selective loss of cholinergic sudomotor fibers causes anhidrosis in ross syndrome

Progressive Loss of CardiacSympathetic Innervation inParkinson’s DiseaseSheng-Ting Li, MD, PhD, Raghuveer Dendi, MD,Courtney Holmes, CMT,and David S. Goldstein, MD, PhD

This study addressed whether cardiac sympatheticdenervation progresses over time in Parkinson’s dis-ease. In 9 patients without orthostatic hypotension,6-[18F]fluorodopamine positron emission tomographyscanning was repeated after a mean of 2 years from thefirst scan. 6-[18F]fluorodopamine-derived radioactivitywas less in the second scan than in the first scan, by31% in the left ventricular free wall and 16% in theseptum. In Parkinson’s disease, loss of cardiac sympa-thetic denervation progresses in a pattern of loss sug-gesting a dying-back mechanism.

Ann Neurol 2002;52:220–223

All of at least a dozen studies have agreed that patientswith Parkinson’s disease have a high prevalence of neu-roimaging evidence for decreased sympathetic innerva-tion of the heart. Low myocardial concentrations of ra-dioactivity have been noted after injection of thesympathoneural imaging agents 123I-metaiodobenzyl-guanidine1–12 and 6-[18F]fluorodopamine.13 Neuro-chemical assessments during right heart catheterizationhave confirmed that low concentrations of radioactivityresult from loss of functional cardiac sympathetic nerveterminals.13

Although all patients with Parkinson’s disease andorthostatic hypotension have diffusely decreased sym-pathetic innervation throughout the left ventricularmyocardium, among patients who do not have ortho-static hypotension, about half have diffusely decreasedinnervation and about half have normal or only locally

decreased innervation.13 The latter finding afforded anopportunity to determine whether the loss of cardiacsympathetic innervation in Parkinson’s diseaseprogresses over time and, if so, with what timing, pat-tern, and consistency across patients. This report de-scribes the results of retesting such patients with6-[18F]fluorodopamine positron emission tomographyscanning after an average of 2 years.

Patients and MethodsThe study protocol was approved by the Intramural ResearchBoard of the National Institute of Neurological Disordersand Stroke. Each patient gave informed, written consent.

PatientsThoracic positron emission tomography scanning was per-formed after intravenous injection of 6-[18F]fluoro-dopamine in 9 patients with Parkinson’s disease (age:mean, 60 years; SEM, 3 years). None of the patients hadorthostatic hypotension, which was defined as a decrease insystolic blood pressure greater than 20mm Hg and decreasein diastolic pressure greater than 5mm Hg between the su-pine position and standing for 5 minutes. Caffeine-containing beverages, cigarettes, and alcohol were not al-lowed for at least 24 hours before the scanning. Patientswere allowed to take their usual medications, includingL-dopa, except for medications known to inhibit neuronaluptake of catecholamines.

Positron Emission Tomography Scanning6-[18F]fluorodopamine, synthesized as described previously,14

was infused intravenously at a constant rate for 3 minutes.Tomography images (35 contiguous transaxial slices 4.25mmapart) were acquired for up to 30 minutes. Three-dimensional positron emission tomography scans were ob-tained with an Advance whole-body scanner (General Elec-tric, Milwaukee, WI). Transmission scans of 2 minutes and8 minutes, with rotating 68Ge/68Ga pin sources, were ob-tained for attenuation correction and for confirming properpositioning in the scanner.

Follow-up scanning was performed 1 to 4 years (mean,2.0 years; SEM, 0.3 years) after the first scan, with the iden-tical scanning procedure. None of the patients had ortho-static hypotension at the time of either test. Of the 9 pa-tients, 2 had normal 6-[18F]fluorodopamine-derivedradioactivity and 7 had locally decreased 6-[18F]-fluorodopamine-derived radioactivity in the left ventricularmyocardium at the time of the first test.

Data Analysis and StatisticsTomography images were reconstructed after correction forattenuation and for physical decay of 18F. Cardiac imageswere analyzed as described previously.14 Briefly, circular re-gions of interest approximately half the ventricular wallthickness were placed on images of the septum, with time-

From the Clinical Neurocardiology Section, National Institute ofNeurological Disorders and Stroke, National Institutes of Health,Bethesda, MD.

Received Nov 28, 2001, and in revised form Feb 28 and Mar 7,2002. Accepted for publication Mar 7, 2002.

Published online Jun 23, 2002, in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/ana.10236

Address correspondence to Dr Li, Building 10, Room 6N252, Na-tional Institute of Neurological Disorders and Stroke, National In-stitutes of Health, 10 Center Drive, MSC-1620, Bethesda, MD20892-1620. E-mail: [email protected]

This article is a US Government work and, as such, is in the public do-main in the United States of America.

BRIEF COMMUNICATIONS

220 Published 2002 by Wiley-Liss, Inc.

averaged pictures of a single slice. Left ventricular septalradioactivity was averaged from two regions of interest forthe 5-minute scanning interval with a midpoint about8 minutes after initiation of the infusion. The sametime interval was used for radioactivity in the liver and kid-ney. For radioactivity in structures of the head and neck,static three-dimensional data were obtained for 10 to 15minutes. Images of noncardiac structures, including theliver, spleen, renal cortex, renal pelvis, salivary glands, na-sopharyngeal mucosa, and thyroid, were reconstructed andanalyzed by manual drawing of the regions of interest out-lining the structures. Radioactivity concentrations werenormalized by correction for the radioactivity concentrationfor the administered dose of radioactive drug per unit ofbody mass of the subject and were expressed as nCi-kg/cc-mCi.14

Mean values for 6-[18F]fluorodopamine-derived radioac-tivity were compared with paired t tests. Differences betweengroups in trends over time of 6-[18F]fluorodopamine-derivedradioactivity were assessed by analyses of variance for re-peated measures. A p value of less than 0.05 defined statis-tical significance.

ResultsAt the time of the first scan, patients had had Parkin-son’s disease for 5.7 � 1.2 years (range, 0.3–13 years).Disease severity averaged 2.2 � 0.2 (range, 1–3) of amaximum of 5. Heart rate and beat-to-beat systolicblood pressure changes during phase II of the Valsalvamaneuver averaged 11 � 2bpm and �43 � 5mm Hgfor a mean arterial baroreflex-cardiovagal gain of 2.3 �0.4ms/mm Hg (normal: mean, 8.5ms/mm Hg; SEM,2.2ms/mm Hg). Plasma catecholamine levels (norepi-nephrine: mean, 2.34nmol/L; SEM, 0.37nmol/L; epi-nephrine: mean, 0.23nmol/L; SEM, 10nmol/L) wereapproximately normal. Most patients had tremor and

urinary frequency. None of the patients developed or-thostatic hypotension between the first and secondscans.

At the time of the first scan, 2 patients had normalmyocardial 6-[18F]fluorodopamine-derived radioactiv-ity, and 7 had decreased myocardial 6-[18F]-fluorodopamine-derived radioactivity confined to thelateral wall or apex, so that no patient had6-[18F]fluorodopamine-derived radioactivity more thantwo standard deviations below the normal mean inboth the lateral wall and interventricular septum.

All 9 patients had lower lateral wall concentrationsof 6-[18F]fluorodopamine-derived radioactivity inthe second scan than in the first scan (Fig 1).Left ventricular myocardial mean concentrations of6-[18F]fluorodopamine-derived radioactivity decreasedby 23% between the first scans (mean, 5,122nCi-kg/cc-mCi; SEM, 564nCi-kg/cc-mCi) and second scans(mean, 6,634nCi-kg/cc-mCi; SEM, 447nCi-kg/cc-mCi;p � 0.003). Lateral wall radioactivity decreased by31% 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;p � 0.003), and septal radioactivity decreased by 16%between the first scans (mean, 6,137nCi-kg/cc-mCi;SEM, 716nCi-kg/cc-mCi) and second scans (mean,7,278nCi-kg/cc-mCi; SEM, 385nCi-kg/cc-mCi; p �0.05; Fig 2, Table). In 1 patient, the lateral ventricularwall was not visualized in the second scan, and the tis-sue concentration was assumed to be equal to the leftventricular chamber concentration.

With the exclusion of data from the patient forwhom the left ventricular wall was not visualized in thesecond scan, the percentage decrease in lateral wall ra-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

32%; SEM, 7%) exceeded the percentage decrease inseptal wall radioactivity (mean, 13%; SEM, 7%; t �2.5, p � 0.04).

Tissue concentrations of 6-[18F]fluorodopamine-derived radioactivity in the liver, renal cortex, renal pel-vis, salivary glands, and thyroid did not change be-tween the two scans (see Table). Radioactivity in thespleen, however, was lower in the second scan (mean,5,020nCi-kg/cc-mCi; SEM, 137nCi-kg/cc-mCi) thanin the first scan (mean, 6,495nCi-kg/cc-mCi; SEM,364nCi-kg/cc-mCi; p � 0.001).

DiscussionThese findings, based on sympathetic neuroimagingwith 6-[18F]fluorodopamine positron emission tomog-raphy scanning, indicate that in patients with Parkin-son’s disease who have normal or only locally decreasedcardiac sympathetic innervation, the loss of innervationprogresses over time, especially in the lateral ventricularwall, in which 6-[18F]fluorodopamine-derived radioac-tivity decreased by about 30% over an average of 2

years. This rate of loss of sympathetic terminals appearsto be at least as high as the rate of loss of nigrostriataldopamine terminals.15

So far in our ongoing series, all patients with Par-kinson’s disease and orthostatic hypotension have hadevidence of diffuse loss of cardiac sympathetic inner-vation at the time of initial testing.13 About half ofpatients with Parkinson’s disease without orthostatichypotension have also had evidence of diffuse cardiacsympathetic denervation, and because of the likeli-hood of a floor effect, data from these patients werenot included in this study. Given these results, andthe present findings, based on the remaining patientswith Parkinson’s disease who did not have either or-thostatic hypotension or diffuse cardiac sympatheticdenervation at the time of initial testing, indicate thatprogressive loss of cardiac sympathetic innervationcharacterizes the disease. As of the time of the secondscan, none of the patients developed orthostatic hy-potension.

The sympathetic innervation of the myocardiumtravels with the coronary arteries. The finding of moreseverely decreased 6-[18F]fluorodopamine-derived ra-dioactivity in the lateral wall than in the interventric-ular septal wall leads to a suggestion of a dying-backmechanism for the loss of sympathetic terminals, asopposed to death of the cell bodies followed by loss ofthe terminals (as in Wallerian degeneration). In agree-ment with this notion, about one half of patients withParkinson’s disease who do not have orthostatic hypo-tension already have decreased concentrations of6-[18F]fluorodopamine-derived radioactivity throughout

Table. Tissue Concentrations of 6-[18F]Fluorodopamine-Derived Radioactivity (nCi-kg/cc-mCi) in Patients withParkinson’s Disease

Organ

First Scan Second Scan

Mean SEM Mean SEM

Left lateral wall 5,991 � 537 4,107 � 535a

Septum 7,278 � 385 6,137 � 716a

Right myocardium 5,889 � 564 5,390 � 485Right chamber 4,799 � 411 4,240 � 263Left chamber 4,971 � 517 4,725 � 423Liver 6,897 � 724 7,165 � 869Spleen 6,495 � 364 5,020 � 137a

Renal cortex 23,564 � 2,412 20,716 � 2,070Renal pelvis 27,667 � 3,874 23,278 � 5,203Nasophanrynx 1,431 � 162 1,501 � 163Parotid 1,832 � 170 1,698 � 221Submandibular gland 1,870 � 239 1,782 � 267Thyroid 1,933 � 187 1,624 � 250

aSignificantly different from first scan.

SEM � standard error of the mean

Fig 2. Concentrations (mean � standard error of the mean) of6-[18F]fluorodopamine-derived radioactivity in the (top) lateralleft ventricular wall and (bottom) interventricular septum innormal control subjects (open circles), patients with Parkin-son’s disease at the time of the first scan (filled squares), andthe same patients at the time of the second scan an average of2 years later (open squares).

222 Annals of Neurology Vol 52 No 2 August 2002

the left ventricular myocardium, and of the remaininghalf, most have decreased 6-[18F]fluorodopamine-derived radioactivity in the lateral ventricular wall orapex, with relative sparing of the interventricular sep-tum.13

Because the patients did not have a progressive lossof 6-[18F]fluorodopamine-derived radioactivity in theliver or renal cortex but did in the heart and spleen,rates of loss of sympathetic innervation appear to varyacross organs. This fits with the notion of cardioselec-tive sympathetic denervation in Parkinson’s dis-ease.11,12

The results lead to the general inference that Parkin-son’s disease features progressive neurodegeneration notonly in the nigrostriatal dopaminergic system but alsoin the sympathetic noradrenergic system.

We gratefully acknowledge the assistance of Sandra Brentzel, RN,and the Positron Emission Tomography Department of the Na-tional Institutes of Health.

References1. Braune S, Reinhardt M, Bathmann J, et al. Impaired cardiac

uptake of meta-[123I]iodobenzylguanidine in Parkinson’s dis-ease 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 systematrophy. Neurology 1999;53:1020–1025.

3. Takatsu H, Nishida H, Matsuo H, et al. Cardiac sympatheticdenervation from the early stage of Parkinson’s disease: clinicaland experimental studies with radiolabeled MIBG. J Nucl Med2000;41:71–77.

4. Takatsu H, Nagashima K, Murase M, et al. Differentiating Par-kinson disease from multiple-system atrophy by measuring car-diac iodine-123 metaiodobenzylguanidine accumulation. JAMA2000;284:44–45.

5. Orimo S, Ozawa E, Nakade S, et al. (123)I-metaiodobenzyl-guanidine myocardial scintigraphy in Parkinson’s disease.J Neurol Neurosurg Psychiatry 1999;67:189–194.

6. Yoshita M, Hayashi M, Hirai S. Iodine 123-labeled meta-iodobenzylguanidine myocardial scintigraphy in the cases of id-iopathic Parkinson’s disease, multiple system atrophy, and pro-gressive supranuclear palsy. Rinsho Shinkeigaku 1997;37:476–482.

7. Yoshita M. Differentiation of idiopathic Parkinson’s diseasefrom striatonigral degeneration and progressive supranuclearpalsy using iodine-123 meta-iodobenzylguanidine myocardialscintigraphy. 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 diseasestages by means of I-123-MIBG-SPECT. J Neurol Sci 2000;175:3–12.

9. Satoh A, Serita T, Tsujihata M. Total defect of metaiodoben-zylguanidine (MIBG) imaging on heart in Parkinson’s disease:assessment of cardiac sympathetic denervation. Nippon Rinsho1997;55:202–206.

10. Satoh A, Serita T, Seto M, et al. Loss of 123I-MIBG uptake bythe heart in Parkinson’s disease: assessment of cardiac sympa-thetic denervation and diagnostic value. J Nucl Med 1999;40:371–375.

11. Reinhardt MJ, Jungling FD, Krause TM, Braune S. Scinti-graphic differentiation between two forms of primary dysauto-nomia early after onset of autonomic dysfunction: value of car-diac and pulmonary iodine-123 MIBG uptake. Eur J Nucl Med2000;27:595–600.

12. Taki J, Nakajima K, Hwang EH, et al. Peripheral sympatheticdysfunction in patients with Parkinson’s disease without auto-nomic failure is heart selective and disease specific. Eur J NuclMed 2000;27:566–573.

13. Goldstein DS, Holmes C, Li ST, et al. Cardiac sympatheticdenervation in Parkinson disease. Ann Intern Med 2000;133:338–347.

14. Goldstein DS, Eisenhofer G, Dunn BB, et al. Positron emissiontomographic imaging of cardiac sympathetic innervation using6-[18F]fluorodopamine: initial findings in humans. J Am CollCardiol 1993;22:1961–1971.

15. Poewe WH. The natural history of Parkinson’s disease. AnnNeurol 1998;44(suppl 1):1–9.

Li et al: Sympathetic Innervation in Parkinson’s Disease 223

No Mutation in the TRKA(NTRK1) Gene Encoding aReceptor Tyrosine Kinasefor Nerve Growth Factor ina Patient with HereditarySensory and AutonomicNeuropathy Type VEnnio Toscano, MD, PhD,1 Alessandro Simonati, MD,2

Yasuhiro Indo, MD, PhD,3 and Generoso Andria, MD1

Hereditary sensory and autonomic neuropathy type IV(HSAN-IV) and type V (HSAN-V) are autosomal reces-sive genetic disorders, both characterized by a lack ofpain sensation. We report a girl with clinical and neuro-physiological findings consistent with a diagnosis ofHSAN-V. We sequenced her TRKA gene, encoding a re-ceptor tyrosine kinase for nerve growth factor and re-sponsible for HSAN-IV, but we could not detect any mu-tation. These data indicate that a gene (or genes) otherthan TRKA is probably responsible for HSAN-V in somepatients.

Ann Neurol 2002;52:224–227

Hereditary sensory and autonomic neuropathy(HSAN) are classified into 5 different types accordingto Dyck.1 We have demonstrated that the TRKA(NTRK1) gene, encoding a receptor tyrosine kinasethat is phosphorylated in response to nerve growth fac-tor, is responsible for HSAN-IV.2 HSAN-IV is charac-terized by febrile episodes, anhidrosis, insensitivity topain, self-mutilating behavior, and mental retardation.Pathological features of HSAN-IV are severe reductionof small-diameter afferent neurons, which are activatedby tissue-damaging stimuli,3,4 and a loss of sympatheticneurons innervating eccrine sweat glands.5

HSAN-V also is characterized by absent reaction to

noxious stimuli but usually lacks anhidrosis and mentalretardation. Some patients with possible HSAN-V weredescribed before 1960 (see Landrieu and colleagues6

for references), and 3 families with HSAN-V subse-quently were described in which were demonstrated aselective severe decrease of the small myelinated fi-bers7,8 and a small reduction in unmyelinated fibers9 ofthe sural nerve. HSAN-V is likely an autosomal reces-sive disorder, but Landrieu and colleagues6 reported ina family 2 dominantly transmitted cases with normalnerve biopsy. Therefore, a clinical entity, also calledcongenital indifference to pain, is genetically heteroge-neous and probably includes HSAN-IV or HSAN-V,as suggested by Dyck.1

Recently, Houlden and colleagues10 described a boywith febrile episodes and a lack of pain sensation thatthey diagnosed as HSAN-V, according to the typicalfindings in a nerve biopsy.9 DNA analysis showed thathe had a homozygous mutation in the TRKA gene.They stated that HSAN-IV and HSAN-V are likely tobe allelic.

Here we present a girl with clinical features consis-tent with HSAN-V, namely, a lack of pain sensation,but no febrile episodes and normal sural nerve biopsy.We sequenced her TRKA gene and detected no muta-tion. Therefore, these data indicate that a gene (orgenes) other than TRKA is probably responsible forHSAN-V in some patients, arguing against a previouslycited report.10 We also discuss the differential diagnosisof HSAN-IV versus HSAN-V.

Case ReportI.F., a girl, was the second child of consanguineous parents(first cousins). She was born at term (birth weight, 3,400gm)with Apgar scores of 9 and 10. She was found to have in-sensitivity to painful stimuli, and multiple traumatic episodeswere experienced from early life. She bit off the tip of hertongue, and she did not cry when she fell or during bloodsampling. At the ages of 3, 4, and 5 years, she had episodesof left hip dislocation without pain perception. At the age of6 years, she suffered from bilateral osteochondritis of thefeet. Her psychomotor development was apparently normal,and she never exhibited behavioral problems or attentiondeficit problems. She never had thermoregulatory or feedingproblems, vomiting, bowel dysfunction, or other signs orsymptoms of gastrointestinal dysmotility.

