Case ReportValproate Treatment in an ALS Patient Carrying a c194GgtASpastin Mutation and SMN2 Homozygous Deletion
Lucio Tremolizzo1 Gessica Sala1 Elisa Conti1 Virginia Rodriguez-Menendez1
Antonella Fogli2 Angela Michelucci2 Paolo Simi2 Silvana Penco3 Christian Lunetta4
Massimo Corbo45 and Carlo Ferrarese1
1 Section of Neurology Ospedale San Gerardo and DCMT Milan Center for Neuroscience (NeuroMI) University of Milano-BicoccaVia Pergolesi 33 20900 Monza Italy
2Medical Genetics AOU Pisana Ospedale Santa Chiara Pisa Italy3Medical Genetics Department of Laboratory Medicine Fondazione Serena Ospedale Niguarda Carsquo Granda Milano Italy4NEuroMuscular Omnicentre (NEMO) Fondazione Serena Ospedale Niguarda Carsquo Granda Milano Italy5 Department of Neurorehabilitation Sciences Casa Cura Policlinico Milano Italy
Correspondence should be addressed to Lucio Tremolizzo luciotremolizzounimibit
Received 26 February 2014 Revised 25 June 2014 Accepted 29 June 2014 Published 17 July 2014
Academic Editor Chin-Chang Huang
Copyright copy 2014 Lucio Tremolizzo et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Here we report the case of anALS patient found to carry both a novel heterozygous change (c194GgtA)within the spastin gene and ahomozygous deletion of the SMN2 geneThe patient was started on valproic acid (VPA 600mgdie per os) considering the capacityof this drug of increasing survival motor neuron through an epigenetic mechanism Patient clinical course and molecular effectsof VPA on skin fibroblasts obtained from the proband are describedThis c194GgtA spastin mutation might expand the previouslyknown borders of type 4 spastic paraplegia (SPG4) and we suggest the intriguing possibility that the absence of SMN2 might haveacted as a contributory risk factor for starting lower motor neuron damage Exploring the relationship genocopy-phenocopy inselected ALS patients might represent an interesting strategy for understanding its clinical variability
1 Introduction
Amyotrophic lateral sclerosis (ALS) is a neurodegenerativedisorder defined by the involvement of both upper andlower motoneurons (UMN and LMN) [1] Although severalpathological mechanisms have progressively been elucidatedin this disease ALS phenotypic determinants are not com-pletely understood and even genome-wide association basedapproaches have not yet been able to penetrate such hetero-geneity [2] Considering such diversity the possibility thatgenocopies and phenocopies might contribute significantlyto ALS cannot be rejected and is probably still far from beingcompletely elucidated [3]
Interestingly ALS belongs to a clinical continuum involv-ing other selective UMN and LMN diseases with knowngenetic causes such as hereditary spastic paraparesis (HSP)
and spinalmuscular atrophy (SMA) respectively offering thepossibility of investigating the contribution of these factors tothe clinical expression of full-blown ALS [4ndash6]
Here we report a very peculiar ALS case bearing botha novel missense mutation within the spastin gene (SPG4)the commonest causative gene for HSP and at the same timethe homozygous deletion of the SMN2 gene the centromericcopy of the SMA defective gene (SMN1) known to have thepotential to mitigate the clinical phenotype in SMA [7 8]and possibly in ALS [9 10] albeit in this latter case withconflicting results [11] We anyhow hypothesized that thisparticular patient could be considered an unusual ALS com-posite genocopy that is expressing UMN pathology as theresult of a spastinmutation interacting with LMNpathologywhich could have been predisposed by the concurrent lack ofSMN2
Hindawi Publishing CorporationCase Reports in Neurological MedicineVolume 2014 Article ID 216094 7 pageshttpdxdoiorg1011552014216094
Figure 1 (a) Electropherograms from the proband (A) and a control subject (B) The sequence variation is a G to A transition at position194 (GenBank Acc no AJ246001 c194GgtA) causing the substitution of arginine at position 65 with a histidine (pR65H) (b) The residue atposition 65 is highly conserved among different species
Aim of this paper consists not only in describing thisatypical case but also in reporting the clinical and moleculareffects of the treatment with valproate (VPA) that was offeredto the patient considering its potential of increasing SMNlevels by an epigenetic mechanism [12ndash14] Moreover inorder to interpret SMNmolecular data into the correct framethe expression of this protein in skin fibroblasts obtainedfrom our patient was compared to that assessed in a groupof sporadic ALS patients and healthy controls Interestinglythe lack of SMN2 gene copies in our patient offers the uniqueopportunity of selectively studying the impact of the modu-lation of SMN1 promoter on motoneuron disease phenotypean issue that might be relevant when thinking that SMN1duplications have been associated with ALS susceptibility [6]and that the loss of spliceosome integrity (related to SMNfunction) has been postulated to play a role in ALS [15]
2 Case Report
A 50-year-old woman was referred in 2010 to the NEuro-Muscular Omnicentre (NEMO) of Milan Italy due to pro-gressively worsening limb strength deficits causing repeatedfalls She complained of lower limb cramps and fasciculationsassociated with slowly progressing weakness since 2007Laboratory investigations were normal Due to the presenceof clear pyramidal signs with spasticity and increased deep
tendon reflexes at the lower limbs lumbar puncture and brainand spinal cord MR scans were performed with negativeresults Nerve conduction studies were normal while needleEMGshowed spontaneous activity (fibrillation potentials andpositive sharp waves) increased amplitude and long durationmotor unit action potentials associated with reduced recruit-ment in all limbs The bulbar district was involved as wellDuring a one-year follow-up period the diagnosis of ALS wasestablished based on the clinical and neurophysiologic pic-ture Cognitive statuswas unaffectedHowever family historywas positive for dementia in both her father and paternalgrandmother in absence of reported motor dysfunction Theproband gave her consent to specific genetic testing SOD1TARDPB FUS and C9orf72 were all negative for mutationsDue to the predominant UMN presentation HSP-relatedmutations were also assessed SPG7 was negative while aheterozygous mutation (c194GgtA) within exon 1 of thespastin gene (SPG4)was found (Figure 1(a))This novel SPG4missense mutation was also found in the probandrsquos fatherand her two-year-older and neurologically unremarkablesister although it was not found in 100 healthy unrelatedItalian subjects Moreover the residue at position 65 is highlyconserved among different species (Figure 1(b)) and in silicoanalysis using the SIFT software (httpsiftjcviorg) revealedthat the substitution is predicted to affect protein functionwith a score of 001 and therefore could cause loss-of-function
Case Reports in Neurological Medicine 3
or exert a dominant negative effect Later on SMN genecopies were also assessed in the proband discovering thehomozygous deletion of the SMN2 gene (exons 7 and 8)
The patient was then started on valproic acid (VPAsodium salt) at the oral dose of 300mg extended releasebid (total 600mgday) Even if an initial subjective positiveresponse was reported the patient eventually discontinuedthe drug after 12 months due to the perceived lack ofefficacy in contrasting disease progression No significantchanges in disease progression rates were analogously notedby the assessing physicians No significant side effects werereported during treatment The proband consented to skinbiopsy before starting VPA treatment for fibroblast culturingMoreover she also consented to blood sampling both at thebeginning and at the end of the period of VPA administra-tion
3 Methods
31 Fibroblast Cultures Following ethical approval andinformed consent skin fibroblasts were obtained by biopsiesperformed within the forearm by a round needle (diameter4mm) Fibroblasts from the biopsy specimens were culturedin high-glucoseDulbeccorsquosmodifiedEaglersquosmediumcontain-ing 25mM HEPES supplemented with 10 heat-inactivatedfetal calf serum 100UmL penicillin 100 120583gmL strepto-mycin and 2mM glutamine Cell lines were maintained at37∘C in 95 humidified air and 5 CO
2 Fibroblasts were
always used at an equivalent number of passages of growth(ranging from 5 to 10) Culture medium was always renewed24 h before each experiment [16]
Fibroblasts were exposed in vitro to VPA at concentra-tions ranging from05 to 10mM according to theHDAC IC
50
of this drug and to previous reports [12 17] Fibroblasts wereat the same time obtained from 12 sporadic ALS outpatientsrecruited at the San Gerardo Hospital Monza Italy (SALSMF 48 mean age plusmn SD 595 plusmn 12 years disease durationrange 1ndash50months ALSFRS-R score 253plusmn 10) and 9 age- andsex-comparable healthy controls (CTRLMF 45 age 62plusmn10years)
32 Blood Sample Preparation Whole-blood samples wereobtained from the antecubital vein in K
3-EDTA between
0800 and 0900 AM following overnight fasting Peripheralblood mononuclear cells (PBMC) isolation was obtainedby Ficoll-Histopaque density gradient centrifugation asdescribed before [18] PBMC pellets were then stocked atminus80∘C until assessment
33 Whole-Blood Global DNA Methylation Whole-bloodglobal DNA methylation was evaluated by an inverse assay[19] Briefly 1 120583g of whole-blood DNA was cut ON with a10-fold excess of both HpaII (cutting only nonmethylated 51015840-CCGG-31015840 recognition sites New England Biolabs) and MspI(cutting independently frommethylation status at 51015840-CCGG-31015840 recognition sites Fermentas Life Sciences) endonucleasesEach sample was then split in two aliquots incubated with1 120583L of 1 10 diluted [3H]dCTP (60Cimmol Amersham)
at 56∘C for 60min respectively in presence or absence ofTAQDNApolymerase (total volume 20120583L)The reactionwasstopped on ice and radioactivity beta-counted Total DNAmethylation was calculated as 1-(HpaIIMspI values)
34 Western Blotting Fibroblasts were collected and homog-enized in cell extraction buffer containing protease inhibitors(Sigma) and PMSF and protein concentration was assessedby Bradfordrsquos method Serial dilutions of each sample (5 1020120583g) were separated by NuPAGE 4ndash12 Bis-Tris gels (LifeTechnologies) in order to ensure the subsequent readingswithin the linear part of the detection curve and blottedonto Hybond nitrocellulose membranes (Amersham GEHealthcare) After blocking for 1 h at room temperature(RT) samples were incubated ON at 4∘C with either anti-SMN (1 5000 BD Biosciences) or anti-acetyl-histone H3(1 5000 Upstate Biotechnology) antibodies followed by thecorresponding peroxidase-conjugated secondary antibody(1 800090minRT) Following ECL Plus (Amersham GEHealthcare) application chemiluminescence was quantifiedby densitometer and expressed as optical density (OD) ofthe target protein and beta-actin (1 20000 Sigma) used asinternal standard
35 Immunohistochemistry Fibroblast cultures were fixedin 4 paraformaldehyde for 20 minutes and then incu-bated with 3 of triton (10min) and 5 BSA (45min)solutions Primary antibody mouse anti-SMN (BD Trans-duction Laboratories) at 1 100 was incubated ON at 4∘Cand secondary antibody Alexa Fluor 546 goat anti-mousetogether with Alexa Fluor 488-conjugated phalloidin (both at1 200 Invitrogen)was incubated for 1 h at room temperatureImageswere takenwith a confocalmicroscopy Radiance 2100microscope (Biorad Laboratories Hercules CA)
36 ChIP Assay ChIP assay was performed according tothe manufacturerrsquos protocol (EZ-ChIP Millipore) Briefly10e6 cells incubated or not with increasing concentrationof VPA were treated with 1 formaldehyde After cell lysiscross-linked chromatin was sonicated and then immuno-precipitated with antibody against acetylated H3 (Millipore)ProteinDNA complexes were reverted then ChIP enrichedDNA samples were quantified by real-time PCR and dataexpressed as percentage of input The primer pair used toamplify SMN promoter was 51015840-TTAAGGATCTGCCTT-CCTTCCTGC-31015840 and 51015840-ATGTTGCTTAGGCCTCGT-CTCGAA-31015840 PCR condition was 1 cycle at 50∘C for 2min1 cycle at 95∘C for 10min 40 cycles at 95∘C for 15 s 60∘C for30 s
37 Statistical Analysis Unpaired Studentrsquos 119905-test was used toassess the significance of differences between two treatmentregimens or two subject groups as appropriate Correlationwas computed with Pearsonrsquos 119903-test Statistical analysis wasperformed by Prism 400 (GraphPad Software Inc) Data areexpressed throughout the entire paper as mean plusmn SEM
4 Case Reports in Neurological Medicine
4 Results
Fibroblasts obtained from the proband did not display majormorphological alterations and a faint immunoreactive signalfor SMN was present within nuclear aggregates possiblymarking nuclear gems (Figure 2(a)) VPA treatment at theconcentration of 5mMfor 72 hwas able to induce an apparentincrease of SMN-positive nuclear aggregates with respectto vehicle-treated cells (Figure 2(b)) The same treatmentwas able to induce chromatin rearrangement since increasedvalues of acetyl-histone H3 were measured (sim65 119899 = 3data not shown) and ChIP assay documented a significantlyincreased association between acetyl-histone H3 and theSMN promoter (Figure 2(c)) When SMN expression wasassessed following exposure to VPA at concentrations rang-ing from 05 to 10mM for 72 h there was a trend towardan increase of SMN-like immunoreactive signal in total celllysates that became significant only at the concentrationof 10mM (about twofold increase see Figure 2(d)) How-ever the semiquantification of basal SMN-like immunore-active content (with respect to beta-actin) in the fibroblastsobtained from the proband was similar to that obtained inhealthy controls (Figures 2(e) and 2(f)) On the other handSALS patients (119899 = 12) showed about 50 lower SMN-likeimmunoreactive content with respect to the control group(119899 = 9 Figures 2(c) and 2(f)) without any significantrelationship with demographic or clinical variables Finallyglobal DNA methylation in PBMC obtained before and afterthe period of VPA administration (12 months at the doseof 300mg extended release bid per os) documented adecrease of sim50 of methyl-cytosine content consistent withthe demethylating properties of the drug while SMN-likeimmunoreactivity was apparently unchanged (119899 = 3 data notshown)
5 Discussion
The c194GgtA spastin mutation apparently expands thepreviously known borders of type 4 spastic paraplegia (SPG4)[20] causing the substitution within exon 1 of Arg with aHis at position 65 (pR65H) highly conserved across differentspecies as shown by in silico analysis Arguing against thehypothesis that this genetic variation might be pathogenicthere is the lack of segregation with the neuromusculardisease within the familiar group Furthermore current lackof functional assays certainly does not allow establishinga conclusion about the potential pathogenic effects of thereported change Intriguingly however the heterozygousduplication c304 309dupGCCTCG within the spastin genewas previously reported in a patient affected by a veryslowly progressing form of ALS [21] The same authorsfurther reported a case of rapidly progressing ALS bearing aheterozygous missense change (S44L) within the same gene[5] We may conclude hypothesizing that all these spastinvariations might have increased the risk for developing ALSalthough this hypothesis cannot be verified at the momentLower motoneuron involvement has already been sporad-ically described in similar patients (eg SPG17 see [22])and we find intriguing the possibility that the absence of
