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Case Reports
. 2012 Aug 10;91(2):320-9.
doi: 10.1016/j.ajhg.2012年07月01日4.

The TRK-fused gene is mutated in hereditary motor and sensory neuropathy with proximal dominant involvement

Affiliations
Case Reports

The TRK-fused gene is mutated in hereditary motor and sensory neuropathy with proximal dominant involvement

Hiroyuki Ishiura et al. Am J Hum Genet. .

Abstract

Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) is an autosomal-dominant neurodegenerative disorder characterized by widespread fasciculations, proximal-predominant muscle weakness, and atrophy followed by distal sensory involvement. To date, large families affected by HMSN-P have been reported from two different regions in Japan. Linkage and haplotype analyses of two previously reported families and two new families with the use of high-density SNP arrays further defined the minimum candidate region of 3.3 Mb in chromosomal region 3q12. Exome sequencing showed an identical c.854C>T (p.Pro285Leu) mutation in the TRK-fused gene (TFG) in the four families. Detailed haplotype analysis suggested two independent origins of the mutation. Pathological studies of an autopsied patient revealed TFG- and ubiquitin-immunopositive cytoplasmic inclusions in the spinal and cortical motor neurons. Fragmentation of the Golgi apparatus, a frequent finding in amyotrophic lateral sclerosis, was also observed in the motor neurons with inclusion bodies. Moreover, TAR DNA-binding protein 43 kDa (TDP-43)-positive cytoplasmic inclusions were also demonstrated. In cultured cells expressing mutant TFG, cytoplasmic aggregation of TDP-43 was demonstrated. These findings indicate that formation of TFG-containing cytoplasmic inclusions and concomitant mislocalization of TDP-43 underlie motor neuron degeneration in HMSN-P. Pathological overlap of proteinopathies involving TFG and TDP-43 highlights a new pathway leading to motor neuron degeneration.

