LETTERS Alteration of the Serotoninergic System in Fatal Familial Insomnia Pietro Cortelli,1 Ronald Polinsky,2 Pasquale Montagna,3 and Elio Lugaresi3 1 We read with interest that Wanschitz and colleagues found a substantial increase in tryptophan hydroxylase-immunoreactive neurons in the pons (superior central nucleus) and medulla (raphe obscurus nucleus) of fatal familial insomnia (FFI) patients, suggesting that a serotoninergic system impairment represents the functional substrate of some typical FFI symptoms. We report the lumbar cerebrospinal fluid (CSF) concentrations of monoamine metabolites in 3 FFI subjects (1 female) of 2 unrelated Italian families already described.2 We measured 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) by a gas chromatographic, mass spectroscopic method previously described.3,4 The central component of MHPG was calculated according to the formula [total CSF MHPG] ⫺ 0.9 [free plasma MHPG].4 FFI patients had the typical point mutation at codon 178 of the prion protein gene combined with methionine at codon 129 of the mutated allele. Lumbar puncture was performed in a standardized fashion3 13 (in 1 case) and 6 (in 2 cases) months after disease onset. Average ages (⫾ standard error of the mean) at the time of CSF collection for the 3 FFI patients and 24 controls were 55 ⫾ 2 and 47 ⫾ 3 years. The CSF level of 5-HIAA in FFI patients (37.63 ⫾ 4.13ng/ml) was significantly higher ( p ⬍ 0.0001) than that in controls (18.72 ⫾ 1.95ng/ml). There were no significant differences between controls and FFI patients with respect to lumbar CSF levels of HVA (FFI 44 ⫾ 1.2ng/ml, controls 42 ⫾ 2.3ng/ml) and corrected CSF MHPG (FFI 6.46 ⫾ 0.8ng/ml, controls 5.34 ⫾ 0.2ng/ml). Our results in FFI patients are consistent with increased central serotonin [5-hydroxytryptophan (5-HT)] turnover and with the findings of Wanschitz et al.,1 attended by normal dopaminergic activity and a slight increase in central noradrenergic function. However, we caution against an oversimplified explanation of an altered serotoninergic system as the only functional substrate for all symptoms of FFI. The hallmark of FFI is selective degeneration of anteroventral (AV) and mediodorsal (MD) thalamic nuclei, which constitute an important control station in the so-called central autonomic network, including several areas of the telencephalon, hypothalamus, and brain stem interconnected via parallel, functionally specific, and neurochemically complex pathways.5 The 5-HT system is one of these pathways and has been implicated in the regulation of the sleep–wake cycle and cardiovascular system, but its effects are subordinate to the control of higher diencephalic centers and the subtype of 5-HT receptors involved.5 The crucial role of the AV and MD thalamic nuclei in regulating sleep and integrating autonomic and endocrine responses critical for homeostasis has been confirmed by studies showing that wake–sleep and autonomic features comparable to FFI may be reproduced in experimental animals by kainic acid injection into the MD and the reticular nucleus of the thalamus.2 Lesions to the thalamus disrupt the cortical– limbic circuitry with a cascade effect on the functions of many systems. For instance, there is pharmacological evidence of GABAergic system involvement in FFI patients.6 Thus, while confirming increased activity of the serotoninergic system in FFI, we believe that it represents just one of the many biochemical systems involved. 1 University of Modena and Reggio Emilia, Bologna, Italy; AstraZeneca Pharmaceuticals, Wilmington, DE; and 3 University of Bologna, Bologna, Italy 2 References 1. Wanschitz J, Kloppel S, Jarius C, et al. Alteration of the serotonergic system in fatal familial insomnia. Ann Neurol 2000;48: 788 –791. 2. Montagna P, Cortelli P, Avoni P, et al. Clinical features of fatal familial insomnia: phenotypic variability in relation to a polymorphism at codon 129 of the prion protein gene. Brain Pathol 1998;8:515–520. 3. Polinsky RJ, Brown RT, Burns RS, et al. Low lumbar CSF levels of homovanillic acid and 5-hydroxyindoleacetic acid in multiple system atrophy with autonomic failure. J Neurol Neurosurg Psychiatry 1988;51:914 –919. 