Genetic and functional analysis of Raynaud's syndrome implicates loci in vasculature and immunity

Anniina Tervi¹, Markus Ramste², Erik Abner³, Paul Cheng², Jacqueline M Lane⁴, Matthew Maher⁵, Jesse Valliere⁵, Vilma Lammi⁶, Satu Strausz⁶, Juha Riikonen⁶, Trieu Nguyen², Gabriella E Martyn⁷, Maya U Sheth⁷, Fan Xia⁷, Mauro Lago Docampo⁸, Wenduo Gu²; FinnGen, Estonian Biobank research team; Tõnu Esko³, Richa Saxena⁹, Matti Pirinen¹⁰, Aarno Palotie¹¹, Samuli Ripatti¹², Nasa Sinnott-Armstrong¹³, Mark Daly¹¹, Jesse M Engreitz¹⁴, Marlene Rabinovitch¹⁵, Caroline A Heckman⁶, Thomas Quertermous², Samuel E Jones⁶, Hanna M Ollila¹⁶

Affiliations

¹ Institute for Molecular Medicine Finland, FIMM, Helsinki Institute of Life Science - HiLIFE, University of Helsinki, Helsinki, Finland. Electronic address: anniina.tervi@helsinki.fi.
² Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
³ Institute of Genomics, University of Tartu, Tartu, Estonia.
⁴ Division of Sleep and Circadian Disorders, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁵ Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁶ Institute for Molecular Medicine Finland, FIMM, Helsinki Institute of Life Science - HiLIFE, University of Helsinki, Helsinki, Finland.
⁷ Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA; Basic Science and Engineering Initiative, Stanford Children's Health, Betty Irene Moore Children's Heart Center, Stanford, CA, USA.
⁸ Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA; Stanford Children's Health Betty Irene Moore Children's Heart Center, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
⁹ Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
¹⁰ Institute for Molecular Medicine Finland, FIMM, Helsinki Institute of Life Science - HiLIFE, University of Helsinki, Helsinki, Finland; Department of Mathematics and Statistics, University of Helsinki, Helsinki, Finland; Public Health, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
¹¹ Institute for Molecular Medicine Finland, FIMM, Helsinki Institute of Life Science - HiLIFE, University of Helsinki, Helsinki, Finland; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA; Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
¹² Institute for Molecular Medicine Finland, FIMM, Helsinki Institute of Life Science - HiLIFE, University of Helsinki, Helsinki, Finland; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Public Health, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
¹³ Herbold Computational Biology Program, Public Health Sciences Division, Fred Hutch, Seattle, WA, USA.
¹⁴ Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA; Basic Science and Engineering Initiative, Stanford Children's Health, Betty Irene Moore Children's Heart Center, Stanford, CA, USA; The Novo Nordisk Foundation Center for Genomic Mechanisms of Disease, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Gene Regulation Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Stanford Cardiovascular Institute, Stanford University, Stanford, CA, USA.
¹⁵ Stanford Children's Health Betty Irene Moore Children's Heart Center, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
¹⁶ Institute for Molecular Medicine Finland, FIMM, Helsinki Institute of Life Science - HiLIFE, University of Helsinki, Helsinki, Finland; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. Electronic address: hanna.m.ollila@helsinki.fi.

PMID: 39142284
PMCID: PMC11480858
DOI: 10.1016/j.xgen.2024.100630

Genetic and functional analysis of Raynaud's syndrome implicates loci in vasculature and immunity

Anniina Tervi et al. Cell Genom. 2024.

. 2024 Sep 11;4(9):100630.

doi: 10.1016/j.xgen.2024.100630. Epub 2024 Aug 13.

Authors

Affiliations

¹ Institute for Molecular Medicine Finland, FIMM, Helsinki Institute of Life Science - HiLIFE, University of Helsinki, Helsinki, Finland. Electronic address: anniina.tervi@helsinki.fi.
² Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
³ Institute of Genomics, University of Tartu, Tartu, Estonia.
⁴ Division of Sleep and Circadian Disorders, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁵ Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁶ Institute for Molecular Medicine Finland, FIMM, Helsinki Institute of Life Science - HiLIFE, University of Helsinki, Helsinki, Finland.
⁷ Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA; Basic Science and Engineering Initiative, Stanford Children's Health, Betty Irene Moore Children's Heart Center, Stanford, CA, USA.
⁸ Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA; Stanford Children's Health Betty Irene Moore Children's Heart Center, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
⁹ Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
¹⁰ Institute for Molecular Medicine Finland, FIMM, Helsinki Institute of Life Science - HiLIFE, University of Helsinki, Helsinki, Finland; Department of Mathematics and Statistics, University of Helsinki, Helsinki, Finland; Public Health, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
¹¹ Institute for Molecular Medicine Finland, FIMM, Helsinki Institute of Life Science - HiLIFE, University of Helsinki, Helsinki, Finland; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA; Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
¹² Institute for Molecular Medicine Finland, FIMM, Helsinki Institute of Life Science - HiLIFE, University of Helsinki, Helsinki, Finland; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Public Health, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
¹³ Herbold Computational Biology Program, Public Health Sciences Division, Fred Hutch, Seattle, WA, USA.
¹⁴ Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA; Basic Science and Engineering Initiative, Stanford Children's Health, Betty Irene Moore Children's Heart Center, Stanford, CA, USA; The Novo Nordisk Foundation Center for Genomic Mechanisms of Disease, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Gene Regulation Observatory, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Stanford Cardiovascular Institute, Stanford University, Stanford, CA, USA.
¹⁵ Stanford Children's Health Betty Irene Moore Children's Heart Center, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA.
¹⁶ Institute for Molecular Medicine Finland, FIMM, Helsinki Institute of Life Science - HiLIFE, University of Helsinki, Helsinki, Finland; Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. Electronic address: hanna.m.ollila@helsinki.fi.

