|Home | About | Journals | Submit | Contact Us | Français|
The goal of this study was to identify chromosomal regions likely to contain susceptibility loci for obsessive-compulsive disorder (OCD).
We conducted a genome-wide linkage scan, with average marker spacing less than 10 centimorgans (cM), in 121 subjects from 26 families ascertained through probands with early-onset OCD. Best-estimate lifetime psychiatric diagnoses were based on semi-structured interviews and all other available sources of information. Parametric and nonparametric linkage analyses were conducted with GENEHUNTER+ and Allegro. Family-based association analyses were done using 35 single nucleotide polymorphisms (SNPs) in the 10p15 region.
The maximum nonparametric LOD score was 2.43 on chromosome 10p15 at position 4.37. When data from our first genome scan were added to data from this scan, the maximum NLOD score in the 10p15 region was 1.79. Association was detected on 10p15 with three adjacent SNPs, including the amino acid variant rs2271275, in the 3′ region of adenosine deaminase acting on RNA 3 (ADAR3) (p < 0.05).
The results provide suggestive evidence for linkage on chromosome 10p15. Evidence for association was found with three markers in the 3′ end of ADAR3 in the linkage region. Limitations include the lack of significant linkage and association findings when corrected for multiple testing.
Obsessive-compulsive disorder (OCD) is a heterogeneous psychiatric disorder with lifetime prevalence estimates in adolescents and adults ranging from 1% to 3% (Kessler et al 2005a; Weissman et al 1994; Zohar et al 1992). The National Comorbidity Survey Replication (NCS-R) found that OCD has a median age at onset of 19 years, with 21% of cases starting by age 10 (Kessler et al 2005a), and that OCD is the anxiety disorder with the highest percentage (50.6%) of serious cases (Kessler et al 2005b). Several studies indicate that an early age at onset in OCD is associated with a worse outcome (Skoog and Skoog 1999; Stewart et al 2004).
Twin, family, and segregation studies provide evidence that OCD is a complex trait with both genetic and environmental susceptibility factors. Estimates of the heritability of obsessive-compulsive (OC) symptoms in children range from 45% to 65% (van Grootheest et al 2005). Controlled family studies with either adult or pediatric probands have found that the lifetime prevalence of OCD is significantly higher in case compared to control first-degree relatives, and that an early age at onset of OC symptoms is often associated with a more familial form of the disorder (Fyer et al 2005; Hanna et al 2005a; Nestadt et al 2000; Rosario-Campos et al 2005). Segregation analyses indicate that a gene of major effect is involved in the transmission of OCD and that its penetrance is influenced by both age and sex (Hanna et al 2005b).
Our first genome-wide linkage analysis of OCD used seven families ascertained through pediatric probands to identify a region on chromosome 9p24 with suggestive evidence for linkage (Hanna et al 2002). An independent replication study of 50 families with OCD, using markers only on 9p24, identified an overlapping region with evidence for linkage centered only 0.5 cM away from our original finding (Willour et al 2004). Family-based association studies of the gene on 9p24 encoding the neuronal glutamate transporter, SLC1A1, indicate that the 3′ region of that gene may contain a susceptibility allele for early-onset OCD, with differential effects in males and females (Arnold et al 2006; Dickel et al 2006). In contrast, a recent genome-wide linkage analysis of 219 families with OCD found evidence for susceptibility loci on chromosomes 1q, 3q, 6q, 7p, and 15q with negligible evidence for a susceptibility locus on 9p (Shugart et al 2006).
Because of the promising results from the previous linkage studies of OCD, we conducted another linkage scan for DNA markers cosegregating with susceptibility loci for OCD using 26 new families ascertained through OCD probands with an onset of OC symptoms by age 18 years. We report suggestive evidence for linkage on chromosome 10p15, with family-based association tests providing evidence for association with three adjacent single nucleotide polymorphisms (SNPs) in the linkage region. The results implicate the third member of the gene family for adenosine deaminase acting on RNA (ADAR3, ADARB2, or hRED2) as a positional candidate gene for early-onset OCD (Chen et al 2000; Mittaz et al 1997).
