Functional characterization of a novel CSF1R mutation causing hereditary diffuse leukoencephalopathy with spheroids

Abstract

Background: Colony-stimulating factor 1 receptor is a tyrosine kinase transmembrane protein that mediates proliferation, differentiation, and survival of monocytes/macrophages and microglia. CSF1R gene mutations cause hereditary diffuse leukoencephalopathy with spheroids (HDLS), an autosomal-dominantly inherited microgliopathy, leading to early onset dementia with high lethality.

Methods: By interdisciplinary assessment of a complex neuropsychiatric condition in a 44-year old female patient, we narrowed down the genetic diagnostic to CSF1R gene sequencing. Flow cytometric analyses of uncultivated peripheral blood monocytes were conducted sequentially to measure the cell surface CSF1 receptor and autophosphorylation levels. Monocyte subpopulations were monitored during disease progression.

Results: We identified a novel heterozygous deletion-insertion mutation c.2527_2530delinsGGCA, p.(Ile843_Leu844delinsGlyIle) in our patient with initial signs of HDLS. Marginally elevated cell surface CSF1 receptor levels with increased Tyr723 autophosphorylation suggest an enhanced receptor activity. Furthermore, we observed a shift in monocyte subpopulations during disease course.

Conclusion: Our data indicate a mutation-related CSF1R gain-of-function, accompanied by an altered composition of the peripheral innate immune cells in our patient with HDLS. Since pharmacological targeting of CSF1R with tyrosine kinase inhibitors prevents disease progression in mouse models of neurodegenerative disorders, a potential pharmacological benefit of CSF1R inhibition remains to be elucidated for patients with HDLS.

Keywords: DNA; clinical diagnostics; disease; gene; molecular biology; mutation.

Figure 1

Magnetic resonance imaging (MRI) of the brain. (a) Initial diffuse white matter lesions and gliosis at the age of 43 years. (b) Follow‐up MRI at age of 46 years showing increasing diffuse white matter lesions and progression of frontotemporal cerebral atrophy. (c) Diffusion weighted imaging (DWI) of the follow‐up examination revealing various isolated spots of hyperintense signal as sign of diffusion restriction and characteristic imaging feature for HDLS

Figure 2

Family pedigree. The accumulation of different neurological disorders over three generations reflects an autosomal dominant inheritance pattern with phenotypic variability. (I:2) polyneuropathy, kidney disease, and diabetes mellitus; (II:1) multiple cerebral infarctions; (II:2) mobilization with walking frame, history of thrombosis, and alcohol abuse; (II:6) aphasia and paresis after cerebral infarction at age of 38; alcohol abuse, smoker, elevated arterial blood pressure; (II:9) clinical diagnosis of Parkinson due to gait disturbance, aphasia, epilepsy; (II:10) cerebral infarction; (III:6) index patient

Figure 3

Molecular etiology. (a) Result of the genetic analysis of the patient revealing the heterozygous sequence variant c.2527_2530delinsGGCA, p.(Ile843_Leu844delinsGlyIle) in exon 19 of the CSF1R gene (NM_005211.3). (b) Mutation map of previously described CSF1R mutations. (c) Protein structural analysis of the CSF1R region containing the tyrosine kinase domain comparing wild‐type and mutated state

Figure 4

Peripheral monocyte subtype distribution and CSF1R surface expression and constitutive intracellular phospo‐CSF1R in HDLS. Gender‐ and age‐matched healthy donors (HD) were used for comparison (n = 5). HDLS patient blood was obtained at three time points. Density gradient‐derived PBMC were used for analysis. (a) Example FACS plots are given for CD14^high and CD14^interm monocyte distribution and for surface CSF1R expression divided for monocyte subpopulations (CD14^high and CD14^interm) for HDLS and HD. (b) Summary of monocyte subpopulation of CD14^interm is given as combined values or as single values for the HDLS patient over time. (c) Surface expression of CSF1R on CD14^high and CD14^interm expressing monocytes was measured and is plotted as median intensity. (d) Intracellular phospho‐CSF1R expression on total CD14 monocytes upon fixation and methanol permeabilization was analyzed directly from PBMC ex vivo. Delta median intensity values with background subtraction are given. Data are obtained by flow cytometry and cells of (a–c) are gated on live cells by propidium iodide exclusion. Floating bars from Min to Max are given and significance refers to p < 0.05