Molecular Psychiatry (2002) 7, 289–301  2002 Nature Publishing Group All rights reserved 1359-4184/02 $25.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Mutation screening and imprinting analysis of four candidate for autism in the 7q32 region E Bonora1, E Bacchelli2, ER Levy1, F Blasi2, A Marlow1, AP Monaco1, E Maestrini2 and the International Molecular Genetic Study of Autism Consortium (IMGSAC)*

1The Wellcome Trust Centre for Genetics, University of Oxford, Oxford, UK; 2University of Bologna, Dipartimento di Biologia Evoluzionistica Sperimentale, Bologna, Italy

Genetic studies indicate that 7q is likely to contain an autism susceptibility locus (AUTS1). We have followed a positional candidate approach to identify the relevant gene and report the analysis of four adjacent genes localised to a 800 kb region in 7q32 that con- tains an imprinted domain: PEG1/MEST, COPG2, CPA1 and CPA5—a previously uncharacter- ised member of the carboxypeptidase gene family. Screening these genes for DNA changes and association analysis using intragenic single nucleotide polymorphisms (SNPs) provided no evidence for an etiological role in IMGSAC families. We also searched for imprinting mutations potentially implicated in autism: analysis of both DNA methylation and replication timing indicated a normal imprinting regulation of the PEG1/COPG2 domain in blood lympho- cytes of all patients tested. The analysis of these four genes strongly suggests that they do not play a major role in autism aetiology, and delineates our strategy to screen additional candidate genes in the AUTS1 locus. Molecular Psychiatry (2002) 7, 289–301. DOI: 10.1038/sj/mp/4001004 Keywords: autism; candidate genes; mutation screening; methlylation; imprinting; PEG1/MEST; COPG2; ; 7q32

Introduction cluster within 7q32. They were selected for investi- gation because several lines of evidence support a role Autism is a severe neurodevelopmental disorder for imprinted genes in brain function.14 In addition, characterised by impairments in reciprocal communi- some evidence of increased paternal allele sharing in cation and social interaction, accompanied by affected sib-pairs and preferential paternal allele trans- unusually restricted and stereotyped patterns of behav- mission to autistic probands has been reported for mar- iours and interests, and an onset in the first three years kers across the region,8,15 suggesting the possibility that of life. Twin and family studies have indicated a com- AUTS1 may show parent of origin-specific effects. plex genetic predisposition to this disorder,1–3 and sev- PEG1/MEST was the first imprinted gene mapped to eral genome scans for autism susceptibility loci have human . The mouse homologue, been recently completed.4–9 These studies, as well as Peg1/Mest, is widely expressed during embryonic cytogenetic data,8,10 provide evidence that the long arm development, particularly in mesodermal tissues, and of human chromosome 7q is likely to contain an autism in areas of the developing brain.16 Targeted inacti- susceptibility locus (AUTS1). vation of the paternally derived Peg1/Mest allele We are examining candidate genes in this region to results in embryonic growth retardation and, signifi- investigate their possible etiological role in autism and cantly, Peg1/Mest deficient females show abnormal we report here the analysis of four genes: the meso- maternal behaviour, providing evidence for a role of derm specific transcript homologue (PEG1/MEST), the Peg1/Mest in the regulation of mammalian behaviour.17 gene encoding the complex subunit Human PEG1/MEST expression has been shown to be gamma 2 (COPG2), the gene for carboxypeptidase A1 imprinted in all fetal tissues.12 In adult lymphocytes (CPA1), and a novel gene similar to CPA1, designated PEG1/MEST is expressed in two different isoforms, carboxypeptidase A5 (CPA5). having distinctive first exons and differential PEG1/MEST11,12 and COPG213 define an imprinting imprinting status: paternal specific expression is lim- ited to the canonical isoform (isoform 1), while the alternative isoform (isoform A) is biallelically Correspondence: E Maestrini, University of Bologna, Diparti- expressed.18 Monoallelic transcription of PEG1/MEST mento di Biologia Evoluzionistica Sperimentale, via Selmi 3, Bol- isoform 1 is paralleled by parent of origin-specific ogna, Italy 40126. E-mail: maestrinȰalma.unibo.it *http://www.well.ox.ac.uk/ෂmaestrin/iat.html methylation of a CpG island surrounding the first exon 12 Received 13 August 2001; revised 28 September 2001; accepted of isoform 1. A second imprinted gene, COPG2 4 October 2001 (coatomer protein complex subunit gamma 2), was Analysis of candidate genes for autism in 7q32 E Bonora et al 290 recently identified adjacent to PEG1/MEST.13 The two escent in situ hybridisation (FISH) to interphase genes are transcribed in opposite directions and over- nuclei.28 Nuclei in which the gene has not yet repli- lap in their 3ЈUTR. Blagitko and colleagues13 reported cated show two single hybridisation spots, whereas paternal specific expression of COPG2 in most fetal nuclei in which the gene has replicated reveal two sets tissues, and biallelic expression in fetal brain, liver and of doublets. In general most gene loci replicate on the adult lymphocytes. COPG2 encodes for the ␥ subunit two homologous in a fairly synchronous of the COPI coatomer protein complex, which is manner with only a small proportion of the nuclei involved in vesicle trafficking of from the showing an asynchronous single/double hybridisation Golgi complex to the endoplasmic reticulum. pattern. Probes for imprinted genes show a higher fre- Sequence analysis of the BAC clone djs201/RP11– quency of cells with both a single and a double hybrid- 2E11 (AC007938), containing the entire PEG1/MEST isation dot compared to non-imprinted genes.26 gene, revealed the presence of two genes belonging to the carboxypeptidase A (CPA) family: the first enco- Materials and methods ding the known pancreatic carboxypeptidase isoform A1 (CPA1), the second encoding a novel CPA isoform. Families Carboxypeptidases are -containing exopeptidases, The identification of families and assessment methods catalysing the release of carboxy-terminal amino acids used by the IMGSAC have been previously from peptides and proteins. They are synthesised as described.15,29 Briefly, in families passing an initial zymogenes that are activated by proteolytic release of screen, parents were administered the ADI-R and the an activation segment. The possible involvement of Vineland Adaptive Behaviour Scales. Potential cases carboxypeptidases in the pathology of autism is raised were assessed using the ADOS or ADOS-G. Physical by several reports of abnormal patterns of urinary pep- examination was undertaken to exclude recognisable tides in some individuals with autism.19–21 Although medical causes, particularly tuberous sclerosis. Kary- these findings have failed to be replicated by other otyping was performed when possible on all affected studies and remain controversial,22 it is possible that individuals and molecular genetic testing for Fragile X altered pancreatic peptidase function could lead to an performed on one case per family. One family meeting unusual accumulation of peptides. Furthermore, the all other criteria was excluded from the study because presence of CPA gene transcripts has been identified at of a chromosomal abnormality −45, XY, der(13;14) low levels in extrapancreatic tissues, namely in brain, (q10;q10), and one family was excluded because the where they could potentially participate in signalling probands were positive for fragile X. events such as biosynthesis or inactivation of peptide messengers.23 Characterisation of the CPA5 gene To investigate the presence of possible etiological CPA5 exons were predicted based on homology to variants in these candidate genes, we screened for DNA other CPAs by a BLASTX search of the AC007938 changes in their entire coding sequence and intron– sequence, leading to the identification of 10 potential exon junctions in a group of 48 individuals with exons, which did not include the initial exon. The autism, selected from those IMGSAC families showing sequence of the first exon was predicted by the Gen- marker allele sharing at the linkage peak on chromo- scan program. We designed a set of nested primers some 7q. To assess the relevance of the identified (Table 1) from the predicted exons and used them to sequence changes to the autistic phenotype, all vari- amplify by RT-PCR the corresponding cDNA from lym- ants with potential functional significance were phoblastoid cell lines and in a multiple tissue cDNA screened in a control sample of 48 non-autistic individ- panel (Clontech, CA, USA, cat No. K1420–1). RNA was uals. In addition, the most frequent polymorphisms extracted from lymphoblastoid cell lines of two normal and their haplotypes identified through this screening individuals with Trizol reagent (GIBCO Life Techno- were tested for association with autism in the full logies, The Netherlands) according to the manufac- IMGSAC family data set. turer’s instructions. Five ␮g of total RNA was subjected Imprinted genes disorders, such as Prader–Willi syn- to reverse transcription using Superscript RT kit drome (MIM 176270), Angelman syndrome (MIM (GIBCO Life Technologies) and random hexamers. 105830) and Beckwith–Wiedemann syndrome (MIM Reactions with no reverse transcriptase were perfor- 130650), are caused in some cases by ‘imprinting med as controls. Five per cent of the RT reaction or 2 mutations’, which result in incorrect resetting of the ␮l of cDNA from the Clontech panel were used for PCR,

