Preservation of skin DNA for oligonucleotide array CGH studies : a feasibility study Array-based comparative genomic hybridization -LRB- a-CGH -RRB- is a promising tool for clinical genomic studies . However , pre-analytical sample preparation methods have not been fully evaluated for this purpose . Parallel sections of normal male human skin biopsy samples were collected and immediately immersed in saline , formalin and a molecular fixative for 8 , 12 and 24 h. Genomic DNA was isolated from the samples and subjected to amplification and labeling . Labeled samples were then co-hybridized with normal reference female DNA to Agilent oligonucleotide-based a-CGH 44k slides . Pre-analytic parameters such as DNA yield , quality of genomic DNA and labeling efficacy were evaluated . Also microarray analytical variables , including the feature signal intensity , data distribution dynamic range , signal to noise ratio and background intensity levels were assessed for data quality . DNA yield and quality of genomic DNA -- as evaluated by spectrophotometry and gel electrophoresis -- were similar for fresh and molecular fixative-exposed samples . In addition , labeling efficacy of dye incorporation was not drastically different . There was no difference between fresh and molecular fixative material comparing scan parameters and stem plot analysis of a-CGH result . Formalin-fixed samples , on the other hand , showed various errors such as oversaturation , non-uniformity in replicates , and decreased signal to noise ratio . Overall , the a-CGH result of formalin samples was not interpretable . DNA extracted from formalin-fixed tissue samples is not suitable for oligonucleotide-based a-CGH studies . On the other hand , the molecular fixative preserves tissue DNA similar to its fresh state with no discernable analytical differences . Introduction Applications of new technologies have resulted in major advancements in laboratory medicine -LSB- 1 -RSB- . Bringing these advances into clinical practice , however , requires careful evaluation and validation . Array-based comparative genomic hybridization -LRB- a-CGH -RRB- is an extremely powerful tool that can generate high resolution mapping of chromosomal abnormalities . Advances in microarray technology and bioinformatics have now made a-CGH easily available and affordable -LSB- 14 , 15 -RSB- . With a-CGH 's potential for clinical application , it is important that guidelines for proper sample preparation and control of quality are developed . Since conventional methods of clinical tissue preparation commonly employ formalin fixation , we studied the suitability of formalin for array CGH studies and compared the results to that of clinical samples that were preserved in a newly developed , molecular-friendly fixative . The study was approved by the University of Miami Institutional Review Board . Three separate normal skin biopsies from one volunteer healthy male were immediately sliced in three parts each 0.1 × 0.2 × 0.2 cm . One part was immersed in normal saline solution , one part in a methanol-based molecular tissue fixative , UMFIX -LRB- Universal Molecular Fixative , marketed as Tissue-Tek ® Xpress ™ Molecular Fixative , Sakura Finetek , Torrance , CA -RRB- , and the third slice was fixed in 10 % neutral buffered formalin . Volume of fixative/preservative was 150 ml and incubation was performed at room temperature . After 8 -LRB- set 1 -RRB- , 12 -LRB- set 2 -RRB- and 24 h -LRB- set 3 -RRB- , the genomic DNA was extracted from the samples using Puregene DNA Purification System tissue kit -LRB- Gentra , Minneapolis , MN -RRB- . One microliter of extracted DNA solution was diluted in Tris -- EDTA -LRB- TE -RRB- buffer and evaluated on ND-1000 spectrophotometer -LRB- NanoDrop Technologies , Rockland , DE -RRB- . Gel electrophoresis -LRB- 0.8 % agarose gel -RRB- was performed to evaluate the quality of genomic DNA . DNA yield and quality of genomic DNA as evaluated by spectrophotometry , were similar between the samples -LRB- Table 1 -RRB- , although UMFIX samples appeared to have better quality . A260/A280 ratio determines presence of contaminating proteins and a ratio of more than 1.8 is generally considered to be indicative of high quality sample . All UMFIX samples consistently had a ratio of more than 1.8 . We further evaluated the quality of DNA by agarose gel electrophoresis that showed presence of high molecular weight -LRB- HMW -RRB- genomic DNA band in all samples -LRB- Fig. 1a -RRB- . While formalin-fixed sample showed higher degradation and lower intensity of genomic DNA , UMFIX-exposed samples did not appear degraded . Table 1Spectrophotometric result of total DNA yield , DNA purity , and labeling efficiency , for fresh , formalin-fixed and UMFIX-exposed samplesSampleTotal DNA -LRB- μg -RRB- A260/A280A260/A230Sample-cy 5 labeling efficiency -LRB- pmol/μg -RRB- Control-cy3 labeling efficiency -LRB- pmol/μg -RRB- Fresh 13.891.771.4370.8111 Fresh 22.941.861.7942.889.6 Fresh 32.931.9252.897 Mean ± SD3 .25 ± 0.551.84 ± 0.071.74 ± 0.2955.47 ± 14.1999.20 ± 10.87 UMFIX 16.4161.85278.6110 UMFIX 22.71381.861.8449.688 UMFIX 34.0121.861.848.281.8 Mean ± SD4 .38 ± 1.881.86 ± 0.011.88 ± 0.1158.80 ± 17.1693.27 ± 14.82 Formalin 15.3661.580.8459.8142.4 Formalin 24.04241.671.1141.273.4 Formalin 34.4271.932.0836.277.4 Mean ± SD4 .61 ± 0.681.73 ± 0.181.34 ± 0.6545.73 ± 12.4497.73 ± 38.731 8 h , 2 12 h , 3 24 hFig . 1Result of gel electrophoresis for genomic DNA a HMW genomic DNA is visible in all samples . There is more DNA degradation in formalin-fixed samples . b Formalin-fixed samples do not show uniform strong smear after linear amplification . c PCR for GAPDH shows a 450 bp amplicon in all samples with a lesser intensity in formalin-fixed samples , L ladder -LRB- numbers indicate bp -RRB- , S fresh control in saline , U UMFIX , F formalin , P positive control , N negative control Genomic DNA from the skin samples and the control female DNA -LRB- Promega , Madison , WI -RRB- were then subjected to amplification according to Agilent 's -LRB- Palo Alto , CA -RRB- protocol for oligonucleotide array-based CGH for genomic DNA -LRB- version 2.0 August 2005 -RRB- . Amplification , of both male genomic DNA and female control DNA , 100 ng each , was performed using Qiagen REPLI-g Amplification Kit . Amplified DNA was digested during a 2 h incubation at 37 °C , with Alu I and Rsa I -LRB- 10 U/μl ; 5μl/reaction -RRB- restriction enzymes -LRB- Promega , Madison , WI -RRB- . Purification of digested DNA was performed with QIAprep Spin Miniprep Kit -LRB- Qiagen , Valencia , CA -RRB- . Digested DNA was subjected to electrophoresis to evaluate quality of amplified DNA by visual inspection of its uniformity and range . The amplification efficiency of DNA from formalin-fixed samples was less than fresh and UMFIX samples , as evidenced by the size and fluorescent intensity of the bands -LRB- Fig. 1b -RRB- . We also used PCR for GAPDH primers to gauge the quality of extracted DNA . PCR was performed using glyceraldehyde-3-phosphate dehydrogenase primers -LRB- GAPDH , Clonetech , Palo Alto , CA -RRB- using 0.5 μg of RNase-treated isolated DNA and Qiagen TaqPCR Mastermix -LRB- Qiagen , Valencia , CA -RRB- . The conditions for DNA PCR were as follows : 95 °C , 15 min ; 35 cycles at 94 °C , 45 s ; 60 °C , 45 s ; 72 °C , 2 min . As seen in Fig. 1c , a 450 bp band was detected in all samples , although the intensity was considerably lower in formalin-fixed material . Amplified Genomic DNA was then labeled using BioPrime Array CGH Genomic Labeling kit -LRB- Invitrogen , Carlsbad , CA -RRB- . Quality analysis and quantitation was performed with ND-1000 -LRB- NanoDrop Technologies , Wilmington , DE -RRB- spectrophotometer to calculate the labeling efficiency . Labeled and purified samples were combined with hybridization master mix and applied to Agilent Human Genome CGH Microarray 44B slides for 40 h at 65 °C . To minimize the impact of environmental oxidants on signal intensities , slides were scanned immediately using Agilent microarray scanner 4800B . Array images were analyzed using Agilent feature extraction software -LRB- v8 .1 -RRB- and CGH explorer -LRB- v2 .51 -RRB- -LSB- 9 -RSB- . Microarray analytical variables , including the feature signal intensity , data distribution dynamic range , signal to noise ratio and background intensity levels , and the number of saturated and undetected features was used to assess microarray quality . One of the steps in microarray quality control is labeling efficacy or dye incorporation . Dye incorporation was not drastically different between fresh and UMFIX-exposed samples but formalin-fixed samples showed less dye incorporation -LRB- Table 1 -RRB- . There were no differences between fresh and UMFIX-exposed material with regard to scan parameters -LRB- Table 2 -RRB- . Formalin-fixed samples , showed various errors such as