4. Polinsky RJ, Jimerson DC, Kopin IJ. Chronic autonomic failure: CSF and plasma 3-methoxy-4-hydroxyphenylglycol. Neurology 1984;34:979 –983. 5. Benarroch EE. Central autonomic network: functional organization and clinical correlations. Armonk, NY: Futura, 1997. 6. Lugaresi E, Medori R, Montagna P, et al. Fatal familial insomnia and dysautonomia with selective degeneration of thalamic nuclei. N Engl J Med 1986;315:997–1003. Reply Julia Wanschitz, MD,1 Marin Guentchev, MD,1 and Herbert Budka, MD1,2 We thank Cortelli and colleagues for their attention to our article. Their report of high cerebrospinal fluid (CSF) levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in 3 patients with fatal familial insomnia (FFI) confirms increased activity of the serotoninergic [5-hydroxytryptophan (5-HT)] system in FFI patients as indicated by our observation of an increased number of tryptophan hydroxylaseimmunoreactive (TH⫹) neurons in median raphe nuclei.1 As Cortelli and colleagues stated, these results must be interpreted with caution. We did not postulate a trivial explanation of the complex and unique clinical features of FFI as simply a serotonin-related disorder. We hypothesized that enhanced serotoninergic neurotransmission might contribute to the profound disturbance of control of autonomic homeostasis in FFI in accordance with the current knowledge about serotoninergic function on sleep, circadian rhythmicity,2 and cardiovascular responses,3 based on the dense innervation of the limbic anteroventral and mediodorsal thalamic nuclei by serotoninergic fibers. The critical role of thalamic nuclei in the pathophysiology of central dysautonomia characteristic of FFI is well established.4 Despite the paucicity of classical histopathological lesions in other areas involved in the control of autonomic functions, such as the hypothalamus and several regions of the brain stem, the importance of subtle changes recorded in these areas5 remained unclear. The changes in the ratio of TH⫹ to TH⫺ neurons in median raphe nuclei of the brain stem in FFI patients most likely represents an impaired neuronal function of the 5-HT system as one of the pathways © 2001 Wiley-Liss, Inc. 421 possibly involved in the disease. We did not observe similar changes in sporadic and familial Creutzfeldt-Jakob disease or cases with a selective ischemic thalamic lesion (Budka et al, unpublished observation). The main question arising from both studies is whether the observed changes are pathogenic or occur as a compensatory phenomenon to other events, such as downregulation of serotonin receptors, loss of the neuronal targets of the serotoninergic system, or alteration of other neurotransmitters (eg, corticotropin-releasing factor, which may modulate 5-HT release6). We believe that such considerations have an important clinical impact as they form the theoretical basis for the potential use of serotonin antagonists in the treatment of FFI. Additional studies should clarify the usefulness of CSF 5-HIAA determination as an early diagnostic marker of FFI. 1 Institute of Neurology, University of Vienna, and 2Austrian Reference Center of Human Prion Diseases, Vienna, Austria References 1. Wanschitz J, Klöppel S, Jarius C, et al. Alteration of the serotonergic nervous system in fatal familial insomnia. Ann Neurol 2000;48:788 –791. 2. Jouvet M. Sleep and serotonin: an unfinished story. Neuropsychopharmacology 1999;21(Suppl):24S–27S. 3. Bell AA, Butz BL, Alper RH. Cardiovascular responses produced by microinjection of serotonin-receptor agonists into the paraventricular nucleus in conscious rats. J Cardiovasc Pharmacol 1999;33:175–180. 4. Benarroch EE, Stotz-Potter EH. Dysautonomia in fatal familial insomnia as an indicator of the potential role of the thalamus in autonomic control. Brain Pathol 1998;8:527–530. 5. Parchi P, Castellani R, Cortelli P, et al. Regional distribution of protease-resistant prion protein in fatal familial insomnia. Ann Neurol 1995;38:21–29. 