parental-specific marks during early gametogenesis, under the following conditions: 2.5 mM MgCl2, 0.2 mM giving rise to silencing of both alleles or double dose dNTPs, 0.4 ␮M primers, 0.25 U Taq Gold Polymerase expression of imprinted genes.24 The maternal and (Applied Biosystems, USA) in standard KCl buffer, and paternal alleles of imprinted genes are generally dis- a ‘touch-down’ protocol (10 cycles of 30 s at 95°C, 30 tinguished by a differential methylation pattern25 and satT1—0.5°C per cycle, 45 s at 72°C, followed by 20 asynchronous replication timing.26,27 Therefore, we cycles of 30 s at 95°C, 30 s at T2, 45 s at 72°C). PCR also searched for potential imprinting mutations products were gel-purified using Qiagen Gel Extraction involved in autism, both by DNA methylation analysis kit and directly sequenced on both strands with Big of PEG1/MEST and COPG2 in autistic patients, and by Dye Terminator kit (Applied Biosystems) on an ABI- assessing asynchronous replication timing by fluor- PRISM 377 semi-automated sequencer.

Molecular Psychiatry Analysis of candidate genes for autism in 7q32 E Bonora et al 291 Table 1 Primers for RT-PCR analysis of CPA5

PCR product Primers Fragment size (bp) T1–T2a

CPA5 1/4 ATGCAGGGCACCCCTGGAGGCGGGAC 472 60–55 TCGGAATGCTCCATTACAAA CPA5 2/4 TTTCACTTCTCGGGGATCTG 313 55–50 TCGGAATGCTCCATTACAAA CPA5 3b/5 TCATCATACCACACCCTGGA 206 60–55 GTGGCATGGGTGATCCACT CPA5 5/8 CCATCTGGATTGACACTGGA 322 60–55 AGGGCCCGTGATAAGTTTCT CPA5 8/10 TTCTAACAGCAACCCCTGCT 305 60–55 AGTCGACGGTGATCCCACT

aT1: annealing temperature of the first PCR cycle; T2: annealing temperature of the 25 last cycles.

