TIE1 as a Candidate Gene for Lymphatic Malformations with or without Lymphedema

TIE1 is a cell surface protein expressed in endothelial cells. Involved in angiogenesis and lymphangiogenesis, including morphogenesis of lymphatic valves, TIE1 is important for lymphatic system functional integrity. The main purpose of this study was to identify different variants in the TIE1 gene that could be associated with lymphatic malformations or dysfunction and predisposition for lymphedema. In a cohort of 235 Italian lymphedema patients, who tested negative for variants in known lymphedema genes, we performed a further test for new candidate genes, including TIE1. Three probands carried different variants in TIE1. Two of these segregated with lymphedema or lymphatic dysfunction in familial cases. Variants in TIE1 could contribute to the onset of lymphedema. On the basis of our findings, we propose TIE1 as a candidate gene for comprehensive genetic testing of lymphedema.


Introduction
The TIE1 gene, located on the short arm of chromosome 1 (1p34.2), encodes a tyrosine kinase receptor with an immunoglobulin and EGF factor homology domain. The TIE1 protein occurs on the surface of endothelial cells and was cloned for the first time in 1992 by Partanen and colleagues [1]. TIE1 is predominantly expressed in endothelial cells of blood and lymphatic vessels and in several lines of hematopoietic cells. TIE1 is important for angiogenesis and lymphangiogenesis, because together with TIE2 and angiopoietins (ANGs), it takes part in the ANG-TIE signaling pathway. Interestingly, no ligand that binds TIE1 has yet been identified, making TIE1 an orphan receptor [2]. However, TIE1 interacts directly with another member of the tyrosine kinase receptor family, TIE2. TIE2 binds various angiopoietins, including Ang1 and Ang2. ANGs can activate the formation of the TIE signaling complex in the junctions of endothelial cells. The TIE complex in turn regulates the survival signal of endothelial cells [3,4]. The normal function of TIE1 is therefore directly and indirectly important for normal vascular development, since it affects the function of TIE2 and angiopoietins [2]. The ANG-TIE signaling pathway is involved in homeostasis, inflammatory response, angiogenesis and lymphangiogenesis; abnormal ANG-TIE signaling can give rise to pathological processes [2].
More insight into the biological functions of TIE1 has come from mouse models (Table 1). First, the role of TIE1 in the vascular system was demonstrated. TIE1 −/− mice showed severe edema and bleeding and died during gestation (E13.5). Death was due to hemorrhage and abnormal formation of microvessels [5,6]. The role of TIE1 in the lymphatic system was subsequently demonstrated. D'Amico et al. showed that deletion of TIE1 in mice has a major impact on the formation of lymphatic vasculature. TIE1 −/− embryos developed edema at E12.5 in the dorsal body and neck area. The authors reported that TIE1 was expressed in lymphatic sacs during embryo development and that the lymphatic sacs showed impaired patterning in TIE1-null mice. TIE1-deficiency also led to impaired development and integrity of the lymphatic capillaries. A similar phenotype was reported in animals with conditional TIE1 mutation [7]. These findings demonstrate that TIE1 is indispensable for lymphangiogenesis during embryogenesis. Lymphatic vessel remodeling failure, abnormal lymphatic valve morphogenesis, impaired maturation of the lymphatic system and failed development of lymphatic collecting vessels [9] In another study, Qu et al. generated hypomorphic mice with low TIE1 expression, as well as conditional TIE1 mutant mice. Insertion of a neo cassette (TIE1 neo/neo ) caused abnormal splicing and reduction of TIE1 expression to~20% of normal. This resulted in abnormalities in the lymphatic vasculature and overgrown jugular lymphatic vessels. Furthermore, insufficient expression of TIE1 led to failure of fluid drainage in the skin of mutant mice, causing edema. Interestingly, an abnormal cardiovascular phenotype was not observed in mice with reduced TIE1 expression [8].
These results clearly demonstrate the importance of TIE1 in the normal development of the lymphatic system, especially the lymphatic vasculature. Recent studies also showed that TIE1 is indispensable for the morphogenesis of lymphatic valves [9]. It was reported that while TIE1 is expressed ubiquitously in early embryonic development of the lymphatic system, there is a significant increase in TIE1 expression in lymphatic endothelial cells at the onset of lymphatic valve formation. TIE1 mutant mice lack lymphatic vessel remodeling capacity, show abnormal lymphatic valve morphogenesis and impaired maturation of the lymphatic system, and fail to develop collecting lymphatic vessels [9,10]. Taken together, TIE1 mouse models demonstrate that TIE1 is crucial for lymphatic system formation in a dose-dependent manner and is especially important for the normal function and formation of lymphatic vessels and valves.
The network of lymphatic vessels and capillaries is an essential part of the lymphatic system. Lymphatic capillaries are permeable and allow protein-rich interstitial fluid to enter the lymphatic system. This is important for fluid homeostasis and normal immune function. Lymphatic valves are an element of lymphatic-collecting vessels and their role is to maintain retrograde lymph flow and prevent blockage of lymph flow [11]. Lymphatic abnormalities can give rise to diseases such as lymphedema [12].
Lymphedema is a lymphatic system disorder caused by accumulation of interstitial fluid. It is a chronic disease with clinical symptoms such as edema, inflammation and fibrosis. The swelling usually affects the extremities and can be accompanied by pain and functional impairment. Fluid accumulates due to lymphatic system malformations such as lymphatic valve abnormalities [13], excessive lymphangiogenesis, or complete or partial absence of lymphatic capillaries and collecting vessels [14].
Several genes are known to be involved in the onset of, or predisposition for, lymphedema. Current genetic testing of lymphedema patients and their family members includes as many as 29 known lymphedema-associated genes [15]. Despite this large number, variants in those genes can usually only explain the etiology of 25-30% patents with lymphedema [16,17]. Therefore, additional genes, as we show, are certainly involved and need to be included to improve test accuracy.
In our study, we tested 246 Italian lymphedema patients for the known lymphedema genes, but 235 did not carry pathogenic variants. We therefore tested these 235 probands for candidate genes including TIE1. Here we demonstrate a link between TIE1 and lymphatic system malformations, indicating the potential of TIE1 as a candidate gene for genetic testing of lymphedema.