over-saturation , non-uniformity in replicates and increased signal to noise ratio . The numbers of non-uniform features were at least ten fold higher in formalin-fixed samples . The signal to noise ratio of replicated probes can be used to evaluate reproducibility of signals . Formalin-fixed samples showed a higher median % CV value , indicating lower reproducibility of signal across the microarray and lower signal to noise ratio . The number of features that were saturated in the scanned image was also significantly higher in formalin-fixed samples . Table 2Representative data of microarray scan quality measurementSampleNon-uniform featureReproducibility : non-control replicated probes median % CVSaturated featureRedGreenRedGreenRedGreenFresh 11133797801Fresh 2511394400Fresh 3381355600Mean ± SD67 ± 40218 ± 1405 ± 16 ± 200UMFIX 1352560131300UMFIX 2851797800UMFIX 3361366700Mean ± SD158 ± 170292 ± 2338 ± 49 ± 300Formalin 11,4023027762851 Formalin 21,98715737123610 Formalin 33,1351709483260 Mean ± SD2 ,175 ± 882210 ± 8069 ± 309 ± 3324 ± 380Red cy5 labeled samples , green cy3 labeled controls , 1 8 h , 2 12 h , 3 24 h These findings may be attributed to erratic and random fragmentation of DNA in formalin-fixed samples . The fragmentation itself results in increase background noise and aberrant signal intensity due to random hybridization . We further analyzed array scan data by a-CGH explorer software using stem-plot analysis for moving averages . Fresh and UMFIX-exposed sample showed a readable plot with the expected difference in XY chromosome regions ; as expected from the female control and male samples . Also , they showed reproducibly amplified and deleted region in our test sample DNA . A-CGH result from formalin-fixed samples was not interpretable -LRB- Fig. 2 -RRB- . Fig. 2Array CGH stem plot of 8-h preserved skin samples -- chromosomes are displayed on x-axis and relative ratios on y-axis . Green -LRB- control XX female , Cy3 labeled -RRB- Red -LRB- test XY male , Cy5 labeled -RRB- . X and Y chromosomes are marked Array CGH studies have great potential for clinical application . Since the technique does not utilize live cells , it is considerably more advantageous when compared to conventional karyotyping techniques . Array CGH has its own limitations , such as failure to detect translocations ; nevertheless it offers unprecedented spatial resolution -LSB- 6 , 11 -RSB- . Recently , it has been shown that low-level gene copy number change is associated with changes in expression level of its transcripts -LSB- 12 -RSB- . Therefore , results of transcriptomics studies can be used to study DNA markers , which are more stable and easier to study than RNA . Array GGH was originally based on BAC -LRB- Bacterial Artificial Chromosomes -RRB- , but more recently it utilizes oligonucleotides , with its ability to provide whole genome-covering resolution . Synthetic oligonucleotides obviously have the advantage because their exact sequence and length are known for each element on the array . Oligonucleotide array CGH -LRB- oa-CGH -RRB- has the benefit of overcoming the difficulties inherent to BAC arrays , such as the amount of available DNA , clone management , probe identity due to PCR contaminations , and mapping inaccuracies . Using oa-CGH , it is also possible to eliminate another source of error , which is the batch-to-batch variability of Cot-1 DNA , used to block repetitive DNA sequences , since the oligonucleotide probes are designed to be repeat-free -LSB- 20 -RSB- . Samples used in nearly all a-CGH studies have been fresh or fresh-frozen tissue . Such material , although useful in research settings , is impractical and cumbersome to use in clinical practice . Furthermore , because diagnostic biopsy samples are relatively small , the amount of residual tissue for additional ancillary testing may be inadequate . Hence there is a great need to develop test strategies that require minimal amounts of tissue and are robust enough to withstand pre-analytical sample preparation . Simplified schemes for sample preservation that allow reliable histomorphology along with preservation of high quality macromolecules are desirable . We have previously described a novel tissue fixation and processing technique that beside providing adequate histomorphology also preserves high quality HMW RNA , akin to fresh samples -LSB- 19 -RSB- . This fixative also protects HMW tissue DNA for use in PCR studies -LSB- 18 -RSB- . Here , we further evaluated the suitability of skin tissue DNA for array CGH studies using same methods applied routinely for fresh samples . This study demonstrates that using the novel fixative it is possible to preserve and extract high molecular weight genomic DNA , supported by high labeling efficiency , comparable to that from fresh tissue . Furthermore , no artifacts were seen using this DNA in microarray scanning or analysis for array CGH . Array CGH studies have been performed on formalin-fixed tissues with variable and irreproducible results . Most of the prior studies were based on low-resolution BAC arrays without detailed description of DNA quality or array metrics -LSB- 10 , 21 -RSB- . Besides , there was often inadequate documentation of fixation time , the volumetric ratio of fixative to tissue , or buffering status . Only few studies have adequately addressed analytical aspects of fixation and processing on array CGH . Ghazani et al. -LSB- 4 -RSB- studied the effect of formalin fixation on MCF-7 cell line and on one breast cancer tissue sample . They used two different BAC clone arrays ; 1.7 k for the cell line and 19k for the breast cancer sample -LSB- 4 -RSB- . There was no mention of fixative volume or quality of genomic DNA . They showed that long term -LRB- 1 week -RRB- fixation results in extensive loss of HMW DNA . In cell lines , the concordance between fresh and formalin-fixed samples was around 85 % . When genomic DNA was amplified , the concordance decreased to 75 % . Johnson et al. -LSB- 5 -RSB- also studied the effect of formalin fixation on a-CGH using BAC arrays . Our results support their conclusion that analysis of DNA samples on agarose gels does not offer any advantage in prediction of suitability of formalin-fixed tissue samples for a-CGH . They also show that the quality of a-CGH depends on the integrity of DNA samples with the requirement that extracted DNA supports the PCR amplification of an amplicon of 300 bp or longer . We showed that it is possible to detect amplicons of up to 450 bp in formalin-fixed sample ; -LRB- albeit with lower intensity when compared to fresh or UMFIX-exposed samples -RRB- . The studies by Johnson and others show the possibility of having acceptable results with DNA from formalin-fixed samples using BAC arrays but there are no data that supports its suitability for oa-CGH arrays . This may be due to larger sequence of probes presented in BAC array . Other investigators have shown that using alternative approaches to DNA extraction , quantification , amplification or labeling may produce improved results . However , all other authors do agree that the procedures for improving DNA quality are neither substitute for high quality DNA nor they could obtain consistent results from formalin-fixed samples -LSB- 7 , 10 -RSB- . This is mostly due to complex chemical effect of formalin on tissue , which is still poorly understood . Formalin has a tissue penetration rate of 2.4 mm in 24 h and adequate fixation requires at least a ten to one volumetric ratio of fixative to tissue -LSB- 3 -RSB- . Therefore , many clinical specimens are only partially fixed by formalin before processing . Furthermore during processing , they are exposed to formalin and alcohol , introducing formalin and alcohol-related tissue artifacts -LSB- 13 -RSB- . Formaldehyde is a dipolar molecule and can react with amino , or imino group of the anionic forms of the amino acids . This reaction is time and temperature dependent -LSB- 2 , 9 , 16 , 17 , 18 -RSB- . More recent findings show that the deleterious effect of formalin fixation might also result from cumulative action of other reagents and processing conditions . Conversely , no DNA or nucleoside reactions have been reported with ethanol and none would be expected under physiological conditions -LSB- 8 -RSB- . By changing the three dimensional structure of the proteins , alcohols prevent protein functions . Therefore , rapid fixation of samples prevents alteration and degradation of biomolecules and preserves them in their native form -LSB- 11 -RSB- . In summary , we show that by using a new molecular fixative it is possible to preserve skin tissue DNA that is suitable for array CGH studies ; identical to fresh tissue . This can be achieved using same methods and protocols used for fresh samples .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

genomics
array comparative genomic hybridization
