Yo-Tsen Liu

Charcot–Marie–Tooth disease: frequency of genetic subtypes and guidelines for genetic testing

Background Charcot–Marie–Tooth disease (CMT) is a clinically and genetically heterogeneous group of diseases with approximately 45 different causative genes described. The aims of this study were to determine the frequency of different genes in a large cohort of patients with CMT and devise guidelines for genetic testing in practice. Methods The genes known to cause CMT were sequenced in 1607 patients with CMT (425 patients attending an inherited neuropathy clinic and 1182 patients whose DNA was sent to the authors for genetic testing) to determine the proportion of different subtypes in a UK population. Results A molecular diagnosis was achieved in 62.6% of patients with CMT attending the inherited neuropathy clinic; in 80.4% of patients with CMT1 (demyelinating CMT) and in 25.2% of those with CMT2 (axonal CMT). Mutations or rearrangements in PMP22, GJB1, MPZ and MFN2 accounted for over 90% of the molecular diagnoses while mutations in all other genes tested were rare. Conclusion Four commonly available genes account for over 90% of all CMT molecular diagnoses; a diagnostic algorithm is proposed based on these results for use in clinical practice. Any patient with CMT without a mutation in these four genes or with an unusual phenotype should be considered for referral for an expert opinion to maximise the chance of reaching a molecular diagnosis.

Correlation of magnetic resonance images with neuropathology in acute Wernicke's encephalopathy

We correlated serial brain MRIs with neuropathological findings in a 16-year-old female whose autopsy was consistent with Wernickes encephalopathy (WE). Diffusion-weighted imaging, diffusion coefficients mapping and neuropathology findings were suggested vasogenic edema in the periaqueductal and peri-the-fourth ventricular areas. This is the first documented case report to make this direct comparison. The characteristic WE changes in the mammillary body was also correlated with the findings of MRI with contrast enhancement. Bilateral cortical lesions revealed by MRI were atypical and rare in WE and were not evidenced by pathological changes.

Hereditary sensory and autonomic neuropathy type 1 (HSANI) caused by a novel mutation in SPTLC2

Objective: To describe the clinical and neurophysiologic phenotype of a family with hereditary sensory and autonomic neuropathy type 1 (HSANI) due to a novel mutation in SPTLC2 and to characterize the biochemical properties of this mutation. Methods: We screened 107 patients with HSAN who were negative for other genetic causes for mutations in SPTLC2. The biochemical properties of a new mutation were characterized in cell-free and cell-based activity assays. Results: A novel mutation (A182P) was found in 2 subjects of a single family. The phenotype of the 2 subjects was an ulcero-mutilating sensory-predominant neuropathy as described previously for patients with HSANI, but with prominent motor involvement and earlier disease onset in the first decade of life. Affected patients had elevated levels of plasma 1-deoxysphingolipids (1-deoxySLs). Biochemically, the A182P mutation was associated with a reduced canonical activity but an increased alternative activity with alanine, which results in largely increased 1-deoxySL levels, supporting their pathogenicity. Conclusion: This study confirms that mutations in SPTLC2 are associated with increased deoxySL formation causing HSANI.

A novel TFG mutation causes Charcot-Marie-Tooth disease type 2 and impairs TFG function

Objective: To describe a novel mutation in TRK-fused gene (TFG) as a new cause of dominant axonal Charcot-Marie-Tooth disease (CMT) identified by exome sequencing and further characterized by in vitro functional studies. Methods: Exome sequencing and linkage analysis were utilized to investigate a large Taiwanese family with a dominantly inherited adult-onset motor and sensory axonal neuropathy in which mutations in common CMT2-implicated genes had been previously excluded. Functional effects of the mutant gene products were investigated in vitro. Results: Exome sequencing of 2 affected individuals in this family revealed a novel heterozygous mutation, c.806G>T (p.Gly269Val), in TFG that perfectly cosegregates with the CMT2 phenotype in all 27 family members. This mutation occurs at an evolutionarily conserved residue and is absent in the 1,140 ethnically matched control chromosomes. Genome-wide linkage study also supported its disease-causative role. Cell transfection studies showed that the TFG p.Gly269Val mutation increased the propensity of TFG proteins to form aggregates, resulting in sequestration of both mutant and wild-type TFG proteins and might thus deplete functional TFG molecules. The secreted Gaussia luciferase reporter assay demonstrated that inhibition of endogenous TFG compromised the protein secretion pathways, which could only be rescued by expressing wild-type TFG but not the p.Gly269Val altered proteins. TFG mutation was not found in 55 additional unrelated patients with CMT2, suggesting its rarity. Conclusion: This study identifies a new cause of dominant CMT2 and highlights the importance of TFG in the protein secretory pathways that are essential for proper functioning of the human peripheral nervous system.

