A Novel Mutation in the Deoxyguanosine Kinase Gene

BRIEFCOMMUNICATIONS

decreasedinnervation.13 Thelatterfindingaffordedan ProgressiveLossofCardiac opportunitytodeterminewhetherthelossofcardiac SympatheticInnervationin sympathetic innervation in Parkinson’s disease progressesovertimeand,ifso,withwhattiming,pat- Parkinson’sDisease tern,andconsistencyacrosspatients.Thisreportde- scribestheresultsofretestingsuchpatientswith Sheng-TingLi,MD,PhD,RaghuveerDendi,MD, 6-[18F]fluorodopaminepositronemissiontomography CourtneyHolmes,CMT, scanningafteranaverageof2years. andDavidS.Goldstein,MD,PhD

PatientsandMethods Thisstudyaddressedwhethercardiacsympathetic denervationprogressesovertimeinParkinson’sdis- ThestudyprotocolwasapprovedbytheIntramuralResearch ease.In9patientswithoutorthostatichypotension, BoardoftheNationalInstituteofNeurologicalDisorders 6-[18F]fluorodopaminepositronemissiontomography andStroke.Eachpatientgaveinformed,writtenconsent. scanningwasrepeatedafterameanof2yearsfromthe firstscan.6-[18F]fluorodopamine-derivedradioactivity waslessinthesecondscanthaninthefirstscan,by Patients 31%intheleftventricularfreewalland16%inthe Thoracicpositronemissiontomographyscanningwasper- septum.InParkinson’sdisease,lossofcardiacsympa- formedafterintravenousinjectionof6-[18F]fluoro- theticdenervationprogressesinapatternoflosssug- dopaminein9patientswithParkinson’sdisease(age: gestingadying-backmechanism. mean,60years;SEM,3years).Noneofthepatientshad orthostatichypotension,whichwasdefinedasadecreasein AnnNeurol2002;52:220–223 systolicbloodpressuregreaterthan20mmHganddecrease indiastolicpressuregreaterthan5mmHgbetweenthesu- pinepositionandstandingfor5minutes.Caffeine- containingbeverages,cigarettes,andalcoholwerenotal- Allofatleastadozenstudieshaveagreedthatpatients lowedforatleast24hoursbeforethescanning.Patients withParkinson’sdiseasehaveahighprevalenceofneu- wereallowedtotaketheirusualmedications,including roimagingevidencefordecreasedsympatheticinnerva- L-dopa,exceptformedicationsknowntoinhibitneuronal tionoftheheart.Lowmyocardialconcentrationsofra- uptakeofcatecholamines. dioactivityhavebeennotedafterinjectionofthe sympathoneuralimagingagents 123I-metaiodobenzyl- guanidine1–12 and6-[18F]fluorodopamine.13 Neuro- PositronEmissionTomographyScanning 18 14 chemicalassessmentsduringrightheartcatheterization 6-[ F]fluorodopamine,synthesizedasdescribedpreviously, haveconfirmedthatlowconcentrationsofradioactivity wasinfusedintravenouslyataconstantratefor3minutes. resultfromlossoffunctionalcardiacsympatheticnerve Tomographyimages(35contiguoustransaxialslices4.25mm 13 apart)wereacquiredforupto30minutes.Three- terminals. dimensionalpositronemissiontomographyscanswereob- AlthoughallpatientswithParkinson’sdiseaseand tainedwithanAdvancewhole-bodyscanner(GeneralElec- orthostatichypotensionhavediffuselydecreasedsym- tric,Milwaukee,WI).Transmissionscansof2minutesand patheticinnervationthroughouttheleftventricular 8minutes,withrotating 68Ge/68Gapinsources,wereob- myocardium,amongpatientswhodonothaveortho- tainedforattenuationcorrectionandforconfirmingproper statichypotension,abouthalfhavediffuselydecreased positioninginthescanner. innervationandabouthalfhavenormaloronlylocally Follow-upscanningwasperformed1to4years(mean, 2.0years;SEM,0.3years)afterthefirstscan,withtheiden- ticalscanningprocedure.Noneofthepatientshadortho- statichypotensionatthetimeofeithertest.Ofthe9pa- FromtheClinicalNeurocardiologySection,NationalInstituteof tients, 2 had normal 6-[18F]fluorodopamine-derived NeurologicalDisordersandStroke,NationalInstitutesofHealth, 18 Bethesda,MD. radioactivity and 7 had locally decreased 6-[ F]- fluorodopamine-derivedradioactivityintheleftventricular ReceivedNov28,2001,andinrevisedformFeb28andMar7, myocardiumatthetimeofthefirsttest. 2002.AcceptedforpublicationMar7,2002. Published online Jun 23, 2002, in Wiley InterScience (www.interscience.wiley.com).DOI:10.1002/ana.10236 DataAnalysisandStatistics AddresscorrespondencetoDrLi,Building10,Room6N252,Na- Tomographyimageswerereconstructedaftercorrectionfor tionalInstituteofNeurologicalDisordersandStroke,NationalIn- 18 stitutesofHealth,10CenterDrive,MSC-1620,Bethesda,MD attenuationandforphysicaldecayof F.Cardiacimages 20892-1620.E-mail:[email protected] wereanalyzedasdescribedpreviously.14 Briefly,circularre- ThisarticleisaUSGovernmentworkand,assuch,isinthepublicdo- gionsofinterestapproximatelyhalftheventricularwall mainintheUnitedStatesofAmerica. thicknesswereplacedonimagesoftheseptum,withtime-

220 Published2002byWiley-Liss,Inc. averaged pictures of a single slice. Left ventricular septal urinary frequency. None of the patients developed or- radioactivity was averaged from two regions of interest for thostatic hypotension between the first and second the 5-minute scanning interval with a midpoint about scans. 8 minutes after initiation of the infusion. The same At the time of the first scan, 2 patients had normal time interval was used for radioactivity in the liver and kid- myocardial 6-[18F]fluorodopamine-derived radioactiv- ney. For radioactivity in structures of the head and neck, 18 static three-dimensional data were obtained for 10 to 15 ity, and 7 had decreased myocardial 6-[ F]- minutes. Images of noncardiac structures, including the fluorodopamine-derived radioactivity confined to the liver, spleen, renal cortex, renal pelvis, salivary glands, na- lateral wall or apex, so that no patient had sopharyngeal mucosa, and thyroid, were reconstructed and 6-[18F]fluorodopamine-derived radioactivity more than analyzed by manual drawing of the regions of interest out- two standard deviations below the normal mean in lining the structures. Radioactivity concentrations were both the lateral wall and interventricular septum. normalized by correction for the radioactivity concentration All 9 patients had lower lateral wall concentrations for the administered dose of radioactive drug per unit of of 6-[18F]fluorodopamine-derived radioactivity in body mass of the subject and were expressed as nCi-kg/cc- mCi.14 the second scan than in the first scan (Fig 1). Mean values for 6-[18F]fluorodopamine-derived radioac- Left ventricular myocardial mean concentrations of 18 tivity were compared with paired t tests. Differences between 6-[ F]fluorodopamine-derived radioactivity decreased groups in trends over time of 6-[18F]fluorodopamine-derived by 23% between the first scans (mean, 5,122nCi-kg/ radioactivity were assessed by analyses of variance for re- cc-mCi; SEM, 564nCi-kg/cc-mCi) and second scans peated measures. A p value of less than 0.05 defined statis- (mean, 6,634nCi-kg/cc-mCi; SEM, 447nCi-kg/cc-mCi; tical significance. p ϭ 0.003). Lateral wall radioactivity decreased by 31% between the first scans (mean, 4,107nCi-kg/ cc-mCi; SEM, 535nCi-kg/cc-mCi) and second scans (mean, 5,991nCi-kg/cc-mCi; SEM, 537nCi-kg/cc-mCi; Results ϭ At the time of the first scan, patients had had Parkin- p 0.003), and septal radioactivity decreased by 16% son’s disease for 5.7 Ϯ 1.2 years (range, 0.3–13 years). between the first scans (mean, 6,137nCi-kg/cc-mCi; Disease severity averaged 2.2 Ϯ 0.2 (range, 1–3) of a SEM, 716nCi-kg/cc-mCi) and second scans (mean, ϭ maximum of 5. Heart rate and beat-to-beat systolic 7,278nCi-kg/cc-mCi; SEM, 385nCi-kg/cc-mCi; p blood pressure changes during phase II of the Valsalva 0.05; Fig 2, Table). In 1 patient, the lateral ventricular maneuver averaged 11 Ϯ 2bpm and Ϫ43 Ϯ 5mm Hg wall was not visualized in the second scan, and the tis- for a mean arterial baroreflex-cardiovagal gain of 2.3 Ϯ sue concentration was assumed to be equal to the left 0.4ms/mm Hg (normal: mean, 8.5ms/mm Hg; SEM, ventricular chamber concentration. 2.2ms/mm Hg). Plasma catecholamine levels (norepi- With the exclusion of data from the patient for nephrine: mean, 2.34nmol/L; SEM, 0.37nmol/L; epi- whom the left ventricular wall was not visualized in the nephrine: mean, 0.23nmol/L; SEM, 10nmol/L) were second scan, the percentage decrease in lateral wall ra- approximately normal. Most patients had tremor and dioactivity between the first and second scans (mean,

Fig 1. Progressive loss of myocardial 6-[18F]fluorodopamine-derived radioactivity in a patient with Parkinson’s disease.

Li et al: Sympathetic Innervation in Parkinson’s Disease 221 years. This rate of loss of sympathetic terminals appears to be at least as high as the rate of loss of nigrostriatal dopamine terminals.15 So far in our ongoing series, all patients with Par- kinson’s disease and orthostatic hypotension have had evidence of diffuse loss of cardiac sympathetic innervation at the time of initial testing.13 About half of patients with Parkinson’s disease without orthostatic hypotension have also had evidence of diffuse cardiac sympathetic denervation, and because of the likeli- hood of a floor effect, data from these patients were not included in this study. Given these results, and the present findings, based on the remaining patients with Parkinson’s disease who did not have either orthostatic hypotension or diffuse cardiac sympathetic denervation at the time of initial testing, indicate that progressive loss of cardiac sympathetic innervation characterizes the disease. As of the time of the second scan, none of the patients developed orthostatic hypotension. The sympathetic innervation of the myocardium travels with the coronary arteries. The finding of more severely decreased 6-[18F]fluorodopamine-derived ra- Fig 2. Concentrations (mean Ϯ standard error of the mean) of dioactivity in the lateral wall than in the interventric- 6-[18F]fluorodopamine-derived radioactivity in the (top) lateral ular septal wall leads to a suggestion of a dying-back left ventricular wall and (bottom) interventricular septum in mechanism for the loss of sympathetic terminals, as normal control subjects (open circles), patients with Parkin- opposed to death of the cell bodies followed by loss of son’s disease at the time of the first scan (filled squares), and the terminals (as in Wallerian degeneration). In agree- the same patients at the time of the second scan an average of ment with this notion, about one half of patients with 2 years later (open squares). Parkinson’s disease who do not have orthostatic hypotension already have decreased concentrations of 6-[18F]fluorodopamine-derived radioactivity throughout

32%; SEM, 7%) exceeded the percentage decrease in septal wall radioactivity (mean, 13%; SEM, 7%; t ϭ 18 2.5, p ϭ 0.04). Table. Tissue Concentrations of 6-[ F]Fluorodopamine- 18 Derived Radioactivity (nCi-kg/cc-mCi) in Patients with Tissue concentrations of 6-[ F]fluorodopamine- Parkinson’s Disease derived radioactivity in the liver, renal cortex, renal pelvis, salivary glands, and thyroid did not change be- First Scan Second Scan tween the two scans (see Table). Radioactivity in the spleen, however, was lower in the second scan (mean, Organ Mean SEM Mean SEM 5,020nCi-kg/cc-mCi; SEM, 137nCi-kg/cc-mCi) than Left lateral wall 5,991 Ϯ 537 4,107 Ϯ 535a in the first scan (mean, 6,495nCi-kg/cc-mCi; SEM, Septum 7,278 Ϯ 385 6,137 Ϯ 716a 364nCi-kg/cc-mCi; p ϭ 0.001). Right myocardium 5,889 Ϯ 564 5,390 Ϯ 485 Right chamber 4,799 Ϯ 411 4,240 Ϯ 263 Left chamber 4,971 Ϯ 517 4,725 Ϯ 423 Liver 6,897 Ϯ 724 7,165 Ϯ 869 Discussion Spleen 6,495 Ϯ 364 5,020 Ϯ 137a These findings, based on sympathetic neuroimaging Renal cortex 23,564 Ϯ 2,412 20,716 Ϯ 2,070 with 6-[18F]fluorodopamine positron emission tomog- Renal pelvis 27,667 Ϯ 3,874 23,278 Ϯ 5,203 raphy scanning, indicate that in patients with Parkin- Nasophanrynx 1,431 Ϯ 162 1,501 Ϯ 163 Ϯ Ϯ son’s disease who have normal or only locally decreased Parotid 1,832 170 1,698 221 Submandibular gland 1,870 Ϯ 239 1,782 Ϯ 267 cardiac sympathetic innervation, the loss of innervation Thyroid 1,933 Ϯ 187 1,624 Ϯ 250 progresses over time, especially in the lateral ventricular wall, in which 6-[18F]fluorodopamine-derived radioac- aSignificantly different from first scan. tivity decreased by about 30% over an average of 2 SEM ϭ standard error of the mean

222 Annals of Neurology Vol 52 No 2 August 2002 the left ventricular myocardium, and of the remaining 10. Satoh A, Serita T, Seto M, et al. Loss of 123I-MIBG uptake by half, most have decreased 6-[18F]fluorodopamine- the heart in Parkinson’s disease: assessment of cardiac sympa- derived radioactivity in the lateral ventricular wall or thetic denervation and diagnostic value. J Nucl Med 1999;40: apex, with relative sparing of the interventricular sep- 371–375. 13 11. Reinhardt MJ, Jungling FD, Krause TM, Braune S. Scinti- tum. graphic differentiation between two forms of primary dysauto- Because the patients did not have a progressive loss nomia early after onset of autonomic dysfunction: value of car- 18 of 6-[ F]fluorodopamine-derived radioactivity in the diac and pulmonary iodine-123 MIBG uptake. Eur J Nucl Med liver or renal cortex but did in the heart and spleen, 2000;27:595–600. rates of loss of sympathetic innervation appear to vary 12. Taki J, Nakajima K, Hwang EH, et al. Peripheral sympathetic across organs. This fits with the notion of cardioselec- dysfunction in patients with Parkinson’s disease without auto- tive sympathetic denervation in Parkinson’s dis- nomic failure is heart selective and disease specific. Eur J Nucl ease.11,12 Med 2000;27:566–573. The results lead to the general inference that Parkin- 13. Goldstein DS, Holmes C, Li ST, et al. Cardiac sympathetic denervation in Parkinson disease. Ann Intern Med 2000;133: son’s disease features progressive neurodegeneration not 338–347. only in the nigrostriatal dopaminergic system but also 14. Goldstein DS, Eisenhofer G, Dunn BB, et al. Positron emission in the sympathetic noradrenergic system. tomographic imaging of cardiac sympathetic innervation using 6-[18F]fluorodopamine: initial findings in humans. J Am Coll Cardiol 1993;22:1961–1971. 15. Poewe WH. The natural history of Parkinson’s disease. Ann Neurol 1998;44(suppl 1):1–9. We gratefully acknowledge the assistance of Sandra Brentzel, RN, and the Positron Emission Tomography Department of the Na- tional Institutes of Health.

References 1. Braune S, Reinhardt M, Bathmann J, et al. Impaired cardiac uptake of meta-[123I]iodobenzylguanidine in Parkinson’s disease with autonomic failure. Acta Neurol Scand 1998;97: 307–314. 2. Braune S, Reinhardt M, Schnitzer R, et al. Cardiac uptake of [123I]MIBG separates Parkinson’s disease from multiple system atrophy. Neurology 1999;53:1020–1025. 3. Takatsu H, Nishida H, Matsuo H, et al. Cardiac sympathetic denervation from the early stage of Parkinson’s disease: clinical and experimental studies with radiolabeled MIBG. J Nucl Med 2000;41:71–77. 4. Takatsu H, Nagashima K, Murase M, et al. Differentiating Par- kinson disease from multiple-system atrophy by measuring cardiac iodine-123 metaiodobenzylguanidine accumulation. JAMA 2000;284:44–45. 5. Orimo S, Ozawa E, Nakade S, et al. (123)I-metaiodobenzylguanidine myocardial scintigraphy in Parkinson’s disease. J Neurol Neurosurg Psychiatry 1999;67:189–194. 6. Yoshita M, Hayashi M, Hirai S. Iodine 123-labeled metaiodobenzylguanidine myocardial scintigraphy in the cases of idiopathic Parkinson’s disease, multiple system atrophy, and progressive supranuclear palsy. Rinsho Shinkeigaku 1997;37: 476–482. 7. Yoshita M. Differentiation of idiopathic Parkinson’s disease from striatonigral degeneration and progressive supranuclear palsy using iodine-123 meta-iodobenzylguanidine myocardial scintigraphy. J Neurol Sci 1998;155:60–67. 8. Druschky A, Hilz MJ, Platsch G et al. Differentiation of Par- kinson’s disease and multiple system atrophy in early disease stages by means of I-123-MIBG-SPECT. J Neurol Sci 2000; 175:3–12. 9. Satoh A, Serita T, Tsujihata M. Total defect of metaiodobenzylguanidine (MIBG) imaging on heart in Parkinson’s disease: assessment of cardiac sympathetic denervation. Nippon Rinsho 1997;55:202–206.

Li et al: Sympathetic Innervation in Parkinson’s Disease 223 noxiousstimulibutusuallylacksanhidrosisandmental NoMutationintheTRKA retardation.SomepatientswithpossibleHSAN-Vwere (NTRK1)GeneEncodinga describedbefore1960(seeLandrieuandcolleagues6 forreferences),and3familieswithHSAN-Vsubse- ReceptorTyrosineKinase quentlyweredescribedinwhichweredemonstrateda selectiveseveredecreaseofthesmallmyelinatedfi- forNerveGrowthFactorin bers7,8 andasmallreductioninunmyelinatedfibers9 of thesuralnerve.HSAN-Vislikelyanautosomalreces- aPatientwithHereditary sivedisorder,butLandrieuandcolleagues6 reportedin SensoryandAutonomic afamily2dominantlytransmittedcaseswithnormal nervebiopsy.Therefore,aclinicalentity,alsocalled NeuropathyTypeV congenitalindifferencetopain,isgeneticallyheteroge- 1 2 neousandprobablyincludesHSAN-IVorHSAN-V, EnnioToscano,MD,PhD, AlessandroSimonati,MD, 1 YasuhiroIndo,MD,PhD,3 andGenerosoAndria,MD1 assuggestedbyDyck. Recently,Houldenandcolleagues10 describedaboy withfebrileepisodesandalackofpainsensationthat HereditarysensoryandautonomicneuropathytypeIV theydiagnosedasHSAN-V,accordingtothetypical 9 (HSAN-IV)andtypeV(HSAN-V)areautosomalreces- findingsinanervebiopsy. DNAanalysisshowedthat sivegeneticdisorders,bothcharacterizedbyalackof hehadahomozygousmutationintheTRKAgene. painsensation.Wereportagirlwithclinicalandneuro- TheystatedthatHSAN-IVandHSAN-Varelikelyto physiologicalfindingsconsistentwithadiagnosisof beallelic. HSAN-V.WesequencedherTRKAgene,encodingare- Herewepresentagirlwithclinicalfeaturesconsis- ceptortyrosinekinasefornervegrowthfactorandre- tentwithHSAN-V,namely,alackofpainsensation, sponsibleforHSAN-IV,butwecouldnotdetectanymu- butnofebrileepisodesandnormalsuralnervebiopsy. tation.Thesedataindicatethatagene(orgenes)other thanTRKAisprobablyresponsibleforHSAN-Vinsome WesequencedherTRKAgeneanddetectednomuta- patients. tion.Therefore,thesedataindicatethatagene(or genes)otherthanTRKAisprobablyresponsiblefor AnnNeurol2002;52:224–227 HSAN-Vinsomepatients,arguingagainstapreviously citedreport.10 Wealsodiscussthedifferentialdiagnosis ofHSAN-IVversusHSAN-V. Hereditary sensory and autonomic neuropathy (HSAN)areclassifiedinto5differenttypesaccording 1 CaseReport toDyck. WehavedemonstratedthattheTRKA I.F.,agirl,wasthesecondchildofconsanguineousparents (NTRK1)gene,encodingareceptortyrosinekinase (firstcousins).Shewasbornatterm(birthweight,3,400gm) thatisphosphorylatedinresponsetonervegrowthfac- withApgarscoresof9and10.Shewasfoundtohavein- 2 tor,isresponsibleforHSAN-IV. HSAN-IVischarac- sensitivitytopainfulstimuli,andmultipletraumaticepisodes terizedbyfebrileepisodes,anhidrosis,insensitivityto wereexperiencedfromearlylife.Shebitoffthetipofher pain,self-mutilatingbehavior,andmentalretardation. tongue,andshedidnotcrywhenshefellorduringblood PathologicalfeaturesofHSAN-IVareseverereduction sampling.Attheagesof3,4,and5years,shehadepisodes ofsmall-diameterafferentneurons,whichareactivated oflefthipdislocationwithoutpainperception.Attheageof bytissue-damagingstimuli,3,4 andalossofsympathetic 6years,shesufferedfrombilateralosteochondritisofthe neuronsinnervatingeccrinesweatglands.5 feet.Herpsychomotordevelopmentwasapparentlynormal, andsheneverexhibitedbehavioralproblemsorattention HSAN-Valsoischaracterizedbyabsentreactionto deficitproblems.Sheneverhadthermoregulatoryorfeeding problems,vomiting,boweldysfunction,orothersignsor symptomsofgastrointestinaldysmotility.

