Structures and Molecular Mechanisms for Common
15q13.3 Microduplications Involving CHRNA7: Benign
Przemyslaw Szafranski,1yChristian P. Schaaf,1yRichard E. Person,1Ian B. Gibson,1Zhilian Xia,1Sangeetha Mahadevan,1
Joanna Wiszniewska,1Carlos A. Bacino,1Seema Lalani,1Lorraine Potocki,1Sung-Hae Kang,1Ankita Patel,1
Sau Wai Cheung,1Frank J. Probst,1Brett H. Graham,1Marwan Shinawi,1Arthur L. Beaudet,1and Pawel Stankiewicz1,2?
1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas;2Department of Medical Genetics, Institute
of Mother and Child, Warsaw, Poland
Communicated by Jacques S. Beckmann
Received 27 January 2010; accepted revised manuscript 26 April 2010.
Published online 10 May 2010 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/humu.21284
ABSTRACT: We have investigated four ?1.6-Mb micro-
duplications and 55 smaller 350–680-kb microduplica-
tions at 15q13.2–q13.3 involving the CHRNA7 gene that
were detected by clinical microarray analysis. Applying
high-resolution array-CGH, we mapped all 118 chromo-
somal breakpoints of these microduplications. We also
sequenced 26 small microduplication breakpoints that
were clustering at hotspots of nonallelic homologous
recombination (NAHR). All four large microduplica-
tions likely arose by NAHR between BP4 and BP5 LCRs,
and 54 small microduplications arose by NAHR between
two CHRNA7-LCR copies. We identified two classes of
?1.6-Mb microduplications and five classes of small
microduplications differing in duplication size, and show
that they duplicate the entire CHRNA7. We propose that
size differences among small microduplications result
from preexisting heterogeneity of the common BP4–BP5
inversion. Clinical data and family histories of 11 patients
with small microduplications involving CHRNA7 suggest
that these microduplications might be associated with
developmental delay/mental retardation, muscular hypo-
tonia, and a variety of neuropsychiatric disorders.
However, we conclude that these microduplications and
their associated potential for increased dosage of the
CHRNA7-encoded a7 subunit of nicotinic acetylcholine
receptors are of uncertain clinical significance at present.
Nevertheless, if they prove to have a pathological effects,
their high frequency could make them a common risk
factor for many neurobehavioral disorders.
Hum Mutat 31:840–850, 2010. & 2010 Wiley-Liss, Inc.
KEY WORDS: microduplication; CHRNA7; NAHR; hy-
potonia; autism spectrum disorder
Genomic regions flanked by low-copy repeats (LCRs, or
segmental duplications) with DNA sequence identity greater than
95–97% are prone to recurrent microdeletions, microduplications,
and inversions mediated by nonallelic homologous recombination
(NAHR) [Lupski, 1998; Stankiewicz and Lupski, 2002]. The LCR-
rich proximal region of chromosome 15 (15q11–q14) is one of the
most unstable regions in the human genome [Knoll et al., 1993;
Makoff and Flomen, 2007; Toth-Fejel et al., 1995]. DNA copy-
number variations (CNVs) in this region include deletions,
peri- and para-centric inversions, duplications, triplications,
translocations, and supernumerary inv dup(15) chromosomes
[Jauch et al., 1995; Rivera et al., 1990; Schinzel et al., 1994;
Woodage et al., 1994]. Breakpoints of these rearrangements are
located within complex sets of LCRs named BP1 to BP6 (Fig. 1).
Deletions between BP1 or BP2 on the proximal side and BP3 on
the distal side result in Prader-Willi or Angelman syndromes
[Amos-Landgraf et al., 1999; Carrozzo et al., 1997; Christian et al.,
1999; Robinson et al., 1998]. Duplications of the region flanked by
BP1 or BP2 and BP3 are associated with learning disabilities,
autism, and seizures [Cook et al., 1997; Dennis et al., 2006].
An ?1.6Mb recurrent microdeletion of a region between more
distal LCRs, BP4, and BP5, and harboring six RefSeq genes:
MTMR15, MTMR10, TRPM1 (MIM] 603561), KLF13 (MIM]
605328), OTUD7A (MIM] 612024), CHRNA7 (MIM] 118511), and
one miRNA hsa-mir211 gene, has been found in patients with
developmental delay/mental retardation, epilepsy, autism, schizo-
phrenia, and other neurocognitive disorders [Ben-Shachar et al.,
2009; Dibbens et al., 2009; International Schizophrenia Consortium,
2008; Helbig et al., 2009; Miller et al., 2009; Pagnamenta et al., 2009;
Sharp et al., 2008; Stefansson et al., 2008; van Bon et al., 2009].
In many human populations, the BP4 LCR in this genomic
region also harbors the chimeric CHRFAM7A gene. CHRFAM7A
has not been found in nonhuman primates, and its occurrence in
individuals of African descent is significantly lower than in
Caucasian populations [Gault et al., 1998; Stassen et al., 2000].
