Heterozygous Mutations of Clpb As a Newly Identified and Frequent Cause of Severe Congenital Neutropenia

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. Although many cases of SCN are associated with mutations in ELANE, encoding the neutrophil elastase, roughly one-third of cases do not have an identifiable genetic cause. In collaboration with the Severe Chronic Neutropenia International Registry (SCNIR), we performed exome sequencing on 90 cases of congenital neutropenia. Heterozygous missense mutations of CLPB were identified in six patients with SCN. None of these patients had mutations in other genes known to cause SCN. A total of 5 different mutations were identified that clustered within the ATPase domain. Of note, all of these mutations were predicted to be functionally deleterious and had a frequency of <0.002% in the ExAC and gnomAD databases. We subsequently identified heterozygous CLPB mutations in an additional 3 cases of SCN that were not part of our original cohort.

Prior studies showed that biallelic mutations of CLPB are associated with a syndrome defined by 3-methylglutaconic aciduria (3-MGA), cataracts, neurologic disease, and variable neutropenia. In our original cohort of 6 SCN patients with heterozygous CLPB mutations, 3-MGA was not present in the three cases where urine samples were available and none had reported cataracts. In total, 3 had neurologic abnormalities (2 seizures and 1 developmental delay). Of note, the CLPB mutations present in syndromic cases were distinct from those seen in our SCN cohort, and none of the mutations in our series are biallelic. CLPB encodes for caseinolytic peptidase B, a protein implicated in protein folding in bacteria and yeast but with an unknown role in human granulopoiesis. Based on these observations, we hypothesize that CLPB is required for normal basal granulopoiesis and that the heterozygous CLPB mutations identified in our study act in a dominant-negative fashion to disrupt granulopoiesis.

To test this hypothesis, two complementary genetic approaches were employed. First, we used CRISPR-Cas9 gene editing to generate null mutations in CLPB in human cord blood-derived CD34+ hematopoietic stem/progenitor cells (HSPCs). Using this approach, we are able to achieve greater than 90% editing efficiency as assessed by next generation sequencing. The genetically modified HSPCs were cultured for 14 days under conditions that promote granulocytic differentiation. Modified HSPCs were also seeded into methylcellulose cultures to measure CFU-G. The percentage and absolute number of mature granulocytes, but not early granulocytic precursors (promyelocytes and myelocytes), were significantly reduced in cultures of gene-edited cord blood CD34+ cells. Moreover, the frequency of edited cells decreases over time in our culture system indicating that CLPB-knockout cells have a competitive disadvantage. A significant decrease in CFU-G also was observed. Second, we generated lentivirus expressing all 5 of the neutropenia-associated heterozygous CLPB mutations identified in our SCN cohort (N496K, E557K, R561G, R603H, and R620C). Expression of all of these mutants (except R603H) in cord blood-derived CD34+ cells was associated with a significant decrease in mature neutrophils and corresponding increase in early granulocytic precursors. These four CLPB mutants also resulted in a decrease in CFU-G. Collectively, these data strongly suggest that heterozygous mutations of CLPB are a new cause of congenital neutropenia. Indeed, in the North American population, CLPB mutations appear to be the second most common cause of congenital neutropenia, behind ELANE mutations. Studies are underway to examine the molecular mechanisms by which mutant CLPB disrupts granulopoiesis.