Probands were recruited from clinics in the University of Michigan Health System and from local chapters of the Obsessive-Compulsive Foundation. All probands were directly interviewed to determine whether they met DSM-IV criteria for a lifetime diagnosis of OCD (American Psychiatric Association 2000). The proband inclusion criteria were: 1) a lifetime diagnosis of definite OCD with onset of OC symptoms by age 18 years and 2) a sibling or second-degree relative with a lifetime diagnosis of definite OCD who was available for interviewing and blood sampling. The proband exclusion criteria were: 1) a lifetime DSM-IV diagnosis of autistic disorder, schizophrenia, bipolar I disorder, or moderate to severe mental retardation, 2) adoption, 3) if less than 18 years old, currently living away from both biological parents, and 4) a first-degree relative with a lifetime DSM-IV diagnosis of autistic disorder, schizophrenia, or bipolar I disorder. Age at OC symptom onset was restricted in the probands and the diagnoses of autistic disorder, schizophrenia, and bipolar I disorder were excluded in both probands and their first-degree relatives in an effort to minimize the genetic heterogeneity of the families. Early-onset OCD was broadly defined as onset by age 18 years because previous family studies of OCD found that probands with onset after the age of 18 years generally have no affected relatives (Nestadt et al. 2000; Pauls et al 1995), and because the median age at onset of OCD in the NCS-R was 19 years (Kessler et al 2005a). The study was approved by the Institutional Review Board of the University of Michigan Medical School.
The probands were 11 males and 15 females ranging in age from 7 to 64 years (28.4 ± 17.5 years, mean ± SD). Age at OC symptom onset in the probands ranged from 3 to 18 years (8.0 ± 3.7 years, mean ± SD). Eleven probands had a history of Tourette’s disorder or another tic disorder. Probands with a history of tics had a significantly earlier age at OC symptom onset than did those without tics (6.4 ± 2.6 versus 9.3 ± 4.0 years, mean ± SD; t24 = 2.12, p < 0.05).
Direct interviews were completed with 85 relatives (32 males, 53 females) ranging in age from 5 to 85 years (41.5 ± 17.7 years, mean ± SD). They consisted of 57 first-degree relatives, 20 second-degree relatives, 6 third-degree relatives, and 2 individuals related by marriage. Of the directly interviewed relatives, 47 had a lifetime diagnosis of definite OCD (13 males, 34 females), 6 had a lifetime diagnosis of subthreshold OCD (4 males, 2 females), 22 were unaffected (13 males, 9 females), and 10 were indeterminate (2 males, 8 females). Of those with definite OCD, 19 were siblings, 11 were parents, 11 were second-degree relatives, 5 were third-degree relatives, and 1 was related by marriage. The relatives with definite OCD ranged in age from 6 to 71 years (36.0 ± 15.7 years, mean ± SD). Age at OC symptom onset in the relatives with definite OCD ranged from 3 to 50 years (11.5 ± 9.2 years, mean ± SD). The unaffected relatives ranged in age from 18 to 85 years (52.3 ± 85 years, mean ± SD). Eleven relatives had a history of Tourette’s disorder or another tic disorder. All six relatives with a history of chronic tics had either definite or subthreshold OCD.
Of the 73 probands and relatives with a lifetime diagnosis of definite OCD, 49 (67%) were female. Age at OC symptom onset in all subjects with definite OCD ranged from 3 to 50 years (10.3 ± 2.9 years, mean ± SD). Of the 22 probands and relatives with a lifetime diagnosis of tic disorder, 11 (50%) were female. Blood samples were obtained from all probands and directly interviewed relatives and from 10 relatives without direct interviews.
After providing informed consent and assent, probands and relatives younger than 18 years were interviewed using the Schedule for Affective Disorders and Schizophrenia for School Age Children-Epidemiologic Version-5 (Orvaschel 1995). This interview was completed independently with a parent of the subject and with the subject. Probands and relatives 18 years and older were interviewed with the Structured Clinical Interview for DSM-IV (First et al 1998). Both interviews were supplemented with sections on OCD and tic disorders derived from the Schedule for Tourette and Other Behavioral Syndromes (Pauls and Hurst 1991; Pauls et al 1995). The section on OCD included a series of questions modified to cover all the criteria for a lifetime DSM-IV diagnosis of OCD (Pauls et al 1995) and a checklist from the Yale-Brown Obsessive Compulsive Scale (Goodman et al 1989) modified to obtain information about the lifetime occurrence of specific obsessions and compulsions. Further information on relatives 18 years and older was obtained, from either adult probands or the parents of younger probands, using the Family Informant Schedule and Criteria (FISC) (Mannuza et al 1985) supplemented with additional questions for OCD and tic disorders. Hence, two types of data were collected on adult subjects: 1) information from direct structured interviews and 2) personal history information from a biological relative and/or spouse.