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Figures

Figure 1
Figure 1
Pedigree Charts and Linkage Analysis (A) Pedigree charts of families 1 and 2 (Kansai kindreds) and families 3 and 4 (Okinawan kindreds) are shown. Squares and circles indicate males and females, respectively. Affected persons are designated with filled symbols. A diagonal line through a symbol represents a deceased person. A person with an arrow is an index patient. Genotypes of TFG c.854 are shown in individuals in whom genomic DNA was analyzed. Individuals genotyped with SNP arrays for linkage analysis and haplotype reconstruction are indicated by dots. (B) Cumulative parametric multipoint LOD scores on chromosome 3 of all the families are shown.
Figure 2
Figure 2
Haplotype Analysis and Minimum Candidate Region of HMSN-P (A) Haplotypes were reconstructed for all the families with the use of SNP array data and microsatellite markers. Previously reported candidate regions are shown as "Kansai 2007" and "Okinawa 2007." Because families 1 and 2 are distantly related, an extended shared common haplotype was observed on chromosome 3, as indicated by a previous study. A reassessment of linkage analysis with high-density SNP markers revealed a recombination between rs4894942 and rs1104964 in family 2, thus refining the telomeric boundary of the candidate region in Kansai families (designated as "Common haplotype shared between families 1 and 2). Furthermore, a shared common haplotype (3.3 Mb with boundaries at rs16840796 and rs1284730) between families 3 and 4 was found, defining the minimum candidate region. (B) Disease haplotypes in the Kansai and Okinawan kindreds are indicated below. Local recombination rates, RefSeq genes, and the linkage disequilibrium map from HapMap JPT (Japanese in Tokyo, Japan) and CHB (Han Chinese in Beijing, China) samples are shown above the disease haplotypes. When disease haplotypes of the Kansai and Okinawan kindreds are compared, the markers nearest to TFG are discordant at markers 48.5 kb centromeric and 677 bp telomeric to the mutation within a haploblock, strongly supporting the interpretation that the mutations have independent origins.
Figure 3
Figure 3
Identification of Causative Mutation (A) Exome sequencing revealed that only one novel nonsynonymous variant is located within the minimum candidate region. Direct nucleotide-sequence analysis confirmed the mutation, c.854C>T (p.Pro285Leu), in TFG in both Kansai and Okinawan families. The mutation cosegregated with the disease (Figure 1A). (B) Schematic representation of TFG isoform 1. The alteration (p.Pro285Leu) detected in this study is shown below. (C) Cross-species homology search of the partial TFG amino acid sequence containing the p.Pro285Leu alteration revealed that Pro285 is evolutionally conserved among species.
Figure 4
Figure 4
TFG-Related Neuropathological Findings (A) TFG immunostaining (with hematoxylin counterstaining) of a motor neuron in the spinal cord of a neurologically normal control. A high-power magnified photomicrograph (inset) shows fine granular staining of TFG in the cytoplasm. The scale bars represent 20 μm (main panel) and 10 μm (inset). (B–E) TFG-immunopositive inclusions of the neurons (with hematoxylin counterstaining) in the hypoglossal nucleus (B), anterior horn of the spinal cord (C), dorsal root ganglion (D, arrows), and motor cortex (E, arrow) of the patient with the TFG mutation. The scale bars represent 20 μm (B–D) and 50 μm (E). (F and F') Serial section analysis of the facial nucleus motor neuron showing an inclusion body colabeled for TFG (F) and ubiquitin (F'). The scale bars represent 20 μm. (G–G′′) Double immunofluorescence microscopy confirming colocalization of TFG (green) and ubiquitin (red) in an inclusion body of a motor neuron in the hypoglossal nucleus. The scale bars represent 20 μm. (H and I) TDP-43-positive skein-like inclusions in the motor neurons of the abducens nucleus (H) and anterior horn of the lumbar cord (I). The scale bars represent 20 μm. (J and K) Phosporylated TDP-43-positive inclusion bodies in the cervical anterior horn (J) and Clarke’s nucleus (K). The scale bars represent 20 μm. (L–L′′) Round inclusions (arrows) positive for TFG (green) but negative for TDP-43 (red). The scale bars represent 20 μm. (M–M′′) An inclusion immunopositive for both TFG (green) and TDP-43 (red) is observed in a small number of neurons. The scale bars represent 20 μm. (N) Normal Golgi apparatus in the neurons of the intact thoracic intermediolateral nucleus. The scale bar represents 20 μm. (O and P) Fragmentation of the Golgi apparatus with small, round, and disconnected profiles in the affected motor neurons of the lumbar anterior horn. The scale bars represent 20 μm. (Q–R′′) Immunohistochemical observations of the Golgi apparatus and TFG-immunopositive inclusions employing antibodies against TGN46 (red) and TFG (green), respectively. The scale bars represent 10 μm. (Q) Normal size and distribution (red) in a motor neuron without inclusions. (R–R′′) The Golgi apparatus was fragmented into various sizes and reduced in number in the lumbar anterior horn motor neuron with TFG-positive inclusions (green). The fragmentation predominates near the inclusion (arrow), whereas the Golgi apparatuses distant from the inclusion showed nearly normal patterns (arrow head).
Figure 5
Figure 5
Formation of Cytoplasmic TDP-43 Aggregation Bodies in Cells Stably Expressing Mutant p.Pro285Leu TFG The coding sequence of TFG cDNA was subcloned into pBluescript (Stratagene). After site-directed mutagenesis with a primer pair shown in Table S9, the mutant cDNAs were cloned into the BamHI and XhoI sites of pcDNA3 (Life Technologies). Stable cell lines were established by Lipofectamine (Life Technologies) transfection according to the manufacturer’s instructions. Established cell lines were cultured under the ordinary cell-culture conditions (37°C and 5% CO2) for 5–6 days and were subjected to immunocytochemical analyses. Neuro-2a cells stably expressing wild-type TFG (A), mutant TFG (p.Pro285Leu) (B), and a mock vector (C) are shown. TDP-43-immunopositive cytoplasmic inclusions are absent in the cells stably expressing wild-type TFG or the mock vector (A and C); however, TDP-43-immunopositive cytoplasmic inclusions were exclusively demonstrated in cells stably expressing mutant TFG (p.Pro285Leu), as indicated by arrows (B). Similar results were obtained with HEK 293 cells (not shown). Scale bars represent 10 μm.

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