Fromthe 1DepartmentofPediatrics,“FedericoII”University,Na- ples,Italy; 2SectionofClinicalNeurology,DepartmentofNeuro- Results logicalandVisualSciences,UniversityofVerona,Verona,Italy;and Clinicalexaminationofthegirlwhenshewas11years 3 DepartmentofPediatrics,KumamotoUniversity,Honjo,Ku- oldshowedreducedresponsetopainfulstimuliand mamoto,Japan. anosmia;thermicsensationappearedtobeconserved. ReceivedAug21,2001,andinrevisedformMarch11,2002.Ac- Fungiformpapillaeofthetongue,cornealreflexes,and ceptedforpublicationMarch11,2002. overflowtearswerepresent.Intelligenceanddeepten- Published online Jun 23, 2002, in Wiley InterScience (www.interscience.wiley.com).DOI:10.1002/ana.10245 donreflexeswerenormal.Bloodpressurewasnormal inbothsupineandstandingpositions.Anintradermal AddresscorrespondencetoDrAndria,DepartmentofPediatrics, “FedericoII”University,ViaS.Pansini5,I-80131Naples,Italy. injectionofhistamineevokedaxonalreflexasobserved E-mail:[email protected] inanormalcontrol.Shecouldcontrolherbodytem-

224 ©2002Wiley-Liss,Inc. perature well under hot environmental conditions. Pi- locarpine ionophoresis and sympathetic skin response were normal. A right sural nerve biopsy was performed and pro- cessed according to routine procedures for both morphological and morphometrical investigations.11 Fea- tures of the nerve fascicle were normal. At the ultrastructural level, unmyelinated fibers were regularly detected; neither denervated Schwann cells nor colla- gen pockets were seen (Fig 1). Myelinated fiber density was within normal ranges; the shape of the histogram was bimodal, showing the small-caliber fiber peak (Fig 2). Skin biopsy was examined on plastic sections only. Glands were present; both myelinated and unmyelinated fibers were present in the intradermal nerve fas- cicles. We sequenced all 17 exons of the TRKA gene of the patient, including their flanking intronic sequences,12 and detected no putative mutation. Furthermore, we analyzed 8 polymorphic sites in this gene and found that the patient is heterozygous at the intragenic poly- 13 Fig 2. Size distribution (X-axis) of the myelinated fibers of the morphic site (c. 1,953 cytosine/thymine). This fur- index case and an age-matched control. Note the normal, bimodal pattern of the histogram; small-caliber fibers are simi- larly represented in both the patient and the control. Numbers Fig 1. Sural nerve biopsy. (A) Semithin section; toluidine blue on the Y-axis refer to the absolute figures of the measured stain. Normal features of the nerve fascicle showing myelinated fibers. fibers of both small and large calibers. Bar ϭ 12.5␮m. (B) Thin section; uranyl acetate and lead citrate stain. Representa- ther supports that the patient has no mutation in the tive clusters of normal unmyelinated fibers. Bar ϭ 3␮m. TRKA locus because the parents are consanguineous.

Discussion The patient reported here probably presents a hereditary autosomal recessive sensory neuropathy with selective loss of pain sensation. Autonomic abnormalities apparently were not observed. Reduced sweating was mentioned once, but she can maintain her body temperature under hot environmental conditions. This is compatible with the findings in her nerve and skin biopsies. These clinical and laboratory data suggest that the patient suffers from HSAN-V. Similar features, to- gether with apparently intact peripheral nerves, were described in some classic reports of congenital indifference to pain.6 The patient reported by Low and colleagues,7 presenting indifference to pain and selective loss of small myelinated fibers without sweating abnormality, was classified as HSAN-V. Dyck and col- leagues9 described a patient with indifference to pain, sweating abnormality, and a severe decrease in small- diameter myelinated fibers with a mild reduction of unmyelinated fibers. It is their view that most earlier cases of indifference to pain may have had either HSAN-IV or HSAN-V.1,9 In contrast, HSAN-IV is a distinct clinical entity characterized by recurrent episodic fever, anhidrosis, insensitivity to pain, self-mutilating behavior, and mental retardation.3,14 Mental retardation is variable,

Toscano et al: No Mutation in TRKA Gene in HSAN-V 225 from severe to mild, and some patients were apparently tion to distinguish overlapping phenotypes of normal, but later mild retardation was showed by a HSAN-IV and HSAN-V, both characterized by a lack formal assessment (patients KI-108 and KI-116 re- of pain sensation. Most patients with HSAN-IV prob- ported by Mardy and colleagues12 and Indo and col- ably have mutations in the TRKA gene,2,12,13 although leagues,15 respectively). Sweating may be variable but we cannot rule out the possibility that a mutation (or should be evaluated cautiously, as we recently report- mutations) in another gene (or genes) is responsible for ed.15 We think that a fundamental phenotype of similar clinical phenotypes. The patient presented here HSAN-IV consists of insensitivity to pain, anhidrosis, suffers from HSAN-V, but not HSAN-IV, because she and mental retardation, each of variable degree. Recur- does not show anhidrosis or mental retardation. The rent hyperpyrexia, self-mutilating behaviors, traumas, presence of myelinated and unmyelinated fibers in and bone fractures can be devastating and often lead to nerve biopsy and the normal intradermal histamine test crippling or fatal consequences. further support this diagnosis. We could not detect any Nerve growth factor supports the survival of sympa- putative mutation in the TRKA gene, whereas we thetic ganglion neurons and nociceptive sensory neu- found that the patient is heterozygous for this locus, rons in dorsal root ganglia and ascending cholinergic despite the parental consanguinity. These findings neurons of the basal forebrain.16 Eccrine sweat glands, strongly indicate that defects of a gene (or genes) other innervated by sympathetic cholinergic fibers, are well than TRKA is likely responsible for at least some pa- developed in humans. Therefore, the nerve growth tients with HSAN-V. factor-TRKA system has a crucial role in the develop- It remains unknown whether peripheral nociceptive ment and function of the nociceptive reception and es- transmission or central processing might be involved in tablishment of thermoregulation via sweating.2 A neg- our case and in some cases with similar phenotypes re- ative result of the intradermal histamine test, an ported as having HSAN-V. Anosmia observed in our important diagnostic criterion in HSAN-IV, probably case remains to be examined and may give us some also can be explained by defective peripheral sympa- clue for studying a mechanism underlying the defect in thetic neurons. Therefore, anhidrosis and associated pain sensation. Some patients diagnosed as having failure to maintain body temperature are characteristic HSAN-V do not show an apparent abnormality of pe- features of HSAN-IV. ripheral nerve fibers. Therefore, their manifestations Recently, Houlden and colleagues10 described a boy might be caused by defects in peripheral nociceptor or 9 years of age, born to healthy consanguineous parents, transduction and transmission of pain sensation or presenting anhidrosis and loss of pain and temperature even central processing, such as a lack of concern for a sensation. Sural nerve biopsy demonstrated severe re- painful stimulus well received by the peripheral ner- duction in small-caliber myelinated fiber density but vous system, according to the classic definition of in- only modest reduction in unmyelinated axons. The au- difference to pain.19 thors did not mention either a skin biopsy or a histamine test, which provide important diagnostic criteria for the group of hereditary peripheral neuropathies.17 References They detected a homozygous missense mutation in the 1. Dyck PJ. Neuronal atrophy and degeneration predominantly af- TRKA gene, changing a tyrosine to a cysteine at codon fecting peripheral sensory and autonomic neurons. In: Dick PJ, Thomas PK, Griffin JW, Low PA, Podreslo JC, eds. Peripheral 359. According to this case, they concluded that the 2 neuropathy. Philadelphia: Saunders, 1993:1065–1093. disorders, HSAN-IV and HSAN-V, are likely to be al- 2. Indo Y, Tsuruta M, Hayashida Y, et al. Mutations in the lelic. However, we propose an alternative interpretation TRKA/NGF receptor gene in patients with congenital insensi- of Houlden and colleagues’ report. Their patient may tivity to pain with anhidrosis. Nat Genet 1996;13:485–488. suffer from HSAN-IV, but not HSAN-V. They argued 3. Swanson AG, Buchan GG, Alvord EC Jr, et al. Autonomic changes in congenital insensitivity to pain: absence of small pri- for the diagnosis of HSAN-V, mainly basing their ar- mary sensory neurons in ganglia, roots and Lissauer’s tract. gument on the finding of nerve biopsy with modest Arch Neurol 1965;12:12–18. reduction of unmyelinated fibers. However, anhidrosis 4. Rafel E, Alberca R, Bautista J, et al. Congenital insensitivity to observed in the patient strongly indicates dysfunction pain with anhidrosis. Muscle Nerve 1980;3:216–220. of the most distal intradermal portions of the sympa- 5. Langer J, Goebel HH, Veit S. Eccrine sweat gland are not innervated in hereditary sensory neuropathy type IV: an electron- thetic axons. That also might account for the normal microscopic study. Acta Neuropathol (Berl) 1981;54:199–202. appearance of the more proximal unmyelinated fibers 6. Landrieu P, Said G, Allaire C. Dominantly transmitted congen- observed after sural nerve. Furthermore, it would be ital indifference to pain. Ann Neurol 1990;27:574–578. important to confirm the functional significance of the 7. Low PA, Burke WJ, McLeod JG. Congenital sensory neuropa- missense mutation by an expression study, such as that thy with selective loss of small myelinated fibers. Ann Neurol 18 1978;3:179–182. reported recently. 8. Donaghy M, Hakin RN, Bamford JM, et al. Hereditary sensory We stress the importance of the molecular analysis and autonomic neuropathy with neurotrophic keratitis. Brain of the TRKA gene in all cases with lack of pain sensa- 1987;110:563–583.

226 Annals of Neurology Vol 52 No 2 August 2002 9.DyckPJ,MellingerJF,ReaganTJ,etal.Not“indifferenceto pain”butvarietiesofhereditarysensoryandautonomicneurop- X-LinkedCreatine athy.Brain1983;106:373–390. 10.HouldenH,KingRHM,Hashemi-NejadA,etal.Anovel DeficiencySyndrome:A TRKA(NTRK1)mutationassociatedwithhereditarysensory andautonomicneuropathytypeV.AnnNeurol2001;49: NovelMutationinCreatine 521–525. 11.SimonatiA,FabriziGM,PasquinelliA,etal.Congenitalhy- TransporterGeneSLC6A8 pomyelinationneuropathywithSer72Leusubstitutionin 1 2 PMP22.NeuromusculDisord1999;9:257–261. AlbertoBizzi,MD, MariannaBugiani,MD, GajjaS.Salomons,PhD,3 DonaldH.Hunneman,PhD,4 12.MardyS,MiuraY,EndoF,etal.Congenitalinsensitivityto 2 2 painwithanhidrosis:novelmutationsintheTRKA(NTRK1) IsabellaMoroni,MD, MargheritaEstienne,MD, UgoDanesi,PhD,1 CornelisJakobs,PhD,3 geneencodingahigh-affinityreceptorfornervegrowthfactor. 2 AmJHumGenet1999;64:1570–1579. andGraziellaUziel,MD 13.MiuraY,MardyS,AwayaY,etal.Mutationandpolymor- phismanalysisoftheTRKA(NTRK1)geneencodingahigh- affinityreceptorfornervegrowthfactorincongenitalinsensi- Amongcreatinedeficiencysyndromes,anX-linkedcon- tivitytopainwithanhidrosis(CIPA)families.HumGenet ditionrelatedtoadefectivecreatinetransportintothe 2000;106:116–124. centralnervoussystemhasbeendescribedrecently.Hall- 14.SwansonAG.Congenitalinsensitivitytopainwithanhidrosis. marksofthediseasearetheabsenceofacreatinesignal ArchNeurol1963;8:299–306. 15.IndoY,MardyS,MiuraY,etal.Congenitalinsensitivityto atbrainspectroscopy,increasedcreatinelevelsinblood painwithanhidrosis(CIPA):novelmutationsofTRKA andurine,ineffectivenessoforalsupplementation,anda (NTRK1)geneencodingthereceptortyrosinekinasefornerve mutationintheSLC6A8(OnlineMendelianInheritance growthfactor,aputativeuniparentaldisomyandalinkageof inMan[OMIM]300036)creatinetransportergene.We themutantTRKAandPKLRgenesinafamilywithCIPAand reportonapatientinwhomanovelmutation(1221- pyruvatekinasedeficiency.HumMutat2001;18:308–318. 1223delTTC)wasidentified. 16.LeviMontalciniR.Thenervegrowthfactor:thirty-fiveyears later.EMBOJ1987;6:1145–1154. AnnNeurol2002;52:227–231 17.AxelrodFB,PearsonJ.Congenitalsensoryneuropathies.AmJ DisChild1984;138:947–954. 18.MardyS,MiuraY,EndoF,etal.Congenitalinsensitivityto Creatinedeficiencysyndromesarerecentlyidentified painwithanhidrosis(CIPA):effectofTRKA(NTRK1)mis- sensemutationsonautophosphorylationofthereceptorty- inbornerrorsofmetabolismresultinginaprogressive rosinekinasefornervegrowthfactor.HumMolGenet2001; encephalopathywithearlyonsetandmentalretarda- 10:179–188. tion,extrapyramidalfeatures,anddrug-resistantepi- 19.JewesburryECO.Congenitalindifferencetopain.In:Vinken lepsy.1–3 Symptomsarerelatedtoadepletionofthe PJ,BruynGW,eds.Handbookofclinicalneurology.Vol8. creatine/phosphocreatinepoolwithinthecentralner- Amsterdam:Elsevier,1979:187–204. voussystem,makingthisconditioneasilydetectableby brainspectroscopy.Mostofthecasesreportedsofar werecausedbyadefectofthesecondenzymeinvolved increatinebiosynthesis:guanidino-acetatemethyltrans- ferase(GAMT;OMIM601240).Thisdefectresultsin increasedguanidino-acetate(GAA)andreducedcreat- inelevelsinbloodandurine.4,5 GAMT-deficientpa- tientsbenefitfromoralcreatinemonohydratesupple- mentation,whichhelpstocontrolmovementdisorders andepilepsy,partiallyrecoversmentalimpairment,and restoresneurologicaldevelopmentovertime.6 Theab-

Fromthe 1DepartmentsofNeuroradiologyand 2ChildNeurology, IstitutoNazionaleNeurologico“C.Besta,”Milano,Italy; 3Metabol- icUnit,VUMedicalCenter,Amsterdam,TheNetherlands;and 4UniversityKinderklinik,Gottingen,Germany. ReceivedNov6,2001,andinrevisedformMar11,2002.Accepted forpublicationMar11,2002. Published online Jun 21, 2002, in Wiley InterScience (www.interscience.wiley.com).DOI:10.1002/ana.10246 AddresscorrespondencetoDrUziel,DepartmentofChildNeurol- ogy,IstitutoNazionaleNeurologico“C.Besta,”ViaCeloria11, 20133Milano,Italy.E-mail:[email protected]

©2002Wiley-Liss,Inc. 227 sence of complete recovery can be explained by neuro- to the hospital after a febrile seizure. An electroencephalo- toxic GAA accumulation or by an imbalance of brain gram recording and a magnetic resonance imaging scan were creatine and high-energy phosphates.7,8 Very recently, normal. Routine blood and urine analysis and investigation Item and colleagues9,10 described 2 sisters with mental for infectious (screening for rubella, cytomegalovirus, toxo- impairment, low GAA levels in urine, and undetectable plasma, herpes simplex, Coxsackie virus, and Mycoplasma activity of arginine to glycine amidinotransferase pneumoniae) and neurometabolic disorders (plasma lactate and metabolic screening of a 24-hour urine sample with an (AGAT; OMIM 602360), the first enzyme involved in assessment of amino acids, organic acids, and mucopolysac- creatine biosynthesis. A homozygous nonsense muta- charides) were normal. Since 16 months of age, he experi- tion in AGAT gene was detected. Clinical symptoms enced complex partial seizures with secondary generalization, and brain creatine deficiency were partially recovered responding to sodium valproate. Serial electroencephalogram by means of creatine supplementation. A different dis- tracings showed a progressive instability of background activ- ease due to impairment of creatine transport into the ity with abundant fast activity and epileptic discharges from brain was reported by Salomons and colleagues11,12 in frontal and temporal leads during sleep. Physical examination a boy with language delay, short attention span, epi- at 3 years and 9 months showed a severe delay in speech and lepsy, and increased creatine levels in blood and urine. language functions with behavioral disturbances in agreement A nonsense mutation in the X-linked creatine trans- with an autistic disorder. He did not follow commands or porter gene SLC6A8 was demonstrated. Creatine sup- speak, he presented with stereotypical motor behaviors, and he could not engage in any structured play. Gross and fine plementation in this boy was totally ineffective. This motor functions were normal. article reports on a second case caused by defective cre- A second magnetic resonance imaging showed mild atro- atine transport into the central nervous system. phy with signal abnormality in the right hippocampus, suggesting mesial temporal sclerosis. Proton magnetic resonance Case Report spectroscopic imaging (H-MRSI) was performed as part of The patient was the second-born son of healthy, nonconsan- the diagnostic workup for mesial temporal sclerosis con- guineous parents. The child’s prenatal and perinatal history ducted at our institution and showed a normal N-acetylas- was unremarkable. Since the first months of life, he pre- partate/choline ratio in both hippocampi without abnormal- sented with motor delay, reduced interest in surroundings, ity in the asymmetry index. The surprising feature was the and no language acquisition. At 8 months, he was admitted absence of the creatine peak in the whole brain (Fig), indi-

Fig. Proton magnetic resonance spectroscopic imaging shows a complete absence of the creatine peak in both white and gray matter (1A, 2A, 3A, 4A). The position of selected voxels is indicated on the T2-weighted magnetic resonance image at the level of the centrum semiovale. There was no restoration of the creatine pool after 3 months of oral creatine monohydrate supplementation at 400mg/kg/day (1B) and after 8 months of supplementation at 700mg/kg/day (1C). There were no significant changes in choline and N-acetylaspartate levels. A normal spectrum from an age-matched control is given for comparison: the peaks of choline (Cho; 3.2ppm), creatine (Cr; 3.02ppm), and N-acetylaspartate (NAA; 2.02ppm) are indicated.

228 Annals of Neurology Vol 52 No 2 August 2002 cating a creatine deficiency syndrome, without any abnormal NM 005629). This mutation resulted in the deletion peak in the expected frequency of GAA (3.8ppm). Creatine of a single phenylalanine at residue 408 of the protein and GAA, therefore, were measured in plasma and urine, (delF408). The patient’s mother was heterozygous for and oral creatine monohydrate supplementation (400mg/kg/ the mutation. day) was started. Because H-MRSI showed no creatine recovery after 3 months of therapy, supplementation was in- Discussion creased to 700mg/kg/day. Eight months later, a follow-up The failure of creatine supplementation to restore brain H-MRSI examination confirmed no appearance of brain creatine. By this time, clinical symptoms were not improved, creatine by H-MRSI and to improve clinical symptoms and therapy was discontinued. Currently, the patient is 5 and normal plasma and urine GAA levels ruled out a years old, and his clinical symptoms are grossly unchanged. defect of creatine biosynthesis and prompted a search 12,14 The very recent identification of the first patient with a cre- for molecular defects in the SLC6A8 gene. A novel atine transporter defect11,12 suggested that our patient suf- hemizygous deletion located in a short repeat of 3 phe- fered from a creatine uptake defect as well, which was in line nylalanines in exon 8 was detected. The repeat is part with the ineffectiveness of creatine supplementation and with of transmembrane domain VIII, which is a very con- ϩ Ϫ biochemical data. Mutational analysis of the SLC6A8 gene, served region among the Na - and Cl -dependent therefore, was performed. neurotransmitter family.15 The mutation, resulting in a phenylalanine deletion at position 408, most likely Methods causes a partial or even complete loss of creatine trans- Before therapy, H-MRSI (two-dimensional phase encoding port function. The activity of the transporter could not point resolved spectroscopy repetition (PRESS) technique: be tested in fibroblasts because the parents refused con- recovery time/echo time, 1,500/136msec; field of view, 160mm; matrix, 16 ϫ 16; 20mm slice thickness) was per- sent to a skin biopsy. formed from 3 separate sections at the level of the centrum To our knowledge, this is the first creatine deficiency semiovale, basal nuclei, and hippocampi. During therapy, case studied with the multivoxel H-MRSI technique, H-MRSI was performed at the level of the centrum semi- which has better spatial resolution and allows an evalu- ovale. Single-voxel spectra (PRESS: recovery time/echo delay ation of lesion heterogeneity in the shortest amount of time, 1,500/34msec) were acquired from a 30ml volume in time. We did not find any significant difference in cre- the frontoparietal parasagittal cortical gray matter before and atine levels across the brain regions examined, suggesting during therapy. Raw data were transferred to a SUN work- that the transporter defect does not spare any brain area. 13 station and reconstructed with custom-made software. Symptoms of creatine deficiency have been related Mutational analysis complementary DNA–based sequence previously to a defect in creatine transport by analysis was performed according to methods described else- Salomons and colleagues,11,12 who described a male where.12 DNA was isolated from blood cells with a QIAamp blood kit (Qiagen, Chatsworth, CA) for conformation of the patient with mental retardation and severe delay in ex- mutation at the genomic level. Primers specific for exon 8 of pressive speech and language function presenting a the SLC6A8 gene were designed: forward 5ЈTCCCAGC- hemizygous nonsense mutation in the SLC6A8 gene. CCCTGCCGCAC and reverse 5ЈTACAAACTGTGGC- As in our case, a lack of creatine transport into the CAGGGC. central nervous system resulted in the failure of creatine supplementation to reverse clinical symptoms and Results brain spectroscopy abnormality. Very recently, another Creatine and GAA levels before, during, and after the unrelated family with a creatine transporter defect was withdrawal of creatine supplementation are shown in recognized by the same authors.16 The biochemical Table 1. Sequence analysis of the creatine transporter profile of our patient showed a massive loss of creatine gene SLC6A8 identified an hemizygous 3bp deletion in in urine, but in contrast with the first reported index exon 8 involving nucleotides TTC in position 1221- case, creatine levels in plasma were normal even during 1223 (1221-1223delTTC; GenBank accession number, creatine supplementation.

