A Frameshift Deletion in Peripherin Gene Associated with Amyotrophic Lateral Sclerosis

Peripherin is a neuronal intermediate filament associated with inclusion bodies in motor neurons of patients with amyotrophic lateral sclerosis (ALS). A possible peripherin involvement in ALS pathogenesis has been suggested based on studies with transgenic mouse overexpressors and with a toxic splicing variant of the mouse peripherin gene. However, the existence of peripherin gene mutations in human ALS has not yet been documented. Therefore, we screened for sequence variants of the peripherin gene (PRPH) in a cohort of ALS patients including familial and sporadic cases. We identified 18 polymorphic variants of PRPH detected in both ALS and age-matched control populations. Two additional PRPH variants were discovered in ALS cases but not in 380 control individuals. One variant consisted of a nucleotide insertion in intron 8 (PRPHIVS8–36insA), whereas the other one consisted of a 1-bp deletion within exon 1 (PRPH228delC), predicting a truncated peripherin species of 85 amino acids. Remarkably, expression of this frameshift peripherin mutant in SW13 cells resulted in disruption of neurofilament network assembly. These results suggest that PRPH mutations may be responsible for a small percentage of ALS, cases and they provide further support of the view that neurofilament disorganization may contribute to pathogenesis. Peripherin is a neuronal intermediate filament associated with inclusion bodies in motor neurons of patients with amyotrophic lateral sclerosis (ALS). A possible peripherin involvement in ALS pathogenesis has been suggested based on studies with transgenic mouse overexpressors and with a toxic splicing variant of the mouse peripherin gene. However, the existence of peripherin gene mutations in human ALS has not yet been documented. Therefore, we screened for sequence variants of the peripherin gene (PRPH) in a cohort of ALS patients including familial and sporadic cases. We identified 18 polymorphic variants of PRPH detected in both ALS and age-matched control populations. Two additional PRPH variants were discovered in ALS cases but not in 380 control individuals. One variant consisted of a nucleotide insertion in intron 8 (PRPHIVS8–36insA), whereas the other one consisted of a 1-bp deletion within exon 1 (PRPH228delC), predicting a truncated peripherin species of 85 amino acids. Remarkably, expression of this frameshift peripherin mutant in SW13 cells resulted in disruption of neurofilament network assembly. These results suggest that PRPH mutations may be responsible for a small percentage of ALS, cases and they provide further support of the view that neurofilament disorganization may contribute to pathogenesis. Amyotrophic lateral sclerosis (ALS) 1The abbreviation used are: ALS, amyotrophic lateral sclerosis; FALS, familial ALS; SALS, sporadic ALS; PRPH, peripherin; IF, intermediate filament; NF-H, neurofilament heavy; NF-L, neurofilament light; NF-M, neurofilament medium. is a common, usually fatal, adult onset neurodegenerative disorder characterized by degeneration of motor neurons in the cortex, brainstem, and spinal cord (1Tandan R. Bradley W.G. Ann. Neurol. 1985; 18: 271-280Crossref PubMed Scopus (355) Google Scholar). Dysfunction and death of these motor neurons lead to progressive muscle weakness, atrophy, and ultimately paralysis and death, usually within 3–5 years after disease onset (2Mulder D.W. Kurland L.T. Offord K.P. Beard C.M. Neurology. 1986; 36: 511-517Crossref PubMed Scopus (352) Google Scholar). The disease occurs in sporadic (SALS) and familial (FALS) forms with an estimated incidence of ∼2 per 100,000. Although most cases of ALS are sporadic, some families demonstrate a clinically indistinguishable form of ALS with clear Mendelian inheritance and high penetrance (FALS). Mutations in the copper/zinc superoxide dismutase gene (SOD1), a ubiquitously expressed and highly conserved metalloenzyme, are responsible for ∼15% of FALS (3Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. Rahmani Z. Krizus A. McKenna-Yasek D. Cayabyab A. Gaston S.M. Berger R. Tanzi R.E. Halperin J.J. Herzfeldt B. Van den Bergh R. Hung W.Y. Bird T. Deng G. Mulder D.W. Smyth C. Laing N.G. Soriano E. Pericak-Vance M.A. Haines J. Rouleau G.A. Gusella J.S. Horvitz H.R. Brown R.H. Nature. 