Possible Role of the RORC Gene in Primary and Secondary Lymphedema: Review of the Literature and Genetic Study of Two Rare Causative Variants

Abstract

Background:

RAR-related Orphan Receptor C (RORC) is a DNA-binding transcription factor and the key transcription factor responsible for differentiation of T helper 17 cells. The RORC gene plays a role in lymphoid organogenesis, thymopoiesis, and lymph node organogenesis. The aim of our study was to determine the possible role of RORC in the development of lymphatic system malformations by combining data from the scientific literature and next-generation sequencing of RORC in lymphedema patients negative for known causative genes.

Methods and Results:

We sequenced RORC in 235 lymphedema patients negative for known lymphedema-associated genes. We found two probands carrying nonsense RORC variants.

Conclusions:

We show that RORC is important for normal function of the lymphatic system and that a rare variant with a possible causative effect may imply predisposition for lymphedema.

Introduction

RORC (RAR-Related Orphan Receptor C, also known as RORγ or NR1F3) is a 24 kb protein-coding gene located on chromosome 1 (1q21.3). It can be found in two isoforms. The protein encoded by this gene is a DNA-binding transcription factor and member of the large NR1 subfamily of nuclear hormone receptors. It contains a conserved DNA-binding domain and two zinc finger domains.¹

RORC has prominent roles in lymphoid organogenesis, such as thymopoiesis and lymph node (LN) organogenesis, since it is the key transcription factor responsible for differentiation of T helper 17 (Th17) cells and production of interleukin 17 (IL-17).² In addition, RORC is responsible for development of type 3 innate lymphoid cells (ILC3).^3,4 These cells are linked to lymphatic capillary growth around the nascent LN. Since this immunological commitment is responsible for autoimmunity and inflammation, lymphangiogenesis of this kind is considered to be related to several autoimmune diseases, such as asthma,⁵ rheumatoid arthritis,³ and multiple sclerosis.⁴ Human phenotype associated with RORC reported by OMIM are synthetized in Table 1.

Table 1.

Human Phenotype Associated with RORC

Gene	Function	OMIM disease	Lymphatic phenotype	Genome-wide association study
RORC	Transcription factor	Immunodeficiency-42	Overexpression: overgrowth of lymphatic vessels, inflammation, blocked lymph flow^15,16 Mutation: aplasia or absence of lymph nodes¹⁸	Possible genetic predisposition to secondary lymphedema after breast cancer treatment¹⁰

The lymphatic system is composed of a large network of lymphatic vessels, LNs, Peyer's patches, spleen, and thymus. It is an open system, the main function of which is to drain protein-rich fluid and cells from the interstitium.² The different types of lymphatic vessels play roles in homeostasis, immune surveillance, and lipid absorption. Lymphatic vessels can be remodeled during development and disease, including growth of new lymphatic vessels (lymphangiogenesis).⁶

Lymphedema is a lymphatic transport dysfunction. It is a progressive disease, caused by accumulation of protein-rich fluid in the interstitium. Lymphatic vessel hypoplasia or obstruction of lymph flow by a structural alteration of the architecture of lymphatic vessels and capillaries, or by LN hypoplasia/aplasia results in lymphedema.⁷ This leads to inflammation, swelling of the extremities, and fibrosis.⁸ Primary lymphedema is caused by mutations in different genes,⁹ and secondary lymphedema is caused by damage to the lymphatic system from surgical, physical, chemical, infectious, and biological causes. Many polymorphisms have been reported in the genes involved in predisposition for secondary lymphedema.^9,10

The importance of RORC in the lymphatic system has also been demonstrated by disruption of the RORC gene. Mice homozygotes for the null variant of RORC show absence of peripheral and mesenteric LNs and Peyer's patches, indicating complete arrest of LN development. Other changes in phenotype include decrease in number of thymocytes and higher susceptibility to apoptosis, without the lymphatic vessels being affected.¹¹

RORC^+/− mice are reported to have fewer thymocytes and increased expression of proapoptotic factor than wild-type mice.^12,13 Study of the autoimmune dry eye disease model shows that IL-17 from Th17 cells induces lymphangiogenesis. IL-17 is directly linked to lymphatic vessel growth by triggering VEGF-D expression and proliferation of lymphatic endothelial cells in vitro. In addition, in vivo blockade of IL-17 in a model of Th17-dominant autoimmune eye disease results in a reduction in lymphangiogenesis.¹⁴ These findings demonstrate that RORC is crucial for LN organogenesis (Massoud et al.).⁵

