The TRK-Fused Gene Is Mutated in Hereditary Motor and Sensory Neuropathy with Proximal Dominant Involvement
2012; Elsevier BV; Volume: 91; Issue: 2 Linguagem: Inglês
10.1016/j.ajhg.2012.07.014
ISSN1537-6605
AutoresHiroyuki Ishiura, Wataru Sako, Mari Yoshida, Toshitaka Kawarai, Osamu Tanabe, Jun Goto, Yuji Takahashi, Hidetoshi Date, Jun Mitsui, Budrul Ahsan, Yaeko Ichikawa, Atsushi Iwata, Hiide Yoshino, Yuishin Izumi, Koji Fujita, Kouji Maeda, Satoshi Goto, Hidetaka Koizumi, Ryoma Morigaki, Masako Ikemura, Naoko Yamauchi, Shigeo Murayama, Garth A. Nicholson, Hidefumi Ito, Gen Sobue, Masanori Nakagawa, Ryuji Kaji, Shoji Tsuji,
Tópico(s)Neurogenetic and Muscular Disorders Research
ResumoHereditary 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. 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. Hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P [MIM 604484]) is an autosomal-dominant disease characterized by predominantly proximal muscle weakness and atrophy followed by distal sensory disturbances.1Takashima H. Nakagawa M. Nakahara K. Suehara M. Matsuzaki T. Higuchi I. Higa H. Arimura K. Iwamasa T. Izumo S. Osame M. A new type of hereditary motor and sensory neuropathy linked to chromosome 3.Ann. Neurol. 1997; 41: 771-780Crossref PubMed Scopus (73) Google Scholar HMSN-P was first described in patients from the Okinawa Islands of Japan, where more than 100 people are estimated to be affected.2Nakagawa M. [Wide spectrum of hereditary motor sensory neuropathy (HMSN)].Rinsho Shinkeigaku. 2009; 49: 950-952Crossref PubMed Scopus (3) Google Scholar Two Brazilian HMSN-P-affected families of Okinawan ancestry have also been reported.3Maeda K. Sugiura M. Kato H. Sanada M. Kawai H. Yasuda H. Hereditary motor and sensory neuropathy (proximal dominant form, HMSN-P) among Brazilians of Japanese ancestry.Clin. Neurol. Neurosurg. 2007; 109: 830-832Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 4Patroclo C.B. Lino A.M. Marchiori P.E. Brotto M.W. Hirata M.T. Autosomal dominant HMSN with proximal involvement: new Brazilian cases.Arq. Neuropsiquiatr. 2009; 67: 892-896Crossref PubMed Scopus (11) Google Scholar The disease onset is usually in the 40s and is followed by a slowly progressive course. Painful muscle cramps and abundant fasciculations are observed, particularly in the early stage of the disease. In contrast to the clinical presentations of other hereditary motor and sensory neuropathies (HMSNs) presenting with predominantly distal motor weakness reflecting axonal-length dependence, the clinical presentation of HMSN-P is unique in that it involves proximal predominant weakness with widespread fasciculations resembling those of amyotrophic lateral sclerosis (ALS).5Fujita K. Yoshida M. Sako W. Maeda K. Hashizume Y. Goto S. Sobue G. Izumi Y. Kaji R. Brainstem and spinal cord motor neuron involvement with optineurin inclusions in proximal-dominant hereditary motor and sensory neuropathy.J. Neurol. Neurosurg. Psychiatry. 2011; 82: 1402-1403Crossref PubMed Scopus (19) Google Scholar Distal sensory loss is accompanied later in the disease course, but the degree of the sensory involvement varies among patients. Neuropathological findings revealed severe neuronal loss and gliosis in the spinal anterior horns and mild neuronal loss and gliosis in the hypoglossal and facial nuclei of the brainstem, which indicates that the primary pathological feature of HMSN-P is a motor neuronopathy involving motor neurons, but not a motor neuropathy involving axons.1Takashima H. Nakagawa M. Nakahara K. Suehara M. Matsuzaki T. Higuchi I. Higa H. Arimura K. Iwamasa T. Izumo S. Osame M. A new type of hereditary motor and sensory neuropathy linked to chromosome 3.Ann. Neurol. 