Table 1. Laboratory Results before, during, and after Withdrawal of Oral Creatine Monohydrate Supplementation (700mg/kg/day)

Blood Urine

GAA Creatine GAA Creatine Result (␮mol/L) (␮mol/L) (mmol/mol creatinine) (mmol/mol creatinine)

Normal values 0.4–310–200 10–125 40–360 Prior 1.8 83 79 4,519 During ND 174 28 21,315 After ND 66 46 3,102

GAA ϭ guanidino-acetate; ND ϭ not determined.

Bizzi et al: Creatine Transporter Defect 229 Table 2. Creatine and GAA Levels in GAMT and AGAT Defects and in Creatine Transporter Gene (SLC6A8) Mutations

Blood Urine Brain

Defect or mutation Creatine GAA Creatine GAA Creatine GAA

GAMT Decrease Increase Decrease Increase Decrease Undetectable/Increase AGAT Normal Normal ND Decrease Decrease Undetectable SLC6A8 Normal/Increase Normal Increase Normal Decrease Undetectable

AGAT ϭ arginine to glycine amidinotransferase; GAA ϭ guanidino-acetate; GAMT ϭ guanidino-acetate methyltransferase; ND ϭ not determined.

The mother underwent a brain H-MRSI that dem- time of conventional magnetic resonance imaging and onstrated a mildly reduced creatine signal compared may practically disclose creatine or other metabolites de- with that of age-matched controls. Moreover, the re- pletion: just recently, the first case of N-acetylaspartate sults showed that she was heterozygous for the muta- brain deficiency was reported in a patient with mental tion identified in her son. However, no learning dis- retardation and severe language impairment.17 abilities were reported for the patient’s female relatives, Clinical features of mental impairment, language de- in contrast with what was encountered in 2 of 3 female lay, and autistic behavior with epilepsy frequently are carriers in the first family described.12 These data agree encountered in infancy. Conceivably, creatine defi- with skewed X inactivation (mosaic expression of mu- ciency could be underdiagnosed, and if brain spectros- tant and wild-type alleles), resulting in a variably favor- copy cannot be easily achieved, an assessment of blood able mosaic expression of the wild-type allele. and urinary creatine should always be performed. The 3 creatine deficiency syndromes described so far (ie, GAMT and AGAT defects and impairment of cre- We thank Dr S. J. M. van Dooren and D. Brunea for excellent tech- atine transport) share overlapping symptoms of mental nical support to Dr G. S. Salomons. retardation with severe language impairment, autistic behavior, and epilepsy. Movement disorders have been re- Electronic Database Information: OMIM. www.ncbi.nlm.nih.gov/ ported only in patients with GAMT deficiency, suggest- omim; GenBank. www.ncbi.nlm.nih.gov/genbank. ing that extrapyramidal features may result from neurotoxic GAA accumulation rather than from reduced References creatine availability in brain. The major involvement of 1. Stockler S, Isbrandt D, Hanefeld F, et al. Guanidinoacetate higher cortical functions and the frequent finding of ep- methyltransferase deficiency: the first inborn error of creatine metabolism in man. Am J Hum Genet 1996;58:914–922. ilepsy suggest that the cerebral cortex may be selectively 2. Stockler S, Marescau B, De Deyn PP, et al. Guanidinoacetate vulnerable to creatine deficiency. How creatine defi- methyltransferase deficiency, a new inborn error of creatine syn- ciency adversely affects cortical functions is still to be thesis. Metabolism 1997;46:1189–1193. established. It is conceivable that creatine plays a role in 3. Ganesan V, Johnson A, Connelly A, et al. Guanidinoacetate the latest stages of cortical organization, including syn- methyltransferase deficiency: new clinical features. Pediatr Neu- rol 1997;17:155–157. aptogenesis, a process that continues after birth. This 4. Schulze A, Hess T, Wevers R, et al. Creatine deficiency syn- could explain the homogeneity of clinical presentation drome caused by guanidinoacetate methyltransferase deficiency: and age at onset in the 3 creatine deficiency syndromes, diagnostic tools for a new inborn error of metabolism. J Pediatr even though in children with biosynthesis defects, crea- 1997;131:626–631. tine is supplied through the placenta during fetal life, 5. Ilas J, Muhl A, Stockler-Ipsiroglu S. Guanidinoacetate methyltransferase deficiency: non-invasive enzymatic diagnosis of a whereas patients with a transporter defect suffer from newly recognized inborn error of metabolism. Clin Chim Acta creatine depletion already in utero. 2000;290:179–188. Spectroscopy alone cannot always distinguish between 6. Stockler S, Hanefeld F, Frahm J. Creatine replacement therapy synthesis and transport defects. In GAMT deficiency, in guanidinoacetate methyltransferase deficiency, a novel inborn brain spectroscopy at a short echo time can identify an error of metabolism. Lancet 1996;348:789–790. 7. van der Knaap MS, Verhoeven NM, Maaswinkel-Mooij P, et al. abnormal peak that is assigned to GAA (3.8ppm), but Mental retardation and behavioural problems as presenting signs this elevation may be subtle, and it has been reported of a creatine synthesis defect. Ann Neurol 2000;47:540–543. only in a few cases. Therefore, all patients in whom a 8. Leuzzi V, Bianchi MC, Tosetti M, et al. Brain creatine diagnosis of creatine deficiency is reached should un- depletion: guanidinoacetate methyltransferase deficiency (im- dergo a careful biochemical evaluation to assess creatine proving with creatine supplementation). Neurology 2000;55: 1407–1409. and GAA levels in blood and urine (Table 2). Brain 9. Bianchi MC, Tosetti M, Fornai F, et al. Reversible brain crea- spectroscopy is becoming more available; a quick auto- tine deficiency in two sisters with normal blood creatine level. mated spectrum acquisition can be performed at the Ann Neurol 2000;47:511–513.

230 Annals of Neurology Vol 52 No 2 August 2002 10.ItemCB,Stockler-IpsirogluS,StrombergerC,etal.Arginine: TheBlalock-Taussigshunt,firstperformedin1944at glycineamidinotransferasedeficiency:thethirdinbornerrorof theJohnsHopkinsHospital,1 isasurgicalanastomosis creatinemetabolisminhumans.AmJHumGenet2001;69: establishedbetweenasubclavianarteryandtheipsilat- 1127–1133. 11.CecilKM,SalomonsGS,BallWS,etal.Irreversiblebraincre- eralpulmonaryartery.Inpatientswithatetralogyof atinedeficiencywithelevatedserumandurinecreatine:acre- Fallot,thisprocedureallowsdefermentofthedefinitive atinetransporterdefect?AnnNeurol2001;49:401–404. cardiacrepairtoamorerobuststageoflifebybypass- 12.SalomonsGS,vanDoorenSJM,VerhoevenNM,etal. ingthepulmonaryarterystenosis.TheBlalock-Taussig X-linkedcreatine-transportergene(SLC6A8)defect:anew shunt,however,createsthepermanentanatomicalcon- creatine-deficiencysyndrome.AmJHumGenet2001;68: 1497–1500. ditionofasubclavianstealphenomenon(Fig1).The 13.SoherBJ,vanZijlPCM,DuynJH,etal.Quantitativeproton subclavianstealphenomenon,initiallydescribedby spectroscopicimagingofthehumanbrain.MagnResonMed Reivichandcolleaguesin1961,2 isrelatedtoaproxi- 1996;35:356–363. malsubclavianarterystenosisorocclusionresponsible 14.GregorP,NashSR,CaronMG,etal.Assignmentofthecre- fortheoccurrenceofretrogradecollateralflowinthe atinetransportergene(SLC6A8)tohumanchromosomeXq28 telomerictoG6PD.Genomics1995;25:332–333. ipsilateralvertebralartery.Afewreportshavesuggested 15.NashSR,GirosB,KingsmoreSF,etal.Cloning,pharmacolog- apossiblelinkbetweenaBlalock-Taussigshuntand icalcharacterizationandgenomiclocalizationofthehumancre- thelateoccurrenceofcerebrovasculardisease.Were- atinetransporter.ReceptorsChannels1994;2:165–174. portthefirstdocumentedcaseofvertebrobasilarstroke 16.SalomonsGS,DoorenSJ,VerhoevenNM,etal.X-linkedcre- occurringasalatecomplicationofaBlalock-Taussig atinetransporterdefect:thesecondfamily.JInheritMetabDis 2001;24(suppl1):119. shuntfollowedbytotalcardiacrepairduringchild- 17.MartinE,CaponeA,SchneiderJ,etal.Absenceof hood. N-acetylaspartateinthehumanbrain:impactonneurospectros- copy?AnnNeurol2001;49:518–521. CaseReport A39-year-oldmanwasadmittedtoourhospitalbecauseof anacuteepisodeofnauseawithoutvomiting,vertigo,and decreasedhearingontherightside,rapidlyfollowedbyright facialnumbnessaswellasweaknessanddecreasedsensation intheleftarm.Thepatientalsodescribedavisualdistur- banceconsistentwithnystagmus.Thesesymptomsappeared VertebrobasilarStrokeasa withoutanidentifiableprecipitatingfactorandlastedforap- proximately20to30minutes.Thepatientwasknownto LateComplicationofa havetetralogyofFallottreatedbyaBlalock-Taussigshuntat theageof2yearsfollowedbydefinitivesurgicalrepairatthe Blalock-TaussigShunt ageof9years.Hehadbeeningoodhealthsincethen.The reviewofriskfactorsforcerebrovasculardisease,including PhilippeGailloud,MD,1 ArgyeHillis,MD,2 obesity,tobaccouse,hyperlipidemia,andhypertension,was BrucePerler,MD,3 andKieranJ.Murphy,MD1 negative.Thesocialandfamilialhistoriesshowedonlyhy- pertensionandhyperlipidemiainbothparents. Onadmission,thegeneralandneurologicalexaminations Wedescribea39-year-oldpatientwithacerebellarin- werenormalexceptforasymmetricalupperextremityblood farctandahistoryofatetralogyofFallotcorrecteddur- pressuremeasurements(125/80mmHgontheright,146/ ingchildhood.Thisisthefirstdocumentedcaseofver- 95mmHgontheleft)andunpalpabledistalarterialpulses tebrobasilarstrokeoccurringasalatecomplicationofa intherightarm.Accordingtothepatient,thisdiscrepancy Blalock-Taussigshuntfollowedbytotalcardiacrepair. hadbeenknownsincehissecondcardiacsurgery.Thelab- AnnNeurol2002;52:231–234 oratoryvalueswereunremarkable;inparticular,thelipid profilewasnormal,andtherewasnoevidenceofahyper- coagulablestate(theproteinCandSantigens,antithrom- binIIIactivity,diluteRussell’svipervenomtimetest,and homocysteinelevelwerewithinnormalranges;thefactorV Leidenmutationwasnegative).Computedtomographyof FromtheDepartmentof 1RadiologyandRadiologicalSciences, 2Neurology,and 3VascularSurgery,JohnsHopkinsHospital,Balti- thebrainwasnormal.Magneticresonanceimagingshowed more,MD. signalanomaliesintheinferioraspectoftherightcerebellar ReceivedMay15,2000,andinrevisedformFeb28,2002.Ac- hemisphereconsistentwithanacuteischemiclesioninthe ceptedforpublicationMar12,2002. rightposteriorinferiorcerebellararteryterritory(Fig2A). Magneticresonanceimagingincidentallyshowedinflamma- Published online Jun 21, 2002, in Wiley InterScience (www.interscience.wiley.com).DOI:10.1002/ana.10249 torychangesinthemaxillarysinusandethmoidcellsbilat- erally,whereasthesphenoidandfrontalsinuseswere AddresscorrespondencetoDrGailloud,DepartmentofRadiology, JohnsHopkinsUniversitySchoolofMedicine,600N.Wolfe unremarkable.Thepatientwasnottreatedforthisasymp- Street,RadiologyB-100,Baltimore,MD21287. tomaticinflammatorysinusdisease.Atransesophageal E-mail:[email protected] echocardiogram,includingabubblestudyperformedto

©2002Wiley-Liss,Inc. 231 tery branches, including the right vertebral artery and the right ascending and deep cervical arteries (see Fig 2C). The cerebral vasculature was otherwise unremarkable. There was in particular no evidence of intracranial or extracranial ath- eromatous disease and no arterial or venous anomalies po- tentially associated with stroke. The patient was discharged without sequelae from his stroke. However, he continued to report frequent episodes of dizziness, associated once with facial numbness and dip- lopia, despite being anticoagulated with warfarin at a therapeutic level. At this stage, surgical correction of his vascu- lar anomaly was advised to the patient, who wished to proceed with this option after the exposition of its potential risks and benefits. Interposition of a synthetic graft (Hemoshield Dacron Graft, Boston Scientific Corporation, Natick, MA) between the right common carotid and subclavian arteries was performed uneventfully, allowing antegrade flow to be reestablished in the right vertebral artery. After surgery, his episodic symptoms attributable to brain- stem ischemia resolved. He has remained asymptomatic for more than 1 year.

Discussion We report the case of a 39-year-old man presenting with a vertebrobasilar stroke in the absence of identifi- able cerebrovascular risk factors. The symptoms described by our patient pointed to a lesion in the territory of the right posterior inferior cerebellar artery, which was confirmed by neuroimaging. In our patient, the co-occurrence of a vertebrobasilar infarct and a subclavian steal phenomenon strongly suggest a caus- ative relationship between the 2 events, that is, a sub- Fig 1. Schematic representation of the morphological changes clavian steal syndrome secondary to the cardiac surgery associated with a Blalock-Taussig shunt. The pulmonary trunk undergone during childhood. Although an embolic and arteries are represented in light gray; the aortic arch (1) mechanism cannot be completely excluded because of and supra-aortic trunks are in dark gray. The Blalock-Taussig normal transesophageal echocardiogram and transcra- shunt is established between the proximal right subclavian nial Doppler, both with negative bubble tests, that the artery (6) and the right pulmonary artery (8) by interposition of a synthetic graft (7). The subclavian steal phenomenon, involved branch (the right posterior inferior cerebellar that is, antegrade flow in the left vertebral artery and retro- artery) originates from the vertebral artery with reverse grade flow in the right vertebral artery (4), provides blood flow renders this explanation very unlikely. Conversely, supply to the right arm (direction of blood flow as indicated this anatomical situation is consistent with an increased by the arrows). The figure also shows the right common ca- risk of preferential hypoperfusion in the right posterior rotid artery (2), the right carotid bifurcation (3), and the inferior cerebellar artery territory. basilar artery (5). The possible association between a Blalock-Taussig shunt and a subclavian steal phenomenon initially was proposed by Folger and Shah in 1965.3 By reviewing rule out potential cardioembolic sources, was unremarkable 123 cardiac angiograms performed on patients with a except for a mildly dilated right ventricular cavity. Reverse Blalock-Taussig shunt, these authors could identify late flow in the right vertebral artery was by documented trans- opacification of a subclavian artery suggestive of a sub- cranial Doppler. The transcranial Doppler bubble test was clavian steal phenomenon in 12 instances. The steal negative for embolic events at rest and during Valsalva ma- phenomenon was associated with dizziness in 3 pa- neuver and coughing. Cerebral digital subtraction angiography then was performed. The aortic arch study showed tients and with visual disturbances in 2 patients. How- interruption of the right subclavian artery at its origin (see ever, in those patients and in other case reports de- Fig 2B). Selective injections of the left vertebral artery con- scribing early cerebrovascular events after a Blalock- 4,5 firmed the presence of a left-to-right subclavian steal phe- Taussig shunt, the vertebrobasilar manifestations nomenon. Revascularization of the right upper extremity occurred after the creation of the shunt but before de- was provided by retrograde filling of several subclavian ar- finitive repair of the tetralogy of Fallot. In these pa-

232 Annals of Neurology Vol 52 No 2 August 2002 Fig 2. (A) Magnetic resonance imaging study of the brain. This axial T2-weighted image shows increased signal in the lower portion of the right cerebellar hemisphere associated with mini- mal mass effect on the right posterior-lateral aspect of the brain- stem and subtle leftward midline shift. A small area of hypersig- nal also is seen in the right side of the myelencephalon (arrowhead). This pattern of abnormal signal is consistent with an ischemic lesion in the territory of the right posterior-inferior cerebellar artery. Note the incidental finding of chronic maxil- lary sinus inflammation. (B) Digital subtraction angiography of the aortic arch, left anterior oblique view. The aortic arch, the right common carotid artery (RCC), the left common carotid artery (LCC), and the left subclavian artery (LSC) are unremarkable. The right subclavian artery is interrupted close to its origin from the innominate artery (arrow). (C) Selective digital subtraction angiography of the left vertebral artery, anteroposte- rior view. The catheter tip (arrowhead) is placed at the origin at the left vertebral artery (LV). Retrograde flow in the right vertebral artery (RV) and in several cervical branches allows for late opacification of the right subclavian artery (arrow).

tients, contributing factors related to cardiac dysfunc- after total cardiac repair of the tetralogy of Fallot was tion, such as hypoxia, polycythemia, bacterial suggested in 1984 by Kurlan and colleagues.6 These endocarditis, and mural or valvular thrombosis, have to authors described a 38-year-old patient developing be considered.6 The association of a Blalock-Taussig transient vertebrobasilar ischemia 31 years after a shunt with delayed ischemic complications occurring Blalock-Taussig shunt and 4 years after total repair of