ResultsClinical examination of the girl when she was 11 yearsold showed reduced response to painful stimuli andanosmia; thermic sensation appeared to be conserved.Fungiform papillae of the tongue, corneal reflexes, andoverflow tears were present. Intelligence and deep ten-don reflexes were normal. Blood pressure was normalin both supine and standing positions. An intradermalinjection of histamine evoked axonal reflex as observedin a normal control. She could control her body tem-

From the 1Department of Pediatrics, “Federico II” University, Na-ples, Italy; 2Section of Clinical Neurology, Department of Neuro-logical and Visual Sciences, University of Verona, Verona, Italy; and3Department of Pediatrics, Kumamoto University, Honjo, Ku-mamoto, Japan.

Received Aug 21, 2001, and in revised form March 11, 2002. Ac-cepted for publication March 11, 2002.


Address correspondence to Dr Andria, Department of Pediatrics,“Federico II” University, Via S. Pansini 5, I-80131 Naples, Italy.E-mail: [email protected]

224 © 2002 Wiley-Liss, Inc.

perature well under hot environmental conditions. Pi-locarpine ionophoresis and sympathetic skin responsewere normal.

A right sural nerve biopsy was performed and pro-cessed according to routine procedures for both mor-phological and morphometrical investigations.11 Fea-tures of the nerve fascicle were normal. At theultrastructural level, unmyelinated fibers were regularlydetected; neither denervated Schwann cells nor colla-gen pockets were seen (Fig 1). Myelinated fiber densitywas within normal ranges; the shape of the histogramwas bimodal, showing the small-caliber fiber peak (Fig2). Skin biopsy was examined on plastic sections only.Glands were present; both myelinated and unmyeli-nated fibers were present in the intradermal nerve fas-cicles.

We sequenced all 17 exons of the TRKA gene of thepatient, including their flanking intronic sequences,12

and detected no putative mutation. Furthermore, weanalyzed 8 polymorphic sites in this gene and foundthat the patient is heterozygous at the intragenic poly-morphic site (c. 1,953 cytosine/thymine).13 This fur-

ther supports that the patient has no mutation in theTRKA locus because the parents are consanguineous.

DiscussionThe patient reported here probably presents a heredi-tary autosomal recessive sensory neuropathy with selec-tive loss of pain sensation. Autonomic abnormalitiesapparently were not observed. Reduced sweating wasmentioned once, but she can maintain her body tem-perature under hot environmental conditions. This iscompatible with the findings in her nerve and skin bi-opsies. These clinical and laboratory data suggest thatthe patient suffers from HSAN-V. Similar features, to-gether with apparently intact peripheral nerves, weredescribed in some classic reports of congenital indiffer-ence to pain.6 The patient reported by Low and col-leagues,7 presenting indifference to pain and selectiveloss of small myelinated fibers without sweating abnor-mality, 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 ofunmyelinated fibers. It is their view that most earliercases of indifference to pain may have had eitherHSAN-IV or HSAN-V.1,9

In contrast, HSAN-IV is a distinct clinical entitycharacterized by recurrent episodic fever, anhidrosis,insensitivity to pain, self-mutilating behavior, andmental retardation.3,14 Mental retardation is variable,

Fig 1. Sural nerve biopsy. (A) Semithin section; toluidine bluestain. Normal features of the nerve fascicle showing myelinatedfibers of both small and large calibers. Bar � 12.5�m. (B)Thin section; uranyl acetate and lead citrate stain. Representa-tive clusters of normal unmyelinated fibers. Bar � 3�m.

Fig 2. Size distribution (X-axis) of the myelinated fibers of theindex case and an age-matched control. Note the normal, bi-modal pattern of the histogram; small-caliber fibers are simi-larly represented in both the patient and the control. Numberson the Y-axis refer to the absolute figures of the measuredfibers.

Toscano et al: No Mutation in TRKA Gene in HSAN-V 225

from severe to mild, and some patients were apparentlynormal, but later mild retardation was showed by aformal assessment (patients KI-108 and KI-116 re-ported by Mardy and colleagues12 and Indo and col-leagues,15 respectively). Sweating may be variable butshould be evaluated cautiously, as we recently report-ed.15 We think that a fundamental phenotype ofHSAN-IV consists of insensitivity to pain, anhidrosis,and mental retardation, each of variable degree. Recur-rent hyperpyrexia, self-mutilating behaviors, traumas,and bone fractures can be devastating and often lead tocrippling or fatal consequences.

Nerve growth factor supports the survival of sympa-thetic ganglion neurons and nociceptive sensory neu-rons in dorsal root ganglia and ascending cholinergicneurons of the basal forebrain.16 Eccrine sweat glands,innervated by sympathetic cholinergic fibers, are welldeveloped in humans. Therefore, the nerve growthfactor-TRKA system has a crucial role in the develop-ment and function of the nociceptive reception and es-tablishment of thermoregulation via sweating.2 A neg-ative result of the intradermal histamine test, animportant diagnostic criterion in HSAN-IV, probablyalso can be explained by defective peripheral sympa-thetic neurons. Therefore, anhidrosis and associatedfailure to maintain body temperature are characteristicfeatures of HSAN-IV.

Recently, Houlden and colleagues10 described a boy9 years of age, born to healthy consanguineous parents,presenting anhidrosis and loss of pain and temperaturesensation. Sural nerve biopsy demonstrated severe re-duction in small-caliber myelinated fiber density butonly modest reduction in unmyelinated axons. The au-thors did not mention either a skin biopsy or a hista-mine test, which provide important diagnostic criteriafor the group of hereditary peripheral neuropathies.17

They detected a homozygous missense mutation in theTRKA gene, changing a tyrosine to a cysteine at codon359. According to this case, they concluded that the 2disorders, HSAN-IV and HSAN-V, are likely to be al-lelic. However, we propose an alternative interpretationof Houlden and colleagues’ report. Their patient maysuffer from HSAN-IV, but not HSAN-V. They arguedfor the diagnosis of HSAN-V, mainly basing their ar-gument on the finding of nerve biopsy with modestreduction of unmyelinated fibers. However, anhidrosisobserved in the patient strongly indicates dysfunctionof the most distal intradermal portions of the sympa-thetic axons. That also might account for the normalappearance of the more proximal unmyelinated fibersobserved after sural nerve. Furthermore, it would beimportant to confirm the functional significance of themissense mutation by an expression study, such as thatreported recently.18

We stress the importance of the molecular analysisof the TRKA gene in all cases with lack of pain sensa-

tion to distinguish overlapping phenotypes ofHSAN-IV and HSAN-V, both characterized by a lackof pain sensation. Most patients with HSAN-IV prob-ably have mutations in the TRKA gene,2,12,13 althoughwe cannot rule out the possibility that a mutation (ormutations) in another gene (or genes) is responsible forsimilar clinical phenotypes. The patient presented heresuffers from HSAN-V, but not HSAN-IV, because shedoes not show anhidrosis or mental retardation. Thepresence of myelinated and unmyelinated fibers innerve biopsy and the normal intradermal histamine testfurther support this diagnosis. We could not detect anyputative mutation in the TRKA gene, whereas wefound that the patient is heterozygous for this locus,despite the parental consanguinity. These findingsstrongly indicate that defects of a gene (or genes) otherthan TRKA is likely responsible for at least some pa-tients with HSAN-V.

It remains unknown whether peripheral nociceptivetransmission or central processing might be involved inour case and in some cases with similar phenotypes re-ported as having HSAN-V. Anosmia observed in ourcase remains to be examined and may give us someclue for studying a mechanism underlying the defect inpain sensation. Some patients diagnosed as havingHSAN-V do not show an apparent abnormality of pe-ripheral nerve fibers. Therefore, their manifestationsmight be caused by defects in peripheral nociceptor ortransduction and transmission of pain sensation oreven central processing, such as a lack of concern for apainful stimulus well received by the peripheral ner-vous system, according to the classic definition of in-difference to pain.19

References1. Dyck PJ. Neuronal atrophy and degeneration predominantly af-

fecting peripheral sensory and autonomic neurons. In: Dick PJ,Thomas PK, Griffin JW, Low PA, Podreslo JC, eds. Peripheralneuropathy. Philadelphia: Saunders, 1993:1065–1093.

2. Indo Y, Tsuruta M, Hayashida Y, et al. Mutations in theTRKA/NGF receptor gene in patients with congenital insensi-tivity to pain with anhidrosis. Nat Genet 1996;13:485–488.

3. Swanson AG, Buchan GG, Alvord EC Jr, et al. Autonomicchanges in congenital insensitivity to pain: absence of small pri-mary sensory neurons in ganglia, roots and Lissauer’s tract.Arch Neurol 1965;12:12–18.

4. Rafel E, Alberca R, Bautista J, et al. Congenital insensitivity topain with anhidrosis. Muscle Nerve 1980;3:216–220.

5. Langer J, Goebel HH, Veit S. Eccrine sweat gland are not in-nervated in hereditary sensory neuropathy type IV: an electron-microscopic study. Acta Neuropathol (Berl) 1981;54:199–202.

6. Landrieu P, Said G, Allaire C. Dominantly transmitted congen-ital indifference to pain. Ann Neurol 1990;27:574–578.

7. Low PA, Burke WJ, McLeod JG. Congenital sensory neuropa-thy with selective loss of small myelinated fibers. Ann Neurol1978;3:179–182.

8. Donaghy M, Hakin RN, Bamford JM, et al. Hereditary sensoryand autonomic neuropathy with neurotrophic keratitis. Brain1987;110:563–583.


9. Dyck PJ, Mellinger JF, Reagan TJ, et al. Not “indifference topain” but varieties of hereditary sensory and autonomic neurop-athy. Brain 1983;106:373–390.

10. Houlden H, King RHM, Hashemi-Nejad A, et al. A novelTRK A (NTRK1) mutation associated with hereditary sensoryand autonomic neuropathy type V. Ann Neurol 2001;49:521–525.

11. Simonati A, Fabrizi GM, Pasquinelli A, et al. Congenital hy-pomyelination neuropathy with Ser72Leu substitution inPMP22. Neuromuscul Disord 1999;9:257–261.

12. Mardy S, Miura Y, Endo F, et al. Congenital insensitivity topain with anhidrosis: novel mutations in the TRKA (NTRK1)gene encoding a high-affinity receptor for nerve growth factor.Am J Hum Genet 1999;64:1570–1579.

13. Miura Y, Mardy S, Awaya Y, et al. Mutation and polymor-phism analysis of the TRKA (NTRK1) gene encoding a high-affinity receptor for nerve growth factor in congenital insensi-tivity to pain with anhidrosis (CIPA) families. Hum Genet2000;106:116–124.

14. Swanson AG. Congenital insensitivity to pain with anhidrosis.Arch Neurol 1963;8:299–306.

15. Indo Y, Mardy S, Miura Y, et al. Congenital insensitivity topain with anhidrosis (CIPA): novel mutations of TRKA(NTRK1) gene encoding the receptor tyrosine kinase for nervegrowth factor, a putative uniparental disomy and a linkage ofthe mutant TRKA and PKLR genes in a family with CIPA andpyruvate kinase deficiency. Hum Mutat 2001;18:308–318.

16. Levi Montalcini R. The nerve growth factor: thirty-five yearslater. EMBO J 1987;6:1145–1154.

17. Axelrod FB, Pearson J. Congenital sensory neuropathies. Am JDis Child 1984;138:947–954.

18. Mardy S, Miura Y, Endo F, et al. Congenital insensitivity topain with anhidrosis (CIPA): effect of TRKA (NTRK1) mis-sense mutations on autophosphorylation of the receptor ty-rosine kinase for nerve growth factor. Hum Mol Genet 2001;10:179–188.

19. Jewesburry ECO. Congenital indifference to pain. In: VinkenPJ, Bruyn GW, eds. Handbook of clinical neurology. Vol 8.Amsterdam: Elsevier, 1979:187–204.

X-Linked CreatineDeficiency Syndrome: ANovel Mutation in CreatineTransporter Gene SLC6A8Alberto Bizzi, MD,1 Marianna Bugiani, MD,2

Gajja S. Salomons, PhD,3 Donald H. Hunneman, PhD,4

Isabella Moroni, MD,2 Margherita Estienne, MD,2

Ugo Danesi, PhD,1 Cornelis Jakobs, PhD,3

and Graziella Uziel, MD2

Among creatine deficiency syndromes, an X-linked con-dition related to a defective creatine transport into thecentral nervous system has been described recently. Hall-marks of the disease are the absence of a creatine signalat brain spectroscopy, increased creatine levels in bloodand urine, ineffectiveness of oral supplementation, and amutation in the SLC6A8 (Online Mendelian Inheritancein Man [OMIM] 300036) creatine transporter gene. Wereport on a patient in whom a novel mutation (1221-1223delTTC) was identified.

Ann Neurol 2002;52:227–231

Creatine deficiency syndromes are recently identifiedinborn errors of metabolism resulting in a progressiveencephalopathy with early onset and mental retarda-tion, extrapyramidal features, and drug-resistant epi-lepsy.1–3 Symptoms are related to a depletion of thecreatine/phosphocreatine pool within the central ner-vous system, making this condition easily detectable bybrain spectroscopy. Most of the cases reported so farwere caused by a defect of the second enzyme involvedin creatine biosynthesis: guanidino-acetate methyltrans-ferase (GAMT; OMIM 601240). This defect results inincreased guanidino-acetate (GAA) and reduced creat-ine levels in blood and urine.4,5 GAMT-deficient pa-tients benefit from oral creatine monohydrate supple-mentation, which helps to control movement disordersand epilepsy, partially recovers mental impairment, andrestores neurological development over time.6 The ab-

From the 1Departments of Neuroradiology and 2Child Neurology,Istituto Nazionale Neurologico “C. Besta,” Milano, Italy; 3Metabol-ic Unit, VU Medical Center, Amsterdam, The Netherlands; and4University Kinderklinik, Gottingen, Germany.

Received Nov 6, 2001, and in revised form Mar 11, 2002. Acceptedfor publication Mar 11, 2002.


Address correspondence to Dr Uziel, Department of Child Neurol-ogy, Istituto Nazionale Neurologico “C. Besta,” Via Celoria 11,20133 Milano, Italy. E-mail: [email protected]

© 2002 Wiley-Liss, Inc. 227

sence of complete recovery can be explained by neuro-toxic GAA accumulation or by an imbalance of braincreatine and high-energy phosphates.7,8 Very recently,Item and colleagues9,10 described 2 sisters with mentalimpairment, low GAA levels in urine, and undetectableactivity of arginine to glycine amidinotransferase(AGAT; OMIM 602360), the first enzyme involved increatine biosynthesis. A homozygous nonsense muta-tion in AGAT gene was detected. Clinical symptomsand brain creatine deficiency were partially recoveredby means of creatine supplementation. A different dis-ease due to impairment of creatine transport into thebrain was reported by Salomons and colleagues11,12 ina boy with language delay, short attention span, epi-lepsy, and increased creatine levels in blood and urine.A nonsense mutation in the X-linked creatine trans-porter gene SLC6A8 was demonstrated. Creatine sup-plementation in this boy was totally ineffective. Thisarticle reports on a second case caused by defective cre-atine transport into the central nervous system.

Case ReportThe patient was the second-born son of healthy, nonconsan-guineous parents. The child’s prenatal and perinatal historywas unremarkable. Since the first months of life, he pre-sented with motor delay, reduced interest in surroundings,and no language acquisition. At 8 months, he was admitted

to the hospital after a febrile seizure. An electroencephalo-gram recording and a magnetic resonance imaging scan werenormal. Routine blood and urine analysis and investigationfor infectious (screening for rubella, cytomegalovirus, toxo-plasma, herpes simplex, Coxsackie virus, and Mycoplasmapneumoniae) and neurometabolic disorders (plasma lactateand metabolic screening of a 24-hour urine sample with anassessment of amino acids, organic acids, and mucopolysac-charides) were normal. Since 16 months of age, he experi-enced complex partial seizures with secondary generalization,responding to sodium valproate. Serial electroencephalogramtracings showed a progressive instability of background activ-ity with abundant fast activity and epileptic discharges fromfrontal and temporal leads during sleep. Physical examinationat 3 years and 9 months showed a severe delay in speech andlanguage functions with behavioral disturbances in agreementwith an autistic disorder. He did not follow commands orspeak, he presented with stereotypical motor behaviors, andhe could not engage in any structured play. Gross and finemotor functions were normal.

A second magnetic resonance imaging showed mild atro-phy with signal abnormality in the right hippocampus, sug-gesting mesial temporal sclerosis. Proton magnetic resonancespectroscopic imaging (H-MRSI) was performed as part ofthe diagnostic workup for mesial temporal sclerosis con-ducted at our institution and showed a normal N-acetylas-partate/choline ratio in both hippocampi without abnormal-ity in the asymmetry index. The surprising feature was theabsence 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 cen-trum semiovale. There was no restoration of the creatine pool after 3 months of oral creatine monohydrate supplementation at400mg/kg/day (1B) and after 8 months of supplementation at 700mg/kg/day (1C). There were no significant changes in cholineand 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.


cating a creatine deficiency syndrome, without any abnormalpeak in the expected frequency of GAA (3.8ppm). Creatineand GAA, therefore, were measured in plasma and urine,and oral creatine monohydrate supplementation (400mg/kg/day) was started. Because H-MRSI showed no creatine re-covery after 3 months of therapy, supplementation was in-creased to 700mg/kg/day. Eight months later, a follow-upH-MRSI examination confirmed no appearance of brain cre-atine. By this time, clinical symptoms were not improved,and therapy was discontinued. Currently, the patient is 5years old, and his clinical symptoms are grossly unchanged.The very recent identification of the first patient with a cre-atine transporter defect11,12 suggested that our patient suf-fered from a creatine uptake defect as well, which was in linewith the ineffectiveness of creatine supplementation and withbiochemical data. Mutational analysis of the SLC6A8 gene,therefore, was performed.

MethodsBefore therapy, H-MRSI (two-dimensional phase encodingpoint resolved spectroscopy repetition (PRESS) technique:recovery time/echo time, 1,500/136msec; field of view,160mm; matrix, 16 � 16; 20mm slice thickness) was per-formed from 3 separate sections at the level of the centrumsemiovale, basal nuclei, and hippocampi. During therapy,H-MRSI was performed at the level of the centrum semi-ovale. Single-voxel spectra (PRESS: recovery time/echo delaytime, 1,500/34msec) were acquired from a 30ml volume inthe frontoparietal parasagittal cortical gray matter before andduring therapy. Raw data were transferred to a SUN work-station and reconstructed with custom-made software.13

Mutational analysis complementary DNA–based sequenceanalysis was performed according to methods described else-where.12 DNA was isolated from blood cells with a QIAampblood kit (Qiagen, Chatsworth, CA) for conformation of themutation at the genomic level. Primers specific for exon 8 ofthe SLC6A8 gene were designed: forward 5�TCCCAGC-CCCTGCCGCAC and reverse 5�TACAAACTGTGGC-CAGGGC.

ResultsCreatine and GAA levels before, during, and after thewithdrawal of creatine supplementation are shown inTable 1. Sequence analysis of the creatine transportergene SLC6A8 identified an hemizygous 3bp deletion inexon 8 involving nucleotides TTC in position 1221-1223 (1221-1223delTTC; GenBank accession number,

NM 005629). This mutation resulted in the deletionof a single phenylalanine at residue 408 of the protein(delF408). The patient’s mother was heterozygous forthe mutation.