fixatives
tissue preservation


Environmental Factors in Obsessive-Compulsive Behavior : Evidence from Discordant and Concordant Monozygotic Twins To investigate environmental factors that protect against or exacerbate obsessive-compulsive -LRB- OC -RRB- symptoms , we selected 25 monozygotic -LRB- MZ -RRB- twin pairs discordant , 17 MZ twin pairs concordant high and 34 MZ pairs concordant low on OC symptoms from a large longitudinal Dutch sample of adult twin pairs and their family members , applying stringent criteria for OC symptomatology . Data were collected on psychopathology , family structure , health , lifestyle , birth complications and life events . Unique environmental factors were studied using within-discordant MZ pair comparisons , whereas between-concordant MZ pair comparisons were used to study environmental factors that are shared by the twins of an MZ pair . The high-scoring MZ twins of the discordant group reported more life events -LRB- especially sexual abuse -RRB- than their low-scoring twin-siblings . The between-pair comparisons showed lower birth weight in the discordant MZ pairs than in the concordant MZ pairs . Further , the concordant high MZ pairs as well as their spouses had a lower educational level than the two other groups . On scale scores of anxious-depression , neuroticism , and somatic complaints , concordant high MZ pairs showed highest scores , and the discordant MZ pairs scored intermediate , except for neuroticism , on which the high-scoring twins of discordant MZ pairs were equal to the concordant high pairs . Discordance on psychological scale scores between the concordant MZ pairs was evident from 1991 onward , and within the discordant MZ pairs from 1997 onward , confirming previous reports of an association of early-onset OC symptoms with higher genetic load . Parent scores of OC symptoms and anxious-depression suggested intermediate genetic load in the discordant MZ group . In conclusion , this study reports on both unique and shared environmental factors associated with OC symptomatology . Whether these factors operate in addition to or in interaction with genetic disposition is to be elucidated in future studies . Introduction Obsessive-compulsive disorder -LRB- OCD -RRB- is characterized by repetitive distressing and anxiety-provoking intrusive thoughts , mostly in combination with time-consuming repetitive actions designed to reduce tension or anxiety caused by the disturbing thoughts -LRB- American Psychiatric Association 1994 -RRB- . OCD can run , especially if untreated , a chronic and disabling course -LRB- Nestadt et al. 1998 -RRB- . Family studies have quite convincingly shown that early-onset OCD is familial -LRB- Pauls et al. 1995 ; Nestadt et al. 2000 -RRB- . Studies in 7 - to 12-year-old twins have indicated that between 47 and 58 % of the variance in obsessive-compulsive -LRB- OC -RRB- behavior is explained by additive genetic factors -LRB- Hudziak et al. 2004 -RRB- . The remaining variance is almost entirely explained by unique environment , with a small contribution of shared environmental factors -LRB- 16 % -RRB- at age 12 . In adults , twin studies have indicated a more modest contribution of genetic factors -LRB- van Grootheest 2005 and others -RRB- . One twin study in women suggested heritability of 33 and 26 % respectively for obsessions and compulsions -LRB- Jonnal et al. 2000 -RRB- . Further , a recent twin study in 5,893 mono - and dizygotic twins , and 1,304 additional siblings from the population-based Netherlands Twin Register -LRB- NTR ; Boomsma et al. 2002 -RRB- , indicated heritability estimates of 47 % for both men and women -LRB- van Grootheest et al. 2007a , b -RRB- . The course of OCD is moderately stable : longitudinal twin studies as well as epidemiological and clinical studies have indicated that on average 50 % of cases remit over time -LRB- van Grootheest et al. 2007a , b ; Angst et al. 2004 ; Skoog and Skoog 1999 -RRB- . Environmental factors explain about half of persistence in boys and two-third of persistence in girls . Thus , environmental factors are of substantial importance in the likelihood to obtain and persist or remit with respect to OC symptomatology . To date , only few studies have addressed the specific nature of these environmental factors in OC phenomenology . Which environmental influences can be detected from the literature ? Family studies have revealed that parents of children with OCD suffer from poorer mental health and have fewer coping strategies than parents of healthy children -LRB- Derisley et al. 2005 -RRB- . A-specific risk factors for -LRB- the persistence of -RRB- OCD include : earlier age at onset , presence of co-morbid conditions and low socio-economic status -LRB- Skoog and Skoog 1999 ; Stewart et al. 2004 , 2006 ; Angst et al. 2004 -RRB- . Further , OCD patients report more often than healthy controls to have been overprotected or emotionally neglected by their parents -LRB- Cavedo and Parker 1994 -RRB- . Patients with the hoarding subtype of OCD in particular , report a lack of parental emotional warmth -LRB- Alonso et al. 2004 -RRB- . Perinatal risk factors , such as prolonged labor and edema during pregnancy , have been reported to increase the risk of later OCD -LRB- Vasconcelos et al. 2007 -RRB- . Childhood sexual abuse appears to be an important mediator for later OCD , especially in women -LRB- Lochner et al. 2002 -RRB- . The relationship between religiosity and OCD is unclear . Some authors find increased frequencies of religious obsessions and hand washing among highly religious protestants in comparison with less or non-religious subjects -LRB- Abramowitz et al. 2004 -RRB- , while others find no relation between religiosity and an increase in OC symptoms -LRB- Assarian et al. 2006 -RRB- , and argue that religiosity is merely a form in which OC symptoms can be displayed -LRB- religious obsessions -RRB- -LRB- Tek and Ulug 2001 -RRB- . Finally , β-hemolytic streptococcal infections have been reported to be associated with OC symptom exacerbation -LRB- March et al. 1990 -RRB- . The comparison of monozygotic -LRB- MZ -RRB- twins who score high on a trait with their low-scoring co-twins , comprises a powerful method to identify environmental factors involved in a disorder -LRB- Martin et al. 1997 -RRB- . MZ twins have identical genomes and are born and raised at the same time in the same family , thus sharing a very similar family environment . Consequently , discordance on the trait is mostly explained by differences in the non-shared -LRB- i.e. , unique -RRB- environment that act either directly on the phenotype , or by epigenetic mechanisms -LRB- Fraga et al. 2005 -RRB- . Environmental factors that are shared by both members of a twin pair -LRB- such as maternal smoking during pregnancy , or parental divorce -RRB- can be studied by comparing MZ twins who are concordant high on the trait with MZ twins who are concordant low . Comparisons within discordant MZ pairs or between concordant MZ pairs have not been employed to study environmental factors involved in OC phenomenology . In other psychiatric disorders , such as schizophrenia and ADHD -LRB- Stabenau and Pollin 1993 ; Lehn et al. 2007 -RRB- , as well as in somatic disorders such as diabetes mellitus -LRB- Bo et al. 2000 -RRB- , this method has been successfully used . One twin study on a disorder related to OCD , i.e. , Gilles de la Tourettes ' Syndrome , has studied basal ganglia D2-receptorbinding in five MZ twins who were discordant on tic severity , and found that caudate nucleus D2 receptor binding increased by up to 17 % in the more severely affected twins when compared with their less severely affected twin siblings -LRB- Wolf et al. 1996 -RRB- . This within-MZ twin discordance reflects unique environmental influences on D2-caudate receptor density . In this study , we used prospective data of adult twins from the NTR , who have been followed between 1991 and 2002 , and about whom information on a wide range of variables was collected every 2 -- 3 years -LRB- Boomsma et al. 2000 -RRB- . Differences between the MZ concordant and discordant groups were described using measures of anxiety and depression co-occurring with OC behavior . The aim of this explorative study was to replicate and extend the information from previous studies on both unique and shared environmental influences that might protect against or exacerbate OC behavior . Unique environmental factors were studied using within-discordant MZ twin pair comparisons . To study environmental factors shared by both twins of a pair , between-MZ pair comparisons were used . Parent data on level of education and on drinking and smoking behavior were used to compare the groups of twin pairs on these common environment influences . Further , measures of anxiety , depression and personality were compared between the parents of the concordant