Gailloud et al: Blalock-Taussig Shunt and Stroke 233 histetralogyofFallot.Our39-year-oldpatientpre- 5.SokolS,NarkiewiczM,BillewiczO.Subclavianstealsyndrome sentedwithacerebellarinfarct37yearsafterthecre- afterBlalock-Taussiganastomoses.JCardiovascSurg(Torino) ationoftheshuntand30yearsaftertotalcardiacre- 1969;10:350–354. 6.KurlanR,KrallRL,DeweeseJA.Vertebrobasilarischemiaafter pair.Thisobservation,thefirstreportofadocumented totalrepairoftetralogyofFallot:significanceofsubclaviansteal strokeafterBlalock-Taussigshuntandtotalcardiacre- createdbyBlalock-Taussiganastomosis.Stroke1984;15: pair,appearstoconfirmthatpatientswhounderwenta 359–362. Blalock-Taussigprocedureinchildhoodhaveanin- 7.MurphyJG,GershBJ,MairDD,etal.Long-termoutcomein creasedriskofvertebrobasilarinsufficiencylaterinlife. patientsundergoingsurgicalrepairoftetralogyofFallot.NEngl JMed1993;329:593–599. Thefewcasesreportedsofarthathaveattributedalate cerebrovasculareventtoaBlalock-Taussigshuntmight reflectonlytherelativelyyoungageoftheconcerned population.Blalock-Taussigshuntshavebeenper- formedoninfantsandsmallchildrenforapproximately 50years,somuchofthatpopulationisnowapproach- CerebralX-Linked ingmiddleage.Itisconceivablethatthemorphologi- calriskfactorconstitutedbythesurgicallycreatedsub- Adrenoleukodystrophy clavianstealphenomenongenerallyisnotsignificant enoughtobecomesymptomaticinisolation,asappears inaGirlwith tohavebeenthecaseforourpatient.However,the associationoftheBlalock-Taussiganatomicalanoma- Xq27-TerDeletion EliHershkovitz,MD,1 GinatNarkis,BA,2 lieswithage-relatedprocessessuchashypertensionand 1 3 atheromatousdiseasemightpredisposethesepatientsto ZamirShorer,MD, AnnB.Moser,BA, PaulA.Watkins,MD,PhD,3 HugoW.Moser,MD,3 moreprecociousdevelopmentofcerebrovasculardis- andEstherManor,PhD2 eases.Ifthisassumptioniscorrect,patientswithahis- toryofBlalock-Taussigshuntconstituteanoverlooked groupatriskforcerebrovasculardisease.Furthermore, An8.5-year-oldgirlwithapathogenicmutation thesepatientsarenowingoodgeneralhealth;mostof (515insC)oftheATP-bindingcassette,subfamilyD, themhavelostcontactwiththeircardiologistandare member1gene(ABCD1)onhermaternallyderivedX notfolloweduponasystematicbasis. chromosomeshowedclinical,biochemical,andmagnetic Inviewofthepotentiallydevastatingconsequences resonanceimagingabnormalitiessimilartothoseinaf- ofavertebrobasilarstroke,patientswhounderwenta fectedmales.Cytogeneticstudiesledtothesurprisefind- Blalock-Taussigshuntmaybenefitfromprophylactic ingofadenovodeletionofXq27onthepaternallyde- measures,suchastheadministrationofplateletanti- rivedXchromosome.Abonemarrowtransplanthadan aggregantagents.Moreaggressivetreatmentsmay apparentlyfavorableeffect.Cytogeneticstudiesshouldbe performedinallseverelysymptomaticX-linkedadreno- benecessaryforpatientswhoremainsymptomatic leukodystrophyheterozygotes. underconservativemanagement,suchasthecarotid- subclavianbypassperformedinourpatient.Follow-up AnnNeurol2002;52:234–237 studiesofpatientswithaBlalock-Taussigshunthave beenconductedmainlyfromacardiologicalper- spective.7 Thereis,asfarasweknow,nopublished X-linkedadrenoleukodystrophy(X-ALD)isaprogressive long-termevaluationofthispopulationfromaspe- disorderthataffectsmyelinbecauseofadefectinthe cificneurologicalviewpoint.Suchastudyisrequired genefortheadenosinetriphosphate(ATP)-bindingcas- 1 tobetterdefinethelatecerebrovascularriskpoten- sette,subfamilyD,member1(ABCD1). ABCD1islo- 2 tiallyassociatedwithahistoryofBlalock-Taussig catedonXq28.ItcodesforALDprotein(ALDP), a shunt. References Fromthe 1PediatricDepartmentand 2GeneticInstitute,Soroka 1.BlalockA,TaussigHB.Thesurgicaltreatmentoftheheartin UniversityMedicalCentre,FacultyofHealthSciences,Ben-Gurion whichthereispulmonarystenosisorpulmonaryatresia.JAMA UniversityoftheNegev,BeerSheva,Israel,and 3KennedyKrieger 1945;128:189–202. Institute,JohnsHopkinsUniversity,Baltimore,MD. 2.ReivichM,HollingHE,RobertsB,TooleJF.Reversalofblood flowthroughthevertebralarteryanditseffectoncerebralcircu- ReceivedDec14,2001,andinrevisedformMar12,2002.Ac- ceptedforpublicationMar12,2002. lation.NEnglJMed1961;265:878–885. 3.FolgerGM,ShahKD.SubclavianstealinpatientswithBlalock- Published online Jun 23, 2002, in Wiley InterScience Taussiganastomosis.Circulation1965;31:241–248. (www.interscience.wiley.com).DOI:10.1002/ana.10248 4.NaitoH,KurokawaK,KannoT,etal.Statusepilepticusand AddresscorrespondencetoDrHershkovitz,PediatricDepartment, corticalblindnessduetosubclavianstealsyndromeinagirlwith SorokaUniversityMedicalCentre,POB151,BeerSheva84101, Blalock’soperation.SurgNeurol1973;1:46–49. Israel.E-mail:[email protected]

234 ©2002Wiley-Liss,Inc. peroxisomal membrane protein that belongs to the ATP- Case Report binding cassette superfamily of membrane transport pro- The patient is the first child of unrelated parents. Two ma- teins.3 X-ALD is associated with the accumulation of ternal uncles have X-ALD, 1 with the adolescent cerebral saturated, very long chain fatty acid in tissues, cultured phenotype and the other with the adrenomyeloneuropathy skin fibroblasts,4 and plasma.5 In males, phenotypic ex- phenotype. Pregnancy and early development were normal. She started to walk at 14 months and to speak at 1 year. pression ranges from the severe childhood or adolescent Several behavioral problems were noted in childhood. These cerebral forms, which are associated with a white matter included separation anxiety, with refusal to stay alone in the 6 inflammatory reaction, to slowly progressive adrenomy- kindergarten and school; oversensitivity to noises; and isola- eloneuropathy, which manifests most commonly in tion from peers and kindergarten staff. She continued to de- young adults and mainly affects the long tracts in the velop neurologically in agreement with her age, and she spinal cord, with the inflammatory reaction mild or ab- started to read at 5 to 6 years of age. At that time, she was sent.7 Approximately 70% of affected males have pri- referred to formal psychological assessment because of mary adrenocortical insufficiency. Adrenal steroid ther- “school phobia,” but no diagnosis was made at that time. The deterioration of her academic performance and behavior apy is effective for the endocrine dysfunction but does was noted at 8.5 years. Physical and neurological examina- not appear to alter neurological progression. Bone mar- tions were normal. Formal psychological assessment (Wech- row transplantation (BMT) can be of long-term benefit sler intelligence scale for children-revised) showed that she to boys and adolescents with the cerebral forms of the functioned at the low normal range. The verbal intelligence disease when provided at a time when brain involvement quotient was 87, the performance intelligence quotient was is still relatively mild.8 There is no specific therapy for 83, and the total intelligence quotient was 83. Her achieve- adrenomyeloneuropathy. ments were adversely affected by easy distractibility and visuospatial defects. Cortisol response to 1␮g of intravenous Approximately 50% of women who are heterozygous adrenocorticotropic hormone was normal at 30 minutes for X-ALD develop a progressive syndrome that resem- (26.2␮g/dl), ruling out clinically significant adrenal insuffi- bles adrenomyeloneuropathy but is milder and usually ciency. Brain magnetic resonance imaging demonstrated dif- does not manifest until 35 years of age or later. Adre- fuse white matter involvement that was most prominent in nocortical insufficiency is rare. Approximately 1% of the frontal regions. heterozygous women have a more severe phenotype, in which there is progressive cerebral involvement, adre- Results nocortical insufficiency, and earlier onset9 and which Biochemical and genetic studies were performed after resembles what is seen in boys or adolescents with the informed consent had been obtained from the parents. cerebral phenotypes (H.W.M., unpublished observa- Levels of very long chain fatty acid in plasma and cul- tion). tured skin fibroblasts were measured by capillary gas- 4,5 The cause of the severe manifestations in this small liquid chromatography and found to be elevated proportion of heterozygous women has not been deter- (Table 1). DNA analysis of the patient’s white blood mined definitively. Skewed inactivation of the normal cells and her mother’s white blood cells showed a cy- X chromosome has been considered to be the most tosine insertion in codon 515 (515insC) resulting in a 9,10 frameshift after amino acid 171 (tyrosine). Immunocy- likely explanation. X-inactivation patterns in cul- 11 tured skin fibroblasts or white blood cells can be as- tochemical studies of the cultured skin fibroblasts sessed by an examination of the expression of ALDP by showed that ALDP-reactive material was lacking in 11 99% of the patient’s cells (100 cells counted) and in all immunocytochemical techniques in women who are of the cells of the affected maternal uncles. Cytogenetic members of families in which affected males are known analyses showed a deletion at Xq27.23ter in the pa- to lack immunoreactive material. In 15 heterozygous tient’s peripheral blood lymphocytes. Her karyotype women, 23 to 86% of cells expressed ALDP.12 In con- was 46X;Xdel(q27-tel) (data not shown). Both parents’ trast, ALDP was expressed in only 0 to 2% of cells karyotype analyses were normal. A deletion of the dis- from 2 women who had the adolescent cerebral phe- 10 tal part of Xq, including the telomeric region in the notype, suggesting that in these patients the normal patient’s fibroblasts, also was observed by fluorescence X chromosome failed to exert its protective effect. This in situ hybridization with a specific Xq telomeric conclusion must be considered tentative because find- probe. This region was present in the X chromosomes ings in fibroblasts may not reflect those in the nervous of both parents (data not shown). DNA analysis with system. We now report a patient with the severe phe- 23 polymorphic markers spanning the q26-28 region notype who was known to be heterozygous for X-ALD of the patient’s and her parents’ X chromosomes was because of a pathogenic mutation in the maternal cell performed to define the extent of the deletion, using line but who had in addition a deletion of Xq27-ter in methods that have been described previously.13 An the paternal chromosome, thereby rendering her totally analysis of 13 informative markers showed that a de deficient in ALDP. novo deletion had occurred in the paternal chromo-

Hershkovitz et al: Cerebral X-ALD 235 Table 1. Very Long Chain Fatty Acid Levels in Patient’s Plasma and Cultured Skin Fibroblasts

Location Patient X-ALD Male X-ALD Heterozygote Control

Plasmaa C26:0 1.81 ND ND Ͻ1 C24:0/C22:0 0.91 ND ND 0.68 Ϯ 0.15 C26:0/C22:0 0.043 ND ND 0.018 Ϯ 0.009 Cultured skin fibroblastsb C22:0 0.762 0.64 Ϯ 0.32 0.72 Ϯ 0.26 0.90 Ϯ 0.50 C26:0 0.434 0.41 Ϯ 0.15 0.27 Ϯ 0.17 0.09 Ϯ 0.07 C26:0/C22:0 0.457 0.69 Ϯ 0.19 0.40 Ϯ 0.23 0.08 Ϯ 0.03

The plasma assays were performed at the Sharee Zedek Hospital in Jerusalem (Dr Orly Elpeleg) and those in fibroblasts at the Kennedy Krieger Institute. The C26:0 levels in fibroblasts are similar to those of X-ALD male, and the C26:0/C22:0 ratios in fibroblasts are intermediate between those in male and heterozygous X-ALD patients and compatible with either genetic status. aExpressed as micrograms per milliliter of plasma. bExpressed as micrograms per milligram of protein. ND ϭ not done; X-ALD ϭ X-linked adrenoleukodystrophy. some. The deleted region is distal to marker DXS1227, finding is of practical significance because under these which maps to Xq27.2 (Table 2). Therefore, the de- circumstances therapeutic approaches are similar to leted segment spans part of Xq27.2 and all of Xq27.3 those that would be used in affected males. Given this and Xq28. reasoning and because the patient met current criteria At 8.9 years of age, she received a bone marrow for BMT in affected males,8 we decided to perform the transplant. Her human leukocyte antigen–identical transplant. Her condition was stable 18 months later, normal younger sister was the donor. The procedure and we hope that her long-term outcome will be favor- was tolerated well. Full engraftment was documented. able, as has been the case for male patients.8 Although some deterioration in concentration and We recommend that cytogenetic studies be per- short-term memory were noticed during the early pe- formed in the approximately 1% of heterozygotes who riod after transplantation, her neurological and neuro- show evidence of cerebral involvement by magnetic res- psychological status were stable 18 months after BMT. onance imaging. A karyotype study should be the first step. A normal result should be followed by a search Discussion for microdeletions in chromosome X with specific The abnormal magnetic resonance imaging results and DNA markers spanning the ALD gene region. This in- moderately abnormal neuropsychological dysfunction formation is clinically significant because patients with in this 8.5-year-old girl known to be heterozygous for cerebral involvement may be candidates for BMT. At X-ALD on the basis of family history, mutation anal- this time, BMT is considered only for patients who ysis, and very long chain fatty acid studies indicate that have evidence of cerebral involvement.8 It is never rec- she is among the few heterozygotes with a severe phe- ommended for males or females who have spinal cord notype similar to that in boys with the cerebral forms of the disease. Mutation analysis indicates that the ma- Table 2. Haplotype DNA Analysis of Xq26-28 Using ternal side of her family has a mutation that abolishes Informative DNA Markers the expression of ALDP. Studies in other heterozygotes have led to the hypothesis that severe disability, such as Maternal Paternal cerebral involvement in childhood, is caused by skewed Marker Band Allele Allele 9,10 X inactivation, and the absence of ALDP immuno- DXS8098 Xq26.1 Present Present reactive material would be compatible with this. How- DXS1047 Xq26.3 Present Present ever, cytogenetic studies led to a different conclusion, DXS994 Xq26.3 Present Present namely, the unexpected demonstration of a de novo DXS8050 Xq26.3 Present Present deletion in her paternal X chromosome that involves DXS8072 Xq26.3 Present Present DXS1062 Xq27.1 Present Present all of Xq28 and part of Xq27. Combined with the ab- DXS8094 Xq27.1 Present Present normality on the maternal X chromosome, this leads to DXS1192 Xq27.2 Present Present failure to express functional ALDP, similar to what oc- DXS1227 Xq27.2 Present Absent curs in affected males. Cytogenetic studies are per- DXS8084 Xq27.3 Present Absent formed only rarely in patients with X-ALD. To our DXS8043 Xq27.3 Present Absent DXS1200 Xq27.3 Present Absent knowledge, this type of abnormality has not been re- DXS8091 Xq28 Present Absent ported before in women heterozygous for X-ALD. The

236 Annals of Neurology Vol 52 No 2 August 2002 involvementonly.Forfemalepatientswithcerebralin- volvement,BMTwouldberecommendedonlyfor ANovelMutationinthe thoseinwhombothallelesareinvolved,therebymak- DeoxyguanosineKinase ingthemequivalenttohemizygotes.Wedonotbelieve thatBMTwouldbeindicatedforwomenwhodohave GeneCausingDepletionof 1normalallele,wherethepathogenesisofbrainin- volvementisunknown(althoughskewedinactivationis MitochondrialDNA themostlikelycause),andwedonotbelievethatin Jan-WillemTaanman,PhD,1 IhabKateeb,MD,2 3 4 suchpatientstherisk-benefitratiowouldwarrant AniaC.Muntau,MD, MichaelaJaksch,MD, NadineCohen,MD,2 andHannaMandel,MD5 BMT.Althoughitmightbeofsomeinteresttodo cytogeneticstudiesonallheterozygotes,theaddedex- penseisnotwarrantedinwomenwithoutcerebralin- Recently,ahomozygoussingle-nucleotidedeletionin volvement. exon2ofthedeoxyguanosinekinasegene(DGUOK)was identifiedasthedisease-causingmutationin3apparently unrelatedIsraeli-Druzefamilieswithdepletedhepatoce- References rebralmitochondrialDNA.Wehavediscoveredanovel 1.MoserHW,SmithKD,WatkinsPA,etal.X-linkedadreno- homozygousnonsensemutationinexon3ofDGUOK leukodystrophy.In:ScriverCR,BeaudetAL,SlyWS,ValleD, (313C3T)fromapatientborntononconsanguineous eds.Themetabolicandmolecularbasesofinheriteddisease.8th Germanparents.Thisfindingshowsthatmutationsin ed.NewYork:McGraw-Hill,2001:3257–3301. DGUOKcausingmitochondrialDNAdepletionarenot 2.MosserJ,DouarAM,SardeCO,etal.PutativeX-linkedadre- confinedtoasingleethnicgroup. noleukodystrophygenesharesunexpectedhomologywithABC transporters.Nature1993;361:726–730. AnnNeurol2002;52:237–239 3.HigginsCF,GallagherMP,MimmackML,PearceSR.Afam- ilyofcloselyrelatedATP-bindingsubunitsfromprokaryotic andeukaryoticcells.Bioessays1988;8:111–116.fs In1991,Moraesandcolleagues1 identifiedagroupof 4.MoserHW,MoserAB,KawamuraN,etal.Adrenoleuko- infantswithmarkeddepletionofmitochondrialDNA dystrophy:elevatedC26fattyacidinculturedskinfibroblasts. (mtDNA)inassociationwithdefectivemitochondrial AnnNeurol1980;7:542–549. respiratorychainfunction.Thiscondition,oftencalled 5.MoserAB,KreiterN,BezmanL,etal.Plasmaverylongchain fattyacidsin3,000peroxisomediseasepatientsand29,000 mtDNAdepletionsyndrome(OnlineMendelianInheri- controls.AnnNeurol1999;45:100–110. tanceinMan[OMIM]251880),nowhasbeende- 2–11 6.PowersJM,LiuY,MoserAB,MoserHW.Theinflammatory scribedformorethan50patients; thissuggeststhat myelinopathyofadreno-leukodystrophy:cells,effectormole- itmaybeanimportantcauseofmitochondrialdys- cules,andpathogeneticimplications.JNeuropatholExpNeu- functioninneonatesandinfants.Mostofthereported rol1992;51:630–643. patientspresentintheneonatalperiodwithmuscle 7.PowersJM,DeCieroDP,ItoM,etal.Adrenomyeloneu- weakness,liverfailure,andneurologicalabnormalities ropathy:aneuropathologicreviewfeaturingitsnoninflamma- torymyelopathy.JNeuropatholExpNeurol2000;59:89–102. associatedwithlacticacidemiaanddiebefore12 8.ShapiroE,KrivitW,LockmanL,etal.Long-termbeneficial effectofbonemarrowtransplantationforchildhoodonsetce- rebral X-linked adrenoleukodystrophy. Lancet 2000;356: 713–718. Fromthe 1UniversityDepartmentofClinicalNeurosciences,Royal 9.HeffungsW,HameisterH,RopersHH.Addisondiseaseand FreeandUniversityCollegeMedicalSchool,UniversityCollege cerebralsclerosisinanapparentlyheterozygousgirl:evidence London,London,UnitedKingdom; 2DepartmentofGenetics, forinactivationoftheadrenoleukodystrophylocus.ClinGenet TamkinHumanMolecularGeneticsResearchFacility,Technion- 1980;18:184–188. IsraelInstituteofTechnology,BruceRappaportFacultyofMedi- cine,Haifa,Israel; 3DrvonHaunerChildren’sHospital,Ludwig- 10.NaiduS,WashingtonC,ThirumalaiS,etal.X-chromosome Maximilians-Universita¨t,Munich,Germany; 4Stoffwechselzentrum inactivationinsymptomaticheterozygotesinX-linkedadreno- undInstitutfu¨rKlinische-Chemie,KrankenhausMu¨nchen- leukodystrophy.AnnNeurol1997;42:498(Abstract). Schwabing,Munich,Germany;and 5MetabolicDiseaseUnit,De- 11.WatkinsPA,GouldSJ,SmithMA,etal.Alteredexpressionof partmentofPediatrics,RambamMedicalCenter,Technion-Israel ALDPinX-linkedadrenoleukodystrophy.AmJHumGenet InstituteofTechnology,BruceRappaportFacultyofMedicine, 1995;57:292–301. Haifa,Israel. 12.FeigenbaumV,Lombard-PlatetG,GuidouxS,etal.Muta- ReceivedDec13,2001,andinrevisedformMar12,2002.Ac- tionalandproteinanalysisofpatientsandheterozygouswomen ceptedforpublicationMar13,2002. withX-linkedadrenoleukodystrophy.AmJHumGenet1996; Published online Jun 23, 2002, in Wiley InterScience 58:1135–1144. (www.interscience.wiley.com).DOI:10.1002/ana.10247 13.ParvariR,MummS,GalilA,etal.Deletionof8.5Mb,in- AddresscorrespondencetoDrTaanman,UniversityDepartmentof cludingtheFMR1gene,inamalewiththefragileXsyndrome ClinicalNeurosciences,RoyalFreeandUniversityCollegeMedical phenotypeandovergrowth.AmJMedGenet1999;83: School,UniversityCollegeLondon,RowlandHillStreet,London 302–307. NW32PF,UnitedKingdom.E-mail:[email protected]

