Analysis of CHRNA7 rare variants in autism spectrum disorder susceptibility

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 1 AMERICAN JOURNAL OF MEDICAL GENETICS PART A 2 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 (20<IQ<34) 0 2 Moderate (35<IQ<49) 11 3 Mild (50<IQ<69) 27 11 BCI (70<IQ<85) 20 3 Normal (IQ>85) 17 7 Unknown 23 8 2 1 0 0 0 0 1 1 1 106 29 2 2 14 38 24 25 32 Total 98 34 Male 76 28 3 135 BCI, borderline cognitive impairments. 3 BACCHELLI ET AL. All data from either affected patients or their parents and controls, including informed consent, were handled in accordance with the local ethical committee’s approved protocols and in compliance with the Helsinki declaration. Clinical Assessment of Proband 3474_3 With CHRNA7 Duplication The propositus of family 3474 was the only child of healthy nonconsanguineous parents. The paternal grandfather’s sister was affected by ID, and the paternal grandfather’s brother had speech delay. During pregnancy there was no exposure or history of alcohol, tobacco, or drug abuse. The child was born at term by caesarean. Birth weight was 3550 g (50th centile), and length 50 cm (50th centile). He was breast-fed with good suction. He sat unaided at age eight months, and started walking independently at 16 months. Babbling was noted at age nine months, and at age 12 months he pronounced his first words. At 18 months, his parents observed a regression of his language skills, associated with refusal of physical contact, selective feeding, and hyperactivity. When seen by us, at age five years five months, he exhibited moderate cognitive impairment; was able to pronounce only single words with a reduced gesture repertoire, and limited comprehension. On examination, there were bilateral epicanthal folds, broad-hypoplasic nasal bridge, and prominent digit pads. Weight was 26 kg (97th centile), height 114 cm (50th–75th centile), and OFC 55.5 cm (>90th centile). Metabolic work-up, high resolution karyotype, molecular analyses of the FRAXA/E loci, thyroid function, Brainstem Auditory Evoked Potentials (BAEPs), Electrocardiogram (EKG), Electroencephalography (EEG), and Magnetic Resonance brain Imaging (MRI) were reported to be normal. ADIR, and ADOSG scores met criteria for a diagnosis of autism. He was not available for a follow-up visit. However, his mother reported, on a phone call, that he had developed complex partial seizures with secondary generalization at age 13 years. His father, who transmitted the microdeletion, as well as his mother are both apparently normal. CNV Analysis All ASD probands were analyzed by Quantitative Real Time PCR (qPCR) using Fast SYBR-green (Life Technologies) to amplify a region of 111 bp partially overlapping exon 3, specific for CHRNA7 (Primer forward: 50 -GGCTGCAAATGGTAAGTTAAGAG-3’ and primer reverse: 50 -AACAGGACCTCTCAGAAGCAAG-30 ). The data were normalized to the reference gene FOXP2. Each assay was conducted in three replicates for the target region and for the control region. Relative levels of region dosage were determined using the comparative CT method assuming that there were two copies of DNA in the control region. The relative copy number for each target region was calculated as 2-DDCT with confidence interval as 2-(DDCTSD). The validation and the resolution of the extension of the CHRNA7 duplication identified in proband 3474_3, was carried out using Genome Wide SNP arrays data (Illumina 1M-duo array) generated as part of a large genome-wide scan for CNVs carried out by the Autism Genome Project (AGP)[Pinto et al., 2014]. CHRNA7 Sequencing The genomic sequence comprising CHRNA7 exons 5–10 is duplicated and nearly identical (>99%) in the CHRFAM7A gene, complicating the mutation screening [Gault et al., 1998]. The first four exons, which are specific for CHRNA7, were amplified using exon specific primers corresponding to flanking intronic sequences and then subjected to Sanger sequencing. We aligned the sequences of the duplicated segment of CHRNA7 as reported in human genome reference sequence (GRCh37, hg19) and we identified three small insertions present in CHRNA7 and absent in CHRFAM7A , namely: 36 bp at the 30 end of CHRNA7 (chr15: 32,462,638–32,462,673); 4 bp in intron 5 (chr15: 32,448,389– 32,448,392); 5 bp in intron 9 (chr15: 32,458,725–32,458,729) (Fig. S1). To selectively amplify CHRNA7 exons 5–10, we took advantage of the 3’UTR 36 bp insertion to design a primer specific for CHRNA7. Two long range PCRs (LR-PCRs) were performed to amplify: a) a