The University of Arizona

ABNORMAL LYMPHATIC PHENOTYPE IN A CRISPR MOUSE MODEL OF THE HUMAN LYMPHEDEMA-CAUSING CONNEXIN47 R260C POINT MUTATION

D.J. Mustacich, R.I. Kylat, M.J. Bernas, R.J. Myles, J.A. Jones, J.D. Kanady, A.M. Simon, T.G. Georgieva, M.H. Witte, R.P. Erickson, P.W. Pires

Abstract


Connexin proteins form gap junctions
controlling exchange of ions and small molecules
between cells and play an important role
in movement of lymph within lymphatic vessels.
Connexin47 (CX47) is highly expressed in
lymphatic endothelial cells and CX47 missense
mutations, i.e., R260C, cosegregate with primary
lymphedema in humans. However, studies
utilizing CX47 knockout mice have failed
to demonstrate any lymphatic anomalies. To
unravel the lymphatic consequences of expressing
a mutant CX47 protein, we used CRISPR
technology to create a mouse carrying a Cx47
missense mutation (Cx47R259C) equivalent to
the human CX47R260C missense mutation
associated with human primary lymphedema.
Intradermal Evans Blue dye injection identified
a 2-fold increase in regional lymph nodes
in homozygous Cx47R259C mice compared to
wildtype, particularly in the jugular region (4.8
± 0.4 and 2.0 ± 0.0, respectively, p<0.01).
Associated lymphatic channels were increased
in Cx47R259C mice and mesenteric lymph
reflux occurred in homozygous Cx47R259C
mice but not in wildtype. Contractility of
superficial cervical lymphatics, assessed by
pressure myography, was reduced in homozygous
Cx47R259C mice compared to wildtype.
In conclusion, our data are the first to demonstrate
a role for the Cx47 protein in lymphatic
anatomy and function. This phenotype is similar
to that found with other valve deficient
mouse mutants, e.g., in Foxc2. Of significance,
this study is the first to use CRISPR technology
to develop a pre-clinical model of primary
lymphedema and demonstrates the importance
of distinguishing between lack of and presence
of mutant protein when developing clinically
relevant animal models for translation of
pre-clinical findings.


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