1997; 41: 771-780Crossref PubMed Scopus (73) Google Scholar, 5Fujita K. Yoshida M. Sako W. Maeda K. Hashizume Y. Goto S. Sobue G. Izumi Y. Kaji R. Brainstem and spinal cord motor neuron involvement with optineurin inclusions in proximal-dominant hereditary motor and sensory neuropathy.J. Neurol. Neurosurg. Psychiatry. 2011; 82: 1402-1403Crossref PubMed Scopus (19) Google Scholar The posterior column, corticospinal tract, and spinocerebellar tract showed loss of myelinated fibers and gliosis. Neuronal loss and gliosis were found in Clarke's nucleus. Dorsal root ganglia showed mild to marked neuronal loss.1Takashima H. Nakagawa M. Nakahara K. Suehara M. Matsuzaki T. Higuchi I. Higa H. Arimura K. Iwamasa T. Izumo S. Osame M. A new type of hereditary motor and sensory neuropathy linked to chromosome 3.Ann. Neurol. 1997; 41: 771-780Crossref PubMed Scopus (73) Google Scholar, 5Fujita K. Yoshida M. Sako W. Maeda K. Hashizume Y. Goto S. Sobue G. Izumi Y. Kaji R. Brainstem and spinal cord motor neuron involvement with optineurin inclusions in proximal-dominant hereditary motor and sensory neuropathy.J. Neurol. Neurosurg. Psychiatry. 2011; 82: 1402-1403Crossref PubMed Scopus (19) Google Scholar These observations suggest that HMSN-P shares neuropathological findings in part with those observed in familial ALS.6Takahashi H. Makifuchi T. Nakano R. Sato S. Inuzuka T. Sakimura K. Mishina M. Honma Y. Tsuji S. Ikuta F. Familial amyotrophic lateral sclerosis with a mutation in the Cu/Zn superoxide dismutase gene.Acta Neuropathol. 1994; 88: 185-188Crossref PubMed Scopus (66) Google Scholar Previous studies on Okinawan kindreds mapped the disease locus to chromosome 3q.1Takashima H. Nakagawa M. Nakahara K. Suehara M. Matsuzaki T. Higuchi I. Higa H. Arimura K. Iwamasa T. Izumo S. Osame M. A new type of hereditary motor and sensory neuropathy linked to chromosome 3.Ann. Neurol. 1997; 41: 771-780Crossref PubMed Scopus (73) Google Scholar Subsequently, we identified two large families (families 1 and 2 in Figure 1A) affected by quite a similar phenotype in the Kansai area of Japan, located in the middle of the main island of Japan and far distant from the Okinawa Islands. We mapped the disease locus to chromosome 3q,7Maeda K. Kaji R. Yasuno K. Jambaldorj J. Nodera H. Takashima H. Nakagawa M. Makino S. Tamiya G. Refinement of a locus for autosomal dominant hereditary motor and sensory neuropathy with proximal dominancy (HMSN-P) and genetic heterogeneity.J. Hum. Genet. 2007; 52: 907-914Crossref PubMed Scopus (20) Google Scholar overlapping with the previously defined locus, which strongly indicates that these diseases are indeed identical. In addition to the large Kansai HMSN-P-affected families, we found two new Okinawan HMSN-P-affected families (families 3 and 4 in Figure 1A) in our study. In total, 9 affected and 15 unaffected individuals from the Kansai area and four affected and four unaffected individuals from the Okinawa Islands were enrolled in the study. Written informed consent was obtained from all participants. This study was approved by the institutional review boards at the University of Tokyo and the Tokushima University Hospital. Genomic DNA was extracted from peripheral-blood leukocytes or an autopsied brain according to standard procedures. The clinical presentations of the patients from the four families are summarized in Table 1 and Table S1, available online. Characteristic painful cramps and fasciculations were noted at the initial stage of the disease in all the patients from the four families. Whereas some of the patients showed painful cramps in their 20s, the ages of onset of motor weakness (41.6 ± 2.9 years old) were quite uniform. These patients presented slowly progressive, predominantly