Clinical Results
We retrospectively enrolled 235 Italian patients diagnosed with lymphedema in our study. These patients had previously been tested for known lymphedema-associated genes and were found negative. We performed a second genetic test for new candidate genes, including TIE1. We found TIE1 variants in three out of 235 probands. The clinical features of probands and tested family members are summarized in Table 2.
The proband of the first family (female, 23 years) was diagnosed with edema of the lower limbs at age 13 and carried a missense heterozygous TIE1 variant NM_001253357.1:c.1306C>T. The variant causes a change from arginine to cysteine at position 436 of the protein. According to GnomAD, the frequency of this allele is 0.0000319 and the variant is known in dbSNP as rs139244400. Polyphen and SIFT predict the variant to be deleterious and possibly damaging, respectively. We also tested both parents, the brother and a grandmother of the proband ( Figure 1). We found that while the father and brother were negative for TIE1 variants, the mother and grandmother carried the same variant as the proband. We performed lymphoscintigraphy of the mother, defined as healthy, to investigate her lymphatic system function. The results showed mild deficits of the lymphatic system development, compatible however with good overall clinical compliance. In addition, the grandmother reported episodes of cyclic edema.   The second proband (female, 52 years) has had right lower limb edema since age 25 years. Since this is a sporadic case, no other family members were tested. The proband was found to carry a heterozygous single nucleotide missense variant in TIE1 (NM_001253357.1:c.3046G>A) that results in a change of glutamine 1016 to lysine. The frequency of this allele is 0.0000489 (GnomAD), and it is listed in dbSNP as rs760492428. SIFT predicts the variant to be deleterious, and according to PolyPhen it is possibly damaging.
In the third family, we tested the proband and both parents ( Figure 2). The proband (female, 47 years) suffers from lymphedema of lower limbs, which started at the knees, diagnosed at age 15. She carries a heterozygous missense variant NM_001253357.1:c.3191G>A that causes a change of arginine 1064 to histidine. The case is familial: the proband's mother has lymphedema and was found to carry the same TIE1 variant as the proband. SIFT and PolyPhen predictions characterized this variant as deleterious and probably damaging, respectively. GnomAD reports the frequency of this variant as 0.000772 and its dbSNP ID is rs34993202. All the TIE1 variants identified in this study are shown in Table 3.

