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Linkage and association of Tourette Syndrome (TS) and Attention-Deficit/Hyperactivity Disorder (ADHD) have previously been reported in the 11q24 chromosomal region. To identify the risk gene within the region we studied the potassium inwardly-rectifying channel J5 (KCNJ5) gene in a sample of 170 nuclear families with TS. We genotyped eight markers across the gene and observed biased transmission of haplotypes from parents to probands in this sample.
We then tested these markers in an independent sample of 242 nuclear families with ADHD and found the same haplotype was significantly over transmitted to ADHD probands. Screening of the coding region of KCNJ5 in 48 probands with TS did not identify any variation that could explain the association of the haplotype. We also genotyped two microsatellite markers, one in the promoter and the other in the 3' region and found no evidence for association for either marker for TS, however, we found significant evidence for association with the 3' repeat and ADHD. A small gene (c11orf45) of unknown function lies within the first intron of KCNJ5 that is transcribed in the opposite orientation and this gene may regulate the expression of KCNJ5. We studied the correlation of the expression of KCNJ5 and the antisense transcript in brain tissues from control individuals and found that the antisense transcript and the short KCNJ5 isoform are co-expressed in three brain regions.
The results of this study indicate that KCNJ5 is associated with TS and ADHD in our samples, however, the functional variant(s) remain to be identified. Linkage and association of Tourette Syndrome (TS) and Attention-Deficit/Hyperactivity Disorder (ADHD) have previously been reported in the 11q24 chromosomal region. To identify the risk gene within the region we studied the potassium inwardly-rectifying channel J5 ( KCNJ5) gene in a sample of 170 nuclear families with TS. We genotyped eight markers across the gene and observed biased transmission of haplotypes from parents to probands in this sample. We then tested these markers in an independent sample of 242 nuclear families with ADHD and found the same haplotype was significantly over transmitted to ADHD probands. Screening of the coding region of KCNJ5 in 48 probands with TS did not identify any variation that could explain the association of the haplotype. We also genotyped two microsatellite markers, one in the promoter and the other in the 3′ region and found no evidence for association for either marker for TS, however, we found significant evidence for association with the 3′ repeat and ADHD.
A small gene ( c11orf45) of unknown function lies within the first intron of KCNJ5 that is transcribed in the opposite orientation and this gene may regulate the expression of KCNJ5. We studied the correlation of the expression of KCNJ5 and the antisense transcript in brain tissues from control individuals and found that the antisense transcript and the short KCNJ5 isoform are co-expressed in three brain regions.
The results of this study indicate that KCNJ5 is associated with TS and ADHD in our samples, however, the functional variant(s) remain to be identified. Tourette Syndrome (TS) is a neuropsychiatric disorder characterized by motor and vocal tics with onset in childhood. Family studies have demonstrated that genetic factors play an important role in the manifestation of TS and segregation analysis have led to the consensus that TS is a genetically complex multigenic disorder involving a high degree of locus and allelic heterogeneity (;; ). Linkage and association studies of TS have suggested a risk locus in the 11q24 chromosomal region.
We note that this region was originally identified as 11q23, however, the current genome annotation (hg19) indicates the location of these markers as 11q24. Linkage to the chromosome 11q24 region was reported in a single large multigenerational pedigree (127 members) from the French Canadian population. That study focused on the 24 markers that were previously identified by Simonic and colleagues as being significantly associated in the South African Afrikaner population (, ). The most significant result in the French Canadian family was in the 11q24 region, found using multipoint analysis (LOD score of 3.24, or 3.18 after correction for multiple testing) across the markers D11S1377 (Mfd316) and D11S933 (11q24.1–24.2). Interestingly, one of the markers in the linked region (D11S933) is located 7 cM from the marker D11S912 (11q24.3) that resulted in a LOD score greater than 1 in the first Tourette Syndrome Association (TSA) linkage genome scan. This region has therefore been implicated by studies using independent TS samples using association (case–control and family based controls) and linkage. Also of interest is suggestive evidence for linkage from two, independent genome scans for ADHD in this region overlapping in 11q24 (; ).A strong candidate gene on chromosome 11q24 is the gene for the potassium inwardly-rectifying channel J5 ( KCNJ5) selected based on its function.