PRRT2 mutations lead to neuronal dysfunction and neurodevelopmental defects

Mutations in the proline-rich transmembrane protein 2 (PRRT2) gene cause a wide spectrum of neurological diseases, ranging from paroxysmal kinesigenic dyskinesia (PKD) to mental retardation and epilepsy. Previously, seven PKD-related PRRT2 heterozygous mutations were identified in the Taiwanese population: P91QfsX, E199X, S202HfsX, R217PfsX, R217EfsX, R240X and R308C. This study aimed to investigate the disease-causing mechanisms of these PRRT2 mutations. We first documented that Prrt2 was localized at the pre- and post-synaptic membranes with a close spatial association with SNAP25 by synaptic membrane fractionation and immunostaining of the rat neurons. Our results then revealed that the six truncating Prrt2 mutants were accumulated in the cytoplasm and thus failed to target to the cell membrane; the R308C missense mutant had significantly reduced protein expression, suggesting loss-of function effects generated by these mutations. Using in utero electroporation of shRNA into cortical neurons, we further found that knocking down Prrt2 expression in vivo resulted in a delay in neuronal migration during embryonic development and a marked decrease in synaptic density after birth. These pathologic effects and novel disease-causing mechanisms may contribute to the severe clinical symptoms in PRRT2–related diseases.

Characterization of CADASIL among the Han Chinese in Taiwan: Distinct Genotypic and Phenotypic Profiles.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is originally featured with a strong clustering of mutations in NOTCH3 exons 3–6 and leukoencephalopathy with frequent anterior temporal pole involvement. The present study aims at characterizing the genotypic and phenotypic profiles of CADASIL in Taiwan. One hundred and twelve patients with CADASIL from 95 families of Chinese descents in Taiwan were identified by Sanger sequencing of exons 2 to 24 of NOTCH3. Twenty different mutations in NOTCH3 were uncovered, including 3 novel ones, and R544C in exon 11 was the most common mutation, accounting for 70.5% of the pedigrees. Haplotype analyses were conducted in 14 families harboring NOTCH3 R544C mutation and demonstrated a common haplotype linked to NOTCH3 R544C at loci D19S929 and D19S411. Comparing with CADASIL in most Caucasian populations, CADASIL in Taiwan has several distinct features, including less frequent anterior temporal involvement, older age at symptom onset, higher incidence of intracerebral hemorrhage, and rarer occurrence of migraine. Subgroup analyses revealed that the R544C mutation is associated with lower frequency of anterior temporal involvement, later age at onset and higher frequency of cognitive dysfunction. In conclusion, the present study broadens the spectrum of NOTCH3 mutations and provides additional insights for the clinical and molecular characteristics of CADASIL patients of Han-Chinese descents.

Novel HSAN1 Mutation in Serine Palmitoyltransferase Resides at a Putative Phosphorylation Site That Is Involved in Regulating Substrate Specificity

Abstract 1-Deoxysphingolipids (1-deoxySL) are atypical sphingolipids that are formed by the enzyme serine palmitoyltransferase (SPT) due to a promiscuous use of l-alanine over its canonical substrate l-serine. Several mutations in SPT are associated with the hereditary sensory and autonomic neuropathy type I (HSAN1). The current hypothesis is that these mutations induce a permanent shift in the affinity from l-serine toward l-alanine which results in a pathologically increased 1-deoxySL formation in HSAN1 patients. Also, wild-type SPT forms 1-deoxySL under certain conditions, and elevated levels were found in individuals with the metabolic syndrome and diabetes. However, the molecular mechanisms which control the substrate shift of the wild-type enzyme are not understood. Here, we report a novel SPTLC2–S384F variant in two unrelated HSAN1 families. Affected patients showed elevated plasma 1-deoxySL levels and expression of the S384F mutant in HEK293 cells increased 1-deoxySL formation. Previously, S384 has been reported as one of the two (S384 and Y387) putative phosphorylation sites in SPTLC2. The phosphorylation of wild-type SPTLC2 was confirmed by isoelectric focusing. The impact of an S384 phosphorylation on SPT activity was tested by creating mutants mimicking either a constitutively phosphorylated (S384D, S384E) or non-phosphorylated (S384A, Y387F, Y387F+S384A) protein. The S384D but not the S384E variant was associated with increased 1-deoxySL formation. The other mutations had no influence on activity and substrate affinity. In summary, our data show that S384F is a novel mutation in HSAN1 and that the substrate specificity of wild-type SPT might by dynamically regulated by a phosphorylation at this position.