Hybridization to a Clontech Multiple Tissue North- change. In all cases where a non-synonymous substi- ern Blot (No. 7760–1) was performed using a probe tution was detected (CPA1 ex6, CPA4 ex3, and CPA4 obtained by PCR with primers 5ЈCCCCTACTAGCAG ex8), the corresponding fragment was further examined CACGACT3Ј and 5ЈGGAGAGTGCAGAGGCTTCC3Ј. in all 48 autistic subjects and a control group of 48 Caucasian individuals by sequencing, to compare the Mutation screening by DHPLC variant’s frequency in the two samples. PCR amplifications were performed in a final volume of 50 ␮l containing 40 ng of genomic DNA extracted Genotyping of SNPs and microsatellites ␮ from blood, 1.5–2.5 mM MgCl2, 0.2 mM dNTPs, 0.2 M The three SNPs COPG2 ex17, COPG2 int20, and CPA1 each primer and a mixture of 0.9 U Taq Gold Poly- ex3 were genotyped by restriction digestion using the merase and 0.05 U Pfu Turbo DNA Polymerase BsaI, BseRI, ApaI, respectively. The SNPs (Stratagene, UK) in a standard KCl buffer. For GC-rich PEG1-int2, PEG1 3ЈUTR and CPA5 ex3a could not be regions PCR reactions were performed with the distinguished by a commercial restriction , addition of 5% DMSO and 100 ␮M 7-deaza-2Ј-deoxy- therefore we designed the following mismatch primers guanosine-5Ј-triphosphate (Amersham Pharmacia using the program CRFLP (kindly provided by Prof Biotech, UK). Thirty-nine cycles were performed with Hongua Li, University of Medicine and Dentistry, NJ, a touch-down protocol (14 cycles of 30 s at 95°C, 30 s USA): 5ЈCATTTTAATGTCAATTTGGTCAC3Ј and at T1—0.5°C per cycle, 30 s at 72°C, followed by 25 5ЈTCTCTGGACTTCCAACCACACC3Ј for PEG1-int2 cycles of 30 s at 95°C, 30 s at T2, 30 s at 72°C). Primers creating a PvuII site, 5ЈCTGTATTACCTCCCCTACT for PEG1/MEST, CPA1 and CPA5 were designed using CCCTTA3Ј and 5ЈAGCTCCTGCTGGCTTCTTCCTA3Ј the program Primer 3 (http://www.genome.wi. for PEG1 3ЈUTR creating a NdeI site, 5ЈAGGA mit.edu/cgi-bin/primer/primer3Fwww.cgi), while for TTGTGTGGAAGGACATGGAG3Ј and 5ЈGATAAGC COPG2 we used the same primers described by Blag- TTTGATGTCTTTCAGTCC3Ј for CPA5 ex3a, creating a itko et al.13 Sequences and PCR conditions of all primer BspEI site. pairs are available from the IMGSAC web site Crude PCR products were digested in a 20-␮l reac- (http://www. well.ox.ac.uk/ෂmaestrin/iat.html). tion with the appropriate enzyme (New England DHPLC analysis was carried out using the WAVE Biolabs), according to the manufacturer’s instructions. DNA Fragment Analysis System (Transgenomic, UK). Fragments were separated on 3:1 NuSieve GTG agarose Crude PCR products were subjected to denaturation at (FMCBioproducts, USA)/standard agarose gel. The 95°C for 4 min followed by gradual reannealing from PAC clone 144H10 was obtained from the RCPI Human 95°Cto25°C over 35 min, and were injected into a PAC library (Research Genetics). By STS analysis we DNASepR Column. The column mobile phase con- determined that it contains the genomic sequence sisted of a linear acetonitrile gradient in a 0.1 M tri- spanning CPA1 exon 4 to the 3Ј end of PEG1/MEST. ethylamine acetate buffer (TEAA), achieved by mixing Two dinucleotide repeat polymorphisms, named of buffer A (0.1 M TEAA) and buffer B (25% acetoni- M144CA1AC and M144CA2DB, were isolated from the trile in 0.1 M TEAA). All the gradients and the relative PAC by subcloning and hybridisation to a labelled CA temperature(s) for the elution of the fragments were repeat probe. The following primers were designed to designed using the WAVEMaker 3.4 program. The cal- amplify these markers from genomic DNA: 5ЈTCT culated gradient at a flow rate of 0.9 ml min−1 was run GCATGTTCCTGATTGGG3Ј,5ЈCACACATCAAAAAC for all the amplicons at the relevant column CCATGG3Ј (M144CA1AC); 5ЈGACACAGAAGAG temperature(s) for each fragment. CCCAGCAA3Ј,5ЈTCACAAATTAAACTTTATCAT Samples showing a variant DHPLC pattern were GGGTC3Ј (M144CA2DB). The two microsatellites are sequenced to determine the nature of the heterozygous localised between the CPA1 and PEG1/MEST genes at

