Identification of new genetic causes of congenital neutropenia

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The long-term goal of this of this research is to identify new genetic causes of congenital neutropenia and characterize their molecular mechanisms of disease pathogenesis. Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by severe chronic neutropenia from birth, premature death secondary to infectious complications, and transformation to myeloid malignancy. Approximately one-third of cases do not have a known genetic cause. We performed whole exome sequencing of 85 cases of congenital neutropenia. Heterozygous missense mutations of CLPB, encoding caseinolytic peptidase B, were identified in 6 of 45 (13%) ELANE-negative SCN cases; we subsequently identified heterozygous CLPB mutations in an additional 3 cases of SCN that were not part of our original cohort. CLPB encodes for caseinolytic peptidase B, an ATPase implicated in protein folding and mitochondrial function. Prior studies showed that biallelic mutations of CLPB are associated with a syndrome of 3-methylglutaconic aciduria, cataracts, neurologic disease, and variable neutropenia. However, these mutations are distinct from those seen in our series, which are heterozygous and cluster near the ATP binding pocket. Preliminary data show that CRISPR-Cas9 gene editing to inactivate CLPB or lentiviral-mediated overexpression of mutant CLPB in human CD34+ cells results in impaired granulocytic differentiation. Collectively, these data suggest that heterozygous mutations of CLPB are a new and relatively common cause of SCN. In this proposal, we will examine molecular mechanisms by which mutant CLPB disrupts granulopoiesis. We also will identify and biologically validate, using a similar approach, other potential neutropenia-causing gene mutations identified by current and ongoing sequencing of congenital neutropenia cases. The following specific aims are proposed. Aim 1: To define the spectrum of CLPB mutations that disrupt granulopoiesis. We will use lentiviral- mediated overexpression in human CD34+ cells to systematically assess the impact on granulopoiesis of CLPB mutations identified in patients with congenital neutropenia, including the biallelic mutations found in syndromic cases. Aim 2. To characterize mechanisms by which mutations of CLPB disrupt granulopoiesis. We will test the hypothesis that mutant CLPB acts in a dominant fashion to disrupt the chaperone function of CLPB, resulting in impaired activation of the mitochondrial unfolded protein response (UPRMT) and induction of apoptosis in granulocytic precursors. Aim 3. To identify and validate new genetic causes of congenital neutropenia. We will perform exome sequencing of at least 100 additional cases of congenital neutropenia with no known cause. These data will be interrogated to identify candidate genes for functional validation.