proximal weakness and atrophy with diminished tendon reflexes in the lower extremities. Sensory impairment was generally mild. Indeed, one patient (III-4 in family 4) has been diagnosed with very slowly progressive ALS. Although frontotemporal dementia (FTD) is an occasionally observed clinical presentation in patients with ALS, dementia was not observed in these patients. Laboratory tests showed mildly elevated serum creatine kinase levels. Electrophysiological studies showed similar results in all the patients investigated and revealed a decreased number of motor units with abundant positive sharp waves, fibrillation, and fasciculation potentials. Sensory-nerve action potentials of the sural nerve were lost in the later stage of the disease. All these clinical findings were similar to those described in previous reports.1Takashima H. Nakagawa M. Nakahara K. Suehara M. Matsuzaki T. Higuchi I. Higa H. Arimura K. Iwamasa T. Izumo S. Osame M. A new type of hereditary motor and sensory neuropathy linked to chromosome 3.Ann. Neurol. 1997; 41: 771-780Crossref PubMed Scopus (73) Google Scholar, 3Maeda K. Sugiura M. Kato H. Sanada M. Kawai H. Yasuda H. Hereditary motor and sensory neuropathy (proximal dominant form, HMSN-P) among Brazilians of Japanese ancestry.Clin. Neurol. Neurosurg. 2007; 109: 830-832Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 4Patroclo C.B. Lino A.M. Marchiori P.E. Brotto M.W. Hirata M.T. Autosomal dominant HMSN with proximal involvement: new Brazilian cases.Arq. Neuropsiquiatr. 2009; 67: 892-896Crossref PubMed Scopus (11) Google ScholarTable 1Clinical Characteristics of Patients with HMSN-P from Families 1 and 2 from Kansai and Families 3 and 4 from OkinawaFamilies 1 and 2Family 3Family 4III-12III-14III-15III-4Age at examination (years)40s–50s54525054Age at onset (years)37.5 ± 84440early 20s41Initial symptomsshoulder dislocation and difficulty walkingproximal leg weaknesspainful crampspainful cramps and fasciculationpainful cramps and calf atrophyMotorProximal muscle weakness and atrophy++mild++Painful cramps+++++Fasciculations+++++Motor abilitybedridden after 10–20 years from disease onsetunable to walk; wheelchaironly mild difficulty climbing stairswalk with effortunable to walk; wheelchairBulbar symptoms− ∼ +−−−−SensoryDysesthesia++mild++Decreased tactile sensation++−mild+Decreased vibratory sensation+mildmildmild+ReflexesTendon reflexesdiminisheddiminisheddiminisheddiminisheddiminishedPathological reflexes−−−−−Laboratory Tests and Electrophysiological FindingsSerum creatine kinase level270 ± 101 IU/l761 IU/lnot measured625 IU/l399 IU/lHyperglycemia4/13 patients−−−+Hyperlipidemia3/13 patients+−++Nerve conduction studymotor and sensory axonal degenerationmotor and sensory axonal degenerationnot examinednot examinedmotor and sensory axonal degenerationNeedle electromyographyneurogenic changes with fibrillation potentials and positive sharp wavesneurogenic changes with fibrillation potentials and positive sharp wavesnot examinednot examinednot examinedThe clinical characteristics of the patients from families 1 and 2 were summarized in accordance with the previous studies.5Fujita K. Yoshida M. Sako W. Maeda K. Hashizume Y. Goto S. Sobue G. Izumi Y. Kaji R. Brainstem and spinal cord motor neuron involvement with optineurin inclusions in proximal-dominant hereditary motor and sensory neuropathy.J. Neurol. Neurosurg. Psychiatry. 2011; 82: 1402-1403Crossref PubMed Scopus (19) Google Scholar, 6Takahashi H. Makifuchi T. Nakano R. Sato S. Inuzuka T. Sakimura K. Mishina M. Honma Y. Tsuji S. Ikuta F. Familial amyotrophic lateral sclerosis with a mutation in the Cu/Zn superoxide dismutase gene.Acta Neuropathol. 