In-Silico Analysis, Template Selection and Model Building
A template search with BLAST and HHBlits was performed against the SWISS-MODEL template library (SMTL, last update: 24 October 2019, last included PDB release: 18 October 2019). The target sequence (Table 4) was searched against the primary amino acid sequence contained in   (Table 4) was searched against the primary amino acid sequence contained in the SMTL. A total of 13,529 templates matching with different sequence identity and quality percentages were found. Details of the top 10 templates are shown in Table 5.  Based on the percentage of sequence identity, similarity and best quality square, the 1fvr.1.A chain was selected to align the template and query sequences for model building. The final model is shown in Figure 2. Then, we entered the model in Discovery studio visualizer to generate Arg436Cys

Discussion
Lymphedema is a progressive disease that affects approximately 1 in 100,000 individuals. It is characterized by accretion of lymphatic fluid in tissues, causing swelling, inflammation and fibrosis [18]. Despite the many studies into the genetic background of lymphedema, the underlying molecular mechanisms are still unclear. We used Next-Generation Sequencing to determine the genotype of 246 Italian lymphedema patients with regard to known lymphedema genes [15]. Surprisingly, 235 of the patients tested negative for variants in known genes. We therefore decided to investigate new candidate genes, including TIE1 (OMIM 600222).
The protein TIE1 is a receptor tyrosine kinase, with a role in cardiovascular and lymphatic system development. It is an orphan receptor since no direct binding of ligands by TIE1 has been discovered [1,2]. TIE1 forms a complex with TIE2, another member of the TIE family, which despite its structural similarities with TIE1, directly binds various ligands. Angiopoietins (ANG) have been shown to bind TIE2 and activate the ANG-TIE signaling pathway [19]. ANG-TIE signaling is important for lymphatic vessel network development, especially for the remodeling and maintenance of lymphatic collecting vessels [20].
Development of the mammalian lymphatic system is a highly conserved process and the importance of TIE1 function has been demonstrated by mouse models in different situations [7][8][9][10]21]. All in all, since animal models of TIE1 clearly show its function in the morphogenesis, remodeling and maturation of lymphatic vessels, TIE1 has been suggested as a gene involved in the development of lymphedema [9].
In our study, we identified three different heterozygous missense TIE1 variants in 235 lymphedema patients (3/235; 1.28%), who tested negative for known lymphedema genes. All three variants caused a change in amino acids in the resulting protein. The probands were all female and diagnosed with lymphedema of one or both lower limbs at an early age (before 25 years).
In the first family we also found the same rs139244400 variant carried by the proband in the mother and grandmother, who do not have overt lymphedema, although lymphoscintigraphy of the mother confirmed mild deficits of the lymphatic system and the grandmother reported episodes of lymphatic impairment.
For the second proband, a sporadic case carrying the variant rs760492428, no family members were tested.
In the third family, the case is familial as the mother of the proband also suffers from lymphedema. The rs34993202 variant appears to segregate with lymphedema in this family.
To further evaluate the effects of the variants on the overall structure and function of the resulting protein, we performed comprehensive bioinformatic analysis. Our in-silico analysis showed that the TIE1 gene codes a structure in which Arg436 is much more stable than the variant Cys436: the gene coded with Arg436 shows 11 interactions whereas the variant shows only five, and all except two interactions, i.e. those with Arg388 and Glu425, are quite different from each other and even the two similar interactions show a slight difference in bond length. The same was observed in the case of Glu1016Lys, where Glu1016 has 11 interactions, whereas Lys1016 only shows nine, but compared to the Arg436Cys variant, Lys1016 in the Glu1016Cys variant shows six similar interactions, like Glu1016, but varying in bond length. In case of the Arg1064His variant, Arg1064 shows five interactions whereas His1064 shows only three, two quite similar to Arg1064, i.e. Ala1060 and Glu1061 have the same bond length. These results suggest that the overall protein structure is somehow altered by these different interactions with nearby residues, leading to functional defects in the protein.