©2002Wiley-Liss,Inc. 237 months of age. Others present in infancy with isolated After informed parental consent, in accordance with the myopathy associated with motor regression or a slowly guidelines of the local institution, DNA was extracted from progressive encephalomyopathy. Inheritance appears to liver of the proband, blood of the parents, and cultured fi- 6 be autosomal recessive, suggesting that nuclear gene de- broblasts of the second child. All exons of DGUOK were amplified by polymerase chain reaction, purified, and se- fects are responsible for mtDNA depletion. In agree- 13 ment with this notion, cybrid studies have shown that quenced exactly as described earlier. Sequencing was per- 3,12 formed in both directions. The human DGUOK messenger the defect is not transmitted by patient mtDNA, RNA sequence, with accession number U41668, and the hu- and partial sequencing of patient mtDNA has failed to 1,4 man chromosome 2 working draft sequence segment, with identify mutations. accession number NT025651, were used to determine the Recently, homozygosity mapping followed by candi- intron/exon structure of DGUOK. date gene sequence analysis in 3 kindreds of Israeli- Druze origin with the early-onset hepatocerebral vari- Results ant of the disease showed a homozygous mutation in Sequencing of the 7 exons and the intron/exon bound- the gene for deoxyguanosine kinase (DGUOK)on 13 aries of the DGUOK gene from the proband showed a chromosome 2p13. In addition, mutations in the homozygous C3T transition at nucleotide position gene for mitochondrial thymidine kinase (TK2)on 313 (numbering according to Johansson and Karls- chromosome 16q22 were identified in patients with 16 14 son ) of exon 3. The same homozygous mutation was the late-onset muscle-specific variant of the disease. found in the clinically affected sibling. Both parents Deoxyguanosine kinase is responsible for phosphoryla- were heterozygous for the mutation (Fig). The mutation of purine deoxyribonucleosides in the mitochon- 15 tion changes the arginine CGA codon 105 into a TGA drial matrix compartment. The 3 apparently unre- stop codon (see Fig). This base change is predicted to lated Israeli-Druze families carried the same single- 13 result in a 173–amino acid residue truncation at the C nucleotide deletion in exon 2 of DGUOK. This terminus of the DGUOK protein product. The muta- mutation is expected to result in premature termina- tion was not found in 15 control subjects of European tion of translation. To investigate whether mutations origin. in DGUOK are also present in patients from a different ethnic origin, we sequenced the exons and intron/exon boundaries of DGUOK from a German patient with Discussion the hepatocerebral form of the disease. The patient was In an earlier study, we identified an infant, born to found to harbor a novel homozygous nonsense muta- healthy, nonconsanguineous German parents, with tion in DGUOK. This shows that mutations in early-onset encephalopathy and rapidly progressing DGUOK causing mtDNA depletion are not restricted liver failure associated with severely depleted levels of to patients belonging to one particular ethnic group. mtDNA.11 The recent discovery of a single-nucleotide deletion in exon 2 of DGUOK in 3 unrelated Israeli- Druze families with depleted hepatocerebral mtDNA Case Report prompted us to screen the German patient for muta- The clinical details of the patient and histochemical findings 11 tions in the gene. We found a homozygous nonsense are documented in detail elsewhere. In brief, the patient mutation in exon 3 of the proband and his affected was the first child of healthy, nonconsanguineous German younger brother, whereas both parents were carriers. parents (birth weight, 2,570gm). The boy presented with lac- tic acidosis, hepatomegaly, hypoglycemia, and jaundice The mutation will lead to a shortening of the DGUOK shortly after birth. He had a severe encephalopathy, charac- protein product by more than half. The truncation in- terized by marked muscle hypotonia, hyperreflexia, irritabil- cludes 3 domains that are evolutionarily conserved be- ity, and pendular horizontal nystagmus. At the age of 2 tween nucleoside kinases and are thought to be essen- 16 months, neonatal giant cell hepatitis was diagnosed by light tial for catalysis. It is, therefore, highly unlikely that microscopy. Electron microscopy of the liver showed an ac- the mutated DGUOK gene is still functionally active in cumulation of abnormal mitochondria and steatosis. Histo- the patient. The absence of the mutation in control chemistry and immunohistochemistry for cytochrome-c oxi- subjects of European origin further supports the patho- dase demonstrated a mosaic pattern of normal and deficient genicity of the mutation. hepatocytes. Skeletal muscle was normal on both light and The mutation in the DGUOK gene that we identi- electron microscopy, but biochemical assays showed a minor fied in a German family indicates that mutations in cytochrome-c oxidase deficiency. Southern blot analysis of liver biopsies, taken at 2 and 3 months of age, showed that DGUOK causing mtDNA depletion are not limited to mtDNA was of normal size, but mtDNA levels were only 17 Israeli-Druze patients. However, mutation screening of and 18%, respectively, of the mean of 6 age-matched control DGUOK in 11 additional families with early-onset en- specimens (range controls, 66–140%).11 The patient died of cephalopathy, liver failure, and mtDNA depletion of hepatic failure at the age of 5 months. A younger brother British (5 patients), Greek-Cypriot (3 patients), Ger- shows similar symptoms. man (1 patient), French (1 patient), and Turkish (1

238 Annals of Neurology Vol 52 No 2 August 2002 This study was supported by the Wellcome Trust (grant 048410, J.W.T.), the DFG (grant Ja 802/2-1, M.J.), and the Joseph Elias Fund/Technion VPR Fund (grant 181-421, H.M.). We thank Dr A. H. V. Schapira and Dr J. V. Leonard for helpful discussions.

References 1. Moraes CT, Shanske S, Trischler H-J, et al. mtDNA depletion with variable tissue expression: a novel genetic abnormality in mitochondrial diseases. Am J Hum Genet 1991;48:492–501. 2. Tritschler H-J, Andreetta F, Moraes CT, et al. Mitochondrial myopathy of childhood associated with depletion of mitochondrial DNA. Neurology 1992;42:209–217. 3. Bodnar AG, Cooper JM, Holt IJ, et al. Nuclear complementa- tion restores mtDNA levels in cultured cells from a patient with mtDNA depletion. Am J Hum Genet 1993;53:663–669. 4. Mariotti C, Uziel G, Carrara F, et al. Early-onset encephalomyopathy associated with tissue-specific mitochondrial DNA depletion: a morphological, biochemical and molecular-genetic study. J Neurol 1995;242:547–556. 5. Macmillan CJ, Shoubridge EA. Mitochondrial DNA depletion: prevalence in a pediatric population referred for neurologic evaluation. Pediatr Neurol 1996;14:203–210. 6. Morris AAM, Taanman J-W, Blake J, et al. Liver failure associated with mitochondrial DNA depletion. J Hepatol 1998;28: 556–563. 7. Vu TH, Sciacco M, Tanji K, et al. Clinical manifestations of mitochondrial DNA depletion. Neurology 1998;50:1783–1790. 8. Blake JC, Taanman J-W, Morris AMM, et al. Mitochondrial DNA depletion syndrome is expressed in amniotic fluid cell cultures. Am J Pathol 1999;155:67–70. 9. Barthe´le´ my C, Ogier de Baulny H, Diaz J, et al. Late-onset mitochondrial DNA depletion: DNA copy number, multiple deletions, and compensation. Ann Neurol 2001;49:607–617. 10. Mandel H, Hartman C, Berkowitz D, et al. The hepatic mitochondrial DNA depletion syndrome: ultrastructural changes in Fig. Electropherograms showing part of the exon 3 sequences of liver biopsies. Hepatology 2001;34:776–784. the deoxyguanosine kinase gene (DGUOK) of a control, both 11. Mu¨ller-Ho¨cker J, Muntau AC, Scha¨fer S, et al. Depletion of parents, the proband (1st child), and his brother (2nd child). mitochondrial DNA in the liver of an infant with neonatal gi- The position of the mutation is indicated with an arrow. ant cell hepatitis. Hum Pathol 2002;33:247–253. Numbering is according to Johansson and Karlsson.16 12. Taanman J-W, Bodnar AG, Cooper JM, et al. Molecular mechanisms in mitochondrial DNA depletion syndrome. Hum Mol patient) origin, including the 6 families described by us Genet 1997;6:935–942. 3,6,8 13. Mandel H, Szargel R, Labay V, et al. The deoxyguanosine ki- previously, did not show any mutations in the 7 nase gene is mutated in individuals with depleted hepatocere- exons and the intron/exon boundaries of DGUOK (not bral mitochondrial DNA. Nat Genet 2001;29:337–341. shown). The clinical, biochemical, and molecular find- 14. Saada A, Shaag A, Mandel H, et al. Mutant mitochondrial thy- ings of these patients were not markedly different from midine kinase in mitochondrial DNA depletion myopathy. Nat the case described here. These results suggest that mu- Genet 2001;29:342–344. 15. Ju¨llig, M, Eriksson S. Mitochondrial and submitochondrial lo- tations in the coding region of DGUOK are not com- calization of human deoxyguanosine kinase. Eur J Biochem mon in patients with hepatocerebral mtDNA deple- 2000;267:5466–5472. tion. Although the disease may be genetically 16. Johansson M, Karlsson A. Cloning and expression of human heterogeneous, our results do not rule out that these 11 deoxyguanosine kinase cDNA. Proc Natl Acad SciUSA1996; families carry mutations elsewhere in DGUOK that af- 93:7258–7262. fect gene expression. This possibility is likely in 2 additional families of Druze and Moroccan origin, respectively, in which the disease has been linked to DGUOK but in which mutations in the coding region of the gene could not be identified.13 More detailed mutation analysis and expression studies of DGUOK, therefore, are necessary to investigate the genetic heterogeneity of hepatocerebral mtDNA depletion.

Taanman et al: A Novel Mutation Depleting mtDNA 239 L-Dopa–induceddyskinesias(LIDs)andmotorfluctu- DyskinesiasandGrip ationsaremajorcomplicationsoftherapyinlong- standingParkinson’sdisease(PD).Onceapatientde- ControlinParkinson’s velops LID, the sensitization to dopaminergic DiseaseAreNormalizedby treatmentcannotbereversedbydrugs.1 Deep-brain stimulationofthesubthalamicnucleus(STNDBS)is ChronicStimulationofthe aneffectivetreatmentforbothfluctuationsanddyski- 2,3 nesiasinL-dopa–responsivePD. Itsbeneficialeffects SubthalamicNucleus onakinesia,rigidity,andtremorresemblethatof 4 RolandWenzelburger,MD,1 Bao-RongZhang,MD,1 L-dopa, exceptforLIDs,whicharepromotedbyacute MeikePoepping,MD,1 BettinaSchrader,MD,2 STNstimulation,whereastheyimprovewithchronic DieterMu¨ller,PhD,3 FlorianKopper,MD,1 stimulation.5 ThissubstantialimprovementofLIDby UrbanFietzek,MD,1 HubertusM.Mehdorn,PhD,2 1 1 STNDBSevenintheon-drugstate(desensitization)is Gu¨ntherDeuschl,PhD, andPaulKrack,PhD notfullyunderstood.2,3,6 Ithasbeenascribedtoare- ductionofdopaminergicdosageoftenallowedbythe motorbenefitsoftheprocedure,butitspathophysiol- Deep-brainstimulationofthesubthalamicnucleusap- ogyhasremainedunclear.Wefollowedthehypothesis pearstoreducelevodopa-induceddyskinesias,but thataninhibitionoftheexcessofcommandsentto whetherthiseffectiscausedbythereductionofthetotal themusclecouldunderlietheantidyskineticeffectsof levodopaingestionorrepresentsadirecteffectonthe chronicSTNDBSandthereforemeasuredthecalibra- motorsystemisunknown.Precisiongripforceofgrasp- tionofforceintheprecisiongrip,afunctiongoverned ingmovementsandlevodopa-induceddyskinesiaswas 7,8 analyzedin10parkinsonianpatientsbeforeandafter3 mainlybythemotorcortex. Anovershootingofgrip monthsofdeep-brainstimulationofthesubthalamicnu- forceinlate-stagePDhasbeendescribedinthiscon- cleus.Peakgripforcewasabnormallyincreasedbefore dition.Patientsoftenapplyexcessiveforceintheon- 9–11 surgeryintheoff-drugstateand,particularly,intheon- drugstatewhengraspingtoliftasmallobject. drugstate(sensitization).Thisgripforceupregulation Thisforceexcessiscloselyrelatedtotheseverityof 12 normalizedwithchronicdeep-brainstimulationinboth LIDinPD andthereforeissuitedtoobjectively conditions(desensitization).Peak-dosedyskinesiasalso studythebeneficialeffectofSTNDBSonasurrogate improved,andoff-dystoniawascompletelyabolished. parameterforLID.Wealsotriedtoelucidateifdirect Meandosageofdopaminergicdrugswasreduced,but (relatedtostimulation)orindirect(relatedtoreduction forceoverflowanddyskinesiaswereequallyimprovedin ofdopaminergicdrugs)effectsaccountfortherapeutic 2patientswithoutareduction.Despitethesamesingle effects. levodopatestdose,forceexcessandlevodopa-induced dyskinesiasweredrasticallyreducedafter3monthsof deep-brainstimulationofthesubthalamicnucleus.This PatientsandMethods indicatesthatdirecteffectsofdeep-brainstimulationof Westudiedaseriesof10advanced-stagepatientswithPD thesubthalamicnucleusonlevodopa-induceddyskinesias whounderwentSTNDBS.Allsufferedfromseveremotor arelikelytooccur.Gripforceoverflowisapromising fluctuationsandLID(Table).Writteninformedconsentwas parametertostudythedesensitizingeffectofchronic givenbyallpatients.Tenage-matchedhealthycontrolsalso deep-brainstimulationonlevodopa-induceddyskinesias. wereincludedinthestudy,whichwasapprovedbythelocal ethicscommittee.Theassessmentwasconductedaftera12- AnnNeurol2002;52:240–243 hourovernightwithdrawalofdopaminergicdrugs.Patients wereassessedin2treatmentconditionsbefore(off-drugand on-drug)andin4conditions3monthsaftersurgery(off- drug/off-stimulation,on-drug/off-stimulation,off-drug/on- stimulation,andon-drug/on-stimulation).Asuprathreshold 4 FromtheDepartmentsof 1Neurologyand 2Neurosurgery,Christian doseofL-dopawasapplied. ThemotorscoreoftheUnified AlbrechtsUniversity,Kiel;andthe 3DepartmentofNeurosurgery, Parkinson’sDiseaseRatingScale(UPDRS)andadyskinesia UniversityHamburg,Hamburg,Germany. scoreof7bodyregions(scorerange0–28)wereratedinall 5 ReceivedDec27,2001,andinrevisedformMar14,2002.Ac- conditions. Theamplitudeofmotorfluctuationswasde- ceptedforpublicationMar23,2002. finedasthedifferencebetweenUPDRSmotorscoreoff-drug Published online Jun 23, 2002, in Wiley InterScience andon-drugbothbeforesurgeryandaftersurgery(on- (www.interscience.wiley.com).DOI:10.1002/ana.10254 stimulation).TheL-dopaequivalentdailydose(LEDD)was 4 DrZhang’scurrentaddressistheDepartmentofNeuology,Second calculatedasdescribedpreviously. Thesubjectsgraspedan AffiliatedHospital,ZhejiangUniversity,Hangzhou,P.R.China. object(220gm,equippedwith3-dimensionalforcesensors) AddresscorrespondencetoProfDrDeuschl,NeurologischeKlinik betweenthethumbandindexfingerandliftedit15timesat oftheUniversityHospitalKiel,Niemannsweg147,24105Kiel, anaturalpace.Wefocusedonthepeakgripforce(GFPEAK), Germany.E-mail:[email protected] ameasurewithaprovensensitivityforLID.12 Meanvalues

Condition Motor Score ( Ϯ SD) Dyskinesia Score ( Ϯ SD)

Before surgery Off drug 50.3 Ϯ 12.8 3.4 Ϯ 5.4 On drug 20.4 Ϯ 6.3 9.1 Ϯ 5.6 ⌬ Off-on 29.9 Ϯ 14.4 After surgery Off drug/off stim 42 Ϯ 17.2 0a On drug/off stim 17.7 Ϯ 7.1 2.9 Ϯ 2.9c Off drug/on stim 19.9 Ϯ 10.6b 2.9 Ϯ 1.9 On drug/on stim 13.8 Ϯ 5.7c 4.9 Ϯ 4.2c ⌬ Off-on drug 6.1 Ϯ 7.4d ap Ͻ 0.001, bp Ͻ 0.01, compared with presurgery off drug condition. cp Ͻ 0.01 compared with presurgery drug condition. dp Ͻ 0.01 compared with amplitude of motor fluctuations before surgery (⌬ off-on drug, on stim). SD ϭ standard deviation; stim ϭ stimulation.

of GFPEAK of the thumb were calculated from the lifting enced by treatment. After 3 months of chronic stimu- trials 6 to 15 of both sides (Fig 1). lation, the force overflow vanished. No excess of Grip force was compared between groups and treatment GFPEAK was observed any longer regardless of the state conditions by a general linear model (SPSS 10). Significance of drug or stimulation, and the grip force was equiva- was assumed for p values less than 0.05 after Bonferroni cor- lent to controls (see Figs 1 and 2). The presurgical ex- rection. Clinical scores and LEDD were compared using the Wilcoxon test with a p level of less than 0.01. cess of GFPEAK in the off-drug condition was abolished. In the on-drug state GFPEAK decreased by Ϫ Results 110%. In the combined treatment state GFPEAK was The LEDD was reduced in all but 2 patients, on av- still significantly lower than before surgery (see Fig 2). erage by 45% (p Ͻ 0.01). The UPDRS motor score A reduction of initially exaggerated GFPEAK was ob- was improved by Ϫ60% off-drug and by Ϫ32% on- served not only in the 8 patients with a reduced LEDD drug. The amplitude of motor fluctuations decreased after surgery, but also in the 2 patients on-stimulation by Ϫ80% (see Table). whose LEDD was not reduced because of remaining Off-dystonia was abolished in all 9 patients involved. minor fluctuations and positive effects on mood. In On-phase dyskinesias of the total body were completely on-drug condition, the peak grip force decreased by abolished in 1 patient, and the score was reduced in all Ϫ51% and by Ϫ48% in the first and second patient, the other patients, on average by Ϫ67%. Dyskinesias respectively. The same was true in the off-drug state Ϫ Ϫ tended to increase on combined challenge with L-dopa (GFPEAK, 45% and 25%). Thus, grip force excess and STN DBS, but the mean score was still Ϫ44% resolved, although LEDD was not decreased in either lower than it was preoperatively. of the patients. Furthermore, GFPEAK was not corre- Peak grip force (GFPEAK) was significantly influ- lated significantly with LEDD, neither before nor after

Fig 1. Peak grip force (GFPEAK in Newtons) in a healthy control subject and a representative patient who suffered from severe L-Dopa–induced dyskinesia. Force overshooting in on-state was abolished after chronic stimulation. (thick lines) Mean; (thin lines) standard error of mean from 10 trials. Scale is identical for all conditions. PD ϭ Parkinson’s disease; N ϭ Newton; S ϭ seconds.

Wenzelburger et al: Desensitization by DBS of the STN in PD 241 Fig 2. Mean peak grip force ϩ (GFPEAK SD) before and 3 months after surgery. The overshooting of force was abolished in all conditions after surgery. (single dagger) p Ͻ 0.05, (double dagger) p Ͻ 0.01 compared with on-drug state before surgery. (asterisk) p Ͻ 0.05 compared with presurgical off- drug state. (single circle) p Ͻ 0.05, (double circle) p Ͻ 0.01 compared with controls. surgery (Spearman coefficient, Ͻ 0.4; p Ͼ 0.05). The all the parkinsonian symptoms because switching off reduction of GFPEAK closely matched the effect of the stimulator led to a reoccurrence of severe akinesia STN DBS on peak-dose dyskinesias. and rigidity but left LID and force excess unchanged. Therefore, the LID-suppressing effect is more likely to Discussion reflect a desensitizing long-term effect of chronic STN We observed a remarkable normalization of force cali- DBS on LID and force regulation that outlasts even a bration in dexterous movements induced by chronic temporary interruption of stimulation. The develop- STN DBS which was found both during on-state and ment of response fluctuations is believed to be caused off-state. The preoperative L-dopa challenge caused an by the discontinuous pharmacological stimulation of overshooting of grip force in line with previous find- 11,12 the dopamine-receptors by drug treatment. In contrast, ings, but this was no longer the case in the post- STN DBS is continuously stimulating the motor sys- operative challenge with the same dosage. The initial tem. We propose that such continuous stimulation grip force excess vanished almost completely under may explain desensitization of both LID and grip force chronic STN DBS. In parallel with the reduction of excess. This desensitization of involuntary motor activ- grip force overshoot, the LID score during a suprath- ity might be explained by plastic changes in the motor reshold L-dopa challenge was reduced substantially, system directly related to chronic STN DBS. It is not which was within the range expected from recent reports.2,3,5,6 These findings extend our earlier observa- yet clear if this desensitization takes place within the tion that grip force excess in on-state is found in par- basal ganglia or the cortex. The profound changes of kinsonian patients with motor fluctuations only, and cortical metabolism when comparing the on-state and overshooting of force correlates with the severity of the off-state with functional imaging might support the LID.12 The close relationship between LID and force latter possibility but is far from proving this interpre- regulation is now supported by similar benefits of STN tation. DBS on both. The reduction of LID in patients with STN DBS has been ascribed to a reduction of dopaminergic dos- This research was supported by the Deutsche Forschungsgemeinss- 3,5,6 age, and a direct effect of DBS has been discussed chaft (01KO9811/7, R.W.) and the Kompetenznetzwerk Parkinson. 5 mainly for off-dystonia. These observations make this B.-R.Z. was on sabbatical leave from the Department of Neurology, view unlikely. We observed substantial benefits on LID Second Affiliated Hospital, Zhejiang University, Hangzhou, P.R. in all patients, regardless whether the dopaminergic China, and was supported by the Kiel University. drugs were reduced. Furthermore, the LID-suppressing We thank Mrs Witt for the excellent support of this investigation, effect of STN is not simply part of a general effect on Dr Johansson and Mr Ba¨ckstro¨m for advice concerning the grip