All interviews were audiotaped and coded on paper to assess reliability, maintain quality control, and achieve diagnostic consensus. All interviewers had at least a master’s degree with clinical training in either child or adult psychopathology. They were trained to at least 90% diagnostic agreement with the individual instruments. Depending upon their clinical expertise, the interviewers were confined to interviewing either children and adolescents or adults. After completion of all interviews for an individual, all available materials (personal interview data, family history data, and clinical records) were collated.
Best-estimate lifetime diagnoses were made independently by two investigators (M.V.E., D.J.F., J.A.H., and G.L.H. with G.L.H. reviewing the diagnostic information for all subjects) using DSM-IV criteria (American Psychiatric Association 2000). Definite OCD was diagnosed if an individual met all the diagnostic criteria. Subthreshold OCD was diagnosed if a subject met all criteria for obsessions and/or compulsions, but lacked compelling evidence for any of the following criteria: 1) marked distress, 2) duration of OC symptoms for more than one hour a day, or 3) significant interference in the person’s normal routine, occupational (or academic) functioning, or usual social activities or relationships with others. A subject was considered indeterminate if there was a history of thoughts or behaviors suggestive of OC symptoms that met most, but not all criteria for obsessions and/or compulsions. No diagnosis was made if a subject had no history of any OC symptoms.
To avoid forcing closure on inadequate diagnostic information, subjects were reinterviewed if necessary to clarify incomplete or contradictory information. When major disagreements occurred between two diagnosticians, consensus diagnoses were reached with the assistance of a third diagnostician following procedures developed for other psychiatric diagnoses (Roy et al 1997). The interrater reliability of this diagnostic process was studied in a sample of 108 subjects. There was good diagnostic agreement, as evidenced by a κ = 0.91 for OCD, a κ = 0.91 for tic disorder, and an intraclass correlation coefficient of 0.94 for age at OC symptom onset age. In the linkage and association analyses, subjects with definite OCD were considered affected and subjects with no diagnosis were considered unaffected. Subjects with subthreshold OCD or an indeterminate diagnosis were considered unknown.
Peripheral blood samples were obtained by venipuncture from consenting individuals. DNA was extracted using the PureGene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). Genome-wide genotyping was performed with 121 individuals from 26 families using 401 microsatellite markers from the Applied Biosystems prism Linkage Mapping Set, version 2.5 (Foster City, Calif.), with an average between-marker distance of 10 cM and an average heterozygosity of 0.81. Twenty markers were eliminated because of problems with quality control and excessive Mendelian errors. Completion rate for genotypes was 98.1%, and Mendelian incompatibility rate for the 381 markers was 2.0%.
Primer sequences for these markers are available at The Genome Database with one exception, SLC1A1, which has been described previously (Veenstra-VanderWeele et al 2001). For markers that were on the Marshfield genetic map (Broman et al 1998), the sex-averaged distances were used. For the few markers not on the Marshfield map, the nucleotide positions of the markers were located in the National Center for Biotechnology Information map viewer (http://www.ncbi.nlm.nih.gov/mapview/map_searchcgi?), and the flanking markers with defined positions on the Marshfield map and map viewer were identified. The genetic distance was then estimated by using the approximation that 1 megabase = 1 centimorgan (1Mb = 1cM). Polymerase chain reaction, genotyping, and allele calling have been described previously (Hanna et al 2002). A total of 35 SNPs in the 10p15 region were chosen from Taqman® SNP Genotyping Assays that were available from Applied Biosystems (Foster City, CA, www.appliedbiosystems.com).
Parametric and nonparametric linkage analyses were conducted with GENEHUNTER+ (Kong and Cox 1997) and Allegro (Gudbjartsson et al 2000). In the parametric analyses, we assumed reduced, age-dependent penetrance and sporadic cases, with both penetrance and sporadic case rate increasing with age, and the proportion of sporadic cases among all cases increasing with age (Hanna et al 2002). Age at OC symptom was specified as a linear function from 2 to 25 years. The lifetime prevalence of the affected phenotype was estimated to be 2%. Sex ratio was assumed to be 1:1. Two simple genetic models were used in the parametric analyses: a dominant model in which 1 > f(DD;a) = f(Dd;a) > f(dd:a) > 0 and a recessive model in which 1 > f(DD;a) > f(Dd:a) = f(dd;a) > 0. Haploview version 3.32 was used to determine pairwise linkage disequilibrium (r2), generate a corresponding plot, determine haplotype blocks (using the default setting), and perform the Transmission Disequilibrium Test (TDT) for single markers (Barrett et al 2005).