Studies show that correct function of RORC in humans is important, since down- and upregulation are both linked to the development of diseases. Rajasekaran et al. reported the role of RORC-induced Th17 cells in filariasis lymphedema (FL).¹⁵ This specific type of lymphedema is caused by a nematode-infected mosquito bite¹⁶; symptoms include inflammation of LNs and lymphatic vessels and subsequent swelling of parts of the lower or upper limbs. The authors showed that at messenger RNA (mRNA) level, patients with FL express higher levels of RORC. Expression of cytokines associated with T-helper 17 cells is also significantly elevated. Based on the evidence, T-helper 17 cells, regulated by RORC, are important for the immune response, maintaining homeostasis and, therefore, for normal function of the lymphatic system.^15–18

In this study, we investigated the role of RORC as a potential candidate gene for lymphedema. We conducted a search in the literature and analyzed the gene in 235 patients with lymphedema negative to genetic testing for known lymphedema-associated genes.

Polymorphisms in RORC are associated with several diseases in humans; main of these are reported in Table 2. Newman et al. investigated candidate genes for predisposition to secondary lymphedema after breast cancer surgery.¹⁰ They discovered several RORC variants, one of which showed a statistically significant association with secondary lymphedema. This variant, rs11801866, is in intron 1 at the 5′ end of RORC.¹⁰

Table 2.

Reported RORC Variants

Polymorphism	dbSNP	Type of mutation	Frequency (gnomAD)	Description	References
NC_000001.10:g.151804213G>A	rs17582155	Nonsense	0.00211, TAF—0.002067	Primary lymphedema	This study
NC_000001.10:g.151789754G>A	/	Nonsense	/	Primary lymphedema	This study
NC_000001.10:g.151789647T>G	rs12144914	Intronic variant	0.45279, TAF—0.4532	Primary lymphedema	Ferrell et al.²²
NC_000001.10:g.151809066A>G	rs11204897	TF binding site	0.0885, TAF—0.08852	Predisposition for secondary lymphedema	Newman et al.¹⁰
NC_000001.10:g.151802356T>A	rs11801866	Intronic variant	0.0738, TAF—0.07384	Predisposition for secondary lymphedema	Newman et al.¹⁰
NC_000001.10:g.151801680G>A	rs4845604	Intronic variant	0.149, TAF—0.1626	Potential protection against allergies	Ehm et al.¹²
NC_000001.10:g.151801680G>A	rs4845604	Intronic variant	0.149, TAF—0.1626	Immune activation in inflammatory bowel disease	De Lange et al.¹³

gnomAD, Genome Aggregation Database; TAF, total allele frequency as reported by gnomAD.

In another association study, Okada et al. determined genotypes of patients with immunodeficiency-42. They identified three loss-of-function mutations (rs774357869, rs863225091, and rs863225092) in RORC that lead to lymphocyte impairment due to lower production of interleukins (IL17A, IL17F, IL22) at mRNA and protein level. Their probands showed symptoms similar to those of lymphedema, such as inflammation and absence of palpable axillary and cervical LNs.¹⁹

Materials and Methods

Clinical evaluations

Samples from 246 Caucasian patients diagnosed with lymphedema in different hospitals across Italy were included in our retrospective study. No consanguinity was reported in families. Clinical diagnosis of lymphedema was based on generally approved criteria.²⁰ Genetic testing was performed on germline DNA extracted from saliva or blood of probands.

Detailed pretest genetic counseling was provided to all subjects, who were then invited to sign specific informed consent to use of their anonymized genetic results for research.

Genetic analysis

A custom-made oligonucleotide probe library was designed to capture all coding exons and flanking exon/intron boundaries (±15 bp) of 29 genes known to be associated with lymphedema. In our panel we addressed the candidate gene RORC in proband DNA. Variants with likely clinical significance were confirmed by bidirectional Sanger sequencing on a CEQ8800 Sequencer (Beckman Coulter).

We searched the international databases dbSNP and Human Gene Mutation Database Professional for all nucleotide changes. Minor allele frequencies were checked in the Genome Aggregation Database (gnomAD). All variants were evaluated according to American College of Medical Genetics and Genomics guidelines.²¹

Results

Clinical and genetic evaluations

Among 235 patients negative to genetic testing, we identified 2 patients with 2 heterozygous variants in RORC. Both cases were sporadic (no family history of lymphedema). Table 3 shows clinical features.

Table 3.

Clinical Features of Probands from Two Families

Gender	Age	Clinical features	Age of onset	Family history	Variant nomenclature
F	60	Lymphatic malformations	Not available	No	NM_001001523.1; c.10C>T; p.(Gln4Ter)
F	32	Lower limb lymphedema	21	No	NM_005060.3:c.28C>T; p.Arg10Ter

The first proband carries a nonsense mutation NM_005060.3:c.28C>T, which creates an early terminus and, therefore, a truncated protein. The proband is a female with sporadic lower-limb lymphedema and age of onset was 21 years. Allele frequency according to gnomAD database is 0.002067 (rs17582155).

