Novel association approach for variable number tandem repeats (VNTRs) identifies DOCK5 as a susceptibility gene for severe obesity

Abstract

Variable number tandem repeats (VNTRs) constitute a relatively under-examined class of genomic variants in the context of complex disease because of their sequence complexity and the challenges in assaying them. Recent large-scale genome-wide copy number variant mapping and association efforts have highlighted the need for improved methodology for association studies using these complex polymorphisms. Here we describe the in-depth investigation of a complex region on chromosome 8p21.2 encompassing the dedicator of cytokinesis 5 (DOCK5) gene. The region includes two VNTRs of complex sequence composition which flank a common 3975 bp deletion, all three of which were genotyped by polymerase chain reaction and fragment analysis in a total of 2744 subjects. We have developed a novel VNTR association method named VNTRtest, suitable for association analysis of multi-allelic loci with binary and quantitative outcomes, and have used this approach to show significant association of the DOCK5 VNTRs with childhood and adult severe obesity (P(empirical)= 8.9 × 10(-8) and P= 3.1 × 10(-3), respectively) which we estimate explains ~0.8% of the phenotypic variance. We also identified an independent association between the 3975 base pair (bp) deletion and obesity, explaining a further 0.46% of the variance (P(combined)= 1.6 × 10(-3)). Evidence for association between DOCK5 transcript levels and the 3975 bp deletion (P= 0.027) and both VNTRs (P(empirical)= 0.015) was also identified in adipose tissue from a Swedish family sample, providing support for a functional effect of the DOCK5 deletion and VNTRs. These findings highlight the potential role of DOCK5 in human obesity and illustrate a novel approach for analysis of the contribution of VNTRs to disease susceptibility through association studies.

Figure 1.

The DOCK5 region. (A) Schematic overview of the DOCK5 CNV region. Position of DOCK5 is shown by the black arrow, with exons shown as black boxes. The position of probe rs6997760 (chr8: 25 101 829) is indicated relative to VNTRs A and B and a 3975 bp deletion on chr8p21.2 in intron 1–2 of DOCK5. (B and D) Cluster plots of the first (x-axis) versus second (y-axis) principal components of the LRR across three and six intensity-only probes within each of VNTRs A and B, respectively. Probe positions located between chr8: 25 085 709–25 085 826 (VNTR A) and chr8: 25 129 632–25 130 278 (VNTR B). Red closed circles: obesity cases; black closed circles: normal-weight controls. (C) CGH analytics (Agilent Technologies) view of the 3975 bp CNV on chr8p21.2. Array CGH was carried out on 9 child obese cases, 10 adult obese cases and 9 child controls, using Agilent 8 × 15 k custom arrays. Log₂ ratios are ∼0 for homozygous deleted samples, and ∼2 for heterozygous samples, as the reference sample appears to have a homozygous deletion at this locus. Samples homozygous for the deletion are shown in black, whereas samples with two copies are shown in red. Both the 3975 bp deletion and VNTR B lie within intron 1–2 of the DOCK5 gene (represented by the black arrow in A). Exons are represented as black boxes in (A).

Figure 2.

Association of DOCK5 VNTR bins with obesity. The heat map indicates the level of association (−log₁₀ P-value) between VNTR bins and obesity in a logistic regression model, after regressing out gender and the DOCK5 3795 bp deletion (see Materials and Methods). (A) VNTR A bins; (B) VNTR B bins. Top left triangle: association in child cohort; middle triangle: association in adult cohort; lower triangle: association in combined child and adult cohort.

Figure 3.

Log odds ratios of variants significantly associated with obesity in both the child and adult obesity samples. For the 3975 bp DOCK5 deletion, the log odds of disease risk for homozygote undeleted relative to homozygote deleted is shown. For VNTR bins, the log odds of disease risk for heterozygote relative to homozygote is shown.