Defects of mutant DNMT1 are linked to a spectrum of neurological disorders

Abstract

We report a broader than previously appreciated clinical spectrum for hereditary sensory and autonomic neuropathy type 1E (HSAN1E) and a potential pathogenic mechanism for DNA methyltransferase (DNMT1) mutations. The clinical presentations and genetic characteristics of nine newly identified HSAN1E kinships (45 affected subjects) were investigated. Five novel mutations of DNMT1 were discovered; p.C353F, p.T481P, p.P491L, p.Y524D and p.I531N, all within the target-sequence domain, and two mutations (p.T481P, p.P491L) arising de novo. Recently, HSAN1E has been suggested as an allelic disorder of autosomal dominant cerebellar ataxia, deafness and narcolepsy. Our results indicate that all the mutations causal for HSAN1E are located in the middle part or N-terminus end of the TS domain, whereas all the mutations causal for autosomal dominant cerebellar ataxia, deafness and narcolepsy are located in the C-terminus end of the TS domain. The impact of the seven causal mutations in this cohort was studied by cellular localization experiments. The binding efficiency of the mutant DNMT proteins at the replication foci and heterochromatin were evaluated. Phenotypic characterizations included electromyography, brain magnetic resonance and nuclear imaging, electroencephalography, sural nerve biopsies, sleep evaluation and neuropsychometric testing. The average survival of HSAN1E was 53.6 years. [standard deviation = 7.7, range 43-75 years], and mean onset age was 37.7 years. (standard deviation = 8.6, range 18-51 years). Expanded phenotypes include myoclonic seizures, auditory or visual hallucinations, and renal failure. Hypersomnia, rapid eye movement sleep disorder and/or narcolepsy were identified in 11 subjects. Global brain atrophy was found in 12 of 14 who had brain MRI. EEGs showed low frequency (delta waves) frontal-predominant abnormality in five of six patients. Marked variability in cognitive deficits was observed, but the majority of patients (89%) developed significant cognitive deficit by the age of 45 years. Cognitive function decline often started with personality changes and psychiatric manifestations. A triad of hearing loss, sensory neuropathy and cognitive decline remains as the stereotypic presentation of HSAN1E. Moreover, we show that mutant DNMT1 proteins translocate to the cytoplasm and are prone to form aggresomes while losing their binding ability to heterochromatin during the G2 cell cycle. Our results suggest mutations in DNMT1 result in imbalanced protein homeostasis through aggresome-induced autophagy. This mechanism may explain why mutations in the sole DNA maintenance methyltransferase lead to selective central and peripheral neurodegeneration.

Keywords: REM sleep behaviour disorder; narcolepsy; neurodegeneration; protein aggregation; sensory neuropathy.

Figure 1

Pedigrees of nine recently identified HSAN1E kindreds. Kindreds 1–4 and 6 have novel mutations in the TS domain of DNMT1, and four kindreds have mutations at Y495. Black symbols represent affected subjects. The age of death is marked under the deceased patients.

Figure 2

Imaging results. (A) Brain MRI in Patient III-2 from Kindred 7, a 54-year-old male with significant cognitive impairment and diffuse slowing on EEG: sagittal T₁ image showing global supra- and infra-tentorial brain atrophy (Philips, 1.5 T). (B) Transversal FLAIR image showing multiple white matter lesions and global atrophy (Philips, 1.5 T). (C) Morphological analysis of glutaraldehyde-fixed sural nerve biopsy tissue embedded in epoxy resin of Patient II-1 from Kindred 3: showing severely reduced numbers of myelinated nerve fibres. Semi-thin section, toluidine blue. Scale bar = 25 µm. (D) Remak bundles containing several bundles of collagen fibres encircled by Schwann cell processes (collagen pockets, indicated by arrowheads) indicative of unmyelinated axon loss. Electron microscopy of ultrathin section. Scale bar = 1 µm. (E) Twenty-four hours of continuous video-EEG monitoring in Patient II-2 from Kindred 1 showing irregular myoclonic movements associated with runs of higher voltage, irregular theta activity. (F) EEG in the same patient showing abnormal diffuse rhythmic delta frequency (1–4 Hz) background slowing especially in the bifrontal regions, correlated with a mild to moderate diffuse encephalopathy.

Figure 3

Targeting sequence (TS) domain structure with mutations of DNMT1 shown. The TS domain (grey) is the site for all known causal mutations for HSAN1E (red) and ADCA-DN (blue). Images were created using PyMol (www.pymol.org) and crystal structures of the human DNMT1 TS domain.

Figure 4

Representative images from confocal microscopy in HEK293 cells co-transfected with RFP-PCNA and GFP-wild-type DNMT1, or various mutant GFP-DNMT1. Wild-type and mutant DNMT1 appear in green in the left panels, PCNA appears in red in the middle panels and merged images are shown in the right panels. Scale bars = 5 µm. In the S phase, PCNA locates at the toroidal structures of the replication foci; in the G2 phase, PCNA shows a diffused pattern in the nucleus. Both wild-type DNMT1 and mutant DNMT1 proteins co-localize with PCNA at replication foci during the S phase. However, during G2 phase (when PCNA diffuses) wild-type DNMT1 stays in the nucleus and binds to heterochromatin while mutant DNMT1 proteins mislocated into cytoplasm and formed aggregates.

Figure 5

DNMT1 mutants. (A) Representative images from fluorescence aggresomes marker staining of HEK293 cells transfected with either enhanced GFP-wild type DNMT1 or various mutant enhanced GFP-DNMT1. Wild-type DNMT1 stays in the nucleus and binds to heterochromatin whereas mutant DNMT1 aggregates in cytosol overlaid with red fluorescence aggresome markers. Scale bars = 5 µm. (B) Quantification of GFP-DNMT1 proteins in HEK293 cells transfected with eGFP-wild type DNMT1, or various mutant enhanced GFP-DNMT1. All mutant DNMT1 proteins showed degradation comparing to wild-type DNMT protein. Data are derived from three independent experiments, asterisk represents there is statistically significant change between wild-type and mutant proteins.