Potassium channels are membrane-spanning proteins that selectively conduct K+ ions across the cell membrane. These channels play an important role in cellular signaling processes and are critical to neurotransmission.
The inward rectifier K+ channels (Kirs) belong to a superfamily of channels with four subunits each containing two transmembrane segments with a pore loop in between (;; ). These channels conduct K+ currents more in the inward direction than outward, and they are important in setting the resting membrane potential.In this study we examined the association of the KCNJ5 gene with TS based on the location and biology of the gene. We tested for association in a sample of 170 nuclear families (228 affected children) with TS and identified trends for association with a haplotype of markers selected to tag the major haplotypes.
On the basis of this association finding, and because of the previous linkage findings for ADHD to this region (; ), we also examined a subset of the markers for association to ADHD in an independent sample of 242 nuclear families with 277 children diagnosed with ADHD. In the ADHD sample, we observed association for ADHD with the same haplotype showing trends for biased transmission for TS in the TS sample.
On the basis of this association findings, we then sought to identify the functional DNA changes contributing to risk. We screened the coding regions of the gene for non-synonymous coding region changes. We then genotyped two microsatellites markers, either of which could influence gene expression. One of these is located in the promoter and the other located in the 3′ region. Considering the data showing regulatory roles of natural antisense transcripts (NATs) in the gene expression of the corresponding sense transcript, we also screened the antisense transcript ( c11orf45) that is located within the first intron of the KCNJ5 gene. We then studied the correlation between expression of both transcripts in three different regions of the human brain to determine if the two transcripts were co-expressed.
We found they were co-expressed in the three brain regions examined. Tourette Syndrome sampleThe sample consisted of 170 nuclear families from Ontario, Canada with one or more affected siblings for a total of 228 affected children. All families were recruited from The Tourette Syndrome Clinic at The Toronto Western Hospital. This study was approved by the research ethics of the University Health Network.
Written informed parental consent and verbal assent for younger children or written patient consent was obtained for all participants.The diagnostic assessment of subjects for this study has been previously described. Briefly, information about symptoms associated with TS and obsessive–compulsive disorder was collected using a self- and family-report based on the tic inventory and ordinal severity scales of the Yale Global Tic Severity Scale and the symptom checklist and ordinal scales of the Yale-Brown Obsessive–Compulsive scale. The information was checked by an experienced neuropsychiatrist and complemented by the direct examination of subjects using the same scales.The majority of the sample (92%) described their ethnicity by self report as European Caucasian, 3% as non-European and 5% as mixed European and Non-European.
ADHD sampleThe sample consisted of 242 nuclear families from Toronto, Canada recruited following referral to the Child Development or Neuropsychiatry Clinics at the Hospital for Sick Children in Toronto. It included 242 probands and 35 affected siblings.The diagnostic assessment has been described in previous studies of this sample. Briefly, probands and their siblings between 7 and 16 years old were included if they met DSM-IV criteria for one of the three ADHD subtypes (predominantly inattentive, predominantly hyperactive/impulsive or combined) based on semi-structured parent and teacher interviews.
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Children with TS or chronic tics were excluded from this sample.The majority of the sample (90%) describe their ethnicity as European ancestry, with the remaining 10% describing their ethnicity as either African, Chinese, Indian, native Canadian or of mixed descent.This protocol was approved by the Hospital for Sick Children’s Research Ethics Board and informed written consent or verbal assent was obtained for all participants. GenotypingDNA was extracted from blood using a high salt method. The single nucleotide polymorphism (SNP) assays were manufactured by Applied Biosystems Inc., Life Technologies (Foster City, CA, USA) as either Assays-On-Demand (predesigned) or as Assays-by-Design (made to order).