Molecular Psychiatry Analysis of candidate genes for autism in 7q32 E Bonora et al 292 the following distances: CPA1 −19 kb—M144CA1AC SSC, 0.1 SDS%. The same filters were successively −51 kb—M144CA2DB −33 kb PEG1/MEST. Genotyping hybridized to the probes described below. The was performed on ABI sequencing machines as PEG1/MEST probe was obtained by PCR amplification described previously.4 For each marker genotyping was of a control genomic DNA using primers successful in over 90% of the individuals. AGTCTGCCCACTTGATCCAT and TGGCCCCTAA- TAAATACTGCTT. For COPG2, probe A was obtained Association analysis by EcoRI/PstI digestion of the 6-kb subclone described The 169 IMGSAC multiplex families used for associ- above. Probe B was obtained by subcloning an ation analysis have been described elsewhere.15,29 EcoRI/PstI fragment from the BAC DJ972012. Briefly, the data set included 146 sibling pair families contributing 152 sib-pairs, and 23 other extended rela- Replication timing analysis tive-pair families, for a total of 341 affected individ- Replication analyses were performed on phytohae- uals. moagglutinin (PHA) stimulated T lymphocytes pre- 30 Transmission disequilibrium testing (TDT) was pared for karyotypic analysis.34 performed using the sib tdt program from the ASPEX PAC clone 144H10 (RCPI Human PAC library, 31 package. This program calculates probabilities for Research Genetics) containing the sequence for chi-squared statistics by permuting parental alleles MEST/PEG1, cosmid clone ICRFc113M216Q1 contain- whilst fixing the IBD status of siblings within a family, ing the sequence for COPG2 and chromosome 9 BAC thereby allowing the use of multiple siblings within a clone 273N4 (RPCI-11 Human BAC Library, Research nuclear family; this is a valid test for association in the Genetics) were directly labelled with SpectrumRed presence of linkage. For the 23 extended relative-pair dUTP using Vysis Nick translation kit. A commercial families, only one affected individual was included in preparation of SpectrumOrange labelled DNA for the analysis. Sex-specific TDT was carried out using SNRPN was obtained from Vysis UK Ltd. Slides and the ‘sexFsplit’ option. TDT analysis of two-point and probes were prepared for FISH as described.35 Follow- three-point multilocus haplotypes of flanking markers ing post-hybridisation washes, slides were rinsed in 32 was performed using the TDTPHASE program; PBS and mounted in Vectorshield containing DAPI empirical probabilities were calculated on 1000 repli- (4,6-diamidino2-phenyl indole) (Vector Labs, USA) cates and the ‘tsu’ option was used to apply a correc- and examined under a Nikon optiphot microscope 33 tion for linkage in sib-pairs. with appropriate filters. Interphase cells were scored for three replication pat- Cloning and sequencing of COPG2 promoter region terns: two singlets, a singlet plus a doublet, or two The BAC clone DJ972012 containing the COPG2 gene doublets. Since singlets in late replicating BAC and and promoter region was isolated from the BAC RPCI- PAC probes can also produce hybridisation signal as 5 library (obtained from the YAC Screening Centre, clusters of two or three dots of reduced intensity, DiBiT, Milan, Italy) by PCR screening, utilising COPG2 interphase cells showing this phenomenon were not Ј exon-1 specific primers. A restriction map of the 5 scored. Where possible a minimum of 50 cells were region of the gene was obtained by digestion of the scored. BAC with several restriction endonucleases, followed by Southern blot hybridisation with an exon-1 specific Data accession probe. A 6-kb EcoRI/PvuII fragment containing the pro- Sequence data from this article have been deposited moter region and exon-1 was cloned into EcoRI/SmaI with the EMBL/GenBank Data Libraries under digested pBluescript SK II+, and sequenced on both Accession No. AF324497 and AF384667. strands by primer walking (GenBank accession num- ber AF324497). Results Methylation analysis The mat-UPD7 cell line was obtained from Coriell Cell Characterisation and genomic structure of the Repositories (GM11496). Seven ␮g of DNA from 46 carboxypeptidase A genes patient and 10 control blood DNA samples were The CPA1 gene was localised adjacent to PEG1/MEST digested with the restriction endonucleases EcoRI and in the BAC clone djs201/RP11–2E11 (AC007938) by HpaII, or EcoRI and MspI. MspI and HpaII recognise BLAST analysis. The intron–exon organisation of the same sequence but HpaII does not cut in the pres- CPA1 was obtained by comparison of the cDNA ence of methylation. Therefore, the methylation status sequence with the genomic sequence of clone djs201. of a sequence can be inferred by comparison of their The CPA2 gene was previously reported to map in restriction pattern. The digested samples were fraction- the genomic region just proximal to CPA1.36 Sequence ated on a 1% agarose gel and transferred to a Hybond analysis of the djs201 BAC clone predicted the pres- N+ nylon membrane, using a standard protocol. Probes ence of a gene similar to CPA1, however distinct from were labelled using the Megaprime DNA Labelling Sys- CPA2. This gene corresponds to a novel CPA isoform, tem (Amersham Pharmacia Biotech) and hybridised in which we designated CPA5. By searching the EST data- 5 × SSPE, 5X Denhardt’s, 2% SDS, 0.5 mg ml−1 yeast base we found several ESTs belonging to the Unigene RNA, for 20 h, at 65°C. The filters were washed in 0.2 × cluster Hs.144699 identical to the 3Ј end of the CPA5

Molecular Psychiatry Analysis of candidate genes for autism in 7q32 E Bonora et al 293

Figure 1 cDNA and deduced amino acid sequence of CPA5. Arrows indicate the positions of intron–exon boundaries; a prob- able polyadenylation signal is underlined.