1993; 362: 59-62Crossref PubMed Scopus (5654) Google Scholar, 4Pramatarova A. Figlewicz D.A. Krizus A. Han F.Y. Ceballos-Picot I. Nicole A. Dib M. Meininger V. Brown R.H. Rouleau G.A. Am. J. Hum. Genet. 1995; 56: 592-596PubMed Google Scholar). 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Lerman-Sagie T. Lev D. Hayden M.R. Clin. Genet. 2003; 64: 210-215Crossref PubMed Scopus (89) Google Scholar, 9Gros-Louis F. Meijer I.A. Hand C.K. Dube M.P. MacGregor D.L. Seni M.H. Devon R.S. Hayden M.R. Andermann F. Andermann E. Rouleau G.A. Ann. Neurol. 2003; 53: 144-145Crossref PubMed Scopus (98) Google Scholar). Transgenic mice overexpressing mutant SOD1 genes develop a neurodegenerative disease with many pathological features found in both familial and sporadic ALS (10Gurney M.E. Pu H. Chiu A.Y. Dal Canto M.C. Polchow C.Y. Alexander D.D. Caliendo J. Hentati A. Kwon Y.W. Deng H.X. Chen W. Zhai P. Sufit R.L. Siddique T. Science. 1994; 264: 1772-1775Crossref PubMed Scopus (3537) Google Scholar, 11Wong P.C. Pardo C.A. Borchelt D.R. Lee M.K. Copeland N.G. Jenkins N.A. Sisodia S.S. Cleveland D.W. Price D.L. Neuron. 1995; 14: 1105-1116Abstract Full Text PDF PubMed Scopus (1268) Google Scholar, 12Bruijn L.I. Becher M.W. Lee M.K. Anderson K.L. Jenkins N.A. Copeland N.G. Sisodia S.S. Rothstein J.D. Borchelt D.R. Price D.L. Cleveland D.W. Neuron. 1997; 18: 327-338Abstract Full Text Full Text PDF PubMed Scopus (1137) Google Scholar). However, the mechanism by which mutant SOD1 induces motor neuron cell death remains unclear. A hallmark of motor neurons in ALS is the presence of perikaryal and axonal inclusion bodies composed of intermediate filament (IF) proteins, namely neurofilament and peripherin proteins (13Carpenter S. Neurology. 1968; 18: 841-851Crossref PubMed Google Scholar, 14Corbo M. Hays A.P. J. Neuropathol. Exp. Neurol. 1992; 51: 531-537Crossref PubMed Scopus (137) Google Scholar, 15Migheli A. Pezzulo T. Attanasio A. Schiffer D. Lab. Investig. 1993; 68: 185-191PubMed Google Scholar). Neurofilaments, the major type of IFs expressed in adult neurons, are formed by the co-assembly of three type IV IF proteins, the neurofilament light (NF-L), neurofilament medium (NF-M), and neurofilament heavy (NF-H) subunits. Peripherin is a type III IF protein expressed predominantly in the peripheral nervous system with low levels detectable in spinal motor neurons (16Escurat M. Djabali K. Gumpel M. Gros F. Portier M.M. J. Neurosci. 1990; 10: 764-784Crossref PubMed Google Scholar, 17Brody B.A. Ley C.A. Parysek L.M. J. Neurosci. 1989; 9: 2391-2401Crossref PubMed Google Scholar, 18Parysek L.M. Chisholm R.L. Ley C.A. Goldman R.D. Neuron. 1988; 1: 395-401Abstract Full Text PDF PubMed Scopus (78) Google Scholar). Peripherin expression is increased after neuronal injury (19Troy C.M. Muma N.A. Greene L.A. Price D.L. Shelanski M.L. Brain Res. 1990; 529: 232-238Crossref PubMed Scopus (133) Google Scholar, 20Wong J. Oblinger M.M. J. Neurosci. Res. 1990; 27: 332-341Crossref PubMed Scopus (104) Google Scholar). The peripherin gene (PRPH) comprises nine exons spanning a 3.5-kb region of chromosome 12. The full-length peripherin protein contains 471 amino acids. Peripherin shows similarity in sequence to proteins of the IF family. The IF proteins share a central rod domain comprising 310 residues characterized by the presence of coiled-coil α-helices (1A, 1B, 2A, and 2B) connected by short linkers. The rod domain is flanked by an N-terminal nonhelical head domain of variable length and a C-terminal tail domain tail, which is also non-helical with great length variation between different IF proteins. Currently, it remains unclear to what extent IF abnormalities contribute to ALS pathogenesis. Codon deletions or insertions in the KSP phosphorylation domain of NF-H have been detected in a small number of sporadic cases of ALS, ∼1% of total cases (21Figlewicz D.A. Krizus A. Martinoli M.G. Meininger V. Dib M. Rouleau G.A. Julien J.P. Hum. Mol. Genet. 