SMN2 might have acted as a further contributory risk factorin our hypothesis synergistically spreading motor neurondamage In fact the ultimate phenotypic expression of theproband was clinically indistinguishable from that of severaltypical ALS cases although initially the preponderance ofupper motor neuron dysfunction prompted looking forfurther non-ALS specific genetic characterization CertainlySMN2 deletion involves a limited albeit defined quote of thepopulation (about 8-9 in [11]) and its significance is notclarified since it has been proposed either as a protectivefactor [11] a noninfluent factor [23 24] or as a risk factor fordeveloping ALS [9 25]
We anyhow hypothesized that this particular patientcould be considered an ALS composite genocopy that isexpressing UMN pathology due to this novel spastin muta-tion that interacted with a LMN pathology whose risk ofappearance could have been increased by a concurrent minordecrease in SMN levels Certainly this is a speculation thatcannot be directly verified but exploring the relationshipbetween genotype and phenotype in selected ALS patientsincluding different genocopies and phenocopies might rep-resent an interesting strategy for understanding the clinicalvariability strongly characterizing this disorder Even if theexact clinical expression of this novel SPG4 mutation isnot yet known altered SMN expression has already beenhypothesized to play a role in ALS with quite conflictingresults [6 9 11] starting from the original hypothesis thatthe lack of this gene product might induce the same well-known dysfunction that determines and modulates SMAclinical phenotype [7 8] Consistent with the accessory rolecharacterizing SMN2 SMN total levels were not significantlydecreased in our patient fibroblasts On the other handour SALS patients displayed a significant decrease of thisprotein despite the limited number of recruited subjects asalready suggested [9] Further studies addressing SMN levelsin peripheral cells from ALS patients with respect to theirspecific SMN1 and SMN2 genotype might certainly clarifythis issue [26]
VPA was offered to our patient in an open label courseconsidering the epigenetic properties of this drug in differentneuropsychiatric conditionmodels and cell types [16 27]Thesystemic administration ofVPA is in fact able to increase SNClevels of acetyl-histone H3 [28] and to prevent the increasein 5-methyl-cytosine content following the administration ofhypermethylating agents [29] Furthermore SMN levels areselectively increased by the administration of VPAmodifyingSMN12 promoter structure [12 14] VPA has already beentested on SMA and ALS patients [30ndash32] albeit mainly withnegative results As a matter of fact our patientrsquos absence ofclinical response to VPA parallels the findings of these trials[30ndash33] and might also be consistent with the apparentlyhigh-enough residual levels of SMN in our patient althoughalternative biological explanations might apply [34] Forexample the proposed VPA dose was of 600mgdie con-ceivably corresponding to a plasma concentration rangingfrom sub- to just above the lower border of the recommendedtherapeutic window for epilepsy plausibly close to a valueroughly equivalent to the IC
50of this HDAC inhibitor
(sim04mM) [17] This dose could have been producing a quite
Case Reports in Neurological Medicine 5
VEH
(a)
VPA5mM72h
(b)
VEH 05 500
25
50
75
100
SMN
( o
f inp
ut)
lowast
VPA mM72h
(c)
VEH 05 5 100
1
2
3
4 ∘
SMN
120573-a
ctin
VPA mM72h
(d)
CTRL ALS
4
3
2
1
0
lowast
SMN
120573-a
ctin
(e)
120573-Actin
SMN
CTRL ALS
(f)
Figure 2 (a b) SMN immunoreactivity in fibroblasts obtained from the proband (scale bar = 10 120583m) a faint (red) staining was present withinnuclear structures (actin filaments counterstained in green) in vehicle-treated cells (VEH) while VPA 5mM72 h induced an apparent densityincrease of SMN-positive nuclear aggregates (c) acetyl-histone H3 association with SMN promoter was increased in fibroblast obtained fromthe proband following exposure to VPA 5mM72 h as semiquantified by ChIP assay (lowast119875 lt 005) (d) SMN-like immunoreactive content infibroblasts obtained from the proband is increased following in vitro exposure to VPA as semiquantified by Western blotting (∘119875 lt 005)(e) (Western blotting) SMN-like immunoreactive content in fibroblasts obtained from 12 SALS patients is reduced with respect to 9 matchedcontrols (lowast119875 = 001) as also shown by (f) representative immunoreactive signals (10 120583g of total protein) obtained in 5 different SALS patientsand 4 CTRL subjects
6 Case Reports in Neurological Medicine
small effect not eventually reaching clinical threshold Forthis reason we decided to include in ex vivo experiments adose one order of magnitude higher in order to magnify theexpected outcomes [12]
However recent suggestions that modifications of theepigenome might be of interest for understanding clinicalvariability in ALS [19 35] might further justify the attemptsof testing in the future other compounds able to modifyhistone acetylation or histone andor DNAmethylation [36]In fact in spite of the fact that ALS clinical heterogeneitymight imply differences in the involved pathophysiology acommon downstream pathway converging on the regulationof mRNA expression has been repeatedly demonstratedsince the discovery of TARDP43 and FUSTLS mutationsin ALS [37 38] Moreover an epigenetic dysfunction hasbeen hypothesized in several complex disorders for whichone single clear-cut mechanism cannot be demonstrated[39] such as in the case of ALS Interestingly TDP-43 andFUSTLS localize to nuclear gems through an associationwith SMN [15 40] implying that defective integrity of thespliceosome might be started by an expression or functionaldefect in any of these gene products leading at least to LMNdysfunction
6 Conclusion
The case we report here is carrier of both a novel spastinmutation and plausibly a minor defective SMN productionpossibly leading to a composite ALS genocopy Describingpeculiar ALS genocopies and phenocopies might be ofinterest for the field since dissecting the roads of phenotypiccomplexity inmotor neuron disorders conceivably representsone way to eventually find an effective cure for ALS
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors would like to thank the Italian Association forAmyotrophic Lateral Sclerosis (AISLA)
References
[1] B R Brooks R G Miller M Swash and T L MunsatldquoEl Escorial revisited revised criteria for the diagnosis ofamyotrophic lateral sclerosisrdquoAmyotrophic Lateral Sclerosis vol1 no 5 pp 293ndash299 2000
[2] L C Kwee Y Liu C Haynes et al ldquoA high-density genome-wide association screen of sporadic ALS in US veteransrdquo PLoSONE vol 7 no 3 Article ID e32768 2012
[3] M Sabatelli A Conte and M Zollino ldquoClinical and geneticheterogeneity of amyotrophic lateral sclerosisrdquo Clinical Genet-ics vol 83 no 5 pp 408ndash416 2013
[4] M J Strong and P H Gordon ldquoPrimary lateral sclerosishereditary spastic paraplegia and amyotrophic lateral sclerosis
discrete entities or spectrumrdquo Amyotrophic Lateral Sclerosisand Other Motor Neuron Disorders vol 6 no 1 pp 8ndash16 2005
[5] C Munch A Rolfs and T Meyer ldquoHeterozygous S44Lmissense change of the spastin gene in amyotrophic lateralsclerosisrdquo Amyotrophic Lateral Sclerosis vol 9 no 4 pp 251ndash253 2008
[6] H M Blauw C P Barnes P W J van Vught et al ldquoSMN1 geneduplications are associated with sporadic ALSrdquo Neurology vol78 no 11 pp 776ndash780 2012
[7] F Petit J Cuisset N Rouaix-Emery et al ldquoInsights intogenotype-phenotype correlations in spinal muscular atrophy aretrospective study of 103 patientsrdquoMuscle amp Nerve vol 43 no1 pp 26ndash30 2011
[8] G Y Zheleznyakova A V Kiselev V G Vakharlovsky et alldquoGenetic and expression studies of SMN2 gene in Russianpatients with spinal muscular atrophy type II and IIIrdquo BMCMedical Genetics vol 12 article 96 2011
[9] J H Veldink S Kalmijn A H van der Hout et al ldquoSMNgenotypes producing less SMN protein increase susceptibilityto and severity of sporadic ALSrdquo Neurology vol 65 no 6 pp820ndash825 2005
[10] J Kim S Lee Y Choi et al ldquoAssociation between survivormotor neuron 2 (SMN2) gene homozygous deletion and spo-radic lower motor neuron disease in a Korean populationrdquoAnnals of Clinical and Laboratory Science vol 40 no 4 pp 368ndash374 2010
[11] P Corcia C Ingre H Blasco et al ldquoHomozygous SMN2deletion is a protective factor in the Swedish ALS populationrdquoEuropean Journal of Human Genetics vol 20 no 5 pp 588ndash5912012
[12] C J Sumner T N Huynh J A Markowitz et al ldquoValproic acidincreases SMN levels in spinal muscular atrophy patient cellsrdquoAnnals of Neurology vol 54 no 5 pp 647ndash654 2003
[13] L Tremolizzo V Rodriguez-Menendez G Sala J C diFrancesco and C Ferrarese ldquoValproate and HDAC inhibitiona new epigenetic strategy to mitigate phenotypic severity inALSrdquo Amyotrophic Lateral Sclerosis and Other Motor NeuronDisorders vol 6 no 3 pp 185ndash186 2005
[14] L E Kernochan M L Russo N S Woodling et al ldquoThe role ofhistone acetylation in SMN gene expressionrdquoHumanMolecularGenetics vol 14 no 9 pp 1171ndash1182 2005
[15] H Tsuiji Y Iguchi A Furuya et al ldquoSpliceosome integrity isdefective in the motor neuron diseases ALS and SMArdquo EMBOMolecular Medicine vol 5 no 2 pp 221ndash234 2013
[16] G Sala F Trombin L Mattavelli et al ldquoLack of evidencefor oxidative stress in sporadic amyotrophic lateral sclerosisfibroblastsrdquoNeurodegenerative Diseases vol 6 no 1-2 pp 9ndash152008
[17] L Tremolizzo J C DiFrancesco V Rodriguez-Menendezet al ldquoValproate induces epigenetic modifications in lym-phomonocytes from epileptic patientsrdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 39 no 1 pp47ndash51 2012
[18] G Sala L Tremolizzo L Melchionda et al ldquoA panel ofmacroautophagymarkers in lymphomonocytes of patients withamyotrophic lateral sclerosisrdquoAmyotrophic Lateral Sclerosis vol13 no 1 pp 119ndash124 2012
[19] L Tremolizzo P Messina E Conti et al ldquoWhole-blood globalDNA methylation is increased in amyotrophic lateral sclerosisindependently of age of onsetrdquo Amyotroph Lateral Scler Fron-totemporal Degener vol 15 pp 98ndash105 2014
Case Reports in Neurological Medicine 7
[20] C J McDermott C E Burness J Kirby et al ldquoClinical featuresof hereditary spastic paraplegia due to spastin mutationrdquoNeurology vol 67 no 1 pp 45ndash51 2006
[21] T Meyer A Schwan J S Dullinger et al ldquoEarly-onset ALSwith long-term survival associated with spastin genemutationrdquoNeurology vol 65 no 1 pp 141ndash143 2005
[22] J Irobi P van den Bergh L Merlini et al ldquoThe phenotypeof motor neuropathies associated with BSCL2 mutations isbroader than Silver syndrome and distal HMN type Vrdquo Brainvol 127 no 9 pp 2124ndash2130 2004
[23] P Corcia W Camu J-M Halimi et al ldquoSMN1 gene but notSMN2 is a risk factor for sporadic ALSrdquo Neurology vol 67 no7 pp 1147ndash1150 2006
[24] X B Wang N H Cui J J Gao X P Qiu and F ZhengldquoSMN1 duplications contribute to sporadic amyotrophic lateralsclerosis susceptibility evidence from a meta-analysisrdquo Journalof the Neurological Sciences vol 340 no 1-2 pp 63ndash68 2014
[25] J H Veldink L H van den Berg J M Cobben et alldquoHomozygous deletion of the survival motor neuron 2 gene is aprognostic factor in sporadic ALSrdquoNeurology vol 56 no 6 pp749ndash752 2001
[26] S Piepers J Cobben P Sodaar et al ldquoQuantification of SMNprotein in leucocytes from spinal muscular atrophy patientseffects of treatment with valproic acidrdquo Journal of NeurologyNeurosurgery and Psychiatry vol 82 no 8 pp 850ndash852 2011
[27] L Tremolizzo V Rodriguez-Menendez E Conti C P ZoiaG Cavaletti and C Ferrarese ldquoNovel therapeutic targetsin neuropsychiatric disorders the neuroepigenomerdquo CurrentPharmaceutical Design vol 20 no 11 pp 1831ndash1839 2014
[28] L Tremolizzo G Carboni W B Ruzicka et al ldquoAn epigeneticmouse model for molecular and behavioral neuropatholo-gies related to schizophrenia vulnerabilityrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 99 no 26 pp 17095ndash17100 2002
[29] L Tremolizzo M S Doueiri E Dong et al ldquoValproatecorrects the schizophrenia-like epigenetic behavioral modifica-tions induced bymethionine inmicerdquo Biological Psychiatry vol57 no 5 pp 500ndash509 2005
[30] C C Weihl A M Connolly and A Pestronk ldquoValproate mayimprove strength and function in patients with type IIIIVspinal muscle atrophyrdquo Neurology vol 67 no 3 pp 500ndash5012006
[31] I A Darbar P G Plaggert M B D Resende E Zanoteli andU C Reed ldquoEvaluation of muscle strength and motor abilitiesin children with type II and III spinal muscle atrophy treatedwith valproic acidrdquo BMC Neurology vol 11 article 36 2011
[32] S Piepers J H Veldink S W de Jong et al ldquoRandomizedsequential trial of valproic acid in amyotrophic lateral sclerosisrdquoAnnals of Neurology vol 66 no 2 pp 227ndash234 2009
[33] J T Kissel B Elsheikh W M King et al ldquoSMA valiant triala prospective double-blind placebo-controlled trial of valproicacid in ambulatory adults with spinalmuscular atrophyrdquoMuscleamp Nerve vol 49 no 2 pp 187ndash192 2013
[34] L Garbes L Heesen I Holker et al ldquoVPA response in SMAis suppressed by the fatty acid translocase CD36rdquo HumanMolecular Genetics vol 22 no 2 pp 398ndash407 2013
[35] B A Chestnut Q Chang A Price C Lesuisse M Wong andL J Martin ldquoEpigenetic regulation of motor neuron cell deaththrough DNAmethylationrdquoThe Journal of Neuroscience vol 31no 46 pp 16619ndash16636 2011
[36] E Beghi E Pupillo V Bonito et al ldquoRandomized double-blind placebo-controlled trial of acetyl-L-carnitine for ALSrdquoAmyotrophic Lateral Sclerosis and Frontotemporal Degenerationvol 14 no 5-6 pp 397ndash405 2013
[37] A Verma and R Tandan ldquoRNA quality control and proteinaggregates in amyotrophic lateral sclerosis a reviewrdquo Muscleand Nerve vol 47 no 3 pp 330ndash338 2013
[38] S C Ling M Polymenidou and D W Cleveland ldquoConvergingmechanisms in als and FTD disrupted RNA and proteinhomeostasisrdquo Neuron vol 79 no 3 pp 416ndash438 2013
[39] A Petronis ldquoEpigenetics as a unifying principle in the aetiologyof complex traits and diseasesrdquo Nature vol 465 no 7299 pp721ndash727 2010
[40] T Yamazaki S Chen Y Yu et al ldquoFUS- SMN protein inter-actions link the motor neuron diseases ALS and SMArdquo CellReports vol 2 no 4 pp 799ndash806 2012
Figure 1 (a) Electropherograms from the proband (A) and a control subject (B) The sequence variation is a G to A transition at position194 (GenBank Acc no AJ246001 c194GgtA) causing the substitution of arginine at position 65 with a histidine (pR65H) (b) The residue atposition 65 is highly conserved among different species
