Antecedents of Adult Attachment 1
Supplemental Materials
Designed for web-based archive
Part A. Additional information on genotyping methods
Part B. Table of correlations among all study variables
Part C. Analyses based on the Strange Situation
Part A
Genotyping Web-based Supplement
Extraction. Extraction for all polymorphisms was based on adaptations to Freeman et al. (Freeman et al., 2003). Specifically, buccal mucosa cells were collected with cotton swabs by the subject. The swabs were placed in 15-ml centrifuge tubes containing 2.5 mls of lysis buffer. The tubes were incubated in a water bath at 65°C for 2 hr to activate the proteinase K. After incubation the tubes were centrifuged at 300g for 4 min and the supernatant added to 4ml of isopropanol. Tubes were centrifuged again for 30 min. The supernatant was poured off, the pellet dried and 1 ml of lysis buffer without proteinase K was added. Pellets were resuspended by shaking overnight. The liquid was transferred to a 1,5 ml microfuge tube and 200 µl of an organic deproteinization reagent (ODPR) were added to each tube. The tubes were capped and shaken vigorously by hand. The denatured debris and remaining organic mix were then centrifuged at 5000g for10 min. Supernatant from the tube was transferred to a fresh 1.5-ml tube and 800 ul of isopropanol was added and mixed gently for approximately 1 min. The DNA was collected by centrifugation at 5000g for 10 min. The pellets were dried and washed with 1 ml ethanol 70% (v/v) by centrifugation at 5000g for 10 min. The ethanol wash was discarded, the tubes were inverted, and the pellets were dried for 60 min. The DNA was re-suspended in 250 ul of Tris EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) by rotation in an incubator at 37oC. The DNA was quantified by measuring the absorbance at 260 nm using a Nanodrop spectrophotometer. Samples were aliquoted into storage vials and placed in a -80oC freezer.
OXTR rs53576 genotyping. For OXTR rs53576, Taqman SNP Genotyping Assays were performed using an Allelic Discrimination Assay (Applied Biosystems, Foster City, CA) protocol. Forty nanograms of DNA were combined in a volume of 5 microliters with 2X Universal PCR Mix (Applied Biosystems) and 1/20 the volume of the Taqman SNP assay in a 384 well plate. A Pre-Read was performed and then PCR as follows: a 10 min hold at 95 C, followed by 40 to 45 cycles of 15 sec at 92 C and then 1 min at 60 C in a 7900HT PCR System. After amplification, a Post-Read was performed to analyze. Automatic and manual calls were made. Reliability genotyping was also conducted as described immediately above.
OXTR rs2254298 genotyping. Finally, for OXTR rs2254298 Taqman SNP Genotyping Assays were performed using an Allelic Discrimination Assay (Applied Biosystems, Foster City, CA) protocol. Forty nanograms of DNA were combined in a volume of 5 microliters with 2X Universal PCR Mix (Applied Biosystems) and 1/20 the volume of the Taqman SNP assay in a 384 well plate. A Pre-Read was performed and then PCR as follows: a 10 min hold at 95 C, followed by 40 to 45 cycles of 15 sec at 92 C and then 1 min at 60 C in a 7900HT PCR System. After amplification, a Post-Read was performed to analyze. Automatic and manual calls were made. Reliability genotyping was also conducted as described immediately above.
DRD2 rs1800497 genotyping. In order to genotype DRD2, Taqman SNP Genotyping Assays were performed using an Allelic Discrimination Assay (Applied Biosystems, Foster City, CA) protocol. Forty nanograms of DNA were combined in a volume of 5 microliters with 2X Universal PCR Mix (Applied Biosystems) and 1/20 the volume of the Taqman SNP assay in a 384 well plate. A Pre-Read was performed and then PCR as follows: a 10 min hold at 95 C, followed by 40 to 45 cycles of 15 sec at 92 C and then 1 min at 60 C in a 7900HT PCR System. After amplification, a Post-Read was performed to analyze. Automatic and manual calls were made. Reliability genotyping for this SNP was conducted on an Open Array (see description under HTR2A rs6313 and OPRM1 rs1799971 below).