6. Price ML, Lucki I. Regulation of serotonin release in the lateral septum and striatum by corticotropin-releasing factor. J Neurosci 2001;21:2833–2841. Effects of Seasons on Magnetic Resonance Imaging-Measured Disease Activity in Patients with Multiple Sclerosis Helen Tremlett, BPharm Hons, PhD I am writing in response to the study by Rovaris and colleagues,1 which concluded that MRI in MS patients does not exhibit seasonal variation. Whilst such an effect may or may not exist, their study does not refute this possibility for a number of reasons. First, the MS patients recruited were a highly selected group. Inclusion criteria required each patient to have at least one active lesion and one relapse in the past 2 years. These patients were therefore in a very active phase of disease, as acknowledged by the authors of the original study who stated, “Clearly, this makes this cohort of patients difficult to compare with those of previous trials.”2 This quite possibly means that the effects of seasonal variation, if it exists, or if indeed it exists in this group of highly selected patients, may be tempered and “lost.” Second, the study included patients from Canada as well as Europe; the spread of patients in each country was not stated. Geographical location is important as seasonal varia- 422 Annals of Neurology Vol 50 No 3 September 2001 tion of MRI scans maybe associated with 25-hydroxyvitamin D (25(OH)D) levels, as found in Germany.3 This phenomenon may not be so pronounced in North American and Scandinavian countries. This is because seasonal variation of 25(OH)D levels is more evident in central and western Europe than in North America and Scandinavia.4 Whilst studies in all these countries have shown seasonal variation of 25(OH)D levels in young adults, the effect was not so pronounced in central and Western European countries.4 Indeed, central and western European 25(OH)D levels in young adults were significantly lower compared to North American and Scandinavian countries in winter and spring/ fall ( p ⬍ 0.01) but not summer ( p ⬎ 0.05).4 This appeared primarily because of increased consumption of vitamin D containing supplements and foodstuffs (notably fortified foodstuffs in North America and oily fish in Scandinavia).4 Third, data in the study1 was only collected over a 10month period. To sensibly assess a seasonal variation, data should surely be collected over 12 months and preferably over a number of years for comparison. In conclusion, whilst the study by Rovaris and colleagues1 appears to demonstrate a minimal effect of seasonal variation on their data set, possible seasonal variation of MRI in MS should not be disregarded. Only well-designed studies with patients in defined geographical areas will confirm or refute such an association. Medicines Research Unit, Cardiff University, Cardiff, United Kingdom References 1. Rovaris M, Comi G, Sormani MP, et al. Effects of seasons on magnetic resonance imaging-measured disease activity in patients with multiple sclerosis. Ann Neurol 2001;49:415– 416. 2. Comi G, Filippi M, Wolinsky JS, the European/Canadian glatiramer acetate study group. European/Canadian multicenter, double-blind, randomized, placebo-controlled study of the effects of glatiramer acetate on magnetic resonance imaging-measured disease activity and burden in patients with relapsing multiple sclerosis. Ann Neurol 2001;49:290 –297. 3. Auer D, Schumann E, Kumpfel T, et al. Seasonal fluctuations of gadolinium-enhancing magnetic resonance imaging lesions in multiple sclerosis [letter]. Ann Neurol 2000;47:276 –277. 4. McKenna M. Differences in vitamin D status between countries in young adults and elderly. Am J Med 1992;93:69 –77. Reply Marco Rovaris, MD,1 Giancarlo Comi, MD,2 Maria Pia Sormani, PhD,1 Jerry S. Wolinsky, MD,3 David Ladkani, PhD,4 and Massimo Filippi, MD1 In our letter1 we reported that in 120 patients with relapsing-remitting (RR) multiple sclerosis (MS), who were part of the placebo arm of a double-blind, randomized, controlled trial assessing the efficacy of glatiramer acetate2 and underwent monthly, gadolinium (Gd)-enhanced magnetic resonance imaging (MRI) scans of the brain, the number of enhancing lesions varied in the different seasons, but the magnitude of this fluctuation was not statistically significant.1 As a consequence, we concluded that “the