1994; 88: 185-188Crossref PubMed Scopus (66) Google Scholar Open table in a new tab The clinical characteristics of the patients from families 1 and 2 were summarized in accordance with the previous studies.5Fujita K. Yoshida M. Sako W. Maeda K. Hashizume Y. Goto S. Sobue G. Izumi Y. Kaji R. Brainstem and spinal cord motor neuron involvement with optineurin inclusions in proximal-dominant hereditary motor and sensory neuropathy.J. Neurol. Neurosurg. Psychiatry. 2011; 82: 1402-1403Crossref PubMed Scopus (19) Google Scholar, 6Takahashi H. Makifuchi T. Nakano R. Sato S. Inuzuka T. Sakimura K. Mishina M. Honma Y. Tsuji S. Ikuta F. Familial amyotrophic lateral sclerosis with a mutation in the Cu/Zn superoxide dismutase gene.Acta Neuropathol. 1994; 88: 185-188Crossref PubMed Scopus (66) Google Scholar To further narrow the candidate region, we conducted detailed genotyping by employing the Genome-Wide Human SNP array 6.0 (Affymetrix). Multipoint parametric linkage analysis and haplotype reconstruction were performed with the pipeline software SNP-HiTLink8Fukuda Y. Nakahara Y. Date H. Takahashi Y. Goto J. Miyashita A. Kuwano R. Adachi H. Nakamura E. Tsuji S. SNP HiTLink: A high-throughput linkage analysis system employing dense SNP data.BMC Bioinformatics. 2009; 10: 121Crossref PubMed Scopus (27) Google Scholar and Allegro v.29Gudbjartsson D.F. Thorvaldsson T. Kong A. Gunnarsson G. Ingolfsdottir A. Allegro version 2.Nat. Genet. 2005; 37: 1015-1016Crossref PubMed Scopus (170) Google Scholar (Figure 1A). In addition to the SNP genotyping, we also used newly discovered polymorphic dinucleotide repeats for haplotype comparison (microsatellite marker 1 [MS1], chr3: 101,901,207–101,901,249; and MS2, chr3: 102,157,749–102,157,795 in hg18) around TFG (see Table S2 for primer sequences). The genome-wide linkage study revealed only one chromosome 3 region showing a cumulative LOD score exceeding 3.0 (Figure 1B), confirming the result of our previous study.7Maeda K. Kaji R. Yasuno K. Jambaldorj J. Nodera H. Takashima H. Nakagawa M. Makino S. Tamiya G. Refinement of a locus for autosomal dominant hereditary motor and sensory neuropathy with proximal dominancy (HMSN-P) and genetic heterogeneity.J. Hum. Genet. 2007; 52: 907-914Crossref PubMed Scopus (20) Google Scholar An obligate recombination event was observed between rs4894942 and rs1104964, thus further refining the telomeric boundary of the candidate region in Kansai families (Figure 2A). The Okinawan families (families 3 and 4) shared an extended disease haplotype spanning 3.3 Mb, consistent with a founder effect reported in the Okinawan HMSN-P-affected families,1Takashima H. Nakagawa M. Nakahara K. Suehara M. Matsuzaki T. Higuchi I. Higa H. Arimura K. Iwamasa T. Izumo S. Osame M. A new type of hereditary motor and sensory neuropathy linked to chromosome 3.Ann. Neurol. 1997; 41: 771-780Crossref PubMed Scopus (73) Google Scholar thus defining the 3.3 Mb region as the minimum candidate region. We then performed exon capture (Sequence Capture Human Exome 2.1 M Array [NimbleGen]) of the index patient from family 3 and subsequent passively parallel sequencing by using two lanes of GAIIx (100 bp single end [Illumina]) and a one-fifth slide of SOLiD 4 (50 bp single end [Life Technologies]). GAIIx and SOLiD4 yielded 2.60 and 2.76 Gb of uniquely mapped reads,10Li H. Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics. 2009; 25: 1754-1760Crossref PubMed Scopus (26611) Google Scholar respectively. The average coverages were 29.0× and 26.8× in GAIIx and SOLiD4, respectively (Table S3 and Figure S1). In summary, 175,236 single nucleotide variants (SNVs) and 25,987 small insertions/deletions were called.11Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (31514) Google Scholar The numbers of exonic and splice-site variants were 14,189 and 127, respectively. In the minimum candidate region of 3.3 Mb, only 11 exonic SNVs were found, and only one was novel (i.e., not found in dbSNP) and nonsynonymous. Direct nucleotide-sequence analysis confirmed the presence of heterozygous SNV c.854C>T (p.Pro285Leu) in TRK-fused gene (TFG [NM_006070.5]) in all the patients from families 3 and 4 (Figure 3A and Figure S212Robinson J.T. Thorvaldsdóttir H. Winckler W. Guttman M. Lander E.S. Getz G. Mesirov J.P. Integrative genomics viewer.Nat. Biotechnol. 2011; 29: 24-26Crossref PubMed Scopus (7579) Google Scholar). Intriguingly, direct nucleotide-sequence analysis of all TFG exons (see Table S4 for primer sequences) of one patient from each of families 1 and 2 from the Kansai area revealed an identical c.854C>T (p.Pro285Leu) TFG mutation cosegregating with the disease (Figure 1A and Figure 3A). The base substitution was not observed in 482 Japanese controls (964 chromosomes), dbSNP, the 1000 Genomes Project Database, or the Exome Sequencing Project Database. Pro285 is located in the P/Q-rich domain in the C-terminal region of TFG (Figure 3B) and is evolutionally conserved (Figure 3C). PolyPhen predicts it to be "probably damaging." Because some of the exonic sequences were not sufficiently covered by exome sequencing (i.e., their read depths were no more than 10×) (Figure S1), direct nucleotide-sequence analysis was further conducted for these exonic sequences (Table S5). However, it did not reveal any other novel nonsynonymous variants, confirming that c.854C>T (p.Pro285Leu) is the only mutation exclusively present in the candidate region of 3.3 Mb. All together, we concluded that it was the disease-causing mutation. Because we found an identical mutation in both Kansai (families 1 and 2) and Okinawan (families 3 and 4) families, we then compared the haplotypes with the c.854C>T (p.Pro285Leu) mutation in the Kansai and Okinawan families in detail. To obtain high-resolution haplotypes, we included custom-made markers, including MS1 and MS2, and new SNVs identified by our exome analysis, in addition to the high-density SNPs used in the linkage analysis. The two Kansai families shared as long as 24.0 Mb of haplotype, and the two Okinawan families shared 3.3 Mb, strongly supporting a common ancestry in each region. When the haplotypes of the Kansai and Okinawan families were compared, it turned out that these families do not share the same haplotype because the markers nearest to TFG are discordant at markers 48.5 kb centromeric and 677 bp telomeric to the mutation within a haploblock (Figure 2B). Although the possibility of rare recombination events just distal to the mutation cannot be completely excluded, as suggested by the population-based recombination map (Figure 2B), these findings strongly support the interpretation that the mutations have independent origins and provide further evidence that TFG contains the causative mutation for this disease. Mutational analyses of TFG were further conducted in patients with other diseases affecting lower motor neurons (including familial ALS [n = 18], axonal HMSN [n = 26], and hereditary motor neuropathy [n = 3]) and revealed no mutations in TFG, indicating that c.854C>T (p.Pro285Leu) in TFG is highly specific to HMSN-P. In this study, we identified in all four families a single variant that appears to have developed on two different haplotypes. The mutation disrupts the PXXP motif, also known as the Src homology 3 (SH3) domain, which might affect protein-protein interactions. In addition, substitution of leucine for proline is expected to markedly alter the protein's secondary structure, which might substantially compromise the physiological