Molecular Psychiatry Analysis of candidate genes for autism in 7q32 E Bonora et al 294 gene. The complete coding sequence of CPA5 was non-synonymous substitutions was compared between deduced by homology to other CPAs and through exon the autism sample and a control group of 48 non-autis- prediction programs and was confirmed by RT-PCR tic subjects (Table 2). No significant increased fre- amplification and sequencing of the resulting frag- quency was detected in the autism sample, suggesting ments (Figure 1). that these substitutions are neutral polymorphisms CPA5 is composed of 11 exons, its intron-exon struc- rather than etiological variants. ture is conserved with the human and rat CPA1 and CPA2 genes, except that both human CPA5 and rat Association analysis CPA2 contain an extra intron dividing the third exon We isolated two microsatellite markers from the PAC into two parts (exons 3A and 3B). Comparison of the clone 144H10 (RCPI Human PAC library, Research translated sequence of CPA5 with other known metal- Genetics), located in the intergenic region between locarboxypeptidases showed the greatest similarity to CPA1 and PEG1/MEST. The two microsatellites and human CPA1 (60.3% amino acid identity), followed by six of the identified SNPs (CPA5-ex3a, CPA1-ex3, CPA2 (53.8%) and CPA4 (51.2%). PEG1-int2, PEG1–3ЈUTR, COPG2-ex17 and COPG2- CPAs exist as procarboxypeptidases with a structure int20) were genotyped in 169 multiplex families from containing an amino-terminal signal peptide, an acti- the IMGSAC collection and tested for association with vation segment, and a carboxypeptidase domain. As autism using the Transmission Disequilibrium Test shown in Figure 2, CPA5 shares significant structural (TDT).30 similarity with other members of the CPA family. All Association was also tested for two-point and three- residues known to be important in zinc binding, sub- point haplotypes of flanking markers. No significant strate anchoring and positioning, and catalysis are con- transmission disequilibrium was detected at any of the served. The amino terminal region of the deduced markers or haplotypes. CPA5 protein is longer compared to other CPAs and Given the possibility of a differential effect of was predicted to be a signal peptide by computer paternal and maternal allele transmissions, we exam- analysis programs (SigCleave and SignalP37). ined transmission according to parental sex. Only The expression of CPA5 was studied by RT-PCR on COPG2-ex17 showed a trend toward transmission dis- a multiple tissue cDNA panel. CPA5 was found to be equilibrium of maternal alleles (36 transmissions vs 19 expressed at low levels in all human tissues examined, non-transmissions, P = 0.039). However, given the including brain, kidney, liver, pancreas, placenta and number of tests performed this result has probably skeletal muscle. Hybridisation to a Clontech Multiple arisen by chance. Tissue Northern Blot containing RNA from heart, brain, placenta, lung, liver, skeletal muscle, kidney and Methylation analysis pancreas was performed using a probe from the 3Ј To test for potential imprinting mutations, leading to untranslated (3ЈUTR) region with low homology to an abnormal methylation pattern, we performed DNA other CPAs, but no signal was detected, suggesting that methylation studies in the 5Ј CpG island regions of CPA5 is a rare transcript in most human tissues. PEG1/MEST and COPG2. The parent-specific methylation pattern of the 5Ј Mutation screening CpG island of PEG1/MEST has been previously Forty-eight unrelated individuals with autism from the described.12 The CpG island is unmethylated on the IMGSAC family collection were screened for sequence active paternal chromosome and methylated on the variants using Denaturing High Performance Liquid maternal chromosome. We performed Southern blot Chromatography (DHPLC).38 The patients were selec- hybridisation using DNA extracted from lymphocytes ted from affected sib-pair families on the basis of shar- of 46 individuals with autism from the IMGSAC family ing either one or two alleles identical by descent (IBD) collection, and 10 normal controls. Genomic DNA was in the region surrounding the candidate genes, based digested with EcoRI followed by digestion with either on flanking microsatellite marker analysis. Primers were designed to cover all exons and intron–exon boundaries of the four genes, as well as the putative Figure 2 Multiple alignment of several members of the car- promoter sequences of PEG1/MEST, COPG2, and boxypeptidase A family (accession No. NPF001859, CPA1. Genomic DNA from the patients was amplified NPF058694, AAD17690, P00730, P19222, NPF001860, by PCR and subjected to analysis by DHPLC to identify NPF057436, AF384667). Dots represent identities with human heteroduplexes. Samples that showed variant DHPLC CPA1 and dashes represent insertion of a gap. The standard patterns were reamplified and directly sequenced. We numbering of human CPA1 amino acid sequence is shown on identified a total of 41 sequence variants: four in top, with position 1 set to the start of the mature enzyme. PEG1/MEST, 14 in COPG2, five in CPA1 and 18 in Dotted lines indicate the boundaries between the signal pep- tide, activation segment and mature enzyme. Arrows indicate CPA5 (Table 2). All variants detected in PEG1/MEST conserved residues known to be involved in catalysis, zinc and COPG2 are in intronic regions or lead to synony- binding and substrate anchoring and positioning. Residues mous substitutions in the translated protein sequence. thought to be critical in determining substrate specificity are Four variants detected in CPA1 and CPA5 lead to non- boxed. Circled amino acids in CPA1 and CPA5 correspond to synonymous substitutions and possibly could have an the non-synonymous substitutions detected through our impact on protein function. The frequency of the four screening.

Molecular Psychiatry Analysis of candidate genes for autism in 7q32 E Bonora et al 295

Molecular Psychiatry Analysis of candidate genes for autism in 7q32 E Bonora et al 296 Table 2 Sequence variants in PEG1/MEST, COPG2, CPA1 and CPA5

