Sample demographics for the 1044 individuals are given in Table . There were 698 females and 346 males. The subjects from GENOA were on average older (average age 59.6) than the HyperGEN subjects (average age 49.4). Within each network, the average body mass index (BMI), HDL-C and LDL-C were generally higher in females than in males, whereas TG levels showed slightly reversed tendencies. The male and female subjects from GENOA had higher HDL-C and TG levels than their corresponding counterparts from HyperGEN. There was little variation in BMI between networks. Average European ancestry varied modestly between the two networks, as was previously noted (36
Demographic characteristics of unrelated study participants: Family Blood Pressure Program
LDL-C levels were modestly correlated with HDL-C and TG levels (correlation coefficient −0.13 with HDL-C and 0.19 with TG). HDL-C and TG, as observed in other studies, showed a stronger inverse correlation (−0.28). The correlation between the traits was virtually unaffected by the transformations. By analysis of variance, all three lipid traits were strongly associated with BMI and age (Table ). HDL-C was strongly associated with sex. TG and LDL-C differed significantly between networks.
Analysis of variance results on LDL, HDL and TG
LDL-C was not found to be correlated with overall European individual ancestry (IA) (P = .80). On the other hand, HDL-C was strongly negatively correlated with European IA (t = −2.76, P=0.0059) and TG was strongly positively correlated with European IA (t = 3.29, P = .0010).
A Q–Q plot of the rl values (standardized regression coefficients) for the 284 loci for LDL-C, TG and HDL-C showed a reasonably good fit to normality (Fig. ) except for some outlier points, in particular at the low (negative) end of the distribution for HDL-C.
Q–Q plot comparing rl with a standard normal distribution and the corresponding histograms for the trait LDL-C, HDL-C and TG.
According to normal distribution theory, we expected 14 (~5% of 284) marker positions to have an absolute standardized regression coefficient |rl
| value > 1.96, or seven in each tail. However, we found an excess of points in the tails in each of the three distributions. For LDL-C, there were seven rl
values > 1.96 as expected but 10 in the left tail (i.e. values less than −1.96) (Supplementary Material, Table S1
). For HDL-C, there were 11 rl
values > 1.96 (i.e. in the right tail) and 10 in the left tail (less than −1.96) (Supplementary Material, Table S2
). For TG, there were 12 rl
values > 1.96 (i.e. in the right tail) and eight in the left tail (Supplementary Material, Table S3
For LDL-C, the two most promising chromosomal regions were at 3q and 4q, both showing negative correlation with European ancestry. On 3q, five markers covering 64 cM had Z-scores less than −2.0, with a peak of −2.35 at 188.3 cM (marker D3S2427). On chromosome 4q, three markers had Z-scores less than −2.0, with a peak of −2.33 at 78 cM (marker D4S2367).
For HDL-C, two significant regions on chromosomes 8q and 14q showed negative correlation with European ancestry, whereas a region on chromosome 9q showed positive correlation with European ancestry. Chromosome 8q had four markers covering 27 cM with Z-scores less than −2.0, with a peak of −4.13 at 82.3 cM (marker D8S1136). On chromosome 14q, three markers had Z-scores less than −2, with a peak of −3.00 at 95.9 cM (marker GATA193A07). On chromosome 9q, there were three markers with Z-scores > 2, with a peak of 2.69 at 110.9 cM.
The two most promising locations for TG included one extended region positively correlated with European ancestry on chromosome 8q and another negatively correlated with European ancestry on chromosome 15q. On chromosome 15q, the peak Z-score of −2.75 occurred at 90.0 cM (marker D15S652), whereas two neighboring markers also had Z-scores less than −2. On chromosome 8q, an extended region of 87 cM stretching from 77.9 to 164.5 cM contained seven markers with Z-scores > 2. Two separate peaks occurred in this interval, one at 135.1 cM with a Z-score of 2.58 (marker D8S1179) and the other at 77.9–82.3 cM (markers D8S1113 and D8S1136) with a Z-score of 2.31. We note that this is precisely the same location harboring the most significant result for HDL-C (marker D8S1136).
To determine the statistical significance of our findings, we employed a permutation test (see Materials and Methods). For LDL-C and TG, none of the markers was significant at P < 0.05. For HDL-C, however, in 5000 permutations, the minimum of 284 rl scores crossed −4.13 only seven times, −3.01 seventy-three times and −2.99 ninety times. Hence, the result associated with the marker D8S1136 is highly significant on a genome-wide level (P = 0.0014), whereas that for D8S1113 and GATA193A07 are also formally significant (P < 0.02).
We also looked at possible significant interactions of BMI, age, sex, network, usage of lipid-lowering medication, and hypertension status with local ancestry at the significant genomic marker locations for all three lipid traits and did not find any. The Z
-scores for HDL-C and TG across chromosomes 8 and 14 are plotted in Figure , which also gives a visual impression of the inverse relationship between the two traits. We also examined the possibility of outlier points driving the Z
-scores for the three significant loci (Supplementary Material, Fig. S1
) but found no evidence of outliers.
Overview of the Z-scores and its inverse relationship for HDL-C and TG across chromosomes 8 and 14.
Furthermore, as another check on the robustness of our conclusions, we examined results of multiple additional analyses based on different randomly selected sets of unrelated individuals from the African-American families in our study. The results for these subsets were essentially identical to those we obtained in the original analysis, indicating that our results were not dependent on the original selection of unrelated individuals from these families.
Our results also indicated an overall genome-wide ancestry deviation in opposite directions for HDL-C and TG. To determine whether multiple loci are influential in the joint distribution of these lipid traits, we looked at the correlation between the regression coefficients of the 284 markers for the two lipids. If a common set of genes is responsible, for example, for both high HDL-C and low TG values, then we would observe a large negative correlation between the ancestry regression coefficients.
For the 284 markers, the correlation between the regression coefficients for HDL-C and TG was −0.55. To determine statistical significance of this correlation, we performed 5000 permutations (as described in Materials and Methods), and for each permutation calculated the pairwise correlation between the ancestry regression coefficients for the two lipid traits. Out of 5000 permutations, the correlation exceeded −0.55 only 70 times, which corresponds to a P-value of .014. After removing the two chromosomes eight markers with highly negative rl scores for HDL-C and highly positive rl scores for TG from the analysis, the correlation declined only to −0.53, which was still statistically significant (P = 0.028). After removal of the markers on chromosomes 8q and 15q, the correlation still trended in the same direction but was no longer significant (P = .063). To assess whether the regressions of HDL-C and TG on total European IA could be attributed solely to ancestry at the peak location on chromosome 8q, we performed a regression analysis of HDL-C (and TG) including both 8q locus-specific European ancestry and total European ancestry in the model. For both lipids, total European IA was no longer significant (and regression coefficients were close to 0), once the locus-specific European ancestry was included in the model. Hence, the overall ancestry effect we observed for both lipids could be explained simply by a locus on chromosome 8q, although the situation may be more complex, with other loci contributing.