Aim of this paper consists not only in describing thisatypical case but also in reporting the clinical and moleculareffects of the treatment with valproate (VPA) that was offeredto the patient considering its potential of increasing SMNlevels by an epigenetic mechanism [12ndash14] Moreover inorder to interpret SMNmolecular data into the correct framethe expression of this protein in skin fibroblasts obtainedfrom our patient was compared to that assessed in a groupof sporadic ALS patients and healthy controls Interestinglythe lack of SMN2 gene copies in our patient offers the uniqueopportunity of selectively studying the impact of the modu-lation of SMN1 promoter on motoneuron disease phenotypean issue that might be relevant when thinking that SMN1duplications have been associated with ALS susceptibility [6]and that the loss of spliceosome integrity (related to SMNfunction) has been postulated to play a role in ALS [15]
2 Case Report
A 50-year-old woman was referred in 2010 to the NEuro-Muscular Omnicentre (NEMO) of Milan Italy due to pro-gressively worsening limb strength deficits causing repeatedfalls She complained of lower limb cramps and fasciculationsassociated with slowly progressing weakness since 2007Laboratory investigations were normal Due to the presenceof clear pyramidal signs with spasticity and increased deep
tendon reflexes at the lower limbs lumbar puncture and brainand spinal cord MR scans were performed with negativeresults Nerve conduction studies were normal while needleEMGshowed spontaneous activity (fibrillation potentials andpositive sharp waves) increased amplitude and long durationmotor unit action potentials associated with reduced recruit-ment in all limbs The bulbar district was involved as wellDuring a one-year follow-up period the diagnosis of ALS wasestablished based on the clinical and neurophysiologic pic-ture Cognitive statuswas unaffectedHowever family historywas positive for dementia in both her father and paternalgrandmother in absence of reported motor dysfunction Theproband gave her consent to specific genetic testing SOD1TARDPB FUS and C9orf72 were all negative for mutationsDue to the predominant UMN presentation HSP-relatedmutations were also assessed SPG7 was negative while aheterozygous mutation (c194GgtA) within exon 1 of thespastin gene (SPG4)was found (Figure 1(a))This novel SPG4missense mutation was also found in the probandrsquos fatherand her two-year-older and neurologically unremarkablesister although it was not found in 100 healthy unrelatedItalian subjects Moreover the residue at position 65 is highlyconserved among different species (Figure 1(b)) and in silicoanalysis using the SIFT software (httpsiftjcviorg) revealedthat the substitution is predicted to affect protein functionwith a score of 001 and therefore could cause loss-of-function
Case Reports in Neurological Medicine 3
or exert a dominant negative effect Later on SMN genecopies were also assessed in the proband discovering thehomozygous deletion of the SMN2 gene (exons 7 and 8)
The patient was then started on valproic acid (VPAsodium salt) at the oral dose of 300mg extended releasebid (total 600mgday) Even if an initial subjective positiveresponse was reported the patient eventually discontinuedthe drug after 12 months due to the perceived lack ofefficacy in contrasting disease progression No significantchanges in disease progression rates were analogously notedby the assessing physicians No significant side effects werereported during treatment The proband consented to skinbiopsy before starting VPA treatment for fibroblast culturingMoreover she also consented to blood sampling both at thebeginning and at the end of the period of VPA administra-tion
3 Methods
31 Fibroblast Cultures Following ethical approval andinformed consent skin fibroblasts were obtained by biopsiesperformed within the forearm by a round needle (diameter4mm) Fibroblasts from the biopsy specimens were culturedin high-glucoseDulbeccorsquosmodifiedEaglersquosmediumcontain-ing 25mM HEPES supplemented with 10 heat-inactivatedfetal calf serum 100UmL penicillin 100 120583gmL strepto-mycin and 2mM glutamine Cell lines were maintained at37∘C in 95 humidified air and 5 CO
2 Fibroblasts were
always used at an equivalent number of passages of growth(ranging from 5 to 10) Culture medium was always renewed24 h before each experiment [16]
Fibroblasts were exposed in vitro to VPA at concentra-tions ranging from05 to 10mM according to theHDAC IC
50
of this drug and to previous reports [12 17] Fibroblasts wereat the same time obtained from 12 sporadic ALS outpatientsrecruited at the San Gerardo Hospital Monza Italy (SALSMF 48 mean age plusmn SD 595 plusmn 12 years disease durationrange 1ndash50months ALSFRS-R score 253plusmn 10) and 9 age- andsex-comparable healthy controls (CTRLMF 45 age 62plusmn10years)
32 Blood Sample Preparation Whole-blood samples wereobtained from the antecubital vein in K
3-EDTA between
0800 and 0900 AM following overnight fasting Peripheralblood mononuclear cells (PBMC) isolation was obtainedby Ficoll-Histopaque density gradient centrifugation asdescribed before [18] PBMC pellets were then stocked atminus80∘C until assessment
33 Whole-Blood Global DNA Methylation Whole-bloodglobal DNA methylation was evaluated by an inverse assay[19] Briefly 1 120583g of whole-blood DNA was cut ON with a10-fold excess of both HpaII (cutting only nonmethylated 51015840-CCGG-31015840 recognition sites New England Biolabs) and MspI(cutting independently frommethylation status at 51015840-CCGG-31015840 recognition sites Fermentas Life Sciences) endonucleasesEach sample was then split in two aliquots incubated with1 120583L of 1 10 diluted [3H]dCTP (60Cimmol Amersham)
at 56∘C for 60min respectively in presence or absence ofTAQDNApolymerase (total volume 20120583L)The reactionwasstopped on ice and radioactivity beta-counted Total DNAmethylation was calculated as 1-(HpaIIMspI values)
34 Western Blotting Fibroblasts were collected and homog-enized in cell extraction buffer containing protease inhibitors(Sigma) and PMSF and protein concentration was assessedby Bradfordrsquos method Serial dilutions of each sample (5 1020120583g) were separated by NuPAGE 4ndash12 Bis-Tris gels (LifeTechnologies) in order to ensure the subsequent readingswithin the linear part of the detection curve and blottedonto Hybond nitrocellulose membranes (Amersham GEHealthcare) After blocking for 1 h at room temperature(RT) samples were incubated ON at 4∘C with either anti-SMN (1 5000 BD Biosciences) or anti-acetyl-histone H3(1 5000 Upstate Biotechnology) antibodies followed by thecorresponding peroxidase-conjugated secondary antibody(1 800090minRT) Following ECL Plus (Amersham GEHealthcare) application chemiluminescence was quantifiedby densitometer and expressed as optical density (OD) ofthe target protein and beta-actin (1 20000 Sigma) used asinternal standard
35 Immunohistochemistry Fibroblast cultures were fixedin 4 paraformaldehyde for 20 minutes and then incu-bated with 3 of triton (10min) and 5 BSA (45min)solutions Primary antibody mouse anti-SMN (BD Trans-duction Laboratories) at 1 100 was incubated ON at 4∘Cand secondary antibody Alexa Fluor 546 goat anti-mousetogether with Alexa Fluor 488-conjugated phalloidin (both at1 200 Invitrogen)was incubated for 1 h at room temperatureImageswere takenwith a confocalmicroscopy Radiance 2100microscope (Biorad Laboratories Hercules CA)
36 ChIP Assay ChIP assay was performed according tothe manufacturerrsquos protocol (EZ-ChIP Millipore) Briefly10e6 cells incubated or not with increasing concentrationof VPA were treated with 1 formaldehyde After cell lysiscross-linked chromatin was sonicated and then immuno-precipitated with antibody against acetylated H3 (Millipore)ProteinDNA complexes were reverted then ChIP enrichedDNA samples were quantified by real-time PCR and dataexpressed as percentage of input The primer pair used toamplify SMN promoter was 51015840-TTAAGGATCTGCCTT-CCTTCCTGC-31015840 and 51015840-ATGTTGCTTAGGCCTCGT-CTCGAA-31015840 PCR condition was 1 cycle at 50∘C for 2min1 cycle at 95∘C for 10min 40 cycles at 95∘C for 15 s 60∘C for30 s
37 Statistical Analysis Unpaired Studentrsquos 119905-test was used toassess the significance of differences between two treatmentregimens or two subject groups as appropriate Correlationwas computed with Pearsonrsquos 119903-test Statistical analysis wasperformed by Prism 400 (GraphPad Software Inc) Data areexpressed throughout the entire paper as mean plusmn SEM
4 Case Reports in Neurological Medicine
4 Results
Fibroblasts obtained from the proband did not display majormorphological alterations and a faint immunoreactive signalfor SMN was present within nuclear aggregates possiblymarking nuclear gems (Figure 2(a)) VPA treatment at theconcentration of 5mMfor 72 hwas able to induce an apparentincrease of SMN-positive nuclear aggregates with respectto vehicle-treated cells (Figure 2(b)) The same treatmentwas able to induce chromatin rearrangement since increasedvalues of acetyl-histone H3 were measured (sim65 119899 = 3data not shown) and ChIP assay documented a significantlyincreased association between acetyl-histone H3 and theSMN promoter (Figure 2(c)) When SMN expression wasassessed following exposure to VPA at concentrations rang-ing from 05 to 10mM for 72 h there was a trend towardan increase of SMN-like immunoreactive signal in total celllysates that became significant only at the concentrationof 10mM (about twofold increase see Figure 2(d)) How-ever the semiquantification of basal SMN-like immunore-active content (with respect to beta-actin) in the fibroblastsobtained from the proband was similar to that obtained inhealthy controls (Figures 2(e) and 2(f)) On the other handSALS patients (119899 = 12) showed about 50 lower SMN-likeimmunoreactive content with respect to the control group(119899 = 9 Figures 2(c) and 2(f)) without any significantrelationship with demographic or clinical variables Finallyglobal DNA methylation in PBMC obtained before and afterthe period of VPA administration (12 months at the doseof 300mg extended release bid per os) documented adecrease of sim50 of methyl-cytosine content consistent withthe demethylating properties of the drug while SMN-likeimmunoreactivity was apparently unchanged (119899 = 3 data notshown)
5 Discussion
The c194GgtA spastin mutation apparently expands thepreviously known borders of type 4 spastic paraplegia (SPG4)[20] causing the substitution within exon 1 of Arg with aHis at position 65 (pR65H) highly conserved across differentspecies as shown by in silico analysis Arguing against thehypothesis that this genetic variation might be pathogenicthere is the lack of segregation with the neuromusculardisease within the familiar group Furthermore current lackof functional assays certainly does not allow establishinga conclusion about the potential pathogenic effects of thereported change Intriguingly however the heterozygousduplication c304 309dupGCCTCG within the spastin genewas previously reported in a patient affected by a veryslowly progressing form of ALS [21] The same authorsfurther reported a case of rapidly progressing ALS bearing aheterozygous missense change (S44L) within the same gene[5] We may conclude hypothesizing that all these spastinvariations might have increased the risk for developing ALSalthough this hypothesis cannot be verified at the momentLower motoneuron involvement has already been sporad-ically described in similar patients (eg SPG17 see [22])and we find intriguing the possibility that the absence of
SMN2 might have acted as a further contributory risk factorin our hypothesis synergistically spreading motor neurondamage In fact the ultimate phenotypic expression of theproband was clinically indistinguishable from that of severaltypical ALS cases although initially the preponderance ofupper motor neuron dysfunction prompted looking forfurther non-ALS specific genetic characterization CertainlySMN2 deletion involves a limited albeit defined quote of thepopulation (about 8-9 in [11]) and its significance is notclarified since it has been proposed either as a protectivefactor [11] a noninfluent factor [23 24] or as a risk factor fordeveloping ALS [9 25]
We anyhow hypothesized that this particular patientcould be considered an ALS composite genocopy that isexpressing UMN pathology due to this novel spastin muta-tion that interacted with a LMN pathology whose risk ofappearance could have been increased by a concurrent minordecrease in SMN levels Certainly this is a speculation thatcannot be directly verified but exploring the relationshipbetween genotype and phenotype in selected ALS patientsincluding different genocopies and phenocopies might rep-resent an interesting strategy for understanding the clinicalvariability strongly characterizing this disorder Even if theexact clinical expression of this novel SPG4 mutation isnot yet known altered SMN expression has already beenhypothesized to play a role in ALS with quite conflictingresults [6 9 11] starting from the original hypothesis thatthe lack of this gene product might induce the same well-known dysfunction that determines and modulates SMAclinical phenotype [7 8] Consistent with the accessory rolecharacterizing SMN2 SMN total levels were not significantlydecreased in our patient fibroblasts On the other handour SALS patients displayed a significant decrease of thisprotein despite the limited number of recruited subjects asalready suggested [9] Further studies addressing SMN levelsin peripheral cells from ALS patients with respect to theirspecific SMN1 and SMN2 genotype might certainly clarifythis issue [26]
VPA was offered to our patient in an open label courseconsidering the epigenetic properties of this drug in differentneuropsychiatric conditionmodels and cell types [16 27]Thesystemic administration ofVPA is in fact able to increase SNClevels of acetyl-histone H3 [28] and to prevent the increasein 5-methyl-cytosine content following the administration ofhypermethylating agents [29] Furthermore SMN levels areselectively increased by the administration of VPAmodifyingSMN12 promoter structure [12 14] VPA has already beentested on SMA and ALS patients [30ndash32] albeit mainly withnegative results As a matter of fact our patientrsquos absence ofclinical response to VPA parallels the findings of these trials[30ndash33] and might also be consistent with the apparentlyhigh-enough residual levels of SMN in our patient althoughalternative biological explanations might apply [34] Forexample the proposed VPA dose was of 600mgdie con-ceivably corresponding to a plasma concentration rangingfrom sub- to just above the lower border of the recommendedtherapeutic window for epilepsy plausibly close to a valueroughly equivalent to the IC
50of this HDAC inhibitor
(sim04mM) [17] This dose could have been producing a quite
Case Reports in Neurological Medicine 5
VEH
(a)
VPA5mM72h
(b)
VEH 05 500
25
50
75
100
SMN
( o
f inp
ut)
lowast
VPA mM72h
(c)
VEH 05 5 100
1
2
3
4 ∘
SMN
120573-a
ctin
VPA mM72h
(d)
CTRL ALS
4
3
2
1
0
lowast
SMN
120573-a
ctin
(e)
120573-Actin
SMN
CTRL ALS
(f)