(a) NON-CODING SNPs

Varianta Primers Sizeb (bp) Posc (bp) DNA change Het. Autismd

PEG1 int2 AGGCCTGTTCTCACCTAACG 439 80 G→T27 CATGCCTGGCCAATTTTTAT PEG1 int8 GGCAAAACAAAATGCCAAGT 610 339 del(G) 3 AAACACGTCTCTGGAAACCAA PEG1 int10 GTGTTCCTTGTGGCTCGTTA 258 217 C→T2 TTGCTAAATCTGGTAACCCTAATTG PEG1 3ЈUTR CTCCAGCCTCAAGTTCACCT 572 206 G→C24 CCCCTTCTCCTCCTCCTTACTAT COPG2 int9 CTGTGCTTTTAATCCCTATGC 255 31 C→G10 CAAAAACTTAGGAACATAAAAGCC C0PG2 int10 ATAGTTCTCTTAAGCCAGGAG 234 27 A→G1 GAGAGAAAACCAAATGCTTCAC COPG2 int10 ATAGTTCTCTTAAGCCAGGAG 234 28 C→T3 GAGAGAAAACCAAATGCTTCAC COPG2 int11 ATAGTTCTCTTAAGCCAGGAG 234 212 T→C3 GAGAGAAAACCAAATGCTTCAC COPG2 int17 GGCAAGAGGTTATAATGCTG 257 182 G→A4 AGTGACAATTAAATGAGTATTAGAG COPG2 int17 GCTAGAAATGTCTGCTCTC 207 35 C→T4 TACAATGCCTAGGGGAAAG COPG2 int17 GCTAGAAATGTCTGCTCTC 207 39 G→C3 TACAATGCCTAGGGGAAAG COPG2 int18 GCTAGAAATGTCTGCTCTC 207 181 G→A19 TACAATGCCTAGGGGAAAG COPG2 int20 CTTGCACTCCCCACTGAT 220 38 C→T24 GAAAAATGTTCTCTCAAGGCA COPG2 int21 CTTGCACTCCCCACTGAT 220 176 ins(G) 3 GAAAAATGTTCTCTCAAGGCA COPG2 3ЈUTR CCAGTGCTTGTTTTGGGT 333 271 T→C1 TGCTTGATCTGCTGGCTC CPA1 int5 AGGCCTCAGCTGTGAAATTG 498 329 C→G1 AGAGGCCCATCCTGCTTC CPA1 int7 CTCTCTGCAGCCTCTGAACC 250 187 C→G28 GAGCAAGAGCAGTGGTGAGG CPA5 5Ј TTCCTCCTTCTCTCTTCCCTCT 463 130 T→A14 ATCCTGACTCTCCGCCTTGAT CPA5 5Ј TTCCTCCTTCTCTCTTCCCTCT 463 140 T→C14 ATCCTGACTCTCCGCCTTGAT CPA5 int1 CTTCCAAGTGACACAGCCTTCCAG 226 61 G→A1 GGCAGGACAAGGAGCTAAATGTGT CPA5 int2 CTTCCAAGTGACACAGCCTTCCAG 226 198 G→T7 GGCAGGACAAGGAGCTAAATGTGT CPA5 int2 CTTCCAAGTGACACAGCCTTCCAG 226 186 C→T4 GGCAGGACAAGGAGCTAAATGTGT CPA5 int6 ACACACACCCTCTTACCCAGTGCT 749 444 C→G8 CTTTTCTCCATCCCCCTAGAGCTG CPA5 int8 CAGGATATCAGAGGCTCCCATCC 301 270 A→G13 GCCGTGACTCATGCATATTAGCTG CPA5 int9 GGAAATGTAGGGGAACACACAGGT 397 339 G→A12 ACCCCTGCATTCAGAGTAACAACC CPA5 int10 TGCTAAATCCATGCTTCTCACCAG 318 252 A→T17 CTGGGTACCACTGTTCACCCTTCT CPA5 int10 TGCTAAATCCATGCTTCTCACCAG 318 264 T→G15 CTGGGTACCACTGTTCACCCTTCT

Molecular Psychiatry Analysis of candidate genes for autism in 7q32 E Bonora et al 297 Table 2 Continued

(b) CODING SNPs – SYNONYMOUS

Varianta Primers Sizeb (bp) Posc (bp) DNA change Het. Autismd

COPG ex17 GGCAAGAGGTTATAATGCTG 257 50 A→G10 AGTGACAATTAAATGAGTATTAGAG COPG2 ex23 CACCCTGTCTTCATCATCT 378 269 G→A1 TGATGGGACAACAGCAAGGC COPG2 ex24 CCAGTGCTTGTTTTGGGT 333 93 G→A1 TGCTTGATCTGCTGGCTC CPA1 ex3 TATGAGAACTTCTGGCACCAACAGC 408 129 G→C29 CGTTCTACCAGCCATGATGAAGAG CPA1 ex6 AGGCCTCAGCTGTGAAATTG 498 358 C→T13 AGAGGCCCATCCTGCTTC CPA5 ex1 TTCCTCCTTCTCTCTTCCCTCT 463 242 C→T14 ATCCTGACTCTCCGCCTTGAT CPA5 ex3a AGGATTGTGTGGAAGGACATGGAG 301 121 T→C22 ACCCAGAAACCACATGTCCACTTT CPA5 ex5 ACACACACCCTCTTACCCAGTGCT 749 173 T→C9 CTTTTCTCCATCCCCCTAGAGCTG CPA5 ex10 CTTCTTCCTTTTGTGGGGCACATT 397 78 T→C13 ATAGGGGTGGTAGCCAACACGAGA

(c) CODING SNPs – NONSYNONYMOUS

Varianta Primers Sizeb Posc DNA/aa Het/Home Het/Home (bp) (bp) change Autism Controls

CPA1 ex6 AGGCCTCAGCTGTGAAATTG 498 380 G→A 3 G/A 2 G/A AGAGGCCCATCCTGCTTC A208T 0 A/A 0 A/A CPA5 ex3a AGGATTGTGTGGAAGGACATGGAG 301 95 C→T 4 C/T 4 C/T ACCCAGAAACCACATGTCCACTTT P79S 0 T/T 0 T/T CPA5 ex8 GGAAATGTAGGGGAACACACAGGT 397 261 T→C 11 T/C 16 T/C ACCCCTGCATTCAGAGTAACAACC L336S 3 C/C 1 C/C CPA5 ex8 GGAAATGTAGGGGAACACACAGGT 397 268 G→T 12 G/T 16 G/T ACCCCTGCATTCAGAGTAACAACC E338D 4 T/T 1 T/T aUnderlined SNPs were tested for association by TDT; bsize of PCR fragment; cposition of the nucleotide change starting from the 5Ј end of the PCR fragment; dnumber of heterozygotes detected in 48 autism cases; enumber of heterozygotes and homozy- gotes for the minor allele detected in 48 autism cases and 48 controls, respectively.

