Posted October 23, 2015
Marshall S. Horwitz, M.D., Ph.D., University of Washington
With support from a 2012 Department of Defense Bone Marrow Failure Research Program Idea Award, Dr. Marshall Horwitz is making major breakthroughs in understanding the genetic background of severe congenital neutropenia (SCN), a rare and serious hematological condition that causes affected individuals to be prone to recurrent infections. Inherited forms of neutropenia often progress to myelodysplasia, resulting in generalized bone marrow failure. Heritable mutations in several genes can cause neutropenia, although most often ELANE, the gene that encodes the protease neutrophil elastase, is responsible. Other mutations are known such as those in the HAX1 gene may lead to neutropenia as well, but it remains unclear how mutations in neutrophil elastase or other genes can cause neutropenia.
In order to investigate the role of neutrophil elastase in SCN, Dr. Horwitz and colleagues identified a particular subset of SCN patients that all had mutations disrupting the canonical translational start-site of ELANE. Dr. Horwitz hypothesized that, instead of preventing translation of neutrophil elastase, mutations of ELANE's translational start site may result in translation commencing from an internal start site within the ELANE gene (i.e., alternative ATG sites further downstream that satisfy the minimal requirements for initiation of translation). Through a series of elegantly planned and well-executed experiments to test this hypothesis, Dr. Horwitz and his team at the University of Washington demonstrated that mutations inactivating the translation start site of ELANE caused translation to start from a downstream in-frame initiation start site, resulting in the production of truncated neutrophil elastase isoforms. Specifically, Dr. Horwitz found that these truncated isoforms mislocalize in vitro, probably due to the lack of localizing signal sequences. The mislocalization of neutrophil elastase to the nucleus likely contributes to the pathogenicity of ELANE start site mutations. Even though the truncated isoforms only retain minimal proteolytic activity, residual activity could be damaging to the cell, if not properly compartmentalized. These observations suggest that drugs inhibiting neutrophil elastase enzymatic activity could ultimately prove therapeutic for neutropenia.
Currently, Dr. Horwitz is using patient-derived induced pluripotent stem cell (iPSC) models to study whether other types of ELANE mutations lead to translational expression of incorrectly translated neutrophil elastase. In addition to studying ELANE mutations, Dr. Horwitz is also using iPSC models to study the HAX1 gene. Mutations in the HAX1 gene can also cause neutropenia and Dr. Horwitz is interested in investigating whether mutations in the HAX1 are responsible for regulating the production of neutrophil elastase. Murine models have largely failed to recapitulate key clinical features of inherited forms of neutropenia; therefore, iPSC models offer an exciting and relevant new tool for understanding these diseases, as well as potentially serving as a platform for gene-correction therapies. Myelopoiesis is the process by which innate immune cells develop from myeloid precursors and Dr. Horwitz has successfully developed and validated several patient-derived iPSC myelopoiesis models. Dr. Horwitz has been able to demonstrate that the stem cells in his models faithfully recapitulate the molecular, genetic, and cellular features naturally occurring in the bone marrow, making these models excellent candidate platforms from which to study neutropenia and other forms of hereditary blood disorders.
Taken together, Dr. Horwitz's work has illuminated unconventional ways through which coding sequence mutations may have unexpected effects on translation. Given the large volume of "variants of undetermined significance" now pouring in from whole genome and whole exome sequencing studies, Dr. Horwitz's novel models and results help establish a new paradigm for the evaluation of synonymous variants across disease states.
Tidwell T, Wechsler J, Nayak RC, Trump L, Salipante SJ, Cheng JC, Donadieu J, Glaubach T, Corey SJ, Grimes HL, Lutzko C, Cancelas JA, and Horwitz MS. 2014. Neutropenia-associated ELANE mutations disrupting translation initiation produce novel neutrophil elastase isoforms. Blood 123(4):562-569. doi: 10.1182/blood-2013-07-513242. Epub 2013 Nov 1.
Borregaard N. 2014. Comment--Severe congenital neutropenia: New lane for ELANE. Blood 123(4):462-463. doi: 10.1182/blood-2013-11-537068. PubMed PMID: 24458273.
Nayak RC, Trump LR, Aronow BJ, Myers K, Mehta P, Kalfa T, Wellendorf AM, Valencia CA, Paddison PJ, Horwitz MS, Grimes HL, Lutzko C, and Cancelas JA. 2015. Pathogenesis of ELANE-mutant severe neutropenia revealed by induced pluripotent stem cells. Journal of Clinical Investigation 125(8):3103-31016.