Recently, Sinkus et al.  suggested that CHRFAM7A has
arisen during human evolution. CHRFAM7A is composed of a
copy of the exon 5–10 portion of the CHRNA7 gene fused with a
copy of the FAM7A gene. Its expression pattern and function are
not well known.
Sharp et al.  have shown that the BP4–BP5 region is
inverted in 44% of individuals of varied ethnicities in the study
population. This inversion likely results from NAHR between
& 2010 WILEY-LISS, INC.
yPrzemyslaw Szafranski and Christian P. Schaaf contributed equally to this work.
Current address for Marwan Shinawi: Division of Genetics and Genomic Medicine,
Department of Pediatrics, Washington University School of Medicine, St. Louis, MO.
?Correspondence to: Pawel Stankiewicz, Department of Molecular & Human
Genetics, Baylor College of Medicine, One Baylor Plaza, Rm. R809, Houston,
TX 77030. E-mail: email@example.com
Additional Supporting Information may be found in the online version of this article.
oppositely oriented BP4 and BP5 LCRs, and apparently predis-
poses to 15q13.3 microdeletions. Recently, we have described
recurrent small 680-kb deletions and suggested that haploinsuffi-
ciency of CHRNA7 is causative for the majority of neurodevelop-
mental phenotypes observed in the 15q13.3 microdeletion
syndrome [Shinawi et al., 2009]. CHRNA7 encodes the a7 subunit
of the neuronal nicotinic acetylcholine receptor (nAChR), which
forms pentameric ligand-gated cation channels.
Little is known, however, about the reciprocal 15q13.3
microduplications. Van Bon et al.  reported four patients
with cognitive impairment and psychiatric disorders coinciding
with a BP4–BP5 microduplication. However, due to the limited
number of individuals enrolled in those studies, it is difficult to
conclude that this microduplication contributes to the etiology of
Here, we report the molecular characterization of four ?1.6Mb
BP4–BP5 and 55 small 350–680kb microduplications involving
CHRNA7, along with clinical phenotyping of 11 index patients
with small microduplications and analysis of their extended
Subjects and Methods
Fifty-five small 15q13.3 microduplications involving CHRNA7
[Shinawi et al., 2009] and four large BP4–BP5 microduplications
were detected in 8,832 unrelated subjects referred for Chromo-
somal Microarray Analysis (CMA) at the Medical Genetics
Laboratories (MGL) at Baylor College of Medicine (BCM). In
this series, there were predominantly children with developmental
delay/mental retardation, multiple congenital anomalies, dys-
morphic features, autism or autistic spectrum, seizures, or others.
CMA was performed using array-based comparative genomic
hybridization (aCGH) with oligonucleotide-based version 6 (V6
OLIGO, 44K) (?4,000 patients), version 7 (V7 OLIGO, 105K)
(?4,000 patients), and version 8 (V8 OLIGO, 180K) (?900
patients), designed by Baylor Medical Genetics Laboratories
(http://www.bcm.edu/geneticlabs/) and manufactured by Agilent
Technology (Santa Clara, CA) as previously described [El-Hattab
et al., 2009; Lu et al., 2007; Ou et al., 2008]. Anonymized DNA
microarrays (NimbleGen). BP1, BP2, and BP3 are LCRs flanking the common recurrent Prader-Willi and Angelman syndrome deletions; P and D
are proximal and distal copies of the CHRNA7-LCR, respectively. A: One copy of the B segment of BP4 or BP5 is deleted. B: One copy of both A
and B segments of BP4 or BP5 is deleted. C: One copy of the A segment of BP4 or BP5 is duplicated. D: Neither BP4 nor BP5 show a change in
copy number compared to the control. E: A smaller sized CHRNA7 microduplication with proximal breakpoint mapping closer to the 50end of the
OTUD7A gene. All gains and losses are relative to the control sample used.
Results of aCGH analysis of 55 small microduplications in 15q13.3 using region-specific high-resolution 12?135K oligonucleotide
HUMAN MUTATION, Vol. 31, No. 7, 840–850, 2010
samples were obtained from 55 individuals with small micro-
duplications and four subjects with BP4–BP5 microduplications.
Eleven probands with small microduplications were examined by
C.P.S. at the outpatient Genetics Clinic at Texas Children’s
Hospital and informed consent was obtained (approved by the
Institutional Review Board for Human Subject Research at Baylor
College of Medicine, H-12971 and H-24566). Five of these
patients were of European, four of Hispanic and two of Asian
descent. In 15 of 46 patients, additional CNVs were identified
(Table 1, Supp. Table S1).
DNA was prepared from peripheral blood using the Puregene
DNA isolation kit (Gentra Systems, Minneapolis, MN).
aCGH. The 12?135K custom-designed microarrays (NimbleGen
Systems, Madison, WI) were hybridized according to the
manufacturer’s protocol. Briefly, 0.5mg of patient genomic DNA
and 0.5mg of a normal male reference DNA sample were labeled
by random priming with Cy3 and Cy5, respectively, using the
NimbleGen Dual Color labeling kit. Microarrays were scanned on
the GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA).
Scans were processed using NimbleScan v2.5, and analyzed with
the SignalMap v1.9 (NimbleGen Systems). Array data have been
deposited in GEO database under accession number GSE21268.
