New phenotype of DCTN1-related spectrum: early-onset dHMN plus congenital foot deformity.

OBJECTIVE: To describe the clinical and genetic features of two patients with different phenotypes due to various Dynactin 1 (DCTN1) gene mutations and further explore the phenotype-genotype relationship. METHODS: Patient 1 is a 23-year-old man with congenital foot deformity and life-long distal muscle weakness and atrophy. Patient 2 is a 48-year-old woman with adult-onset progressive weakness, lower limbs atrophy, and pyramid bundle signs. Electrophysiology test showed normal nerve conduction velocity of both patients and neurogenic changes in needle electromyography. Open sural nerve biopsy for Patient 1 showed slight loss of myelinated nerve fibers. Both patients were performed with whole-exome sequencing followed by functional study of identified variants. RESULTS: Two mutations in DCTN1 gene were identified in Patient 1 (c.626dupC) and Patient 2 (c.3823C>T), respectively. In vitro, the wild type mostly located in cytoplasm and colocalized with α-tubulin. However, c.626dupC tended to be trapped into nuclear and the c.3823C>T formed cytoplasmic aggregates, both losing colocalization with α-tubulin. Western blotting showed a truncated mutant with less molecular weight of c.626dupC was expressed. INTERPRETATION: We identify two novel DCTN1 mutations causing different phenotypes: (1) early-onset distal hereditary motor neuropathy plus congenital foot malformation and (2) amyotrophic lateral sclerosis, respectively. We provide the initial evidence that foot developmental deficiency probably arises from subcellular localizing abnormality of Dynactin 1, revealing DCTN1-related spectrum is still expanding.

Primary familial brain calcification presenting as paroxysmal kinesigenic dyskinesia: Genetic and functional analyses.

BACKGROUND: Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder characterized by calcium deposition in bilateral and symmetric brain. Evidence suggested that PFBC might be associated with paroxysmal kinesigenic dyskinesia (PKD). We aim to investigate the genetic causes in PFBC patients manifested as PKD, and further to explore the pathogenic impact of the identified mutations. METHODS: 4 PKD-mimic PFBC patients were investigated in the study. Clinical assessment including laboratory tests, head computed tomography (CT) were conducted and followed by exome sequencing. Variants of PFBC genes were screened, and Sanger sequencing, segregation analysis were applied to confirm the findings. Functional assessment of the identified mutations was further analyzed. RESULTS: Among the 4 PKD-mimic PFBC patients, 3 presented with brain calcification, and 1 was identified carrying a PFBC mutation but without brain calcification. The clinical characteristics were summarized. Three heterozygous variants (2 novel, 1 documented) in PFBC genes were found. Further functional study showed abnormal accumulation and reduced uptake of Pi of the mutant protein, and the aggregated PDGFB failing to induce membrane ruffles compared with wild-type. CONCLUSIONS: PKD can be a manifestation of PFBC, and brain calcification may be a cause of secondary PKD. So thoroughly evaluation including head CT or genetic screening for paroxysmal dyskinesia and PFBC should be applied before the diagnosis of PKD or PFBC. Moreover, negative brain calcification may not exclude the possibility of PFBC. The possible pathogenesis of primary calcification lie in the dysfunction of the protein or defective signal transduction caused by the mutations.

Clinicopathologic characterization and abnormal autophagy of CSF1R-related leukoencephalopathy.

BACKGROUND: CSF1R-related leukoencephalopathy, also known as hereditary diffuse leukoencephalopathy with spheroids (HDLS), is a rare white-matter encephalopathy characterized by motor and neuropsychiatric symptoms due to colony-stimulating factor 1 receptor (CSF1R) gene mutation. Few of CSF1R mutations have been functionally testified and the pathogenesis remains unknown. METHODS: In order to investigate clinical and pathological characteristics of patients with CSF1R-related leukoencephalopathy and explore the potential impact of CSF1R mutations, we analyzed clinical manifestations of 15 patients from 10 unrelated families and performed brain biopsy in 2 cases. Next generation sequencing was conducted for 10 probands to confirm the diagnosis. Sanger sequencing, segregation analysis and phenotypic reevaluation were utilized to substantiate findings. Functional examination of identified mutations was further explored. RESULTS: Clinical and neuroimaging characteristics were summarized. The average age at onset was 35.9 ± 6.4 years (range 24-46 years old). Younger age of onset was observed in female than male (34.2 vs. 39.2 years). The most common initial symptoms were speech dysfunction, cognitive decline and parkinsonian symptoms. One patient also had marked peripheral neuropathy. Brain biopsy of two cases showed typical pathological changes, including myelin loss, axonal spheroids, phosphorylated neurofilament and activated macrophages. Electron microscopy disclosed increased mitochondrial vacuolation and disorganized neurofilaments in ballooned axons. A total of 7 pathogenic variants (4 novel, 3 documented) were identified with autophosphorylation deficiency, among which c.2342C > T remained partial function of autophosphorylation. Western blotting disclosed the significantly lower level of c.2026C > T (p.R676*) than wild type. The level of microtubule associated protein 1 light chain 3-II (LC3-II), a classical marker of autophagy, was significantly lower in mutants expressed cells than wild type group by western blotting and immunofluorescence staining. CONCLUSIONS: Our findings support the loss-of-function and haploinsufficiency hypothesis in pathogenesis. Autophagy abnormality may play a role in the disease. Repairing or promoting the phosphorylation level of mutant CSF1R may shed light on therapeutic targets in the future. However, whether peripheral polyneuropathy potentially belongs to CSF1R-related spectrum deserves further study with longer follow-up and more patients enrolled. TRIAL REGISTRATION: ChiCTR, ChiCTR1800015295. Registered 21 March 2018.

Lysosomal degradation of GMPPB is associated with limb-girdle muscular dystrophy type 2T.

OBJECTIVE: GDP-mannose pyrophosphorylase B (GMPPB) related phenotype spectrum ranges widely from congenital myasthenic syndrome (CMS), limb-girdle muscular dystrophy type 2T (LGMD 2T) to severe congenital muscle-eye-brain syndrome. Our study investigates the clinicopathologic features of a patient with novel GMPPB mutations and explores the pathogenetic mechanism. METHODS: The patient was a 22-year-old woman with chronic proximal limb weakness for 9 years without cognitive deterioration. Weakness became worse after fatigue. Elevated serum creatine kinase and decrements on repetitive nerve stimulation test were recorded. MRI showed fatty infiltration in muscles of lower limbs and shoulder girdle on T1 sequence. Open muscle biopsy and genetic analysis were performed. RESULTS: Muscle biopsy showed myogenic changes. Two missense mutations in GMPPB gene (c.803T>C and c.1060G>A) were identified in the patient. Western blotting and immunostaining showed GMPPB and α-dystroglycan deficiency in the patient's muscle. In vitro, mutant GMPPB forming cytoplasmic aggregates completely colocalized with microtubule-associated protein 1 light chain 3-II (LC3-II), a classical marker of autophagosome. Degradation of GMPPB was accompanied by an upregulation of LC3-II, which could be restored by lysosomal inhibitor leupeptin. INTERPRETATION: We identified two novel GMPPB mutations causing overlap phenotype between LGMD 2T and CMS. We provided the initial evidence that mutant GMPPB colocalizes with autophagosome at subcellular level. GMPPB mutants degraded by autophagy-lysosome pathway is associated with LGMD 2T. This study shed the light into the enzyme replacement which could become one of the therapeutic targets in the future study.