and discordant twin pairs , with the following reasoning : concordance between MZ twin pairs on OC behavior most likely results from genetic similarity between the twins of a pair . Thus , the contrasts between twin pairs who are concordant high and low reflect differences in genetic vulnerability to OC behavior . As a consequence , the parent scores on OC symptoms , on anxious depression and on neuroticism -LRB- the latter characteristics are known to be related to OC symptoms -RRB- are expected to reflect these differences in genetic vulnerability and therefore to be highest in the parents of the concordant high MZ pairs , to be intermediate in the parents of the discordant MZ pairs and to be low in the parents of the concordant low MZ pairs . Finally , longitudinal measures of psychopathology were studied to investigate age at onset of OC symptoms , anxiety and depressive symptoms in the concordant and discordant groups . Family studies have suggested that lower age at onset is associated with higher familiarity , possibly reflecting higher genetic load -LRB- Delorme 2005 -RRB- . We hypothesized that the concordant high MZ twin pairs , in whom the OC symptoms are theoretically more genetically determined , would show lower age at onset than the high-scoring twins of the discordant group in whom unique environmental factors might be more important . Method Sample selection The data of this study originate from a longitudinal study in twin families registered with the NTR -LRB- Boomsma et al. 2002 -RRB- . Since 1991 , twins and their families received a survey by mail every 2 -- 3 years containing questionnaires about health , personality , life events , perinatal circumstances and lifestyle . The 2002 survey formed the starting point of this study . The Padua Inventory Abbreviated -LRB- PI ABBR -RRB- was added to the 2002 wave of data collection and was derived from the Padua Inventory-Revised version -LRB- PI-R -RRB- , a widely used self-report inventory on OC symptoms -LRB- Sanavio 1988 ; van Oppen 1992 -RRB- . The PI-R is a 41-item self-report instrument that measures OC symptoms on a 0 -- 4 scale , and contains five subscales : washing , checking , rumination , precision and impulses -LRB- van Oppen et al. 1995 -RRB- . It has been validated in the Netherlands , shows good psychometric qualities , and moderately correlates with the Y-BOCS symptom checklist , a clinician-derived checklist on OC symptoms -LRB- Denys et al. 2004 -RRB- . For the purpose of this epidemiological twin study , the PI-R was reduced to 12 items . Item choice was based on two items of each subscale with highest factor loadings in a previous validation study -LRB- van Oppen et al. 1995 -RRB- , and with one additional item for each of the more equivocal obsession subscales : rumination and impulses . The PI-R ABBR is shown in Table 1 . To investigate its psychometric qualities psychometric analyses have been conducted in three groups derived from an earlier study by van Oppen et al. -LRB- 1995 -RRB- . These groups encompassed a population-based control group -LRB- n = 428 -RRB- , a psychiatric control group -LRB- n = 272 -RRB- and a clinical OCD group -LRB- n = 120 -RRB- ; for an extensive description of the study groups -LRB- see van Oppen et al. 1995 -RRB- . Cronbachs ' α of the scale was 0.73 , which is an indication of good internal consistency . Analyses of variance -LRB- ANOVAs -RRB- of PI-R ABBR scores within the three groups revealed a significant main between-group effect -LRB- p < 0.0001 -RRB- . Post-hoc t-tests showed that the mean PI-R ABBR OC score for the OCD group -LRB- 20.7 ± 8.1 -RRB- was significantly higher than scores of the psychiatric control group -LRB- 12.4 ± 7.4 -RRB- as well as the population control group -LRB- 6.6 ± 5.6 ; p < 0.0001 in both comparisons -RRB- . To investigate whether the PI-R ABBR can accurately screen for OCD , and to establish cut-points of OC behavior , receiver operating characteristic -LRB- ROC -RRB- analyses were carried out . ROC analyses use the association between sensitivity and specificity to derive an area under the curve -LRB- AUC -RRB- , which indicates how well a measure distinguishes between case positives -LRB- i.e. , OCD group -RRB- and case negatives -LRB- i.e. , psychiatric controls or population controls -RRB- irrespective of the base rate . A value of 0.50 of the AUC indicates chance level and 1.0 indicates a perfect diagnostic tool -LRB- Swets 1996 ; McFall and Treat 1999 -RRB- . The AUC for the PI-R ABBR when compared with clinical controls was 0.78 -LRB- 95 % CI = 0.73 -- 0.83 -RRB- . When compared with the population controls , the AUC was 0.93 -LRB- 95 % CI = 0.90 -- 0.95 -RRB- . At the best cut-off point of 16 -LRB- i.e. , maximum difference between sensitivity and 1-specificity -RRB- , the sensitivity was 0.74 with a specificity of 0.72 , when compared with clinical controls . -LRB- A detailed description of the ROC analyses on the PI-R ABBR is available upon request through the first author . -RRB- Table 1The Padua Inventory-Revised abbreviated -LRB- PI-R ABBR -RRB- PI-R ABBROriginal factor1In certain situations , I am afraid of losing my self-control and doing embarrassing thingsImpulses2I check and recheck gas and water taps and light switches after turning them offChecking3I feel obliged to follow a particular order in dressing , undressing and washing myselfPrecision4When I see a train approaching I sometimes think I could throw myself under its wheelsImpulses5I return home to check doors , windows , drawers etc. , to make sure they are properly shutChecking6When I start thinking of certain things , I become obsessed with themRumination7I feel I have to repeat certain numbers for no reasonPrecision8Unpleasant thoughts come into my mind against my will and I can not get rid of themRumination9My thoughts constantly go astray , therefore I find it difficult to attend to what is happening around meRumination10I sometimes have to wash or clean myself dimply because I think I may be dirty or ` contaminated ' Washing11I get upset and worried at the sight of knives , daggers and other pointed objectsImpulses12If I touch something which I think is ` contaminated ' , I immediately have to wash or clean myselfWashingFig . 1The 2002 NTR wave using the PI-R abbreviated Of the adult twins , 2,672 pairs , their family members and -- in some instances -- their spouses -LRB- a total of 9,950 individuals -RRB- returned the survey . Monozygotic twin pairs were selected on the basis of high or low scores on the PI-R ABBR . Using the stringent criteria derived from the analyses described above , discordant , concordant high and concordant low MZ twin pairs were selected . Twin pairs were considered to be discordant when one twin scored > 17 -LRB- in the clinical range -RRB- , and his/her MZ twin sibling scored < 7 -LRB- population control range -RRB- . Pairs were considered to be concordant high when both twins scored > 17 , and concordant low when both twins scored < 7 . Information on zygosity from DNA polymorphisms was available in 19 MZ twin pairs -LRB- 25 % -RRB- of the final sample . When DNA polymorphisms were not available zygosity was determined from questions about physical similarity of the twins and confusion of the twins by family members , friends and strangers . Overall , agreement between zygosity diagnoses based on questionnaire and DNA data is 97 % -LRB- Willemsen et al. 2005 -RRB- . After exclusion of incomplete pairs , 25 MZ discordant pairs , 17 MZ concordant high pairs and 521 MZ concordant low pairs were identified . Concordant low pairs were matched on age and sex with concordant high pairs and oversampled , so that 34 concordant low pairs were finally retained -LRB- Fig. 1 -RRB- . Of the final 76 MZ twin pairs selected for this study , 18 pairs participated in wave 2002 only , 28 pairs participated in two waves , 4 pairs in three , 8 pairs in four , 13 pairs in five , and 4 pairs in all six waves . Measures and instruments The NTR survey contains a broad range of longitudinal measurements taken at six time points between 1991 and 2002 , as well as cross-sectional measurements . Information is obtained on life events , perinatal adversities , physical and mental health , lifestyle factors such as physical activity , religiosity , drinking , smoking and drug behavior , and on demographic variables such as relationships , number of children , level of education , living situation , and work status . Since this is an exploratory study , all available information was taken into account . Religiosity was assessed by asking whether the respondent had had a religious upbringing -LRB- yes/no -RRB- , the person 's current religion , and whether the respondent currently was an active church member . On alcohol and smoking behavior , respondents were questioned about their consumption ever , in the past year and past month , as well as