Verification of this analysis was performed using high-
resolution 60K microarrays designed with the use of eArray
(Agilent Technologies, Santa Clara, CA). The array consisted of
50,508 variable length probes with uniform Tm mapping between
chr15:27,779,649 and 30,810,128 (hg18), or approximately one
probe every 60bp. Two mg of test sample and control DNA in
100ml of H2O, was sheared to an average size of 300bp using a
Fisher Scientific Sonic Dismembrator Model 500 (three times for
15sec at 14% power interrupted by 30-sec pause intervals). Test
and control samples were labeled with Cy5 and Cy3, respectively,
using the Bioprime Array CGH Genomic Labelling System
(Invitrogen, Carlsbad, CA). From each sample, 900ng were
cohybridized for 20hr. Slides were washed and scanned using an
Agilent DNA Microarray Scanner. Data were normalized and
plotted using Origin 8 software (OriginLab, Northampton, MA).
were mapped usingthe
Multiplex Ligation-Dependent Probe Amplification (MLPA)
Probes for MLPA analysis were designed using the freely
available H-MAPD Web server (http://genomics01.arcan.stony-
brook.edu/mlpa/cgi-bin/mlpa.cgi) and mapped in exons for the
following genes: APBA2, NDNL2, TJP1, ARHGAP11B, MTMR15,
MTMR10, TRPM1, KLF13, and OTUD7A. For CHRNA7, probes
were placed in exons 2 and 4. For CHRNA7/CHRFAM7A,
probes were placed in exons 5 and 10 (Supp. Table S2). MLPA
reactions were carried out using SALSA MLPA reagents and P300
reference probe mix as per instructions (MRC-Holland, Amster-
dam). MLPA product (1.1ml) and 0.25ml of GS500 Liz Size
Standard was added to 10ml of formamide and loaded onto an
ABI 3730xl capillary electrophoresis machine (Applied Biosys-
tems, Foster City, CA). Data were analyzed using GeneMarker
MLPA analysis software (SoftGenetics, State College, PA).
Long-Range PCR and DNA Sequencing
Long-range PCR primers were designed to harbor at least three
to four nucleotides specific in one primer to proximal CHRNA7-
LCR and in the other primer to distal CHRNA7-LCR, to allow
preferential amplification of the predicted chimeric fragment
containing the junction between parts of proximal and distal
CHRNA7-LCRs, but not fragments of the original CHRNA7-LCRs
[Shinawi et al., 2009]. The primers were design using Primer 3
software (http://www.frodo.wi.mit.edu/primer3). Amplification of
10–20kb fragments was performed using Takara LA Taq
Polymerase (TaKaRa Bio USA, Madison, WI) following the
manufacturer’s protocol. Briefly, we used 25ml reaction mixtures
containing 100ng genomic DNA, 0.4mM dNTP (each), 0.2mM
primers (each), and 1.25U of Taq polymerase. PCR conditions
were: 941C for 1min, followed by 30 cycles at 941C for 30sec, and
681C for 12min, and 721C for 10min. In cases with residual
amplification of the normal alleles from the normal control DNA,
we adjusted the specificity of PCR conditions by increasing
the annealing temperature to 701C. The PCR products were
treated with ExoSAP-IT (USB, Cleveland, OH) to remove
unconsumed dNTPs and primers, and directly sequenced by the
dye-terminator method (Lone Star Labs, Houston, TX) using the
primers used to amplify these DNA fragments and primers
specific for both proximal and distal copies of the CHRNA7-LCR
(Supp. Table S3).
DNA Sequence Analysis
The genomic sequences defined by coordinates identified in the
aCGH experiments, were downloaded from the UCSC genome
browser (NCBI build 36, March 2006, http://www.genome.ucsc.
edu) and assembled using the Sequencher v4.8 software (Gene
Codes, Ann Arbor, MI). Interspersed repeat sequences were
identified using RepeatMasker (http://www.repeatmasker.org).
DNA secondary structure was analyzed with Mfold (http://
mfold.bioinfo.rpi.edu). The NAHR site sequences have been
deposited in GenBank database with the accession numbers
Structural Heterogeneity of Microduplications
Microduplications were initially detected using Agilent clinical
targeted arrays covering the BP4–BP5 region at 15q13.2–q13.3.
Chromosomal breakpoints of the microduplications were more
precisely mapped using two separate custom-designed high
resolution microarray platforms: 135K NimbleGen and 60K
Agilent oligonucleotide arrays. Additionally, the microduplica-
tions were further verified using MLPA. An example of the data
for one class of microduplication is shown in Supp. Figure S1.
Fifty-four of the 55 small microduplications were ?430–680kb
in size and included the interval between the distal CHRNA7-LCR
and BP5 (Fig. 1, Table 2). The majority of both the small and the
large microduplications were accompanied by deletion or
duplication of the A or B segment of BP4 or BP5 (Figs. 1–3).