DiscussionThe failure of creatine supplementation to restore braincreatine by H-MRSI and to improve clinical symptomsand normal plasma and urine GAA levels ruled out adefect of creatine biosynthesis and prompted a searchfor molecular defects in the SLC6A8 gene.12,14 A novelhemizygous deletion located in a short repeat of 3 phe-nylalanines in exon 8 was detected. The repeat is partof transmembrane domain VIII, which is a very con-served region among the Na�- and Cl�-dependentneurotransmitter family.15 The mutation, resulting in aphenylalanine deletion at position 408, most likelycauses a partial or even complete loss of creatine trans-port function. The activity of the transporter could notbe tested in fibroblasts because the parents refused con-sent to a skin biopsy.

To our knowledge, this is the first creatine deficiencycase studied with the multivoxel H-MRSI technique,which has better spatial resolution and allows an evalu-ation of lesion heterogeneity in the shortest amount oftime. We did not find any significant difference in cre-atine levels across the brain regions examined, suggestingthat the transporter defect does not spare any brain area.

Symptoms of creatine deficiency have been relatedpreviously to a defect in creatine transport bySalomons and colleagues,11,12 who described a malepatient with mental retardation and severe delay in ex-pressive speech and language function presenting ahemizygous nonsense mutation in the SLC6A8 gene.As in our case, a lack of creatine transport into thecentral nervous system resulted in the failure of creat-ine supplementation to reverse clinical symptoms andbrain spectroscopy abnormality. Very recently, anotherunrelated family with a creatine transporter defect wasrecognized by the same authors.16 The biochemicalprofile of our patient showed a massive loss of creatinein urine, but in contrast with the first reported indexcase, creatine levels in plasma were normal even duringcreatine supplementation.

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

Result

Blood Urine

GAA(�mol/L)

Creatine(�mol/L)

GAA(mmol/mol creatinine)

Creatine(mmol/mol creatinine)

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

GAA � guanidino-acetate; ND � not determined.

Bizzi et al: Creatine Transporter Defect 229

The mother underwent a brain H-MRSI that dem-onstrated a mildly reduced creatine signal comparedwith that of age-matched controls. Moreover, the re-sults showed that she was heterozygous for the muta-tion identified in her son. However, no learning dis-abilities were reported for the patient’s female relatives,in contrast with what was encountered in 2 of 3 femalecarriers in the first family described.12 These data agreewith skewed X inactivation (mosaic expression of mu-tant and wild-type alleles), resulting in a variably favor-able mosaic expression of the wild-type allele.

The 3 creatine deficiency syndromes described so far(ie, GAMT and AGAT defects and impairment of cre-atine transport) share overlapping symptoms of mentalretardation with severe language impairment, autistic be-havior, and epilepsy. Movement disorders have been re-ported only in patients with GAMT deficiency, suggest-ing that extrapyramidal features may result fromneurotoxic GAA accumulation rather than from reducedcreatine availability in brain. The major involvement ofhigher cortical functions and the frequent finding of ep-ilepsy suggest that the cerebral cortex may be selectivelyvulnerable to creatine deficiency. How creatine defi-ciency adversely affects cortical functions is still to beestablished. It is conceivable that creatine plays a role inthe latest stages of cortical organization, including syn-aptogenesis, a process that continues after birth. Thiscould explain the homogeneity of clinical presentationand age at onset in the 3 creatine deficiency syndromes,even though in children with biosynthesis defects, crea-tine is supplied through the placenta during fetal life,whereas patients with a transporter defect suffer fromcreatine depletion already in utero.

Spectroscopy alone cannot always distinguish betweensynthesis and transport defects. In GAMT deficiency,brain spectroscopy at a short echo time can identify anabnormal peak that is assigned to GAA (3.8ppm), butthis elevation may be subtle, and it has been reportedonly in a few cases. Therefore, all patients in whom adiagnosis of creatine deficiency is reached should un-dergo a careful biochemical evaluation to assess creatineand GAA levels in blood and urine (Table 2). Brainspectroscopy is becoming more available; a quick auto-mated spectrum acquisition can be performed at the

time of conventional magnetic resonance imaging andmay practically disclose creatine or other metabolites de-pletion: just recently, the first case of N-acetylaspartatebrain deficiency was reported in a patient with mentalretardation and severe language impairment.17

Clinical features of mental impairment, language de-lay, and autistic behavior with epilepsy frequently areencountered in infancy. Conceivably, creatine defi-ciency could be underdiagnosed, and if brain spectros-copy cannot be easily achieved, an assessment of bloodand urinary creatine should always be performed.

We thank Dr S. J. M. van Dooren and D. Brunea for excellent tech-nical support to Dr G. S. Salomons.

Electronic Database Information: OMIM. www.ncbi.nlm.nih.gov/omim; GenBank. www.ncbi.nlm.nih.gov/genbank.

References1. Stockler S, Isbrandt D, Hanefeld F, et al. Guanidinoacetate

methyltransferase deficiency: the first inborn error of creatinemetabolism in man. Am J Hum Genet 1996;58:914–922.

2. Stockler S, Marescau B, De Deyn PP, et al. Guanidinoacetatemethyltransferase deficiency, a new inborn error of creatine syn-thesis. Metabolism 1997;46:1189–1193.

3. Ganesan V, Johnson A, Connelly A, et al. Guanidinoacetatemethyltransferase deficiency: new clinical features. Pediatr Neu-rol 1997;17:155–157.

4. Schulze A, Hess T, Wevers R, et al. Creatine deficiency syn-drome caused by guanidinoacetate methyltransferase deficiency:diagnostic tools for a new inborn error of metabolism. J Pediatr1997;131:626–631.

5. Ilas J, Muhl A, Stockler-Ipsiroglu S. Guanidinoacetate methyl-transferase deficiency: non-invasive enzymatic diagnosis of anewly recognized inborn error of metabolism. Clin Chim Acta2000;290:179–188.

6. Stockler S, Hanefeld F, Frahm J. Creatine replacement therapyin guanidinoacetate methyltransferase deficiency, a novel inbornerror of metabolism. Lancet 1996;348:789–790.

7. van der Knaap MS, Verhoeven NM, Maaswinkel-Mooij P, et al.Mental retardation and behavioural problems as presenting signsof a creatine synthesis defect. Ann Neurol 2000;47:540–543.

8. Leuzzi V, Bianchi MC, Tosetti M, et al. Brain creatinedepletion: guanidinoacetate methyltransferase deficiency (im-proving with creatine supplementation). Neurology 2000;55:1407–1409.

9. Bianchi MC, Tosetti M, Fornai F, et al. Reversible brain crea-tine deficiency in two sisters with normal blood creatine level.Ann Neurol 2000;47:511–513.

Table 2. Creatine and GAA Levels in GAMT and AGAT Defects and in Creatine Transporter Gene (SLC6A8) Mutations

Defect or mutation

Blood Urine Brain

Creatine GAA Creatine GAA Creatine GAA

GAMT Decrease Increase Decrease Increase Decrease Undetectable/IncreaseAGAT Normal Normal ND Decrease Decrease UndetectableSLC6A8 Normal/Increase Normal Increase Normal Decrease Undetectable

AGAT � arginine to glycine amidinotransferase; GAA � guanidino-acetate; GAMT � guanidino-acetate methyltransferase; ND � not de-termined.


10. Item CB, Stockler-Ipsiroglu S, Stromberger C, et al. Arginine:glycine amidinotransferase deficiency: the third inborn error ofcreatine metabolism in humans. Am J Hum Genet 2001;69:1127–1133.

11. Cecil KM, Salomons GS, Ball WS, et al. Irreversible brain cre-atine deficiency with elevated serum and urine creatine: a cre-atine transporter defect? Ann Neurol 2001;49:401–404.

12. Salomons GS, van Dooren SJM, Verhoeven NM, et al.X-linked creatine-transporter gene (SLC6A8) defect: a newcreatine-deficiency syndrome. Am J Hum Genet 2001;68:1497–1500.

13. Soher BJ, van Zijl PCM, Duyn JH, et al. Quantitative protonspectroscopic imaging of the human brain. Magn Reson Med1996;35:356–363.

14. Gregor P, Nash SR, Caron MG, et al. Assignment of the cre-atine transporter gene (SLC6A8) to human chromosome Xq28telomeric to G6PD. Genomics 1995;25:332–333.

15. Nash SR, Giros B, Kingsmore SF, et al. Cloning, pharmacolog-ical characterization and genomic localization of the human cre-atine transporter. Receptors Channels 1994;2:165–174.

16. Salomons GS, Dooren SJ, Verhoeven NM, et al. X-linked cre-atine transporter defect: the second family. J Inherit Metab Dis2001;24(suppl 1):119.

17. Martin E, Capone A, Schneider J, et al. Absence ofN-acetylaspartate in the human brain: impact on neurospectros-copy? Ann Neurol 2001;49:518–521.

Vertebrobasilar Stroke as aLate Complication of aBlalock-Taussig ShuntPhilippe Gailloud, MD,1 Argye Hillis, MD,2

Bruce Perler, MD,3 and Kieran J. Murphy, MD1

We describe a 39-year-old patient with a cerebellar in-farct and a history of a tetralogy of Fallot corrected dur-ing childhood. This is the first documented case of ver-tebrobasilar stroke occurring as a late complication of aBlalock-Taussig shunt followed by total cardiac repair.

Ann Neurol 2002;52:231–234

The Blalock-Taussig shunt, first performed in 1944 atthe Johns Hopkins Hospital,1 is a surgical anastomosisestablished between a subclavian artery and the ipsilat-eral pulmonary artery. In patients with a tetralogy ofFallot, this procedure allows deferment of the definitivecardiac repair to a more robust stage of life by bypass-ing the pulmonary artery stenosis. The Blalock-Taussigshunt, however, creates the permanent anatomical con-dition of a subclavian steal phenomenon (Fig 1). Thesubclavian steal phenomenon, initially described byReivich and colleagues in 1961,2 is related to a proxi-mal subclavian artery stenosis or occlusion responsiblefor the occurrence of retrograde collateral flow in theipsilateral vertebral artery. A few reports have suggesteda possible link between a Blalock-Taussig shunt andthe late occurrence of cerebrovascular disease. We re-port the first documented case of vertebrobasilar strokeoccurring as a late complication of a Blalock-Taussigshunt followed by total cardiac repair during child-hood.

Case ReportA 39-year-old man was admitted to our hospital because ofan acute episode of nausea without vomiting, vertigo, anddecreased hearing on the right side, rapidly followed by rightfacial numbness as well as weakness and decreased sensationin the left arm. The patient also described a visual distur-bance consistent with nystagmus. These symptoms appearedwithout an identifiable precipitating factor and lasted for ap-proximately 20 to 30 minutes. The patient was known tohave tetralogy of Fallot treated by a Blalock-Taussig shunt atthe age of 2 years followed by definitive surgical repair at theage of 9 years. He had been in good health since then. Thereview of risk factors for cerebrovascular disease, includingobesity, tobacco use, hyperlipidemia, and hypertension, wasnegative. The social and familial histories showed only hy-pertension and hyperlipidemia in both parents.

On admission, the general and neurological examinationswere normal except for asymmetrical upper extremity bloodpressure measurements (125/80mm Hg on the right, 146/95mm Hg on the left) and unpalpable distal arterial pulsesin the right arm. According to the patient, this discrepancyhad been known since his second cardiac surgery. The lab-oratory values were unremarkable; in particular, the lipidprofile was normal, and there was no evidence of a hyper-coagulable state (the protein C and S antigens, antithrom-bin III activity, dilute Russell’s viper venom time test, andhomocysteine level were within normal ranges; the factor VLeiden mutation was negative). Computed tomography ofthe brain was normal. Magnetic resonance imaging showedsignal anomalies in the inferior aspect of the right cerebellarhemisphere consistent with an acute ischemic lesion in theright posterior inferior cerebellar artery territory (Fig 2A).Magnetic resonance imaging incidentally showed inflamma-tory changes in the maxillary sinus and ethmoid cells bilat-erally, whereas the sphenoid and frontal sinuses wereunremarkable. The patient was not treated for this asymp-tomatic inflammatory sinus disease. A transesophagealechocardiogram, including a bubble study performed to

From the Department of 1Radiology and Radiological Sciences,2Neurology, and 3Vascular Surgery, Johns Hopkins Hospital, Balti-more, MD.

Received May 15, 2000, and in revised form Feb 28, 2002. Ac-cepted for publication Mar 12, 2002.


Address correspondence to Dr Gailloud, Department of Radiology,Johns Hopkins University School of Medicine, 600 N. WolfeStreet, Radiology B-100, Baltimore, MD 21287.E-mail: [email protected]


rule out potential cardioembolic sources, was unremarkableexcept for a mildly dilated right ventricular cavity. Reverseflow in the right vertebral artery was by documented trans-cranial Doppler. The transcranial Doppler bubble test wasnegative for embolic events at rest and during Valsalva ma-neuver and coughing. Cerebral digital subtraction angiog-raphy then was performed. The aortic arch study showedinterruption of the right subclavian artery at its origin (seeFig 2B). Selective injections of the left vertebral artery con-firmed the presence of a left-to-right subclavian steal phe-nomenon. Revascularization of the right upper extremitywas provided by retrograde filling of several subclavian ar-

tery branches, including the right vertebral artery and theright ascending and deep cervical arteries (see Fig 2C). Thecerebral vasculature was otherwise unremarkable. There wasin 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 hisstroke. However, he continued to report frequent episodesof dizziness, associated once with facial numbness and dip-lopia, despite being anticoagulated with warfarin at a ther-apeutic level. At this stage, surgical correction of his vascu-lar anomaly was advised to the patient, who wished toproceed with this option after the exposition of its potentialrisks and benefits. Interposition of a synthetic graft(Hemoshield Dacron Graft, Boston Scientific Corporation,Natick, MA) between the right common carotid and sub-clavian arteries was performed uneventfully, allowing ante-grade flow to be reestablished in the right vertebral artery.After surgery, his episodic symptoms attributable to brain-stem ischemia resolved. He has remained asymptomatic formore than 1 year.

DiscussionWe report the case of a 39-year-old man presentingwith a vertebrobasilar stroke in the absence of identifi-able cerebrovascular risk factors. The symptoms de-scribed by our patient pointed to a lesion in the terri-tory of the right posterior inferior cerebellar artery,which was confirmed by neuroimaging. In our patient,the co-occurrence of a vertebrobasilar infarct and asubclavian steal phenomenon strongly suggest a caus-ative relationship between the 2 events, that is, a sub-clavian steal syndrome secondary to the cardiac surgeryundergone during childhood. Although an embolicmechanism cannot be completely excluded because ofnormal transesophageal echocardiogram and transcra-nial Doppler, both with negative bubble tests, that theinvolved branch (the right posterior inferior cerebellarartery) originates from the vertebral artery with reverseflow renders this explanation very unlikely. Conversely,this anatomical situation is consistent with an increasedrisk of preferential hypoperfusion in the right posteriorinferior cerebellar artery territory.

The possible association between a Blalock-Taussigshunt and a subclavian steal phenomenon initially wasproposed by Folger and Shah in 1965.3 By reviewing123 cardiac angiograms performed on patients with aBlalock-Taussig shunt, these authors could identify lateopacification of a subclavian artery suggestive of a sub-clavian steal phenomenon in 12 instances. The stealphenomenon was associated with dizziness in 3 pa-tients and with visual disturbances in 2 patients. How-ever, in those patients and in other case reports de-scribing early cerebrovascular events after a Blalock-Taussig shunt,4,5 the vertebrobasilar manifestationsoccurred after the creation of the shunt but before de-finitive repair of the tetralogy of Fallot. In these pa-

Fig 1. Schematic representation of the morphological changesassociated with a Blalock-Taussig shunt. The pulmonary trunkand arteries are represented in light gray; the aortic arch (1)and supra-aortic trunks are in dark gray. The Blalock-Taussigshunt is established between the proximal right subclavianartery (6) and the right pulmonary artery (8) by interpositionof a synthetic graft (7). The subclavian steal phenomenon,that is, antegrade flow in the left vertebral artery and retro-grade flow in the right vertebral artery (4), provides bloodsupply to the right arm (direction of blood flow as indicatedby the arrows). The figure also shows the right common ca-rotid artery (2), the right carotid bifurcation (3), and thebasilar artery (5).


tients, contributing factors related to cardiac dysfunc-tion, such as hypoxia, polycythemia, bacterialendocarditis, and mural or valvular thrombosis, have tobe considered.6 The association of a Blalock-Taussigshunt with delayed ischemic complications occurring

after total cardiac repair of the tetralogy of Fallot wassuggested in 1984 by Kurlan and colleagues.6 Theseauthors described a 38-year-old patient developingtransient vertebrobasilar ischemia 31 years after aBlalock-Taussig shunt and 4 years after total repair of

Fig 2. (A) Magnetic resonance imaging study of the brain. Thisaxial T2-weighted image shows increased signal in the lowerportion 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 withan ischemic lesion in the territory of the right posterior-inferiorcerebellar artery. Note the incidental finding of chronic maxil-lary sinus inflammation. (B) Digital subtraction angiography ofthe aortic arch, left anterior oblique view. The aortic arch, theright common carotid artery (RCC), the left common carotidartery (LCC), and the left subclavian artery (LSC) are unre-markable. The right subclavian artery is interrupted close to itsorigin from the innominate artery (arrow). (C) Selective digitalsubtraction angiography of the left vertebral artery, anteroposte-rior view. The catheter tip (arrowhead) is placed at the originat the left vertebral artery (LV). Retrograde flow in the rightvertebral artery (RV) and in several cervical branches allows forlate opacification of the right subclavian artery (arrow).

Gailloud et al: Blalock-Taussig Shunt and Stroke 233

his tetralogy of Fallot. Our 39-year-old patient pre-sented with a cerebellar infarct 37 years after the cre-ation of the shunt and 30 years after total cardiac re-pair. This observation, the first report of a documentedstroke after Blalock-Taussig shunt and total cardiac re-pair, appears to confirm that patients who underwent aBlalock-Taussig procedure in childhood have an in-creased risk of vertebrobasilar insufficiency later in life.The few cases reported so far that have attributed a latecerebrovascular event to a Blalock-Taussig shunt mightreflect only the relatively young age of the concernedpopulation. Blalock-Taussig shunts have been per-formed on infants and small children for approximately50 years, so much of that population is now approach-ing middle age. It is conceivable that the morphologi-cal risk factor constituted by the surgically created sub-clavian steal phenomenon generally is not significantenough to become symptomatic in isolation, as appearsto have been the case for our patient. However, theassociation of the Blalock-Taussig anatomical anoma-lies with age-related processes such as hypertension andatheromatous disease might predispose these patients tomore precocious development of cerebrovascular dis-eases. If this assumption is correct, patients with a his-tory of Blalock-Taussig shunt constitute an overlookedgroup at risk for cerebrovascular disease. Furthermore,these patients are now in good general health; most ofthem have lost contact with their cardiologist and arenot followed up on a systematic basis.

In view of the potentially devastating consequencesof a vertebrobasilar stroke, patients who underwent aBlalock-Taussig shunt may benefit from prophylacticmeasures, such as the administration of platelet anti-aggregant agents. More aggressive treatments maybe necessary for patients who remain symptomaticunder conservative management, such as the carotid-subclavian bypass performed in our patient. Follow-upstudies of patients with a Blalock-Taussig shunt havebeen conducted mainly from a cardiological per-spective.7 There is, as far as we know, no publishedlong-term evaluation of this population from a spe-cific neurological viewpoint. Such a study is requiredto better define the late cerebrovascular risk poten-tially associated with a history of Blalock-Taussigshunt.

References1. Blalock A, Taussig HB. The surgical treatment of the heart in

which there is pulmonary stenosis or pulmonary atresia. JAMA1945;128:189–202.