functions of TFG. By employing the primers shown in Table S6, we obtained full-length cDNAs by PCR amplification of the cDNAs prepared from a cDNA library of the human fetal brain (Clontech). During this process, four species of cDNA were identified (Figure S3A). To determine the relative abundance of these cDNA species, we used the primers shown in Table S7 to conduct fragment analysis of the RT-PCR products obtained from RNAs extracted from various tissues; these primers were designed to discriminate four cDNA species on the basis of the size of the PCR products. The analysis revealed that TFG is ubiquitously expressed, including in the spinal cord and dorsal root ganglia, which are the affected sites of HMSN-P (Figure S3B). Neuropathological studies were performed in a TFG-mutation-positive patient (IV-25 in family 1) who died of pneumonia at 67 years of age.5Fujita K. Yoshida M. Sako W. Maeda K. Hashizume Y. Goto S. Sobue G. Izumi Y. Kaji R. Brainstem and spinal cord motor neuron involvement with optineurin inclusions in proximal-dominant hereditary motor and sensory neuropathy.J. Neurol. Neurosurg. Psychiatry. 2011; 82: 1402-1403Crossref PubMed Scopus (19) Google Scholar Immunohistochemical observations employing a TFG antibody (Table S8) revealed fine granular immunostaining of TFG in the cytoplasm of motor neurons in the spinal cord of neurologically normal controls (n = 3; age at death = 58.7 ± 19.6 years old) (Figure 4A). In the HMSN-P patient, in contrast, TFG-immunopositive inclusion bodies were detected in the motor neurons of the facial, hypoglossal, and abducens nuclei and the spinal cord, as well as in the sensory neurons of the dorsal root ganglia, but were not detected in glial cells (Figures 4B–4D). A small number of cortical neurons in the precentral gyrus also showed TFG-immunopositive inclusion bodies (Figure 4E). Serial sections stained with antibodies against ubiquitin or TFG (Figure 4F) and double immunofluorescence staining (Figure 4G) demonstrated that TFG-immunopositive inclusions colocalized with ubiquitin deposition. Inclusion bodies were immunopositive for optineurin in motor neurons of the brainstem nuclei and the anterior horn of the spinal cord,5Fujita K. Yoshida M. Sako W. Maeda K. Hashizume Y. Goto S. Sobue G. Izumi Y. Kaji R. Brainstem and spinal cord motor neuron involvement with optineurin inclusions in proximal-dominant hereditary motor and sensory neuropathy.J. Neurol. Neurosurg. Psychiatry. 2011; 82: 1402-1403Crossref PubMed Scopus (19) Google Scholar as well as in sensory neurons of the dorsal root ganglia (data not shown). These data strongly indicate that HMSN-P is a proteinopathy involving TFG. Because HMSN-P and ALS share some clinical characteristics, we then examined whether neuropathological findings of HMSN-P shared cardinal features with those of sporadic ALS.13Neumann M. Sampathu D.M. Kwong L.K. Truax A.C. Micsenyi M.C. Chou T.T. Bruce J. Schuck T. Grossman M. Clark C.M. et al.Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Science. 2006; 314: 130-133Crossref PubMed Scopus (4467) Google Scholar, 14Arai T. Hasegawa M. Akiyama H. Ikeda K. Nonaka T. Mori H. Mann D. Tsuchiya K. Yoshida M. Hashizume Y. Oda T. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Biochem. Biophys. Res. Commun. 2006; 351: 602-611Crossref PubMed Scopus (1872) Google Scholar, 15Hasegawa M. Arai T. Nonaka T. Kametani F. Yoshida M. Hashizume Y. Beach T.G. Buratti E. Baralle F. Morita M. et al.Phosphorylated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Ann. Neurol. 