MspI or its methylation sensitive isoschizomer HpaII. we first subcloned a 6-kb EcoRI-PvuII fragment from Using the probe shown in Figure 3, the unmethylated the BAC clone DJ972012 containing COPG2 and paternal allele gives rise to a 540-bp band, and the sequenced the region upstream and downstream of maternal allele gives rise to a 3.9-kb band, due to exon 1 (GenBank accession No AF324497). The puta- methylation of the CpG islands and resistance to HpaII tive promoter sequence is typical of a CpG island, lacks digestion. All patients showed a methylation pattern a TATA and CAAT box and presents several MspI sites. identical to the control DNAs indicating a normal Methylation analysis was performed as described for imprinting status of PEG1 (Figure 3). The methylation PEG1/MEST, using two EcoRI-PstI fragments as probes status of an upstream CpG island surrounding the alter- (Figure 4); probe A (4.8 kb) covers the region upstream native exon corresponding to PEG1/MEST isoform 218 of exon 1, and probe B (3.4 kb) encompasses exon 1 was also investigated in DNA of control individuals. and part of intron 1. Probe A detected a partially meth- HpaII sites in this CpG island appeared unmethylated ylated HpaII/MspI site: digestion with MspI + EcoRI on both chromosomes (data not shown), in agreement resulted in two fragments of 0.9 and 3.6 kb. The same with the biallelic expression of PEG1/MEST isoform 2. fragments were present in the HpaII + EcoRI digestion To study the methylation pattern of the COPG2 gene, as well as the undigested fragment of 4.4 kb, raising

Molecular Psychiatry Analysis of candidate genes for autism in 7q32 E Bonora et al 298

Figure 3 Methylation analysis of the 5Ј end of PEG1/MEST. (a) Restriction map, showing the positions of MspI/HpaII sites (circles) on the paternal (top) and maternal (bottom) chromosomes; open circles = unmethylated, black circles = methylated; E, EcoRI. (b) Southern Blot hybridisation to DNA isolated from blood and digested with EcoRI (E), EcoRI + HpaII (H), and EcoRI + MspI (M); C, control; 1 and 2, patients.

the possibility that this site may be differentially meth- regulated by imprinting (chromosome 9 BAC clone ylated on the maternal and paternal chromosome 273N4) was also performed as internal control. Inter- (Figure 4). Hybridisation of probe A to DNA from a phase cells were scored for the presence of maternal uniparental disomy of chromosome 7 (mat- single/double (SD) hybridisation dots vs single/single UPD7) lymphoblastoid cell line39 revealed the presence (SS) and double/double (DD) dots.28 There was an aver- of the 3.6- and 0.9-kb bands only, suggesting that this age of 2–6% DD seen in all samples. The average pro- site may be unmethylated on the maternal chromosome portion of SD dots detected was 15.1% for and methylated on the paternal chromosome (Figure PEG1/MEST, 18.9% for COPG2, 15.4% for SNRPN and 4). The rest of the HpaII/MspI sites in the COPG2 CpG 5.4% for the chromosome 9 probe. Data for the chromo- island appeared to be completely unmethylated. some 9 probe were not scored in three patients due to Hybridisation of probe A to Southern blots contain- poor hybridisation quality. In all cases the proportions ing DNA from 46 individuals with autism revealed no of SD detected with PEG1 and COPG2 probes was com- abnormalities in the methylation pattern compared to parable to those detected for SNRPN, and greater than controls (Figure 4). those revealed by the chromosome 9 control probe (Figure 5). Replication timing analysis FISH analysis was perfomed on T lymphocytes from Discussion 12 patients and one normal individual, to test for pres- ence of anomalous replication timing of PEG1/MEST The genetics of autism is complex, probably involving and COPG2. Analysis with a probe for a known the action of several genes. Linkage studies and cyto- imprinted gene, SNRPN, and for a genomic region not genetic data have implicated several regions of interest

Molecular Psychiatry Analysis of candidate genes for autism in 7q32 E Bonora et al 299

Figure 4 Methylation analysis of the 5Ј end of COPG2. (a) Restriction map showing the positions of MspI/HpaII sites, open circles = unmethylated, black circles = methylated; the arrow indicates the differentially methylated site. E, EcoRI. (b) and (c) Southern Blot hybridisation to DNA digested with EcoRI + HpaII (H), and EcoRI + MspI (M). (b) DNA extracted from blood, C, control; 1 2 and 3, patients. (c) Lymphoblastoid cell line DNA from a control and a matUPD7 subject.

identified in a number of other studies. At least 190 known genes are contained within this interval. The difficulties in localising the linkage signal to a small interval, a common problem in nonparametric linkage studies of complex disorders,41 make the identification of the susceptibility gene in the 7q locus a challenging task, and a combination of approaches may be neces- sary to achieve this goal. In an attempt to refine the location of the disease locus the IMGSAC is following a linkage disequilib- rium (LD) mapping strategy, by testing a dense grid of polymorphic markers throughout the 7q22–7q34 region for association with the disease.15 Linkage dis- equilibrium (LD) mapping can provide a tool to further Figure 5 Percentage of nuclei with single/double hybridis- ation signals for 12 autism cases (a–l) and one control (contr); refine the location of a disease locus, by effectively nd: not determined. exploiting historic recombination events in a population. However, at this stage it is not possible to predict whether LD will be detectable between the in genetic susceptibility to autism, the long arm of susceptibility gene and nearby polymorphisms, as this 15,40 chromosome 7 being one of the most significant. may be hampered by a high degree of allelic com- The region of interest on 7q is very broad; the interval plexity or by other factors, such as the age of the etio- 15 of linkage reported by the IMGSAC (D7S527– logical variant, markers frequency, population history, D7S2513) covers around 50 Mb and overlaps regions and region-specific recombination rates. Since we can