The most common was the association of the ?430kb
microduplication with the deletion of a copy of the B segment
of BP4 or BP5. It was found in 45 of 55 microduplication cases
(class 1, Fig. 1). The mechanisms of formation of these
microduplications proposed in Figure 2A and B assume that
HUMAN MUTATION, Vol. 31, No. 7, 840–850, 2010
Phenotypic Features in 11 Unrelated Individuals with Small Microduplication of CHRNA7
Class of CHRNA7
Other CMA abnormalities
none dup Xq25 (mat)b
del 8q24.13q24.21 (de
dup 6q27 (not mat, father
ASD, Disruptive behavior
ADHD, Bipolar disorder
M, male; F, female; yo, years old; pat, paternal; mat, maternal; CMA, chromosome microarray; dup, duplication; del, deletion; DD, developmental delay; MR, mental retardation; GDD, global developmental delay; ASD, autism spectrum disorder;
ADHD, attention-deficit/hyperactivity disorder.
aarr 5p15.2(9021335-9243037)?3.nuc ish 5p15.2(RP11-109L5?3)mat Gain in copy number in the 5p15.2 region of the short arm of chromosome 5, spanning a minimum of 0.222Mb and a maximum of 0.261Mb.
barr Xq25(123313165-123849841)?2.nuc ish Xq25(RP11-107O17?2)mat Gain in copy number in the long arm of chromosome X, spanning a minimum of 0.537Mb and a maximum of 0.571Mb.
carr 4q22.3(RP11-369I16)?1.ish del(4)(q22.3q22.3)(RP11-369I16dim)mat. This loss is approximately 140kb in the proximal region covered by the clone. The nearest adjacent clone with no copy number change is 5.4Mb proximal to the deleted
darr 8q24.13q24.21(127185755-128672802)?1.ish del(8)(q24.13q24.13)(RP11-248A1-)dn. Loss in copy number in the 8q24.13q24.21 region of the long arm of chromosome 8, spanning a minimum of 1.487Mb and a maximum of 1.564Mb.
earr 6q27(168458422-168622241)?3.nuc ish 6q27(RP11-673P11?3) Gain in copy number in the 6q27 region of the long arm of chromosome 6, spanning a minimum of 0.164Mb and a maximum of 0.932Mb. FISH analysis with clone RP11-
673P11 on chromosome 6 showed no evidence of the same gain in the mother.
HUMAN MUTATION, Vol. 31, No. 7, 840–850, 2010
BP4–BP5 region, both in the common and inverted orientation,
carry a copy of CHRFAM7A gene. Matters are even more
complicated as explained in the Discussion, because there is
limited information as to the proportion of chromosomes with
the common or inverted BP4–BP5 region that do or do not carry a
copy of CHRFAM7A.
The class 1 microduplications could arise on the background of
two slightly different inversions. One of the inversions would
result in a novel fusion of at least first four exons of CHRNA7 with
the remaining paralogous CHRNA7 exons that are part of the
fusion CHRFAM7A gene (class 1?). We designate this novel fusion
Selected Small and BP4–BP5 Microduplications in 15q13.3
Representing All Different Classes Derived from Region-Specific
High-Resolution Array-CGH Analyses
Genomic Coordinates (hg 18) of the Randomly
MicroduplicationClassPatient Proximal breakpoint Distal breakpoint
BP4-BP51 19 28,156,762
events leading to the variety of small microduplications involving
CHRNA7. CHRNA7-LCRs are depicted in red. The yellow rectangle
represents the ?90-kb segment in BP4 that is duplicated in patients
with small microdeletion and deleted in patients with reciprocal
microduplication (class 1 or 3). A, B: The most common class 1/1?
microduplication is shown. A: Class 1 microduplication with inver-
sion breakpoints mapping within the A segment of BP4 and BP5
(brackets), but further downstream of CHRFAM7A and CHRNA7,
respectively. NAHR between proximal and distal CHRNA7-LCRs
leads to the loss of a copy of the BP4B repeat. B: Class 1?
lines) mapping in intron 4 of CHRNA7 and its homologous part of
CHRFAM7A within the A portion of BP5 and BP4, respectively. Note
that the entire true copy of CHRNA7 is duplicated in A and C, and the
fusion of the upstream portion of the true copy of CHRNA7 (exons 1–4)
with the downstream portion of the CHRFAM7A gene (homologous
to exons 5–10 of CHRNA7) is generated due to BP4–BP5 inversion.
C: Class 3 of the small microduplications is depicted. NAHR between
proximal and distal CHRNA7-LCRs leads to the duplication of the A
repeat of BP5.