Figure 2 (a b) SMN immunoreactivity in fibroblasts obtained from the proband (scale bar = 10 120583m) a faint (red) staining was present withinnuclear structures (actin filaments counterstained in green) in vehicle-treated cells (VEH) while VPA 5mM72 h induced an apparent densityincrease of SMN-positive nuclear aggregates (c) acetyl-histone H3 association with SMN promoter was increased in fibroblast obtained fromthe proband following exposure to VPA 5mM72 h as semiquantified by ChIP assay (lowast119875 lt 005) (d) SMN-like immunoreactive content infibroblasts obtained from the proband is increased following in vitro exposure to VPA as semiquantified by Western blotting (∘119875 lt 005)(e) (Western blotting) SMN-like immunoreactive content in fibroblasts obtained from 12 SALS patients is reduced with respect to 9 matchedcontrols (lowast119875 = 001) as also shown by (f) representative immunoreactive signals (10 120583g of total protein) obtained in 5 different SALS patientsand 4 CTRL subjects
6 Case Reports in Neurological Medicine
small effect not eventually reaching clinical threshold Forthis reason we decided to include in ex vivo experiments adose one order of magnitude higher in order to magnify theexpected outcomes [12]
However recent suggestions that modifications of theepigenome might be of interest for understanding clinicalvariability in ALS [19 35] might further justify the attemptsof testing in the future other compounds able to modifyhistone acetylation or histone andor DNAmethylation [36]In fact in spite of the fact that ALS clinical heterogeneitymight imply differences in the involved pathophysiology acommon downstream pathway converging on the regulationof mRNA expression has been repeatedly demonstratedsince the discovery of TARDP43 and FUSTLS mutationsin ALS [37 38] Moreover an epigenetic dysfunction hasbeen hypothesized in several complex disorders for whichone single clear-cut mechanism cannot be demonstrated[39] such as in the case of ALS Interestingly TDP-43 andFUSTLS localize to nuclear gems through an associationwith SMN [15 40] implying that defective integrity of thespliceosome might be started by an expression or functionaldefect in any of these gene products leading at least to LMNdysfunction
6 Conclusion
The case we report here is carrier of both a novel spastinmutation and plausibly a minor defective SMN productionpossibly leading to a composite ALS genocopy Describingpeculiar ALS genocopies and phenocopies might be ofinterest for the field since dissecting the roads of phenotypiccomplexity inmotor neuron disorders conceivably representsone way to eventually find an effective cure for ALS
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors would like to thank the Italian Association forAmyotrophic Lateral Sclerosis (AISLA)
References
[1] B R Brooks R G Miller M Swash and T L MunsatldquoEl Escorial revisited revised criteria for the diagnosis ofamyotrophic lateral sclerosisrdquoAmyotrophic Lateral Sclerosis vol1 no 5 pp 293ndash299 2000
[2] L C Kwee Y Liu C Haynes et al ldquoA high-density genome-wide association screen of sporadic ALS in US veteransrdquo PLoSONE vol 7 no 3 Article ID e32768 2012
[3] M Sabatelli A Conte and M Zollino ldquoClinical and geneticheterogeneity of amyotrophic lateral sclerosisrdquo Clinical Genet-ics vol 83 no 5 pp 408ndash416 2013
[4] M J Strong and P H Gordon ldquoPrimary lateral sclerosishereditary spastic paraplegia and amyotrophic lateral sclerosis
discrete entities or spectrumrdquo Amyotrophic Lateral Sclerosisand Other Motor Neuron Disorders vol 6 no 1 pp 8ndash16 2005
[5] C Munch A Rolfs and T Meyer ldquoHeterozygous S44Lmissense change of the spastin gene in amyotrophic lateralsclerosisrdquo Amyotrophic Lateral Sclerosis vol 9 no 4 pp 251ndash253 2008
[6] H M Blauw C P Barnes P W J van Vught et al ldquoSMN1 geneduplications are associated with sporadic ALSrdquo Neurology vol78 no 11 pp 776ndash780 2012
[7] F Petit J Cuisset N Rouaix-Emery et al ldquoInsights intogenotype-phenotype correlations in spinal muscular atrophy aretrospective study of 103 patientsrdquoMuscle amp Nerve vol 43 no1 pp 26ndash30 2011
[8] G Y Zheleznyakova A V Kiselev V G Vakharlovsky et alldquoGenetic and expression studies of SMN2 gene in Russianpatients with spinal muscular atrophy type II and IIIrdquo BMCMedical Genetics vol 12 article 96 2011
[9] J H Veldink S Kalmijn A H van der Hout et al ldquoSMNgenotypes producing less SMN protein increase susceptibilityto and severity of sporadic ALSrdquo Neurology vol 65 no 6 pp820ndash825 2005
[10] J Kim S Lee Y Choi et al ldquoAssociation between survivormotor neuron 2 (SMN2) gene homozygous deletion and spo-radic lower motor neuron disease in a Korean populationrdquoAnnals of Clinical and Laboratory Science vol 40 no 4 pp 368ndash374 2010
[11] P Corcia C Ingre H Blasco et al ldquoHomozygous SMN2deletion is a protective factor in the Swedish ALS populationrdquoEuropean Journal of Human Genetics vol 20 no 5 pp 588ndash5912012
[12] C J Sumner T N Huynh J A Markowitz et al ldquoValproic acidincreases SMN levels in spinal muscular atrophy patient cellsrdquoAnnals of Neurology vol 54 no 5 pp 647ndash654 2003
[13] L Tremolizzo V Rodriguez-Menendez G Sala J C diFrancesco and C Ferrarese ldquoValproate and HDAC inhibitiona new epigenetic strategy to mitigate phenotypic severity inALSrdquo Amyotrophic Lateral Sclerosis and Other Motor NeuronDisorders vol 6 no 3 pp 185ndash186 2005
[14] L E Kernochan M L Russo N S Woodling et al ldquoThe role ofhistone acetylation in SMN gene expressionrdquoHumanMolecularGenetics vol 14 no 9 pp 1171ndash1182 2005
[15] H Tsuiji Y Iguchi A Furuya et al ldquoSpliceosome integrity isdefective in the motor neuron diseases ALS and SMArdquo EMBOMolecular Medicine vol 5 no 2 pp 221ndash234 2013
[16] G Sala F Trombin L Mattavelli et al ldquoLack of evidencefor oxidative stress in sporadic amyotrophic lateral sclerosisfibroblastsrdquoNeurodegenerative Diseases vol 6 no 1-2 pp 9ndash152008
[17] L Tremolizzo J C DiFrancesco V Rodriguez-Menendezet al ldquoValproate induces epigenetic modifications in lym-phomonocytes from epileptic patientsrdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 39 no 1 pp47ndash51 2012
[18] G Sala L Tremolizzo L Melchionda et al ldquoA panel ofmacroautophagymarkers in lymphomonocytes of patients withamyotrophic lateral sclerosisrdquoAmyotrophic Lateral Sclerosis vol13 no 1 pp 119ndash124 2012
[19] L Tremolizzo P Messina E Conti et al ldquoWhole-blood globalDNA methylation is increased in amyotrophic lateral sclerosisindependently of age of onsetrdquo Amyotroph Lateral Scler Fron-totemporal Degener vol 15 pp 98ndash105 2014
Case Reports in Neurological Medicine 7
[20] C J McDermott C E Burness J Kirby et al ldquoClinical featuresof hereditary spastic paraplegia due to spastin mutationrdquoNeurology vol 67 no 1 pp 45ndash51 2006
[21] T Meyer A Schwan J S Dullinger et al ldquoEarly-onset ALSwith long-term survival associated with spastin genemutationrdquoNeurology vol 65 no 1 pp 141ndash143 2005
[22] J Irobi P van den Bergh L Merlini et al ldquoThe phenotypeof motor neuropathies associated with BSCL2 mutations isbroader than Silver syndrome and distal HMN type Vrdquo Brainvol 127 no 9 pp 2124ndash2130 2004
[23] P Corcia W Camu J-M Halimi et al ldquoSMN1 gene but notSMN2 is a risk factor for sporadic ALSrdquo Neurology vol 67 no7 pp 1147ndash1150 2006
[24] X B Wang N H Cui J J Gao X P Qiu and F ZhengldquoSMN1 duplications contribute to sporadic amyotrophic lateralsclerosis susceptibility evidence from a meta-analysisrdquo Journalof the Neurological Sciences vol 340 no 1-2 pp 63ndash68 2014
[25] J H Veldink L H van den Berg J M Cobben et alldquoHomozygous deletion of the survival motor neuron 2 gene is aprognostic factor in sporadic ALSrdquoNeurology vol 56 no 6 pp749ndash752 2001
[26] S Piepers J Cobben P Sodaar et al ldquoQuantification of SMNprotein in leucocytes from spinal muscular atrophy patientseffects of treatment with valproic acidrdquo Journal of NeurologyNeurosurgery and Psychiatry vol 82 no 8 pp 850ndash852 2011
[27] L Tremolizzo V Rodriguez-Menendez E Conti C P ZoiaG Cavaletti and C Ferrarese ldquoNovel therapeutic targetsin neuropsychiatric disorders the neuroepigenomerdquo CurrentPharmaceutical Design vol 20 no 11 pp 1831ndash1839 2014
[28] L Tremolizzo G Carboni W B Ruzicka et al ldquoAn epigeneticmouse model for molecular and behavioral neuropatholo-gies related to schizophrenia vulnerabilityrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 99 no 26 pp 17095ndash17100 2002
[29] L Tremolizzo M S Doueiri E Dong et al ldquoValproatecorrects the schizophrenia-like epigenetic behavioral modifica-tions induced bymethionine inmicerdquo Biological Psychiatry vol57 no 5 pp 500ndash509 2005
[30] C C Weihl A M Connolly and A Pestronk ldquoValproate mayimprove strength and function in patients with type IIIIVspinal muscle atrophyrdquo Neurology vol 67 no 3 pp 500ndash5012006
[31] I A Darbar P G Plaggert M B D Resende E Zanoteli andU C Reed ldquoEvaluation of muscle strength and motor abilitiesin children with type II and III spinal muscle atrophy treatedwith valproic acidrdquo BMC Neurology vol 11 article 36 2011
[32] S Piepers J H Veldink S W de Jong et al ldquoRandomizedsequential trial of valproic acid in amyotrophic lateral sclerosisrdquoAnnals of Neurology vol 66 no 2 pp 227ndash234 2009
[33] J T Kissel B Elsheikh W M King et al ldquoSMA valiant triala prospective double-blind placebo-controlled trial of valproicacid in ambulatory adults with spinalmuscular atrophyrdquoMuscleamp Nerve vol 49 no 2 pp 187ndash192 2013
[34] L Garbes L Heesen I Holker et al ldquoVPA response in SMAis suppressed by the fatty acid translocase CD36rdquo HumanMolecular Genetics vol 22 no 2 pp 398ndash407 2013
[35] B A Chestnut Q Chang A Price C Lesuisse M Wong andL J Martin ldquoEpigenetic regulation of motor neuron cell deaththrough DNAmethylationrdquoThe Journal of Neuroscience vol 31no 46 pp 16619ndash16636 2011
[36] E Beghi E Pupillo V Bonito et al ldquoRandomized double-blind placebo-controlled trial of acetyl-L-carnitine for ALSrdquoAmyotrophic Lateral Sclerosis and Frontotemporal Degenerationvol 14 no 5-6 pp 397ndash405 2013
[37] A Verma and R Tandan ldquoRNA quality control and proteinaggregates in amyotrophic lateral sclerosis a reviewrdquo Muscleand Nerve vol 47 no 3 pp 330ndash338 2013
[38] S C Ling M Polymenidou and D W Cleveland ldquoConvergingmechanisms in als and FTD disrupted RNA and proteinhomeostasisrdquo Neuron vol 79 no 3 pp 416ndash438 2013
[39] A Petronis ldquoEpigenetics as a unifying principle in the aetiologyof complex traits and diseasesrdquo Nature vol 465 no 7299 pp721ndash727 2010
[40] T Yamazaki S Chen Y Yu et al ldquoFUS- SMN protein inter-actions link the motor neuron diseases ALS and SMArdquo CellReports vol 2 no 4 pp 799ndash806 2012
or exert a dominant negative effect Later on SMN genecopies were also assessed in the proband discovering thehomozygous deletion of the SMN2 gene (exons 7 and 8)
The patient was then started on valproic acid (VPAsodium salt) at the oral dose of 300mg extended releasebid (total 600mgday) Even if an initial subjective positiveresponse was reported the patient eventually discontinuedthe drug after 12 months due to the perceived lack ofefficacy in contrasting disease progression No significantchanges in disease progression rates were analogously notedby the assessing physicians No significant side effects werereported during treatment The proband consented to skinbiopsy before starting VPA treatment for fibroblast culturingMoreover she also consented to blood sampling both at thebeginning and at the end of the period of VPA administra-tion
3 Methods
31 Fibroblast Cultures Following ethical approval andinformed consent skin fibroblasts were obtained by biopsiesperformed within the forearm by a round needle (diameter4mm) Fibroblasts from the biopsy specimens were culturedin high-glucoseDulbeccorsquosmodifiedEaglersquosmediumcontain-ing 25mM HEPES supplemented with 10 heat-inactivatedfetal calf serum 100UmL penicillin 100 120583gmL strepto-mycin and 2mM glutamine Cell lines were maintained at37∘C in 95 humidified air and 5 CO
2 Fibroblasts were
always used at an equivalent number of passages of growth(ranging from 5 to 10) Culture medium was always renewed24 h before each experiment [16]
Fibroblasts were exposed in vitro to VPA at concentra-tions ranging from05 to 10mM according to theHDAC IC
50
of this drug and to previous reports [12 17] Fibroblasts wereat the same time obtained from 12 sporadic ALS outpatientsrecruited at the San Gerardo Hospital Monza Italy (SALSMF 48 mean age plusmn SD 595 plusmn 12 years disease durationrange 1ndash50months ALSFRS-R score 253plusmn 10) and 9 age- andsex-comparable healthy controls (CTRLMF 45 age 62plusmn10years)
32 Blood Sample Preparation Whole-blood samples wereobtained from the antecubital vein in K
3-EDTA between
0800 and 0900 AM following overnight fasting Peripheralblood mononuclear cells (PBMC) isolation was obtainedby Ficoll-Histopaque density gradient centrifugation asdescribed before [18] PBMC pellets were then stocked atminus80∘C until assessment
33 Whole-Blood Global DNA Methylation Whole-bloodglobal DNA methylation was evaluated by an inverse assay[19] Briefly 1 120583g of whole-blood DNA was cut ON with a10-fold excess of both HpaII (cutting only nonmethylated 51015840-CCGG-31015840 recognition sites New England Biolabs) and MspI(cutting independently frommethylation status at 51015840-CCGG-31015840 recognition sites Fermentas Life Sciences) endonucleasesEach sample was then split in two aliquots incubated with1 120583L of 1 10 diluted [3H]dCTP (60Cimmol Amersham)
at 56∘C for 60min respectively in presence or absence ofTAQDNApolymerase (total volume 20120583L)The reactionwasstopped on ice and radioactivity beta-counted Total DNAmethylation was calculated as 1-(HpaIIMspI values)
34 Western Blotting Fibroblasts were collected and homog-enized in cell extraction buffer containing protease inhibitors(Sigma) and PMSF and protein concentration was assessedby Bradfordrsquos method Serial dilutions of each sample (5 1020120583g) were separated by NuPAGE 4ndash12 Bis-Tris gels (LifeTechnologies) in order to ensure the subsequent readingswithin the linear part of the detection curve and blottedonto Hybond nitrocellulose membranes (Amersham GEHealthcare) After blocking for 1 h at room temperature(RT) samples were incubated ON at 4∘C with either anti-SMN (1 5000 BD Biosciences) or anti-acetyl-histone H3(1 5000 Upstate Biotechnology) antibodies followed by thecorresponding peroxidase-conjugated secondary antibody(1 800090minRT) Following ECL Plus (Amersham GEHealthcare) application chemiluminescence was quantifiedby densitometer and expressed as optical density (OD) ofthe target protein and beta-actin (1 20000 Sigma) used asinternal standard
35 Immunohistochemistry Fibroblast cultures were fixedin 4 paraformaldehyde for 20 minutes and then incu-bated with 3 of triton (10min) and 5 BSA (45min)solutions Primary antibody mouse anti-SMN (BD Trans-duction Laboratories) at 1 100 was incubated ON at 4∘Cand secondary antibody Alexa Fluor 546 goat anti-mousetogether with Alexa Fluor 488-conjugated phalloidin (both at1 200 Invitrogen)was incubated for 1 h at room temperatureImageswere takenwith a confocalmicroscopy Radiance 2100microscope (Biorad Laboratories Hercules CA)