Molecular Psychiatry Analysis of candidate genes for autism in 7q32 E Bonora et al 300 not completely rely on LD mapping to define the sus- specific transmission disequilibrium, suggesting that ceptibility locus, we simultaneously undertook a the genes tested here do not display a parent of origin- complementary positional candidate approach, scan- specific effect. The trend toward preferential trans- ning individual candidate genes in the AUTS1 locus mission of maternal alleles observed for a SNP in for genetic variation and assessing their relevance to COPG2 is likely to have arisen by chance and is in con- autism. trast to the putative paternal expression of COPG2 tran- In this paper we report a detailed analysis of the first scripts. four candidate genes examined by the IMGSAC, local- The PEG1/MEST gene is paternally expressed in ised to a region in 7q32 containing an imprinted human fetal tissues as a result of genomic imprinting. domain, close to the maximum peak of linkage orig- Monoallelic expression of PEG1/MEST has been inally reported by the IMGSAC.4 PEG1/MEST, COPG2 shown to be correlated to maternal specific methyl- and CPA1 were previously known genes, while CPA5, ation of a CpG island surrounding exon 1.12 The a novel member of the carboxypeptidase family, has COPG2 gene has also been reported to be a maternally been characterised in this study. imprinted gene.13 However, a more recent study, pub- Our mutation screening targeted gene regions most lished during the course of this work,44 demonstrated likely to harbour functionally significant variants biallelic expression of COPG2 in all tested fetal and (coding sequences, intronic regions critical for splic- adult tissues, while an antisense transcript in COPG2 ing, and putative promoters). The screening was car- intron 20 (CIT1) was found to be paternally expressed ried out in a sample of 48 unrelated individuals with in fetal tissues. These data indicate the presence of autism chosen from the subgroup of IMGSAC families additional imprinted transcripts in the region adjacent contributing to the linkage peak on 7q, thereby sel- to PEG1/MEST, but argue against monoallelic ecting for individuals who are more likely to carry eti- expression of COPG2. We have characterised the pro- ological variants at this locus. Using DHPLC, a very moter region of the COPG2 gene containing a CpG sensitive method for mutation scanning, we presum- island; in lymphocyte DNA we identified an upstream ably identified the near totality of DNA variants present HpaII site likely to be unmethylated on the maternal in our autism sample. Our DHPLC screening method chromosome and methylated on the paternal chromo- would have missed variants which are exclusively some. The parent of origin-specific methylation of this present in homozygous individuals and not in hetero- site could be correlated to the monoallelic expression zygotes, however, we considered this an unlikely of the CIT1 transcript, or of another imprinted tran- event. script not yet characterised. Methylation studies of the We identified several sequence changes, the majority critical CpG island upstream of PEG1/MEST and of the being in non-coding regions or leading to synonymous 5Ј region of COPG2 in lymphocyte DNA from 46 indi- substitutions. Four non-synonymous substitutions viduals with autism showed methylation patterns were identified in CPA1 and CPA5, and all of them identical to those of control DNA. Similarly, repli- were also found in a control group of non-autistic indi- cation timing analysis in 12 patients revealed that the viduals at a similar frequency. Accordingly, the corre- genomic region containing PEG1/MEST and COPG2 sponding amino-acids are in non-conserved positions exhibits a replication pattern typical of imprinted in the family of zinc-carboxypeptidase proteins, sug- genes. Taken together, our data indicate normal gesting that these changes have no major impact on the imprinting regulation of the PEG1/COPG2 domain in protein function. lymphocytes of the tested individuals. Although the We can not exclude that the remaining DNA changes possibility of imprinting abnormalities in tissues other with no apparent functional significance could play a than blood can not be excluded, our data suggest that role in autism susceptibility by subtly affecting gene imprinting mutations of the PEG1 domain are not fre- expression or regulation. To investigate this possi- quently found in autism. bility, the most informative SNPs identified through In conclusion, the lack of sequence variants that our screen, as well as two microsatellite markers, were could be related to the autistic phenotype in the four tested for presence of association with autism in the genes PEG1, COPG2, CPA1 and CPA5, together with total collection of 169 IMGSAC multiplex families. the absence of obvious abnormalities in the regulation This sample size should be sufficient to detect a locus of imprinting, strongly suggests that these genes do not with a small effect: it can be calculated42 that a sample play a major role in the aetiology of autism. The analy- size of 130–160 affected sib-pairs would provide 80% sis of additional candidate genes mapping to the 7q power at the 10−5 significance level to detect a locus locus is currently in progress. having a genotypic risk ratio of 2 and a moderate allele frequency (P = 0.1–0.4), assuming a fully informative Acknowledgements and closely linked marker and a multiplicative model. No significant results were detected with single mark- This work would not have been possible without the ers or haplotypes, indicating that these polymorphisms co-operation of both the subjects and their families and do not influence susceptibility, nor are they in LD with the many referring professionals. We would like to the actual causal variation. thank Zoe Docherty from Guy’s Hospital for providing Moreover, previous reports have suggested a paternal samples from blood lymphocytes, Pat Scudder for tech- effect in the AUTS1 locus.15,43 We could not detect sex- nical support, Janine A Lamb, Gabrielle Barnby and

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