Schematic representation of the proposed recombination
HUMAN MUTATION, Vol. 31, No. 7, 840–850, 2010
gene as CHRNA7?. In the second inversion, the entire true copy of
CHRNA7 would be inverted (class 1). Depending on whether the
breakpoint in the inversion chromosome occurred in intron 4
fusing all of the 5–10 exons of the CHRFAM7A to CHRNA7 exons
1–4, occurred downstream of exon 10 leaving the entire CHRNA7
gene intact, or occurred in an intermediate site, the inverted
CHRNA7 can be comprised entirely of exons derived from exons
5–10 of either CHRNA7 or CHRFAM7A or of varying combina-
tions of proximal exons from CHRNA7 and distal exons from
CHRFAM7A. The microarray data presented in Figure 1 cannot
distinguish between class 1 and class 1?microduplications. We do
not know the relative proportion of class 1 versus class 1?
microduplications among the 45 microduplications of the broadly
defined class 1. Class 1 microduplications might be more likely to
overexpress CHRNA7, while the CHRNA7?copy in class 1?
microduplication might produce a protein of mutant or normal
Deletion of the A and B segments of BP4 or BP5 was found in
three small ?430kb microduplications (class 2, Figs. 1 and 2Aand B),
and duplications of the A segment of BP5 were identified in five
?680-kb microduplication cases (class 3, Figs. 1 and 2C). The
class 3 microduplications likely represent the reciprocal products
of the small ?680-kb microdeletions that occurred through
NAHR between the proximal and distal CHRNA7-LCRs on the
normal and inverted chromosomes 15 [Shinawi et al., 2009].
Moreover, we found two classes of small microduplications not
associated with deletions or duplications within BP4, BP5, or
other regions within at least 1Mb upstream and downstream of
CHRNA7 (classes 4 and 5, Fig. 1). Interestingly, in one of these
cases, the duplicated region was ?80kb shorter (class 5) than that
in the most common ?430kb microduplications (Fig. 1).
In three out of four ?1.6Mb BP4–BP5 microduplications, in
which the entire CHRNA7 was duplicated, we observed also
duplication of BP4 or BP5 LCRs (Fig. 3, Table 2).
The microduplication was inherited in all 21 small micro-
duplication and two BP4–BP5 microduplication cases, in which
both parents were studied (data not shown).
NAHR Hotspots in the Proximal and Distal CHRNA7-LCRs
In 11 microduplication cases, DNA sequencing of the long-
range PCR products amplified from the patients’ DNA enabled us
to narrow the recombination sites to 100–400bp regions (flanked
by two informative cis-morphic nucleotides) (Fig. 4). In two cases
(patients 38 and 46), the NAHR region containing the
recombination site could not be narrowed to less than 600bp.
In addition, we mapped and sequenced the NAHR site of the
previously published small ?680-kb microdeletion (patient 4 in
cases, only the upstream portion of CHRNA7 is duplicated (B). Note that BP4 and BP5 segments are either (A) duplicated or (B) unchanged.
aCGH analysis of four BP4–BP5 microduplications using 12?135K oligonucleotide microarrays (NimbleGen). In one out of four
HUMAN MUTATION, Vol. 31, No. 7, 840–850, 2010
[Shinawi et al., 2009]) (Fig. 4). Consistent with the previous data
for small ?680-kb microdeletions, the majority of NAHR sites of
small microduplications cluster in the same two recombination
hotspots, regions 1 and 6 (Fig. 4). In two cases, the patterns of
alternating sequences of proximal and distal LCR-specific cis-
morphisms in the chimeric LCRs (recombination site 2, 3, and 5)
likely represent the results of earlier crossovers or gene conversion
events (Fig. 4).
We analyzed the DNA structure of the two recombination
hotspots for the presence of structural features that might have
contributed to meiotic recombinogenic activity. NAHR hotspot 1
resides at the end of a retrotransposon LINE1 and hotspot 6 maps to
a DNA segment harboring the LTR81AB of a Gypsy retrotransposon.
We also found that the 7-mer CCTCCCT motif, often associated
with meiotic recombination hotspots in humans [Myers et al.,
2008], is located 125bp downstream of the NAHR site 2. Further, we
found several copies of this motif, carrying just a single nucleotide
mismatch, located in the NAHR site regions 1, 5, and 6.
Interestingly, two of the five imperfect copies of the recombination
motif found in the hotspot region 6 match the 50half of a larger
13-mer, CCNCCNTNNCCNC, that has been suggested to be critical
in recruiting crossover in 40% of human recombination hotspots
[Myers et al., 2008]. However, CCTCCCT motifs and their
derivatives are also scattered along the entire CHRNA7-LCRs, and
their presence cannot, by themselves, serve as a predictor of NAHR
To investigate whether there is a common feature of the DNA
secondary structures shared by the identified NAHR hotspots, we
analyzed them in silico using the Mfold software. We found that
the predicted thermodynamically most stable secondary structures
of these recombination hotspot regions contain centrally localized
palindromes that are more complex and longer than the
neighboring palindromes (Supp. Fig. S2). Interestingly, the
identified variants of CCTCCCT recombination motif reside
consistently within the loop (head) regions of these palindromes.
Clinical Phenotyping and Pedigree Analysis
Eleven of 55 index patients (ages 3–12 years) with small
microduplications involving CHRNA7 were examined, and
extended pedigrees were obtained (Supp. Fig. S3). These 11
patients represent all cases for which consent could be obtained.
All 11 patients displayed mild to moderate developmental delay or
mental retardation (Table 1). Muscular hypotonia was present in
six of 11 probands, with marked hypotonia in some of the younger
patients and associated delays in motor development. No
common dysmorphic facial features were noted in this patient
population. Patient 1 had a history of congenital heart disease
(hypoplastic left heart and coarctation of the aorta).