seasonal fluctuations of subclinical activity in RRMS patients should not affect the interpretation of the results from MRI- monitored clinical trials a great deal.” We have not at all concluded that “MS patients do not exhibit seasonal variations of MRI activity.” As pointed out, the inclusion criteria for the original trial2 required the presence of at least one Gd-enhancing lesion on a screening MRI scan and one or more clinical relapses in the prior 2 years. Clearly, these criteria increased the likelihood for detecting further disease activity over the subsequent months.3 Although we commented that our results cannot be compared with those of previous trials where no selection criteria for MRI activity was used,2 we cannot see how this may prevent detecting a seasonal fluctuation of MRI activity, if any. On the contrary, an increased MRI activity should enhance the probability of detecting significant fluctuations over time. Our sample is also more homogeneous than that of the study by Auer and colleagues,4 where patients with either RR or secondary progressive (SP) MS were included. This, by reducing interpatient variability, should again increase the likelihood to detect seasonal fluctuations. As specified in the paper reporting the results of this trial,2 we included patients from Canada (n ⫽ 17) and from the following European countries: Belgium (n ⫽ 8), England (n ⫽ 38), France (n ⫽ 11), Germany (n ⫽ 4), Holland (n ⫽ 6) and Italy (n ⫽ 36). There were no Scandinavian patients. Although we are not aware of any evidence supporting the concept that 25-hydroxyvitamin D levels influence MS activity as detected by Gd-enhanced MRI, we analyzed separately data from Canadian and European patients and repeated the original analysis1 after correcting for patients’ geographical origin. The patterns of MRI enhancement did not differ between Canadian and European patients. The frequency of MRI activity was similar among the subgroups of patients from different countries and the seasonal fluctuation of Gd-enhancing lesion numbers was still not statistically significant. As far as the patient recruitment is concerned, we believe that 10 consecutive months can be considered representative of the whole of the year. In addition, all the scans were collected between February 1997 and August 1998, thus covering a 11⁄2 year period. We also performed additional analysis to patients with at least one scan for each of the four seasons, thus limiting the variability in the number of scans obtained during each season. This again did not yield significant results. We also believe that the collection of data over longer periods of time is likely to increase dramatically the influence of other factors known to affect MRI-detected MS activity, such as changes of the disease phenotype (for instance, from RR to SP), patients’ aging,5 and major upgrades or changes of the MR scanners,6 thus making the interpretability of the analysis even more challenging. We agree that the possibility of seasonal variations of MRI activity in RRMS patients should not be disregarded. To our knowledge, this issue has been addressed only by two preliminary studies,1,4 including ours, and as a consequence deserves further consideration. However, we believe that our study was designed in the appropriate way to provide evidence that the fluctuation of subclinical MS activity over the time periods needed for MRI-monitored clinical trials should not affect the interpretation of their results. 1 Neuroimaging Research Unit and 2Clinical Trials Unit, Department of Neuroscience, Scientific Institute and University Ospedale San Raffaele, Milan, Italy; 3Department of Neurology, University of Texas Houston, Health Science Center, Houston, TX; and 4TEVA Pharmaceutical Industries Ltd, Kyriat Nordau, Netanya, Israel References 1. Rovaris M, Comi G, Sormani MP, et al. Effects of seasons on magnetic resonance imaging-measured disease activity in patients with multiple sclerosis [letter]. Ann Neurol 2001;49:415– 416. 