2. Reivich M, Holling HE, Roberts B, Toole JF. Reversal of bloodflow through the vertebral artery and its effect on cerebral circu-lation. N Engl J Med 1961;265:878–885.

3. Folger GM, Shah KD. Subclavian steal in patients with Blalock-Taussig anastomosis. Circulation 1965;31:241–248.

4. Naito H, Kurokawa K, Kanno T, et al. Status epilepticus andcortical blindness due to subclavian steal syndrome in a girl withBlalock’s operation. Surg Neurol 1973;1:46–49.

5. Sokol S, Narkiewicz M, Billewicz O. Subclavian steal syndromeafter Blalock-Taussig anastomoses. J Cardiovasc Surg (Torino)1969;10:350–354.

6. Kurlan R, Krall RL, Deweese JA. Vertebrobasilar ischemia aftertotal repair of tetralogy of Fallot: significance of subclavian stealcreated by Blalock-Taussig anastomosis. Stroke 1984;15:359–362.

7. Murphy JG, Gersh BJ, Mair DD, et al. Long-term outcome inpatients undergoing surgical repair of tetralogy of Fallot. N EnglJ Med 1993;329:593–599.

Cerebral X-LinkedAdrenoleukodystrophyin a Girl withXq27-Ter DeletionEli Hershkovitz, MD,1 Ginat Narkis, BA,2

Zamir Shorer, MD,1 Ann B. Moser, BA,3

Paul A. Watkins, MD, PhD,3 Hugo W. Moser, MD,3

and Esther Manor, PhD2

An 8.5-year-old girl with a pathogenic mutation(515insC) of the ATP-binding cassette, subfamily D,member 1 gene (ABCD1) on her maternally derived Xchromosome showed clinical, biochemical, and magneticresonance imaging abnormalities similar to those in af-fected males. Cytogenetic studies led to the surprise find-ing of a de novo deletion of Xq27 on the paternally de-rived X chromosome. A bone marrow transplant had anapparently favorable effect. Cytogenetic studies should beperformed in all severely symptomatic X-linked adreno-leukodystrophy heterozygotes.

Ann Neurol 2002;52:234–237

X-linked adrenoleukodystrophy (X-ALD) is a progressivedisorder that affects myelin because of a defect in thegene for the adenosine triphosphate (ATP)-binding cas-sette, subfamily D, member 1 (ABCD1).1 ABCD1 is lo-cated on Xq28. It codes for ALD protein (ALDP),2 a

From the 1Pediatric Department and 2Genetic Institute, SorokaUniversity Medical Centre, Faculty of Health Sciences, Ben-GurionUniversity of the Negev, Beer Sheva, Israel, and 3Kennedy KriegerInstitute, Johns Hopkins University, Baltimore, MD.

Received Dec 14, 2001, and in revised form Mar 12, 2002. Ac-cepted for publication Mar 12, 2002.


Address correspondence to Dr Hershkovitz, Pediatric Department,Soroka University Medical Centre, POB 151, Beer Sheva 84101,Israel. E-mail: [email protected]


peroxisomal membrane protein that belongs to the ATP-binding cassette superfamily of membrane transport pro-teins.3 X-ALD is associated with the accumulation ofsaturated, very long chain fatty acid in tissues, culturedskin fibroblasts,4 and plasma.5 In males, phenotypic ex-pression ranges from the severe childhood or adolescentcerebral forms, which are associated with a white matterinflammatory reaction,6 to slowly progressive adrenomy-eloneuropathy, which manifests most commonly inyoung adults and mainly affects the long tracts in thespinal cord, with the inflammatory reaction mild or ab-sent.7 Approximately 70% of affected males have pri-mary adrenocortical insufficiency. Adrenal steroid ther-apy is effective for the endocrine dysfunction but doesnot appear to alter neurological progression. Bone mar-row transplantation (BMT) can be of long-term benefitto boys and adolescents with the cerebral forms of thedisease when provided at a time when brain involvementis still relatively mild.8 There is no specific therapy foradrenomyeloneuropathy.

Approximately 50% of women who are heterozygousfor X-ALD develop a progressive syndrome that resem-bles adrenomyeloneuropathy but is milder and usuallydoes not manifest until 35 years of age or later. Adre-nocortical insufficiency is rare. Approximately 1% ofheterozygous women have a more severe phenotype, inwhich there is progressive cerebral involvement, adre-nocortical insufficiency, and earlier onset9 and whichresembles what is seen in boys or adolescents with thecerebral phenotypes (H.W.M., unpublished observa-tion).

The cause of the severe manifestations in this smallproportion of heterozygous women has not been deter-mined definitively. Skewed inactivation of the normalX chromosome has been considered to be the mostlikely explanation.9,10 X-inactivation patterns in cul-tured skin fibroblasts or white blood cells can be as-sessed by an examination of the expression of ALDP byimmunocytochemical techniques11 in women who aremembers of families in which affected males are knownto lack immunoreactive material. In 15 heterozygouswomen, 23 to 86% of cells expressed ALDP.12 In con-trast, ALDP was expressed in only 0 to 2% of cellsfrom 2 women who had the adolescent cerebral phe-notype,10 suggesting that in these patients the normalX chromosome failed to exert its protective effect. Thisconclusion must be considered tentative because find-ings in fibroblasts may not reflect those in the nervoussystem. We now report a patient with the severe phe-notype who was known to be heterozygous for X-ALDbecause of a pathogenic mutation in the maternal cellline but who had in addition a deletion of Xq27-ter inthe paternal chromosome, thereby rendering her totallydeficient in ALDP.

Case ReportThe patient is the first child of unrelated parents. Two ma-ternal uncles have X-ALD, 1 with the adolescent cerebralphenotype and the other with the adrenomyeloneuropathyphenotype. Pregnancy and early development were normal.She started to walk at 14 months and to speak at 1 year.Several behavioral problems were noted in childhood. Theseincluded separation anxiety, with refusal to stay alone in thekindergarten and school; oversensitivity to noises; and isola-tion from peers and kindergarten staff. She continued to de-velop neurologically in agreement with her age, and shestarted to read at 5 to 6 years of age. At that time, she wasreferred to formal psychological assessment because of“school phobia,” but no diagnosis was made at that time.The deterioration of her academic performance and behaviorwas noted at 8.5 years. Physical and neurological examina-tions were normal. Formal psychological assessment (Wech-sler intelligence scale for children-revised) showed that shefunctioned at the low normal range. The verbal intelligencequotient was 87, the performance intelligence quotient was83, and the total intelligence quotient was 83. Her achieve-ments were adversely affected by easy distractibility andvisuospatial defects. Cortisol response to 1�g of intravenousadrenocorticotropic hormone was normal at 30 minutes(26.2�g/dl), ruling out clinically significant adrenal insuffi-ciency. Brain magnetic resonance imaging demonstrated dif-fuse white matter involvement that was most prominent inthe frontal regions.

ResultsBiochemical and genetic studies were performed afterinformed consent had been obtained from the parents.Levels of very long chain fatty acid in plasma and cul-tured skin fibroblasts were measured by capillary gas-liquid chromatography4,5 and found to be elevated(Table 1). DNA analysis of the patient’s white bloodcells and her mother’s white blood cells showed a cy-tosine insertion in codon 515 (515insC) resulting in aframeshift after amino acid 171 (tyrosine). Immunocy-tochemical studies of the cultured skin fibroblasts11

showed that ALDP-reactive material was lacking in99% of the patient’s cells (100 cells counted) and in allof the cells of the affected maternal uncles. Cytogeneticanalyses showed a deletion at Xq27.23ter in the pa-tient’s peripheral blood lymphocytes. Her karyotypewas 46X;Xdel(q27-tel) (data not shown). Both parents’karyotype analyses were normal. A deletion of the dis-tal part of Xq, including the telomeric region in thepatient’s fibroblasts, also was observed by fluorescencein situ hybridization with a specific Xq telomericprobe. This region was present in the X chromosomesof both parents (data not shown). DNA analysis with23 polymorphic markers spanning the q26-28 regionof the patient’s and her parents’ X chromosomes wasperformed to define the extent of the deletion, usingmethods that have been described previously.13 Ananalysis of 13 informative markers showed that a denovo deletion had occurred in the paternal chromo-

Hershkovitz et al: Cerebral X-ALD 235

some. The deleted region is distal to marker DXS1227,which maps to Xq27.2 (Table 2). Therefore, the de-leted segment spans part of Xq27.2 and all of Xq27.3and Xq28.

At 8.9 years of age, she received a bone marrowtransplant. Her human leukocyte antigen–identicalnormal younger sister was the donor. The procedurewas tolerated well. Full engraftment was documented.Although some deterioration in concentration andshort-term memory were noticed during the early pe-riod after transplantation, her neurological and neuro-psychological status were stable 18 months after BMT.

DiscussionThe abnormal magnetic resonance imaging results andmoderately abnormal neuropsychological dysfunctionin this 8.5-year-old girl known to be heterozygous forX-ALD on the basis of family history, mutation anal-ysis, and very long chain fatty acid studies indicate thatshe is among the few heterozygotes with a severe phe-notype similar to that in boys with the cerebral formsof the disease. Mutation analysis indicates that the ma-ternal side of her family has a mutation that abolishesthe expression of ALDP. Studies in other heterozygoteshave led to the hypothesis that severe disability, such ascerebral involvement in childhood, is caused by skewedX inactivation,9,10 and the absence of ALDP immuno-reactive material would be compatible with this. How-ever, cytogenetic studies led to a different conclusion,namely, the unexpected demonstration of a de novodeletion in her paternal X chromosome that involvesall of Xq28 and part of Xq27. Combined with the ab-normality on the maternal X chromosome, this leads tofailure to express functional ALDP, similar to what oc-curs in affected males. Cytogenetic studies are per-formed only rarely in patients with X-ALD. To ourknowledge, this type of abnormality has not been re-ported before in women heterozygous for X-ALD. The

finding is of practical significance because under thesecircumstances therapeutic approaches are similar tothose that would be used in affected males. Given thisreasoning and because the patient met current criteriafor BMT in affected males,8 we decided to perform thetransplant. Her condition was stable 18 months later,and we hope that her long-term outcome will be favor-able, as has been the case for male patients.8

We recommend that cytogenetic studies be per-formed in the approximately 1% of heterozygotes whoshow evidence of cerebral involvement by magnetic res-onance imaging. A karyotype study should be the firststep. A normal result should be followed by a searchfor microdeletions in chromosome X with specificDNA markers spanning the ALD gene region. This in-formation is clinically significant because patients withcerebral involvement may be candidates for BMT. Atthis time, BMT is considered only for patients whohave evidence of cerebral involvement.8 It is never rec-ommended for males or females who have spinal cord

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 �1C24:0/C22:0 0.91 ND ND 0.68 � 0.15C26:0/C22:0 0.043 ND ND 0.018 � 0.009

Cultured skin fibroblastsb

C22:0 0.762 0.64 � 0.32 0.72 � 0.26 0.90 � 0.50C26:0 0.434 0.41 � 0.15 0.27 � 0.17 0.09 � 0.07C26:0/C22:0 0.457 0.69 � 0.19 0.40 � 0.23 0.08 � 0.03

The plasma assays were performed at the Sharee Zedek Hospital in Jerusalem (Dr Orly Elpeleg) and those in fibroblasts at the Kennedy KriegerInstitute. The C26:0 levels in fibroblasts are similar to those of X-ALD male, and the C26:0/C22:0 ratios in fibroblasts are intermediatebetween 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 � not done; X-ALD � X-linked adrenoleukodystrophy.

Table 2. Haplotype DNA Analysis of Xq26-28 UsingInformative DNA Markers

Marker BandMaternal

AllelePaternal

Allele

DXS8098 Xq26.1 Present PresentDXS1047 Xq26.3 Present PresentDXS994 Xq26.3 Present PresentDXS8050 Xq26.3 Present PresentDXS8072 Xq26.3 Present PresentDXS1062 Xq27.1 Present PresentDXS8094 Xq27.1 Present PresentDXS1192 Xq27.2 Present PresentDXS1227 Xq27.2 Present AbsentDXS8084 Xq27.3 Present AbsentDXS8043 Xq27.3 Present AbsentDXS1200 Xq27.3 Present AbsentDXS8091 Xq28 Present Absent


involvement only. For female patients with cerebral in-volvement, BMT would be recommended only forthose in whom both alleles are involved, thereby mak-ing them equivalent to hemizygotes. We do not believethat BMT would be indicated for women who do have1 normal allele, where the pathogenesis of brain in-volvement is unknown (although skewed inactivation isthe most likely cause), and we do not believe that insuch patients the risk-benefit ratio would warrantBMT. Although it might be of some interest to docytogenetic studies on all heterozygotes, the added ex-pense is not warranted in women without cerebral in-volvement.

References1. Moser HW, Smith KD, Watkins PA, et al. X-linked adreno-

leukodystrophy. In: Scriver CR, Beaudet AL, Sly WS, Valle D,eds. The metabolic and molecular bases of inherited disease. 8thed. New York: McGraw-Hill, 2001:3257–3301.

2. Mosser J, Douar AM, Sarde CO, et al. Putative X-linked adre-noleukodystrophy gene shares unexpected homology with ABCtransporters. Nature 1993;361:726–730.

3. Higgins CF, Gallagher MP, Mimmack ML, Pearce SR. A fam-ily of closely related ATP-binding subunits from prokaryoticand eukaryotic cells. Bioessays 1988;8:111–116. fs

4. Moser HW, Moser AB, Kawamura N, et al. Adrenoleuko-dystrophy: elevated C26 fatty acid in cultured skin fibroblasts.Ann Neurol 1980;7:542–549.

5. Moser AB, Kreiter N, Bezman L, et al. Plasma very long chainfatty acids in 3,000 peroxisome disease patients and 29,000controls. Ann Neurol 1999;45:100–110.

6. Powers JM, Liu Y, Moser AB, Moser HW. The inflammatorymyelinopathy of adreno-leukodystrophy: cells, effector mole-cules, and pathogenetic implications. J Neuropathol Exp Neu-rol 1992;51:630–643.

7. Powers JM, DeCiero DP, Ito M, et al. Adrenomyeloneu-ropathy: a neuropathologic review featuring its noninflamma-tory myelopathy. J Neuropathol Exp Neurol 2000;59:89–102.

8. Shapiro E, Krivit W, Lockman L, et al. Long-term beneficialeffect of bone marrow transplantation for childhood onset ce-rebral X-linked adrenoleukodystrophy. Lancet 2000;356:713–718.

9. Heffungs W, Hameister H, Ropers HH. Addison disease andcerebral sclerosis in an apparently heterozygous girl: evidencefor inactivation of the adrenoleukodystrophy locus. Clin Genet1980;18:184–188.

10. Naidu S, Washington C, Thirumalai S, et al. X-chromosomeinactivation in symptomatic heterozygotes in X-linked adreno-leukodystrophy. Ann Neurol 1997;42:498 (Abstract).

11. Watkins PA, Gould SJ, Smith MA, et al. Altered expression ofALDP in X-linked adrenoleukodystrophy. Am J Hum Genet1995;57:292–301.

12. Feigenbaum V, Lombard-Platet G, Guidoux S, et al. Muta-tional and protein analysis of patients and heterozygous womenwith X-linked adrenoleukodystrophy. Am J Hum Genet 1996;58:1135–1144.

13. Parvari R, Mumm S, Galil A, et al. Deletion of 8.5 Mb, in-cluding the FMR1 gene, in a male with the fragile X syndromephenotype and overgrowth. Am J Med Genet 1999;83:302–307.

A Novel Mutation in theDeoxyguanosine KinaseGene Causing Depletion ofMitochondrial DNAJan-Willem Taanman, PhD,1 Ihab Kateeb, MD,2

Ania C. Muntau, MD,3 Michaela Jaksch, MD,4

Nadine Cohen, MD,2 and Hanna Mandel, MD5

Recently, a homozygous single-nucleotide deletion inexon 2 of the deoxyguanosine kinase gene (DGUOK) wasidentified as the disease-causing mutation in 3 apparentlyunrelated Israeli-Druze families with depleted hepatoce-rebral mitochondrial DNA. We have discovered a novelhomozygous nonsense mutation in exon 3 of DGUOK(313C3T) from a patient born to nonconsanguineousGerman parents. This finding shows that mutations inDGUOK causing mitochondrial DNA depletion are notconfined to a single ethnic group.

Ann Neurol 2002;52:237–239

In 1991, Moraes and colleagues1 identified a group ofinfants with marked depletion of mitochondrial DNA(mtDNA) in association with defective mitochondrialrespiratory chain function. This condition, often calledmtDNA depletion syndrome (Online Mendelian Inheri-tance in Man [OMIM] 251880), now has been de-scribed for more than 50 patients;2–11 this suggests thatit may be an important cause of mitochondrial dys-function in neonates and infants. Most of the reportedpatients present in the neonatal period with muscleweakness, liver failure, and neurological abnormalitiesassociated with lactic acidemia and die before 12

From the 1University Department of Clinical Neurosciences, RoyalFree and University College Medical School, University CollegeLondon, London, United Kingdom; 2Department of Genetics,Tamkin Human Molecular Genetics Research Facility, Technion-Israel Institute of Technology, Bruce Rappaport Faculty of Medi-cine, Haifa, Israel; 3Dr von Hauner Children’s Hospital, Ludwig-Maximilians-Universitat, Munich, Germany; 4Stoffwechselzentrumund Institut fur Klinische-Chemie, Krankenhaus Munchen-Schwabing, Munich, Germany; and 5Metabolic Disease Unit, De-partment of Pediatrics, Rambam Medical Center, Technion-IsraelInstitute of Technology, Bruce Rappaport Faculty of Medicine,Haifa, Israel.



Address correspondence to Dr Taanman, University Department ofClinical Neurosciences, Royal Free and University College MedicalSchool, University College London, Rowland Hill Street, LondonNW3 2PF, United Kingdom. E-mail: [email protected]


months of age. Others present in infancy with isolatedmyopathy associated with motor regression or a slowlyprogressive encephalomyopathy. Inheritance appears tobe autosomal recessive, suggesting that nuclear gene de-fects are responsible for mtDNA depletion. In agree-ment with this notion, cybrid studies have shown thatthe defect is not transmitted by patient mtDNA,3,12

and partial sequencing of patient mtDNA has failed toidentify mutations.1,4

Recently, homozygosity mapping followed by candi-date gene sequence analysis in 3 kindreds of Israeli-Druze origin with the early-onset hepatocerebral vari-ant of the disease showed a homozygous mutation inthe gene for deoxyguanosine kinase (DGUOK) onchromosome 2p13.13 In addition, mutations in thegene for mitochondrial thymidine kinase (TK2) onchromosome 16q22 were identified in patients withthe late-onset muscle-specific variant of the disease.14

Deoxyguanosine kinase is responsible for phosphoryla-tion of purine deoxyribonucleosides in the mitochon-drial matrix compartment.15 The 3 apparently unre-lated Israeli-Druze families carried the same single-nucleotide deletion in exon 2 of DGUOK.13 Thismutation is expected to result in premature termina-tion of translation. To investigate whether mutationsin DGUOK are also present in patients from a differentethnic origin, we sequenced the exons and intron/exonboundaries of DGUOK from a German patient withthe hepatocerebral form of the disease. The patient wasfound to harbor a novel homozygous nonsense muta-tion in DGUOK. This shows that mutations inDGUOK causing mtDNA depletion are not restrictedto patients belonging to one particular ethnic group.