HTR2A rs6313 and OPRM1 rs1799971genotyping. These SNPs (including relevant reliability genotyping) were genotyped on an Open Array. Specifically, SNPs were determined using the OpenArray© Real-Time PCR System (Applied Biosystems, Foster City, CA) as in User Guides PN #4458837 Rev. A and PN #4458840A. Briefly, genomic DNA was added to 96 well plates at the recommended concentration and then transferred to 384 well plates along with the TaqMan® OpenArray® Gene Expression Master Mix.The 384 well plates and OpenArray© slides were placed in an OpenArray© AutoLoader (Applied Biosystems, Foster City, CA) that automatically transferred the solution from the plate to the slide. The slide was placed in the Genotyping Case and then in an Applied Biosystems GeneAmp© PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA). The OpenArray© Run Method was 93o C for 10:00 min followed by 60 cycles of 95o C for 0:45 min, 94o C for 0:13 min and 53o C for 2:14 min. A final step of 25o C for 2:00 min was run before setting the module to 4o C forever.A csv file composed of sample names was prepared and uploaded into the OpenArray© Image software along with the serial number of the slide. After cycling, the slide was placed in the OpenArray© NT Imager and imaged. OpenArray© SNP Genotyping Analysis Software v 1.0.3 was used to analyze the data.
Reference
Freeman, B., Smith, N., Curtis, C., Huckett, L., Mill, J., & Craig, I. W. (2003). DNA from buccal swabs recruited by mail: evaluation of storage effects on long-term stability and suitability for multiplex polymerase chain reaction genotyping. Behavioral Genetics, 33, 67-72.
Antecedents of Adult Attachment 1
Part B. Supplemental Material – Table 1 - Correlations among all study variables, pairwise deletion.
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 151 RSQ Avoidance / 1
2 RSQ Anxiety / .49 / 1
3 ECR-R Avoidance / .57 / .28 / 1
4 ECR-R Anxiety / .34 / .70 / .33 / 1
5 Gender / -.01 / -.01 / .05 / -.00 / 1
6 Ethnicity / -.14 / -.05 / -.08 / .02 / -.03 / 1
7 Maternal education / -.02 / -.03 / .06 / .06 / -.05 / .26 / 1
8 Family income / -.07 / -.07 / .01 / .04 / .02 / .32 / .59 / 1
9 Sensitivity (intercepts) / -.09 / -.04 / .03 / .10 / -.08 / .41 / .51 / .53 / 1
10 Sensitivity (slopes) / -.23 / -.15 / -.23 / -.12 / -.04 / -.04 / -.05 / -.06 / -.23 / 1
11 Depression (intercepts) / .08 / .04 / -.00 / -.08 / -.01 / -.20 / -.31 / -.41 / -.35 / .04 / 1
12 Depression (slopes) / .14 / .20 / .14 / .27 / -.01 / .07 / .04 / .02 / .04 / -.10 / -.20 / 1
13 Father absence / .16 / .08 / .03 / -.01 / -.02 / -.31 / -.34 / -.41 / -.35 / .00 / .30 / .03 / 1
14 Social competence mother ratings (intercepts) / -.18 / -.03 / -.07 / .03 / .06 / .14 / .23 / .26 / .31 / .09 / -.32 / -.02 / -.16 / 1
15 Social competence mother ratings (slopes) / -.04 / -.18 / -.03 / -.14 / -.10 / .01 / .02 / .01 / .01 / .12 / .01 / -.13 / -.07 / -.29 / 1
(Continued on next page)
1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 1516 Social competence teacher ratings (intercepts) / -.09 / -.07 / .02 / .09 / .03 / .21 / .33 / .35 / .38 / .04 / -.25 / -.01 / -.28 / .40 / .01
17 Social competence teacher ratings (slopes) / -.32 / -.18 / -.40 / -.25 / -.07 / -.04 / -.13 / -.11 / -.15 / .11 / .08 / -.12 / .07 / -.15 / .18
18 Friendship quality (intercepts) / .00 / -.06 / -.06 / -.03 / -.29 / -.03 / -.03 / -.06 / -.05 / .09 / .04 / .00 / .03 / .06 / .05