2. Comi G, Filippi M, Wolinsky JS, the European/Canadian Glatiramer Acetate Study Group. The European/Canadian multicenter, double blind, randomized, placebo-controlled study of the effects of glatiramer acetate on magnetic resonance imagingmeasured disease activity and burden in patients with relapsing multiple sclerosis. Ann Neurol 2001;49:290 –297. 3. Kappos L, Moeri D, Radue EW, et al. Predictive value of gadolinium-enhanced MRI for relapse rate and changes in disability/impairment in multiple sclerosis: a meta-analysis. Lancet 1999;353:964 –969. 4. Auer DP, Schumann EM, Kumpfel T, et al. Seasonal fluctuations of gadolinium-enhancing magnetic resonance imaging lesions in multiple sclerosis [letter]. Ann Neurol 2000;47:276 –277. 5. Filippi M, Wolinsky JS, Sormani MP, et al. Enhancement frequency decreases with increasing age in relapsing-remitting multiple sclerosis. Neurology 2001;56:422. 6. Filippi M, van Waesberghe JH, Horsfield MA, et al. Interscanner variation in brain MRI lesion load measurements in MS: implications for clinical trials. Neurology 1997;49:371–377. Borna Disease Virus RNA Is Absent in Chronic Multiple Sclerosis Claus G. Haase, MD,1 Sergei Viazov, MD,2 Melanie Fiedler, MD,2 Nikolaus Koenig, MD,3 Pedro M. Faustmann, MD,4 and Michael Roggendorf, MD2 Multiple sclerosis (MS) is a demyelinating inflammatory disease of the central nervous system (CNS) of unknown etiology, although the involvement of viral infections as a possible triggering event is suspected.1 In MS patients, relatively high prevalence of antibodies (13%) to Borna Disease Virus (BDV) or viral antigen (10.5%) allowed speculation on a possible involvement of this virus in initiating the development of MS or driving a progression from a relapsing form to a chronic state.2,3 This hypothesis is supported by certain BDV characteristics: BDV is a neurotropic, negative-stranded, single-stranded RNA virus that causes inflammatory CNS disease in naturally infected hosts (several species of vertebrates). Association of BDV infection with other human neurological disorders, including major depression and schizophrenia was repeatedly suggested but never proven.2– 4 To clarify the question whether BDV plays a role as a potential trigger in chronic progressive MS, we assessed the prevalence of BDV RNA in blood of 94 persons. The MS patient group included 34 individuals with either a primary chronic progressive course (n ⫽ 17, age 58 ⫾ 10 years, disease duration 15.5 ⫾ 8.3 years, EDSS 6.6 ⫾ 1.1) or a secondary chronic progressive course (n ⫽ 17, age 49 ⫾ 10 years, disease duration 18.5 ⫾ 10.3 years, EDSS 6.2 ⫾ 1.3). Patients with Annals of Neurology Vol 50 No 3 September 2001 423 schizophrenia (n ⫽ 20) and healthy blood donors (n ⫽ 40) served as age- and sex-matched control groups. For the detection of viral RNA, the reverse transcriptase polymerase chain reaction procedure was used, as described in full detail elsewhere.5 Amplification results demonstrated the presence of BDV sequences in only one sample of a schizophrenic patient but in none of the other 93 patients or healthy donors. Sequencing of the amplified DNA from this single positive sample revealed its complete identity with the sequence obtained from the positive control used. Thus the positive result with this single sample was probably a result of laboratory contamination. In conclusion, the negative result of the current investigation complement earlier reports5,6 and supports the evidence against an association between BDV infection and chronic MS. 1 Department of Neurology, University Hospital Essen, Essen; Institute of Virology, University of Essen, Essen; 3Multiple Sclerosis Center Berg, Berg; and 4Institute of Neuroanatomy, University of Bochum, Bochum, Germany 2 References 1. Hohlfeld R. Biotechnological agents for the immunotherapy of multiple sclerosis. Principles, problems and perspectives. Brain 1997;120:865–916. 2. Bode L, Riegel S, Lange W, Ludwig H. Human infections with Borna disease virus: seroprevalence in patients with chronic diseases and healthy individuals. J Med Virol 1992;36:309 –315. 3. Deuschle M, Bode L, Heuser I, et al. Borna disease virus proteins in cerebrospinal fluid of patients with recurrent depression and multiple sclerosis. Lancet 1998;352:1828 –1829. 