Patients with severe congenital neutropenia (SCN, also known as Kostmann syndrome) often die in childhood because they fail to produce adequate numbers of a type of white blood cell, neutrophils, important in defending against bacterial and fungal infections. Those patients who do not succumb to infectious complications unfortunately frequently develop myelodysplasia (MDS) or acute myeloid leukemia (AML). While there are several different genes that can cause SCN, most cases are caused by mutations in the gene, ELANE (formerly known as ELA2), whose product is a protease, neutrophil elastase, responsible for degrading other proteins. Dozens of different mutations have been identified in hundreds of different SCN patients. However, it is not known how the various different mutations in neutrophil elastase cause the same disease, because each mutation has a different effect on the function of the protein. Less commonly, mutations in a different gene, HAX1, can also cause SCN. It is presently not understood if neutrophil elastase and HAX1 interact with each other or how mutations in these two different genes produce the same disease.

We recently identified several SCN patients with new types of ELANE mutations that may provide a window into the pathogenesis of this disorder. This new category of mutations affects the initial sequence of the gene involved in signaling "translation" of the protein. The effect of these mutations is to produce smaller forms of the protein that initiate from more distant points in the gene and that therefore lack some of the features found in the beginning of the protein, including signals directing neutrophil elastase's localization within the cell and preventing premature activation of its protease activity. At the same time, new work from other laboratories has shown that HAX1, in addition to other functions, can bind messenger RNA molecules, which function as a working copy of the gene before it is translated into a protein. We have found that HAX1 protein actually binds to ELANE messenger RNA and that HAX1's normal function is to promote the faithful initiation of protein synthesis from the usual start site. Mutations in either HAX1 or in ELANE disrupt the interaction between these two molecules and also lead to production of the smaller forms of neutrophil elastase. We hypothesize that different mutations in ELANE and HAX1 may actually all do the same thing: promote the production of a shorter form(s) of neutrophil elastase that has toxic effects on the cells in the bone marrow that produce neutrophils.

We propose to identify the messenger RNAs produced from other genes that are also bound by HAX1 and determine whether they contain conserved sequences or structure and share similar functions in regulating cell growth. We aim to determine if the many different mutations found in ELANE and HAX1 among different patients with SCN all have the common effect of producing this shorter form of neutrophil elastase. We then plan to characterize the biochemical properties of this short form of neutrophil elastase and determine how it is toxic to the cell toward an ultimate goal (beyond the scope of this proposal) of targeting a drug against it.

Much of the talk in medicine, particularly cancer, now focuses on personalized theory, in which drugs are developed to target unique mutations particular to individual patients. Here we take a different, reductionist approach. We postulate that, in the case of SCN, even though most patients will have different particular mutations in one of two different genes (ELANE and HAX1), the effect of many of these mutations is actually the same. Rather than disturb the sequence of the protein encoded by ELANE, many of the different mutations may actually lead to production of a short form of neutrophil elastase that is particularly toxic to the cell. Therefore, if the properties of the short form of neutrophil elastase can be defined, then a single drug may be able to specifically target disease in many different patients, regardless of their underlying mutation in ELANE or HAX1.