Case ReportThe clinical details of the patient and histochemical findingsare documented in detail elsewhere.11 In brief, the patientwas the first child of healthy, nonconsanguineous Germanparents (birth weight, 2,570gm). The boy presented with lac-tic acidosis, hepatomegaly, hypoglycemia, and jaundiceshortly after birth. He had a severe encephalopathy, charac-terized by marked muscle hypotonia, hyperreflexia, irritabil-ity, and pendular horizontal nystagmus. At the age of 2months, neonatal giant cell hepatitis was diagnosed by lightmicroscopy. Electron microscopy of the liver showed an ac-cumulation of abnormal mitochondria and steatosis. Histo-chemistry and immunohistochemistry for cytochrome-c oxi-dase demonstrated a mosaic pattern of normal and deficienthepatocytes. Skeletal muscle was normal on both light andelectron microscopy, but biochemical assays showed a minorcytochrome-c oxidase deficiency. Southern blot analysis ofliver biopsies, taken at 2 and 3 months of age, showed thatmtDNA was of normal size, but mtDNA levels were only 17and 18%, respectively, of the mean of 6 age-matched controlspecimens (range controls, 66–140%).11 The patient died ofhepatic failure at the age of 5 months. A younger brothershows similar symptoms.

After informed parental consent, in accordance with theguidelines of the local institution, DNA was extracted fromliver of the proband, blood of the parents, and cultured fi-broblasts of the second child.6 All exons of DGUOK wereamplified by polymerase chain reaction, purified, and se-quenced exactly as described earlier.13 Sequencing was per-formed in both directions. The human DGUOK messengerRNA sequence, with accession number U41668, and the hu-man chromosome 2 working draft sequence segment, withaccession number NT025651, were used to determine theintron/exon structure of DGUOK.

ResultsSequencing of the 7 exons and the intron/exon bound-aries of the DGUOK gene from the proband showed ahomozygous C3T transition at nucleotide position313 (numbering according to Johansson and Karls-son16) of exon 3. The same homozygous mutation wasfound in the clinically affected sibling. Both parentswere heterozygous for the mutation (Fig). The muta-tion changes the arginine CGA codon 105 into a TGAstop codon (see Fig). This base change is predicted toresult in a 173–amino acid residue truncation at the Cterminus of the DGUOK protein product. The muta-tion was not found in 15 control subjects of Europeanorigin.

DiscussionIn an earlier study, we identified an infant, born tohealthy, nonconsanguineous German parents, withearly-onset encephalopathy and rapidly progressingliver failure associated with severely depleted levels ofmtDNA.11 The recent discovery of a single-nucleotidedeletion in exon 2 of DGUOK in 3 unrelated Israeli-Druze families with depleted hepatocerebral mtDNAprompted us to screen the German patient for muta-tions in the gene. We found a homozygous nonsensemutation in exon 3 of the proband and his affectedyounger brother, whereas both parents were carriers.The mutation will lead to a shortening of the DGUOKprotein product by more than half. The truncation in-cludes 3 domains that are evolutionarily conserved be-tween nucleoside kinases and are thought to be essen-tial for catalysis.16 It is, therefore, highly unlikely thatthe mutated DGUOK gene is still functionally active inthe patient. The absence of the mutation in controlsubjects of European origin further supports the patho-genicity of the mutation.

The mutation in the DGUOK gene that we identi-fied in a German family indicates that mutations inDGUOK causing mtDNA depletion are not limited toIsraeli-Druze patients. However, mutation screening ofDGUOK in 11 additional families with early-onset en-cephalopathy, liver failure, and mtDNA depletion ofBritish (5 patients), Greek-Cypriot (3 patients), Ger-man (1 patient), French (1 patient), and Turkish (1


patient) origin, including the 6 families described by uspreviously,3,6,8 did not show any mutations in the 7exons and the intron/exon boundaries of DGUOK (notshown). The clinical, biochemical, and molecular find-ings of these patients were not markedly different fromthe case described here. These results suggest that mu-tations in the coding region of DGUOK are not com-mon in patients with hepatocerebral mtDNA deple-tion. Although the disease may be geneticallyheterogeneous, our results do not rule out that these 11families carry mutations elsewhere in DGUOK that af-fect gene expression. This possibility is likely in 2 ad-ditional families of Druze and Moroccan origin, re-spectively, in which the disease has been linked toDGUOK but in which mutations in the coding regionof the gene could not be identified.13 More detailedmutation analysis and expression studies of DGUOK,therefore, are necessary to investigate the genetic heter-ogeneity of hepatocerebral mtDNA depletion.

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 EliasFund/Technion VPR Fund (grant 181-421, H.M.).

We thank Dr A. H. V. Schapira and Dr J. V. Leonard for helpfuldiscussions.

References1. Moraes CT, Shanske S, Trischler H-J, et al. mtDNA depletion

with variable tissue expression: a novel genetic abnormality inmitochondrial diseases. Am J Hum Genet 1991;48:492–501.

2. Tritschler H-J, Andreetta F, Moraes CT, et al. Mitochondrialmyopathy of childhood associated with depletion of mitochon-drial 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 withmtDNA depletion. Am J Hum Genet 1993;53:663–669.

4. Mariotti C, Uziel G, Carrara F, et al. Early-onset encephalo-myopathy associated with tissue-specific mitochondrial DNAdepletion: a morphological, biochemical and molecular-geneticstudy. J Neurol 1995;242:547–556.

5. Macmillan CJ, Shoubridge EA. Mitochondrial DNA depletion:prevalence in a pediatric population referred for neurologicevaluation. Pediatr Neurol 1996;14:203–210.

6. Morris AAM, Taanman J-W, Blake J, et al. Liver failure asso-ciated with mitochondrial DNA depletion. J Hepatol 1998;28:556–563.

7. Vu TH, Sciacco M, Tanji K, et al. Clinical manifestations ofmitochondrial DNA depletion. Neurology 1998;50:1783–1790.

8. Blake JC, Taanman J-W, Morris AMM, et al. MitochondrialDNA depletion syndrome is expressed in amniotic fluid cellcultures. Am J Pathol 1999;155:67–70.

9. Barthele my C, Ogier de Baulny H, Diaz J, et al. Late-onsetmitochondrial DNA depletion: DNA copy number, multipledeletions, and compensation. Ann Neurol 2001;49:607–617.

10. Mandel H, Hartman C, Berkowitz D, et al. The hepatic mito-chondrial DNA depletion syndrome: ultrastructural changes inliver biopsies. Hepatology 2001;34:776–784.

11. Muller-Hocker J, Muntau AC, Schafer S, et al. Depletion ofmitochondrial DNA in the liver of an infant with neonatal gi-ant cell hepatitis. Hum Pathol 2002;33:247–253.

12. Taanman J-W, Bodnar AG, Cooper JM, et al. Molecular mech-anisms in mitochondrial DNA depletion syndrome. Hum MolGenet 1997;6:935–942.

13. Mandel H, Szargel R, Labay V, et al. The deoxyguanosine ki-nase gene is mutated in individuals with depleted hepatocere-bral mitochondrial DNA. Nat Genet 2001;29:337–341.

14. Saada A, Shaag A, Mandel H, et al. Mutant mitochondrial thy-midine kinase in mitochondrial DNA depletion myopathy. NatGenet 2001;29:342–344.

15. Jullig, M, Eriksson S. Mitochondrial and submitochondrial lo-calization of human deoxyguanosine kinase. Eur J Biochem2000;267:5466–5472.

16. Johansson M, Karlsson A. Cloning and expression of humandeoxyguanosine kinase cDNA. Proc Natl Acad Sci U S A 1996;93:7258–7262.

Fig. Electropherograms showing part of the exon 3 sequences ofthe deoxyguanosine kinase gene (DGUOK) of a control, bothparents, the proband (1st child), and his brother (2nd child).The position of the mutation is indicated with an arrow.Numbering is according to Johansson and Karlsson.16

Taanman et al: A Novel Mutation Depleting mtDNA 239

Dyskinesias and GripControl in Parkinson’sDisease Are Normalized byChronic Stimulation of theSubthalamic NucleusRoland Wenzelburger, MD,1 Bao-Rong Zhang, MD,1

Meike Poepping, MD,1 Bettina Schrader, MD,2

Dieter Muller, PhD,3 Florian Kopper, MD,1

Urban Fietzek, MD,1 Hubertus M. Mehdorn, PhD,2

Gunther Deuschl, PhD,1 and Paul Krack, PhD1

Deep-brain stimulation of the subthalamic nucleus ap-pears to reduce levodopa-induced dyskinesias, butwhether this effect is caused by the reduction of the totallevodopa ingestion or represents a direct effect on themotor system is unknown. Precision grip force of grasp-ing movements and levodopa-induced dyskinesias wasanalyzed in 10 parkinsonian patients before and after 3months of deep-brain stimulation of the subthalamic nu-cleus. Peak grip force was abnormally increased beforesurgery in the off-drug state and, particularly, in the on-drug state (sensitization). This grip force upregulationnormalized with chronic deep-brain stimulation in bothconditions (desensitization). Peak-dose dyskinesias alsoimproved, and off-dystonia was completely abolished.Mean dosage of dopaminergic drugs was reduced, butforce overflow and dyskinesias were equally improved in2 patients without a reduction. Despite the same singlelevodopa test dose, force excess and levodopa-induceddyskinesias were drastically reduced after 3 months ofdeep-brain stimulation of the subthalamic nucleus. Thisindicates that direct effects of deep-brain stimulation ofthe subthalamic nucleus on levodopa-induced dyskinesiasare likely to occur. Grip force overflow is a promisingparameter to study the desensitizing effect of chronicdeep-brain stimulation on levodopa-induced dyskinesias.

Ann Neurol 2002;52:240–243

L-Dopa–induced dyskinesias (LIDs) and motor fluctu-ations are major complications of therapy in long-standing Parkinson’s disease (PD). Once a patient de-velops LID, the sensitization to dopaminergictreatment cannot be reversed by drugs.1 Deep-brainstimulation of the subthalamic nucleus (STN DBS) isan effective treatment for both fluctuations and dyski-nesias in L-dopa–responsive PD.2,3 Its beneficial effectson akinesia, rigidity, and tremor resemble that ofL-dopa,4 except for LIDs, which are promoted by acuteSTN stimulation, whereas they improve with chronicstimulation.5 This substantial improvement of LID bySTN DBS even in the on-drug state (desensitization) isnot fully understood.2,3,6 It has been ascribed to a re-duction of dopaminergic dosage often allowed by themotor benefits of the procedure, but its pathophysiol-ogy has remained unclear. We followed the hypothesisthat an inhibition of the excess of command sent tothe muscle could underlie the antidyskinetic effects ofchronic STN DBS and therefore measured the calibra-tion of force in the precision grip, a function governedmainly by the motor cortex.7,8 An overshooting of gripforce in late-stage PD has been described in this con-dition. Patients often apply excessive force in the on-drug state when grasping to lift a small object.9–11

This force excess is closely related to the severity ofLID in PD12 and therefore is suited to objectivelystudy the beneficial effect of STN DBS on a surrogateparameter for LID. We also tried to elucidate if direct(related to stimulation) or indirect (related to reductionof dopaminergic drugs) effects account for therapeuticeffects.

Patients and MethodsWe studied a series of 10 advanced-stage patients with PDwho underwent STN DBS. All suffered from severe motorfluctuations and LID (Table). Written informed consent wasgiven by all patients. Ten age-matched healthy controls alsowere included in the study, which was approved by the localethics committee. The assessment was conducted after a 12-hour overnight withdrawal of dopaminergic drugs. Patientswere assessed in 2 treatment conditions before (off-drug andon-drug) and in 4 conditions 3 months after surgery (off-drug/off-stimulation, on-drug/off-stimulation, off-drug/on-stimulation, and on-drug/on-stimulation). A suprathresholddose of L-dopa was applied.4 The motor score of the UnifiedParkinson’s Disease Rating Scale (UPDRS) and a dyskinesiascore of 7 body regions (score range 0–28) were rated in allconditions.5 The amplitude of motor fluctuations was de-fined as the difference between UPDRS motor score off-drugand on-drug both before surgery and after surgery (on-stimulation). The L-dopa equivalent daily dose (LEDD) wascalculated as described previously.4 The subjects grasped anobject (220gm, equipped with 3-dimensional force sensors)between the thumb and index finger and lifted it 15 times ata natural pace. We focused on the peak grip force (GFPEAK),a measure with a proven sensitivity for LID.12 Mean values

From the Departments of 1Neurology and 2Neurosurgery, ChristianAlbrechts University, Kiel; and the 3Department of Neurosurgery,University Hamburg, Hamburg, Germany.



Dr Zhang’s current address is the Department of Neuology, SecondAffiliated Hospital, Zhejiang University, Hangzhou, P.R. China.

Address correspondence to Prof Dr Deuschl, Neurologische Klinikof the University Hospital Kiel, Niemannsweg 147, 24105 Kiel,Germany. E-mail: [email protected]


of GFPEAK of the thumb were calculated from the liftingtrials 6 to 15 of both sides (Fig 1).

Grip force was compared between groups and treatmentconditions by a general linear model (SPSS 10). Significancewas assumed for p values less than 0.05 after Bonferroni cor-rection. Clinical scores and LEDD were compared using theWilcoxon test with a p level of less than 0.01.

ResultsThe LEDD was reduced in all but 2 patients, on av-erage by 45% (p � 0.01). The UPDRS motor scorewas improved by �60% off-drug and by �32% on-drug. The amplitude of motor fluctuations decreasedby �80% (see Table).

Off-dystonia was abolished in all 9 patients involved.On-phase dyskinesias of the total body were completelyabolished in 1 patient, and the score was reduced in allthe other patients, on average by �67%. Dyskinesiastended to increase on combined challenge with L-dopaand STN DBS, but the mean score was still �44%lower than it was preoperatively.

Peak grip force (GFPEAK) was significantly influ-

enced by treatment. After 3 months of chronic stimu-lation, the force overflow vanished. No excess ofGFPEAK was observed any longer regardless of the stateof drug or stimulation, and the grip force was equiva-lent to controls (see Figs 1 and 2). The presurgical ex-cess of GFPEAK in the off-drug condition was abol-ished. In the on-drug state GFPEAK decreased by�110%. In the combined treatment state GFPEAK wasstill significantly lower than before surgery (see Fig 2).

A reduction of initially exaggerated GFPEAK was ob-served not only in the 8 patients with a reduced LEDDafter surgery, but also in the 2 patients on-stimulationwhose LEDD was not reduced because of remainingminor fluctuations and positive effects on mood. Inon-drug condition, the peak grip force decreased by�51% and by �48% in the first and second patient,respectively. The same was true in the off-drug state(GFPEAK, �45% and �25%). Thus, grip force excessresolved, although LEDD was not decreased in eitherof the patients. Furthermore, GFPEAK was not corre-lated significantly with LEDD, neither before nor after

Fig 1. Peak grip force (GFPEAK

in Newtons) in a healthy controlsubject and a representative pa-tient who suffered from severeL-Dopa–induced dyskinesia.Force overshooting in on-statewas abolished after chronic stim-ulation. (thick lines) Mean;(thin lines) standard error ofmean from 10 trials. Scale isidentical for all conditions.PD � Parkinson’s disease; N �Newton; S � seconds.

Table. Mean Scores of the UPDRS Motor Score and the Dyskinesias Score Before and 3 Months after Surgery

Condition Motor Score ( � SD) Dyskinesia Score ( � SD)

Before surgery Off drug 50.3 � 12.8 3.4 � 5.4On drug 20.4 � 6.3 9.1 � 5.6 Off-on 29.9 � 14.4

After surgery Off drug/off stim 42 � 17.2 0a

On drug/off stim 17.7 � 7.1 2.9 � 2.9c

Off drug/on stim 19.9 � 10.6b 2.9 � 1.9On drug/on stim 13.8 � 5.7c 4.9 � 4.2c

Off-on drug 6.1 � 7.4d

ap � 0.001, bp � 0.01, compared with presurgery off drug condition.cp � 0.01 compared with presurgery drug condition.dp � 0.01 compared with amplitude of motor fluctuations before surgery ( off-on drug, on stim).

SD � standard deviation; stim � stimulation.

Wenzelburger et al: Desensitization by DBS of the STN in PD 241

surgery (Spearman coefficient, � 0.4; p 0.05). Thereduction of GFPEAK closely matched the effect ofSTN DBS on peak-dose dyskinesias.

DiscussionWe observed a remarkable normalization of force cali-bration in dexterous movements induced by chronicSTN DBS which was found both during on-state andoff-state. The preoperative L-dopa challenge caused anovershooting of grip force in line with previous find-ings,11,12 but this was no longer the case in the post-operative challenge with the same dosage. The initialgrip force excess vanished almost completely underchronic STN DBS. In parallel with the reduction ofgrip force overshoot, the LID score during a suprath-reshold L-dopa challenge was reduced substantially,which was within the range expected from recent re-ports.2,3,5,6 These findings extend our earlier observa-tion that grip force excess in on-state is found in par-kinsonian patients with motor fluctuations only, andovershooting of force correlates with the severity ofLID.12 The close relationship between LID and forceregulation is now supported by similar benefits of STNDBS on both.

The reduction of LID in patients with STN DBShas been ascribed to a reduction of dopaminergic dos-age,3,5,6 and a direct effect of DBS has been discussedmainly for off-dystonia.5 These observations make thisview unlikely. We observed substantial benefits on LIDin all patients, regardless whether the dopaminergicdrugs were reduced. Furthermore, the LID-suppressingeffect of STN is not simply part of a general effect on

all the parkinsonian symptoms because switching offthe stimulator led to a reoccurrence of severe akinesiaand rigidity but left LID and force excess unchanged.Therefore, the LID-suppressing effect is more likely toreflect a desensitizing long-term effect of chronic STNDBS on LID and force regulation that outlasts even atemporary interruption of stimulation. The develop-ment of response fluctuations is believed to be causedby the discontinuous pharmacological stimulation ofthe dopamine-receptors by drug treatment. In contrast,STN DBS is continuously stimulating the motor sys-tem. We propose that such continuous stimulationmay explain desensitization of both LID and grip forceexcess. This desensitization of involuntary motor activ-ity might be explained by plastic changes in the motorsystem directly related to chronic STN DBS. It is notyet clear if this desensitization takes place within thebasal ganglia or the cortex. The profound changes ofcortical metabolism when comparing the on-state andthe off-state with functional imaging might support thelatter possibility but is far from proving this interpre-tation.

This research was supported by the Deutsche Forschungsgemeinss-chaft (01KO9811/7, R.W.) and the Kompetenznetzwerk Parkinson.B.-R.Z. was on sabbatical leave from the Department of Neurology,Second Affiliated Hospital, Zhejiang University, Hangzhou, P.R.China, and was supported by the Kiel University.

We thank Mrs Witt for the excellent support of this investigation,Dr Johansson and Mr Backstrom for advice concerning the grip

Fig 2. Mean peak grip force(GFPEAK � SD) before and 3months after surgery. The over-shooting of force was abolishedin all conditions after surgery.(single dagger) p � 0.05,(double dagger) p � 0.01 com-pared with on-drug state beforesurgery. (asterisk) p � 0.05compared with presurgical off-drug state. (single circle) p �0.05, (double circle) p � 0.01compared with controls.


paradigm and the SC/ZOOM software, and Dr Pohl for valuablehelp with the statistics.

References1. Nutt JG. Clinical pharmacology of levodopa-induced dyskine-

sia. Ann Neurol 2000;47(suppl 1):S160–S164; discussion,S164–S166.

2. Limousin P, Krack P, Pollak P, et al. Electrical stimulation ofthe subthalamic nucleus in advanced Parkinson’s disease.N Engl J Med 1998;339:1105–1111.

3. Krack P, Limousin P, Benabid AL, Pollak P. Chronic stimula-tion of subthalamic nucleus improves levodopa-induced dyski-nesias in Parkinson’s disease. Lancet 1997;350:1676.