Interestingly, six of 11 index patients displayed neuropsychiatric
abnormalities, including autism spectrum disorder in four
patients, and bipolar disorder, anxiety disorder, disruptive
behavior disorder, and severe pica in one patient each. Analysis
of extended pedigrees showed that multiple family members
carrying CHRNA7 small microduplication were affected with
neuropsychiatric disorders, including major depressive disorder,
bipolar disorder, anxiety, and alcoholism. The youngest individual
with a microduplication involving CHRNA7 diagnosed with
bipolar disorder is the 5-year-old brother of propositus 2; the
5-year-old brother manifested irritable mood, racing thoughts,
distractibility, aggressive and hypersexual behaviors, pyromania,
and suicidal ideation.
Pedigree analysis showed that there is significant variability in
the expression of phenotypes in individuals with a microduplica-
tion involving CHRNA7. Penetrance was incomplete, as there are
several family members, who have been identified to be carriers of
Genomic coordinates (hg18) of the proximal CHRNA7-LCR are shown in parenthesis.
Localization of NAHR hotspots within the proximal and distal CHRNA7-LCRs. NAHR site regions 1–6 are shown as black boxes.
HUMAN MUTATION, Vol. 31, No. 7, 840–850, 2010
CHRNA7 microduplications with normal intellectual performance
and no history of neuropsychiatric abnormalities. Of note, five of
11 index patients carried at least one additional different CNV of
potential clinical relevance, representing a complex genomic load
[Lupski, 2007] that could be in line with the recently proposed
‘‘two-hit model’’ for severe developmental delay [Girirajan et al.,
Whereas epilepsy has been strongly associated with microdele-
tions of CHRNA7 [Helbig et al., 2009; Shinawi et al., 2009], it is
present only in one of 11 index patients with small microduplica-
tion involving CHRNA7 (Table 1). Patient 2, a 3-year-old male,
has generalized myoclonic epilepsy and infantile spasms. Further-
more, out of the 10 family members to our index patients shown
to carry a small microduplication involving CHRNA7, none has a
history of seizures.
Both parents were available for microduplication testing in six
of 11 families and the testing of the mother alone was possible in
three families. The microduplication was inherited in six of six
families where both parents were available for testing and in two
families where only the mother was available (Table 1). The
inheritance was maternal in five of eight cases and paternal in
three of eight index cases. No de novo case of small
microduplication of CHRNA7 has been identified to date. In five
families (F2, F5, F8, F9, F11), the microduplication was inherited
from a parent who had a history of neuropsychiatric problems
themselves (four with major depressive disorder, one with a
history of alcohol abuse). The mothers of patients 1 and 11 had
learning disabilities by report. In two families (F3, F7), the
microduplication was inherited from an apparently normal
parent, and these parents were perceived by themselves, family
members, and physicians as being normal based on education,
employment, and rearing of a family. They did not have a history
of cognitive impairment or neuropsychiatric disorder, although
Families of the carriers of BP4–BP5 microduplications were not
available for clinical examination.
and behaviorwere not
The multiple large and highly homologous LCRs in chromo-
somal region 15q13.2–q13.3 are responsible for its extensive
structural variability, including microdeletions, microduplica-
tions, and inversions. The opposite orientation of BP4 and BP5
LCRs is likely responsible for a recurrence of the paracentric
?1.6Mb inversion between BP4 and BP5, found in 44% of
randomly selected individuals [Sharp et al., 2008]; however, it
does not explain the origin of the recurrent BP4–BP5 microdele-
tions and reciprocal microduplications. Recently, Makoff and
Flomen  postulated that a small inversion within the A
portion of BP4 results in BP4A and BP5A segments being in direct
orientation relative to each other; this in turn, could predispose to
NAHR-mediated recurrent reciprocal BP4–BP5 microdeletions
Phenotypic and mechanistic analyses of BP4–BP5 and small
microduplications at 15q13.2–q13.3 are complicated by the
presence of the chimeric CHRFAM7A gene at the junction of
the A and B segments in BP4. Recently, a 2-bp deletion in the
CHRNA7 derived segment (exon 6) of CHRFAM7A, which results
in a downstream premature stop codon, was found to be
associated with schizophrenia [Sinkus et al., 2009], but no
replication is yet available.
In classes 1/1?, and 3 of small microduplications, the NAHR
recombination sites of the BP4–BP5 inversion are likely located
within A segments for 1/1?or B segments for 3 of BP4 and BP5.