1994; 3: 1757-1761Crossref PubMed Scopus (419) Google Scholar, 22Al-Chalabi A. Andersen P.M. Nilsson P. Chioza B. Andersson J.L. Russ C. Shaw C.E. Powell J.F. Leigh P.N. Hum. Mol. Genet. 1999; 8: 157-164Crossref PubMed Scopus (303) Google Scholar, 23Tomkins J. Usher P. Slade J.Y. Ince P.G. Curtis A. Bushby K. Shaw P.J. Neuroreport. 1998; 9: 3967-3970Crossref PubMed Scopus (145) Google Scholar). However, it is unknown how these NF-H variants might predispose to ALS. There is growing evidence for potential peripherin involvement in human disease. Overexpression of peripherin induced motor neuron disease in transgenic mice (24Beaulieu J.-M. Nguyen M.D. Julien J.-P. J. Cell Biol. 1999; 147: 531-544Crossref PubMed Scopus (203) Google Scholar), and it provoked the death of cultured motor neurons (25Robertson J. Beaulieu J.M. Doroudchi M.M. Durham H.D. Julien J.P. Mushynski W.E. J. Cell Biol. 2001; 155: 217-226Crossref PubMed Scopus (106) Google Scholar). Moreover, an up-regulation of peripherin mRNA has recently been detected in a familial ALS case (26Robertson J. Doroudchi M.M. Nguyen M.D. Durham H.D. Strong M.J. Shaw G. Julien J.P. Mushynski W.E. J. Cell Biol. 2003; 160: 939-949Crossref PubMed Scopus (103) Google Scholar). Under certain conditions, including in mice expressing a mutant form of superoxide dismutase linked to ALS, the mouse peripherin gene was found to produce a splicing peripherin species that is assembly-incompetent and toxic when expressed in cultured neuronal cells (26Robertson J. Doroudchi M.M. Nguyen M.D. Durham H.D. Strong M.J. Shaw G. Julien J.P. Mushynski W.E. J. Cell Biol. 2003; 160: 939-949Crossref PubMed Scopus (103) Google Scholar). However, the involvement of peripherin in ALS has recently been called into question because neither up-regulation nor suppression of all peripherin isoforms had any effect on disease onset, mortality, and loss of motor neurons in mice expressing mutant SOD1 (27Lariviere R.C. Beaulieu J.M. Nguyen M.D. Julien J.P. Neurobiol. Dis. 2003; 13: 158-166Crossref PubMed Scopus (32) Google Scholar). To further examine the possible involvement of peripherin in ALS, we screened the peripherin gene (PRPH) for sequence variations in sporadic and familial ALS cases from North American and European origins. Out of 20 identified PRPH variants, two were detected only in ALS cases and not in age-matched controls. Of particular interest is the discovery of a PRPH frameshift deletion mutant, the expression of which disrupted neurofilament assembly in cultured cells. Patient and Control Samples—All patients were assessed by expert clinicians, and they gave written informed consent. Diagnosis of ALS was made according to El Escorial ALS diagnostic criteria (35Brooks B.R. J. Neurol. Sci. 1994; 124: 96-107Abstract Full Text PDF PubMed Scopus (2038) Google Scholar). The FALS panel comprised French (n = 23) samples as well as French-Canadian (n = 10) and North American (n = 7) patients. The SALS sample set comprised French (n = 95) and French-Canadian (n = 54) samples. The FALS cohort exhibited a mean age of onset of 49 years, and the SALS cohort exhibited a mean age of onset of 56 years. Both SALS and FALS cohorts showed a mean disease duration of 4.5 years (see Table I). An initial set of age-match control samples (n = 95) came from non-ALS French individuals. Additional control samples were screened to increase to the power of our analysis and comprised another set of age-match French controls (n = 95).Table IClinical information of the studied ALS cohortPatientTotalMale/FemaleAge of onsetaMean age of onset (with lowest and highest values in parentheses).Spinal onsetbSpinal/bulbar onset information were not available for all the patient.Bulbar onsetbSpinal/bulbar onset information were not available for all the patient.DurationSALS15098/5256 (23-82)62234.5FALS4018/2249 (37-60)944.5a Mean age of onset (with lowest and highest values in parentheses).b Spinal/bulbar