the number of cigarettes or glasses of alcohol per week . Alcohol dependence was assessed by the CAGE -LRB- four questions -RRB- -LRB- Bush et al. 1987 -RRB- . The occurrence of negative life events throughout the lifespan was measured in the 2002 survey , using an adapted version of the Dutch life event scale -LRB- Schokverwerkings Inventarisatie Lijst = SchIL -RRB- -LRB- van der Velden et al. 1992 -RRB- . This scale gathers information on : death of a spouse , father , mother , child , sibling or significant other ; serious illness or injury of self or a significant other ; divorce/break-up of a relationship ; traffic accident ; violent and sexual assault or rape , and robbery . Response categories are : never experienced ; 0 -- 6 months ago ; 6 -- 12 months ago ; 1 -- 5 years ago , and more than 5 years ago . Data on anxiety and depression were available at most time points between wave 1 and 6 , and were assessed with : the Spielberger State-Trait Anxiety Inventory , trait scale -LRB- Spielberger et al. 1970 ; van der Ploeg 1979 -RRB- , and the young adult self-report -LRB- YASR -RRB- , anxious-depressed subscale -LRB- Achenbach 2000 ; Verhulst et al. 1997 -RRB- . Questionnaires between waves 1 and 4 -LRB- between 1991 and 1997 -RRB- also contained the 8-item OC symptom subscale of the YASR -LRB- Nelson et al. 2001 ; Geller et al. 2006 -RRB- . Neuroticism , somatic complaints and extraversion were measured with the Amsterdamse Biografische Vragenlijst -LRB- ABV ; Amsterdam Biographical Questionnaire -RRB- -LRB- Wilde 1970 -RRB- . The ABV neuroticism and extraversion scales are very similar to the Eysenck Personality Questionnaire neuroticism and extraversion subscales -LRB- Eysenck and Eysenck 1964 -RRB- , and contain answer categories : yes/no/don ' t know . The satisfaction with life scale and the subjective happiness scale -LRB- Lewis and Joseph 1995 -RRB- , a combined 10-item scale with scoring possibilities between 1 and 7 were taken , and the Rosenberg self-efficacy scale , a 10-item scale scoring between 1 and 4 -LRB- Rosenberg 1965 ; Helbing 1982 -RRB- . Socio Economic Status -LRB- SES -RRB- in 2002 was assessed using a full description of the occupation of the twins according to the descriptions provided by the Central Office for Statistics in the Netherlands . The work level was coded into three levels based on the mental complexity of the work , ranging from low skilled -LRB- 1 -RRB- to academic work -LRB- 3 -RRB- . Living situation was coded between 1 and 3 -LRB- 1 = with parents ; 2 = alone ; 3 = with partner -RRB- . Finally , we used child-derived information on their parents ' level of education . Direct parent information was used to collect information on their smoking and drinking behavior and on their scores of OC behavior , anxiety and depression . Statistical analyses Within-pair analyses Within-discordant pair differences between the high and low-scoring twins on the PI-R ABBR were calculated using paired t-tests -LRB- t-tests for two related samples -RRB- for continuous data , Wilcoxon signed - rank tests for ordinal data and McNemar χ2 tests of matched pairs for nominal data . Between-pair analyses Variables that measure psychological health , as well as measures of environmental influences on OC symptoms were compared between the concordant high , the concordant low and the discordant MZ twin pairs , using one-way ANOVAs for continuous data , Kruskal -- Wallis tests for ordinal data and χ2 tests for nominal data . Post hoc comparisons were conducted using post hoc Scheffé 's -LRB- continuous data -RRB- and Mann -- Whitney U-tests -LRB- ordinal data -RRB- . Post hoc Scheffé testing , although more liberal than Bonferroni correction , provide some correction of type I error . Two-tailed probabilities were used in all analyses , since we had no clear expectation of the direction of the findings . To adjust for correlated error in the between-group comparisons of common environment variables , separate regression analyses -LRB- multiple regression for continuous measures and logistic regression for categorical measures -RRB- were conducted in STATA 9.2 for these variables -LRB- StatCorp , College Station , TX , USA -RRB- . The robust cluster option was used to account for nonindependence of the twin pairs on the variables that reflected common environmental influences -LRB- i.e. , caesarean section , birth weight , and religious upbringing of the twin ; parental death and divorce , death of a sibling , and level of education , alcohol use and smoking behavior of the parents -RRB- . Alpha was set at 0.05 . Results Within-pair analyses of discordant pairs Twenty-five MZ twin pairs discordant on OC behavior were included , of whom 18 pairs were female . Their mean age was 29.6 years -LRB- SD 6.8 years -RRB- . Mean PI-R ABBR OC scores in the high-scoring twins of the discordant pairs were 21.4 -LRB- SD 5.9 -RRB- , in the low scoring twins 4.5 -LRB- SD 2.0 -RRB- . Health and lifestyle characteristics High-scoring twins of the discordant pairs experienced lower general health -LRB- p = 0.03 -RRB- and more impediments in physical activity -LRB- p < 0.001 -RRB- than the low-scoring co-twins . The duration of the current relationship of the high-scoring twins tended to be shorter -LRB- p = 0.06 -RRB- , and they tended to have fewer children -LRB- p = 0.07 -RRB- . They were less satisfied with life -LRB- p = 0.02 -RRB- , less happy -LRB- p = 0.001 -RRB- and had lower self-efficacy scores -LRB- p = 0.006 -RRB- . They showed no differences with respect to birth weight , birth order , church participation , drinking or smoking behavior , nor on level of education , work status , living situation , or number of -LRB- mental -RRB- health contacts . The high-scoring twins of the discordant pairs scored significantly higher on the YASR anxious-depressed subscale -LRB- p < 0.001 -RRB- , on the neuroticism subscale -LRB- p < 0.001 -RRB- , on the ABV subscale of somatic complaints -LRB- p < 0.001 -RRB- , and on STAI-trait -LRB- p < 0.001 -RRB- . On the ABV extraversion scale , no within-pair differences were found -LRB- Table 2 -RRB- . Table 22002 wave of data collection : within-discordant twin pair characteristicsLow risk twin , mean -LRB- SD -RRB- High risk twin , mean -LRB- SD -RRB- Test statistic * p-ValueBirth weight -LRB- g -RRB- 2,189 -LRB- 806 -RRB- 2,028 -LRB- 667 -RRB- 0.9 n.s.Birth order -LRB- first born -RRB- n = 10n = 141.7 n.s.General health -LRB- 1 -- 5 -RRB- 4.1 -LRB- 0.7 -RRB- 3.8 -LRB- 0.6 -RRB- 2.30.03 Mental health contacts ever yesn = 7n = 90.5 n.s.Sumscore impediments physical activity43 .5 -LRB- 12.5 -RRB- 53.9 -LRB- 12.4 -RRB- − 4.2 < 0.001 Number of persons drinking evern = 23n = 230n . s.Number of drinks per week -LRB- past 12 months -RRB- 11.1 -LRB- 1.4 -RRB- 13.4 -LRB- 0.7 -RRB- 0.19 n.s.CAGE score alcohol dependence4 .0 -LRB- 0.2 -RRB- 4.3 -LRB- 0.7 -RRB- − 1.4 n.s.Duration current relation -LRB- years -RRB- 5.9 -LRB- 7.4 -RRB- 3.0 -LRB- 0 -RRB- 1.97 n.s.Number of children1 .1 -LRB- 1.2 -RRB- 0.7 -LRB- 0.6 -RRB- 1.87 n.s.Education level self -LRB- 1 -- 13 -RRB- 7.9 -LRB- 3.0 -RRB- 8.3 -LRB- 2.6 -RRB- − 0.8 n.s.Education level partner -LRB- 1 -- 13 -RRB- 8.3 -LRB- 3.6 -RRB- 8.1 -LRB- 3.1 -RRB- − 1.2 n.s.Living situation -LRB- 1 -- 4 -RRB- 2.8 -LRB- 0.8 -RRB- 2.7 -LRB- 0.8 -RRB- − 1.3n.s.PI-R ABBR OC scale4 .5 -LRB- 2.0 -RRB- 21.4 -LRB- 5.9 -RRB- − 13.7 < 0.001 YASR anxious depression scale4 .3 -LRB- 2.8 -RRB- 11.6 -LRB- 4.3 -RRB- − 6.4 < 0.001 ABV extraversion51 .0 -LRB- 16.7 -RRB- 45.6 -LRB- 12.5 -RRB- 1.1 n.s.ABV neuroticism48 .6 -LRB- 23.1 -RRB- 85.3 -LRB- 27.4 -RRB- − 5.8 < 0.001 ABV somatic complaints16 .6 -LRB- 5.1 -RRB- 24.4 -LRB- 10.3 -RRB- − 4.0 < 0.001STAI-trait31.6 -LRB- 4.7 -RRB- 46.4 -LRB- 10.8 -RRB- − 6.5 < 0.001 Satisfaction with life scale scores27 .4 -LRB- 4.1 -RRB- 23.8 -LRB- 7.0 -RRB- 2.30.02 Happiness scores22 .7 -LRB- 3.9 -RRB- 17.9 -LRB- 5.8 -RRB- 230.001Self-efficacy scores31 .3 -LRB- 4.0 -RRB- 27.5 -LRB- 4.7 -RRB- 2.90.006PI-R ABBR , Padua Inventory-Revised Abbreviated ; OC , obsessive-compulsive ; YASR , young adult self-report ; ABV , Amsterdamse Biografische vragenlijst ; STAI , State trait Anxiety Inventory ; SBL , Spannings behoefte Lijst -LRB- sensation seeking list -RRB- ; CI , confidence interval ; n.s. , not significant Unique environment influences The only within-pair difference found on unique life events , was the tendency of the high-scoring twins of the discordant pairs to have experienced more sexual assault than the low-scoring twins -LRB- p = 0.08 -RRB- . All persons who had experienced sexual assault were women . Two low-scoring twins of the discordant pairs reported on sexual assault , versus five high-scoring twins . The low-scoring twins and four of the five high-scoring twins of the discordant pairs reported to have experienced the assault more than 5 years ago , versus one twin who had experienced