Myh9
Q1443L Is a Novel Mouse Model of MYH9-Related Disorders
Authors: Anderson Nicole M , Berndl Elizabeth , Berberovic Zorana , Reheman Adili , Brown Trecia A , Bailey Monica L , Flenniken Ann M , Osborne Lucy R , Adamson S. Lee , Rossant Janet , Wang Chen , Paulson Robert F , McNagny Kelly , Minden Mark D. , Quaggin Susan E , Harrison Robert V , Ni Heyu , McPherson John D , Stanford William L. , Barber Dwayne L 
Published: October 2019

Abstract
Abstract 2527
May-Hegglin Anomaly was originally defined as patients with macrothrombocytopenia. It is now recognized that the phenotypes of May-Hegglin Anomaly and the related syndromes described by Epstein, Fechtner and Sebastian overlap comprising macrothrombocytopenia, neutrophil inclusions, deafness, cataract formation and nephritis. These disorders are defined by mutations in the non-muscle myosin heavy chain IIA gene, MYH9 and have been termed MYH9-Related Disorders.
Myh9 is required during murine embryogenesis as Myh9-null embryos die of patterning defects. Megakaryocyte-specific deletion of Myh9 has been performed by the Gachet group and Shivdasani and colleagues examined megakaryocyte function in Myh9-deficient embryonic stem cells induced to differentiate in vitro. The phenotypes of these mice include increased bleeding times, absence of clot retraction and defective Integrin β3 phosphorylation coupling to impaired activation of downstream Rho-Rock signalling and lamellipodia formation. To date, no knock-in MYH9 alleles corresponding to MYH9-related Disorder mutations have been reported. MYH9-related Disorders frequently are misdiagnosed as Chronic Immune Thrombocytopenia, so having enhanced knowledge of the molecular etiology of mutant MYH9 alleles is a priority.
Our group has been interested in utilizing random mutagenesis to identify novel alleles of human hematopoietic disorders. We performed a dominant screen in which 129 male mice were mutagenized with ENU and back-crossing was performed on the C57BL/6 background. We identified a heritable line (7238) with macrothrombocytopenia that mapped to a 4.25 Mb region on mouse Chromosome 15. Due to our inability to further refine the interval through back-crossing, we performed high throughput sequencing by comparing SNPs between 129, C57BL/6, 7238/+ and 7238/7238 mice. We identified 18 novel variations, including 1 that corresponded to a coding change - Myh9 Q1443L. Subsequent Sanger sequencing and back-crossing eliminated the remaining 17 variations, confirming our assignment of the 7238 phenotype to a mutation of Myh9. The Myh9 Q1443L allele is within proximity of the MYH9 D1424Y/N/H mutations found within clinical specimens.
Myh9 Q1443L/Q1443L mice are viable allowing us to examine the effect of mutation of one or both alleles of Myh9. Myh9Q1443L/Q1443L mice had increased bleeding times, whereas Myh9Q1443L/+ mice were identical to wild type mice. Platelet aggregation experiments were performed after ADP or thrombin stimulation. Decreased aggregation was observed in a dosage-dependent manner, with Myh9Q1443L/+ mice displaying intermediate levels when compared with Myh9Q1443L/Q1443L co-hort. Adherence and aggregation of Myh9Q1443L/Q1443L platelets to a collagen matrix was decreased at shear rates of 350/s and 1800/s. Staining of Myh9Q1443L/+ and Myh9Q1443L/Q1443L neutrophils also revealed neutrophil inclusions.
In addition to the hematopoietic phenotypes, we have observed increased cataract formation with a frequency of 36% in wild type animals, 60% in Myh9Q1443L/+ and 92% in Myh9Q1443L/Q1443L mice. Sporadic hematuria and proteinuria is found in Myh9Q1443L mice. We are currently performing albumen loading and lipopolysaccharide activation experiments to determine whether 7238 mice have altered response to nephric stress. We are crossing 7238 mice to the DBA genetic background, which is more sensitive to subtle kidney phenotypes. We performed auditory brain response measurements on aged wild type, Myh9Q1443L/+ and Myh9Q1443L/Q1443L mice and observed no differences in frequency responses ranging from 2 to 24kHz. Myh9Q1443L/+ mice displayed hypertrophy of the preputial gland whereas Myh9Q1443L/Q1443L males showed atrophy of this gland and Myh9Q1443L/Q1443L male mice have reduced fertility. No differences in fertility have been described to date in MYH9 related disorders.
In conclusion, Myh9Q1443L/Q1443L mice display macrothrombocytopenia, neutrophil inclusions and cataract formation described in MYH9-related disorders and represent the first animal model of May-Hegglin Anomaly.

Disclosures:
No relevant conflicts of interest to declare.