2008; 64: 60-70Crossref PubMed Scopus (511) Google Scholar, 16Inukai Y. Nonaka T. Arai T. Yoshida M. Hashizume Y. Beach T.G. Buratti E. Baralle F.E. Akiyama H. Hisanaga S. Hasegawa M. Abnormal phosphorylation of Ser409/410 of TDP-43 in FTLD-U and ALS.FEBS Lett. 2008; 582: 2899-2904Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar Immunohistochemistry with a TDP-43 antibody revealed skein-like inclusions in the remaining motor neurons of the abducens nucleus and the anterior horn of the lumbar cord (Figures 4H–4I). Phosphorylated TDP-43-positive inclusions were also identified in neurons of the anterior horn of the cervical cord and Clarke's nucleus (Figures 4J–4K). In contrast, TFG immunostaining of spinal-cord specimens from four patients with sporadic ALS (their age at death was 72.3 ± 7.4 years old) revealed no pathological staining in the motor neurons (data not shown). Double immunofluorescence staining revealed that many of the TFG-immunopositive round inclusions in the HSMN-P patient were negative for TDP-43 (Figure 4L), whereas a small number of inclusions were positive for both TFG and TDP-43 (Figure 4M). In addition, to investigate morphological Golgi-apparatus changes, which have recently been found in motor neurons of autopsied tissues of ALS patients,17Stieber A. Chen Y. Wei S. Mourelatos Z. Gonatas J. Okamoto K. Gonatas N.K. The fragmented neuronal Golgi apparatus in amyotrophic lateral sclerosis includes the trans-Golgi-network: Functional implications.Acta Neuropathol. 1998; 95: 245-253Crossref PubMed Scopus (71) Google Scholar we conducted immunohistochemical analysis by using a TGN46 antibody. It revealed that the Golgi apparatus was fragmented in approximately 70% of the remaining motor neurons in the lumbar anterior horn. The fragmentation of the Golgi apparatus was prominent near TFG-positive inclusion bodies (Figures 4N–4R). In summary, we found abnormal TDP-43-immunopositive inclusions in the cytoplasm of motor neurons, as well as fragmentation of the Golgi apparatus in HMSN-P, confirming the overlapping neuropathological features between HMSN-P and sporadic ALS. To further investigate the effect of mutant TFG in cultured cells, stable cell lines expressing wild-type and mutant TFG (p.Pro285Leu) were established from neuro-2a and human embryonic kidney (HEK) 293 cells as previously described.18Kuroda Y. Sako W. Goto S. Sawada T. Uchida D. Izumi Y. Takahashi T. Kagawa N. Matsumoto M. Matsumoto M. et al.Parkin interacts with Klokin1 for mitochondrial import and maintenance of membrane potential.Hum. Mol. Genet. 2012; 21: 991-1003Crossref PubMed Scopus (20) Google Scholar 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. The neuro-2a cells stably expressing wild-type or mutant TFG demonstrated no distinct difference in the distribution of endogenous TFG, FUS, or OPTN (data not shown). In contrast, cytoplasmic inclusions containing endogenous TDP-43 were exclusively observed in the neuro-2a cells stably expressing untagged mutant TFG, but not in those expressing wild-type TFG (Figure 5). Similar data were obtained from HEK 293 cells (data not shown). Thus, the expression of mutant TFG leads to mislocalization and inclusion-body formation of TDP-43 in cultured cells. TFG was originally identified as a part of fusion oncoproteins (NTRK1-T3 in papillary thyroid carcinoma,19Greco A. Mariani C. Miranda C. Lupas A. Pagliardini S. Pomati M. Pierotti M.A. The DNA rearrangement that generates the TRK-T3 oncogene involves a novel gene on chromosome 3 whose product has a potential coiled-coil domain.Mol. Cell. Biol. 1995; 15: 6118-6127Crossref PubMed Scopus (181) Google Scholar TFG-ALK in anaplastic large cell lymphoma,20Hernández L. Pinyol M. Hernández S. Beà S. Pulford K. Rosenwald A. Lamant L. Falini B. Ott G. 