4. Krack P, Pollak P, Limousin P, et al. Subthalamic nucleus orinternal pallidal stimulation in young onset Parkinson’s disease.Brain 1998;121:451–457.

5. Krack P, Pollak P, Limousin P, et al. From off-period dystoniato peak-dose chorea. The clinical spectrum of varying subtha-lamic nucleus activity. Brain 1999;122:1133–1146.

6. Bejjani BP, Arnulf I, Demeret S, et al. Levodopa-induced dys-kinesias in Parkinson’s disease: is sensitization reversible? AnnNeurol 2000;47:655–658.

7. Jeannerod M. The formation of finger grip during prehension.A cortically mediated visuomotor pattern. Behav Brain Res1986;19:99–116.

8. Lemon RN, Johansson RS, Westling G. Corticospinal controlduring reach, grasp, and precision lift in man. J Neurosci 1995;15:6145–6156.

9. Gordon AM, Ingvarsson PE, Forssberg H. Anticipatory controlof manipulative forces in Parkinson’s disease. Exp Neurol 1997;145:477–488.

10. Fellows SJ, Noth J, Schwarz M. Precision grip and Parkinson’sdisease. Brain 1998;12:1771–1784.

11. Gordon AM, Reilmann R. Getting a grasp on research: doestreatment taint testing of parkinsonian patients? Brain 1999;122:1597–1598.

12. Wenzelburger R, Zhang BR, Pohle S, et al. Force overflow andlevodopa-induced dyskinesias in Parkinson’s disease. Brain2002;125:871–879.

Demonstration of AcuteIschemic Lesions in the FetalBrain by Diffusion MagneticResonance ImagingCristina Baldoli, MD,1 Andrea Righini, MD,2

Cecilia Parazzini, MD,2 Giuseppe Scotti, MD,1

and Fabio Triulzi, MD2

The possibility of detecting acute hypoxic-ischemic brainlesions by prenatal magnetic resonance imaging or ultra-sound is low. We present a case of a fetus with a vein ofGalen arteriovenous malformation in whom prenataldiffusion-weighted magnetic resonance imaging at 33weeks of gestation clearly detected cerebral acute isch-emic lesions, associated with remarkable decrease of theaverage apparent diffusion coefficient, whereas T2-weighted imaging was still not informative.

Ann Neurol 2002;52:243–246

Prenatal magnetic resonance imaging (MRI) is widelyused to confirm ultrasound findings in cases of com-plex fetal brain malformations.1 Prenatal MRI and ul-trasound also can evaluate the end-stage sequelae ofhypoxic-ischemic fetal brain damage2; however, thepossibility of detecting these lesions by prenatal MRIor ultrasound in the acute phase is low.3 Diffusion-weighted MRI (DWI) has been shown to be very sen-sitive in detecting hyperacute and acute hypoxic-ischemic brain damage in adults4 and in premature5 orterm6–10 newborns. We present the case of a fetus witha vein of Galen arteriovenous malformation (VGAM),in whom prenatal DWI at 33 weeks of gestation clearlydetected acute ischemic lesions within the brain,whereas T2-weighted imaging was still not informative.

Case ReportA 27-year-old pregnant woman was referred to our institu-tion at 33 weeks of gestation because of suspected fetalVGAM at Doppler ultrasonography. A prenatal MRI studyat 1.5T (Eclipse; Marconi, Cleveland, OH) using the flex

From the 1Neuroradiology Departments, Universita Salute e VitaIRCCS–San Raffaele and 2Istituti Clinici di Perfezionamento, Mi-lan, Italy.

Received Jan 25, 2002, and in revised form Mar 20. Accepted forpublication Mar 23, 2002.


Address correspondence to Dr Righini, Radiologia e Neuroradiolo-gia, Ospedale dei Bambini V. Buzzi, Via castelvetro 32, 20154 Mi-lan, Italy. E-mail: [email protected]


body surface coil and T2-weighted 5mm-thick single-shotfast spin-echo sequences was performed.

ResultsT2-weighted images confirmed the presence of theVGAM, which appeared as an interhemispheric flowvoid mass in the region of the vein of Galen, con-nected to the torcular. Some irregular and prominentvessels were noted around the third ventricle; they werecompatible with choroidal arteries feeding the malfor-mation. The brain parenchyma did not exhibit clearsignal alterations, and there was no ventricular enlarge-ment (Fig 1). Because hypoxic-ischemic brain lesionscan be associated with VGAM, a DWI acquisition wasperformed in the same occasion. The DWI study wasbased on an echo-planar 3-axis diffusion sensitized se-quence, and 5mm-thick slices were acquired at fetalbrain level (matrix, 128 � 128; field of view, 30cm;b-factor, 0–1,000sec/mm2). DWI showed areas of hy-perintense abnormal signal within the parietal and oc-cipital lobe of the right hemisphere, and there weresimilar findings in the left temporal lobe; these foci ofhigh signal encompassed not only the cortex, but alsopart of the adjacent subcortical and periventricularwhite matter (see Fig 1); in these areas, the rotationallyaveraged apparent diffusion coefficient (trace-ADC)was remarkably reduced (average value, 0.97; standarddeviation [SD], 0.038�m2/msec) both for apparently

normal frontal lobe areas (average value, 1.83; SD,0.18�m2/msec) or for literature data on ADC in pre-mature neonates.11,12

At 38 weeks of gestation, a cesarean section was per-formed, and a baby girl was delivered. She presentedwith severe muscular hypotonia and signs of moderateleft ventricle failure at Doppler ultrasonography(23mm end diastolic diameter and 41% ejection frac-tion), whereas she did not show significant polypnea orpulmonary edema at chest x-rays. Four days after birth,an MRI study showed severe diffuse atrophy of bothcerebral hemispheres with drastic enlargement of corti-cal liquoral spaces and ex vacuo dilatation of lateralventricles; MRI showed also severe general cortical ne-crosis, most of the cortex being reduced to a thin rim,and diffuse loss of white matter, which presented sev-eral areas of high T1 signal. These were probably a signof diffuse calcification, as they were too confluent to behemorrhagic (Fig 2). No DWI or fluid-attenuated in-version recovery sequences were performed on this oc-casion. These findings were compatible with the end-stage sequelae of severe ischemic insults.

DiscussionThis report shows that DWI studies of fetal brain arefeasible. This case highlights the potential of DWI indetecting 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 33weeks of gestation, showing no clear signal alterations within brain parenchima. The presence of the a vein of Galen arteriovenousmalformation (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 lobeof the right hemisphere (arrows). Some abnormal hyperintensity is visible also in the left temporal lobe (curved arrow). Both corti-cal and adjacent white matter areas are affected. (Bottom row) Corresponding trace–apparent diffusion coefficient (ADC) mapsshowing a clear ADC decrease in the same areas (arrows) of diffusion-weighted magnetic resonance imaging signal alterations.


confirming the well-known high accuracy of DWI inacute stroke diagnosis both in adults and in children.Although we cannot provide a direct demonstration ofthe ischemic nature of the lesions we detected, previousreports13–15 have extensively documented that ischemicand malacic lesions are the main complications that oc-cur in subjects with VGAM, in both the prenatal andpostnatal period. Moreover, the remarkable reduction inADC that we noticed in the lesions has been reportedonly in a few other brain diseases, such as encephalitis,mature abscess, and status epilepticus16; in our case, thelocation of the lesions, their evolution, and the clinicalcourse were poorly compatible with these conditions.

Three mechanisms have been postulated for thepathogenesis of the hypoxic-ischemic brain injuries de-tectable in VGAM patients15: a steal phenomenoncaused by blood shunting from the normal parenchy-mal arteries into those feeding the malformation; pa-renchymal hypoperfusion secondary to intracranial ve-nous hypertension and congestion; and multiple organhypoperfusion and hypoxia due to congestive heart fail-ure. One or more of these mechanisms, acting to-gether, might have produced the brain lesions that weobserved in the fetus. We believe that the lesions werecaused first by a steal phenomenon and second by avenous congestion mechanism, because the degree ofcardiac failure was not so clinically important afterbirth. For these reasons, it is likely that the lesions ofthe cortex and white matter were mainly ischemic (be-cause of reduce blood flow) rather than hypoxic (be-cause of arrival of less oxygenated blood). The postna-tal MRI showed diffuse cortical atrophy andleukomalacia, well beyond the areas of initial gray and

white matter DWI alteration. A possible explanationfor this discrepancy is that episodes of more extensivebrain hypoperfusion repeatedly occurred after the pre-natal MRI. The cerebral hemodynamics of our fetuswas probably unstable, and the intracranial blood flowautoregulation mechanism was weak, so the prenatalMRI provided a snapshot of still evolving and spread-ing damage. The end stage of the damage includedsigns of calcific degeneration of the residual white mat-ter at postnatal MRI; such calcifications are known todevelop within the brain of newborns who have suf-fered hypoxic-ischemic insults.17

The explanation for the ADC decrease observed inour case resembles what is commonly reported foracute ischemia in adult humans or animal models18,19;it is based on energy failure of the cell membrane, withdevelopment of cytotoxic edema and consequent rela-tive decrease of the extracellular water and increase ofthe intracellular one, which is more restricted and lessdiffusible. However, water diffusion in the brain differsaccording to maturity: the normal averaged ADC val-ues in the white matter of premature neonates aremuch higher (range, approximately 1.8–1.9�m2/msec)11,12 than those of adults (range, approximately0.7–0.9�m2/msec)12,20; this finding could be relatedto the higher water content, to the lower macromole-cules concentrations, or to the poorer neuronal andglial “packing” in developing and premyelinated braintissue.12,21 This difference might account also for theabnormal ADC variations that were observed in ourcase. We noticed an ADC decrease to an average valueof 0.97�m2/msec (SD, 0.038�m2/msec); similar ADCreductions have been reported recently in the acute

Fig 2. (Top row) Two T2-weighted axialsections (top left and top middle) and 1axial T1-weighted section (top right) fromthe postnatal magnetic resonance imaging(MRI) study showing diffuse severe atrophyof both hemispheres, lateral ventricles exvacuo dilatation, extensive white matterloss, and large areas of T1 hyperintensitywithin it, compatible with diffuse calcifi-cations (arrows). (Bottom row) Two sagit-tal T1-weighted sections from the postnatalMRI study showing diffuse drastic enlarge-ment of cortical liquoral spaces and thin-ning of corpus callosum. The presence ofabnormal choroidal arteries feeding theVGAM is confirmed (arrows).

Baldoli et al: Diffusion MRI of Fetal Brain Ischemia 245

stage of periventricular leukomalacia affecting prema-ture babies.5 These values are almost double thosecommonly reached by mean diffusivity decrease inacute ischemic lesions of adults.20 However, in thiscase and in those of premature babies, the approxi-mately 50% reduction in ADC for normal areas wassimilar to what has been observed with ischemia inadults. We hypothesize that the same tissular features(eg, water content, macromolecules concentrations,neuronal and glial “packing”), which produce the dif-ferences in ADC between the normal developing brainand adult brain, can play a role also in producing dif-ferent ADC value decreases in pathological conditions,such as ischemia. To better understand these aspects,we need to study more cases of acute fetal or prematureneonatal brain ischemia by using DWI, possibly corre-lating the results with neuropathological findings. Pre-vious work8,9 on timing of the ADC modifications af-ter acute perinatal hypoxic-ischemic damage suggeststhat a first ADC reduction can be noticed within 6 to72 hours after the acute event, with a possible delayedand more widespread reduction in the following days,when conventional MRI also shows signal alterationsdue to developed vasogenic edema. Although in ourcase, we present only 1 time point of the ADC changetime course, we hypothesize that we imaged mostly le-sions associated with the first ADC decrease, becauseprenatal T2-weighted images did not show significantsignal alterations.

Although the prenatal detection of VGAM is rare,the early diagnosis by DWI of any associated destruc-tive brain lesions can be important in therapeutic de-cision making, for example, in the decision to performtherapeutic intravascular procedures. Fetal brainhypoxic-ischemic damage also can occur in severalother conditions, such as twin-to-twin transfusion syn-drome, abruptio placentae, preeclampsia, severe mater-nal anemia, and severe hypovolemia. Under these cir-cumstances, prenatal DWI is a promising techniquethat can detect acute hypoxic-ischemic complications,which could influence clinical and parental decisions.

References1. Girard N, Raybaud C, Gambarelli D, et al. Fetal brain MR

imaging. Magn Reson Imaging Clin N Am 2001;9:19–56.2. De Laveaucoupet J, Audibert F, Guis F, et al. Fetal magnetic

resonance imaging (MRI) of ischemic brain injury. Prenat Di-agn 2001;21:729–736.

3. Langer B, Boudier E, Gasser B, et al. Antenatal diagnosis ofbrain damage in the survivor after second trimester death of amonochorionic monoamniotic co-twin. Fetal Diagn Ther 1997;12:286–291.

4. Warach S, Chien D, Li W, et al. Fast magnetic resonancediffusion-weighted imaging of acute human stroke. Neurology1992;42:1717–1723.

5. Inder T, Huppi PS, Zientara GP, et al. Early detection ofperiventricular leukomalacia by diffusion-weighted magneticresonance imaging techniques. J Pediatr 1999;134:631–634.

6. Cowan FM, Pennock JM, Hanrahan JD, et al. Early detectionof cerebral infarction and hypoxic-ischemic encephalopathy inneonates using diffusion-weighted magnetic resonance imaging.Neuropediatrics 1994;25:172–175.

7. Johnson AJ, Lee BC, Lin WL. Echoplanar diffusion-weightedimaging in neonates and infants with suspected hypoxic-ischemic injury: correlation with patient outcome. Am J Roent-genol 1999;172:219–226.

8. Robertson RL, Ben-Sira L, Barnes PD, et al. MR line scan dif-fusion weighted imaging of term neonates with perinatal brainischemia. AJNR Am J Neuroradiol 1999;20:1658–1670.

9. Soul JS, Robertson RL, Tzika AA, et al. Time course of changesin diffusion-weighted magnetic resonance imaging in a case ofneonatal encephalopathy with defined onset and duration ofhypoxic-ischemic insult. Pediatrics 2001;108:1211–1214.

10. Barkovich AJ, Westmark KD, Bedi HS, et al. Proton spectros-copy and diffusion imaging of the first day of life after perinatalasphyxia: preliminary report. AJNR Am J Neuroradiol 2001;22:1786–1794.

11. Huppi PS, Maier SE, Peled S, et al. Microstructural develop-ment of human newborn cerebral white matter assessed in vivoby diffusion tensor magnetic resonance imaging. Pediatr Res1998;44:584–590.

12. Tanner SF, Ramenghi LA, Ridgway JP, et al. Quantitativecomparison of intrabrain diffusion in adults and preterm andterm neonates and infants. AJR Am J Roentgenol 2000;174:1643–1649.

13. De Koning TJ, Gooskens R, Veenhoven R, et al. Arteriovenousmalformation of vein of Galen in three neonates: emphasis onassociated early ischaemic brain damage. Eur J Pediatr 1997;156:228–229.

14. Baeziger O, Martin E, Willi U, et al. Prenatal brain atrophydue to a giant vein of Galen malformation. Neuroradiology1993;35:105–106.

15. Brunelle F. Arteriovenous malformation of the vein of Galen inchildren. Pediatr Radiol 1997;27:501–513.

16. Righini A, Pierpaoli C, Alger JR, Di Chiro G. Brain paren-chyma apparent diffusion coefficient alterations associated withexperimental complex partial staus epilepticus. Magn Reson Im-aging 1994;12:865–871.

17. Ansari MQ, Chincanchan CA, Armstrong DL. Brain calcifica-tion in hypoxic-ischemic lesions: an autopsy review. PediatrNeurol 1990;6:94–101.

18. Pierpaoli C, Righini A, Linfante I, et al. Histopathologic cor-relates of abnormal water diffusion in cerebral ischemia: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 utilityof diffusion weighted magnetic resonance imaging in the assess-ment of ischemic stroke. Ann Neurol 1997;41:574–580.

20. Warach S, Gaa J, Siewert B, et al. Acute human stroke studiedby whole brain echo planar diffusion-weighted magnetic reso-nance imaging. Ann Neurol 1995;37:231–241.

21. Baratti C, Barnett AS, Pierpaoli C. Comparative MR imagingstudy of brain maturation in kittens with T1, T2, and the traceof the diffusion tensor. Radiology 1999;210:133–142.


Selective Loss of CholinergicSudomotor Fibers CausesAnhidrosis in RossSyndromeClaudia Sommer, MD,1 Thies Lindenlaub, MD,1

Detlef Zillikens, MD,2 Klaus V. Toyka, MD,1

and Markus Naumann, MD1

Ross syndrome consists of segmental hyperhidrosis withwidespread anhidrosis, Adie syndrome, and areflexia. Thecause of this disorder is unknown. Selective degenerationof cholinergic fibers or of neural crest–derived structureshas been suggested. We present clinical and skin biopsydata of 4 patients, providing evidence of reduced cholin-ergic sweat gland innervation in hypohidrotic skin bymorphometric analysis. These findings indicate a selectivedegenerative process of the cholinergic sudomotor neu-rons.

Ann Neurol 2002;52:247–250

The pathogenesis of Ross syndrome1 (tonic pupils,areflexia, and segmental hyperhidrosis) is as yet un-known. Wide overlap between Ross syndrome, Adiesyndrome, Harlequin syndrome (isolated progressivesegmental hypohidrosis), and a more widespread auto-nomic disease has been suggested.2 Here, we presentfindings from 4 patients with anhidrosis and segmentalhyperhidrosis and variable expression of Adie’s pupils,hyporeflexia, and pathological neurophysiological find-ings. Importantly, the underlying pathology was thesame in all patients, with reduction of cholinergicsweat gland innervation and normal epidermal andsubepidermal nerve fibers.

Patients and MethodsSkin biopsies were obtained from a hyperhidrotic and an an-hidrotic area of the patients’ back after informed consent.After fixation in 4% paraformaldehyde, 40�m frozen sec-tions were stained with antibodies to the panneuronal

marker protein gene product 9.5 (PGP 9.5, 1:800; Ultra-clone, Wellow, UK) and to vasoactive intestinal peptide(VIP, 1:500; Peninsula, San Carlos, CA). Immunofluores-cence was performed using Cy3 or Cy2-labeled secondaryantibodies (1:100; Amersham, Arlington Heights, IL). AnABC system (Vector, Burlingame, CA) was used with thesame primary antibodies to verify the staining. For analysisof skin morphology (hematoxylin and eosin stain) and im-mune mediators, 6�m-thick frozen sections were reactedwith primary antibodies to T cells (CD3, CD4, CD8,CD45RO), B cells (CD20), and macrophages (CD68) orfluorescein isothiocyanate–labeled antibodies to human im-munoglobulin (Ig) G, IgM, IgA, and complement compo-nent C3 (Dako, Hamburg, Germany). The sections wereviewed with a Zeiss Axiophot 2 (Zeiss, Gottingen, Germany)microscope equipped with internal z-focus and a motorizedscanning table by Marzhauser, Wetzlar, Germany. Using Im-age Pro Plus 4.0 software, we generated 3-dimensional re-constructions of the epidermis and the sweat glands. Epider-mal innervation was quantified by counting the nerveendings per millimeter of epidermal length and by determi-nation of the epidermal nerve area with optical densitometry.Nerve endings were required to have a visible length greaterthan 20�m to be considered. The subepidermal nerve plexuswas measured in an area of 100�m � 0.3mm along thesubepidermal/epidermal border. Sweat gland innervation wasquantified by measuring the complete nerve fiber area asso-ciated with sweat glands. Values from the ipsilateral and con-tralateral side were compared using Student t test; p values ofless than 0.05 were considered significant.

