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.

Keywords: CM-AVM; indocyanine green imaging; lymphatics; near-infrared fluorescence imaging.

Fig. 1.

Clinical photographs of the patient and his father. (A) Symmetrical overgrowth of left lower extremity with pale red cutaneous capillary stain. (B) Patient’s left foot demonstrates swelling with lymphatic vesicles on digits (arrows). (C) Patient’s right hand demonstrates capillary stain typical of CM-AVM (RASA1 mutation). (D) Father’s left lower extremity shows typical CM. Note pale margin.

Fig. 2.

Dorsal (A) and medial (B and C) views of stain on left leg with circles indicating fields of view for corresponding dorsal (D) and medial (E and F) views from NIRFLI.

Fig. 3.

(A–C) Typical NIRFLI depicting the normal collecting lymphatics on the dorsum of the foot of a 48-y-old female (Movie S1), medial views of ankle/calf of a 47-y-old male (Movie S2), and the knee of a 43-y-old male (Movie S3). (D–F) NIRFLI on the right, unaffected leg of the PKWS subject depicting lymphatics on the dorsum of the foot and medial views of dilated lymphatic vessels in the ankle/calf and knee. See Movies S4, S5, and S6 for sluggish propulsive flow.

Fig. 4.

ICG injection sites on the medial ankle (A), lateral calf (B), and thigh (C) on the left affected limb showing hyperplasia of presumed initial lymphatics. To prevent oversaturation of the camera system, the injection sites are covered with an adhesive bandage.

Fig. 5.

(A) NIRFLI montage of right (unaffected) and left (affected) limbs. (B) NIRFLI montage showing abnormal lymph pooling (arrows) in right unaffected limb. (C) NIRFLI montage showing abnormal lymphatic on the lateral side of the affected limb (Movie S7). (D) NIRFLI overlaid on white light to show lymphatic drainage into groin and pelvic region with lymphoceles displaying bright fluorescence (Movie S8).

Fig. 6.

Radiographic imaging of collecting lymphatic channels of left extremity. Injection of water-soluble contrast medium after ultrasound guided cannulation of dilated lymphatic channel in the left foot demonstrates marked dilation of lymphatic channels in the foot (A) and in the left lateral calf (B). (C) Injection of oily contrast medium (Lipiodol) after cannulation of scrotal lymphatic channel with sonographic guidance is shown (note dilated lymphatic channels and retrograde filling of lymphatic cyst). (D) Pelvis after injection of additional Lipiodol through more proximal lymphatic channel; left pelvic lymphatics are dilated with reflux (contrast filled syringe is visible). (E) image of abdomen after opacification of cisterna chyli (arrow), which is normal in size. Thoracic duct was not well visualized.

Fig. 7.

(A) Normal lymphatic vasculature of a Rasa1^fl/fl mouse 26 s after ICG administration at the base of the tail (Movie S9). (B) Abnormal lymphatic architecture of inducible Rasa1 knockout 117 s after ICG administration at the base of the tail (Movie S10). The phenotype occurs months before onset of chylous ascites and chylothorax (8). Results shown are typical of n= 10 Rasa1^fl/fl and n = 10 Rasa1^fl/flUb^ert2cre mice. (C) Ventral view showing hyperplasia in lower abdominal region and leaked ICG in the gut (arrow), indicating chylous ascites (Movie S11).