36 ChIP Assay ChIP assay was performed according tothe manufacturerrsquos protocol (EZ-ChIP Millipore) Briefly10e6 cells incubated or not with increasing concentrationof VPA were treated with 1 formaldehyde After cell lysiscross-linked chromatin was sonicated and then immuno-precipitated with antibody against acetylated H3 (Millipore)ProteinDNA complexes were reverted then ChIP enrichedDNA samples were quantified by real-time PCR and dataexpressed as percentage of input The primer pair used toamplify SMN promoter was 51015840-TTAAGGATCTGCCTT-CCTTCCTGC-31015840 and 51015840-ATGTTGCTTAGGCCTCGT-CTCGAA-31015840 PCR condition was 1 cycle at 50∘C for 2min1 cycle at 95∘C for 10min 40 cycles at 95∘C for 15 s 60∘C for30 s
37 Statistical Analysis Unpaired Studentrsquos 119905-test was used toassess the significance of differences between two treatmentregimens or two subject groups as appropriate Correlationwas computed with Pearsonrsquos 119903-test Statistical analysis wasperformed by Prism 400 (GraphPad Software Inc) Data areexpressed throughout the entire paper as mean plusmn SEM
4 Case Reports in Neurological Medicine
4 Results
Fibroblasts obtained from the proband did not display majormorphological alterations and a faint immunoreactive signalfor SMN was present within nuclear aggregates possiblymarking nuclear gems (Figure 2(a)) VPA treatment at theconcentration of 5mMfor 72 hwas able to induce an apparentincrease of SMN-positive nuclear aggregates with respectto vehicle-treated cells (Figure 2(b)) The same treatmentwas able to induce chromatin rearrangement since increasedvalues of acetyl-histone H3 were measured (sim65 119899 = 3data not shown) and ChIP assay documented a significantlyincreased association between acetyl-histone H3 and theSMN promoter (Figure 2(c)) When SMN expression wasassessed following exposure to VPA at concentrations rang-ing from 05 to 10mM for 72 h there was a trend towardan increase of SMN-like immunoreactive signal in total celllysates that became significant only at the concentrationof 10mM (about twofold increase see Figure 2(d)) How-ever the semiquantification of basal SMN-like immunore-active content (with respect to beta-actin) in the fibroblastsobtained from the proband was similar to that obtained inhealthy controls (Figures 2(e) and 2(f)) On the other handSALS patients (119899 = 12) showed about 50 lower SMN-likeimmunoreactive content with respect to the control group(119899 = 9 Figures 2(c) and 2(f)) without any significantrelationship with demographic or clinical variables Finallyglobal DNA methylation in PBMC obtained before and afterthe period of VPA administration (12 months at the doseof 300mg extended release bid per os) documented adecrease of sim50 of methyl-cytosine content consistent withthe demethylating properties of the drug while SMN-likeimmunoreactivity was apparently unchanged (119899 = 3 data notshown)
5 Discussion
The c194GgtA spastin mutation apparently expands thepreviously known borders of type 4 spastic paraplegia (SPG4)[20] causing the substitution within exon 1 of Arg with aHis at position 65 (pR65H) highly conserved across differentspecies as shown by in silico analysis Arguing against thehypothesis that this genetic variation might be pathogenicthere is the lack of segregation with the neuromusculardisease within the familiar group Furthermore current lackof functional assays certainly does not allow establishinga conclusion about the potential pathogenic effects of thereported change Intriguingly however the heterozygousduplication c304 309dupGCCTCG within the spastin genewas previously reported in a patient affected by a veryslowly progressing form of ALS [21] The same authorsfurther reported a case of rapidly progressing ALS bearing aheterozygous missense change (S44L) within the same gene[5] We may conclude hypothesizing that all these spastinvariations might have increased the risk for developing ALSalthough this hypothesis cannot be verified at the momentLower motoneuron involvement has already been sporad-ically described in similar patients (eg SPG17 see [22])and we find intriguing the possibility that the absence of
SMN2 might have acted as a further contributory risk factorin our hypothesis synergistically spreading motor neurondamage In fact the ultimate phenotypic expression of theproband was clinically indistinguishable from that of severaltypical ALS cases although initially the preponderance ofupper motor neuron dysfunction prompted looking forfurther non-ALS specific genetic characterization CertainlySMN2 deletion involves a limited albeit defined quote of thepopulation (about 8-9 in [11]) and its significance is notclarified since it has been proposed either as a protectivefactor [11] a noninfluent factor [23 24] or as a risk factor fordeveloping ALS [9 25]
We anyhow hypothesized that this particular patientcould be considered an ALS composite genocopy that isexpressing UMN pathology due to this novel spastin muta-tion that interacted with a LMN pathology whose risk ofappearance could have been increased by a concurrent minordecrease in SMN levels Certainly this is a speculation thatcannot be directly verified but exploring the relationshipbetween genotype and phenotype in selected ALS patientsincluding different genocopies and phenocopies might rep-resent an interesting strategy for understanding the clinicalvariability strongly characterizing this disorder Even if theexact clinical expression of this novel SPG4 mutation isnot yet known altered SMN expression has already beenhypothesized to play a role in ALS with quite conflictingresults [6 9 11] starting from the original hypothesis thatthe lack of this gene product might induce the same well-known dysfunction that determines and modulates SMAclinical phenotype [7 8] Consistent with the accessory rolecharacterizing SMN2 SMN total levels were not significantlydecreased in our patient fibroblasts On the other handour SALS patients displayed a significant decrease of thisprotein despite the limited number of recruited subjects asalready suggested [9] Further studies addressing SMN levelsin peripheral cells from ALS patients with respect to theirspecific SMN1 and SMN2 genotype might certainly clarifythis issue [26]
VPA was offered to our patient in an open label courseconsidering the epigenetic properties of this drug in differentneuropsychiatric conditionmodels and cell types [16 27]Thesystemic administration ofVPA is in fact able to increase SNClevels of acetyl-histone H3 [28] and to prevent the increasein 5-methyl-cytosine content following the administration ofhypermethylating agents [29] Furthermore SMN levels areselectively increased by the administration of VPAmodifyingSMN12 promoter structure [12 14] VPA has already beentested on SMA and ALS patients [30ndash32] albeit mainly withnegative results As a matter of fact our patientrsquos absence ofclinical response to VPA parallels the findings of these trials[30ndash33] and might also be consistent with the apparentlyhigh-enough residual levels of SMN in our patient althoughalternative biological explanations might apply [34] Forexample the proposed VPA dose was of 600mgdie con-ceivably corresponding to a plasma concentration rangingfrom sub- to just above the lower border of the recommendedtherapeutic window for epilepsy plausibly close to a valueroughly equivalent to the IC
50of this HDAC inhibitor
(sim04mM) [17] This dose could have been producing a quite
Case Reports in Neurological Medicine 5
VEH
(a)
VPA5mM72h
(b)
VEH 05 500
25
50
75
100
SMN
( o
f inp
ut)
lowast
VPA mM72h
(c)
VEH 05 5 100
1
2
3
4 ∘
SMN
120573-a
ctin
VPA mM72h
(d)
CTRL ALS
4
3
2
1
0
lowast
SMN
120573-a
ctin
(e)
120573-Actin
SMN
CTRL ALS
(f)
Figure 2 (a b) SMN immunoreactivity in fibroblasts obtained from the proband (scale bar = 10 120583m) a faint (red) staining was present withinnuclear structures (actin filaments counterstained in green) in vehicle-treated cells (VEH) while VPA 5mM72 h induced an apparent densityincrease of SMN-positive nuclear aggregates (c) acetyl-histone H3 association with SMN promoter was increased in fibroblast obtained fromthe proband following exposure to VPA 5mM72 h as semiquantified by ChIP assay (lowast119875 lt 005) (d) SMN-like immunoreactive content infibroblasts obtained from the proband is increased following in vitro exposure to VPA as semiquantified by Western blotting (∘119875 lt 005)(e) (Western blotting) SMN-like immunoreactive content in fibroblasts obtained from 12 SALS patients is reduced with respect to 9 matchedcontrols (lowast119875 = 001) as also shown by (f) representative immunoreactive signals (10 120583g of total protein) obtained in 5 different SALS patientsand 4 CTRL subjects
6 Case Reports in Neurological Medicine
small effect not eventually reaching clinical threshold Forthis reason we decided to include in ex vivo experiments adose one order of magnitude higher in order to magnify theexpected outcomes [12]
However recent suggestions that modifications of theepigenome might be of interest for understanding clinicalvariability in ALS [19 35] might further justify the attemptsof testing in the future other compounds able to modifyhistone acetylation or histone andor DNAmethylation [36]In fact in spite of the fact that ALS clinical heterogeneitymight imply differences in the involved pathophysiology acommon downstream pathway converging on the regulationof mRNA expression has been repeatedly demonstratedsince the discovery of TARDP43 and FUSTLS mutationsin ALS [37 38] Moreover an epigenetic dysfunction hasbeen hypothesized in several complex disorders for whichone single clear-cut mechanism cannot be demonstrated[39] such as in the case of ALS Interestingly TDP-43 andFUSTLS localize to nuclear gems through an associationwith SMN [15 40] implying that defective integrity of thespliceosome might be started by an expression or functionaldefect in any of these gene products leading at least to LMNdysfunction
6 Conclusion
The case we report here is carrier of both a novel spastinmutation and plausibly a minor defective SMN productionpossibly leading to a composite ALS genocopy Describingpeculiar ALS genocopies and phenocopies might be ofinterest for the field since dissecting the roads of phenotypiccomplexity inmotor neuron disorders conceivably representsone way to eventually find an effective cure for ALS
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors would like to thank the Italian Association forAmyotrophic Lateral Sclerosis (AISLA)
References
[1] B R Brooks R G Miller M Swash and T L MunsatldquoEl Escorial revisited revised criteria for the diagnosis ofamyotrophic lateral sclerosisrdquoAmyotrophic Lateral Sclerosis vol1 no 5 pp 293ndash299 2000
[2] L C Kwee Y Liu C Haynes et al ldquoA high-density genome-wide association screen of sporadic ALS in US veteransrdquo PLoSONE vol 7 no 3 Article ID e32768 2012
[3] M Sabatelli A Conte and M Zollino ldquoClinical and geneticheterogeneity of amyotrophic lateral sclerosisrdquo Clinical Genet-ics vol 83 no 5 pp 408ndash416 2013
[4] M J Strong and P H Gordon ldquoPrimary lateral sclerosishereditary spastic paraplegia and amyotrophic lateral sclerosis
discrete entities or spectrumrdquo Amyotrophic Lateral Sclerosisand Other Motor Neuron Disorders vol 6 no 1 pp 8ndash16 2005
[5] C Munch A Rolfs and T Meyer ldquoHeterozygous S44Lmissense change of the spastin gene in amyotrophic lateralsclerosisrdquo Amyotrophic Lateral Sclerosis vol 9 no 4 pp 251ndash253 2008
[6] H M Blauw C P Barnes P W J van Vught et al ldquoSMN1 geneduplications are associated with sporadic ALSrdquo Neurology vol78 no 11 pp 776ndash780 2012
[7] F Petit J Cuisset N Rouaix-Emery et al ldquoInsights intogenotype-phenotype correlations in spinal muscular atrophy aretrospective study of 103 patientsrdquoMuscle amp Nerve vol 43 no1 pp 26ndash30 2011
[8] G Y Zheleznyakova A V Kiselev V G Vakharlovsky et alldquoGenetic and expression studies of SMN2 gene in Russianpatients with spinal muscular atrophy type II and IIIrdquo BMCMedical Genetics vol 12 article 96 2011
[9] J H Veldink S Kalmijn A H van der Hout et al ldquoSMNgenotypes producing less SMN protein increase susceptibilityto and severity of sporadic ALSrdquo Neurology vol 65 no 6 pp820ndash825 2005
[10] J Kim S Lee Y Choi et al ldquoAssociation between survivormotor neuron 2 (SMN2) gene homozygous deletion and spo-radic lower motor neuron disease in a Korean populationrdquoAnnals of Clinical and Laboratory Science vol 40 no 4 pp 368ndash374 2010
[11] P Corcia C Ingre H Blasco et al ldquoHomozygous SMN2deletion is a protective factor in the Swedish ALS populationrdquoEuropean Journal of Human Genetics vol 20 no 5 pp 588ndash5912012
[12] C J Sumner T N Huynh J A Markowitz et al ldquoValproic acidincreases SMN levels in spinal muscular atrophy patient cellsrdquoAnnals of Neurology vol 54 no 5 pp 647ndash654 2003
[13] L Tremolizzo V Rodriguez-Menendez G Sala J C diFrancesco and C Ferrarese ldquoValproate and HDAC inhibitiona new epigenetic strategy to mitigate phenotypic severity inALSrdquo Amyotrophic Lateral Sclerosis and Other Motor NeuronDisorders vol 6 no 3 pp 185ndash186 2005
[14] L E Kernochan M L Russo N S Woodling et al ldquoThe role ofhistone acetylation in SMN gene expressionrdquoHumanMolecularGenetics vol 14 no 9 pp 1171ndash1182 2005
[15] H Tsuiji Y Iguchi A Furuya et al ldquoSpliceosome integrity isdefective in the motor neuron diseases ALS and SMArdquo EMBOMolecular Medicine vol 5 no 2 pp 221ndash234 2013
[16] G Sala F Trombin L Mattavelli et al ldquoLack of evidencefor oxidative stress in sporadic amyotrophic lateral sclerosisfibroblastsrdquoNeurodegenerative Diseases vol 6 no 1-2 pp 9ndash152008
[17] L Tremolizzo J C DiFrancesco V Rodriguez-Menendezet al ldquoValproate induces epigenetic modifications in lym-phomonocytes from epileptic patientsrdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 39 no 1 pp47ndash51 2012
[18] G Sala L Tremolizzo L Melchionda et al ldquoA panel ofmacroautophagymarkers in lymphomonocytes of patients withamyotrophic lateral sclerosisrdquoAmyotrophic Lateral Sclerosis vol13 no 1 pp 119ndash124 2012
[19] L Tremolizzo P Messina E Conti et al ldquoWhole-blood globalDNA methylation is increased in amyotrophic lateral sclerosisindependently of age of onsetrdquo Amyotroph Lateral Scler Fron-totemporal Degener vol 15 pp 98ndash105 2014
Case Reports in Neurological Medicine 7
[20] C J McDermott C E Burness J Kirby et al ldquoClinical featuresof hereditary spastic paraplegia due to spastin mutationrdquoNeurology vol 67 no 1 pp 45ndash51 2006
[21] T Meyer A Schwan J S Dullinger et al ldquoEarly-onset ALSwith long-term survival associated with spastin genemutationrdquoNeurology vol 65 no 1 pp 141ndash143 2005
[22] J Irobi P van den Bergh L Merlini et al ldquoThe phenotypeof motor neuropathies associated with BSCL2 mutations isbroader than Silver syndrome and distal HMN type Vrdquo Brainvol 127 no 9 pp 2124ndash2130 2004
[23] P Corcia W Camu J-M Halimi et al ldquoSMN1 gene but notSMN2 is a risk factor for sporadic ALSrdquo Neurology vol 67 no7 pp 1147ndash1150 2006
