Homozygous Mutations in PHRF1 and PPARG in a Patient with Congenital Neutropenia and Profound Monocytosis: Understanding the Pathway Beyond Monocyte Versus Granulocyte Cell-Fate Choice

Myelopoiesis is a strictly regulated process of monocytes and granulocytes production, originating from common myeloid progenitors. Studies on patients with inherited severe congenital neutropenia (CN) could help to better elucidate myelopoiesis regulation. In CN, maturation arrest of granulocytic precursors at the promyelocytic stage leads to a shift towards monopoiesis and an ineffective granulopoiesis.

Hereby we report on a particular CN patient with a typical "maturation arrest" of granulopoiesis at the stage of promyelocytes, very low levels of neutrophils but extremely high levels of monocytes in the peripheral blood and bone marrow. Upon G-CSF treatment (5μg/kg/day) this patient produces high levels of monocytes (up to 24.8x10³/μL, more than 50 % of total leukocytes) and only up to 1x10³/μl neutrophils. Sanger sequencing of DNA from this patient revealed no mutations in CN-related genes such as ELANE, HAX1 and G6PC3. Therefore we performed whole genome sequencing (Complete Genomics. Inc, Mountain View, CA) of DNA from blood of this patient and his mother to discover causative gene mutations. We identified a homozygous deletion in PHRF1 (PHD and Ring Finger Domain-Containing Protein 1) (p.R1015-G1019, NP_065952.2; rs144630030) and a homozygous missense mutation in PPARG (Peroxisome proliferator-activated receptor gamma) (p.P12A, NP_0569553.2; rs1801282). Both mutations are heterozygote in the patient's mother. Population frequency for heterozygote allele of these two variations was reported to be 13% and 7%, respectively, but no homozygote variants were reported till date.

PHRF1 functions as an essential component of the TGF-ß tumour suppressor pathway by triggering degradation of the homeodomain repressor factor TGIF (TG-Interacting Factor) and a consequent retinoic acid signalling activation in haematopoiesis and monopoiesis. PPARG interacts with Retinoid X Receptors (RXR) and controls the expansion of macrophages.

In order to evaluate the functional role of the detected mutations on disturbed G-CSF-triggered myelopoiesis in reference CN patient, CD33⁺ bone marrow myeloid progenitor cells of two healthy controls and this patient were treated with G-CSF in vitro and mRNA expression profiles were analysed in an Affymetrix Microarray platform, followed by Ingenuity Pathway Analysis (IPA). We found, that 'TREM1 signalling' was among the top three pathways with most significant differences (p<9x10^-7) between this CN patient and healthy individuals. 'Granulocyte adhesion and diapedesis' and 'LXR/RXR pathway' were the next two significantly affected gene sets (p<8x10^-6 and p<1.3x10^-5, respectively).

TREM1 (Triggering Receptor Expressed on Myeloid cells 1) is a chemokine receptor that is expressed by neutrophils and monocytes, however the ligand that activates this receptor is yet unknown. TREM-1 is involved in neutrophil apoptosis and is known to positively regulate monopoiesis by activation of M-CSF synthesis. Intriguingly, M-CSF was 4.2-fold upregulated in myeloid cells of patient, in comparison to healthy individuals. Other known components of TREM 1 signalling were also among the top 10 differentially expressed genes identified by IPA: HSD11B1 (+20 fold), ATP1B2 (+14 fold) and THBS1 (-10 fold). Functional connections between PPARG and TREM1 is known. PPARG mutation could lead to TREM1 signalling activation that consequently lead to M-CSF over-expression (+4.2 fold).

In addition to the activation of TREM1 signalling, deletion in the PHFR1 gene could be the causative effect of marked upregulation of ALDH1A2 (+17 fold), which also could lead to an increase in M-CSF levels and in a retinoid acid signalling activation ultimately leading to increased monocyte production.

Together, PPARG and PHRF1 mutations could hyper-activate the secretion of M-CSF by myeloid progenitors leading to a strong shift towards monopoiesis upon G-CSF treatment.