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TFG-1 acts in a hexameric form that binds the scaffolding protein Sec16 complex assembly and plays an important role in protein secretion with COPII-coated vesicles.22Witte K. Schuh A.L. Hegermann J. Sarkeshik A. Mayers J.R. Schwarze K. Yates 3rd, J.R. Eimer S. Audhya A. TFG-1 function in protein secretion and oncogenesis.Nat. Cell Biol. 2011; 13: 550-558Crossref PubMed Scopus (129) Google Scholar It is noteworthy that mutations in genes involved in vesicle trafficking23Dion P.A. Daoud H. Rouleau G.A. Genetics of motor neuron disorders: New insights into pathogenic mechanisms.Nat. Rev. Genet. 2009; 10: 769-782Crossref PubMed Scopus (232) Google Scholar, 24Andersen P.M. Al-Chalabi A. 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Neurosci. 2012; 13: 38-50Google Scholar Furthermore, recent discovery of hexanucleotide repeat expansions in C9ORF72 in familial and sporadic ALS/FTD (MIM 105550)29DeJesus-Hernandez M. Mackenzie I.R. Boeve B.F. Boxer A.L. Baker M. Rutherford N.J. Nicholson A.M. Finch N.A. Flynn H. Adamson J. et al.Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Abstract Full Text Full Text PDF PubMed Scopus (3380) Google Scholar, 30Renton A.E. Majounie E. Waite A. Simón-Sánchez J. Rollinson S. Gibbs J.R. Schymick J.C. Laaksovirta H. van Swieten J.C. Myllykangas L. et al.ITALSGEN ConsortiumA hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD.Neuron. 2011; 72: 257-268Abstract Full Text Full Text PDF PubMed Scopus (3080) Google Scholar emphasizes the RNA-mediated toxicities as the causal mechanisms of neurodegeneration. Observations of TDP-43-positive cytoplasmic inclusions in the motor neurons of the patient with HMSN-P raise the possibility that RNA-mediated mechanisms might also be involved in motor neuron degeneration in HMSN-P. In summary, we have found that TFG mutations cause HMSN-P. The presence of TFG/ubiquitin- and/or TDP-43-immunopositive cytoplasmic inclusions in motor neurons and cytosolic aggregation composed of TDP-43 in cultured cells expressing mutant TFG indicate a novel pathway of motor neuron death. The authors thank the families for participating in the study. We also thank the doctors who obtained clinical information of the patients. This work was supported in part by Grants-in-Aid for Scientific Research on Innovative Areas (22129002); the Global Centers of Excellence Program; the Integrated Database Project; Scientific Research (A) (B21406026) and Challenging Exploratory Research (23659458) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; a Grant-in-Aid for Research on Intractable Diseases and Comprehensive Research on Disability Health and Welfare from the Ministry of Health, Labour, and Welfare, Japan; Grants-in-Aid from the Research Committee of CNS Degenerative Diseases; the Ministry of Health, Labour, and Welfare of Japan; the Charcot-Marie-Tooth Association; and the National Medical Research Council of Australia. H.I. was supported by a Research Fellowship from the Japan Society for the Promotion of Science for Young Scientists. We also thank S. Ogawa (Cancer Genomics Project, The University of Tokyo) for his kind help in the analyses employing GAIIx and SOLiD4. Download .pdf (1.24 MB) Help with pdf files Document S1. Figures S1–S3 and Tables S1–S9 The URLs for data presented herein are as follows.1000 Genomes Project Database, http://www.1000genomes.org/dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/HapMap, http://hapmap.ncbi.nlm.nih.gov/NHLBI GO Exome Sequencing Project, https://esp.gs.washington.edu/drupal/Online Mendelian Inheritance in Man (OMIM), http://www.omim.orgPolyPhen, http://genetics.bwh.harvard.edu/pph/RefSeq, http://www.ncbi.nlm.nih.gov/projects/RefSeq/UCSC Human Genome Browser, http://genome.ucsc.edu/
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