The 10 μl polymerase chain reaction (PCR) reactions contained 30 ng of genomic DNA, 10 μmol of TaqMan ® Universal PCR Master Mix (Applied Biosystems Inc., Life Technologies) and 0.25 μl of the allelic discrimination mix which is a premade mix containing the specific primers (18 μm) and probes (4 μm; Applied Biosystems Inc., Life Technologies). The thermal cycling conditions were 95° C for 10 min followed by 40 cycles of 94°C for 15 seconds and an annealing temperature of 59°C for 1 min. Included on each 96-well plate were two negative controls. The end-point data, for each plate, were collected using the ABI PRISM 7900HT Sequence Detection System (SDS; Applied Biosystems Inc. Life Technologies) with the allelic discrimination analysis mode of the SDS software package version 2.0 (Applied Biosystems Inc., Life Technologies).A total of six SNPs were genotyped across the KCNJ5 gene in the TS sample. The initial panel of SNPs was selected to tag the major haplotypes using Tagger Pairwise Method as implemented on the International HapMap Project Browser. With this selection of markers we captured 70% of all markers in the region at r 2 ≥ 0.80 and minor allele frequency (MAF) ≥0.10 in the Centre d’Etude du Polymorphisme Humain (CEU) population.
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Two additional SNPs in the intergenic region shared with P53S1P1 were also genotyped in the TS sample.Five of the tag SNPs were genotyped in the ADHD sample after finding trends for biased transmission of haplotypes in the TS sample. After finding association in the ADHD sample, we screened the gene for DNA variants (below) and genotyped two additional SNPs and two repeat polymorphisms.
The non-synonymous SNP, rs7102584, was genotyped after we detected the least frequent allele present in three of the 48 probands that were resequenced for the coding region of the gene. An additional marker (rs11221507) in the 5′ untranslated region (UTR) of the antisense transcript was genotyped in the TS sample after we found that the frequency of the C allele in the screened probands was twice that of the reported frequency for this marker in the Caucasian population (0.133).Two repeat polymorphisms, a (CA)n(GA)n (D11S4150) repeat in the promoter region and a (GTTTT)n repeat motif in the 3′ UTR of the gene, were tested after association was identified. These variants were genotyped using the ABI PRISM ® 3100-Avant Genetic Analyzer (Applied Biosystems Inc., Life Technologies). ResequencingThe promoter region, the three exons of the KCNJ5 gene and the entire antisense transcript ( c11orf45) were resequenced in 48 TS probands.
Primers were designed using Primer Express Software, version 3.0 (PE Applied Biosystems, Life Technologies, Carlsbad CA) for a total of 22 fragments, 15 of them covering the antisense transcript (7091 bp). For the PCR amplification we used 60 ng of genomic DNA, 1.5 mM MgSO 4, 1× PCR Enhancer solution (Invitrogen Corporation, Life Technologies, Carlsbad, CA, USA) and the PCR buffer provided. The PCR reactions were carried out with an initial 5 min denaturing step at 95°C followed by 35 cycles of 95°C for 1 min, annealing temperatures between 59 and 70°C for 1 min according to the pair of primers for each fragment, 72°C for 1 min, and a final extension phase of 72°C for 10 min. Amplification products were sequenced using the Big Dye Terminator v3.0 Cycle Sequencing System (Applied Biosystems Inc., Life Technologies).
Sequences were analyzed using the ContigExpress program provided in the Vector NTI Advance 9.0 software package (Invitrogen Corporation, Life Technologies). Expression analysisPostmortem brain tissues from 40 control individuals (without documented psychiatric disorder) were used for the expression analysis of KCNJ5 and the antisense transcript ( c11orf45). Brain tissues were obtained from the NICHD Brain Bank for Developmental Disorders. Ethics approval was obtained from the University Health Network and The Hospital for Sick Children for use of the tissues. RNA was extracted from three different regions of the brain, dorsolateral pre-frontal cortex (DLPFC), caudate nucleus and hippocampus.
