-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

2533 Heterozygous Variants in the Clpb gene Associated with Neutropenia Affect Neutrophil Function

Program: Oral and Poster Abstracts
Session: 201. Granulocytes, Monocytes, and Macrophages: Poster II
Hematology Disease Topics & Pathways:
Diseases, Immune Disorders, neutropenia
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Weronika Dobrewa, MSc1*, Justyna Folkert, PhD2*, Stanisław Malicki, PhD2*, Barbara Chruscicka-Smaga, PhD2*, Danuta Bryzek, PhD2*, Ewelina Dobosz, PhD2*, Iwona Dachowska-Kalwak, MD, PhD3*, Krzysztof Kalwak, MD, PhD4*, Marcin Hennig, MD, PhD5*, Joanna Renke, MD, PhD5*, Katarzyna Babol-Pokora, PhD6*, Zietkiewicz Szymon, PhD7*, Joanna Koziel, PhD2*, Zuzanna Rydzynska, MSc6*, Wojciech Mlynarski, MD, PhD6*, Jan Potempa, PhD8*, Veillard Florian, PhD2* and Joanna Madzio, PhD6*

1Department of Pediatrics, Oncology and Hematology, Medical University of Lodz, Lódz, Poland
2Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
3Clinical Department of Paediatric Bone Marrow Transplantation, Oncology and Haematology, Wroclaw Medical University, Wroclaw, Poland
4Department of Pediatric Bone Marrow Transplantation, Oncology, and Hematology, Wroclaw Medical University, Wroclaw, Poland
5Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland
6Department of Pediatrics, Oncology and Hematology, Medical University of Lodz, Lodz, Poland
7Intercollegiate Faculty of Biotechnology, Biotechnology University of Gdansk and Medical University of Gdansk, Gdansk, Poland
8Department of Oral Immunology and Infectious Diseases, School of Dentistry, University of Louisville, Louisville, KY


The CLPB gene encodes a caseinolytic peptidase belonging to the ATPase family and acts as a mitochondrial chaperone. While biallelic mutations in CLPB are associated with the 3-methylglutaconic aciduria and severe neutropenia, (MIM number: 616271 3), some heterozygous variants seem to induce neutropenia exclusively. In the current study, we evaluated for the first time a function of neutrophils among neutropenic carriers of CLPB mutation.


Among 282 individuals with neutropenia studied with targeted Next-Generation Sequencing (panel of 725 genes related to hematological disorders), three neutropenic probands with heterozygous CLPB amino acid substitutions were identified. Neutrophils from patients and control healthy donors were isolated from peripheral blood with a Pancoll density gradient. The percentages of freshly isolated neutrophils undergoing apoptosis or necrosis were determined by flow cytometry using Annexin V - FITC and Propidium Iodide (PI) staining. Phagocytic capacity was evaluated using pHrodo Green S. aureus BioParticles Conjugates. A Boyden’s chamber model was used to determine the level of the neutrophil’s mobility and chemotaxis response to 10% human serum. Formation of Neutrophils Extracellular Traps (NETs) and induction of the oxidative burst were studied upon phorbol 12-myristate 13-acetate (PMA) stimulation (25 nM) with SYTOX™ Green Nucleic Acid and flow cytometry with CellROX Green assay, respectively. The intracellular and circulating concentrations of anti-microbial compounds (Myeloperoxidase MPO, Lactotransferrin LTF, Cathelicidin-LL37, and defensins) were measured using commercially available ELISA assays. The results were evaluated with an unpaired t-test using GraphPad Prism 9. Quantitative data presented as medians (25%-75%).


Genetic analysis identified three heterozygous variants of CLPB gene p.Arg327Trp, p.Arg628Cys, p.Arg629Cys in three different families. Two of these mutations were novel p.Arg327Trp and p.Arg629Cys. Interestingly, the p.Arg327Trp variant was located outside the ATPase domain. In total 7 family members who suffered from neutropenia and were carriers of the CLPB variants were available for neutrophil functional assays (Figure 1).

Neutrophils collected from CLPB-deficient individuals have a higher rate of apoptosis as compared to healthy neutrophils: 57(52-66)% vs 23(20-27)%, P<0.0001, and necrosis: 0.56(0.31-1.07)% vs 0.13(0.10-0.21)%, P=0.02. CLPB-deficient neutrophils display reduced mobility in the absence of stimulation: 298(191-413) vs 1011(936-1201) migrating neutrophils, P<0.0001, and after 10% human serum treatment: 2090(1746-2458) vs 3736(3071-4828) migrating neutrophils, P=0.0014. Similarly, reduced phagocytosis of S. aureus bioparticles was observed in CLPB-deficient neutrophils: 1890(1679-2063) vs 3352(2472-4440) Fluorescence Units, respectively, P=0.0029. Upon PMA stimulation, neutrophils from CLPB patients produced less reactive oxygen species compared to controls: 311(175-542)FMI vs 775(766-1150)FMI, P=0.08, and their ability to produce NETs is almost entirely abolished: 0.5(0.21-0.79)ng/µl vs 1.3(1.08-3.05)ng/µl, P=0.0075. Moreover, CLPB-deficient neutrophils have reduced intracellular concentration of myeloperoxidase: 35(31-39)ng/mg vs 55(41-63)ng/mg, P=0.0043 and cathelicidin LL-37: 5.5(5-6.3)ng/µg vs 19.22(14.21-23.28)ng/µg, P=0.0003. The concertation of the LL-37 is also reduced in patients’ plasma 1.34(0.84-1.78)ng/ml vs 7.17(5.38-8.03)ng/ml, P<0.0001. Both intracellular and circulating concentrations of LTF and defensins did not differ between study groups.


Neutrophils from neutropenic carriers of CLPB mutations are functionally impaired, especially concerning their ability for NETs formation and cathelicidin LL-37 production.

Disclosures: Dachowska-Kalwak: Neovi: Other: travel grant. Kalwak: medac: Speakers Bureau; Novartis: Speakers Bureau; Pierre Fabre: Other: travel grants, Speakers Bureau. Mlynarski: Novartis: Membership on an entity's Board of Directors or advisory committees. Potempa: PerioTrap Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Naional Institutes of Health: Research Funding.

Previous Abstract | Next Abstract >>
*signifies non-member of ASH