242 Annals of Neurology Vol 52 No 2 August 2002 paradigmandtheSC/ZOOMsoftware,andDrPohlforvaluable helpwiththestatistics. DemonstrationofAcute IschemicLesionsintheFetal References 1.NuttJG.Clinicalpharmacologyoflevodopa-induceddyskine- BrainbyDiffusionMagnetic sia.AnnNeurol2000;47(suppl1):S160–S164;discussion, S164–S166. 2.LimousinP,KrackP,PollakP,etal.Electricalstimulationof ResonanceImaging 1 2 thesubthalamicnucleusinadvancedParkinson’sdisease. CristinaBaldoli,MD, AndreaRighini,MD, 2 1 NEnglJMed1998;339:1105–1111. CeciliaParazzini,MD, GiuseppeScotti,MD, 2 3.KrackP,LimousinP,BenabidAL,PollakP.Chronicstimula- andFabioTriulzi,MD tionofsubthalamicnucleusimproveslevodopa-induceddyski- nesiasinParkinson’sdisease.Lancet1997;350:1676. 4.KrackP,PollakP,LimousinP,etal.Subthalamicnucleusor Thepossibilityofdetectingacutehypoxic-ischemicbrain internalpallidalstimulationinyoungonsetParkinson’sdisease. lesionsbyprenatalmagneticresonanceimagingorultra- Brain1998;121:451–457. 5.KrackP,PollakP,LimousinP,etal.Fromoff-perioddystonia soundislow.Wepresentacaseofafetuswithaveinof topeak-dosechorea.Theclinicalspectrumofvaryingsubtha- Galenarteriovenousmalformationinwhomprenatal lamicnucleusactivity.Brain1999;122:1133–1146. diffusion-weightedmagneticresonanceimagingat33 6.BejjaniBP,ArnulfI,DemeretS,etal.Levodopa-induceddys- weeksofgestationclearlydetectedcerebralacuteisch- kinesiasinParkinson’sdisease:issensitizationreversible?Ann emiclesions,associatedwithremarkabledecreaseofthe Neurol2000;47:655–658. averageapparentdiffusioncoefficient,whereasT2- 7.JeannerodM.Theformationoffingergripduringprehension. weightedimagingwasstillnotinformative. Acorticallymediatedvisuomotorpattern.BehavBrainRes 1986;19:99–116. AnnNeurol2002;52:243–246 8.LemonRN,JohanssonRS,WestlingG.Corticospinalcontrol duringreach,grasp,andprecisionliftinman.JNeurosci1995; 15:6145–6156. Prenatalmagneticresonanceimaging(MRI)iswidely 9.GordonAM,IngvarssonPE,ForssbergH.Anticipatorycontrol ofmanipulativeforcesinParkinson’sdisease.ExpNeurol1997; usedtoconfirmultrasoundfindingsincasesofcom- 1 145:477–488. plexfetalbrainmalformations. PrenatalMRIandul- 10.FellowsSJ,NothJ,SchwarzM.PrecisiongripandParkinson’s trasoundalsocanevaluatetheend-stagesequelaeof disease.Brain1998;12:1771–1784. hypoxic-ischemicfetalbraindamage2;however,the 11.GordonAM,ReilmannR.Gettingagrasponresearch:does possibilityofdetectingtheselesionsbyprenatalMRI treatmenttainttestingofparkinsonianpatients?Brain1999; 3 122:1597–1598. orultrasoundintheacutephaseislow. Diffusion- 12.WenzelburgerR,ZhangBR,PohleS,etal.Forceoverflowand weightedMRI(DWI)hasbeenshowntobeverysen- levodopa-induceddyskinesiasinParkinson’sdisease.Brain sitiveindetectinghyperacuteandacutehypoxic- 2002;125:871–879. ischemicbraindamageinadults4 andinpremature5 or term6–10 newborns.Wepresentthecaseofafetuswith aveinofGalenarteriovenousmalformation(VGAM), inwhomprenatalDWIat33weeksofgestationclearly detectedacuteischemiclesionswithinthebrain, whereasT2-weightedimagingwasstillnotinformative.

CaseReport A27-year-oldpregnantwomanwasreferredtoourinstitu- tionat33weeksofgestationbecauseofsuspectedfetal VGAMatDopplerultrasonography.AprenatalMRIstudy at1.5T(Eclipse;Marconi,Cleveland,OH)usingtheflex

Fromthe 1NeuroradiologyDepartments,Universita`SaluteeVita IRCCS–SanRaffaeleand 2IstitutiClinicidiPerfezionamento,Mi- lan,Italy. ReceivedJan25,2002,andinrevisedformMar20.Acceptedfor publicationMar23,2002. Published online Jun 23, 2002, in Wiley InterScience (www.interscience.wiley.com).DOI:10.1002/ana.10255 AddresscorrespondencetoDrRighini,RadiologiaeNeuroradiolo- gia,OspedaledeiBambiniV.Buzzi,Viacastelvetro32,20154Mi- lan,Italy.E-mail:[email protected]

©2002Wiley-Liss,Inc. 243 body surface coil and T2-weighted 5mm-thick single-shot normal frontal lobe areas (average value, 1.83; SD, fast spin-echo sequences was performed. 0.18␮m2/msec) or for literature data on ADC in premature neonates.11,12 Results At 38 weeks of gestation, a cesarean section was per- T2-weighted images confirmed the presence of the formed, and a baby girl was delivered. She presented VGAM, which appeared as an interhemispheric flow with severe muscular hypotonia and signs of moderate void mass in the region of the vein of Galen, con- left ventricle failure at Doppler ultrasonography nected to the torcular. Some irregular and prominent (23mm end diastolic diameter and 41% ejection frac- vessels were noted around the third ventricle; they were tion), whereas she did not show significant polypnea or compatible with choroidal arteries feeding the malfor- pulmonary edema at chest x-rays. Four days after birth, mation. The brain parenchyma did not exhibit clear an MRI study showed severe diffuse atrophy of both signal alterations, and there was no ventricular enlarge- cerebral hemispheres with drastic enlargement of corti- ment (Fig 1). Because hypoxic-ischemic brain lesions cal liquoral spaces and ex vacuo dilatation of lateral can be associated with VGAM, a DWI acquisition was ventricles; MRI showed also severe general cortical ne- performed in the same occasion. The DWI study was crosis, most of the cortex being reduced to a thin rim, based on an echo-planar 3-axis diffusion sensitized se- and diffuse loss of white matter, which presented sev- quence, and 5mm-thick slices were acquired at fetal eral areas of high T1 signal. These were probably a sign brain level (matrix, 128 ϫ 128; field of view, 30cm; of diffuse calcification, as they were too confluent to be 2 b-factor, 0–1,000sec/mm ). DWI showed areas of hy- hemorrhagic (Fig 2). No DWI or fluid-attenuated in- perintense abnormal signal within the parietal and oc- version recovery sequences were performed on this occipital lobe of the right hemisphere, and there were casion. These findings were compatible with the end- similar findings in the left temporal lobe; these foci of stage sequelae of severe ischemic insults. high signal encompassed not only the cortex, but also part of the adjacent subcortical and periventricular white matter (see Fig 1); in these areas, the rotationally Discussion averaged apparent diffusion coefficient (trace-ADC) This report shows that DWI studies of fetal brain are was remarkably reduced (average value, 0.97; standard feasible. This case highlights the potential of DWI in deviation [SD], 0.038␮m2/msec) both for apparently detecting acute fetal hypoxic-ischemic brain damage,

Fig 1. (Top row) Single-shot fast spin-echo T2-weighted axial sections from the prenatal magnetic resonance imaging study at 33 weeks of gestation, showing no clear signal alterations within brain parenchima. The presence of the a vein of Galen arteriovenous malformation (arrowheads) and of possible abnormal choroidal arteries (arrow) is clearly noticeable. (Middle row) Diffusion- weighted axial sections at similar levels depicting areas of markedly abnormal hyperintense signal in the occipital and parietal lobe of the right hemisphere (arrows). Some abnormal hyperintensity is visible also in the left temporal lobe (curved arrow). Both cortical and adjacent white matter areas are affected. (Bottom row) Corresponding trace–apparent diffusion coefficient (ADC) maps showing a clear ADC decrease in the same areas (arrows) of diffusion-weighted magnetic resonance imaging signal alterations.

244 Annals of Neurology Vol 52 No 2 August 2002 Fig 2. (Top row) Two T2-weighted axial sections (top left and top middle) and 1 axial T1-weighted section (top right) from the postnatal magnetic resonance imaging (MRI) study showing diffuse severe atrophy of both hemispheres, lateral ventricles ex vacuo dilatation, extensive white matter loss, and large areas of T1 hyperintensity within it, compatible with diffuse calcifications (arrows). (Bottom row) Two sagit- tal T1-weighted sections from the postnatal MRI study showing diffuse drastic enlargement of cortical liquoral spaces and thin- ning of corpus callosum. The presence of abnormal choroidal arteries feeding the VGAM is confirmed (arrows). confirming the well-known high accuracy of DWI in white matter DWI alteration. A possible explanation acute stroke diagnosis both in adults and in children. for this discrepancy is that episodes of more extensive Although we cannot provide a direct demonstration of brain hypoperfusion repeatedly occurred after the pre- the ischemic nature of the lesions we detected, previous natal MRI. The cerebral hemodynamics of our fetus reports13–15 have extensively documented that ischemic was probably unstable, and the intracranial blood flow and malacic lesions are the main complications that oc- autoregulation mechanism was weak, so the prenatal cur in subjects with VGAM, in both the prenatal and MRI provided a snapshot of still evolving and spread- postnatal period. Moreover, the remarkable reduction in ing damage. The end stage of the damage included ADC that we noticed in the lesions has been reported signs of calcific degeneration of the residual white mat- only in a few other brain diseases, such as encephalitis, ter at postnatal MRI; such calcifications are known to mature abscess, and status epilepticus16; in our case, the develop within the brain of newborns who have suf- location of the lesions, their evolution, and the clinical fered hypoxic-ischemic insults.17 course were poorly compatible with these conditions. The explanation for the ADC decrease observed in Three mechanisms have been postulated for the our case resembles what is commonly reported for pathogenesis of the hypoxic-ischemic brain injuries de- acute ischemia in adult humans or animal models18,19; tectable in VGAM patients15: a steal phenomenon it is based on energy failure of the cell membrane, with caused by blood shunting from the normal parenchy- development of cytotoxic edema and consequent rela- mal arteries into those feeding the malformation; pa- tive decrease of the extracellular water and increase of renchymal hypoperfusion secondary to intracranial ve- the intracellular one, which is more restricted and less nous hypertension and congestion; and multiple organ diffusible. However, water diffusion in the brain differs hypoperfusion and hypoxia due to congestive heart fail- according to maturity: the normal averaged ADC val- ure. One or more of these mechanisms, acting to- ues in the white matter of premature neonates are gether, might have produced the brain lesions that we much higher (range, approximately 1.8–1.9␮m2/ observed in the fetus. We believe that the lesions were msec)11,12 than those of adults (range, approximately caused first by a steal phenomenon and second by a 0.7–0.9␮m2/msec)12,20; this finding could be related venous congestion mechanism, because the degree of to the higher water content, to the lower macromole- cardiac failure was not so clinically important after cules concentrations, or to the poorer neuronal and birth. For these reasons, it is likely that the lesions of glial “packing” in developing and premyelinated brain the cortex and white matter were mainly ischemic (be- tissue.12,21 This difference might account also for the cause of reduce blood flow) rather than hypoxic (be- abnormal ADC variations that were observed in our cause of arrival of less oxygenated blood). The postna- case. We noticed an ADC decrease to an average value tal MRI showed diffuse cortical atrophy and of 0.97␮m2/msec (SD, 0.038␮m2/msec); similar ADC leukomalacia, well beyond the areas of initial gray and reductions have been reported recently in the acute

Baldoli et al: Diffusion MRI of Fetal Brain Ischemia 245 stage of periventricular leukomalacia affecting prema- 6. Cowan FM, Pennock JM, Hanrahan JD, et al. Early detection ture babies.5 These values are almost double those of cerebral infarction and hypoxic-ischemic encephalopathy in commonly reached by mean diffusivity decrease in neonates using diffusion-weighted magnetic resonance imaging. acute ischemic lesions of adults.20 However, in this Neuropediatrics 1994;25:172–175. 7. Johnson AJ, Lee BC, Lin WL. Echoplanar diffusion-weighted case and in those of premature babies, the approxi- imaging in neonates and infants with suspected hypoxic- mately 50% reduction in ADC for normal areas was ischemic injury: correlation with patient outcome. Am J Roent- similar to what has been observed with ischemia in genol 1999;172:219–226. adults. We hypothesize that the same tissular features 8. Robertson RL, Ben-Sira L, Barnes PD, et al. MR line scan dif- (eg, water content, macromolecules concentrations, fusion weighted imaging of term neonates with perinatal brain neuronal and glial “packing”), which produce the dif- ischemia. AJNR Am J Neuroradiol 1999;20:1658–1670. ferences in ADC between the normal developing brain 9. Soul JS, Robertson RL, Tzika AA, et al. Time course of changes and adult brain, can play a role also in producing dif- in diffusion-weighted magnetic resonance imaging in a case of ferent ADC value decreases in pathological conditions, neonatal encephalopathy with defined onset and duration of hypoxic-ischemic insult. Pediatrics 2001;108:1211–1214. such as ischemia. To better understand these aspects, 10. Barkovich AJ, Westmark KD, Bedi HS, et al. Proton spectros- we need to study more cases of acute fetal or premature copy and diffusion imaging of the first day of life after perinatal neonatal brain ischemia by using DWI, possibly corre- asphyxia: preliminary report. AJNR Am J Neuroradiol 2001;22: lating the results with neuropathological findings. Pre- 1786–1794. vious work8,9 on timing of the ADC modifications af- 11. Huppi PS, Maier SE, Peled S, et al. Microstructural develop- ter acute perinatal hypoxic-ischemic damage suggests ment of human newborn cerebral white matter assessed in vivo that a first ADC reduction can be noticed within 6 to by diffusion tensor magnetic resonance imaging. Pediatr Res 72 hours after the acute event, with a possible delayed 1998;44:584–590. 12. Tanner SF, Ramenghi LA, Ridgway JP, et al. Quantitative and more widespread reduction in the following days, comparison of intrabrain diffusion in adults and preterm and when conventional MRI also shows signal alterations term neonates and infants. AJR Am J Roentgenol 2000;174: due to developed vasogenic edema. Although in our 1643–1649. case, we present only 1 time point of the ADC change 13. De Koning TJ, Gooskens R, Veenhoven R, et al. Arteriovenous time course, we hypothesize that we imaged mostly le- malformation of vein of Galen in three neonates: emphasis on sions associated with the first ADC decrease, because associated early ischaemic brain damage. Eur J Pediatr 1997; 156:228–229. prenatal T2-weighted images did not show significant signal alterations. 14. Baeziger O, Martin E, Willi U, et al. Prenatal brain atrophy due to a giant vein of Galen malformation. Neuroradiology Although the prenatal detection of VGAM is rare, 1993;35:105–106. the early diagnosis by DWI of any associated destruc- 15. Brunelle F. Arteriovenous malformation of the vein of Galen in tive brain lesions can be important in therapeutic de- children. Pediatr Radiol 1997;27:501–513. cision making, for example, in the decision to perform 16. Righini A, Pierpaoli C, Alger JR, Di Chiro G. Brain paren- therapeutic intravascular procedures. Fetal brain chyma apparent diffusion coefficient alterations associated with hypoxic-ischemic damage also can occur in several experimental complex partial staus epilepticus. Magn Reson Im- other conditions, such as twin-to-twin transfusion syn- aging 1994;12:865–871. drome, abruptio placentae, preeclampsia, severe mater- 17. Ansari MQ, Chincanchan CA, Armstrong DL. Brain calcifica- nal anemia, and severe hypovolemia. Under these cir- tion in hypoxic-ischemic lesions: an autopsy review. Pediatr Neurol 1990;6:94–101. cumstances, prenatal DWI is a promising technique 18. Pierpaoli C, Righini A, Linfante I, et al. Histopathologic cor- that can detect acute hypoxic-ischemic complications, relates of abnormal water diffusion in cerebral ischemia: which could influence clinical and parental decisions. diffusion-weighted MR imaging and light and electron micros- opic study. Radiology 1993;189:439–448. 19. Lutsep HL, Albers GW, DeCrespigny A, et al. Clinical utility References of diffusion weighted magnetic resonance imaging in the assess- 1. Girard N, Raybaud C, Gambarelli D, et al. Fetal brain MR ment of ischemic stroke. Ann Neurol 1997;41:574–580. imaging. Magn Reson Imaging Clin N Am 2001;9:19–56. 20. Warach S, Gaa J, Siewert B, et al. Acute human stroke studied 2. De Laveaucoupet J, Audibert F, Guis F, et al. Fetal magnetic resonance imaging (MRI) of ischemic brain injury. Prenat Di- by whole brain echo planar diffusion-weighted magnetic reso- agn 2001;21:729–736. nance imaging. Ann Neurol 1995;37:231–241. 3. Langer B, Boudier E, Gasser B, et al. Antenatal diagnosis of 21. Baratti C, Barnett AS, Pierpaoli C. Comparative MR imaging brain damage in the survivor after second trimester death of a study of brain maturation in kittens with T1, T2, and the trace monochorionic monoamniotic co-twin. Fetal Diagn Ther 1997; of the diffusion tensor. Radiology 1999;210:133–142. 12:286–291. 4. Warach S, Chien D, Li W, et al. Fast magnetic resonance diffusion-weighted imaging of acute human stroke. Neurology 1992;42:1717–1723. 5. Inder T, Huppi PS, Zientara GP, et al. Early detection of periventricular leukomalacia by diffusion-weighted magnetic resonance imaging techniques. J Pediatr 1999;134:631–634.

246 Annals of Neurology Vol 52 No 2 August 2002 markerproteingeneproduct9.5(PGP9.5,1:800;Ultra- SelectiveLossofCholinergic clone,Wellow,UK)andtovasoactiveintestinalpeptide (VIP,1:500;Peninsula,SanCarlos,CA).Immunofluores- SudomotorFibersCauses cencewasperformedusingCy3orCy2-labeledsecondary antibodies(1:100;Amersham,ArlingtonHeights,IL).An AnhidrosisinRoss ABCsystem(Vector,Burlingame,CA)wasusedwiththe sameprimaryantibodiestoverifythestaining.Foranalysis Syndrome ofskinmorphology(hematoxylinandeosinstain)andim- 1 1 ClaudiaSommer,MD, ThiesLindenlaub,MD, munemediators,6␮m-thickfrozensectionswerereacted DetlefZillikens,MD,2 KlausV.Toyka,MD,1 1 withprimaryantibodiestoTcells(CD3,CD4,CD8, andMarkusNaumann,MD CD45RO),Bcells(CD20),andmacrophages(CD68)or fluoresceinisothiocyanate–labeledantibodiestohumanim- munoglobulin(Ig)G,IgM,IgA,andcomplementcompo- Rosssyndromeconsistsofsegmentalhyperhidrosiswith nentC3(Dako,Hamburg,Germany).Thesectionswere widespreadanhidrosis,Adiesyndrome,andareflexia.The viewedwithaZeissAxiophot2(Zeiss,Go¨ttingen,Germany) causeofthisdisorderisunknown.Selectivedegeneration microscopeequippedwithinternalz-focusandamotorized ofcholinergicfibersorofneuralcrest–derivedstructures scanningtablebyMa¨rzha¨user,Wetzlar,Germany.UsingIm- hasbeensuggested.Wepresentclinicalandskinbiopsy ageProPlus4.0software,wegenerated3-dimensionalre- dataof4patients,providingevidenceofreducedcholin- constructionsoftheepidermisandthesweatglands.Epider- ergicsweatglandinnervationinhypohidroticskinby malinnervationwasquantifiedbycountingthenerve morphometricanalysis.Thesefindingsindicateaselective endingspermillimeterofepidermallengthandbydetermi- degenerativeprocessofthecholinergicsudomotorneu- nationoftheepidermalnerveareawithopticaldensitometry. rons. Nerveendingswererequiredtohaveavisiblelengthgreater than20␮mtobeconsidered.Thesubepidermalnerveplexus AnnNeurol2002;52:247–250 wasmeasuredinanareaof100␮mϫ0.3mmalongthe subepidermal/epidermalborder.Sweatglandinnervationwas 1 quantifiedbymeasuringthecompletenervefiberareaasso- ThepathogenesisofRosssyndrome (tonicpupils, ciatedwithsweatglands.Valuesfromtheipsilateralandcon- areflexia,andsegmentalhyperhidrosis)isasyetun- tralateralsidewerecomparedusingStudentttest;pvaluesof known.WideoverlapbetweenRosssyndrome,Adie lessthan0.05wereconsideredsignificant. syndrome,Harlequinsyndrome(isolatedprogressive segmentalhypohidrosis),andamorewidespreadauto- nomicdiseasehasbeensuggested.2 Here,wepresent NeurophysiologicalStudiesandSudomotorTesting findingsfrom4patientswithanhidrosisandsegmental Nerveconductionstudiesandsomatosensoryandmagnetic- evokedpotentialswereperformedusingourstandardneuro- hyperhidrosisandvariableexpressionofAdie’spupils, physiologicaltechniques.3 Hyperhidroticandanhidroticar- hyporeflexia,andpathologicalneurophysiologicalfind- easwerevisualizedusingMinor’siodinestarchtest. ings.Importantly,theunderlyingpathologywasthe Quantitativesensorytesting(QST)wasperformedusinga sameinallpatients,withreductionofcholinergic Peltierdevice(Medocthermalanalyzer)andthemethodsof sweatglandinnervationandnormalepidermaland limits.4 Heartratevariabilitywasmeasuredasdescribedpre- subepidermalnervefibers. viously.5