sexual abuse between 1 and 5 years ago -LRB- Table 3 -RRB- . Table 3Within-discordant twin comparisons -- unique life eventsLow OC twin , mean -LRB- SD -RRB- High OC twin , mean -LRB- SD -RRB- p-ValueBirth weight -LRB- g -RRB- 2,189 -LRB- 806 -RRB- 2,028 -LRB- 667 -RRB- n.s.Disease self -LRB- 0 -- 2 -RRB- 0.08 -LRB- 0.4 -RRB- 0.26 -LRB- 0.6 -RRB- n.s.Disease child -LRB- 0 -- 2 -RRB- 0.09 -LRB- 0.4 -RRB- 0.6 -LRB- 0.1 -RRB- n.s.Disease partner -LRB- 0 -- 2 -RRB- 0.09 -LRB- 0.4 -RRB- 0n . s.Disease significant other -LRB- 0 -- 2 -RRB- 0.82 -LRB- 0.9 -RRB- 0.82 -LRB- 0.9 -RRB- n.s.Death child -LRB- 0 -- 2 -RRB- 00n . s.Death partner -LRB- 0 -- 2 -RRB- 00n . s.Death significant other -LRB- 0 -- 2 -RRB- 1.1 -LRB- 0.9 -RRB- 1.0 -LRB- 0.9 -RRB- n.s.Sexual abuse * -LRB- 0 -- 2 -RRB- 0.17 -LRB- 0.5 -RRB- 0.43 -LRB- 0.8 -RRB- 0.08 Violence -LRB- 0 -- 2 -RRB- 0.17 -LRB- 0.6 -RRB- 0.17 -LRB- 0.6 -RRB- n.s.Relationship termination -LRB- 0 -- 2 -RRB- 0.52 -LRB- 0.8 -RRB- 0.82 -LRB- 0.9 -RRB- n.s.Theft -LRB- 0 -- 2 -RRB- 0.47 -LRB- 0.8 -RRB- 0.56 -LRB- 0.8 -RRB- n.s.Traffic accident -LRB- 0 -- 2 -RRB- 0.52 -LRB- 0.8 -RRB- 0.38 -LRB- 0.7 -RRB- n.s.Dismissal -LRB- 0 -- 2 -RRB- 0.30 -LRB- 0.7 -RRB- 0.48 -LRB- 0.7 -RRB- n.s.Total score life events2 .35 -LRB- 1.9 -RRB- 2.76 -LRB- 1.7 -RRB- n.s. Longitudinal data Young adult self-report OC subscale scores , taken in 1991 , 1995 and 1997 , revealed significant differences between high and low-scoring twins of the discordant pairs in 1997 -LRB- p = 0.007 -RRB- . Further , scale scores between 1991 and 2002 revealed significant within-pair differences on the YASR anxious-depressed subscale from 1997 onward -LRB- p = 0.001 -RRB- , on the neuroticism subscale from 1993 onward -LRB- p-value between 0.01 and < 0.001 at wave 2 -- 5 -RRB- , on the ABV subscale of somatic complaints from 1997 onward -LRB- p-value between 0.015 and < 0.001 -RRB- , and on STAI-trait scores from 1997 onward -LRB- p-values between 0.007 and < 0.001 -RRB- . Between-pair analyses of concordant and discordant pairs Seventeen MZ twin pairs were included who were concordant high on OC behavior , of whom 14 pairs were female . Their mean age was 30.0 years -LRB- SD 11.2 years -RRB- , mean PI-R ABBR OC scores were 23.7 -LRB- SD 6.7 -RRB- . Thirty-four MZ twin pairs were included who were concordant low on OC behavior , of whom 28 pairs were female . Their mean age was 30.0 years -LRB- SD 11.3 years -RRB- , mean PI-R ABBR OC scores were 3.8 -LRB- SD 2.2 -RRB- . Health and lifestyle characteristics The concordant low group generally experienced the best health , with the discordant group scoring intermediate between high and low concordant groups . Members of the discordant group more often had a spouse than the concordant high group , and were living with a spouse more often than both concordant groups . No between-group differences were found for smoking behavior . For drinking behavior , the concordant high group showed the highest scores on alcohol dependence -LRB- p = 0.02 and 0.04 in comparison with the concordant low and discordant group -RRB- , although they scored intermediate between the low and discordant groups on current number of drinks per week . On religious upbringing , there were no significant differences between the study groups . Interestingly , the concordant low MZ twin pairs , as well as their spouses , reported to have a higher level of education than the concordant high and discordant twin pairs -LRB- p-values 0.02 in both comparisons -RRB- -LRB- Table 4 -RRB- . Table 42002 wave of data collection : between concordant and discordant twin pair health and lifestyle characteristicsConcordant Low twin pairs , mean -LRB- SD -RRB- Concordant high twin pairs , mean -LRB- SD -RRB- Discordant twin pairs , mean -LRB- SD -RRB- Low -- high p-valueLow -- discordant p-valueHigh -- discordant p-valueGeneral health -LRB- 1 -- 5 -RRB- 4.4 -LRB- 0.7 -RRB- 3.6 -LRB- 1.2 -RRB- 4.1 -LRB- 0.7 -RRB- < 0.0010.007 n.s.Mental health contacts ever yesn = 8 -LRB- 12 % -RRB- n = 21 -LRB- 61 % -RRB- n = 16 -LRB- 32 % -RRB- < 0.0010.0060.009 Impediments physical activity41 .3 -LRB- 12.6 -RRB- 53.0 -LRB- 17.1 -RRB- 43.5 -LRB- 12.5 -RRB- 0.001 n.s. 0.03 Specialized medical treatment ever yesn = 11 -LRB- 16 % -RRB- n = 15 -LRB- 44 % -RRB- n = 11 -LRB- 22 % -RRB- 0.002 n.s. 0.04 Currently active in church -LRB- 1 -- 3 -RRB- 0.8 -LRB- 0.7 -RRB- 0.5 -LRB- 0.7 -RRB- 0.9 -LRB- 0.8 -RRB- n.s.n.s.n.s.Number of persons drinking evern = 62 -LRB- 91 % -RRB- n = 19 -LRB- 56 % -RRB- n = 35 -LRB- 70 % -RRB- n.s. 0.003 n.s.Number of drinks per week -LRB- past 12 months -RRB- 2.7 -LRB- 1.4 -RRB- 2.3 -LRB- 1.5 -RRB- 1.9 -LRB- 1.2 -RRB- n.s. 0.01 n.s.CAGE score alcohol dependence4 .1 -LRB- 0.5 -RRB- 4.5 -LRB- 0.8 -RRB- 4.2 -LRB- 0.5 -RRB- 0.02 n.s. 0.04 Number of persons smoking evern = 20 -LRB- 29 % -RRB- n = 14 -LRB- 41 % -RRB- n = 18 -LRB- 36 % -RRB- n.s.n.s.n.s.Number of cigarettes per day -LRB- 1 -- 7 -RRB- 4.1 -LRB- 1.1 -RRB- 4.7 -LRB- 1.1 -RRB- 3.8 -LRB- 1.0 -RRB- n.sn.s .0.07 Number of persons with partnern = 41 -LRB- 60 % -RRB- n = 17 -LRB- 50 % -RRB- n = 35 -LRB- 70 % -RRB- n.s.n.s. 0.04 Children yesn = 16 -LRB- 23 % -RRB- n = 8 -LRB- 23 % -RRB- n = 25 -LRB- 50 % -RRB- n.s. 0.0030.015 Education level self -LRB- 1 -- 13 -RRB- 9.4 -LRB- 2.3 -RRB- 8.27 -LRB- 2.8 -RRB- 8.24 -LRB- 2.8 -RRB- 0.020.02 n.s.Education level partner -LRB- 1 -- 13 -RRB- 9.1 -LRB- 2.8 -RRB- 6.7 -LRB- 3.7 -RRB- 8.20 -LRB- 3.4 -RRB- 0.006 n.s.n.s.Living situation -LRB- 1 -- 3 -RRB- 2.4 -LRB- 0.8 -RRB- 2.2 -LRB- 1.0 -RRB- 2.8 -LRB- 0.8 -RRB- n.s.0.040.01PI-R ABBR OC scale3 .8 -LRB- 2.2 -RRB- 23.7 -LRB- 6.7 -RRB- 12.9 -LRB- 9.5 -RRB- < 0.001 < 0.001 < 0.001 YASR anxious depression scale2 .9 -LRB- 4.7 -RRB- 14.5 -LRB- 19.3 -RRB- 8.7 -LRB- 12.6 -RRB- < 0.001 < 0.001 < 0.001 ABV extraversion62 .8 -LRB- 16.2 -RRB- 46.4 -LRB- 17.7 -RRB- 48.3 -LRB- 17.7 -RRB- < 0.001 < 0.0010.001 ABV neuroticism36 .2 -LRB- 18.9 -RRB- 92.8 -LRB- 19.7 -RRB- 66.6 -LRB- 31.1 -RRB- < 0.001 < 0.001 n.s.ABV somatic complaints15 .7 -LRB- 3.7 -RRB- 27.5 -LRB- 8.3 -RRB- 20.3 -LRB- 8.8 -RRB- < 0.0010.003 < 0.001STAI-trait29.4 -LRB- 6.4 -RRB- 36.9 -LRB- 7.9 -RRB- 37.6 -LRB- 7.5 -RRB- < 0.001 < 0.001 < 0.001 Satisfaction with life28 .7 -LRB- 4.0 -RRB- 19.8 -LRB- 7.2 -RRB- 25.6 -LRB- 5.9 -RRB- < 0.0010.010.001 Happiness24 .2 -LRB- 3.2 -RRB- 16.2 -LRB- 5.7 -RRB- 20.4 -LRB- 5.4 -RRB- < 0.001 < 0.0010.01Self-efficacy33.7 -LRB- 3.9 -RRB- 25.5 -LRB- 3.9 -RRB- 29.4 -LRB- 4.7 -RRB- < 0.001 < 0.001 < 0.001 On life events , the concordant high MZ twin pairs reported more often that they had been dismissed from work than the concordant low scoring pairs -LRB- p = 0.04 -RRB- , with the discordant pairs scoring between the concordant high and low pairs . Further , the discordant pairs reported more often to have been sexually assaulted in comparison with both the concordant low and high-scoring pairs ; n = 7 individuals in the discordant group versus n = 0 and n = 1 individual in the concordant high and low groups -LRB- p = 0.02 and 0.03 respectively -RRB- . Finally , the discordant pairs reported more traffic accidents than the other groups -LRB- p = 0.05 and 0.02 when compared with the concordant low and high pairs respectively -RRB- . On psychological scale scores , the concordant high group scored , as expected , overall higher on the PI-R-ABBR -LRB- p-values < 0.001 -RRB- , the YASR anxious-depressed scale -LRB- p-values < 0.001 -RRB- , ABV neuroticism -LRB- p < 0.001 in low -- high comparison ; p = n.s. between high and discordant twin pairs -RRB- , somatic complaints -LRB- p-values between < 0.001 and 0.003 -RRB- , and STAI-trait anxiety -LRB- p-values < 0.001 -RRB- . Further , the concordant high group had lower scores on ABV extraversion -LRB- p-values 0.001 -RRB- , satisfaction with life -LRB- p-values between 0.01 and 0.001 -RRB- , happiness -LRB- p-values between 0.01 and < 0.001 -RRB- and self-efficacy -LRB- p-values < 0.001 -RRB- than the concordant low and discordant groups . Shared environment influences Between-group analyses revealed that the discordant group had the lowest rate of caesarean sections -LRB- p-values of 0.005 and 0.006 in comparison with the concordant low and high groups -RRB- , while there was no difference between the concordant groups . The discordant group had the lowest birth weight -LRB- p = 0.008 compared with the concordant low pairs and p < 0.001 compared with the concordant high pairs -RRB- . There were no between-group differences on level of education of the parents -LRB- p-values = n.s. in all comparisons -RRB- . There were no between-pair differences in the occurrence of parental death . The concordant low MZ pairs reported most on death of a sibling -LRB- p = 0.05 between concordant low and high pairs -RRB- . There were no between-group differences with respect to relationship termination of the parents . On both drinking and smoking