This results in duplication of the entire original copy of CHRNA7
in class 1, and in the presence of one copy each for CHRNA7 and
CHRNA7?in class 1?microduplications with no copy of
CHRFAM7A in either class 1 or 1?microduplications (Fig. 2A
and B). For class 3 microduplications, we hypothesize that the
microduplication structure includes two copies of CHRNA7, one
of CHRFAM7A, and none of CHRNA7?(Fig. 2C). The functional
possibilities for microduplications with two copies of CHRNA7
and for those with one copy each of CHRNA7 and CHRNA7?are
quite different. In some cases of class 1?microduplications, where
the inversion breakpoints map within CHRNA7 (between intron 4
and exon 10) (Fig. 2B), an inactivating mutation within the
CHRNA7 portion of CHRFAM7A (e.g., the previously described
2-bp deletion in exon 6 of CHRFAM7A) would alter the new
CHRNA7?fusion transcript. Mutant forms of CHRNA7, derived
either from CHRFAM7A or entirely from CHRNA7, could in turn,
cause significant perturbation of neuronal homeostasis, for
example, by a potential dominant negative effect on the
oligomerization of nicotinic receptors in the brain. Further
studies are needed to identify the pathophysiology of different
classes of microduplications involving CHRNA7. Challenges
include the relatively low expression of the a7 subunit of nAChR
in peripheral tissues and the difficulty of working with antibodies
to this receptor [Moser et al., 2007].
In addition to CHRNA7, small microduplications duplicate
exon 1 of only one isoform of OTUD7A, whereas BP4–BP5
microduplications duplicate the entire OTUD7A gene. However,
duplication of OTUD7A is unlikely to result in disease phenotype
given OTUD7A is a putative deubiquitinase [Kayagaki et al.,
2007], and changes in gene dosage for enzymes are rarely
Benign or Pathological?
Phenotypic characterization of patients with microduplication
of CHRNA7 revealed a similar spectrum of cognitive deficits and
neurobehavioral abnormalities as seen in patients with micro-
deletion of CHRNA7, including developmental delay mental
retardation, depression, bipolar disease, autism spectrum disease,
attention-deficit/hyperactivity disorder (ADHD), and disruptive
behavior disorder. However, this spectrum is not significantly
different from the findings for all patients undergoing clinical
array testing. In contrast to the reported incidences of epilepsy in
patients with microdeletion of CHRNA7 [Dibbens et al., 2009;
Helbig et al., 2009; Shinawi et al., 2009], seizures were only
reported in one of 11 propositi with microduplication of
CHRNA7, and zero of nine family members carrying the small
microduplication, for which past medical history was obtained in
The ability to determine whether particular CNVs are benign or
pathological is often, as for microduplications studied here,
limited by the availability of robust frequency data for carefully
characterized controls. The lower the penetrance and the milder
the phenotypes, the more difficult it is to determine pathological
significance. Here, the limited data for frequency of the small
microduplication in controls (one in 180 in [Helbig et al., 2009])
is not significantly different than that observed in patients. In
addition, the small microduplication is inherited in all informative
HUMAN MUTATION, Vol. 31, No. 7, 840–850, 2010
families to date, suggesting that genetic selection against the
microduplication genotype is low. However, the frequency of mild
neuropsychiatric phenotypes including major depression, ADHD,
obsessive/compulsive disorder (OCD), and alcohol abuse in
microduplication positive relatives of propostii leads us to be
concerned that there may be phenotypic risks associated with the
small microduplication. Control samples ordinarily are not
screened for depression, ADHD, OCD, and alcohol abuse. If such
phenotypic effects do exist, they would be important because of
the relatively high frequency of the microduplications and because
overexpression of CHRNA7 might provide pathophysiological and
therapeutic insights. The situation for the small microduplications
is additionally complicated by the fact that their different subtypes
may have different functional properties, and no subtype
information is yet available for controls. We believe that it will
be necessary to study control populations screened for major
depression, ADHD, OCD, and alcohol abuse, to determine
whether each subclass of small microduplications is benign or
pathological. Comparison of microduplication positive and
microduplication negative first-degree relatives of microduplica-
tion cases may also help to clarify pathogenicity as distinct from
assortative mating effects.
The pathogenic significance of the larger BP4–BP5 micro-
duplication, involving CHRNA7, is reported as uncertain,
although it may be associated with cognitive impairment and
psychiatric disease [Ben-Shachar et al., 2009; van Bon et al., 2009].
Because the 350–680-kb small microduplications (1) were
reported to be common in one control cohort, (2) are inherited
in all cases to date, and (3) are often accompanied by other CNVs
of uncertain significance, it is possible that some or all of the five
classes of 350–680-kb microduplications are entirely benign and
that the findings simply reflect bias of ascertainment. Alterna-
tively, the presence of concomitant CNVs in five of 11 index
patients in our study might suggest that the microduplication of
CHRNA7 could be similar to a digenic effect (double hetero-
zygotes) and cause an imbalance of neuronal homeostasis,
predisposing to neurodevelopmental and neuropsychiatric phe-
notypes influenced by the presence or absence of other genetic
modifiers. If the small microduplications are causing phenotypic
effects in some cases, analysis of extended pedigrees would reveal
what could be variable expressivity and incomplete penetrance of
cognitive deficits and neuropsychiatric disorders in individuals
with microduplication of CHRNA7. Phenomena such as variable
expressivity and incomplete penetrance of the clinical phenotype,
and assortative mating among individuals with psychatric disease
(with or without detectable copy number variants) have been
reported for other CNVs [McCarthy et al., 2009]. The clinical
indications for CMA in 46 patients with small CHRNA7
microduplications referred to MGL are summarized in Supp.