19 Friendship quality (slopes) / -.41 / -.17 / -.39 / -.01 / -.10 / .10 / .07 / .11 / .13 / .10 / -.11 / -.05 / -.12 / .09 / .10
20 Strange Situation – proximity seeking / -.02 / -.10 / -.03 / -.07 / -.03 / .05 / .11 / .15 / .14 / .01 / -.05 / .01 / -.09 / .06 / .01
21 Strange Situation - resistance / .03 / -.03 / .04 / .00 / -.01 / .02 / -.02 / -.02 / -.04 / -.01 / .06 / -.04 / .00 / -.04 / .03
22 CBQ Shyness / -.04 / -.07 / -.06 / -.04 / -.05 / -.03 / .04 / .03 / .07 / .05 / .05 / -.06 / -.08 / -.18 / .13
23 CBQ Attentional focusing / -.05 / -.03 / -.03 / .05 / -.16 / .13 / .23 / .23 / .34 / .04 / -.22 / .04 / -.14 / .44 / -.09
24 CBQ Fear / -.01 / .05 / .03 / .06 / -.07 / .00 / .04 / .03 / .06 / -.02 / .10 / -.04 / .03 / -.02 / .00
25 CBQ Restlessness / .11 / .12 / .04 / .02 / .12 / -.02 / -.12 / -.13 / -.17 / -.07 / .34 / .01 / .10 / -.35 / .00
26 DRD2 (TT [1] vs. other [0]) / .04 / -.01 / -.02 / -.04 / -.01 / -.20 / -.19 / -.12 / -.10 / .01 / .05 / -.03 / .12 / -.05 / .06
27 OXTR76 (GG [1] vs. other [0]) / -.04 / .01 / .01 / .02 / -.01 / -.01 / .00 / -.02 / .00 / -.03 / -.01 / .08 / -.06 / .01 / -.03
28 OXTR98 (GG [1] vs. other [0]) / .03 / -.02 / -.07 / -.00 / -.06 / .15 / -.01 / .08 / .12 / .06 / -.02 / .01 / .02 / .02 / -.01
29 HTR2A (TT [1] vs. CC [0]) / Gillath / -.06 / -.06 / -.09 / .01 / -.14 / -.03 / .08 / .04 / .05 / .11 / -.06 / -.06 / -.07 / .05 / .03
30 HTR2A (CC [1] vs. other [0]) / .07 / .11 / .11 / .05 / .09 / .01 / -.03 / -.01 / -.03 / -.07 / .07 / .05 / .09 / -.04 / -.03
31 OPRM1 (AA [1] vs. other [0]) / .02 / -.07 / .00 / -.06 / .04 / .02 / -.03 / -.10 / -.09 / .00 / .06 / -.14 / .06 / -.02 / .04
(Continued on next page)
16 / 17 / 18 / 19 / 20 / 21 / 22 / 23 / 24 / 25 / 26 / 27 / 28 / 29 / 3016 Social competence teacher ratings (intercepts) / 1
17 Social competence teacher ratings (slopes) / -.63 / 1
18 Friendship quality (intercepts) / .03 / .07 / 1
19 Friendship quality (slopes) / .12 / .21 / -.33 / 1
20 Strange Situation – proximity seeking / .12 / -.08 / .03 / .02 / 1
21 Strange Situation - resistance / -.01 / -.02 / .02 / -.01 / .19 / 1
22 CBQ Shyness / .07 / -.00 / -.05 / .01 / .05 / .00 / 1
23 CBQ Attentional focusing / .27 / -.10 / .08 / .05 / .05 / .04 / .02 / 1
24 CBQ Fear / .05 / -.05 / -.02 / .03 / .00 / .00 / .12 / .04 / 1
25 CBQ Restlessness / -.22 / .08 / -.06 / -.07 / -.08 / .00 / .01 / -.44 / .12 / 1
26 DRD2 (TT [1] vs. other [0]) / -.09 / .06 / -.01 / -.04 / -.05 / -.06 / -.03 / -.02 / -.03 / -.06 / 1
27 OXTR76 (GG [1] vs. other [0]) / .07 / -.09 / .00 / .06 / .00 / .00 / .00 / .02 / -.05 / -.04 / .10 / 1
28 OXTR98 (GG [1] vs. other [0]) / -.01 / .03 / -.02 / -.01 / .07 / .01 / .03 / .02 / -.00 / .01 / .01 / -.01 / 1
29 HTR2A (TT [1] vs. CC [0]) / Gillath / -.03 / .07 / .07 / .10 / .11 / .03 / .02 / .02 / .05 / -.13 / -.03 / -.05 / -.04 / 1
30 HTR2A (CC [1] vs. other [0]) / .02 / -.10 / -.12 / -.05 / -.09 / -.05 / -.03 / -.01 / .02 / .10 / .02 / .02 / -.02 / -1 / 1
31 OPRM1 (AA [1] vs. other [0]) / -.07 / .05 / -.04 / -.01 / -.08 / .03 / .04 / .05 / -.05 / .03 / -.01 / .02 / -.01 / .02 / -.00
Note. Correlations are truncated to two decimal places. Sample sizes for each pairwise correlation range from 652 to 707 among non-genetic variables and 237 to 499 among variables involving genetic markers. Boldface is used to highlight statistically significant correlations (2-tailed). Gender was coded as 0 for female and 1 for male. Ethnicity was coded as 1 for White/non-Hispanic and 0 otherwise.
