TMEM16F Deficiency Causes a Strain-Dependent Lethality in Mice and Reduced Phosphatidylserine Exposure on Heterozygous As Well As Homozygous Deficient Mouse Platelets

Scott Syndrome is a rare bleeding disorder characterized by a defect in platelet phosphatidylserine (PS) exposure. The syndrome has recently been linked to mutations in TMEM16F, a Ca++-activated ion channel. Tmem16f^-/- mice were recently reported to be viable with a prolonged tail snip bleeding time but no spontaneous bleeding (Yang et al, Cell 151: 111-122; 2012). We now report analysis of an additional gene targeted Tmem16f allele generated in C57BL/6 ES cells.

JM8 ES cell were obtained from EUCOMM, and successful Tmem16f gene targeting in intron 1 was confirmed by PCR and sequencing. Genotyping of 120 Tmem16f^+/gt (+/gt) intercross progeny identified no surviving Tmem16f^gt/gt (gt/gt) mice at weaning (p<0.001). However, +/gt intercrosses generated the expected Mendelian genotype ratios at both E10.5 and E17.5, with gt/gt embryo’s exhibiting no morphological abnormalities on gross or routine histologic examination. Though complete deficiency of TMEM16F is lethal in the C57BL/6J genetic background between E17.5 and birth, an F2 intercross of +/gt mice outcrossed one generation to 129x1SvJ resulted in gt/gt mice surviving to weaning, though at reduced numbers (6/75 total progeny compared to ~19 expected, p <0.002). Progeny testing of surviving gt/gt mice suggest a single autosomal dominant 129x1SvJ-associated genetic modifier. Preliminary genetic analysis of these mice appears to map this locus to the proximal region of chromosome 3.

Tail bleeding times for gt/gt were >10min, whereas littermate +/gt and +/+ mice bleeding ceased at 8 ± 1 min and 6 ± 0.8 min, respectively, each significantly different than gt/gt (p<0.05). Notably, platelets from +/gt mice exhibited a trend toward reduced PS exposure, detected with FITC-labelled lactadherin, in response to PAR4 agonist peptide, whereas gt/gt mice had significantly reduced PS exposure (p < 0.05). gt/gt platelets showed a trend toward reduced PS exposure in response to A23187, as well as prolonged platelet rich plasma clotting times, and less efficient lactadherin inhibition of platelet clotting time.

These data suggest the existence of a viability-determining genetic modifier of TMEM16F in the 129x1SvJ mouse strain. Identification of the responsible gene may uncover novel functions for TMEM16F and the regulation of hemostatic function. The observation of a PS exposure and hemostatic phenotype in +/gt mice also suggests the possibility that heterozygous TMEM16F mutations may subtly influence hemostasis or thrombosis in humans.