behavior of the parents , surprisingly the concordant low parents reported more drinking than the discordant parents , although alcohol consumption as well as number of cigarettes were low on average -LRB- Table 5 -RRB- . Table 5Between twin-pair comparisons : comparison of common environment characteristics -LRB- after correction for interrelatedness -RRB- Concordant low twin pairs , mean -LRB- SD -RRB- Concordant high twin pairs , mean -LRB- SD -RRB- Discordant twin pairs , mean -LRB- SD -RRB- Low -- high p-valueLow -- discordant p-valueHigh -- discordant p-valueCaesarean section -LRB- yes -RRB- n = 5 pairsn = 3 pairsn = 0 pairsn.s .0.008 < 0.001 Birth weight -LRB- g -RRB- 2,650 -LRB- 876 -RRB- 2,685 -LRB- 795 -RRB- 2,109 -LRB- 736 -RRB- n.s. 0.0040.009 Religious upbringing yesn = 45 -LRB- 67 % -RRB- n = 16 -LRB- 47 % -RRB- n = 34 -LRB- 69 % -RRB- n.s.n.s.n.s.Education level fathera -LRB- 1 -- 13 -RRB- 7.5 -LRB- 4.0 -RRB- 5.5 -LRB- 3.7 -RRB- 5.7 -LRB- 3.5 -RRB- n.s.n.s.n.s.Education level mothera -LRB- 1 -- 13 -RRB- 6.3 -LRB- 3.7 -RRB- 5.2 -LRB- 3.4 -RRB- 4.7 -LRB- 3.0 -RRB- n.s.n.s.n.s.Death mother -LRB- 0 -- 2 -RRB- yesn = 2 -LRB- 3 % -RRB- n = 3 -LRB- 10 % -RRB- n = 4 -LRB- 8 % -RRB- n.s.n.s.n.s.Death father -LRB- 0 -- 2 -RRB- n = 12 -LRB- 19 % -RRB- n = 7 -LRB- 24 % -RRB- n = 6 -LRB- 12 % -RRB- n.s.n.s.n.s.Death sibling -LRB- 0 -- 2 -RRB- n = 6 -LRB- 10 % -RRB- n = 0n = 1 -LRB- 2 % -RRB- 0.05 n.s.n.s.Relationship termination parents -LRB- 0 -- 2 -RRB- bn = 4 -LRB- 14 % -RRB- n = 2 -LRB- 25 % -RRB- n = 1 -LRB- 7 % -RRB- n.s.n.s.n.s.Number of parents drinking -LRB- ever ; yes -RRB- b91 % 100 % 74 % 0.06 n.s. 0.04 Number of drinks/week parents -LRB- 1 -- 7 -RRB- b3 .5 -LRB- 4 drinks/week -RRB- 2.7 -LRB- 2 -- 3 drinks/week -RRB- 2.3 -LRB- 1 -- 2 drinks/week -RRB- n.s. 0.03 n.s.Number of parents smoking ever -LRB- yes -RRB- b71 % 89 % 48 % n.s. 0.060.07 Number of cigarettes/day parents -LRB- 1 -- 7 -RRB- b4 -LRB- 6 -- 10 cig/day -RRB- 5 -LRB- 11 -- 20 cig/day -RRB- 5 -LRB- 11 -- 20 cig/day -RRB- n.sn.s.n.s.aReported by twin children and by parentsbDirect parent data Longitudinal data Young adult self-report OC scale scores revealed significant differences between low and high-scoring twin pairs in the 1995 -LRB- p = 0.02 -RRB- and 1997 wave -LRB- p < 0.001 -RRB- . YASR anxious-depressed scale scores revealed significant differences between the concordant low and high groups from 1991 on -LRB- p-values < 0.05 in all comparisons -RRB- . ABV extraversion scores revealed significant between-group differences from 1993 onward -LRB- p-values between < 0.001 and 0.008 -RRB- , whereas ABV neuroticism scores revealed significant between-group differences at all waves -LRB- p-values between 0.05 and < 0.001 -RRB- . ABV somatic complaints showed significant between-group differences from 1997 on -LRB- p-values < 0.001 -RRB- . Parent data Parent data were available for 66 persons ; the 34 parents of concordant low twin pairs had a mean age of 53.6 years -LRB- SD 5.9 -RRB- , a PI-R ABBR mean score of 5.2 -LRB- SD 3.8 -RRB- ; 9 parents of concordant high twin pairs had a mean age of 51.6 years -LRB- SD 2.5 -RRB- , and a PI-R ABBR mean score of 11.7 -LRB- SD 3.8 -RRB- ; and 23 parents of discordant twin pairs had a mean age of 57.5 years -LRB- SD 6.9 -RRB- and a PI-R ABBR mean score of 9.9 -LRB- SD 5.7 -RRB- . Between-group analyses of psychological scale scores showed that the parents of the discordant pairs scored between the parents of the concordant low and high pairs on anxious depression , satisfaction with life , happiness and self-efficacy scales . On somatic complaints and extraversion they showed higher scores than the other groups . On the PI-R ABBR , STAI trait and neuroticism they scored equal to the parents of the concordant high groups -LRB- Table 6 -RRB- . Table 62002 wave of data collection : between-parents comparisons of psychological scalesParent concordant low , mean -LRB- SD -RRB- Parent concordant high , mean -LRB- SD -RRB- Parent discordant , mean -LRB- SD -RRB- High -- low p-valueLow -- discordant p-valueHigh -- discordant p-valuePI-R ABBR OC scale5 .2 -LRB- 3.8 -RRB- 11.7 -LRB- 3.8 -RRB- 9.9 -LRB- 5.7 -RRB- 0.0020.002 n.s.YASR anxious depression scale4 .5 -LRB- 3.5 -RRB- 11.7 -LRB- 4.2 -RRB- 7.7 -LRB- 4.3 -RRB- < 0.0010.020.04 ABV extraversion53 .4 -LRB- 17.5 -RRB- 44.9 -LRB- 12.962.4 -LRB- 13.3 -RRB- n.s.n.s. 0.02 ABV neuroticism36 .3 -LRB- 25.3 -RRB- 79.8 -LRB- 26.5 -RRB- 62.7 -LRB- 13.3 -RRB- < 0.001 < 0.001 n.s.ABV somatic complaints16 .3 -LRB- 4.5 -RRB- 21.2 -LRB- 6.6 -RRB- 26.9 -LRB- 2.3 -RRB- 0.01 < 0.0010.005STAI-trait29.5 -LRB- 6.7 -RRB- 44.1 -LRB- 7.1 -RRB- 47.7 -LRB- 4.1 -RRB- < 0.001 < 0.001 n.s.Satisfaction with life28 .1 -LRB- 4.9 -RRB- 18.6 -LRB- 8.5 -RRB- 25.5 -LRB- 7.1 -RRB- 0.001 n.s. 0.03 Happiness23 .5 -LRB- 3.5 -RRB- 16.3 -LRB- 6.9 -RRB- 15.1 -LRB- 3.3 -RRB- < 0.001 < 0.001n.s.Self-efficacy32.1 -LRB- 3.8 -RRB- 28.0 -LRB- 4.2 -RRB- 25.3 -LRB- 2.6 -RRB- 0.01 < 0.001n.s.PI-R ABBR , Padua Invetroy-Revised Abbreviated scale ; OC , obsessive-compulsive ; YASR , young adult self-report ; ABV , Amsterdamse Biografische vragenlijst ; STAI , State trait Inventory Discussion The most important aim of this MZ twin study has been to explore unique and shared environmental factors involved in OC symptoms . Unique and shared environmental factors The within-twin pair comparisons of the MZ discordant pairs were primarily used to study unique environmental factors associated with OC symptoms . Although the discordant pairs were genetically identical , were raised at the same time in the same family , and were selected from an epidemiological sample , the twins differed substantially on several measures across time . The twins who scored low on OC symptoms reported to feel healthier , to be more satisfied with life , happier and more self-efficient than their high scoring MZ twin siblings . They tended to have longer relationships and more children . Further , they had lower scores on anxious depression and on neuroticism , mostly from 1997 onward . The most striking unique environmental factor to explain these within-discordant pair differences was the relatively high frequency of sexual assault experienced by the high-scoring twins of the discordant pairs in comparison with their low-scoring twin siblings , which is in line with previous reports on this issue -LRB- Lochner et al. 2004 -RRB- . However , two of the low-scoring twins of the discordant pairs reported on sexual assault as well , underscoring the complexity of presumed causality in the interplay between environmental and genetic factors in OCD . Interestingly , no sexual assault was reported by the concordant high-scoring MZ twin pairs . Thus , although the high-scoring respondents of the discordant pairs show similar OC symptomatology when compared with the concordant high MZ pairs , the pathways along which similar OC symptoms develop seem to differ between the high-scoring discordant twins on the one hand , and the high-scoring concordant pairs on the other . Although one can only speculate about causal relationships in this explorative study , the OC symptoms in the high-scoring twins of the discordant pairs seem to be associated more with environmental stressors -LRB- i.e. , sexual assault -RRB- than are the OC symptoms in the concordant high-scoring pairs . The between-twin pair comparisons to study environmental factors that are shared by the twins of a pair revealed low birth weight and low rates of caesarean section in the discordant pairs . We were unable to take the relationship between low birth weight and gestational age into account in the analyses , and were therefore unable to distinguish whether the study persons had been pre - or dysmature at birth . However , a recent twin study showed that low birth weight in itself resulted in an increase in problem behavior in later life . Children with low birth weight appeared to be more vulnerable to negative environmental factors than normal birth weight children -LRB- Wichers et al. 2002 -RRB- , possibly in association with a negative interaction between genetic vulnerability for problem behavior and low birth weight . Low birth weight can be indicative of a range of prenatal adversities such as maternal psychological stress , alcohol , drug abuse , or smoking during pregnancy . These adversities cause immunological challenge , and lead through various mechanisms to a diversity of psychopathology , including anxiety and depression -LRB- Meyer et al. 2006 ; Huizink et al. 2004 ; Nigg and Breslau 2007 -RRB- . In this study , we did not find an indication of alcohol , smoking or drug abuse in the parents of the twin pairs , but other sources of prenatal stress can not be ruled out . Further , no discordant twin pairs were born through caesarean section , as opposed to eight concordant pairs . This is remarkable in light of the fact that in general , caesarean section is carried out more often in multiple pregnancies , especially when one suspects low birth weight in the fetuses -LRB- Colla et al. 2001 -RRB- . Although it might be a chance finding , one can speculate that -- since caesarean section is intended to decrease perinatal adversities -- the discordant group of this sample has been ` under treated ' , providing an additional negative environmental factor to explain between-group differences . There were no between-group differences in rates of parental death or death of a sibling , nor in frequency of relationship termination between the parents , life events that reflect shared environmental stressors . In general , rates of these life events were low in this relatively young twin group , possibly hampering detection of between-twin pair differences . Alcohol use by the mother -LRB- especially during pregnancy -RRB- as well as maternal smoking are considered to be common environmental risk factors for problem behavior such as ADHD -LRB- Smidts 2007 -RRB- . However , neither alcohol use nor smoking behavior of the parents was associated with OC symptoms in the concordant high or discordant groups of this study . Further , there was no association between OC symptoms and a religious upbringing in the study groups , which is in line with the literature on the lack of association between religiosity and OCD -LRB- Tek et al. 2004 -RRB- , but deviates from reports of a protective effect of religion on other forms of psychopathology such as alcohol and drug abuse , depression and disruptive behavior -LRB- Kendler et al. 1999 -RRB- . Apparently , different problem behavior is associated with different environmental risk factors . Finally , level of education of the parents -LRB- as a measure of socio-economic status , a risk factor reported in OCD -RRB- was not found to be associated with OC symptoms in this study , although the parents of the twins who were concordant low on OC symptoms tended to have a higher level of education than the other groups , a difference that may have failed to reach significance due to the small sample size . Finally , between-twin pair comparisons on unique life events revealed an elevated rate of dismissal in the concordant high-scoring twin pairs compared with the other pairs . Since dismissal typically represents a unique negative environmental influence on each twin of a pair , instead of being an environmental influence shared between the twins of a pair , its elevated rate among the high-scoring concordant MZ pairs is better explained as being the consequence of OC symptomatology rather than causing OC symptoms ; elevated dismissal rates in these OC twin pairs might result from over-scrupulosity and slowness in work -- characteristics well known in OC symptomatology -- and subsequent dysfunction . Health and lifestyle characteristics Overall , as expected , the concordant low pairs reported highest scores of health , fewest mental and medical health contacts , and lowest scores on OC symptoms , anxiety and depression , neuroticism , and somatic complaints compared with the other groups . Further , they reported to be more extravert , more satisfied with life , happier and more self-efficient , with the discordant pairs scoring in between the concordant low and high pairs . On alcohol use , the concordant high-scoring twin pairs scored in between the low and discordant twin pairs over the past 12 months , with the number of drinks per week well below the quantity required to fulfil criteria for alcohol abuse or dependence according to DSM-IV criteria . However , subjective reports of alcohol withdrawal and dependence -LRB- CAGE scores -RRB- were increased in the high-scoring twin pairs compared with the low-scoring and discordant pairs . This might reflect increased scrupulosity and feelings of guilt , a well-known phenomenon in persons with OC symptoms , -LRB- Olatunji et al. 2006 -RRB- , related to alcohol use and its toxic effects rather than a verifiable alcohol problem in the concordant high-scoring MZ pairs . A protective effect of level of education on OC symptoms was suggested by the finding of a higher level of education in the concordant low-scoring twin pairs than in the concordant high and the discordant twin pairs . Not only the concordant high-scoring twin pairs but also their spouses had a lower level of education , which suggests that low level of education and OC symptomatology might share genetic vulnerability . Deficits in encoding complex information and subsequent memory impairments have been reported in OCD -LRB- Buhlmann et al. 2006 ; Deckersbach et al. 2000 -RRB- . These -LRB- genetically determined -RRB- impairments possibly mediate low educational level . On the other hand , low level of education in the concordant high-scoring group might be a consequence of the OC symptomatology in itself , a notion that is supported by the literature -LRB- Sorensen et al. 2004 -RRB- . The longitudinal data As expected , the longitudinal data on OC symptoms , anxiety and depressive symptoms in the concordant and discordant groups revealed an earlier age at onset of OC and related symptoms in the concordant high group -LRB- from 1991 on -RRB- than in the discordant group -LRB- mostly from 1997 on -RRB- . This confirms data from family-based studies where an earlier age at onset was associated with higher familial load -LRB- do Rosario-Campos et al. 2005 -RRB- . Thus , assuming that OC symptoms in the concordant high-scoring twin pairs are more genetically mediated than in the discordant pairs , this study is in line with clinical studies indicating that age at onset might be an important phenotypic characteristic that reflects differences in genetic characteristics underlying OCD -LRB- Delorme et al. 2005 -RRB- . The parent data As parent scores on OC symptoms and related psychopathology were expected to reflect genetic vulnerability , we expected scores to be highest in the concordant high parents , to be intermediate in the discordant parents and to be low in the concordant low parents . On most measures of psychopathology , this assumption was confirmed . Thus , the intermediate scores in the parents of the discordant twins on OC , anxious-depression and neuroticism scales may be the consequence of the intermediate amount of genetic vulnerability to OC symptoms in this group . Therefore , these parent data suggest that the symptoms in the high-scoring twins of the MZ discordant group are likely to be the consequence of a moderate genetic vulnerability to OC pathology in addition to or in interaction with environmental mediators . Limitations First , sample size is small ; although we sampled from a large group of MZ twins , only a small sample was retained due to the use of rigorous criteria . Consequently , especially in the within-discordant pair comparisons , some of the negative outcomes might in fact be the result of lack of power to detect within-pair differences . Alternatively we could have relaxed the stringent selection criteria , with the disadvantage of including twin pairs not scoring in the clinical range of OCD , thus representing an unclear group of problem behavior . Second , considering the large number of tests relative to the small sample size , we only mildly corrected for type I errors . However , considering the exploratory nature of this study , an increase in the odds of type II errors by correction of type I errors was undesirable . Therefore , we decided to compromise by only applying a mild correction of type I errors -LRB- Perneger 1998 -RRB- . Finally , the database used in this study was not primarily designed to specifically inquire about environmental factors , leaving some questions unanswered , especially with respect to protective environmental mediators of OC symptomatology . Conclusion This study has been a first attempt to identify characteristics of the environment associated with OC symptoms using a twin study design . Some important environmental factors involved in OC symptomatology have been identified . Two crucial questions to be addressed in future studies are : -LRB- 1 -RRB- what is the differential impact of the various environmental mediators on OC symptoms , and under which circumstances and at which age are they most harmful ? -LRB- 2 -RRB- Along which lines do the environmental factors found in this study operate ? Do they add to genetic risk factors , are they causal in themselves , or do they operate through gene -- environment interaction ? Future studies are needed to study the differential effects of environment and genes on phenotypes -LRB- and endophenotypes -RRB- , and to elucidate the nature of the interplay between genes and environment .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O B O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

monozygotic twins
environmental factors
discordant
concordant