[24] X B Wang N H Cui J J Gao X P Qiu and F ZhengldquoSMN1 duplications contribute to sporadic amyotrophic lateralsclerosis susceptibility evidence from a meta-analysisrdquo Journalof the Neurological Sciences vol 340 no 1-2 pp 63ndash68 2014
[25] J H Veldink L H van den Berg J M Cobben et alldquoHomozygous deletion of the survival motor neuron 2 gene is aprognostic factor in sporadic ALSrdquoNeurology vol 56 no 6 pp749ndash752 2001
[26] S Piepers J Cobben P Sodaar et al ldquoQuantification of SMNprotein in leucocytes from spinal muscular atrophy patientseffects of treatment with valproic acidrdquo Journal of NeurologyNeurosurgery and Psychiatry vol 82 no 8 pp 850ndash852 2011
[27] L Tremolizzo V Rodriguez-Menendez E Conti C P ZoiaG Cavaletti and C Ferrarese ldquoNovel therapeutic targetsin neuropsychiatric disorders the neuroepigenomerdquo CurrentPharmaceutical Design vol 20 no 11 pp 1831ndash1839 2014
[28] L Tremolizzo G Carboni W B Ruzicka et al ldquoAn epigeneticmouse model for molecular and behavioral neuropatholo-gies related to schizophrenia vulnerabilityrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 99 no 26 pp 17095ndash17100 2002
[29] L Tremolizzo M S Doueiri E Dong et al ldquoValproatecorrects the schizophrenia-like epigenetic behavioral modifica-tions induced bymethionine inmicerdquo Biological Psychiatry vol57 no 5 pp 500ndash509 2005
[30] C C Weihl A M Connolly and A Pestronk ldquoValproate mayimprove strength and function in patients with type IIIIVspinal muscle atrophyrdquo Neurology vol 67 no 3 pp 500ndash5012006
[31] I A Darbar P G Plaggert M B D Resende E Zanoteli andU C Reed ldquoEvaluation of muscle strength and motor abilitiesin children with type II and III spinal muscle atrophy treatedwith valproic acidrdquo BMC Neurology vol 11 article 36 2011
[32] S Piepers J H Veldink S W de Jong et al ldquoRandomizedsequential trial of valproic acid in amyotrophic lateral sclerosisrdquoAnnals of Neurology vol 66 no 2 pp 227ndash234 2009
[33] J T Kissel B Elsheikh W M King et al ldquoSMA valiant triala prospective double-blind placebo-controlled trial of valproicacid in ambulatory adults with spinalmuscular atrophyrdquoMuscleamp Nerve vol 49 no 2 pp 187ndash192 2013
[34] L Garbes L Heesen I Holker et al ldquoVPA response in SMAis suppressed by the fatty acid translocase CD36rdquo HumanMolecular Genetics vol 22 no 2 pp 398ndash407 2013
[35] B A Chestnut Q Chang A Price C Lesuisse M Wong andL J Martin ldquoEpigenetic regulation of motor neuron cell deaththrough DNAmethylationrdquoThe Journal of Neuroscience vol 31no 46 pp 16619ndash16636 2011
[36] E Beghi E Pupillo V Bonito et al ldquoRandomized double-blind placebo-controlled trial of acetyl-L-carnitine for ALSrdquoAmyotrophic Lateral Sclerosis and Frontotemporal Degenerationvol 14 no 5-6 pp 397ndash405 2013
[37] A Verma and R Tandan ldquoRNA quality control and proteinaggregates in amyotrophic lateral sclerosis a reviewrdquo Muscleand Nerve vol 47 no 3 pp 330ndash338 2013
[38] S C Ling M Polymenidou and D W Cleveland ldquoConvergingmechanisms in als and FTD disrupted RNA and proteinhomeostasisrdquo Neuron vol 79 no 3 pp 416ndash438 2013
[39] A Petronis ldquoEpigenetics as a unifying principle in the aetiologyof complex traits and diseasesrdquo Nature vol 465 no 7299 pp721ndash727 2010
[40] T Yamazaki S Chen Y Yu et al ldquoFUS- SMN protein inter-actions link the motor neuron diseases ALS and SMArdquo CellReports vol 2 no 4 pp 799ndash806 2012
Fibroblasts obtained from the proband did not display majormorphological alterations and a faint immunoreactive signalfor SMN was present within nuclear aggregates possiblymarking nuclear gems (Figure 2(a)) VPA treatment at theconcentration of 5mMfor 72 hwas able to induce an apparentincrease of SMN-positive nuclear aggregates with respectto vehicle-treated cells (Figure 2(b)) The same treatmentwas able to induce chromatin rearrangement since increasedvalues of acetyl-histone H3 were measured (sim65 119899 = 3data not shown) and ChIP assay documented a significantlyincreased association between acetyl-histone H3 and theSMN promoter (Figure 2(c)) When SMN expression wasassessed following exposure to VPA at concentrations rang-ing from 05 to 10mM for 72 h there was a trend towardan increase of SMN-like immunoreactive signal in total celllysates that became significant only at the concentrationof 10mM (about twofold increase see Figure 2(d)) How-ever the semiquantification of basal SMN-like immunore-active content (with respect to beta-actin) in the fibroblastsobtained from the proband was similar to that obtained inhealthy controls (Figures 2(e) and 2(f)) On the other handSALS patients (119899 = 12) showed about 50 lower SMN-likeimmunoreactive content with respect to the control group(119899 = 9 Figures 2(c) and 2(f)) without any significantrelationship with demographic or clinical variables Finallyglobal DNA methylation in PBMC obtained before and afterthe period of VPA administration (12 months at the doseof 300mg extended release bid per os) documented adecrease of sim50 of methyl-cytosine content consistent withthe demethylating properties of the drug while SMN-likeimmunoreactivity was apparently unchanged (119899 = 3 data notshown)
5 Discussion
The c194GgtA spastin mutation apparently expands thepreviously known borders of type 4 spastic paraplegia (SPG4)[20] causing the substitution within exon 1 of Arg with aHis at position 65 (pR65H) highly conserved across differentspecies as shown by in silico analysis Arguing against thehypothesis that this genetic variation might be pathogenicthere is the lack of segregation with the neuromusculardisease within the familiar group Furthermore current lackof functional assays certainly does not allow establishinga conclusion about the potential pathogenic effects of thereported change Intriguingly however the heterozygousduplication c304 309dupGCCTCG within the spastin genewas previously reported in a patient affected by a veryslowly progressing form of ALS [21] The same authorsfurther reported a case of rapidly progressing ALS bearing aheterozygous missense change (S44L) within the same gene[5] We may conclude hypothesizing that all these spastinvariations might have increased the risk for developing ALSalthough this hypothesis cannot be verified at the momentLower motoneuron involvement has already been sporad-ically described in similar patients (eg SPG17 see [22])and we find intriguing the possibility that the absence of
SMN2 might have acted as a further contributory risk factorin our hypothesis synergistically spreading motor neurondamage In fact the ultimate phenotypic expression of theproband was clinically indistinguishable from that of severaltypical ALS cases although initially the preponderance ofupper motor neuron dysfunction prompted looking forfurther non-ALS specific genetic characterization CertainlySMN2 deletion involves a limited albeit defined quote of thepopulation (about 8-9 in [11]) and its significance is notclarified since it has been proposed either as a protectivefactor [11] a noninfluent factor [23 24] or as a risk factor fordeveloping ALS [9 25]
We anyhow hypothesized that this particular patientcould be considered an ALS composite genocopy that isexpressing UMN pathology due to this novel spastin muta-tion that interacted with a LMN pathology whose risk ofappearance could have been increased by a concurrent minordecrease in SMN levels Certainly this is a speculation thatcannot be directly verified but exploring the relationshipbetween genotype and phenotype in selected ALS patientsincluding different genocopies and phenocopies might rep-resent an interesting strategy for understanding the clinicalvariability strongly characterizing this disorder Even if theexact clinical expression of this novel SPG4 mutation isnot yet known altered SMN expression has already beenhypothesized to play a role in ALS with quite conflictingresults [6 9 11] starting from the original hypothesis thatthe lack of this gene product might induce the same well-known dysfunction that determines and modulates SMAclinical phenotype [7 8] Consistent with the accessory rolecharacterizing SMN2 SMN total levels were not significantlydecreased in our patient fibroblasts On the other handour SALS patients displayed a significant decrease of thisprotein despite the limited number of recruited subjects asalready suggested [9] Further studies addressing SMN levelsin peripheral cells from ALS patients with respect to theirspecific SMN1 and SMN2 genotype might certainly clarifythis issue [26]
VPA was offered to our patient in an open label courseconsidering the epigenetic properties of this drug in differentneuropsychiatric conditionmodels and cell types [16 27]Thesystemic administration ofVPA is in fact able to increase SNClevels of acetyl-histone H3 [28] and to prevent the increasein 5-methyl-cytosine content following the administration ofhypermethylating agents [29] Furthermore SMN levels areselectively increased by the administration of VPAmodifyingSMN12 promoter structure [12 14] VPA has already beentested on SMA and ALS patients [30ndash32] albeit mainly withnegative results As a matter of fact our patientrsquos absence ofclinical response to VPA parallels the findings of these trials[30ndash33] and might also be consistent with the apparentlyhigh-enough residual levels of SMN in our patient althoughalternative biological explanations might apply [34] Forexample the proposed VPA dose was of 600mgdie con-ceivably corresponding to a plasma concentration rangingfrom sub- to just above the lower border of the recommendedtherapeutic window for epilepsy plausibly close to a valueroughly equivalent to the IC
50of this HDAC inhibitor
(sim04mM) [17] This dose could have been producing a quite
Case Reports in Neurological Medicine 5
VEH
(a)
VPA5mM72h
(b)
VEH 05 500
25
50
75
100
SMN
( o
f inp
ut)
lowast
VPA mM72h
(c)
VEH 05 5 100
1
2
3
4 ∘
SMN
120573-a
ctin
VPA mM72h
(d)
CTRL ALS
4
3
2
1
0
lowast
SMN
120573-a
ctin
(e)
120573-Actin
SMN
CTRL ALS
(f)
Figure 2 (a b) SMN immunoreactivity in fibroblasts obtained from the proband (scale bar = 10 120583m) a faint (red) staining was present withinnuclear structures (actin filaments counterstained in green) in vehicle-treated cells (VEH) while VPA 5mM72 h induced an apparent densityincrease of SMN-positive nuclear aggregates (c) acetyl-histone H3 association with SMN promoter was increased in fibroblast obtained fromthe proband following exposure to VPA 5mM72 h as semiquantified by ChIP assay (lowast119875 lt 005) (d) SMN-like immunoreactive content infibroblasts obtained from the proband is increased following in vitro exposure to VPA as semiquantified by Western blotting (∘119875 lt 005)(e) (Western blotting) SMN-like immunoreactive content in fibroblasts obtained from 12 SALS patients is reduced with respect to 9 matchedcontrols (lowast119875 = 001) as also shown by (f) representative immunoreactive signals (10 120583g of total protein) obtained in 5 different SALS patientsand 4 CTRL subjects
6 Case Reports in Neurological Medicine
small effect not eventually reaching clinical threshold Forthis reason we decided to include in ex vivo experiments adose one order of magnitude higher in order to magnify theexpected outcomes [12]
However recent suggestions that modifications of theepigenome might be of interest for understanding clinicalvariability in ALS [19 35] might further justify the attemptsof testing in the future other compounds able to modifyhistone acetylation or histone andor DNAmethylation [36]In fact in spite of the fact that ALS clinical heterogeneitymight imply differences in the involved pathophysiology acommon downstream pathway converging on the regulationof mRNA expression has been repeatedly demonstratedsince the discovery of TARDP43 and FUSTLS mutationsin ALS [37 38] Moreover an epigenetic dysfunction hasbeen hypothesized in several complex disorders for whichone single clear-cut mechanism cannot be demonstrated[39] such as in the case of ALS Interestingly TDP-43 andFUSTLS localize to nuclear gems through an associationwith SMN [15 40] implying that defective integrity of thespliceosome might be started by an expression or functionaldefect in any of these gene products leading at least to LMNdysfunction
6 Conclusion
The case we report here is carrier of both a novel spastinmutation and plausibly a minor defective SMN productionpossibly leading to a composite ALS genocopy Describingpeculiar ALS genocopies and phenocopies might be ofinterest for the field since dissecting the roads of phenotypiccomplexity inmotor neuron disorders conceivably representsone way to eventually find an effective cure for ALS
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors would like to thank the Italian Association forAmyotrophic Lateral Sclerosis (AISLA)
References
[1] B R Brooks R G Miller M Swash and T L MunsatldquoEl Escorial revisited revised criteria for the diagnosis ofamyotrophic lateral sclerosisrdquoAmyotrophic Lateral Sclerosis vol1 no 5 pp 293ndash299 2000
[2] L C Kwee Y Liu C Haynes et al ldquoA high-density genome-wide association screen of sporadic ALS in US veteransrdquo PLoSONE vol 7 no 3 Article ID e32768 2012
[3] M Sabatelli A Conte and M Zollino ldquoClinical and geneticheterogeneity of amyotrophic lateral sclerosisrdquo Clinical Genet-ics vol 83 no 5 pp 408ndash416 2013
[4] M J Strong and P H Gordon ldquoPrimary lateral sclerosishereditary spastic paraplegia and amyotrophic lateral sclerosis
discrete entities or spectrumrdquo Amyotrophic Lateral Sclerosisand Other Motor Neuron Disorders vol 6 no 1 pp 8ndash16 2005
[5] C Munch A Rolfs and T Meyer ldquoHeterozygous S44Lmissense change of the spastin gene in amyotrophic lateralsclerosisrdquo Amyotrophic Lateral Sclerosis vol 9 no 4 pp 251ndash253 2008
[6] H M Blauw C P Barnes P W J van Vught et al ldquoSMN1 geneduplications are associated with sporadic ALSrdquo Neurology vol78 no 11 pp 776ndash780 2012
[7] F Petit J Cuisset N Rouaix-Emery et al ldquoInsights intogenotype-phenotype correlations in spinal muscular atrophy aretrospective study of 103 patientsrdquoMuscle amp Nerve vol 43 no1 pp 26ndash30 2011
[8] G Y Zheleznyakova A V Kiselev V G Vakharlovsky et alldquoGenetic and expression studies of SMN2 gene in Russianpatients with spinal muscular atrophy type II and IIIrdquo BMCMedical Genetics vol 12 article 96 2011
[9] J H Veldink S Kalmijn A H van der Hout et al ldquoSMNgenotypes producing less SMN protein increase susceptibilityto and severity of sporadic ALSrdquo Neurology vol 65 no 6 pp820ndash825 2005
[10] J Kim S Lee Y Choi et al ldquoAssociation between survivormotor neuron 2 (SMN2) gene homozygous deletion and spo-radic lower motor neuron disease in a Korean populationrdquoAnnals of Clinical and Laboratory Science vol 40 no 4 pp 368ndash374 2010
[11] P Corcia C Ingre H Blasco et al ldquoHomozygous SMN2deletion is a protective factor in the Swedish ALS populationrdquoEuropean Journal of Human Genetics vol 20 no 5 pp 588ndash5912012
[12] C J Sumner T N Huynh J A Markowitz et al ldquoValproic acidincreases SMN levels in spinal muscular atrophy patient cellsrdquoAnnals of Neurology vol 54 no 5 pp 647ndash654 2003