onset information were not available for all the patient. Open table in a new tab Variant Detection and Genotyping—DNA was extracted from whole blood and/or transformed lymphoblastoid cell lines using standard procedures. Primers were designed using Lazergene software and synthesized by Invitrogen. Thirteen overlapping primer pairs were designed from genomic DNA to amplify each intron and exon of the PRPH gene including the flanking splice sites and promoter as well of the 5′- and 3′-untranslated regions (see Fig. 1). Products were PCR-amplified, checked on agarose gels, and then sequenced using the forward primer for all of the amplicons. The PCR fragment sequence variations were also sequenced on the reverse strands. The PCR primer pair sequences will be given upon request. Restriction Digest of of the and variants was by The presence of the and variants new and PCR were with and to each of these two variations in the control assessed in the of each between the ALS, familial ALS, and by the from which we values with one of DNA fragment was by PCR of the human PRPH gene. PCR was on DNA extracted from a blood sample by using and the and 3′-untranslated for for for and for PCR was using The PRPH PCR fragment was then into the expression between the and the of the multiple The PRPH PCR were sequenced using the described cells IFs were in medium with and The cells were using as described in the One from each of these was and described by Beaulieu J.M. J. Julien J.P. Cell Biol. 1999; PubMed Scopus Google were also into the cells with each of the peripherin to co-assembly with the peripherin gene cells on were in in for 20 and for 1 in in was using peripherin and to and all from and used in intermediate filament from number was used was by after with or to or for and in of Peripherin samples from FALS cases and and SALS cases were screened for peripherin sequence set of samples from SALS cases was also to increase the power of our analysis when it was not possible to or between PRPH and ALS. information of the studied ALS cohort is in Table I. A total of 20 sequence variations were detected within the peripherin gene (PRPH) of ALS patients and Table sequence variations within and one in the mRNA The sequence variants were found within or within the promoter region of the gene. sequence variants consisted of nucleotide Of the PRPH variants, one had a deletion of a in exon and two had a nucleotide insertion of guanine or in the region and intron and genomic of each sequence or Open table in a new tab Of the variants in the sequence nucleotide three are to in amino residues that are highly conserved between and peripherin proteins. These variants are A and A to is a in the head domain of the peripherin to is also a an for a within the rod domain of the The of to amino within the rod domain is also a of a amino for a To any of these three PRPH variants may be mutations or for ALS, we the of each variant in ALS patients controls. The variant and results of analysis are in Table of the variant were found to be increased in ALS patients when with the control However, we identified two additional variants that were not detected the control One of these of a nucleotide deletion in a frameshift within the exon 1 and a The truncated protein of has 85 amino the residues to the protein and the other from the frameshift The ALS having this mutant PRPH the age of years after disease duration of years that had the The other insertion variant is to have a on the an effect on be as the A insertion the or the of by of the of the two variants and on the assembly of we with the PRPH genes into SW13 cells that IF We have the PRPH variant into an expression by the in Fig. of expressing human NF-L, peripherin or or resulted in of proteins into the IF However, of the for only and in SW13 cells with the of the IF network peripherin with J.M. J. Julien J.P. Cell Biol. 1999; PubMed Scopus Google and it is with in motor neurons, we the of of the PRPH species with neurofilament or proteins in SW13 cells. the of mouse or human peripherin with to the of the