PatientsandMethods Results Skinbiopsieswereobtainedfromahyperhidroticandanan- hidroticareaofthepatients’backafterinformedconsent. Case1 Afterfixationin4%paraformaldehyde,40␮mfrozensec- A50-year-oldmanreportedincreasedsweatingonthe tionswerestainedwithantibodiestothepanneuronal rightsideofthetrunkandontheleftfacefor18years. Bothhandsandfeetweredry.Physicalexamination showedhyperhidrosisintherightaxilla,intheder- matomesT6toT10ontherightandT11toT12on FromtheDepartmentsof 1Neurologyand 2Dermatology,Univer- theleft,andontheleftfaceandneck.Anklejerkswere sityofWu¨rzburg,Wu¨rzburg,Germany. absent.Nerveconductionstudieswerenormal.The ReceivedFeb21,2002,andinrevisedformMar18.Acceptedfor H-reflexandthesympatheticskinresponse(SSR)on publicationMar23,2002. bothfeetwereabsent.QSTshowednormalthresholds Published online Jun 23, 2002, in Wiley InterScience inhyperhidroticandanhidroticareasofthebackand (www.interscience.wiley.com).DOI:10.1002/ana.10256 onbothhands,butincreasedwarmthresholdsonthe CurrentaddressforDrLindenlaub:NeurologischeKlinik,Univer- sita¨tsklinikendesSaarlandes,66424Homburg,Germany. dorsumoftherightfoot.Thepatientwastreatedwith 200unitsofbotulinumtoxin(Botox௡)inthehyper- AddresscorrespondencetoDrSommer,NeurologischeUniversita¨- tsklinik,Josef-Schneider-Strasse11,D-97080Wu¨rzburg,Germany. hidroticareainT6toT10,witha50%reductionin E-mail:[email protected] sweatsecretionwithin4days.Treatmentwasrepeated

©2002Wiley-Liss,Inc. 247 3 months later, with a good effect lasting for 4 months right and in the inguinal area on the left. Palms and (Table 1). soles were dry. The SSR was absent. The patient was treated with 1,000 units of botulinum toxin (Dysport) Case 2 in the hyperhidrotic area of the trunk. The area of ex- A 49-year-old man had hyperhidrosis in the right T5 cessive sweating decreased by approximately 60% for 6 to T7 dermatomes and in both knees for 7 years. months. Hands and feet did not sweat. The left pupil was wider, unreactive to light but had a tonic reaction to Skin Biopsy Results convergence, consistent with Adie’s pupil. Tendon No inflammatory infiltrates and no deposition of IgG jerks were elicitable only after reinforcement; right an- or C3 were observed around sweat glands, which them- kle jerk was absent. Latencies of somatosensory-evoked selves appeared normal. Using PGP 9.5 immunohisto- potentials of both tibial nerves were delayed, and the chemistry, we found that epidermal innervation and amplitude of the sural nerve action potential was the subepidermal plexus were normal, whereas sweat slightly reduced. Further nerve conduction studies and gland innervation was reduced in the anhidrotic areas SSRs (palms) were normal. The patient was injected (Fig, a–d). Immunoreactivity for VIP was present in with 1,000 units of botulinum toxin (Dysport) in the the fibers innervating the sweat glands, but not in the hyperhidrotic area of the trunk with marked reduction epidermal fibers, and was markedly reduced around of sweating. sweat glands of the anhidrotic side (Fig, e and f). Morphometry showed normal epidermal and subepi- Case 3 dermal innervation density in hyperhidrotic and an- A 41-year-old woman had Adie’s pupils, areflexia, and hidrotic regions. Sweat gland innervation was reduced hyperhidrosis in the left lower thoracic and gluteal significantly in the anhidrotic compared with the hy- area, and on the right ventral thigh and knee. The rest perhidrotic regions (p Ͻ 0.005; Table 2). of the body was anhidrotic. Hypoesthesia was present on both lower legs; vibration thresholds were reduced Discussion at the ankles and knees. Heart rate variability was at Here, we show selective loss of cholinergic sweat gland the lower limit of normal. Nerve conduction studies innervation in 4 patients with Ross syndrome by using showed delayed latencies of somatosensory-evoked po- the panneuronal marker PGP 9.5 and a marker for tentials of the right tibial nerve. Sensory nerve poten- cholinergic fibers, VIP.6 Because downregulation of tials of the sural nerve could not be elicited. SSR of the neuropeptides such as VIP may occur in nerve lesions right hand was absent. The patient opted for local before degeneration of nerve fibers occurs, we used treatment with aluminum chloride hexahydrate, which PGP 9.5 for quantification of nerve fibers. Interest- yielded satisfactory results. ingly, epidermal innervation remained intact, well in accordance with normal QST. Denervation as the Case 4 cause of anhidrosis in patients with Ross syndrome has A 30-year-old man reported anisocoria since age 17 hitherto been suggested only on theoretical grounds.1 years, hyperhidrosis on the right side of the trunk and We previously presented qualitative data in another pa- the left temporal region since age 23 years, and heat tient with Ross syndrome, also in whom a selective re- intolerance. He had Adie’s pupils, normal tendon jerks, duction of PGP 9.5 immunoreactive fibers around and hyperhidrosis of the dermatomes T4 to T6 on the sweat glands could be found.7 Earlier skin biopsies

Table 1. Clinical and Electrophysiological Data of 4 Patients with Ross Syndrome

Duration of Heart Case Age Symptoms Segmental Adie’s Tendon Rate no. (yr) Gender (yr) Hyperhidrosis Pupils Reflexes NCS/SEP SSR Variability QST

1 50 M 18 T6–T10 R No Ankle jerk Tibial nerve: H-reflex Absent Normal Normal T11 L missing missing 249M 7 T5–T7 R Yes Ankle jerk Sural nerve: A reduced Normal Normal ND missing tSEP: A reduced 3 41 F 6 T10–T12 Yes Areflexia Sural nerve: no response Absent Normal ND Gluteal L tSEP: Lat delayed Knee L 430M 7 T4–T6 Yes Normal Normal Absent Normal Normal

A ϭ amplitude; Lat ϭ latency; L ϭ left; NCS ϭ nerve conduction studies; ND ϭ not done; QST ϭ quantitative sensory testing; R ϭ right; SSR ϭ sympathetic skin response; tSEP ϭ somatosensory-evoked potentials of the tibial nerves.

248 Annals of Neurology Vol 52 No 2 August 2002 Fig. Forty-micrometer-thick frozen sections from skin biopsies of Patient 2, immuno- fluorescence, and secondary antibody Cy3. Sections from the hyperhidrotic side are on the left (a, c, e) and from the anhidrotic side on the right (b, d, f). (a, b) Immuno- reaction with antibodies to the panneuronal marker protein gene 9.5 (PGP 9.5) shows normal epidermal innervation in the hyperhidrotic (a) and in the anhidrotic (b) skin. (c, d) PGP 9.5–immunoreactive fibers innervating sweat glands are abundant on the hyperhidrotic side (c), but depleted on the anhidrotic side (d). (e, f) Immunohistochemistry for vasoactive intes- tinal peptide shows normal cholinergic sweat gland innervation on the hyperhidrotic side (e) and loss of this innervation on the anhidrotic side (f). Bar ϭ 50␮m. from Ross syndrome patients were reported as normal; tional Adie’s pupils and 3 also had areflexia or hypore- however, immunohistochemical methods to visualize flexia. Nerve conduction studies were abnormal in 2 axons were not used.8,9 patients, and SSR was absent in 3 patients. Heart rate Segmental anhidrosis and hyperhidrosis are often the variability was normal in 3 and at the lower level of presenting symptoms in patients with Ross syndrome. normal in 1 patient. QST was normal in the 2 patients Of our 4 patients with these symptoms, 3 had addi- in whom it was performed. Our observations are con-

Table 2. Morphometric Analysis of Skin Biopsies

Area of Fibers Innervating Epidermal Nerve Fibers Subepidemal Nerve Fiber Area Sweat Glands per 1,000␮m2 per mm per mm2 sweat gland area

Case no. Hyperhidrosis Anhidrosis Hyperhidrosis Anhidrosis Hyperhidrosis Anhidrosis

1 16.3 18.4 1266 1194 109.9 39.9 2 18.4 16.3 1165 1261 70.0 40.6 3 16.2 18.5 1209 1231 126.7 39.9 4 17.1 19.0 1356 1563 90.1 51.2 Mean Ϯ SD 17.0 Ϯ 1.0 18.1 Ϯ 1.2 1249 Ϯ 82.4 1312.3 Ϯ 169.4 99.2 Ϯ 24.5 42.9aϮ 5.5 aSignificant difference from contralateral side, p Ͻ 0.005. SD ϭ standard deviation.

Sommer et al: Sudomotor Fiber Loss in Ross Syndrome 249 sistent with previous reports suggesting that segmental 5. Flachenecker P, Wermuth P, Hartung H-P, Reiners K. Quan- anhidrosis is part of the spectrum of distinct auto- titative assessment of cardiovascular autonomic function in nomic disorders due to a generalized injury to auto- Guillain-Barre´ syndrome. Ann Neurol 1997;42:171–179. 2,10–12 6. Schu¨tz B, Scha¨fer MK, Eiden LE, Weihe E. VIP and NPY ex- nomic and dorsal root ganglia neurons. It is un- pression during differentiation of cholinergic and noradrenergic known why these particular structures are involved. sympathetic neurons. Ann N Y Acad Sci 1998;865:537–541. Both the ciliary nerves and the sympathetic innervation 7. Bergmann I, Dauphin M, Naumann M, et al. Selective degen- of sweat glands are cholinergic. However, a selective eration of sudomotor fibers in Ross syndrome and successful degeneration of cholinergic fibers does not explain treatment of compensatory hyperhidrosis with botulinum toxin. Muscle Nerve 1998;21:1790–1793. areflexia, which has been suggested to be caused by loss 8. Bartin RH, Schmutz JL, Cuny JF, et al. Le syndrome de Ross. 13 of large diameter afferent fibers. Shin and colleagues A propos d’une observation. Ann Dermatol Venereol 1990;117: suggested a combined lesion in these structures due to 113–114. their derivation from the neural crest.2 Pigment cells 9. Caparros-Lefebvre D, Hache JC, Hurtevent JF, et al. Unilateral and Schwann cells also are derived from the neural loss of facial flushing and sweating with contralateral anhidrosis: harlequin syndrome or Adie’s syndrome? Clin Auton Res 1993; crest. No Schwann cell pathology has been described in 3:239–241. Ross syndrome so far to our knowledge, but we previ- 10. Jacobson DM, Hiner BC. Asymptomatic autonomic and sweat ously described depigmentation in a patient with Ross dysfunction in patients with Adie’s syndrome. J Neuroophthal- syndrome.7 The sympathetic innervation of sweat mol 1998;18:143–147. glands is unusual, because it is initially catecholamin- 11. Bacon PJ, Smith SE. Cardiovascular and sweating dysfunction in patients with Holmes-Adie syndrome. J Neurol Neurosurg ergic but becomes cholinergic after interactions with Psychiatry 1993;56:1096–1102. the target tissue. Catecholamines are necessary to in- 12. Drummond PD, Lance JW. Site of autonomic deficit in harle- duce secretory responsiveness of the sweat glands.14 quin syndrome: local autonomic failure affecting the arm and The reason for the segmental hyperhidrosis in pa- the face. Ann Neurol 1993;34:814–819. tients with Ross syndrome has not been discovered yet. 13. Pavesi G, Macaluso GM, Medici D, et al. On the cause of tendon areflexia in the Holmes-Adie syndrome. Electromyogr The hyperhidrosis is not likely to be efficient as a ther- Clin Neurophysiol 1994;34:111–115. moregulatory measure, and during treatment symp- 14. Tian H, Habecker B, Guidry G, et al. Catecholamines are re- toms of heat intolerance did not occur to any greater quired for the acquisition of secretory responsiveness by sweat degree than before. The area of hyperhidrosis was al- glands. J Neurosci 2000;20:7362–7369. ways localized in the lower thoracic to upper lumbar 15. Haberberger RV, Bodenbenner M. Immunohistochemical local- ization of muscarinic receptors (M2) in the rat skin. Cell Tissue region. Why these ganglia/fibers are spared from the Res 2000;300:389–396. degenerative process is unclear. In sudomotor fibers of 16. Cavanah DK, Casale TB. Cutaneous responses to the rat, muscarinic M2 receptors are inhibitory presyn- anticholinergics: evidence for muscarinic receptor subtype par- aptic autoreceptors.15 The same function has been pos- ticipation. J Allergy Clin Immunol 1991;87:971–976. tulated for these receptors in human skin.16 We thus speculate that the cholinergic fibers innervating sweat glands first lose their M2 autoreceptors and that hyperhidrosis is caused by diminished presynaptic inhibition, before further degeneration leads to anhidrosis.

This research was supported by research funds of the University of Wu¨rzburg. We thank Dr C. Rose for evaluating skin pathology and B. Dekant for excellent technical help.

References 1. Ross AT. Progressive selective sudomotor denervation. Neurol- ogy 1958;8:809–817. 2. Shin RK, Galetta SL, Ting TY, et al. Ross syndrome plus: beyond horner, Holmes-Adie, and harlequin. Neurology 2000;55: 1841–1846. 3. Naumann M, Schalke B, Klopstock T, et al. Neurological mul- tisystem manifestation in multiple symmetric lipomatosis: a clinical and electrophysiological study. Muscle Nerve 1995;18: 693–698. 4. Claus D, Hilz MJ, Hummer I, Neundorfer B. Methods of mea- surement of thermal thresholds. Acta Neurol Scand 1987;76: 288–296.

250 Annals of Neurology Vol 52 No 2 August 2002 ofnormoKPPconfirmedearliersuspicionsthatthese NormokalemicPeriodic familiesactuallyhadavariantofhyperKPPduetoa ParalysisRevisited:Does SCN4Agenemutation.1,5 However,normoKPPre- mainsenshrinedinstandardmedicaltextbookslargely becausetheoriginaldetailedclinicaldescriptionofthe ItExist? 2 1 disorderbyPoskanzerandKerr inafamilyfromthe PatrickF.Chinnery,PhD,MRCP, 6 TimothyJ.Walls,MD,FRCP,1 northeastofEnglandremainsunchallenged. Thisfam- MichaelG.Hanna,MD,MRCP,2 ilyrecentlybecamethesubjectofstudyagainwhena DavidBates,MA,FRCP,1 memberofthenextgenerationdevelopedperiodicpa- 3 andPeterR.W.Fawcett,BSc,FRCP ralysis,allowingustorevisittheoriginaldiagnosis. CaseReport Normokalemicperiodicparalysis(normoKPP)iswelles- A22-year-oldmanpresentedtotheneurologydepartmentof tablishedintheliterature,buttherearedoubtsasto RoyalVictoriaInfirmary,NewcastleUponTyne,becauseof whetheritexistsasadiscreteentity.Retrospectiveclinical anincreaseinthefrequencyandseverityofhisperiodicmus- andmolecularanalysishasconfirmedsuspicionsthat cleweakness.Hissymptomsbeganat2yearsofage.Heex- mostnormoKPPfamiliesactuallyhaveavariantofhy- periencedepisodesofmuscleweaknessinallfourlimbs, perkalemicperiodicparalysis(hyperKPP)duetoamuta- sometimesassociatedwithpainfulmusclestiffness.Theepi- tionofthemuscle-specificsodiumchannelgene sodestypicallywereprecipitatedbycoldanddampweather (SCN4A).However,theoriginalnormoKPPfamilyde- andwouldbeginduringaperiodofrestafteraperiodof scribedbyPoskanzerandKerr(PoskanzerDC,Kerr strenuousexercise.Therewasnorelationshipwithfastingor DNS.Athirdtypeofperiodicparalysis,withnormokal- foodintake.Asevereattacktypicallywouldlastforseveral emiaandfavourableresponsetosodiumchloride.AmJ days,followedbyaprolongedrecoveryphasetakingdaysto Med1961;31:328–342)hasremainedunchallenged.We weeks.Twoattackshadbeensoseverethatheremainedin identifiedtheMet1592ValmutationofSCN4Ainanaf- bedandcouldonlylifthisheadoffthepillow.Althoughhe fecteddescendentofthisoriginalnormoKPPfamily.This occasionallyexperienceddysphagiaduringanattack,therehad isthefinalpieceinthepuzzle:normoKPPisactuallya neverbeenanyrespiratorysymptoms,andhehadnotnoticed variantofhyperKPPandisnotadistinctdisorder. anypigmenturia.Hismedicalhistoryincludedacamptodac- AnnNeurol2002;52:251–252 tylycorrectiontobothhandsbutwasotherwiseunremarkable. Therewasnohistorytosuggestcardiacdysrhythmias.

Traditionally,threecategoriesofperiodicparalysishave Results beendescribed:hypokalemic(hypoKPP),hyperkalemic Therewerenoabnormalfindingsonsystemicorneu- (hyperKPP),andnormokalemic(normoKPP).1 The rologicalexamination.Routinehematologicalandbio- lastdecadehasseenmajoradvancesinourunderstand- chemicaltestswerenormal(includingserumelectro- ingofthemolecularandelectrophysiologicalbasisof2 lytesandthyroidfunctiontests),withtheexceptionof ofthesegroups.Threepointmutationsintheskeletal anelevatedalaninetransaminase(53U/L,normal Ͻ muscledihydropyridinesensitivecalciumchannel 45)andanelevatedcreatinekinase(807U/L,nor- Ͻ (CACLN1A3)areresponsibleformostcasesofhy- mal 190).A12-leadelectrocardiogramandchest poKPP,andmutationsinthemuscle-specificsodium x-raywerenormal.Aneurophysiologicalstudyshowed channelgene(SCN4A)havebeenidentifiedinaspec- normalperipheralnervefunction,butseveralabnor- trumofdisordersthatincludeshyperKPP,paramyoto- malitiesonconcentricneedleelectromyography.The niacongenita,andpotassiumaggravatedmyotonia.1 moststrikingfeaturewasthefrequentmyotonicdis- Despiteitsprominenceinstandardtexts,normoKPP chargesseenineverymusclesampled(Fig).Therewas hasbeenreportedonlyinafewfamilies.2–4 Clinical alsoexcessinsertionalactivityandspontaneousactivity andmolecularreanalysisofsomeoftheoriginalcases intheformoffibrillationpotentialsandpositivesharp wavesrecordedfrombicepsbrachii,indicatingminimal musclefiberdegenerationandearlymyopathicchanges. Quantitativemulti–motorunitpotentialanalysisofthe Fromthe 1DepartmentofNeurology,UniversityofNewcastleUpon Tyne,NewcastleUponTyne; 2DepartmentofNeurology,Instituteof motorunitpotentialsinbicepsshowednormalmotor Neurology,London;and 3DepartmentofClinicalNeurophysiology, unitpotentialdurations,amplitudes,andnumber. RoyalVictoriaInfirmary,NewcastleUponTyne,UnitedKingdom. FamilyHistory ReceivedFeb15,2002,andinrevisedformMar19,2002.Ac- ceptedforpublicationMar25,2002. ThepatientinthisindexcaseisthesonofcaseV:10 2 Published online Jun 23, 2002, in Wiley InterScience whowasstudiedindetailbyPoskanzerandKerr. (www.interscience.wiley.com).DOI:10.1002/ana.10257 MolecularGeneticStudies AddresscorrespondencetoDrChinnery,DepartmentofNeurology, TheMedicalSchool,FramlingtonPlace,NewcastleUponTyne, Thetypicalpatternoftheepisodesofweakness,the NE24HH,UnitedKingdom.E-mail:[email protected]. musclestiffness,andtheprominentmyotonicdis-