Neurophysiological Studies and Sudomotor TestingNerve conduction studies and somatosensory and magnetic-evoked potentials were performed using our standard neuro-physiological techniques.3 Hyperhidrotic and anhidrotic ar-eas were visualized using Minor’s iodine starch test.Quantitative sensory testing (QST) was performed using aPeltier device (Medoc thermal analyzer) and the methods oflimits.4 Heart rate variability was measured as described pre-viously.5

ResultsCase 1A 50-year-old man reported increased sweating on theright side of the trunk and on the left face for 18 years.Both hands and feet were dry. Physical examinationshowed hyperhidrosis in the right axilla, in the der-matomes T6 to T10 on the right and T11 to T12 onthe left, and on the left face and neck. Ankle jerks wereabsent. Nerve conduction studies were normal. TheH-reflex and the sympathetic skin response (SSR) onboth feet were absent. QST showed normal thresholdsin hyperhidrotic and anhidrotic areas of the back andon both hands, but increased warm thresholds on thedorsum of the right foot. The patient was treated with200 units of botulinum toxin (Botox�) in the hyper-hidrotic area in T6 to T10, with a 50% reduction insweat secretion within 4 days. Treatment was repeated

From the Departments of 1Neurology and 2Dermatology, Univer-sity of Wurzburg, Wurzburg, Germany.

Received Feb 21, 2002, and in revised form Mar 18. Accepted forpublication Mar 23, 2002.


Current address for Dr Lindenlaub: Neurologische Klinik, Univer-sitatskliniken des Saarlandes, 66424 Homburg, Germany.

Address correspondence to Dr Sommer, Neurologische Universita-tsklinik, Josef-Schneider-Strasse 11, D-97080 Wurzburg, Germany.E-mail: [email protected]


3 months later, with a good effect lasting for 4 months(Table 1).

Case 2A 49-year-old man had hyperhidrosis in the right T5to T7 dermatomes and in both knees for 7 years.Hands and feet did not sweat. The left pupil waswider, unreactive to light but had a tonic reaction toconvergence, consistent with Adie’s pupil. Tendonjerks were elicitable only after reinforcement; right an-kle jerk was absent. Latencies of somatosensory-evokedpotentials of both tibial nerves were delayed, and theamplitude of the sural nerve action potential wasslightly reduced. Further nerve conduction studies andSSRs (palms) were normal. The patient was injectedwith 1,000 units of botulinum toxin (Dysport) in thehyperhidrotic area of the trunk with marked reductionof sweating.

Case 3A 41-year-old woman had Adie’s pupils, areflexia, andhyperhidrosis in the left lower thoracic and glutealarea, and on the right ventral thigh and knee. The restof the body was anhidrotic. Hypoesthesia was presenton both lower legs; vibration thresholds were reducedat the ankles and knees. Heart rate variability was atthe lower limit of normal. Nerve conduction studiesshowed delayed latencies of somatosensory-evoked po-tentials of the right tibial nerve. Sensory nerve poten-tials of the sural nerve could not be elicited. SSR of theright hand was absent. The patient opted for localtreatment with aluminum chloride hexahydrate, whichyielded satisfactory results.

Case 4A 30-year-old man reported anisocoria since age 17years, hyperhidrosis on the right side of the trunk andthe left temporal region since age 23 years, and heatintolerance. He had Adie’s pupils, normal tendon jerks,and hyperhidrosis of the dermatomes T4 to T6 on the

right and in the inguinal area on the left. Palms andsoles were dry. The SSR was absent. The patient wastreated with 1,000 units of botulinum toxin (Dysport)in the hyperhidrotic area of the trunk. The area of ex-cessive sweating decreased by approximately 60% for 6months.

Skin Biopsy ResultsNo inflammatory infiltrates and no deposition of IgGor C3 were observed around sweat glands, which them-selves appeared normal. Using PGP 9.5 immunohisto-chemistry, we found that epidermal innervation andthe subepidermal plexus were normal, whereas sweatgland innervation was reduced in the anhidrotic areas(Fig, a–d). Immunoreactivity for VIP was present inthe fibers innervating the sweat glands, but not in theepidermal fibers, and was markedly reduced aroundsweat glands of the anhidrotic side (Fig, e and f).

Morphometry showed normal epidermal and subepi-dermal innervation density in hyperhidrotic and an-hidrotic regions. Sweat gland innervation was reducedsignificantly in the anhidrotic compared with the hy-perhidrotic regions (p � 0.005; Table 2).

DiscussionHere, we show selective loss of cholinergic sweat glandinnervation in 4 patients with Ross syndrome by usingthe panneuronal marker PGP 9.5 and a marker forcholinergic fibers, VIP.6 Because downregulation ofneuropeptides such as VIP may occur in nerve lesionsbefore degeneration of nerve fibers occurs, we usedPGP 9.5 for quantification of nerve fibers. Interest-ingly, epidermal innervation remained intact, well inaccordance with normal QST. Denervation as thecause of anhidrosis in patients with Ross syndrome hashitherto been suggested only on theoretical grounds.1

We previously presented qualitative data in another pa-tient with Ross syndrome, also in whom a selective re-duction of PGP 9.5 immunoreactive fibers aroundsweat glands could be found.7 Earlier skin biopsies

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

Caseno.

Age(yr) Gender

Duration ofSymptoms

(yr)Segmental

HyperhidrosisAdie’sPupils

TendonReflexes NCS/SEP SSR

HeartRate

Variability QST

1 50 M 18 T6–T10 RT11 L

No Ankle jerkmissing

Tibial nerve: H-reflexmissing

Absent Normal Normal

2 49 M 7 T5–T7 R Yes Ankle jerkmissing

Sural nerve: A reducedtSEP: A reduced

Normal Normal ND

3 41 F 6 T10–T12Gluteal LKnee L

Yes Areflexia Sural nerve: no responsetSEP: Lat delayed

Absent Normal ND

4 30 M 7 T4–T6 Yes Normal Normal Absent Normal Normal

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


from Ross syndrome patients were reported as normal;however, immunohistochemical methods to visualizeaxons were not used.8,9

Segmental anhidrosis and hyperhidrosis are often thepresenting symptoms in patients with Ross syndrome.Of our 4 patients with these symptoms, 3 had addi-

tional Adie’s pupils and 3 also had areflexia or hypore-flexia. Nerve conduction studies were abnormal in 2patients, and SSR was absent in 3 patients. Heart ratevariability was normal in 3 and at the lower level ofnormal in 1 patient. QST was normal in the 2 patientsin whom it was performed. Our observations are con-

Table 2. Morphometric Analysis of Skin Biopsies

Case no.

Epidermal Nerve Fibersper mm

Subepidemal Nerve Fiber Areaper mm2

Area of Fibers InnervatingSweat Glands per 1,000�m2

sweat gland area

Hyperhidrosis Anhidrosis Hyperhidrosis Anhidrosis Hyperhidrosis Anhidrosis

1 16.3 18.4 1266 1194 109.9 39.92 18.4 16.3 1165 1261 70.0 40.63 16.2 18.5 1209 1231 126.7 39.94 17.1 19.0 1356 1563 90.1 51.2Mean � SD 17.0 � 1.0 18.1 � 1.2 1249 � 82.4 1312.3 � 169.4 99.2 � 24.5 42.9a� 5.5

aSignificant difference from contralateral side, p � 0.005.

SD � standard deviation.

Fig. Forty-micrometer-thick frozen sectionsfrom skin biopsies of Patient 2, immuno-fluorescence, and secondary antibody Cy3.Sections from the hyperhidrotic side are onthe left (a, c, e) and from the anhidroticside on the right (b, d, f). (a, b) Immuno-reaction with antibodies to the panneuro-nal marker protein gene 9.5 (PGP 9.5)shows normal epidermal innervation inthe hyperhidrotic (a) and in the anhidrotic(b) skin. (c, d) PGP 9.5–immunoreactivefibers innervating sweat glands are abun-dant on the hyperhidrotic side (c), butdepleted on the anhidrotic side (d). (e, f)Immunohistochemistry for vasoactive intes-tinal peptide shows normal cholinergicsweat gland innervation on the hyper-hidrotic side (e) and loss of this innerva-tion on the anhidrotic side (f). Bar �50�m.

Sommer et al: Sudomotor Fiber Loss in Ross Syndrome 249

sistent with previous reports suggesting that segmentalanhidrosis is part of the spectrum of distinct auto-nomic disorders due to a generalized injury to auto-nomic and dorsal root ganglia neurons.2,10–12 It is un-known why these particular structures are involved.Both the ciliary nerves and the sympathetic innervationof sweat glands are cholinergic. However, a selectivedegeneration of cholinergic fibers does not explainareflexia, which has been suggested to be caused by lossof large diameter afferent fibers.13 Shin and colleaguessuggested a combined lesion in these structures due totheir derivation from the neural crest.2 Pigment cellsand Schwann cells also are derived from the neuralcrest. No Schwann cell pathology has been described inRoss syndrome so far to our knowledge, but we previ-ously described depigmentation in a patient with Rosssyndrome.7 The sympathetic innervation of sweatglands is unusual, because it is initially catecholamin-ergic but becomes cholinergic after interactions withthe target tissue. Catecholamines are necessary to in-duce secretory responsiveness of the sweat glands.14

The reason for the segmental hyperhidrosis in pa-tients with Ross syndrome has not been discovered yet.The hyperhidrosis is not likely to be efficient as a ther-moregulatory measure, and during treatment symp-toms of heat intolerance did not occur to any greaterdegree than before. The area of hyperhidrosis was al-ways localized in the lower thoracic to upper lumbarregion. Why these ganglia/fibers are spared from thedegenerative process is unclear. In sudomotor fibers ofthe rat, muscarinic M2 receptors are inhibitory presyn-aptic autoreceptors.15 The same function has been pos-tulated for these receptors in human skin.16 We thusspeculate that the cholinergic fibers innervating sweatglands first lose their M2 autoreceptors and that hyper-hidrosis is caused by diminished presynaptic inhibition,before further degeneration leads to anhidrosis.

This research was supported by research funds of the University ofWurzburg.

We thank Dr C. Rose for evaluating skin pathology and B. Dekantfor excellent technical help.

References1. Ross AT. Progressive selective sudomotor denervation. Neurol-

ogy 1958;8:809–817.2. Shin RK, Galetta SL, Ting TY, et al. Ross syndrome plus: be-

yond 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: aclinical 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.

5. Flachenecker P, Wermuth P, Hartung H-P, Reiners K. Quan-titative assessment of cardiovascular autonomic function inGuillain-Barre syndrome. Ann Neurol 1997;42:171–179.

6. Schutz B, Schafer MK, Eiden LE, Weihe E. VIP and NPY ex-pression during differentiation of cholinergic and noradrenergicsympathetic neurons. Ann N Y Acad Sci 1998;865:537–541.

7. Bergmann I, Dauphin M, Naumann M, et al. Selective degen-eration of sudomotor fibers in Ross syndrome and successfultreatment of compensatory hyperhidrosis with botulinum toxin.Muscle Nerve 1998;21:1790–1793.

8. Bartin RH, Schmutz JL, Cuny JF, et al. Le syndrome de Ross.A propos d’une observation. Ann Dermatol Venereol 1990;117:113–114.

9. Caparros-Lefebvre D, Hache JC, Hurtevent JF, et al. Unilateralloss of facial flushing and sweating with contralateral anhidrosis:harlequin syndrome or Adie’s syndrome? Clin Auton Res 1993;3:239–241.

10. Jacobson DM, Hiner BC. Asymptomatic autonomic and sweatdysfunction in patients with Adie’s syndrome. J Neuroophthal-mol 1998;18:143–147.

11. Bacon PJ, Smith SE. Cardiovascular and sweating dysfunctionin patients with Holmes-Adie syndrome. J Neurol NeurosurgPsychiatry 1993;56:1096–1102.

12. Drummond PD, Lance JW. Site of autonomic deficit in harle-quin syndrome: local autonomic failure affecting the arm andthe face. Ann Neurol 1993;34:814–819.

13. Pavesi G, Macaluso GM, Medici D, et al. On the cause oftendon areflexia in the Holmes-Adie syndrome. ElectromyogrClin Neurophysiol 1994;34:111–115.

14. Tian H, Habecker B, Guidry G, et al. Catecholamines are re-quired for the acquisition of secretory responsiveness by sweatglands. J Neurosci 2000;20:7362–7369.

15. Haberberger RV, Bodenbenner M. Immunohistochemical local-ization of muscarinic receptors (M2) in the rat skin. Cell TissueRes 2000;300:389–396.

16. Cavanah DK, Casale TB. Cutaneous responses toanticholinergics: evidence for muscarinic receptor subtype par-ticipation. J Allergy Clin Immunol 1991;87:971–976.


Normokalemic PeriodicParalysis Revisited: DoesIt Exist?Patrick F. Chinnery, PhD, MRCP,1

Timothy J. Walls, MD, FRCP,1

Michael G. Hanna, MD, MRCP,2

David Bates, MA, FRCP,1

and Peter R. W. Fawcett, BSc, FRCP3

Normokalemic periodic paralysis (normoKPP) is well es-tablished in the literature, but there are doubts as towhether it exists as a discrete entity. Retrospective clinicaland molecular analysis has confirmed suspicions thatmost normoKPP families actually have a variant of hy-perkalemic periodic paralysis (hyperKPP) due to a muta-tion of the muscle-specific sodium channel gene(SCN4A). However, the original normoKPP family de-scribed by Poskanzer and Kerr (Poskanzer DC, KerrDNS. A third type of periodic paralysis, with normokal-emia and favourable response to sodium chloride. Am JMed 1961;31:328–342) has remained unchallenged. Weidentified the Met1592Val mutation of SCN4A in an af-fected descendent of this original normoKPP family. Thisis the final piece in the puzzle: normoKPP is actually avariant of hyperKPP and is not a distinct disorder.

Ann Neurol 2002;52:251–252

Traditionally, three categories of periodic paralysis havebeen described: hypokalemic (hypoKPP), hyperkalemic(hyperKPP), and normokalemic (normoKPP).1 Thelast decade has seen major advances in our understand-ing of the molecular and electrophysiological basis of 2of these groups. Three point mutations in the skeletalmuscle dihydropyridine sensitive calcium channel(CACLN1A3) are responsible for most cases of hy-poKPP, and mutations in the muscle-specific sodiumchannel gene (SCN4A) have been identified in a spec-trum of disorders that includes hyperKPP, paramyoto-nia congenita, and potassium aggravated myotonia.1

Despite its prominence in standard texts, normoKPPhas been reported only in a few families.2–4 Clinicaland molecular reanalysis of some of the original cases

of normoKPP confirmed earlier suspicions that thesefamilies actually had a variant of hyperKPP due to aSCN4A gene mutation.1,5 However, normoKPP re-mains enshrined in standard medical textbooks largelybecause the original detailed clinical description of thedisorder by Poskanzer and Kerr2 in a family from thenortheast of England remains unchallenged.6 This fam-ily recently became the subject of study again when amember of the next generation developed periodic pa-ralysis, allowing us to revisit the original diagnosis.

Case ReportA 22-year-old man presented to the neurology department ofRoyal Victoria Infirmary, Newcastle Upon Tyne, because ofan increase in the frequency and severity of his periodic mus-cle weakness. His symptoms began at 2 years of age. He ex-perienced episodes of muscle weakness in all four limbs,sometimes associated with painful muscle stiffness. The epi-sodes typically were precipitated by cold and damp weatherand would begin during a period of rest after a period ofstrenuous exercise. There was no relationship with fasting orfood intake. A severe attack typically would last for severaldays, followed by a prolonged recovery phase taking days toweeks. Two attacks had been so severe that he remained inbed and could only lift his head off the pillow. Although heoccasionally experienced dysphagia during an attack, there hadnever been any respiratory symptoms, and he had not noticedany pigmenturia. His medical history included a camptodac-tyly correction to both hands but was otherwise unremarkable.There was no history to suggest cardiac dysrhythmias.

ResultsThere were no abnormal findings on systemic or neu-rological examination. Routine hematological and bio-chemical tests were normal (including serum electro-lytes and thyroid function tests), with the exception ofan elevated alanine transaminase (53 U/L, normal�45) and an elevated creatine kinase (807 U/L, nor-mal �190). A 12-lead electrocardiogram and chestx-ray were normal. A neurophysiological study showednormal peripheral nerve function, but several abnor-malities on concentric needle electromyography. Themost striking feature was the frequent myotonic dis-charges seen in every muscle sampled (Fig). There wasalso excess insertional activity and spontaneous activityin the form of fibrillation potentials and positive sharpwaves recorded from biceps brachii, indicating minimalmuscle fiber degeneration and early myopathic changes.Quantitative multi–motor unit potential analysis of themotor unit potentials in biceps showed normal motorunit potential durations, amplitudes, and number.

Family HistoryThe patient in this index case is the son of case V:10who was studied in detail by Poskanzer and Kerr.2

Molecular Genetic StudiesThe typical pattern of the episodes of weakness, themuscle stiffness, and the prominent myotonic dis-

From the 1Department of Neurology, University of Newcastle UponTyne, Newcastle Upon Tyne; 2Department of Neurology, Institute ofNeurology, London; and 3Department of Clinical Neurophysiology,Royal Victoria Infirmary, Newcastle Upon Tyne, United Kingdom.

Received Feb 15, 2002, and in revised form Mar 19, 2002. Ac-cepted for publication Mar 25, 2002.


Address correspondence to Dr Chinnery, Department of Neurology,The Medical School, Framlington Place, Newcastle Upon Tyne,NE2 4HH, United Kingdom. E-mail: [email protected].


charges on electromyography pointed toward a musclesodium channel disorder.1 Molecular genetic analysisby a standard polymerase chain reaction/restriction en-zyme analysis identified the Met1592Val mutation ofSCN4A in the case described above.

DiscussionWe have shown that the original normoKPP familystudied by Poskanzer and Kerr2 harbor a point muta-tion in SCN4A that is found in approximately 30% offamilies with hyperKPP.7 Molecular analysis of otherreported normoKPP families also has shown mutationsin SCN4A (Thr704Met).5 Based on these findings, ourconclusion is that it is highly likely that normoKPP isactually a variant of hyperKPP, and it should not beconsidered as a distinct disease entity.

In retrospect, the clinical and biochemical features ofthe original family of Poskanzer and Kerr are consistentwith a diagnosis of hyperKPP.2 Twenty-one individualswere reported in that study, each developing symptomsin their first decade. The individuals experienced at-tacks of weakness every 1 to 3 months, each lastingfrom 2 days to 3 weeks. The attacks were provoked byrest after exertion, cold and damp conditions, and al-cohol, particularly the local beer, which contained ahigh concentration of potassium. Oral potassium chlo-ride exacerbated the attacks of muscle weakness in 4members of the original family. Potassium sensitivity,the hallmark of muscle sodium channel disorders,1

therefore was clearly described in the original article.2

Although no significant change in serum potassiumwas ever documented during numerous spontaneous orprecipitated attacks of weakness, it is now well recog-nized that this can occur in patients with hyperKPP.1,7

Perhaps the most interesting feature of this family isthat before they received medical attention, they real-ized that a high intake of table salt reduced the fre-quency and severity of the attacks of muscle weakness.

The muscle weakness in patients with hyperKPP iscaused by muscle depolarization. In families withMet1592Val, this arises through impaired fast inactiva-tion 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 givenup to 1,750ml of triple normal saline over a 12-hourperiod. The mechanism responsible for this improve-ment is unclear, and neither the weakness nor the infu-sions were associated with a change in serum Na� orK� levels. Although no control experiments were con-ducted, this observation requires further investigationbecause of the possibility that an increased sodium loadmay also benefit other patients with hyperKPP.