segment encompassing exon 5 to exon 8 (x5–x8 LRPCR) using primer 50 -CACCTGCAGTTCAGTCATTCAA-30 , outside the duplication, and 50 -AAAGTCAAACCTCAAAGCTGAA30 , in the duplicated region; b) a segment encompassing exon 9 to exon 10 (x9–x10 LR-PCR) using primer 50 -AGTGCATGGAAGTGCAATGA-30 , in the duplicated region, and 50 -CACTTCTACTTGTTTCTAAAGACACTG-30 , in the 36 bp CHRNA7 specific region (Fig. 1). LR-PCR was performed using KAPA HiFi HotStart DNA Polymerase (Resnova), following the manufacturer’s protocol, and using a ‘touch-down’ protocol: 95˚C for 5 min, followed by 10 cycles of 20 sec at 98˚C, 15 sec at 65˚C 0.5˚C per cycle, 7 min at 72˚C, followed by 25 cycles of 20 sec at 98˚C, 15 sec at 60˚C, 7 min at 72˚C. All LR-PCR products were purified using a Millipore Multi Screen PCR 96 well and then they were directly sequenced by Sanger method using specific primers for each exon. Primers, used to perform PCR and sequencing, were designed using Primer 3 software (http://bioinfo.ut.ee/primer3–0.4.0/). All PCR and sequence primers are listed in Supplementary Table S1 (see supporting information online). To verify the specificity of our two LR-PCR reactions, we checked for the presence of the two CHRNA7 specific sequences in intron 5 and intron 9 (Fig. S1-in supporting information online). Primers flanking these two regions were used to amplify the genomic DNA of 30 patients, thus leading to co-amplification of both CHRNA7 and CHRFAM7A. For each patient, we sequenced and compared the co-amplification product and the LR-PCR product. For the exon 5-exon 8 fragment, sequencing of the LR-PCR product shows the 4 bp CHRNA7 specific insertion, while sequencing of the coamplification products leads to overlapping sequence peaks, as expected (Figs. S2 A and B-in supporting information online). This result confirmed that our x5-x8 LRPCR is specific for CHRNA7. We did notice, however, that in a minority of individuals the LR-PCR sequence showed overlapping peaks, suggesting that the 4 bp insertion in intron 5 is actually polymorphic in CHRNA7. This hypothesis is supported by comparison of the coamplification sequence in these heterozygote individuals, which is compatible with the presence of one allele with the 4 bp insertion and three alleles without the insertion, thus still confirming the specificity of this LR-PCR (Figs. S2C and D-in supporting information online). AMERICAN JOURNAL OF MEDICAL GENETICS PART A 4 FIG. 1. Genomic structure of the human CHRNA7 gene, with the position of the primers used for the x5–x8 and x9–x10 LR-PCRs and the qPCR probe. The dashed rectangle indicates the region duplicated in CHRFAM7A. [Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN) 1552–4833.] For the exon 9-exon 10 fragment, sequencing of the 5 bp specific region in intron 9 did not reveal the expected differences between the LR-PCR and the co-amplification products, as the 5 bp insertion was always present. However, using the same strategy, we compared the genotypes of two common SNPs in exon 10 (rs1042724, rs2253967), by sequencing the x8-x9 LR-PCR versus the coamplification product in all ASD individuals. Since we obtained different genotypes (Fig S3-in supporting information online), we concluded that our x9-x10 LR-PCR is indeed specific for CHRNA7, while the 5 bp insertion in intron 9 as reported GRCh37 is actually present in both CHRNA7 and CHRFAM7A, at least in most individuals. RESULTS CNV Analysis All 135 ASD individuals were tested by qPCR to identify CNVs in CHRNA7 using a probe mapping in exon 3, specific for CHRNA7 (Fig. 1). No deletions were identified, while a duplication was found in a proband with ASD (3474_3). qPCR was then repeated in all family members, showing that the duplication is inherited from the unaffected father (Fig. 2A). Illumina 1 M-Duo SNP arrays data for all members of family 3474 were used to validate and to define the extension and the boundaries of the duplication. The presence of the duplication was confirmed with high confidence by two algorithms (QuantiSNP [Colella et al., 2007] and PennCNV [Wang et al., 2007]); it spans a region of approximately 500 kb, including exon 1 of the longer isoform of OTUD7A and the entire CHRNA7 gene (chr15: 32,005,348–32,515,973 NCBI build 37 coordinates). The presence of the microduplication was also verified by visual inspection of the 1 M SNP data, by plotting intensities (logR ratio) and allelic ratios (B allele frequency) of all members of the family 3474 for the region encompassing the