A total of 992 DNA samples from 217 families were submitted to CIDR for genotyping, of which 980 were successfully genotyped. Of the 992 samples submitted, 832 were from immortalized lymphocytes at RUCDR, and the remaining 160 were sent directly from UAB. Of these 160 samples, 39 originated from buccal cell samples rather than blood samples. Of the 434 parents of the 217 probands, 140 consented to the study and were included. Of these, between 14 and 19 were affected, depending on the definition of affection (S, SS, or P). The distribution of families according to affection status of relations to the proband is given in . The distribution of pairs of relations informational for either of the linkage analysis methods employed is given in . The overall distribution of genotyped individuals in the sample according to family type and number of affected individuals in the family by all three definitions of affection are given in . The tabulation for was also performed separately for each site, and these results are given in Supplementary Table 1.
Distribution of Siblings and Other Relations of Probands by Number of Affected Siblings in Each Pedigree.
Counts of pairs and founders by affection status.
Distribution of genotyped family members by family type.
The linkage panel consisted of 6,008 SNPs of which 5,981 passed CIDR quality control criteria. Of these 5,981 SNPs, 225 showed atypical clustering behavior for the samples submitted for the PAARTNERS project, leaving 5,756 SNPs for this project. Of these, 339 were on sex chromosomes, and the remaining 5,417 were autosomal. Of the 339 SNPs on sex chromosomes, 42 were located either on the Y chromosome or pseudoautosomal regions, and were not used in the current analysis.
The SNPs had an average genetic marker distance of 0.67 cM (537 Kb physical) with an average heterozygosity of 0.38 in African-Americans. The mean missing genotype rate was 0.23% with the highest, 0.68%, from mouth wash samples and the lowest, 0.20%, for cell lines. The Mendelian consistency rate was 99.92%. Illumina’s BeadStudio software was used to evaluate all genotypes using a quantitative quality score, the GenCall (GC) score. Average call rates for the SNPs was 99.47%. Of the 5,417 markers, only 89 did not meet Hardy-Weinberg expectations with a p-value of <0.005. In order to avoid the possibility of inflating evidence for linkage due to LD between consecutive markers, the program Hclust was used to select tag SNPs. (Rinaldo et al 2005
). We used SNPs that were not in strong pair-wise LD amongst all possible pairs (r2
<0.1) for the linkage analysis. After eliminating the 89 SNPs failing Hardy Weinberg and SNPs in LD, the total number of autosomal SNPs used for the analysis was 4,496, and the total number of X-linked SNPs used for the analysis was 206.
As stated under methods, all recruited probands self-identified as African- American. Among the information provided for the six forebears of each proband – that is, both parents and all four grandparents – over 93.1% of the responses indicated the ethnicity as African-American. The definition of this group indicated in the DIGS interview excluded Hispanic origin. Of the responses given for the forebears, only two other groups accounted for more than 1% of responses; 1.3% of the responses were Anglo-Saxon, and 4.2% were Native American/Alaskan Native. A small number of forebears had African ancestry: 3 mothers, 4 fathers, and 7 grandparents. Also present were a small number of forebears with Caribbean or Afro-Caribbean ancestry: 8 mothers and 4 fathers.
We performed linkage analysis with two different programs that used complimentary information within the data, as indicated in the methods section. For graphical purposes, negative values of the statistics from both programs were set to zero. gives the results for the Sibpal scan for each of the three outcomes: S (narrowly defined Schizophrenia), SS (Schizophrenia with Schizoaffective disorder included), and P (all diagnoses indicating psychosis). Several regions show a decrease in the evidence for linkage as the definition is broadened in this way, notably on chromosomes 8, 16, and 20. The peak on chromosome 8 is located at rs911 at 8q22.1 at 99.26 cM. The Merlin p-value for linkage increases from 0.006 to 0.02 at this marker as the definition of affection is broadened, while the Sibpal p-value increases from 0.0025 to 0.0104. The peak on chromosome 16 is at rs1006547 at 16q24.3 at 130.48 cM. The Merlin p-values at this marker increased from 0.006 to 0.03 between outcomes S and SS; for the P outcome, the p-value again decreased to 0.007. This same behavior was seen in the Sibpal results as the p-values changed from 0.00095 to 0.02432 for outcomes S and SS, and to 0.00853 for outcome P. On chromosome 20 at rs1022689 at 20q13.2 at 81.73 cM, the Merlin p-values increase from 0.013 to 0.3, and the Sibpal p-values increase from 0.00015 to 0.03186 as the definition of affection is broadened. However, one region, 11p. strikingly shows an increase in evidence for linkage. When we examine this region in , which shows the results of the scans based on MERLIN, we see very similar results. This peak is at rs722317, at 11p15.2 at 24.27 cM. As the definition of affection status is broadened, the Merlin p-values decrease from 0.02 to 0.00001 while the Sibpal p-values decrease from 0.00217 to 0.0000003. MERLIN indicates a second region on chromosome 11 with strong evidence for linkage (at rs931127 at 11q13.1 at 71.03 cM, p-value for P outcome = 0.0006), while the Sibpal evidence for linkage at this marker is somewhat weaker (p-value for P outcome = 0.00705). On chromosome 17, Sibpal did not show any strong evidence for linkage, while MERLIN did show some evidence for the S outcome (at marker rs231018, at 17p12, at 44.17 cM, p-value = 0.008). A region just beyond the pseudo-autosomal region of Xpterm shows the greatest evidence for linkage with the SS outcome than with either the narrower S outcome or the broader P outcome. This maximum linkage on Xp occurs at rs1921708 at Xp22.31, at 15.7 cM.
SIBPAL Linkage Results for Three Phenotypes (S=Schizophrenia Excluding SAD; SS=Schizophrenia Including SAD; P = All Diagnoses with Psychotic Features)
MERLIN Linkage Results for Three Phenotypes (S=Schizophrenia Excluding SAD; SS=Schizophrenia Including SAD; P = All Diagnoses with Psychotic Features)
The autosomal regions that are significant at three confidence levels (P<0.01, P<0.001, and P<0.0001) are indicated in through . On each of these figures, regions of the scan with p-values equal to or less than but not exceeding the given threshold are indicated for all six of the scans (as given in through 6). The linkage traces from MERLIN and Sibpal show some consistency of patterns in regions on chromosomes 4, 8, and 11, despite the different sources of linkage information inherent in these two methods (i.e., in terms of affection status, Merlin accounts only for affected individuals, whereas SIBPAL accounts for both affected and unaffected family members.) Only the region on 11p passes the most stringent criterion for linkage, and this region shows significant evidence for linkage from both analyses. Using the gene-dropping simulation capabilities of MERLIN, we found that the empirical p-value for the evidence for linkage in this region for the P outcome was less than 0.001.
Figure 3 Linkage Regions with Evidence Significant at the Level P ≤ 0.01 for Three Phenotypes (S=Schizophrenia Excluding SAD; SS=Schizophrenia Including SAD; P = All Diagnoses with Psychotic Features) Using Two Linkage Analysis Programs (SP=Sibpal, M=MERLIN) (more ...)
Figure 5 Linkage Regions with Evidence Significant at the Level P ≤ 0.0001 for Three Phenotypes (S=Schizophrenia Excluding SAD; SS=Schizophrenia Including SAD; P = All Diagnoses with Psychotic Features) Using Two Linkage Analysis Programs (SP=Sibpal, M=MERLIN) (more ...)