[13] L Tremolizzo V Rodriguez-Menendez G Sala J C diFrancesco and C Ferrarese ldquoValproate and HDAC inhibitiona new epigenetic strategy to mitigate phenotypic severity inALSrdquo Amyotrophic Lateral Sclerosis and Other Motor NeuronDisorders vol 6 no 3 pp 185ndash186 2005
[14] L E Kernochan M L Russo N S Woodling et al ldquoThe role ofhistone acetylation in SMN gene expressionrdquoHumanMolecularGenetics vol 14 no 9 pp 1171ndash1182 2005
[15] H Tsuiji Y Iguchi A Furuya et al ldquoSpliceosome integrity isdefective in the motor neuron diseases ALS and SMArdquo EMBOMolecular Medicine vol 5 no 2 pp 221ndash234 2013
[16] G Sala F Trombin L Mattavelli et al ldquoLack of evidencefor oxidative stress in sporadic amyotrophic lateral sclerosisfibroblastsrdquoNeurodegenerative Diseases vol 6 no 1-2 pp 9ndash152008
[17] L Tremolizzo J C DiFrancesco V Rodriguez-Menendezet al ldquoValproate induces epigenetic modifications in lym-phomonocytes from epileptic patientsrdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 39 no 1 pp47ndash51 2012
[18] G Sala L Tremolizzo L Melchionda et al ldquoA panel ofmacroautophagymarkers in lymphomonocytes of patients withamyotrophic lateral sclerosisrdquoAmyotrophic Lateral Sclerosis vol13 no 1 pp 119ndash124 2012
[19] L Tremolizzo P Messina E Conti et al ldquoWhole-blood globalDNA methylation is increased in amyotrophic lateral sclerosisindependently of age of onsetrdquo Amyotroph Lateral Scler Fron-totemporal Degener vol 15 pp 98ndash105 2014
Case Reports in Neurological Medicine 7
[20] C J McDermott C E Burness J Kirby et al ldquoClinical featuresof hereditary spastic paraplegia due to spastin mutationrdquoNeurology vol 67 no 1 pp 45ndash51 2006
[21] T Meyer A Schwan J S Dullinger et al ldquoEarly-onset ALSwith long-term survival associated with spastin genemutationrdquoNeurology vol 65 no 1 pp 141ndash143 2005
[22] J Irobi P van den Bergh L Merlini et al ldquoThe phenotypeof motor neuropathies associated with BSCL2 mutations isbroader than Silver syndrome and distal HMN type Vrdquo Brainvol 127 no 9 pp 2124ndash2130 2004
[23] P Corcia W Camu J-M Halimi et al ldquoSMN1 gene but notSMN2 is a risk factor for sporadic ALSrdquo Neurology vol 67 no7 pp 1147ndash1150 2006
[24] X B Wang N H Cui J J Gao X P Qiu and F ZhengldquoSMN1 duplications contribute to sporadic amyotrophic lateralsclerosis susceptibility evidence from a meta-analysisrdquo Journalof the Neurological Sciences vol 340 no 1-2 pp 63ndash68 2014
[25] J H Veldink L H van den Berg J M Cobben et alldquoHomozygous deletion of the survival motor neuron 2 gene is aprognostic factor in sporadic ALSrdquoNeurology vol 56 no 6 pp749ndash752 2001
[26] S Piepers J Cobben P Sodaar et al ldquoQuantification of SMNprotein in leucocytes from spinal muscular atrophy patientseffects of treatment with valproic acidrdquo Journal of NeurologyNeurosurgery and Psychiatry vol 82 no 8 pp 850ndash852 2011
[27] L Tremolizzo V Rodriguez-Menendez E Conti C P ZoiaG Cavaletti and C Ferrarese ldquoNovel therapeutic targetsin neuropsychiatric disorders the neuroepigenomerdquo CurrentPharmaceutical Design vol 20 no 11 pp 1831ndash1839 2014
[28] L Tremolizzo G Carboni W B Ruzicka et al ldquoAn epigeneticmouse model for molecular and behavioral neuropatholo-gies related to schizophrenia vulnerabilityrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 99 no 26 pp 17095ndash17100 2002
[29] L Tremolizzo M S Doueiri E Dong et al ldquoValproatecorrects the schizophrenia-like epigenetic behavioral modifica-tions induced bymethionine inmicerdquo Biological Psychiatry vol57 no 5 pp 500ndash509 2005
[30] C C Weihl A M Connolly and A Pestronk ldquoValproate mayimprove strength and function in patients with type IIIIVspinal muscle atrophyrdquo Neurology vol 67 no 3 pp 500ndash5012006
[31] I A Darbar P G Plaggert M B D Resende E Zanoteli andU C Reed ldquoEvaluation of muscle strength and motor abilitiesin children with type II and III spinal muscle atrophy treatedwith valproic acidrdquo BMC Neurology vol 11 article 36 2011
[32] S Piepers J H Veldink S W de Jong et al ldquoRandomizedsequential trial of valproic acid in amyotrophic lateral sclerosisrdquoAnnals of Neurology vol 66 no 2 pp 227ndash234 2009
[33] J T Kissel B Elsheikh W M King et al ldquoSMA valiant triala prospective double-blind placebo-controlled trial of valproicacid in ambulatory adults with spinalmuscular atrophyrdquoMuscleamp Nerve vol 49 no 2 pp 187ndash192 2013
[34] L Garbes L Heesen I Holker et al ldquoVPA response in SMAis suppressed by the fatty acid translocase CD36rdquo HumanMolecular Genetics vol 22 no 2 pp 398ndash407 2013
[35] B A Chestnut Q Chang A Price C Lesuisse M Wong andL J Martin ldquoEpigenetic regulation of motor neuron cell deaththrough DNAmethylationrdquoThe Journal of Neuroscience vol 31no 46 pp 16619ndash16636 2011
[36] E Beghi E Pupillo V Bonito et al ldquoRandomized double-blind placebo-controlled trial of acetyl-L-carnitine for ALSrdquoAmyotrophic Lateral Sclerosis and Frontotemporal Degenerationvol 14 no 5-6 pp 397ndash405 2013
[37] A Verma and R Tandan ldquoRNA quality control and proteinaggregates in amyotrophic lateral sclerosis a reviewrdquo Muscleand Nerve vol 47 no 3 pp 330ndash338 2013
[38] S C Ling M Polymenidou and D W Cleveland ldquoConvergingmechanisms in als and FTD disrupted RNA and proteinhomeostasisrdquo Neuron vol 79 no 3 pp 416ndash438 2013
[39] A Petronis ldquoEpigenetics as a unifying principle in the aetiologyof complex traits and diseasesrdquo Nature vol 465 no 7299 pp721ndash727 2010
[40] T Yamazaki S Chen Y Yu et al ldquoFUS- SMN protein inter-actions link the motor neuron diseases ALS and SMArdquo CellReports vol 2 no 4 pp 799ndash806 2012
Figure 2 (a b) SMN immunoreactivity in fibroblasts obtained from the proband (scale bar = 10 120583m) a faint (red) staining was present withinnuclear structures (actin filaments counterstained in green) in vehicle-treated cells (VEH) while VPA 5mM72 h induced an apparent densityincrease of SMN-positive nuclear aggregates (c) acetyl-histone H3 association with SMN promoter was increased in fibroblast obtained fromthe proband following exposure to VPA 5mM72 h as semiquantified by ChIP assay (lowast119875 lt 005) (d) SMN-like immunoreactive content infibroblasts obtained from the proband is increased following in vitro exposure to VPA as semiquantified by Western blotting (∘119875 lt 005)(e) (Western blotting) SMN-like immunoreactive content in fibroblasts obtained from 12 SALS patients is reduced with respect to 9 matchedcontrols (lowast119875 = 001) as also shown by (f) representative immunoreactive signals (10 120583g of total protein) obtained in 5 different SALS patientsand 4 CTRL subjects
6 Case Reports in Neurological Medicine
small effect not eventually reaching clinical threshold Forthis reason we decided to include in ex vivo experiments adose one order of magnitude higher in order to magnify theexpected outcomes [12]
However recent suggestions that modifications of theepigenome might be of interest for understanding clinicalvariability in ALS [19 35] might further justify the attemptsof testing in the future other compounds able to modifyhistone acetylation or histone andor DNAmethylation [36]In fact in spite of the fact that ALS clinical heterogeneitymight imply differences in the involved pathophysiology acommon downstream pathway converging on the regulationof mRNA expression has been repeatedly demonstratedsince the discovery of TARDP43 and FUSTLS mutationsin ALS [37 38] Moreover an epigenetic dysfunction hasbeen hypothesized in several complex disorders for whichone single clear-cut mechanism cannot be demonstrated[39] such as in the case of ALS Interestingly TDP-43 andFUSTLS localize to nuclear gems through an associationwith SMN [15 40] implying that defective integrity of thespliceosome might be started by an expression or functionaldefect in any of these gene products leading at least to LMNdysfunction
6 Conclusion
The case we report here is carrier of both a novel spastinmutation and plausibly a minor defective SMN productionpossibly leading to a composite ALS genocopy Describingpeculiar ALS genocopies and phenocopies might be ofinterest for the field since dissecting the roads of phenotypiccomplexity inmotor neuron disorders conceivably representsone way to eventually find an effective cure for ALS
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors would like to thank the Italian Association forAmyotrophic Lateral Sclerosis (AISLA)
References
[1] B R Brooks R G Miller M Swash and T L MunsatldquoEl Escorial revisited revised criteria for the diagnosis ofamyotrophic lateral sclerosisrdquoAmyotrophic Lateral Sclerosis vol1 no 5 pp 293ndash299 2000
[2] L C Kwee Y Liu C Haynes et al ldquoA high-density genome-wide association screen of sporadic ALS in US veteransrdquo PLoSONE vol 7 no 3 Article ID e32768 2012
[3] M Sabatelli A Conte and M Zollino ldquoClinical and geneticheterogeneity of amyotrophic lateral sclerosisrdquo Clinical Genet-ics vol 83 no 5 pp 408ndash416 2013
[4] M J Strong and P H Gordon ldquoPrimary lateral sclerosishereditary spastic paraplegia and amyotrophic lateral sclerosis
discrete entities or spectrumrdquo Amyotrophic Lateral Sclerosisand Other Motor Neuron Disorders vol 6 no 1 pp 8ndash16 2005
[5] C Munch A Rolfs and T Meyer ldquoHeterozygous S44Lmissense change of the spastin gene in amyotrophic lateralsclerosisrdquo Amyotrophic Lateral Sclerosis vol 9 no 4 pp 251ndash253 2008
[6] H M Blauw C P Barnes P W J van Vught et al ldquoSMN1 geneduplications are associated with sporadic ALSrdquo Neurology vol78 no 11 pp 776ndash780 2012
[7] F Petit J Cuisset N Rouaix-Emery et al ldquoInsights intogenotype-phenotype correlations in spinal muscular atrophy aretrospective study of 103 patientsrdquoMuscle amp Nerve vol 43 no1 pp 26ndash30 2011
[8] G Y Zheleznyakova A V Kiselev V G Vakharlovsky et alldquoGenetic and expression studies of SMN2 gene in Russianpatients with spinal muscular atrophy type II and IIIrdquo BMCMedical Genetics vol 12 article 96 2011
[9] J H Veldink S Kalmijn A H van der Hout et al ldquoSMNgenotypes producing less SMN protein increase susceptibilityto and severity of sporadic ALSrdquo Neurology vol 65 no 6 pp820ndash825 2005
[10] J Kim S Lee Y Choi et al ldquoAssociation between survivormotor neuron 2 (SMN2) gene homozygous deletion and spo-radic lower motor neuron disease in a Korean populationrdquoAnnals of Clinical and Laboratory Science vol 40 no 4 pp 368ndash374 2010
[11] P Corcia C Ingre H Blasco et al ldquoHomozygous SMN2deletion is a protective factor in the Swedish ALS populationrdquoEuropean Journal of Human Genetics vol 20 no 5 pp 588ndash5912012
[12] C J Sumner T N Huynh J A Markowitz et al ldquoValproic acidincreases SMN levels in spinal muscular atrophy patient cellsrdquoAnnals of Neurology vol 54 no 5 pp 647ndash654 2003
[13] L Tremolizzo V Rodriguez-Menendez G Sala J C diFrancesco and C Ferrarese ldquoValproate and HDAC inhibitiona new epigenetic strategy to mitigate phenotypic severity inALSrdquo Amyotrophic Lateral Sclerosis and Other Motor NeuronDisorders vol 6 no 3 pp 185ndash186 2005
[14] L E Kernochan M L Russo N S Woodling et al ldquoThe role ofhistone acetylation in SMN gene expressionrdquoHumanMolecularGenetics vol 14 no 9 pp 1171ndash1182 2005
[15] H Tsuiji Y Iguchi A Furuya et al ldquoSpliceosome integrity isdefective in the motor neuron diseases ALS and SMArdquo EMBOMolecular Medicine vol 5 no 2 pp 221ndash234 2013
[16] G Sala F Trombin L Mattavelli et al ldquoLack of evidencefor oxidative stress in sporadic amyotrophic lateral sclerosisfibroblastsrdquoNeurodegenerative Diseases vol 6 no 1-2 pp 9ndash152008
[17] L Tremolizzo J C DiFrancesco V Rodriguez-Menendezet al ldquoValproate induces epigenetic modifications in lym-phomonocytes from epileptic patientsrdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 39 no 1 pp47ndash51 2012
[18] G Sala L Tremolizzo L Melchionda et al ldquoA panel ofmacroautophagymarkers in lymphomonocytes of patients withamyotrophic lateral sclerosisrdquoAmyotrophic Lateral Sclerosis vol13 no 1 pp 119ndash124 2012
[19] L Tremolizzo P Messina E Conti et al ldquoWhole-blood globalDNA methylation is increased in amyotrophic lateral sclerosisindependently of age of onsetrdquo Amyotroph Lateral Scler Fron-totemporal Degener vol 15 pp 98ndash105 2014
Case Reports in Neurological Medicine 7
[20] C J McDermott C E Burness J Kirby et al ldquoClinical featuresof hereditary spastic paraplegia due to spastin mutationrdquoNeurology vol 67 no 1 pp 45ndash51 2006
[21] T Meyer A Schwan J S Dullinger et al ldquoEarly-onset ALSwith long-term survival associated with spastin genemutationrdquoNeurology vol 65 no 1 pp 141ndash143 2005
[22] J Irobi P van den Bergh L Merlini et al ldquoThe phenotypeof motor neuropathies associated with BSCL2 mutations isbroader than Silver syndrome and distal HMN type Vrdquo Brainvol 127 no 9 pp 2124ndash2130 2004
[23] P Corcia W Camu J-M Halimi et al ldquoSMN1 gene but notSMN2 is a risk factor for sporadic ALSrdquo Neurology vol 67 no7 pp 1147ndash1150 2006
[24] X B Wang N H Cui J J Gao X P Qiu and F ZhengldquoSMN1 duplications contribute to sporadic amyotrophic lateralsclerosis susceptibility evidence from a meta-analysisrdquo Journalof the Neurological Sciences vol 340 no 1-2 pp 63ndash68 2014
[25] J H Veldink L H van den Berg J M Cobben et alldquoHomozygous deletion of the survival motor neuron 2 gene is aprognostic factor in sporadic ALSrdquoNeurology vol 56 no 6 pp749ndash752 2001
[26] S Piepers J Cobben P Sodaar et al ldquoQuantification of SMNprotein in leucocytes from spinal muscular atrophy patientseffects of treatment with valproic acidrdquo Journal of NeurologyNeurosurgery and Psychiatry vol 82 no 8 pp 850ndash852 2011
[27] L Tremolizzo V Rodriguez-Menendez E Conti C P ZoiaG Cavaletti and C Ferrarese ldquoNovel therapeutic targetsin neuropsychiatric disorders the neuroepigenomerdquo CurrentPharmaceutical Design vol 20 no 11 pp 1831ndash1839 2014
[28] L Tremolizzo G Carboni W B Ruzicka et al ldquoAn epigeneticmouse model for molecular and behavioral neuropatholo-gies related to schizophrenia vulnerabilityrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 99 no 26 pp 17095ndash17100 2002
[29] L Tremolizzo M S Doueiri E Dong et al ldquoValproatecorrects the schizophrenia-like epigenetic behavioral modifica-tions induced bymethionine inmicerdquo Biological Psychiatry vol57 no 5 pp 500ndash509 2005
[30] C C Weihl A M Connolly and A Pestronk ldquoValproate mayimprove strength and function in patients with type IIIIVspinal muscle atrophyrdquo Neurology vol 67 no 3 pp 500ndash5012006
[31] I A Darbar P G Plaggert M B D Resende E Zanoteli andU C Reed ldquoEvaluation of muscle strength and motor abilitiesin children with type II and III spinal muscle atrophy treatedwith valproic acidrdquo BMC Neurology vol 11 article 36 2011
[32] S Piepers J H Veldink S W de Jong et al ldquoRandomizedsequential trial of valproic acid in amyotrophic lateral sclerosisrdquoAnnals of Neurology vol 66 no 2 pp 227ndash234 2009
[33] J T Kissel B Elsheikh W M King et al ldquoSMA valiant triala prospective double-blind placebo-controlled trial of valproicacid in ambulatory adults with spinalmuscular atrophyrdquoMuscleamp Nerve vol 49 no 2 pp 187ndash192 2013