Complementary DNA (cDNA) was synthesized using iScript Reverse Transcription Supermix (Bio-Rad Laboratories Inc., Hercules, CA, USA) for Quantitative reverse transcription PCR (RT-qPCR) from 1 μg of total RNA using random primers. We determined the transcript copy number for KCNJ5 and c11orf45 by absolute quantification with real time quantitative PCR using the SYBR Green PCR Master Mix and primers designed with Primer Express Software. One endogenous control, RPLPO, was used for normalization. All reactions were performed in two replicates on the ABI PRISM 7900HT Sequence Detection System. To compare the expression of the two different KCNJ5 isoforms relative to each other and to the expression of the antisense transcript, we also designed primers for exon 1 of KCNJ5. In total, four different fragments in three different regions of the KCNJ5 locus ( KCNJ5 3′ region, KCNJ5 exon 1, antisense transcript 3′ region) and the RPLPO 3′ region were amplified for each sample. Statistical analysisGenotyping errors were checked by first identifying Mendelian errors using the PedStats program.
Further data checking was performed using the error option of Merlin to identify potential double recombinants as a sensitive check for genotyping errors. All Mendelian errors and double recombinants were either resolved or removed from the analyses. The TDTphase program from the UNPHASED, version −2.404 package was used to test for the biased transmission of alleles for single markers and the TRANSMIT program, version 2.5.4 for the transmission of haplotypes. The robust estimate of the variance option was used which is robust to the inclusion of affected siblings and to prior linkage.
Haplotypes with frequencies less than 0.10 were pooled for the analyses. The degree of linkage disequilibrium (LD) between marker alleles in this study was evaluated using Haploview v3.2.Correction for multiple testing was performed using Single Nucleotide Polymorphism Spectral Decomposition (SNPSpD). This calculates the number of independent SNPs using the LD information. The results showed that the effective number of independent marker loci for the single SNPs analysis in the ADHD sample is 5.4 and the experiment-wide significance threshold required to keep Type I error rate at 5% is 0.009.For the correlation of gene expression between the log-transformed expression levels for the sense and the antisense transcripts we used SAS version 9.3. ResultsTo test for association of the KCNJ5 gene in TS, we genotyped six SNPs covering this gene in a sample of 170 nuclear families with 228 affected children. Transmission disequilibrium test (TDT) analysis showed no significant association for any of the single SNPs tested in this sample. Haplotype analysis for three markers in a region of high LD in this gene identified biased transmission from parents to probands for one haplotype rs7118824 (G), rs11221512 (G), rs2604201 (G); P-value = 0.022, global P value for haplotypes 0.10 = 0.028.
This result would not meet the correction for the number of tests performed. However, based on this trend and the previous evidence for linkage of ADHD to this region, we then tested the relationship of this gene to ADHD. We genotyped five of the six markers covering the KCNJ5 gene in an independent sample of nuclear families with ADHD probands and affected siblings (277 affected children) and tested for association to ADHD. The results from the single marker TDT analysis in this sample showed association of the marker rs2604201 in the 3′ region of the gene ( P = 0.019) that would not meet the calculated experiment-wide significance threshold required to keep Type I error rate at 5% (0.009). However, haplotype analysis revealed biased transmission of the same haplotype observed with TS for ADHD ( P = 0.0006, global P value for haplotypes 0.10 = 0.0016, ). GeneLocation.MarkerAlleleAllele frequencyTransmissions †Non-transmissionsχ 2P valueKCNJ5Intron 1rs7924416 ‡C0.755.561T0.2457669Intron 1rs11221503 ‡C0.802.449T0.1985260Intron 1rs2604212 ‡C0.570.768G0.4309094Intron 1rs11221507T0.888.305C0.1123443Exon 2 (syn)rs7118824 ‡G0.834.173T0.1664660Exon 2 (nonsyn)rs7102584G0.0.477C0.98853Intron 2rs11221512 ‡A0.115.000G0.88536363′rs2604201 ‡A0.279.327G0.7218169Intergenicrs1893142G0.307.299A0.6938572Intergenicrs2155548A0.770.469C0.2307382.
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