. Support to this model comes from a recent study where five
out of 11 patients with small microduplications involving CHRNA7
and showing a variety of neuropsychiatric disorders, carried at least
one additional different CNV of potential clinical relevance
[Szafranski et al., 2010]. However, no other clearly pathogenic
CNV has been identified in proband 3474_3, and he does not carry
any of the CHRNA7 sequence variants listed in Table II, even if we
cannot exclude the presence of a sequence variant elsewhere in the
genome that could act within the same pathway to increase the risk
of ASD. Interestingly, two ASD individuals carrying a small
CHRNA7 duplication and a de novo SHANK2 deletion on distinct
parental chromosomes, were recently observed, suggesting the
presence of epistasis between these two loci [Leblond et al.,
2012]. Another report described a boy with severe ID, language
impairment, and behavioral anomalies, carrying a de novo balanced
translocation disrupting the SHANK2 gene as well as an inherited
duplication of CHRNA7 [Chilian et al., 2013]. Thus CHRNA7
duplications might act as modifier in presence of other variants of
larger effect.
Mutation screening of the coding and putative regulatory
regions of CHRNA7 led to the identification of only one nonsynonymous rare variant (p.E452K), which is predicted benign by
bioinformatics tools and it is found with a similar frequency in
controls, therefore not supporting its role in ASD susceptibility.
However, three rare variants have been identified in the proximal
promoter region, one of which (-241 bp from ATG) was found in
one ASD individual who also carries a more frequent 5’-UTR
variant (-86 bp from ATG) on the other chromosome. Since the
presence of each of these two variants has been previously associated
with CHRNA7 decreased transcription in vitro [Leonard et al.,
2002], it is reasonable to suppose a marked reduction of expression
in presence of the double variant. Unfortunately it was not possible
to test the functional effect of this double variant in the carrier

proband 3377_3, as CHRNA7 expression is too low in blood to be
assessed by RT-PCR.
Our results do not support the hypothesis that rare sequence
variants in CHRNA7 contribute to ASD susceptibility in this
current Italian cohort. It is notable that our ASD sample has an
overall lesser degree of medical comorbidities, e.g., the frequency of
epilepsy in our sample is only 1.5% while it is commonly reported to
occur in 30% of individuals with ASD, but prevalence estimates
have varied widely, ranging from 5% to 46% [Viscidi et al., 2013].
Analysis of much larger cohorts of individuals is thus warranted to
elucidate the role of rare CHRNA7 sequence variants in ASD risk
and to discriminate if CHRNA7 might be mainly implicated in ASD
cases associated to other clinical features that would be consistent
with the significant enrichment of 15q13.3 CNVs observed in
individuals with comorbid phenotypes.
In conclusion, even if the sample size of this study is limited, our
results are valuable as they represent the first sequence analysis of
the CHRNA7 coding region in a sample of idiopathic autism.

ACKNOWLEDGMENTS
We gratefully thank all the families who have participated in the
study and the professionals who made this study possible. We thank
the Autism Genome Project (AGP) for family 3474 CNV data. This
work was supported by University of Bologna (RFO).

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