More studies are warranted to determine the frequency of
CHRNA7 microduplications in healthy individuals, who have
undergone extensive neuropsychiatric testing. Additional genetic
modifiers probably influence the clinical phenotype of the
individual patient. Those genetic modifiers could include
(1) the sum of the expression level of all the copies of CHRNA7
copy number, expression
function, if any, of the CHRFAM7A fusion gene; (3) other
monogenetic modifiers; or (4) the presence of concomitant
copy number variants in the same patient, as seen in 15 of 46
patients enroled in this study (Supp. Table S1) and reported
as a general phenomenon [Girirajan et al., 2010; Veltman and
CHRNA7?; (2) level, and
We were able to sequence the NAHR sites of 13 small
microduplications. In the majority of these cases (Fig. 4), we
found that the microduplication NAHR sites overlap with those
for the small ?680-kb microdeletions, further confirming the
proposed double NAHR model of their formation [Shinawi et al.,
2009]. By in-depth computational analyses of these regions, we
found a positive correlation between the presence of NAHR
hotspots and the relative increase of length and complexity of the
predicted DNA hairpin structures. Moreover, we found imperfect
copies of a common recombination-associated motif (CCTCCCT)
within these hotspots, localizing to the head-part of the hairpins
(Supp. Fig. S2). In addition to being associated with the
CCTCCCT motif, the recombination hotspot 1 resides at the 30
end of a non-LTR repetitive element LINE1, and a part of hotspot
6 maps within an LTR of a Gypsy retrotransposon. Interestingly,
retrotransposons were already reported to be overrepresented in
historical recombination hotspots [Cullen et al., 2002; Jeffreys
et al., 1998; Lindsay et al., 2006], and the presence of the
CCTCCCT motif within retrotransposon THE1A/B would dra-
matically increase probability of locating a historical hotspot
within this mobile element [Myers et al., 2005]. However, distal
sequences or epigenetic factors may contribute to hotspot activity
equally well or better than local sequences [Arnheim et al., 2007;
Bagshaw et al., 2006; Lupski, 2004; Ptak et al., 2005; Wu and
Because the small microduplications most likely occur through
NAHR between the CHRNA7-LCRs located within normal and
inverted BP4–BP5 regions, we propose that the observed
variability of the small microduplications and the associated
variation in copy number of the A and B segments of BP4 and BP5
result from different locations of the inversion breakpoints within
BP4 and BP5. We suggest that the most common variant of the
small microduplications at 15q13.3 (class 1, Fig. 1), associated
with the deletion of BP4B, arose on chromosome 15 with the
inversion breakpoints located within the A segments of BP4 and
BP5 (Fig. 2A and B). The BP5A breakpoint could be located
anywhere within BP5A, including the 30-terminal portion (exons
5–10) of the CHRNA7 gene, which overlaps with BP5A (Fig. 2B).
The BP4A breakpoint might be located within CHRNA7-derived
intron 4 of the CHRFAM7A gene, the CHRNA7 portion of
CHRFAM7A, or further upstream within BP4A. The most
plausible mechanism explaining the small microduplication
associated with duplication of the A segment of BP5 assumes
that the BP4–BP5 inversion breakpoints reside within the B
segments of BP4 and BP5 (Fig. 2C). The mechanisms causing the
small microduplication associated with the remaining (less
common) CNVs within BP4 and BP5 seem more complex and
likely involve different haplotypes of the BP4–BP5 inversions. We
suggest that the BP4–BP5 inversion with breakpoints mapping
within the CHRNA7 and CHRFAM7A genes may also lead to
the functional inactivation of CHRNA7 through the transfer of the
2-bp polymorphism from the CHRFAM7A gene [Sinkus et al.,
Future studies should include larger cohorts of individuals with
microduplications involving CHRNA7 to better understand the
complex genomotype–phenotype correlations in these families. In
particular, identification of de novo cases will be very helpful to
show whether parental BP4–BP5 inversions truly predispose to
CHRNA7 microduplications in their offspring. Biological and
biochemical studies are needed to determine whether partial
duplications of CHRNA7 undergo nonsense mediated decay or are
HUMAN MUTATION, Vol. 31, No. 7, 840–850, 2010
translated to truncated protein products and how this affects the
oligomerization of the different nicotinic receptor subunits.
We thank P. Magoulas, K. Plunkett, P. Furman, P. Zimmerman, and S.
Galvan for their tremendous help and assistance in this study. We thank
J.R. Lupski, H.Y. Zoghbi, C.M.B. Carvalho, W. Gu, and C.G. Gonzaga-
Jauregui for valuable discussions. P.S. was supported in part by grant R13-
0005-04/2008 from the Polish Ministry of Science and Higher Education.
Declaration of Competing Financial Interests
C.A.B., S.L., S.-H.K., A.P., S.W.C., A.L.B., and P.S. are based in the
Department of Molecular and Human Genetics at BCM, which offers
extensive genetic laboratory testing, including use of arrays for genomic
copy number analysis, and derives revenue from this activity.
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