The issue of treatment is highly pertinent to our in-dex case. Our patient’s father (V:10 in the originalstudy2) now has generalized muscle wasting and a se-vere fixed vacuolar myopathy limiting normal daily ac-tivities. His son is currently not weak between attacksbut has myopathic features on electromyography. Ourobjective is to prevent clinical progression in our newpatient, and, although unsubstantiated at present, it islogical to take steps to reduce the frequency and sever-ity of attacks. This could be achieved by alterations inhis behavior and diet, or using a carbonic anhydraseinhibitor such as dichlorphenamide.10

We are very grateful to Dr D. N. S. Kerr for his comments on theoriginal study and on this article.

References1. Ptacek LJ, Bendahhou S. Ion channel disorders of muscle. In:

Karpati G, Hilton-Jones D, Griggs RC, eds. Disorders of vol-untary muscle. 7th ed. Cambridge: Cambridge University Press,2001:604–635.

2. Poskanzer DC, Kerr DNS. A third type of periodic paralysis,with normokalemia and favourable response to sodium chlo-ride. Am J Med 1961;31:328–342.

3. Mayers KR, Gilden DH, Rinaldi CF, Hansen JL. Periodic mus-cle weakness, normokalemia, and tubular aggregates. Neurology1972;22:269–279.

4. Danowski TS, Fisher ER, Vidalon C, et al. Clinical and ultra-structural observations in a kindred with normo-hyperkalemicperiodic paralysis. J Med Genet 1975;12:20–28.

5. Lehmann-Horn F, Rudel R, Ricker K. Workshop report. Non-dystrophic myotonias and periodic paralyses. Neuromuscul Dis-ord 1993;3:161–168.

6. Rudel R, Lehmann-Horn F. Muscle sodium channel and chlo-ride channel diseases. In: Lane RJ, ed. Handbook of muscledisease. New York: Marcel Dekker, 1996:348.

7. Cannon SC. From mutation to myotonia in sodium channeldisorders. Neuromuscul Disord 1997;7:241–249.

8. Cannon SC, Brown RH Jr, Corey DP. A sodium channel de-fect in hyperkalemic periodic paralysis: potassium-induced fail-ure of inactivation. Neuron 1991;6:619–626.

9. Hayward LJ, Sandoval GM, Cannon SC. Defective slow inac-tivation of sodium channels contributes to familial periodic pa-ralysis. Neurology 1999;52:1447–1453.

10. Tawil R, McDermott MP, Brown R Jr, et al. Randomized trailsof dichlorphenamide in the periodic paralyses. Ann Neurol2000;47:46–53.

Fig. Continuous raster display of the concentric needle electro-myography findings in the right biceps brachii of the indexcase. E � end of the myotonic discharge; I � insertion activ-ity; O � onset of a myotonic discharge. Scale bar � 0.1mVper 100 milliseconds (ms).


Human Antibodies againstAmyloid � Peptide:A Potential Treatment forAlzheimer’s DiseaseRichard Dodel, MD,1 Harald Hampel, MD,2

Candan Depboylu, MD,1 Suizhen Lin, MD,3

Feng Gao, MD,3 Sabine Schock, MD,1 Steffi Jackel, MD,1

Xing Wei, MD,3 Katharina Buerger, MD,2

Christine Hoft, BSc, 1 Bernhard Hemmer, MD,1

Hans-Jurgen Moller, MD,2 Martin Farlow, MD,3

Wolfgang H. Oertel, MD,1 Norbert Sommer, MD,1

and Yansheng Du, PhD3

Naturally occurring antibodies directed against�-amyloid (A�) were detected in intravenous immuno-globulin preparations. After intravenous immunoglobulintreatment in patients with different neurological diseases,total A� and A�1-42 in the cerebrospinal fluid was re-duced significantly compared with baseline values. In theserum, total A� levels increased after intravenous immu-noglobulin treatment, whereas no significant change wasobserved in A�1-42 levels. Antibodies against A� werefound to be increased in the serum and cerebrospinalfluid after intravenous immunoglobulin treatment. Thisstudy provides evidence that intravenous immunoglobu-lin or purified A� antibodies may modify A� and A�1-42

levels, suggesting potential utility as a therapy for Alzhei-mer disease.

Ann Neurol 2002;52:253–256

The pathological hallmarks of Alzheimer’s disease (AD)are the occurrence of plaques in the neural parenchymaand the formation of neuronal tangles.1 �-Amyloid(A�), a heterogenous 39 to 42–amino acid peptide, isthe main constituent of senile plaques and cerebrovas-cular amyloid deposits. The origin of the A� depositedin vasculature and human brain is uncertain. Accord-ing to the neuronal theory, A� is locally produced in

the brain.2 In contrast, the vascular theory proposesthat A� originates from the circulation, and that cir-culating soluble A� could contribute to neurotoxicity ifit crosses the blood-brain barrier.3 Production of A�via amyloid precursor protein (APP) processing, how-ever, is not the only factor that can influence the prob-ability of A� brain deposition. Evidence has accumu-lated indicating that factors influencing A� catabolism,clearance4 and aggregation,5 are also critical in regulat-ing A� metabolism.6

Recent data from transgenic mouse models of ADsuggest that clearance via immune-mediated pathwaysmay have a major impact on the development ofplaques.7,8 Immunization against A� has preventedsubsequent deposition of amyloid plaques. Further-more, supportive data have shown that passive immu-nization with antibodies directed against A� also pre-vents amyloid deposition, ameliorates behavioraldeterioration, and may even clear existing plaques.9,10

We hypothesized that if these results derived fromanimal experiments are transferable to humans, animmune-mediated A� degrading pathway may bephysiologically present and its actions clinically signif-icant in humans. Recently, we detected naturally oc-curring human antibodies against A� in both the cere-brospinal fluid (CSF) and serum of healthy subjects.11

These antibodies specifically recognize A�. Further-more, we detected significantly lower CSF titers ofthese anti–A� antibodies in AD patients comparedwith controls.

Following these results, we hypothesized that a treat-ment using these naturally occurring antibodies mightbe beneficial as a therapeutic strategy for AD patients.Because these antibodies are predominantly present inthe immunoglobulin G (IgG) fraction, we investigatedwhether they are detectable in commercially availableIgG products. In addition, we investigated whether theadministration of a selected IgG product (intravenousimmunoglobulin [IVIG]) affects human CSF and se-rum A� levels.

Patients and MethodsPatients treated with IVIG were recruited at the Departmentof Neurology, Philipps University, Marburg. Each patient re-ceived IVIG (Octagam; Octapharma, Langenfeld, Germany)at a total dose of 0.4gm/kg body weight on 3 consecutivedays. Seven patients (4 male, 3 female; mean age, 62.7 � 7.0years) were included. During their regular evaluation andtreatment, CSF and blood samples were withdrawn beforeinfusion of IVIG and at the indicated times after the infu-sions. We took CSF samples in the morning at the L3/L4 orL4/L5 interspace by lumbar puncture after obtaining appro-priate informed consent. We determined CSF leukocytecount and protein levels by standard methods. No contami-nation by erythrocytes was seen in any of the samples. Ali-quots then were stored at �80°C until biochemical analysis.Albumin and IgG concentrations were determined in the se-

From the 1Department of Neurology, Philipps University Marburg;2Dementia Research Section and Memory Clinic, Geriatric Psychi-atry Brand and Alzheimer Memorial Center, Ludwig-MaximilianUniversity, Munich, Germany; and 3Department of Neurology, In-diana University School of Medicine, Indianapolis, IN.



Address correspondence to Dr Dodel, Department of Neurology,Philipps University, Rudolf-Bultmann Strasse 8, 35039 Marburg,Germany. E-mail: [email protected] or Dr Du, Depart-ment of Neurology, Indiana University School of Medicine, India-napolis, IN 46202. E-mail: [email protected]


rum, and the CSF by immunoprecipitation nephelometry.Only patients with an intact blood-brain barrier and regularCSF 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 immunopre-cipitation of A� were performed as described previously.11

The concentration of A� antibodies in commercially avail-able IVIG Flebogamma, (Grifols, Langen), Germany; andOctagam; Octapharma) was determined using the A� anti-body ELISA.11

Mean levels of anti-A� titers were compared by 2-wayanalysis of variance. A log transformation was conducted be-fore statistical analysis to reduce skewness within each patientgroup.12 The mean log antibody titers for each patient groupwere back-transformed to give geometric mean and standarderrors.

ResultsWe purified the anti–A� antibodies from IVIG prepa-rations using affinity chromatography as previously de-scribed.11 A marked decrease in anti–A� antibody titerfrom the IgG fraction was observed when the flow-through was tested in the ELISA assay (data notshown). We confirmed the ability of the affinity-purified anti–A� antibody from plasma to bind A� byimmunoprecipitation of synthetic A� peptide (Fig 1A).Moreover, the affinity-purified anti–A� antibody fromhuman IVIG readily detected a 4kDa A� peptide onWestern blots prepared from PDAPP mouse13 hip-pocampal homogenates but not from cerebellar ho-mogenates (see Fig 1B).

In the next set of experiments, we incubated over-night different commercially available IVIG prepara-tions (Flebogamma, [Grifols] and Octagam; Octap-harma) with A�1-40 (0.4�gm/ml; Fig 2). Thereafter,IVIG was analyzed for anti–A� antibodies usingELISA. In both preparations, we could detect antibod-ies against A�. In both IVIG preparations, we coulddetect a considerable decrease of the ELISA signal afterincubation with A�1-40 and agarose A.

To evaluate our hypothesis that these antibodies mayhave an effect on A�- peptide levels (total A� and A�1-

42), we investigated the effects of IVIG administrationon CSF and serum A� levels in patients with differentneurological diseases. We detected a significant de-crease in CSF levels of total A� and A�1-42 after IVIG(Table 2). Similar to the recent results in PDAPPmice,14 a significant increase in total A� concentrationwas observed in the serum after IVIG treatment (seeTable 2). Although there was a trend toward an in-crease of A�1-42 in the serum, values did not reach sta-tistical difference (p � 0.06). The concentration of

Table 1. Clinical Data of the Patients Included in this Study

Patientno.

Age(yr) Gender Diagnosis QA1b

1 67 F MS 4.52 47 F MS 5.93 61 F PNP unknown origin 3.54 67 M PNP unknown origin 4.35 70 M PNP unknown origin 6.36 34 M Lambert-Eaton syndrome 7.97 78 M Dermatomyositis 7.8

None of the patients showed disturbance of the blood-brain barrier(QALb � 8; QALb � CSFALb/SerumALb).15

MS � Multiple Sclerosis; PNP � Polyneuropathy.

Fig 1. Purification and characterization of human anti–A�antibody from human intravenous immunoglobulin (IVIG)samples (Octagam; Octapharma). (A)Anti–A� antibody afterelution from the A� affinity column. We passed IVIG throughan affinity sepharose column conjugated with the A�1-40 pep-tide. Anti–A� antibody from the purified human plasma IgGwas recovered after elution with buffer 1 (pH 2.5) followedby buffer 2 (pH 1.5). A�1-40 was immunoprecipitated byaffinity-purified human anti–A� antibody. (B) A� fromPDAPP mouse hippocampal homogenates was immunoprecipi-tated by purified human anti–A� antibody and detected bymonoclonal antibodies to A�.13 A� � amyloid �; crb � cer-ebellum; hanti-A� � human purified A� antibody; hippo �hippocampus; IgG � immunoglobulin G (IVIG flowthrough).


A�-antibodies in the serum increased after treatmentwith IVIG.

DiscussionOn the basis of the results by Schenk and colleagues,7

we identified specific anti–A� antibodies (IgG) in both

the serum and the CSF from nonimmunized humans,which may act in an immune-mediated A� clearancepathway.11 In an earlier study, human antibodies reac-tive with A� were isolated and cloned from humanB-cell lines from AD patients; however, the role ofthese antibodies in AD pathogenesis remains unclear.6

In our study, we detected a significant difference in theamount of A� antibodies in AD patients comparedwith controls.11 These observations led us to askwhether these anti–A� antibodies are detectable incommercially available human IVIG preparations. Af-ter purification of these antibodies from IVIG, wefound that they specifically recognize A�. Furthermore,we investigated the effect of IVIG treatment on A�,A�1-42 levels in serum and CSF. Intravenous immuno-globulin is an accepted and routinely used treatmentfor several neurological and nonneurological immune-mediated disorders.17 These patients are treated withIVIG on a routine basis in our department. Althoughwe are aware that the patient selection for this studyhas several limitations (eg, age, immune status, differ-ent diagnoses), this was the most straightforward andfeasible approach to evaluate our hypothesis.

Treatment of IVIG resulted in a significant decreaseof total A� and A�1-42 in the CSF compared withbaseline. Mean A� antibody concentration increased inthe CSF. In contrast, the serum total A� and A� an-tibody concentration increased, but A�1-42 remainedunchanged. A� antibody concentration increased inthe serum.

These findings suggest that A� peptides may passfrom the CSF to the blood and probably are metabo-lized locally. Recently, transporters at the blood-brainbarrier have been reported, which control the centraland peripheral exchange of smaller peptides including

Fig 2. Human anti–A� antibody was preabsorbed with A�peptide. Experiments were performed using intravenous immu-noglobulin (IVIG) before and after incubation with A�1-40.Briefly, 100�l of IVIG was incubated (overnight, 4°C) withprotein A–agarose or A�1-40 at the designated concentration.Protein A–agarose (Sigma, St. Louis, MO) was added toIVIG (1�l, overnight, 4°C) and removed by low-speed cen-trifugation. The IVIG then was analyzed for anti–A� anti-body using enzyme-linked immunoadsorbent assay. Antibodywas recovered by incubation with eluting buffer (pH 1.5).This experiment was repeated 3 times with similar results.A� � amyloid �; Alp � Flebogamma, (Grifols); Oct � Oc-tagam (Octapharma).

Table 2. Total A�, A�1–42, and A�. Antibody Concentrations in the CSF and Serum at Baseline and after IVIG

CSF Serum

BaselineAfterIVIG p Baseline

1–4 DaysAfter IVIG p

7–14 DaysAfter 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 87.2 135.5 123.5A�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 12.7A� 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 themean of triplicate determinations from a single assay.ap � 0.05.bLevels of significance 0.05.

A� � �-amyloid; CSF � cerebrospinal fluid; IVIG � intravenous immunoglobulin; ns � not significant; SEM � 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-ripheral compartment has been found in PDAPP micepassively immunized with antibodies against A�.14

Whether there is a specificity for shorter A� peptidesto cross the blood-brain barrier, as seen in the afore-mentioned experiments, cannot be stated at this point.

No data are available on the quantitative decrease ofA� concentration necessary to reduce A� deposition.Therefore, one can only speculate whether the observedreduction may have an impact on plaque formation.Further studies, including careful dose studies in ani-mals, are necessary. From earlier studies, however,some information can be deduced. First, a relativelymodest A� clearance already reduced memory impair-ment in Tg2576 APP�PS1 mice.10 Second, an in-crease in A� concentration of approximately 1.5 timesin familial AD patients because of mutations in theAPP gene shifts the disease onset earlier by several de-cades. It can be assumed that already small changes inA� concentrations in the CSF may have an impact onA� deposition and plaque development.18

Our findings might have important implications forthe understanding of immune-mediated clearance path-ways of A� in humans. Moreover, our data supportearlier in vitro and animal experiments that antibodiesapplied outside of the blood-brain barrier may serve tofacilitate clearance of soluble peptides such as A� outof the central nervous system.2,19,20 Given the rela-tively large volume of distribution of the peripheral ascompared with the central compartment, the additionof anti–A� antibodies or other antipeptide antibodiesmay considerably alter the clearance of biologically ac-tive peptides in the brain.

Finally, further studies are warranted to investigatethe role of A� antibodies in A� clearance; however,our findings suggest that IVIG-purified A� antibodiesmay be a potential therapeutic approach to AD.

References1. Selkoe DJ. Translating cell biology into therapeutic advances in

Alzheimer’s disease. Nature 1999;399:A23–A31.2. Shibata M, Yamada S, Kumar SR, et al. Clearance of Alzhei-

mer’s amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier. J Clin Invest 2000;106:1489–1499.

3. Mackic JB, Stins M, McComb JG, et al. Human blood-brainbarrier receptors for Alzheimer’s amyloid-beta 1- 40. Asymmet-rical binding, endocytosis, and transcytosis at the apical side ofbrain microvascular endothelial cell monolayer. J Clin Invest1998;102:734–743.

4. Ghersi-Egea JF, Strazielle N. Brain drug delivery, drug metab-olism, and multidrug resistance at the choroid plexus. MicroscRes Tech 2001;52:83–88.

5. Wisniewski T, Ghiso J, Frangione B. Biology of A beta amyloidin Alzheimer’s disease. Neurobiol Dis 1997;4:313–328.

6. Iwata N, Tsubuki S, Takaki Y, et al. Identification of the majorAbeta1–42-degrading catabolic pathway in brain parenchyma:suppression leads to biochemical and pathological deposition.Nat Med 2000;6:143–150.

7. Schenk D, Barbour R, Dunn W, et al. Immunization withamyloid-beta attenuates Alzheimer-disease-like pathology in thePDAPP mouse. Nature 1999;400:173–177.

8. Bard F, Cannon C, Burke R, et al. Peripherally administeredantibodies against amyloid beta-peptide enter the central ner-vous system and reduce pathology in a mouse model of Alzhei-mer disease. Nat Med 2000;6:916–919.

9. Janus C, Pearson J, McLaurin J, et al. A beta peptide immuni-zation reduces behavioural impairment and plaques in a modelof Alzheimer’s disease. Nature 2000;408:979–982.

10. Morgan D, Diamond DM, Gottschall PE, et al. A beta peptidevaccination prevents memory loss in an animal model of Alz-heimer’s disease. Nature 2000;408:982–985.

11. Du Y, Dodel RC, Hampel H, et al. Reduced CSF levels ofamyloid beta-peptide antibody in Alzheimer’s disease. Neurol-ogy 2001;57:801–805.

12. Armitage P, Berry G. Statistical methods in medical research.Oxford: Blackwell Scientific Publications, 1994:389–390.

13. Games D, Adams D, Alessandrini R, et al. Alzheimer-type neu-ropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein. Nature 1995;373:523–527.

14. DeMattos RB, Bales KR, Cummins DJ, et al. Peripheral anti-Abeta antibody alters central nervous system and plasma A betaclearance and decreases brain A beta burden in a mouse modelof Alzheimer’s disease. Proc Natl Acad Sci USA 2001;98:8850–8855.

15. Reiber H, Peter JB. Cerebrospinal fluid analysis: disease-relateddata patterns and evaluation programs. J Neurol Sci 2001;184:101–122.

16. Gaskin F, Finley J, Fang Q, et al. Human antibodies reactivewith beta-amyloid protein in Alzheimer’s disease. J Exp Med1993;177:1181–1186.

17. Bril V, Allenby K, Midroni G, et al. IVIG in neurology—evi-dence and recommendations. Can J Neurol Sci 1999;26:139–152.

18. Hardy J, Allsop D. Amyloid deposition as the central event inthe aetiology of Alzheimer’s disease. Trends Pharmacol Sci1991;12:383–388.

19. Zlokovic BV, Yamada S, Holtzman D, et al. Clearance of amy-loid beta-peptide from brain: transport or metabolism? NatMed 2000;6:718–719.

20. Strazielle N, Ghersi-Egea JF, Ghiso J, et al. In vitro evidencethat beta-amyloid peptide 1–40 diffuses across the blood brainbarrier and affects its permeability. J Neuropathol Exp Neurol2000;59:29–38.


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