Haploinsufficiency of CSF-1R and clinicopathologic characterization in patients with HDLS

Abstract

Objective: To clarify the genetic, clinicopathologic, and neuroimaging characteristics of patients with hereditary diffuse leukoencephalopathy with spheroids (HDLS) with the colony stimulating factor 1 receptor (CSF-1R) mutation.

Methods: We performed molecular genetic analysis of CSF-1R in patients with HDLS. Detailed clinical and neuroimaging findings were retrospectively investigated. Five patients were examined neuropathologically.

Results: We found 6 different CSF-1R mutations in 7 index patients from unrelated Japanese families. The CSF-1R mutations included 3 novel mutations and 1 known missense mutation at evolutionarily conserved amino acids, and 1 novel splice-site mutation. We identified a novel frameshift mutation. Reverse transcription PCR analysis revealed that the frameshift mutation causes nonsense-mediated mRNA decay by generating a premature stop codon, suggesting that haploinsufficiency of CSF-1R is sufficient to cause HDLS. Western blot analysis revealed that the expression level of CSF-1R in the brain from the patients was lower than from control subjects. The characteristic MRI findings were the involvement of the white matter and thinning of the corpus callosum with signal alteration, and sequential analysis revealed that the white matter lesions and cerebral atrophy relentlessly progressed with disease duration. Spotty calcifications in the white matter were frequently observed by CT. Neuropathologic analysis revealed that microglia in the brains of the patients demonstrated distinct morphology and distribution.

Conclusions: These findings suggest that patients with HDLS, irrespective of mutation type in CSF-1R, show characteristic clinical and neuroimaging features, and that perturbation of CSF-1R signaling by haploinsufficiency may play a role in microglial dysfunction leading to the pathogenesis of HDLS.

Figure 1. Identification of CSF-1R mutations, and mRNA and protein expressions of mutant CSF-1R

(A) Schematic illustration of CSF-1R structure. Six different mutations identified in this study are shown below the diagram of electropherograms. The tyrosine kinase domain of CSF-1R is shown in gray. Numbers represent exons in which mutations were identified. Positions of previously reported mutations are shown as triangles above the diagram. IVS = intervening sequence. (B) Sequencing electropherogram of amplified genomic DNA and reverse transcription PCR amplicons of patient with frameshift mutation (p.S688EfsX13), which was predicted to undergo nonsense-mediated mRNA decay. The expression of the mutant allele was hardly detectable, suggesting that this frameshift mutation results in nonsense-mediated decay of mutant mRNA. The predicted amino acid sequences followed by a stop codon are shown in red. (C) Immunoblot analysis of CSF-1R protein using anti-CSF-1R antibody (C-20) in Triton X-100 soluble fraction from frontal cortex of autopsied cases (c.2442+1G>T and p.S688EfsX13) and 4 control subjects (1–4) without neurologic disorder. Note that the full-length CSF-1R (150 kDa and 130 kDa, representing mature protein and immature full-length protein, respectively) and proteolytically cleaved C-terminal fragment of CSF-1R (55 kDa) showed markedly decreased expression levels. The equivalency of protein loading is shown in the actin blot (bottom).

Figure 2. Longitudinal MRI changes of patients with hereditary diffuse leukoencephalopathy with spheroids

(A) Sequential MRI studies of patient VI using fluid-attenuated inversion recovery (FLAIR) images. At the early stage of the disease, white matter hyperintensities were often found in the periventricular area, surrounding the anterior and posterior horns with a tendency to confluence, and in the fiber tract in the internal capsule. Enlargement of the lateral ventricles was also noticeable. Notably, the corpus callosum showed hyperintensities and thinning at the time of onset. The progression was relatively rapid and cortical atrophy became evident as the disease progressed. The MRI taken 5 years before the onset for the evaluation of headache showed subtle asymmetric white matter hyperintensities surrounding the anterior horns and faint signal changes and mild thinning of the corpus callosum. (B–E) Chronological changes in semiquantitative MRI scores in 7 patients with hereditary diffuse leukoencephalopathy with spheroids. MRI finding severity was evaluated from the total score (B, scores 0–57), which combines the white matter lesion (WML) score (C, 1–42) and the atrophy score (D, 0–13), and the presence of lesions in the thalamus and basal ganglia. (E) Correlation analysis between WML score and atrophy score.

Figure 3. Spotty calcifications in white matter on CT images

(A) Multiple lesions caused by calcifications in the brain as revealed by CT. The boxed area is enlarged at the right bottom of the panel. Small spotty calcifications were observed in the affected white matter. (B, C) Histopathologic findings of small lesions in frontal white matter close to the corpus callosum of patient VI carrying splice-site mutation. Calcium deposition and fibrillary gliosis were evident. (B) Hematoxylin & eosin, (C) von Kóssa reaction. Bar = 100 μm for B and C.

Figure 4. Histopathologic features of patients with hereditary diffuse leukoencephalopathy with spheroids

(A–C) White matter lesions of frontal lobe of patient VI. (A) Marked myelin loss of white matter with U-fibers spared. (B) Axonal spheroids (arrows) in white matter. (C) Abundant macrophages (arrowheads) in white matter. (D–L) Microglia in white matter of patients with hereditary diffuse leukoencephalopathy with spheroids and control brains. (D-H) Immunohistochemistry of Iba1 in degenerative white matter. (D, E) Patient VI. (F) Patient III. (G) Patient IHC1. (H) Patient IHC2. (D) The boxed area in (A) is enlarged. Spatially restricted appearance of Iba1-immunopositive activated microglia (upper right corner). (E–H) Characteristic features of microglia. (I–L) CSF-1R immunohistochemistry in degenerative white matter. (I) Patient VI. (J) Patient III. (K) Patient with Alzheimer disease. (L) Patient with adrenoleukodystrophy. Images in I and J were taken from serial sections of images in E and F, respectively. Note very faint or no CSF-1R immunopositivity in activated microglia in images I and J. (A) Klüver-Barrera staining, (B, C), hematoxylin & eosin staining, (D–L) immunohistochemistry of Iba1 (D–H) and CSF-1R (I–L). Bar = 7 mm for A, 33 μm for B and C, 306 μm for D, 50 μm for E, F, and I–L, and 25 μm for G and H.