duplication (Fig. 2B). CNV analysis of Family 3474 led also to the identification of a 55 kb paternal deletion of ARHGAP11B, which is known to accompany the majority of CHRNA7 microduplications [Szafranski et al., 2010], and a rare maternal gain of unknown significance on chromosome 5q13.2(chr5:68,594,539–68,638,941). This duplication involves the first seven exons of CCDC125 (Coiled-coil domain-containing protein 125), a gene with a putative role in controlling the cell motility of immune systems [Araya et al., 2009]. Mutation Screening All 10 exons of CHRNA7 were sequenced in the sample of 135 Italian ASD probands. CHRNA7 exons 5–10, along with a large cassette of DNA (300 kb), are duplicated and map approximately 1.5 Mb proximal to the full-length CHRNA7 gene [Gault et al., 1998]. CHRFAM7A is a chimeric gene derived by the fusion of CHRNA7 (exons 5–10) to one of many copies of a novel gene called FAM7A, that encodes exons A–E [Gault et al., 1998] [Riley et al., 2002]. Exons C–A are the result of a partial duplication of a putative kinase-like gene (ULK4) on chromosome 3p22.1, while exon D is of unknown provenance. CHRNA7 DNA sequence in CHRFAM7A is 99.9% conserved. Therefore, in order to selectively analyze the duplicated region of CHRNA7, long range PCR was used to amplify fragments that contained exons 5–8 and 9–10 (see Methods for details). A total of four putatively functional rare (<1% minor allele frequency) variants were identified in CHRNA7: a non-synonymous variant in exon 10, and three variants located in the proximal promoter region [Leonard et al., 2002] (Table IIa). The non-synonymous variant in exon 10 causes an amino acid substitution from Glutamate to Lysine (p.E452K, rs199504752) and it has been identified in only one ASD proband who inherited it from the unaffected mother. Since this variant is located in CHRNA7 exon10, which is duplicated in the CHRFAM7A gene, it was not possible to estimate the frequency of this non-synonymous variant from public databases. Therefore we have sequenced CHRNA7 exon 10 by LR-PCR in a control sample consisting of 125 Italian unaffected individuals and we detected the p.E452K variant in three unrelated individuals. As the frequency of this non-synonymous variant is not different between our ASD sample and the control sample (1/135 in ASD vs. 3/125 in controls, two sided Fisher exact test P-value ¼ 0.35) and in silico analysis using PolyPhen-2(http://genetics.bwh.harvard.edu/pph2/) 5 BACCHELLI ET AL. FIG. 2. A) qPCR results for the CHRNA7 duplication in family 3474. B) GenomeStudio screenshot showing B-allele frequency and log R ratio for family 3474. The duplication results in an increase to the Log R ratio and deviation in the allelic ratio for heterozygote variants away from the expected 0.5. The SNPs included in the duplication are boxed. [Color figure can be viewed in the online issue, which is available at http:// onlinelibrary.wiley.com/journal/10.1002/(ISSN) 1552–4833.] and SIFT (http://sift.jcvi.org/) does not predict any deleterious effect on the protein, it is unlikely that this variant contributes to ASD risk. The other three rare variants identified by Sanger sequencing of the 135 ASD patients are respectively located in the proximal promoter region, respectively at -241 bp (rs188889623, c.1–129A>G), at 191 bp (c.1–79G>A), and -182 bp from ATG (c.1–70_1–69delG) (Table IIa). One out of the three individual with the -241 bp variant (proband 3377_3) also carries another more frequent variant in the 5’UTR region (g.32322712C>T, rs149637464, -86 bp from ATG) (Table IIb). Interestingly, these AMERICAN JOURNAL OF MEDICAL GENETICS PART A CC ¼ 75; CT ¼ 10 (0.059) CC ¼ 85; CT ¼ 13 (0.066) CC ¼ 151; CT ¼ 23 (0.066) CC ¼ 112; CT ¼ 23 (0.085) g.32460504 G>A p.E452K c.1466G>A (rs199504752) b: 5’-UTR variant found in the same individual carrying the 241 variant 5’-UTR ( 86) g.32322712C>T 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 7 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). REFERENCES Albuquerque EX, Pereira EF, Alkondon M, Rogers SW. 2009. Mammalian nicotinic acetylcholine receptors: from structure to function. Physiol Rev 89:73–120. Araya N, Arimura H, Kawahara K, Yagishita N, Ishida J, Fujii R, Aratani S, Fujita H, Sato T, Yamano Y, Higuchi I, Osame M, Nishioka K, Fukamizu A, Arimura K, Maruyama I, Nakajima T. 2009. Role of Kenae/CCDC125 in cell motility through the deregulation of RhoGTPase. Int J Mol Med 24:605–611. 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