The second proband is a female with malformations of the lymphatic system, diagnosed with lymphedema. The nomenclature of the RORC variant in this case is NM_001001523.1:c.10C>T, it is present in both RORC isoforms. This is a stop-gain splice-region variant, not listed in the dbSNP database and with no known frequency.

SIFT prediction was not available for either variant. Variant details and predictions are shown in Table 4.

Table 4.

Characterization of the Two RORC Variants

Variants	dbSNPid	Mutation taster	Frequency
NM_005060.3:c.28C>T	rs17582155	Prediction disease causing	0.002067
NM_001001523.1:c.10C>T	/	Prediction disease causing	/

Discussion and Conclusions

In a population of 246 patients with primary lymphedema, 235 were negative to genetic testing for known genes associated to this disorder and were analyzed for the RORC candidate gene. To investigate the role of RORC in lymphedema, we studied the genotype of these patients. We also conducted a search of the literature for possible links between the role of RORC and lymphedema.

We found 2 samples (2/235; 0.851%) with variants among the 235 samples analyzed for known genes. Both variants create an early stop codon and were sporadic. Family members of both probands were subsequently contacted but did not wish to participate in this study. Therefore, no other members of the families were tested.

The role of RORC as a possible lymphedema candidate gene was previously investigated by Ferrell et al. who identified three RORC variants in ∼100 samples (3%) from patients with primary lymphedema in the United States and Canada. However, the variant did not segregate with the disease in any case.²² To our knowledge, RORC dysfunction is not a Mendelian cause of lymphedema. However, it may play a role in the susceptibility to the disease.

Our review of the literature showed that RORC is important in the differentiation of Th17 cells and for IL17A and IL17F. RORC was recently implicated in the development of new drugs, such as endogenous inhibitors and new synthetic molecules.^23–25 RORC functions as a ligand-dependent transcription factor and its transcriptional activity can be regulated by a variety of synthetic ligands that bind it and act as agonists or antagonists.

An example is one of the first ligands discovered, retinoic acid. It is used to prevent secondary lymphedema after breast cancer surgery and helped lessen lymphedema symptoms in a mouse model.^26,27 RORC antagonists such as SR1078²⁸ mimic the phenotype of RORC^-/- mice. Differentiation of Th17 cells is inhibited, preventing transcription of IL17 and its subsequent production.²⁹ New synthetic antagonists include hexafluoroisopropanol-aryl-sulfonamides, digoxin,^25,28 and oxasultam³⁰; others are being developed. These compounds have high therapeutic potential for inflammatory and immune diseases²⁹ and we presume that RORC may become a therapeutic target in lymphedema as well.

Our search of the literature revealed that the immune and lymphatic systems influence each other and RORC plays a crucial role in maintaining both systems. Based on the animal studies in the literature, the loss of RORC leads to the arrest of LN development.¹¹ Similarly, in case of high expression of RORC, the inflammation of LNs and lymphatic vessels were observed in patients with FL.¹⁵ Therefore, our hypothesis is supported by the discovery that loss of RORC function and overexpression are extremely critical in the development of LNs and can lead to lymphedema.^10,16 We propose that RORC be considered a candidate gene for susceptibility to lymphedema.

Footnotes

Acknowledgment

We thank Helen Ampt for the English language editing.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by funding from the Provincia Autonoma di Trento within the initiative LP 6/99 (dgp 1045/2017).

References

Jetten

. Retinoid-related orphan receptors (RORs): Critical roles in development, immunity, circadian rhythm, and cellular metabolism. Nucl Recept Signal, 2009; 7:e003.

Warren

, Brorson

, Borud

, Slavin

. Lymphedema: A comprehensive review. Ann Plast Surg, 2007; 59:464–472.

Xue

, Soroosh

, De Leon-Tabaldo

, et al. Pharmacologic modulation of RORγt translates to efficacy in preclinical and translational models of psoriasis and inflammatory arthritis. Sci Rep, 2016; 6:1–17.

Serafini

, Rosicarelli

, Veroni

, Zhou

, Reali

, Aloisi

. RORγt expression and lymphoid neogenesis in the brain of patients with secondary progressive multiple sclerosis. J Neuropathol Exp Neurol, 2016; 75:877–888.

Massoud

, Charbonnier

, Lopez

, Pellegrini

, Phipatanakul

, Chatila

. An asthma-associated IL4R variant exacerbates airway inflammation by promoting conversion of regulatory T cells to Th17-like cells. Nat Med, 2016; 22:1013–1022.

Yuan

, Arcucci

, Levy

, Achen

. Modulation of immunity by lymphatic dysfunction in lymphedema. Front Immunol, 2019; 10:1–6.

