Lymphatic abnormalities are associated with RASA1 gene mutations in mouse and man

Abstract

Mutations in gene RASA1 have been historically associated with capillary malformation-arteriovenous malformation, but sporadic reports of lymphatic involvement have yet to be investigated in detail. To investigate the impact of RASA1 mutations in the lymphatic system, we performed investigational near-infrared fluorescence lymphatic imaging and confirmatory radiographic lymphangiography in a Parkes-Weber syndrome (PKWS) patient with suspected RASA1 mutations and correlated the lymphatic abnormalities against that imaged in an inducible Rasa1 knockout mouse. Whole-exome sequencing (WES) analysis and validation by Sanger sequencing of DNA from the patient and unaffected biological parents enabled us to identify an early-frameshift deletion in RASA1 that was shared with the father, who possessed a capillary stain but otherwise no overt disease phenotype. Abnormal lymphatic vasculature was imaged in both affected and unaffected legs of the PKWS subject that transported injected indocyanine green dye to the inguinal lymph node and drained atypically into the abdomen and into dermal lymphocele-like vesicles on the groin. Dermal lymphatic hyperplasia and dilated vessels were observed in Rasa1-deficient mice, with subsequent development of chylous ascites. WES analyses did not identify potential gene modifiers that could explain the variability of penetrance between father and son. Nonetheless, we conclude that the RASA1 mutation is responsible for the aberrant lymphatic architecture and functional abnormalities, as visualized in the PKWS subject and in the animal model. Our unique method to combine investigatory near-infrared fluorescence lymphatic imaging and WES for accurate phenoptyping and unbiased genotyping allows the study of molecular mechanisms of lymphatic involvement of hemovascular disorders.CM-AVM | lymphatics | near-infrared fluorescence imaging | indocyanine green imaging T he gene RASA1 encodes for protein p120 Ras GTPaseactivating protein (p120 RasGAP or RASA1) that regulates Ras activation and blood vessel growth in humans. Germ-line mutations in RASA1 cause the autosomal dominant vascular disorder capillary malformation-arteriovenous malformation (CM-AVM), with high penetrance and variable expressivity [Online Mendelian Inheritance in Man (OMIM) no. 608354] (1-5). CMs are observed in all patients with RASA1 mutations and present as single or multiple small pink cutaneous lesions. Fast-flow lesions that include AVMs, arteriovenous fistulas, and Parkes-Weber syndrome (PKWS) (OMIM no. 608355) are observed in one-third of cases. PKWS caused by RASA1 mutation typically presents with symmetrical overgrowth of the involved extremity with large cutaneous CM and diffuse microshunting causing cardiac volume overload. Recent reports of upper-and lower-extremity lymphedema (6, 7), together with the finding that a small number of CM-AVM patients develop chylothorax and chylous ascites (5), indicate that lymphatic malformation may be an additional phenotype associated with mutations in RASA1. In mice, deletion of ...

Endothelial Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 Is Critical for Lymphatic Vascular Development and Function

“…Third, RASA1 mutations are associated with lymphatic dysfunction in humans (20). Finally, inducible loss of Rasa1 in mice causes lymphatic vascular proliferation phenotypes, as well as chyle leakage and death, which is a phenotype similar to the one we observed in Map4k4 Cdh5 Cre mice (10,20). Interestingly, mapping of the Y2H clone that interacted with Map4k4 determined that Map4k4 bound the GAP domain of Rasa1 (Fig.…”

“…of the two proteins was indirect (19). Third, RASA1 mutations are associated with lymphatic dysfunction in humans (20). Finally, inducible loss of Rasa1 in mice causes lymphatic vascular proliferation phenotypes, as well as chyle leakage and death, which is a phenotype similar to the one we observed in Map4k4 Cdh5 Cre mice (10,20).…”

RASA1 regulates the function of lymphatic vessel valves in mice

“…In agreement with this possibility, valve defects can be detected as soon as 1 week after RASA1 loss (Figure 3). In contrast, as shown in NIRFLI studies, the earliest time that LV hyperplasia can be detected is 2 weeks after gene disruption (4,11). Furthermore, in light of the present findings, it is possible that any apparent hyperplasia at 2 weeks in NIRFLI studies does not represent hyperplasia per se, but back flow of dye into preexisting LVs.…”

“…In previous near-infrared fluorescence lymphatic imaging (NIRFLI) studies of live induced RASA1-deficient mice and humans with RASA1 mutations, we noticed back-flow of dye at injection sites suggestive of an LV valve defect that could account for leakage (4,11). Collecting LVs contain intraluminal semilunar valves that are essential for propulsive lymph flow in these (B) Shown is the mean pump limit plus SEM for vessels from littermate Rasa1 fl/fl (n = 4) and Rasa1 fl/fl Ub ert2cre mice (n = 5) in pump-function tests.…”

“…Lymphatic vessel (LV) disorders have also been described in some CM-AVM patients. These include chylothorax and chylous ascites (accumulation of lymph in the pleural and peritoneal cavities, respectively), lymphedema, LV malformation, and hyperplasia (2)(3)(4)(5)(6). In 70% of CM-AVM patients, inherited inactivating mutations of the RASA1 gene are responsible for disease development.…”