Clinical Evaluation
We retrospectively enrolled 246 Caucasian patients diagnosed with lymphedema in hospitals across Italy. No consanguinity was reported in their families. Clinical diagnosis of lymphedema was made according to generally accepted criteria. The diagnosis of lymphedema was confirmed by three-phase lymphoscintigraphy according to the protocol of Bourgeois. Lymphoscintigraphy analysis was also performed for the mother of the proband of Family 1. Genetic testing was performed on germline DNA extracted from saliva or blood of the proband. Segregation analysis was performed on DNA extracted from the saliva of the proband's relatives.

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 16 . We added the candidate gene TIE1 to our panel (OMIM 600222). DNA from probands was analyzed for genetic variants. Variants with likely clinical significance were confirmed by bidirectional Sanger sequencing on a CEQ8800 Sequencer (Beckman Coulter).
We developed a Next-Generation Sequencing (NGS) protocol for the screening of the most frequently mutated genes, namely ADAMTS3 (OMIM 605011) We searched the international databases dbSNP and Human Gene Mutation Database professional (QIAGEN, CA, United States) for all nucleotide changes. In-silico evaluation of the pathogenicity of sequence changes in TIE1 was performed using the Variant Effect Predictor tool [22] and MutationTaster [23]. Minor allele frequencies were checked in the Genome Aggregation Database (gnomAD) [24]. All variants were evaluated according to American College of Medical Genetics and Genomics guidelines [25]. Detailed pre-test genetic counseling was provided to all subjects, who were then invited to sign informed consent to use of their anonymized genetic results for research.

In-Silico Analysis
The primary amino acid sequences of TIE1 in FASTA format (Tables 4 and 5) were used as targets to search the Swiss model template library (SMTL) version 2019-10-24 and Protein Data Bank (PDB) release 2019-10-18 [26] for matching evolution-related structures by means of BLAST (Basic Local Alignment Search Tool) [27] and HHBlits [28]. Models were based on target-template alignment using ProMod3 of the SWISS-MODEL server [29]. Coordinates conserved between the target and the template were copied from the template to the model. Insertions and deletions were remodeled using a fragment library. Side chains were then rebuilt. Finally, the geometry of the resulting model was regularized with the CHARMM27 force field [30]. In the case of failure of loop modeling with ProMod3, an alternative model was built with ProMod-II [31]. Global and per-residue model quality were assessed using the QMEAN scoring function [32]. BioVia Discovery Studio Visualizer ver17.2 [33] was used to visualize the modeled protein, to vary the targeted amino acids and to analyze interactions at molecular level.

Conclusions
Despite the efforts and progress made in recent years, the genetics of lymphedema are still not entirely clear. Overall, most lymphedema patients remain without a genetic diagnosis since the genes currently associated with lymphedema account for about 25-30% of patients [16,17]. Understanding the genetics of lymphatic malformations may help to develop better therapies for lymphedema. In this report, we attempted to link data in the literature and NGS analysis of TIE1 variants in lymphedema patients to investigate the role of the TIE1 gene in the development of lymphatic system malformations and predisposition for lymphedema. Based on our results, we propose TIE1 as a candidate gene for genetic testing of lymphedema.