The nonparametric LOD (NLOD) scores from the analyses of 26 families with early-onset OCD are summarized in Figure 1. The results from the parametric analyses are not shown because they added minimal information to the NLOD scores. As shown in Figure 2, the highest NLOD score was 2.43 in the 10p15 region at position 4.37 with marker D10S1745. The second highest NLOD score was 1.54 on chromosome 1 at Marshfield map location 126 cM.
When data from the seven families in our first genome scan were added to data from our second scan, yielding a total of 33 families with 177 individuals, the maximum NLOD score in the 10p15 region was 1.79. The maximum NLOD score on chromosome 1, again at 126cM, was 1.48. Contrary to the suggestive evidence for linkage on chromosome 9p24 in our first genome scan (Hanna et al 2002), the maximum NLOD score on 9p in our second scan was 0.23. With the combined data from both scans, the maximum NLOD score in the 9p24 region was 1.15.
As detailed in Table 1, family-based association tests conducted with 35 SNPs in the 10p15 region provided evidence for association and linkage disequilibrium with three adjacent SNPs in the 3′ region of ADAR3 (p < 0.05), each of which had no Mendelian errors and for which genotyping was at least 97.5% complete. One of these SNPs, rs2271275, results in an amino acid change from threonine to alanine (T626A), and gave evidence for overtransmission of the more common amino acid variant in our sample (χ2 = 3.86, p = 0.0495).
As shown in Figure 3, two haplotype blocks were defined by Haploview (see Figure 3). As expected, given the very high r2 values for markers within each haplotype block, near perfect linkage disequilbrium meant that each of the SNPs within the block tagged the haplotype equally well, with two haplotypes accounting for more than 98% of the haplotypes for the first block and 100% of the haplotypes for the second block.
In our second genome linkage scan of early-onset OCD, we detected no significant genome-wide evidence for linkage on any chromosome according to standard guidelines for linkage results (p < 2 × 10-5) (Lander and Kruglyak 1995). We obtained suggestive evidence for linkage, however, on chromosome 10p15 with a maximum NLOD score of 2.43. In an analysis of our combined data from both scans, the maximum NLOD score in the 10p15 region decreased to 1.79. The second highest NLOD score in our combined sample was 1.48 on chromosome 1p at 126 cM, which is approximately 45-50 cM proximal to a region on chromosome 1q implicated in an independent OCD linkage scan (Shugart et al 2006). Our NLOD score of 0.86 on chromosome 2 at 47 cM in our second scan is very close to a significant linkage signal on 2p at 47.43 cM in a recent linkage scan of Tourette’s disorder (The Tourette Syndrome Association International Consortium for Genetics, 2007). Family and twin studies indicate that a form of OCD may be genetically related to Tourette’s disorder, suggesting that a locus may be expressed as either OCD or Tourette’s disorder (Pauls et al 1995; Rosario-Campos et al 2005).
Family-based association tests found evidence for association on 10p15 with three adjacent SNPs, including an amino acid variant, in the 3′ region of ADAR3 (Mittaz et al 1997). Hence, the linkage and association findings implicate ADAR3 as a positional candidate gene for OCD. Further research is necessary to determine whether the amino acid variant rs2271275 in ADAR3 is involved in the etiology of OCD. It is also possible that, if the SNPs are in linkage disequilibrium with another variant in the 3′ untranslated region, that variant could produce changes in messenger RNA (mRNA) processing (Conne et al 2000).
Members of the ADAR gene family produce enzymes responsible for a form of RNA editing involving the conversion of adenosine into inosine (A-to-I) in double-stranded precursor messenger RNA (pre-mRNA) (Chen et al 2000). The expression of ADAR3 has been detected only in postmitotic neurons in brain regions such as the amygdala and thalamus (Chen et al 2000); however, the transcripts expressed in brain that may be edited by ADAR3 have not been identified (Chen et al 2000; Maas et al 2003). Research on various organisms deficient in ADAR activities provides evidence that pre-mRNA editing has an ancient and primary role in the evolution of nervous system function and behavior (Reenan 2001). Furthermore, Drosophila deletion mutants lacking ADAR activity have been observed to spend an inordinate amount of time grooming throughout their lifespan (Palladino et al 2000). Disturbances in A-to-I RNA editing have been implicated in human diseases as diverse as dyschromatosis symmetrica hereditaria and amyotrophic lateral sclerosis (Maas et al 2006).