network for and peripherin A and results were by the variant with However, the of with human to the disruption of the IF network that the of was in the presence of in the a of the mutant peripherin with proteins. is well that the and NF-H proteins the for neurofilament network J.M. J. Julien J.P. Cell Biol. 1999; PubMed Scopus Google Scholar). The in SW13 cells of peripherin or with resulted in the of A and as reported J.M. J. Julien J.P. Cell Biol. 1999; PubMed Scopus Google Scholar). shows that peripherin with to form the presence of IF network results were by the variant gene with However, IF protein have been detected in any SW13 cells after of with the frameshift mutant A is that this may from of into high by with the mutant peripherin of the short N-terminal head this we have identified 20 PRPH variants in ALS including three variants to amino and one frameshift deletion in exon To the possible involvement of these variants in ALS, we the of each variant in of ALS patients controls and the of the variants with disease in for the two variants and found in ALS the not between ALS and control for any of the other 18 variants We that these PRPH variants that not predispose to ALS. for the variant identified in ALS but not in 380 control there is evidence based on in that this variant had any on peripherin and assembly into the IF network and Therefore, this sequence variation is a we evidence for the of the frameshift mutant in exon 1 is to a to a truncated protein comprising amino of the peripherin head domain this truncated peripherin species that rod coiled-coil sequences was to into IF is that this short peripherin head mutant in SW13 cells was to assembly into IF network and to assembly of into Although of the peripherin N-terminal head domain with IF proteins have been studies with the system on domain of another type III IF between the head domain and other regions J.J. S. W. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). However, it is that the head domain of IF proteins is for filament assembly and that phosphorylation of the head domain neurofilament and IF network J. Cell Sci. 1999; Google Scholar, J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of IF assembly disruption by the PRPH mutant expression in SW13 cells that the effect of this is to a disorganization neurofilament network to a of peripherin from the loss of one Moreover, this be in with the that the peripherin mice not develop motor neuron disease R.C. Nguyen M.D. A. Julien J.P. J. 2002; PubMed Scopus Google Scholar). evidence for the involvement of in pathogenesis came from the discovery of neurofilament gene mutations linked to neurodegenerative mutations and a deletion in the gene have been reported in patients with type P. I. E. J. J.J. Van C. O. V. Ann. Neurol. 2001; PubMed Scopus Google Scholar, Am. J. Hum. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar). Codon deletions and insertion in the KSP region of the neurofilament NF-H gene have been detected in a small number of sporadic ALS ∼1% of total cases (21Figlewicz D.A. Krizus A. Martinoli M.G. Meininger V. Dib M. Rouleau G.A. Julien J.P. Hum. Mol. Genet. 1994; 3: 1757-1761Crossref PubMed Scopus (419) Google Scholar, 22Al-Chalabi A. Andersen P.M. Nilsson P. Chioza B. Andersson J.L. Russ C. Shaw C.E. Powell J.F. Leigh P.N. Hum. Mol. Genet. 1999; 8: 157-164Crossref PubMed Scopus (303) Google Scholar, 23Tomkins J. Usher P. Slade J.Y. Ince P.G. Curtis A. Bushby K. Shaw P.J. Neuroreport. 1998; 9: 3967-3970Crossref PubMed Scopus (145) Google Scholar). However, the of these phosphorylation variants are and these variants are as for the disease. discovery of a frameshift in PRPH that neurofilament assembly the evidence for peripherin involvement in ALS, and it that PRPH mutations may be responsible for a small number of ALS cases. the to neuronal death by neurofilament abnormalities studies with transgenic mice expressing this PRPH frameshift mutant might provide new into the of motor neuron We and for in the of samples in this

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