Raju

, Chang

. Vascularized lymph node transfer for treatment of lymphedema: A comprehensive literature review. Ann Surg, 2015; 261:1013–1023.

Michelini

, Cardone

, Maltese

, Bruson

, Fiorentino

, Bertelli

. Primary lymphedema and genetic implications. EuroBiotech J, 2017; 1(Suppl 2):144–146.

Michelini

, Paolacci

, Manara

, et al. Genetic tests in lymphatic vascular malformations and lymphedema. J Med Genet, 2018; 55:222–232.

10.

Newman

, Lose

, Kedda

, et al. Possible genetic predisposition to lymphedema after breast cancer. Lymphat Res Biol, 2012; 10:2–13.

11.

Kurebayashi

, Ueda

, Sakaue

, et al. Retinoid-related orphan receptor γ (RORγ) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis. Proc Natl Acad Sci U S A, 2000; 97:10132–10137.

12.

Ehm

, Aponte

, Chiano

, et al. Phenome-wide association study using research participants' self-reported data provides insight into the Th17 and IL-17 pathway. PLoS One, 2017; 12:1–14.

13.

De Lange

, Moutsianas

, Lee

, et al. Genome-wide association study implicates immune activation of multiple integrin genes in inflammatory bowel disease. Nat Genet, 2017; 49:256–261.

14.

Chauhan

, Jin

, Goyal

, et al. Brief report: A novel pro-lymphangiogenic function for Th17/IL-17. 2011; 118:4630–4634.

15.

Rajasekaran

, Anuradha

, Manokaran

, Bethunaickan

. An overview of lymphatic filariasis lymphedema. Lymphology, 2017; 50:164–182.

16.

Babu

, Bhat

, Kumar

, et al. Filarial lymphedema is characterized by antigen-specific Th1 and Th17 proinflammatory responses and a lack of regulatory T cells. PLoS Negl Trop Dis, 2009; 3:e420.

17.

Shenoy

. Clinical and pathological aspects of filarial lymphedema and its management. Korean J Parasitol, 2008; 46:119–125.

18.

Stockinger

, Omenetti

. The dichotomous nature of T helper 17 cells. Nat Rev Immunol, 2017; 17:535–544.

19.

Okada

, Markle

, Deenick

, et al. Impairment of immunity to Candida and Mycobacterium in humans with bi-allelic RORC mutations. Science (80-), 2015; 349:606–613.

20.

Kayıran

, La Cruz

, Tane

, Soran

. Lymphedema: From diagnosis to treatment. Turk J Surg, 2017; 33:51–57.

21.

Richards

, Aziz

, Bale

, et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med, 2015; 17:405–424.

22.

Ferrell

, Kimak

, Lawrence

, Finegold

. Candidate gene analysis in primary lymphedema. Lymphat Res Biol, 2008; 6:69–76.

23.

Bramos

, Perrault

, Yang

, Jung

, Hong

, Wong

. Prevention of postsurgical lymphedema by 9-cis retinoic acid. Ann Surg, 2016; 264:353–361.

24.

Olsson

, Xue

, Von Berg

, et al. Benzoxazepines achieve potent suppression of IL-17 release in human T-helper 17 (Th17) cells through an induced-fit binding mode to the nuclear receptor RORγ. ChemMedChem, 2016; 11:207–216.

25.

Fauber

, De Leon Boenig

, Burton

, et al. Structure-based design of substituted hexafluoroisopropanol-arylsulfonamides as modulators of RORc. Bioorg Med Chem Lett, 2013; 23:6604–6609.

26.

Mottaghi

, Ebrahimof

, Angoorani

, Saboor-Yaraghi

. Vitamin A Supplementation reduces IL-17 and RORc gene expression in atherosclerotic patients. Scand J Immunol, 2014; 80:151–157.

27.

Guendisch

, Weiss

, Ecoeur

, et al. Pharmacological inhibition of RORγt suppresses the Th17 pathway and alleviates arthritis in vivo. PLoS One, 2017; 12:1–25.

28.

Fauber

, Magnuson

. Modulators of the nuclear receptor retinoic acid receptor-related orphan receptor-γ (RORγ or RORc). J Med Chem, 2014; 57:5871–5892.

29.

Jetten

, Takeda

, Slominski

, Kang

. Retinoic acid-related orphan receptor γ (RORγ): Connecting sterol metabolism to regulation of the immune system and autoimmune disease. Curr Opin Toxicol, 2018; 8:66–80.

30.

René

, Fauber

, Barnard

, et al. Discovery of oxa-sultams as RORc inverse agonists showing reduced lipophilicity, improved selectivity and favorable ADME properties. Bioorg Med Chem Lett, 2016; 26:4455–4461.