PMID: 39142284
PMCID: PMC11480858
DOI: 10.1016/j.xgen.2024.100630

Abstract

Raynaud's syndrome is a dysautonomia where exposure to cold causes vasoconstriction and hypoxia, particularly in the extremities. We performed meta-analysis in four cohorts and discovered eight loci (ADRA2A, IRX1, NOS3, ACVR2A, TMEM51, PCDH10-DT, HLA, and RAB6C) where ADRA2A, ACVR2A, NOS3, TMEM51, and IRX1 co-localized with expression quantitative trait loci (eQTLs), particularly in distal arteries. CRISPR gene editing further showed that ADRA2A and NOS3 loci modified gene expression and in situ RNAscope clarified the specificity of ADRA2A in small vessels and IRX1 around small capillaries in the skin. A functional contraction assay in the cold showed lower contraction in ADRA2A-deficient and higher contraction in ADRA2A-overexpressing smooth muscle cells. Overall, our study highlights the power of genome-wide association testing with functional follow-up as a method to understand complex diseases. The results indicate temperature-dependent adrenergic signaling through ADRA2A, effects at the microvasculature by IRX1, endothelial signaling by NOS3, and immune mechanisms by the HLA locus in Raynaud's syndrome.

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Conflict of interest statement

Declaration of interests J.M.E. is an inventor on patents and patent applications related to CRISPR screening technologies, has received materials from 10× Genomics unrelated to this study, and has received speaking honoraria from GSK plc.

Figures

**Figure 1**
Meta-analysis (A) A Manhattan plot of RS meta-analysis (11,358 cases and 1,106,871 controls) combining UKB, FinnGen, MGB Biobank, and Estonian Biobank (EstBB) (b38). (B and C) Locus zoom plots for (B) regional association at the *ADRA2A* locus (b38), lead variant rs7090046, and (C) regional association at the *NOS3* locus (b38), lead variant rs3918226.

**Figure 2**
*ADRA2A* expression (A and B) Genotype tissue expression for SNP rs7090046 in (A) tibial arteries and (B) across tissues from GTEx (NES, normalized expression values; m-value = posterior probability, p ≤ 0.05). (C) The RS association co-localizes with eQTL signal in tibial arteries. Created with Biorender.com.

**Figure 3**
*ADRA2A* expression is restricted to SMCs located in distal arterioles (A–D) Uniform manifold approximation and projection (UMAP) plot of (A) human skin and (B) human coronary artery scRNA-seq indicating *ADRA2A* expression, co-expression of *NOTCH3* in the same cluster, and *MYH11* expression as SMC cluster and all clusters in skin (C) and in the coronary artery dataset (D). (E) A schematic of vascular wall and *ADRA2A* expression. (F) RNAscope for *ADRA2A* in a dorsal hand biopsy. (G) RT-PCR quantification of *ADRA2A* and *NOTCH* expression in human arterial SMCs and pulmonary arteriolar. Mean ± SEM, ∗∗∗p < 0.0005, ∗∗p < 0.005, and ∗p < 0.05 (unpaired two-tailed Student’s t test).

**Figure 4**
CRISPRi against rs7090046 supports causal role of *ADRA2A* (A) Schematic of the CRISPRi experiment. (B) RT-PCR quantification of *ADRA2A* expression in rs7090046 guide targeted cells vs. CTRL. Mean ± SEM, ∗∗∗p < 0.0005, ∗∗p < 0.005, and ∗p < 0.05 (one-way ANOVA).

**Figure 5**
*ADRA2A* expression affects SMC contraction upon cold stimulus (+28°C) (A) *ADRA2A* and *ADRA2C* silencing in cold exposure. (B) *ADRA2A* and *ADRA2C* silencing in ambient conditions. (C) *ADRA2A* and *ADRA2C* overexpression in cold exposure. (D) *ADRA2A* and *ADRA2C* overexpression in ambient conditions. Mean ± SEM, ∗∗∗p < 0.0005, ∗∗p < 0.005, and ∗p < 0.05 (unpaired two-tailed Student’s t test).

**Figure 6**
Schematic of the proposed RS-associated pathomechanism We propose a possible mechanism based on our observations from genetic and contraction assays. In this hypothesized model, the pathomechanism of RS is dependent on *ADRA2A* expression. In healthy patients without RS, there are mechanisms to prevent unwanted and excessive vessel contraction. First, the cell constriction (upon cold or stress) can be limited by the increased release of ligand by translocation of the α_2C-adrenergic receptor from the cytosol to the cell surface. Second, α_2A-adrenergic receptors are also expressed on the presynaptic membrane and function there as a negative feedback loop for catecholamine release. In patients with RS, the expression of the *ADRA2A* gene, that encodes the α_2A-adrenergic receptor, is higher, and therefore the number of α_2A-adrenergic receptor available for ligand binding on microvascular SMCs is also higher. Increase in SMC *ADRA2A* expression sensitizes the postsynaptic system that aggravates the adrenergic effects of SMC contraction. In conditions such as cold and stress where more ligand is released, the contraction is further accentuated. Black arrows represent adrenergic downstream signaling strength.

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References

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Genetic and functional analysis of Raynaud's syndrome implicates loci in vasculature and immunity

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Genetic and functional analysis of Raynaud's syndrome implicates loci in vasculature and immunity

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