[34] L Garbes L Heesen I Holker et al ldquoVPA response in SMAis suppressed by the fatty acid translocase CD36rdquo HumanMolecular Genetics vol 22 no 2 pp 398ndash407 2013
[35] B A Chestnut Q Chang A Price C Lesuisse M Wong andL J Martin ldquoEpigenetic regulation of motor neuron cell deaththrough DNAmethylationrdquoThe Journal of Neuroscience vol 31no 46 pp 16619ndash16636 2011
[36] E Beghi E Pupillo V Bonito et al ldquoRandomized double-blind placebo-controlled trial of acetyl-L-carnitine for ALSrdquoAmyotrophic Lateral Sclerosis and Frontotemporal Degenerationvol 14 no 5-6 pp 397ndash405 2013
[37] A Verma and R Tandan ldquoRNA quality control and proteinaggregates in amyotrophic lateral sclerosis a reviewrdquo Muscleand Nerve vol 47 no 3 pp 330ndash338 2013
[38] S C Ling M Polymenidou and D W Cleveland ldquoConvergingmechanisms in als and FTD disrupted RNA and proteinhomeostasisrdquo Neuron vol 79 no 3 pp 416ndash438 2013
[39] A Petronis ldquoEpigenetics as a unifying principle in the aetiologyof complex traits and diseasesrdquo Nature vol 465 no 7299 pp721ndash727 2010
[40] T Yamazaki S Chen Y Yu et al ldquoFUS- SMN protein inter-actions link the motor neuron diseases ALS and SMArdquo CellReports vol 2 no 4 pp 799ndash806 2012
small effect not eventually reaching clinical threshold Forthis reason we decided to include in ex vivo experiments adose one order of magnitude higher in order to magnify theexpected outcomes [12]
However recent suggestions that modifications of theepigenome might be of interest for understanding clinicalvariability in ALS [19 35] might further justify the attemptsof testing in the future other compounds able to modifyhistone acetylation or histone andor DNAmethylation [36]In fact in spite of the fact that ALS clinical heterogeneitymight imply differences in the involved pathophysiology acommon downstream pathway converging on the regulationof mRNA expression has been repeatedly demonstratedsince the discovery of TARDP43 and FUSTLS mutationsin ALS [37 38] Moreover an epigenetic dysfunction hasbeen hypothesized in several complex disorders for whichone single clear-cut mechanism cannot be demonstrated[39] such as in the case of ALS Interestingly TDP-43 andFUSTLS localize to nuclear gems through an associationwith SMN [15 40] implying that defective integrity of thespliceosome might be started by an expression or functionaldefect in any of these gene products leading at least to LMNdysfunction
6 Conclusion
The case we report here is carrier of both a novel spastinmutation and plausibly a minor defective SMN productionpossibly leading to a composite ALS genocopy Describingpeculiar ALS genocopies and phenocopies might be ofinterest for the field since dissecting the roads of phenotypiccomplexity inmotor neuron disorders conceivably representsone way to eventually find an effective cure for ALS
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors would like to thank the Italian Association forAmyotrophic Lateral Sclerosis (AISLA)
References
[1] B R Brooks R G Miller M Swash and T L MunsatldquoEl Escorial revisited revised criteria for the diagnosis ofamyotrophic lateral sclerosisrdquoAmyotrophic Lateral Sclerosis vol1 no 5 pp 293ndash299 2000
[2] L C Kwee Y Liu C Haynes et al ldquoA high-density genome-wide association screen of sporadic ALS in US veteransrdquo PLoSONE vol 7 no 3 Article ID e32768 2012
[3] M Sabatelli A Conte and M Zollino ldquoClinical and geneticheterogeneity of amyotrophic lateral sclerosisrdquo Clinical Genet-ics vol 83 no 5 pp 408ndash416 2013
[4] M J Strong and P H Gordon ldquoPrimary lateral sclerosishereditary spastic paraplegia and amyotrophic lateral sclerosis
discrete entities or spectrumrdquo Amyotrophic Lateral Sclerosisand Other Motor Neuron Disorders vol 6 no 1 pp 8ndash16 2005
[5] C Munch A Rolfs and T Meyer ldquoHeterozygous S44Lmissense change of the spastin gene in amyotrophic lateralsclerosisrdquo Amyotrophic Lateral Sclerosis vol 9 no 4 pp 251ndash253 2008
[6] H M Blauw C P Barnes P W J van Vught et al ldquoSMN1 geneduplications are associated with sporadic ALSrdquo Neurology vol78 no 11 pp 776ndash780 2012
[7] F Petit J Cuisset N Rouaix-Emery et al ldquoInsights intogenotype-phenotype correlations in spinal muscular atrophy aretrospective study of 103 patientsrdquoMuscle amp Nerve vol 43 no1 pp 26ndash30 2011
[8] G Y Zheleznyakova A V Kiselev V G Vakharlovsky et alldquoGenetic and expression studies of SMN2 gene in Russianpatients with spinal muscular atrophy type II and IIIrdquo BMCMedical Genetics vol 12 article 96 2011
[9] J H Veldink S Kalmijn A H van der Hout et al ldquoSMNgenotypes producing less SMN protein increase susceptibilityto and severity of sporadic ALSrdquo Neurology vol 65 no 6 pp820ndash825 2005
[10] J Kim S Lee Y Choi et al ldquoAssociation between survivormotor neuron 2 (SMN2) gene homozygous deletion and spo-radic lower motor neuron disease in a Korean populationrdquoAnnals of Clinical and Laboratory Science vol 40 no 4 pp 368ndash374 2010
[11] P Corcia C Ingre H Blasco et al ldquoHomozygous SMN2deletion is a protective factor in the Swedish ALS populationrdquoEuropean Journal of Human Genetics vol 20 no 5 pp 588ndash5912012
[12] C J Sumner T N Huynh J A Markowitz et al ldquoValproic acidincreases SMN levels in spinal muscular atrophy patient cellsrdquoAnnals of Neurology vol 54 no 5 pp 647ndash654 2003
[13] L Tremolizzo V Rodriguez-Menendez G Sala J C diFrancesco and C Ferrarese ldquoValproate and HDAC inhibitiona new epigenetic strategy to mitigate phenotypic severity inALSrdquo Amyotrophic Lateral Sclerosis and Other Motor NeuronDisorders vol 6 no 3 pp 185ndash186 2005
[14] L E Kernochan M L Russo N S Woodling et al ldquoThe role ofhistone acetylation in SMN gene expressionrdquoHumanMolecularGenetics vol 14 no 9 pp 1171ndash1182 2005
[15] H Tsuiji Y Iguchi A Furuya et al ldquoSpliceosome integrity isdefective in the motor neuron diseases ALS and SMArdquo EMBOMolecular Medicine vol 5 no 2 pp 221ndash234 2013
[16] G Sala F Trombin L Mattavelli et al ldquoLack of evidencefor oxidative stress in sporadic amyotrophic lateral sclerosisfibroblastsrdquoNeurodegenerative Diseases vol 6 no 1-2 pp 9ndash152008
[17] L Tremolizzo J C DiFrancesco V Rodriguez-Menendezet al ldquoValproate induces epigenetic modifications in lym-phomonocytes from epileptic patientsrdquo Progress in Neuro-Psychopharmacology and Biological Psychiatry vol 39 no 1 pp47ndash51 2012
[18] G Sala L Tremolizzo L Melchionda et al ldquoA panel ofmacroautophagymarkers in lymphomonocytes of patients withamyotrophic lateral sclerosisrdquoAmyotrophic Lateral Sclerosis vol13 no 1 pp 119ndash124 2012
[19] L Tremolizzo P Messina E Conti et al ldquoWhole-blood globalDNA methylation is increased in amyotrophic lateral sclerosisindependently of age of onsetrdquo Amyotroph Lateral Scler Fron-totemporal Degener vol 15 pp 98ndash105 2014
Case Reports in Neurological Medicine 7
[20] C J McDermott C E Burness J Kirby et al ldquoClinical featuresof hereditary spastic paraplegia due to spastin mutationrdquoNeurology vol 67 no 1 pp 45ndash51 2006
[21] T Meyer A Schwan J S Dullinger et al ldquoEarly-onset ALSwith long-term survival associated with spastin genemutationrdquoNeurology vol 65 no 1 pp 141ndash143 2005
[22] J Irobi P van den Bergh L Merlini et al ldquoThe phenotypeof motor neuropathies associated with BSCL2 mutations isbroader than Silver syndrome and distal HMN type Vrdquo Brainvol 127 no 9 pp 2124ndash2130 2004
[23] P Corcia W Camu J-M Halimi et al ldquoSMN1 gene but notSMN2 is a risk factor for sporadic ALSrdquo Neurology vol 67 no7 pp 1147ndash1150 2006
[24] X B Wang N H Cui J J Gao X P Qiu and F ZhengldquoSMN1 duplications contribute to sporadic amyotrophic lateralsclerosis susceptibility evidence from a meta-analysisrdquo Journalof the Neurological Sciences vol 340 no 1-2 pp 63ndash68 2014
[25] J H Veldink L H van den Berg J M Cobben et alldquoHomozygous deletion of the survival motor neuron 2 gene is aprognostic factor in sporadic ALSrdquoNeurology vol 56 no 6 pp749ndash752 2001
[26] S Piepers J Cobben P Sodaar et al ldquoQuantification of SMNprotein in leucocytes from spinal muscular atrophy patientseffects of treatment with valproic acidrdquo Journal of NeurologyNeurosurgery and Psychiatry vol 82 no 8 pp 850ndash852 2011
[27] L Tremolizzo V Rodriguez-Menendez E Conti C P ZoiaG Cavaletti and C Ferrarese ldquoNovel therapeutic targetsin neuropsychiatric disorders the neuroepigenomerdquo CurrentPharmaceutical Design vol 20 no 11 pp 1831ndash1839 2014
[28] L Tremolizzo G Carboni W B Ruzicka et al ldquoAn epigeneticmouse model for molecular and behavioral neuropatholo-gies related to schizophrenia vulnerabilityrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 99 no 26 pp 17095ndash17100 2002
[29] L Tremolizzo M S Doueiri E Dong et al ldquoValproatecorrects the schizophrenia-like epigenetic behavioral modifica-tions induced bymethionine inmicerdquo Biological Psychiatry vol57 no 5 pp 500ndash509 2005
[30] C C Weihl A M Connolly and A Pestronk ldquoValproate mayimprove strength and function in patients with type IIIIVspinal muscle atrophyrdquo Neurology vol 67 no 3 pp 500ndash5012006
[31] I A Darbar P G Plaggert M B D Resende E Zanoteli andU C Reed ldquoEvaluation of muscle strength and motor abilitiesin children with type II and III spinal muscle atrophy treatedwith valproic acidrdquo BMC Neurology vol 11 article 36 2011
[32] S Piepers J H Veldink S W de Jong et al ldquoRandomizedsequential trial of valproic acid in amyotrophic lateral sclerosisrdquoAnnals of Neurology vol 66 no 2 pp 227ndash234 2009
[33] J T Kissel B Elsheikh W M King et al ldquoSMA valiant triala prospective double-blind placebo-controlled trial of valproicacid in ambulatory adults with spinalmuscular atrophyrdquoMuscleamp Nerve vol 49 no 2 pp 187ndash192 2013
[34] L Garbes L Heesen I Holker et al ldquoVPA response in SMAis suppressed by the fatty acid translocase CD36rdquo HumanMolecular Genetics vol 22 no 2 pp 398ndash407 2013
[35] B A Chestnut Q Chang A Price C Lesuisse M Wong andL J Martin ldquoEpigenetic regulation of motor neuron cell deaththrough DNAmethylationrdquoThe Journal of Neuroscience vol 31no 46 pp 16619ndash16636 2011
[36] E Beghi E Pupillo V Bonito et al ldquoRandomized double-blind placebo-controlled trial of acetyl-L-carnitine for ALSrdquoAmyotrophic Lateral Sclerosis and Frontotemporal Degenerationvol 14 no 5-6 pp 397ndash405 2013
[37] A Verma and R Tandan ldquoRNA quality control and proteinaggregates in amyotrophic lateral sclerosis a reviewrdquo Muscleand Nerve vol 47 no 3 pp 330ndash338 2013
[38] S C Ling M Polymenidou and D W Cleveland ldquoConvergingmechanisms in als and FTD disrupted RNA and proteinhomeostasisrdquo Neuron vol 79 no 3 pp 416ndash438 2013
[39] A Petronis ldquoEpigenetics as a unifying principle in the aetiologyof complex traits and diseasesrdquo Nature vol 465 no 7299 pp721ndash727 2010
[40] T Yamazaki S Chen Y Yu et al ldquoFUS- SMN protein inter-actions link the motor neuron diseases ALS and SMArdquo CellReports vol 2 no 4 pp 799ndash806 2012
[20] C J McDermott C E Burness J Kirby et al ldquoClinical featuresof hereditary spastic paraplegia due to spastin mutationrdquoNeurology vol 67 no 1 pp 45ndash51 2006
[21] T Meyer A Schwan J S Dullinger et al ldquoEarly-onset ALSwith long-term survival associated with spastin genemutationrdquoNeurology vol 65 no 1 pp 141ndash143 2005
[22] J Irobi P van den Bergh L Merlini et al ldquoThe phenotypeof motor neuropathies associated with BSCL2 mutations isbroader than Silver syndrome and distal HMN type Vrdquo Brainvol 127 no 9 pp 2124ndash2130 2004
[23] P Corcia W Camu J-M Halimi et al ldquoSMN1 gene but notSMN2 is a risk factor for sporadic ALSrdquo Neurology vol 67 no7 pp 1147ndash1150 2006
[24] X B Wang N H Cui J J Gao X P Qiu and F ZhengldquoSMN1 duplications contribute to sporadic amyotrophic lateralsclerosis susceptibility evidence from a meta-analysisrdquo Journalof the Neurological Sciences vol 340 no 1-2 pp 63ndash68 2014
[25] J H Veldink L H van den Berg J M Cobben et alldquoHomozygous deletion of the survival motor neuron 2 gene is aprognostic factor in sporadic ALSrdquoNeurology vol 56 no 6 pp749ndash752 2001
[26] S Piepers J Cobben P Sodaar et al ldquoQuantification of SMNprotein in leucocytes from spinal muscular atrophy patientseffects of treatment with valproic acidrdquo Journal of NeurologyNeurosurgery and Psychiatry vol 82 no 8 pp 850ndash852 2011
[27] L Tremolizzo V Rodriguez-Menendez E Conti C P ZoiaG Cavaletti and C Ferrarese ldquoNovel therapeutic targetsin neuropsychiatric disorders the neuroepigenomerdquo CurrentPharmaceutical Design vol 20 no 11 pp 1831ndash1839 2014
[28] L Tremolizzo G Carboni W B Ruzicka et al ldquoAn epigeneticmouse model for molecular and behavioral neuropatholo-gies related to schizophrenia vulnerabilityrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 99 no 26 pp 17095ndash17100 2002
[29] L Tremolizzo M S Doueiri E Dong et al ldquoValproatecorrects the schizophrenia-like epigenetic behavioral modifica-tions induced bymethionine inmicerdquo Biological Psychiatry vol57 no 5 pp 500ndash509 2005
[30] C C Weihl A M Connolly and A Pestronk ldquoValproate mayimprove strength and function in patients with type IIIIVspinal muscle atrophyrdquo Neurology vol 67 no 3 pp 500ndash5012006
[31] I A Darbar P G Plaggert M B D Resende E Zanoteli andU C Reed ldquoEvaluation of muscle strength and motor abilitiesin children with type II and III spinal muscle atrophy treatedwith valproic acidrdquo BMC Neurology vol 11 article 36 2011
[32] S Piepers J H Veldink S W de Jong et al ldquoRandomizedsequential trial of valproic acid in amyotrophic lateral sclerosisrdquoAnnals of Neurology vol 66 no 2 pp 227ndash234 2009
[33] J T Kissel B Elsheikh W M King et al ldquoSMA valiant triala prospective double-blind placebo-controlled trial of valproicacid in ambulatory adults with spinalmuscular atrophyrdquoMuscleamp Nerve vol 49 no 2 pp 187ndash192 2013
[34] L Garbes L Heesen I Holker et al ldquoVPA response in SMAis suppressed by the fatty acid translocase CD36rdquo HumanMolecular Genetics vol 22 no 2 pp 398ndash407 2013
[35] B A Chestnut Q Chang A Price C Lesuisse M Wong andL J Martin ldquoEpigenetic regulation of motor neuron cell deaththrough DNAmethylationrdquoThe Journal of Neuroscience vol 31no 46 pp 16619ndash16636 2011
[36] E Beghi E Pupillo V Bonito et al ldquoRandomized double-blind placebo-controlled trial of acetyl-L-carnitine for ALSrdquoAmyotrophic Lateral Sclerosis and Frontotemporal Degenerationvol 14 no 5-6 pp 397ndash405 2013
[37] A Verma and R Tandan ldquoRNA quality control and proteinaggregates in amyotrophic lateral sclerosis a reviewrdquo Muscleand Nerve vol 47 no 3 pp 330ndash338 2013
[38] S C Ling M Polymenidou and D W Cleveland ldquoConvergingmechanisms in als and FTD disrupted RNA and proteinhomeostasisrdquo Neuron vol 79 no 3 pp 416ndash438 2013
[39] A Petronis ldquoEpigenetics as a unifying principle in the aetiologyof complex traits and diseasesrdquo Nature vol 465 no 7299 pp721ndash727 2010
[40] T Yamazaki S Chen Y Yu et al ldquoFUS- SMN protein inter-actions link the motor neuron diseases ALS and SMArdquo CellReports vol 2 no 4 pp 799ndash806 2012