MPZ mutation G123S characterization Evidence for a complex pathogenesis in CMT disease

Objectives: To characterize the clinical and cellular phenotypes of a novel MPZ mutation identified in a Chinese family with Charcot–Marie–Tooth (CMT) disease type 1B. Methods: The family was evaluated clinically, electrophysiologically, pathologically, and genetically. The wild-type and mutant P0 fused with fluorescent proteins were expressed in vitro to monitor their intracellular trafficking. Adhesion assay was also performed to evaluate the adhesiveness of cells. Results: The novel MPZ mutation, c.367G>A, is associated with a late-onset demyelinating CMT phenotype with autosomal dominant inheritance. The median motor nerve conduction velocities of patients in this family ranged from 15.7 to 19.6 m/second. The neuropathologic studies from a sural nerve biopsy revealed a severe loss of myelinated fibers, and some onion bulb formation with clusters of regenerative fibers. Fluorescence analysis demonstrated that the mutant protein was retained ectopically in the endoplasmic reticulum and Golgi apparatus. Adhesion assay demonstrated a defective adhesiveness of cells expressing the mutant P0G123S protein. Conclusion: The novel P0G123S mutation is associated with typical findings of late-onset demyelinating polyneuropathy in the electrophysiologic and pathologic studies, putatively resulting from aberrant intracellular trafficking of the mutant P0 protein, which compromises the adhesiveness of the cells.

Transthyretin Ala97Ser in Chinese-Taiwanese patients with familial amyloid polyneuropathy: genetic studies and phenotype expression.

BACKGROUND Transthyretin (TTR) variants of familial amyloid neuropathies (FAP) form a heterogenous group of autosomal dominantly inherited diseases. TTR gene analysis in several nationalities (Japanese, Portuguese, French, and British) has shown many distinguishing characteristics in the genotype-phenotype correlation. In Chinese, there are only a few reports of private TTR gene mutations belonging to single kindred. MATERIALS AND METHODS We collected five patients with autosomal dominant inheritant sensorimotor polyneuropathy and tissue-proved amyloid deposition. The diagnosis of FAP was established on the mutation of the TTR gene detected by direct sequencing. Haplotype analysis was conducted in four of these patients. RESULTS AND CONCLUSIONS These five FAP patients shared an identical missense mutation, Ala97Ser, in the TTR gene. This mutation presented with a constellation of late-onset polyneuropathy, preceding carpal tunnel syndrome, and outstanding autonomic dysfunction. Heart was the most frequently involved vital organ. Haplotype analysis hinted independent origins although the numbers were limited. Our study is the first case series gathering from the Chinese-Taiwanese population. We proposed a possible hot-spot mutation of the TTR gene, Ala97Ser, in this ethnic.

Two Novel De Novo GARS Mutations Cause Early-Onset Axonal Charcot-Marie-Tooth Disease

Background Mutations in the GARS gene have been identified in a small number of patients with Charcot-Marie-Tooth disease (CMT) type 2D or distal spinal muscular atrophy type V, for whom disease onset typically occurs during adolescence or young adulthood, initially manifesting as weakness and atrophy of the hand muscles. The role of GARS mutations in patients with inherited neuropathies in Taiwan remains elusive. Methodology and Principal Findings Mutational analyses of the coding regions of GARS were performed using targeted sequencing of 54 patients with molecularly unassigned axonal CMT, who were selected from 340 unrelated CMT patients. Two heterozygous mutations in GARS, p.Asp146Tyr and p.Met238Arg, were identified; one in each patient. Both are novel de novo mutations. The p.Asp146Tyr mutation is associated with a severe infantile-onset neuropathy and the p.Met238Arg mutation results in childhood-onset disability. Conclusion GARS mutations are an uncommon cause of CMT in Taiwan. The p.Asp146Tyr and p.Met238Arg mutations are associated with early-onset axonal CMT. These findings broaden the mutational spectrum of GARS and also highlight the importance of considering GARS mutations as a disease cause in patients with early-onset neuropathies.