Antecedents of Adult Attachment 1
Part C.
Supplemental Material
Analyses involving the Strange Situation
The Strange Situation procedure was used to assess attachment organization at 15-months of age. The Strange Situation (Ainsworth et al., 1978) is a 25-minute procedure designed to elicit and measure infant attachment behavior. The procedure contains brief episodes of increasing stress for the infant, including two mother-infant separations and reunions. The Strange Situation was administered according to standard procedures and coded independently by pairs of highly trained coders (see NICHD ECCRN, 1997, for detailed information).
We used the interactive scales of the Ainsworth coding procedure to produce scores on two dimensions (see Fraley & Spieker, 2003). The first dimension, Proximity-seeking vs. avoidant strategies, characterizes variability in the degree to which children’s attachment systems are organized around the goal of proximity-maintenance, and was computed as the average of the ratings for the following interactive coding scales: Avoidance Episode 5 (reversed), Avoidance Episode 8 (reversed), Contact Maintenance Episode 5, Contact Maintenance Episode 8, Proximity Seeking Episode 5, and Proximity Seeking Episode 8. The second dimension, Angry and resistant strategies, represents variability in the amount of overt conflict and anger the child expressed toward the caregiver during the strange situation, and was computed as the average of the ratings for the following interactive coding scales: Resistance Episode 5, Resistance Episode 8, and Disorganization.
The four tables that follow report the multiple regression analyses that were reported in the article, but including the two Strange Situation dimensions.
Supplemental Table 2. Global avoidance (RSQ avoidance) as a function of covariates and developmental antecedents.
Predictor / B / SE / / pCovariates
Gender / -.11 / .05 / -.08 / .02
Ethnicity / -.12 / .06 / -.07 / .03
Maternal education / .02 / .01 / .07 / .06
Family income / .09 / .08 / .06 / .24
Global anxiety / .30 / .03 / .37 / .01
Caregiving environnent
Sensitivity (intercept) / -.03 / .03 / -.05 / .25
Sensitivity (slope) / -.08 / .02 / -.12 / .01
Maternal depression (intercept) / -.00 / .02 / -.00 / .96
Maternal depression (slope) / .01 / .02 / .02 / .60
Father absence / .11 / .05 / .08 / .02
Strange situation - proximity seeking / .02 / .02 / .04 / .28
Strange situation - resistance / .02 / .03 / .03 / .42
Social competence
Social competence (intercept) / -.09 / .03 / -.11 / .01
Social competence (slope) / -.10 / .03 / -.11 / .01
Friendship quality
Friendship quality (intercept) / -.06 / .02 / -.09 / .01
Friendship quality (slope) / -.23 / .02 / -.33 / .01
Note. The intercept for the model was 1.51. The R2 of the model was .41, F(16,662) = 29.12, p < .05. The R2 of the same model, excluding attachment-related anxiety as a covariate, was .29. Boldface is used to highlight statistically significant predictors. Gender was coded as 0 for female and 1 for male. Ethnicity was coded as 1 for White/non-Hispanic and 0 otherwise.
Supplemental Table 3. Global anxiety (RSQ anxiety) as a function of covariates and developmental antecedents.
