A Genome-wide Association Study of Dupuytren Disease Reveals 17 Additional Variants Implicated in Fibrosis  Michael Ng, Dipti Thakkar, Lorraine Southam, Paul Werker, Roel Ophoff, Kerstin Becker, Michael Nothnagel, Andre Franke, Peter Nürnberg, Ana Isabel Espirito-Santo, David Izadi, Hans Christian Hennies, Jagdeep Nanchahal, Eleftheria Zeggini, Dominic Furniss  The American Journal of Human Genetics  Volume 101, Issue 3, Pages 417-427 (September 2017) DOI: 10.1016/j.ajhg.2017.08.006 Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Manhattan Plot for the Discovery Association Analysis The horizontal blue line represents p = 1 × 10−5 and the horizontal red line indicates p = 5 × 10−8. Variants colored in cyan are suggestive of association (p ≤ 1 × 10−5) and those colored red have genome-wide significant association (p ≤ 5 × 10−8). The nine previously reported associated loci are indicated by an open circle surrounding the SNP. The American Journal of Human Genetics 2017 101, 417-427DOI: (10.1016/j.ajhg.2017.08.006) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 The High-Risk Genotype at rs16879765 Is Associated with a Reduction in SFRP4 Protein Secretion and Reduces Inhibition of Non-canonical WNT Signaling (A) Annotated regional association plot for the 7p14.1 locus, generated using LocusZoom software.57 Recombination rates were derived from HapMap data. (B) qPCR of genotyped DD-derived myofibroblasts revealed that the high-risk TT genotype was associated with increased mRNA expression of SFRP4 (left) but not EPDR1 (right). Each experiment was conducted in triplicate on cells derived from independent individuals (CC n = 10; CT n = 9; TT n = 7). Error bars represent the standard error of the mean. (C) Immunocytochemistry reveals robust expression of SFRP4 (red) in DD-derived myofibroblasts. Nuclei are stained blue, and F-actin is stained green. (D) Immunohistochemistry in fixed surgically resected DD fibrotic fascia confirms expression of SFRP4 (brown). (E) ELISA of supernatant from DD-derived myofibroblasts shows that decreased extracellular accumulation of SFRP4 protein is associated with the high-risk TT genotype at rs16879765. Each experiment was repeated in duplicate on cells from independent individuals (CC n = 9; TT n = 6), at 7 days from the beginning of culture. Error bars represent the standard error of the mean. (F and G) WNT3A stimulation of DD-derived myofibroblasts upregulates both the canonical (AXIN2) and non-canonical (CTGF) pathways, and also upregulates the expression of α-smooth muscle actin (ACTA2), while having no effect on the expression of β-actin (ACTB) or collagen types I (COL1A1) or III (COL3A1). The addition of SFRP4 or non-specific WNT inhibitor DKK1 alone has no appreciable effect on signaling or expression of any tested gene. The addition of SFRP4 in combination with WNT3A selectively inhibits signaling via the non-canonical pathway, whereas DKK1 inhibits both canonical and non-canonical signaling. Each experiment was repeated in duplicate on cells from independent individuals (CC n = 9; TT n = 6) at 7 days from the beginning of culture. ∗p < 0.05. The American Journal of Human Genetics 2017 101, 417-427DOI: (10.1016/j.ajhg.2017.08.006) Copyright © 2017 The Author(s) Terms and Conditions