We failed to replicate two previous reports of suggestive evidence for linkage on chromosome 9p24 (Hanna et al 2002; Willour et al 2004). However, the linkage pedigrees described here were included in one of two recent family-based association studies that found association in the 3′ region of the SLC1A1 on 9p24 (Arnold et al 2006; Dickel et al 2006). The discrepancies in the results between our two genome linkage scans may be explained by genetic (interlocus or nonallelic) heterogeneity, as suggested by simulation studies demonstrating the difficulty of replicating a true linkage finding for an oligogenic phenotype (Suarez et al 1994). The ascertainment strategies used in our two linkage scans were also somewhat different. Our first linkage scan used a sample of 7 families with 4.6 affected individuals per family in which the largest pedigree contributed much of the linkage signal, whereas our second genome scan used a sample of 26 families with 2.8 affected individuals per family. Simulation studies have indicated that extended pedigrees and affected relative pairs differ in their power to detect linkage, depending upon the frequency of the susceptibility allele (Badner et al 1998).
It is possible that the expression of susceptibility alleles for OCD is associated with sex, age, and different OC symptom dimensions, so that the discrepancies in our results may be resolved only with larger genetic studies of OCD that assess OC symptom dimensions in detail (Hanna et al 2005b; Leckman et al 2003; Rosario-Campos et al 2006). However, the differences between the subjects with definite OCD in our two linkage scans were minimal with respect to sex ratio (first versus second: 56% female versus 67% female), age (first versus second: 37.1 ± 22.2 versus 33.3 ± 16.7 years, mean ± SD), age at onset of OC symptoms (first versus second: 11.4 ± 5.3 versus 10.3 ± 7.9 years, mean ± SD), duration of OC symptoms (first versus second: 21.8 ± 20.0 versus 22.7 ± 15.4 years, mean ± SD), or percentage with a history of chronic tics (first versus second: 16% versus 15%). The sample characteristics of the other OCD linkage scan also appear similar to those of our two linkage scans in that 66% of the affected individuals were female and the mean age at onset of OC symptoms was 9.5 years in the independent scan (Samuels et al 2006; Shugart et al 2006). Consequently, it remains possible that genetic heterogeneity may occur in OCD in the absence of any clinical correlates of that heterogeneity.
The results of our second genome linkage scan of early-onset OCD should be interpreted with caution. Even with the combined sample, the power of the linkage analysis was limited by the relatively small number of affected individuals so that only loci with a large effect may have been detectable. Because of the relatively small sample size and concerns about multiple tests, we used only a narrow affection model of definite OCD in our linkage analyses. However, it is possible that the susceptibility loci for OCD may have variable expressivity and not be specific to that phenotype. That is, one or more of those loci may also increase the risk for subthreshold OCD (Hanna et al 2005a; Nestadt et al 2000) or related disorders such as Tourette’s disorder, other chronic tic disorders (Pauls et al 1995; Rosario-Campos et al 2005), generalized anxiety disorder, agoraphobia (Nestadt et al 2001), hypochondriasis, body dysmorphic disorder, eating disorders, pathologic “grooming” conditions (Bienvenu et al 2000; Hanna et al 2005c), or autistic disorder (Bolton et al 1998). Linkage studies with larger samples are necessary to assess multiple affection models for OCD. However, it should also be recognized that linkage studies may not be powerful enough to consistently detect genes in a complex trait like OCD and that genome-wide association studies may be more productive in that effort (Thomas 2006).
In summary, the results from our second genome linkage scan of early-onset OCD provide suggestive evidence for linkage on chromosome 10p15 that requires replication. However, when data from our two linkage scans were combined, there was a decline in the maximum NLOD score in that region. Family-based association tests provide evidence for association on 10p15 with three adjacent SNPs, including an amino acid variant, in the 3′ region of ADAR3, implicating that gene as a positional candidate gene for OCD. It should be noted, however, that these findings do not withstand correction for multiple testing. Further studies of ADAR3 in OCD are necessary to clarify its potential role in the disorder.
The authors thank Kristin R. Chadha, M.S.W., Diane Q. Koram, M.S.W, and Aileen H. Prout, M.S.W. for their diagnostic interviews and Michael Boehnke, Ph.D. for consultation regarding research design. The authors are especially grateful to the families who participated in the study. This research was supported by grants from the National Institute of Mental Health R01 MH 58376 (GLH), K20 MH 01065 (GLH), and K02 MH01389 (EHC), the Obsessive Compulsive Foundation (EHC, GLH), and the Jean Young and Walden W. Shaw Foundation (BLL, EHC).
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.