Covariates
Gender / -.04 / .06 / -.02 / .58
Ethnicity / .02 / .08 / .01 / .82
Maternal education / -.00 / .02 / -.01 / .77
Family income / -.09 / .11 / -.04 / .41
Global avoidance / .57 / .05 / .46 / .01
Caregiving environnent
Sensitivity (intercept) / .01 / .04 / .02 / .73
Sensitivity (slope) / -.01 / .03 / -.01 / .74
Maternal depression (intercept) / .03 / .03 / .03 / .46
Maternal depression (slope) / .10 / .03 / .12 / .01
Father absence / -.05 / .07 / -.03 / .50
Strange situation - proximity seeking / -.06 / .02 / -.09 / .01
Strange situation - resistance / -.02 / .04 / -.02 / .62
Social competence
Social competence (intercept) / -.02 / .05 / -.01 / .75
Social competence (slope) / -.14 / .05 / -.12 / .01
Friendship quality
Friendship quality (intercept) / -.03 / .03 / -.04 / .36
Friendship quality (slope) / .04 / .04 / .05 / .28
Note. The intercept for the model was .88. The R2 of the model was .28, F(16,662) = 16.27, p < .05. The R2 of the same model, excluding attachment-related avoidance as a covariate, was .13. Boldface is used to highlight statistically significant predictors. Gender was coded as 0 for female and 1 for male. Ethnicity was coded as 1 for White/non-Hispanic and 0 otherwise.
Supplemental Table 4. Romantic avoidance (ECR-R avoidance) as a function of covariates and developmental antecedents.
Covariates
Gender / -.14 / .08 / -.06 / .09
Ethnicity / -.26 / .10 / -.09 / .01
Maternal education / .03 / .02 / .07 / .11
Family income / .05 / .15 / .01 / .74
Romantic anxiety / .26 / .03 / .26 / .01
Caregiving environnent
Sensitivity (intercept) / .02 / .05 / .01 / .76
Sensitivity (slope) / -.14 / .04 / -.12 / .01
Maternal depression (intercept) / -.00 / .05 / -.00 / .96
Maternal depression (slope) / .02 / .04 / .02 / .60
Father absence / -.05 / .09 / -.02 / .58
Strange situation - proximity seeking / -.01 / .03 / -.02 / .64
Strange situation - resistance / .07 / .05 / .04 / .18
Social competence
Social competence (intercept) / -.06 / .06 / -.04 / .38
Social competence (slope) / -.18 / .06 / -.11 / .01
Friendship quality
Friendship quality (intercept) / -.21 / .05 / -.18 / .01
Friendship quality (slope) / -.49 / .05 / -.41 / .01
Note. The intercept for the model was 2.09. The R2 of the model was .33, F(16,650) = 19.71, p < .05. The R2 of the same model, excluding attachment-related anxiety as a covariate, was .27. Boldface is used to highlight statistically significant predictors. Gender was coded as 0 for female and 1 for male. Ethnicity was coded as 1 for White/non-Hispanic and 0 otherwise.
Supplemental Table 5. Romantic anxiety (ECR-R anxiety) as a function of covariates and developmental antecedents.
Covariates
Gender / .02 / .09 / .01 / .85
Ethnicity / -.01 / .12 / -.00 / .93
Maternal education / .00 / .02 / .01 / .86
Family income / -.06 / .16 / -.02 / .70
Romantic avoidance / .32 / .04 / .31 / .01
Caregiving environnent
Sensitivity (intercept) / .09 / .06 / .08 / .11
Sensitivity (slope) / -.00 / .05 / -.00 / .94
Maternal depression (intercept) / -.00 / .05 / .00 / .99
Maternal depression (slope) / .25 / .05 / .20 / .01
Father absence / -.00 / .10 / -.00 / .97
Strange situation - proximity seeking / -.07 / .03 / -.08 / .02
Strange situation - resistance / .03 / .06 / .02 / .64
Social competence
Social competence (intercept) / -.06 / .07 / -.04 / .41
Social competence (slope) / -.30 / .07 / -.19 / .01
Friendship quality
Friendship quality (intercept) / .08 / .05 / .07 / .10
Friendship quality (slope) / .22 / .05 / .18 / .01
Note. The intercept for the model was 1.86. The R2 of the model was .21, F(16,650) = 10.87, p < .05. The R2 of the same model, excluding attachment-related avoidance as a covariate, was .14. Boldface is used to highlight statistically significant predictors. Gender was coded as 0 for female and 1 for male. Ethnicity was coded as 1 for White/non-Hispanic and 0 otherwise.