©2002Wiley-Liss,Inc. 251 The muscle weakness in patients with hyperKPP is caused by muscle depolarization. In families with Met1592Val, this arises through impaired fast inactivation and slow inactivation of the sodium channel.8,9 Poskanzer and Kerr2 documented an objective improve- ment in muscle strength in 5 individuals who were given up to 1,750ml of triple normal saline over a 12-hour period. The mechanism responsible for this improve- ment is unclear, and neither the weakness nor the infu- ϩ sions were associated with a change in serum Na or ϩ K levels. Although no control experiments were conducted, this observation requires further investigation Fig. Continuous raster display of the concentric needle electro- because of the possibility that an increased sodium load myography findings in the right biceps brachii of the index may also benefit other patients with hyperKPP. case. E ϭ end of the myotonic discharge; I ϭ insertion activ- The issue of treatment is highly pertinent to our in- ϭ ϭ ity; O onset of a myotonic discharge. Scale bar 0.1mV dex case. Our patient’s father (V:10 in the original per 100 milliseconds (ms). study2) now has generalized muscle wasting and a severe fixed vacuolar myopathy limiting normal daily ac- charges on electromyography pointed toward a muscle tivities. His son is currently not weak between attacks sodium channel disorder.1 Molecular genetic analysis but has myopathic features on electromyography. Our by a standard polymerase chain reaction/restriction en- objective is to prevent clinical progression in our new zyme analysis identified the Met1592Val mutation of patient, and, although unsubstantiated at present, it is SCN4A in the case described above. logical to take steps to reduce the frequency and severity of attacks. This could be achieved by alterations in Discussion his behavior and diet, or using a carbonic anhydrase We have shown that the original normoKPP family inhibitor such as dichlorphenamide.10 studied by Poskanzer and Kerr2 harbor a point mutation in SCN4A that is found in approximately 30% of We are very grateful to Dr D. N. S. Kerr for his comments on the families with hyperKPP.7 Molecular analysis of other original study and on this article. reported normoKPP families also has shown mutations in SCN4A (Thr704Met).5 Based on these findings, our References conclusion is that it is highly likely that normoKPP is 1. Ptacek LJ, Bendahhou S. Ion channel disorders of muscle. In: actually a variant of hyperKPP, and it should not be Karpati G, Hilton-Jones D, Griggs RC, eds. Disorders of vol- untary muscle. 7th ed. Cambridge: Cambridge University Press, considered as a distinct disease entity. 2001:604–635. In retrospect, the clinical and biochemical features of 2. Poskanzer DC, Kerr DNS. A third type of periodic paralysis, the original family of Poskanzer and Kerr are consistent with normokalemia and favourable response to sodium chlo- with a diagnosis of hyperKPP.2 Twenty-one individuals ride. Am J Med 1961;31:328–342. were reported in that study, each developing symptoms 3. Mayers KR, Gilden DH, Rinaldi CF, Hansen JL. Periodic muscle weakness, normokalemia, and tubular aggregates. Neurology in their first decade. The individuals experienced at- 1972;22:269–279. tacks of weakness every 1 to 3 months, each lasting 4. Danowski TS, Fisher ER, Vidalon C, et al. Clinical and ultra- from 2 days to 3 weeks. The attacks were provoked by structural observations in a kindred with normo-hyperkalemic rest after exertion, cold and damp conditions, and al- periodic paralysis. J Med Genet 1975;12:20–28. cohol, particularly the local beer, which contained a 5. Lehmann-Horn F, Rudel R, Ricker K. Workshop report. Non- dystrophic myotonias and periodic paralyses. Neuromuscul Dis- high concentration of potassium. Oral potassium chlo- ord 1993;3:161–168. ride exacerbated the attacks of muscle weakness in 4 6. Rudel R, Lehmann-Horn F. Muscle sodium channel and chlo- members of the original family. Potassium sensitivity, ride channel diseases. In: Lane RJ, ed. Handbook of muscle the hallmark of muscle sodium channel disorders,1 disease. New York: Marcel Dekker, 1996:348. therefore was clearly described in the original article.2 7. Cannon SC. From mutation to myotonia in sodium channel disorders. Neuromuscul Disord 1997;7:241–249. Although no significant change in serum potassium 8. Cannon SC, Brown RH Jr, Corey DP. A sodium channel de- was ever documented during numerous spontaneous or fect in hyperkalemic periodic paralysis: potassium-induced fail- precipitated attacks of weakness, it is now well recog- ure of inactivation. Neuron 1991;6:619–626. nized that this can occur in patients with hyperKPP.1,7 9. Hayward LJ, Sandoval GM, Cannon SC. Defective slow inac- Perhaps the most interesting feature of this family is tivation of sodium channels contributes to familial periodic paralysis. Neurology 1999;52:1447–1453. that before they received medical attention, they real- 10. Tawil R, McDermott MP, Brown R Jr, et al. Randomized trails ized that a high intake of table salt reduced the fre- of dichlorphenamide in the periodic paralyses. Ann Neurol quency and severity of the attacks of muscle weakness. 2000;47:46–53.

252 Annals of Neurology Vol 52 No 2 August 2002 thebrain.2 Incontrast,thevasculartheoryproposes HumanAntibodiesagainst thatA␤originatesfromthecirculation,andthatcir- ␤ culatingsolubleA␤couldcontributetoneurotoxicityif Amyloid Peptide: 3 itcrossestheblood-brainbarrier. ProductionofA␤ APotentialTreatmentfor viaamyloidprecursorprotein(APP)processing,however,isnottheonlyfactorthatcaninfluencetheprob- Alzheimer’sDisease abilityofA␤braindeposition.Evidencehasaccumu- RichardDodel,MD,1 HaraldHampel,MD,2 latedindicatingthatfactorsinfluencingA␤catabolism, CandanDepboylu,MD,1 SuizhenLin,MD,3 clearance4 andaggregation,5 arealsocriticalinregulat- FengGao,MD,3 SabineSchock,MD,1 SteffiJa¨ckel,MD,1 ␤ 6 3 2 ingA metabolism. XingWei,MD, KatharinaBuerger,MD, RecentdatafromtransgenicmousemodelsofAD ChristineHo¨ft,BSc, 1 BernhardHemmer,MD,1 Hans-Ju¨rgenMo¨ller,MD,2 MartinFarlow,MD,3 suggestthatclearanceviaimmune-mediatedpathways WolfgangH.Oertel,MD,1 NorbertSommer,MD,1 mayhaveamajorimpactonthedevelopmentof andYanshengDu,PhD3 plaques.7,8 ImmunizationagainstA␤hasprevented subsequentdepositionofamyloidplaques.Further- more,supportivedatahaveshownthatpassiveimmu- Naturally occurring antibodies directed against nizationwithantibodiesdirectedagainstA␤alsopre- ␤-amyloid(A␤)weredetectedinintravenousimmuno- ventsamyloiddeposition,amelioratesbehavioral globulinpreparations.Afterintravenousimmunoglobulin deterioration,andmayevenclearexistingplaques.9,10 treatmentinpatientswithdifferentneurologicaldiseases, ␤ ␤ Wehypothesizedthatiftheseresultsderivedfrom totalA andA 1-42 inthecerebrospinalfluidwasre- animalexperimentsaretransferabletohumans,an ducedsignificantlycomparedwithbaselinevalues.Inthe ␤ serum,totalA␤levelsincreasedafterintravenousimmu- immune-mediatedA degradingpathwaymaybe noglobulintreatment,whereasnosignificantchangewas physiologicallypresentanditsactionsclinicallysignif- ␤ ␤ icantinhumans.Recently,wedetectednaturallyoc- observedinA 1-42 levels.AntibodiesagainstA were foundtobeincreasedintheserumandcerebrospinal curringhumanantibodiesagainstA␤inboththecere- 11 fluidafterintravenousimmunoglobulintreatment.This brospinalfluid(CSF)andserumofhealthysubjects. studyprovidesevidencethatintravenousimmunoglobu- TheseantibodiesspecificallyrecognizeA␤.Further- ␤ ␤ ␤ linorpurifiedA antibodiesmaymodifyA andA 1-42 more,wedetectedsignificantlylowerCSFtitersof levels,suggestingpotentialutilityasatherapyforAlzhei- theseanti–A␤antibodiesinADpatientscompared merdisease. withcontrols. AnnNeurol2002;52:253–256 Followingtheseresults,wehypothesizedthatatreat- mentusingthesenaturallyoccurringantibodiesmight bebeneficialasatherapeuticstrategyforADpatients. ThepathologicalhallmarksofAlzheimer’sdisease(AD) Becausetheseantibodiesarepredominantlypresentin aretheoccurrenceofplaquesintheneuralparenchyma theimmunoglobulinG(IgG)fraction,weinvestigated andtheformationofneuronaltangles.1 ␤-Amyloid whethertheyaredetectableincommerciallyavailable (A␤),aheterogenous39to42–aminoacidpeptide,is IgGproducts.Inaddition,weinvestigatedwhetherthe themainconstituentofsenileplaquesandcerebrovas- administrationofaselectedIgGproduct(intravenous cularamyloiddeposits.TheoriginoftheA␤deposited immunoglobulin[IVIG])affectshumanCSFandse- invasculatureandhumanbrainisuncertain.Accord- rumA␤levels. ingtotheneuronaltheory,A␤islocallyproducedin PatientsandMethods PatientstreatedwithIVIGwererecruitedattheDepartment ofNeurology,PhilippsUniversity,Marburg.Eachpatientre- Fromthe 1DepartmentofNeurology,PhilippsUniversityMarburg; ceivedIVIG(Octagam;Octapharma,Langenfeld,Germany) 2DementiaResearchSectionandMemoryClinic,GeriatricPsychi- atatotaldoseof0.4gm/kgbodyweighton3consecutive atryBrandandAlzheimerMemorialCenter,Ludwig-Maximilian days.Sevenpatients(4male,3female;meanage,62.7Ϯ7.0 University,Munich,Germany;and 3DepartmentofNeurology,In- dianaUniversitySchoolofMedicine,Indianapolis,IN. years)wereincluded.Duringtheirregularevaluationand treatment,CSFandbloodsampleswerewithdrawnbefore ReceivedDec11,2001,andinrevisedformMar19,2002.Ac- ceptedforpublicationMar23,2002. infusionofIVIGandattheindicatedtimesaftertheinfu- sions.WetookCSFsamplesinthemorningattheL3/L4or Published online Jun 23, 2002, in Wiley InterScience L4/L5interspacebylumbarpunctureafterobtainingappro- (www.interscience.wiley.com).DOI:10.1002/ana.10253 priateinformedconsent.WedeterminedCSFleukocyte AddresscorrespondencetoDrDodel,DepartmentofNeurology, countandproteinlevelsbystandardmethods.Nocontami- PhilippsUniversity,Rudolf-BultmannStrasse8,35039Marburg, nationbyerythrocyteswasseeninanyofthesamples.Ali- Germany.E-mail:[email protected],Depart- Ϫ mentofNeurology,IndianaUniversitySchoolofMedicine,India- quotsthenwerestoredat 80°Cuntilbiochemicalanalysis. napolis,IN46202.E-mail:[email protected] AlbuminandIgGconcentrationsweredeterminedinthese-

©2002Wiley-Liss,Inc. 253 rum, and the CSF by immunoprecipitation nephelometry. Only patients with an intact blood-brain barrier and regular CSF protein concentrations according to our laboratory ref- erence were included (Table 1). The A␤ antibody enzyme-linked immunoadsorbent assay (ELISA), the purification of A␤ antibodies, and immunoprecipitation of A␤ were performed as described previously.11 The concentration of A␤ antibodies in commercially available IVIG Flebogamma, (Grifols, Langen), Germany; and Octagam; Octapharma) was determined using the A␤ antibody ELISA.11 Mean levels of anti-A␤ titers were compared by 2-way analysis of variance. A log transformation was conducted before statistical analysis to reduce skewness within each patient group.12 The mean log antibody titers for each patient group were back-transformed to give geometric mean and standard errors. Results We purified the anti–A␤ antibodies from IVIG preparations using affinity chromatography as previously described.11 A marked decrease in anti–A␤ antibody titer from the IgG fraction was observed when the flowthrough was tested in the ELISA assay (data not shown). We confirmed the ability of the affinity- purified anti–A␤ antibody from plasma to bind A␤ by immunoprecipitation of synthetic A␤ peptide (Fig 1A). Moreover, the affinity-purified anti–A␤ antibody from human IVIG readily detected a 4kDa A␤ peptide on Western blots prepared from PDAPP mouse13 hippocampal homogenates but not from cerebellar homogenates (see Fig 1B). In the next set of experiments, we incubated over- ␤ night different commercially available IVIG prepara- Fig 1. Purification and characterization of human anti–A tions (Flebogamma, [Grifols] and Octagam; Octap- antibody from human intravenous immunoglobulin (IVIG) ␤ ␮ samples (Octagam; Octapharma). (A)Anti–A␤ antibody after harma) with A 1-40 (0.4 gm/ml; Fig 2). Thereafter, ␤ ␤ elution from the A affinity column. We passed IVIG through IVIG was analyzed for anti–A antibodies using ␤ an affinity sepharose column conjugated with the A 1-40 pep- ELISA. In both preparations, we could detect antibod- ␤ ␤ tide. Anti–A antibody from the purified human plasma IgG ies against A . In both IVIG preparations, we could was recovered after elution with buffer 1 (pH 2.5) followed detect a considerable decrease of the ELISA signal after by buffer 2 (pH 1.5). A␤ was immunoprecipitated by ␤ 1-40 incubation with A 1-40 and agarose A. affinity-purified human anti–A␤ antibody. (B) A␤ from To evaluate our hypothesis that these antibodies may PDAPP mouse hippocampal homogenates was immunoprecipi- ␤ ␤ ␤ ␤ have an effect on A - peptide levels (total A and A 1- tated by purified human anti–A antibody and detected by monoclonal antibodies to A␤.13 A␤ ϭ amyloid ␤; crb ϭ cer- ebellum; hanti-A␤ ϭ human purified A␤ antibody; hippo ϭ Table 1. Clinical Data of the Patients Included in this Study hippocampus; IgG ϭ immunoglobulin G (IVIG flowthrough). Patient Age no. (yr) Gender Diagnosis QA1b 42), we investigated the effects of IVIG administration 1 67 F MS 4.5 on CSF and serum A␤ levels in patients with different 2 47 F MS 5.9 3 61 F PNP unknown origin 3.5 neurological diseases. We detected a significant de- ␤ ␤ 4 67 M PNP unknown origin 4.3 crease in CSF levels of total A and A 1-42 after IVIG 5 70 M PNP unknown origin 6.3 (Table 2). Similar to the recent results in PDAPP 6 34 M Lambert-Eaton syndrome 7.9 mice,14 a significant increase in total A␤ concentration 7 78 M Dermatomyositis 7.8 was observed in the serum after IVIG treatment (see None of the patients showed disturbance of the blood-brain barrier Table 2). Although there was a trend toward an in- Ͻ ϭ 15 ␤ (QALb 8; QALb CSFALb/SerumALb). crease of A 1-42 in the serum, values did not reach sta- MS ϭ Multiple Sclerosis; PNP ϭ Polyneuropathy. tistical difference (p ϭ 0.06). The concentration of

254 Annals of Neurology Vol 52 No 2 August 2002 the serum and the CSF from nonimmunized humans, which may act in an immune-mediated A␤ clearance pathway.11 In an earlier study, human antibodies reactive with A␤ were isolated and cloned from human B-cell lines from AD patients; however, the role of these antibodies in AD pathogenesis remains unclear.6 In our study, we detected a significant difference in the amount of A␤ antibodies in AD patients compared with controls.11 These observations led us to ask whether these anti–A␤ antibodies are detectable in commercially available human IVIG preparations. Af- ter purification of these antibodies from IVIG, we found that they specifically recognize A␤. Furthermore, we investigated the effect of IVIG treatment on A␤, ␤ A 1-42 levels in serum and CSF. Intravenous immunoglobulin is an accepted and routinely used treatment for several neurological and nonneurological immune- ␤ ␤ Fig 2. Human anti–A antibody was preabsorbed with A mediated disorders.17 These patients are treated with peptide. Experiments were performed using intravenous immu- ␤ IVIG on a routine basis in our department. Although noglobulin (IVIG) before and after incubation with A 1-40. Briefly, 100␮l of IVIG was incubated (overnight, 4°C) with we are aware that the patient selection for this study ␤ has several limitations (eg, age, immune status, differ- protein A–agarose or A 1-40 at the designated concentration. Protein A–agarose (Sigma, St. Louis, MO) was added to ent diagnoses), this was the most straightforward and IVIG (1␮l, overnight, 4°C) and removed by low-speed cen- feasible approach to evaluate our hypothesis. trifugation. The IVIG then was analyzed for anti–A␤ anti- Treatment of IVIG resulted in a significant decrease ␤ ␤ body using enzyme-linked immunoadsorbent assay. Antibody of total A and A 1-42 in the CSF compared with was recovered by incubation with eluting buffer (pH 1.5). baseline. Mean A␤ antibody concentration increased in This experiment was repeated 3 times with similar results. the CSF. In contrast, the serum total A␤ and A␤ an- A␤ ϭ amyloid ␤; Alp ϭ Flebogamma, (Grifols); Oct ϭ Oc- tibody concentration increased, but A␤ remained tagam (Octapharma). 1-42 unchanged. A␤ antibody concentration increased in A␤-antibodies in the serum increased after treatment the serum. with IVIG. These findings suggest that A␤ peptides may pass from the CSF to the blood and probably are metabo- Discussion lized locally. Recently, transporters at the blood-brain On the basis of the results by Schenk and colleagues,7 barrier have been reported, which control the central we identified specific anti–A␤ antibodies (IgG) in both and peripheral exchange of smaller peptides including ␤ ␤ ␤ Table 2. Total A ,A 1–42, and A . Antibody Concentrations in the CSF and Serum at Baseline and after IVIG

CSF Serum

After 1–4 Days 7–14 Days Baseline IVIG p Baseline After IVIG p After IVIG p

Total A␤ (pg/ml) Mean 1900.3 1545.7 a 358.1 433.5 a 426.4 a SEM 125.4 290.1 87.2 135.5 123.5 ␤ A 1–42(pg/ml) Mean 209.5 159.1 a 23.7 28.3 nsb 30.7 nsb SEM 37.4 45.0 8.25 11.1 12.7 A␤ antibody (ng/ml) Mean 2.5 10.7 a 189.3 341.7 a 276.6 nsb SEM 3.3 5.1 81.5 201.7 195.7 ␤ ␤ ␤ Total A ,A 1–42, and A -antibody concentrations in the CSF and serum were assessed at baseline and 1 to 4 days (serum), 7 to 14 days (serum), and 7 to 20 days (CSF) after IVIG (Octagam [Octapharma], 0.4 gm/kg body weight for 3 consecutive days). Values represent the mean of triplicate determinations from a single assay. ap Ͻ 0.05. bLevels of significance Ͼ0.05. A␤ϭ␤-amyloid; CSF ϭ cerebrospinal fluid; IVIG ϭ intravenous immunoglobulin; ns ϭ not significant; SEM ϭ standard error of the mean.

Dodel et al: Human Antibodies against A␤ Peptide 255 A␤.2,3 A similar A␤ efflux from the central to the pe- 4. Ghersi-Egea JF, Strazielle N. Brain drug delivery, drug metab- ripheral compartment has been found in PDAPP mice olism, and multidrug resistance at the choroid plexus. Microsc passively immunized with antibodies against A␤.14 Res Tech 2001;52:83–88. Whether there is a specificity for shorter A␤ peptides 5. Wisniewski T, Ghiso J, Frangione B. Biology of A beta amyloid in Alzheimer’s disease. Neurobiol Dis 1997;4:313–328. to cross the blood-brain barrier, as seen in the afore- 6. Iwata N, Tsubuki S, Takaki Y, et al. Identification of the major mentioned experiments, cannot be stated at this point. Abeta1–42-degrading catabolic pathway in brain parenchyma: No data are available on the quantitative decrease of suppression leads to biochemical and pathological deposition. A␤ concentration necessary to reduce A␤ deposition. Nat Med 2000;6:143–150. Therefore, one can only speculate whether the observed 7. Schenk D, Barbour R, Dunn W, et al. Immunization with reduction may have an impact on plaque formation. amyloid-beta attenuates Alzheimer-disease-like pathology in the Further studies, including careful dose studies in ani- PDAPP mouse. Nature 1999;400:173–177. mals, are necessary. From earlier studies, however, 8. Bard F, Cannon C, Burke R, et al. Peripherally administered some information can be deduced. First, a relatively antibodies against amyloid beta-peptide enter the central ner- ␤ vous system and reduce pathology in a mouse model of Alzhei- modest A clearance already reduced memory impair- mer disease. Nat Med 2000;6:916–919. ϩ 10 ment in Tg2576 APP PS1 mice. Second, an in- 9. Janus C, Pearson J, McLaurin J, et al. A beta peptide immuni- crease in A␤ concentration of approximately 1.5 times zation reduces behavioural impairment and plaques in a model in familial AD patients because of mutations in the of Alzheimer’s disease. Nature 2000;408:979–982. APP gene shifts the disease onset earlier by several de- 10. Morgan D, Diamond DM, Gottschall PE, et al. A beta peptide cades. It can be assumed that already small changes in vaccination prevents memory loss in an animal model of Alz- A␤ concentrations in the CSF may have an impact on heimer’s disease. Nature 2000;408:982–985. A␤ deposition and plaque development.18 11. Du Y, Dodel RC, Hampel H, et al. Reduced CSF levels of amyloid beta-peptide antibody in Alzheimer’s disease. Neurol- Our findings might have important implications for ogy 2001;57:801–805. the understanding of immune-mediated clearance path- 12. Armitage P, Berry G. Statistical methods in medical research. ways of A␤ in humans. Moreover, our data support Oxford: Blackwell Scientific Publications, 1994:389–390. earlier in vitro and animal experiments that antibodies 13. Games D, Adams D, Alessandrini R, et al. Alzheimer-type neu- applied outside of the blood-brain barrier may serve to ropathology in transgenic mice overexpressing V717F beta- facilitate clearance of soluble peptides such as A␤ out amyloid precursor protein. Nature 1995;373:523–527. of the central nervous system.2,19,20 Given the rela- 14. DeMattos RB, Bales KR, Cummins DJ, et al. Peripheral anti-A tively large volume of distribution of the peripheral as beta antibody alters central nervous system and plasma A beta clearance and decreases brain A beta burden in a mouse model compared with the central compartment, the addition ␤ of Alzheimer’s disease. Proc Natl Acad Sci USA 2001;98: of anti–A antibodies or other antipeptide antibodies 8850–8855. may considerably alter the clearance of biologically ac- 15. Reiber H, Peter JB. 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