4. Bode L, Zimmermann W, Ferszt R, et al. Borna disease virus genome transcribed and expressed in psychiatric patients. Nat Med 1995;1:233–236. 5. Sauder C, de la Torre JC. Sensitivity and reproducibility of RTPCR to detect Borna disease virus (BDV) in blood: implications for BDV epidemiology. J Virol Meth 1998;71:229 –245. 6. Kitze B, Herzog S, Rieckmann P, et al. No evidence of Borna disease virus-specific antibodies in multiple sclerosis patients in Germany. J Neurol 1996;243:660 – 662. Monozygotic Twins with X-Linked Adrenoleukodystrophy and Different Phenotypes Maja Di Rocco, MD,1 Laura Doria-Lamba, MD,1 and Ubaldo Caruso, PhD2 Adrenoleukodystrophy (ADL) is an X-linked genetic disorder characterized by elevated levels of very-long-chain fatty acids (VLCFAs), caused by a deficiency in peroxisomal VLCFA beta oxidation. The ADL gene encodes a peroxisomal membrane transporter. No correlations between phenotype and genotype have been demonstrated. Clinical findings include severe inflammatory reaction of the cerebral white matter, which leads to demyelination, axonal degeneration (adrenomyeloneuropathy), and isolated adrenal insufficiency. Recently in the Annals, van Geel and colleagues1 reported phenotype evolution in 129 men with X-ADL, demonstrating that adrenomyeloneuropathy and cerebral demyelination emerge independently from each other. Furthermore, adult transgenic mice with targeted inactivation of the ADL gene do not have demyelinating lesions of the central nervous system (CNS), although they do have an abnormal accumulation of VLCFAs in the CNS.2 424 Annals of Neurology Vol 50 No 3 September 2001 Two hypotheses have been suggested that likely account for the clinical heterogeneity of adrenoleukodystrophy, modifyng genes and environmental factors. We report on a pair of monozygotic twins with X-linked adrenoleukodystrophy and different clinical courses. Genotyping of HLA DR and microsatellite loci showed identity. Therefore, the probability of monozygosity was greater than 99.9%. They are 15-year-old boys with pseudoachondroplastic skeletal dysplasia. When they were 12 years old, one of them underwent orthopedic surgery. A few weeks later, when the patient was walking, left hemiplegia was observed, and neuroradiological examinations were performed. Magnetic resonance imaging was consistent with adrenoleukodystrophy; plasmatic VLCFA were studied by selected ion monitoring and stable isotope dilution with a GC-MS Hewlett Packard 5973A system and were found to be increased [C26:0 2.389mol/L (normal values 0.46 – 0.98mol/L), C26:1 0.95mol/L (normal values 0.11–0.47), ratio C24:0/C22:0 1.938 (normal values 0.62–1.01mol/L), ratio C26:0/C22:0 0.078 (normal values 0.008 – 0.0026)]. His twin had adrenal insufficiency and no clinical, neurological, or neuroradiological abnormalities, but had increased VLCFA (C26:0 2.981mol/L, C26:1 1.12mol/L, ratio C24:0/C22:0 1.81, ratio C26:0/ C22:0 0.069). Subsequently the boy with CNS involvement had seizures, with no significant neuroradiological, neurophysiological, or neuropsychological progression. No neurological involvement has been observed in the other twin during the 3 years since diagnosis. Two other pairs of identical male twins with different clinical expressions of this disease have been reported.3,4 One of these sets of twins was also studied for mitochondrial DNA, and no differences were found. Therefore, mitochondrial genome involvement in the pathogenesis of linked adrenoleukodystrophy can be ruled out.5 Discordant adrenoleukodystrophy phenotypes in 3 pairs of monozygotic twins strongly suggest that the modifying genes are not involved in CNS degeneration. On the other hand, identifying environmental factors could be very important for effectively preventing CNS degeneration. 1 II Pediatric Unit, Gaslini Institute, and 2Laboratory for the Study of Metabolic Diseases, DIPE, University of Genoa, Genoa, Italy References 1. van Geel BM, Bezman L, Loes DJ, et al. Evolution of phenotypes in adult male patients with X-linked adrenoleukodystrophy. Ann Neurol 2001;49:186 –194. 2. Forss-Petter S, Werner H, Berger J, et al. Targeted inactivation of the X-linked adrenoleukodystrophy in mice. J Neurosci Res 1997;50:829 – 843. 3. Sobue G, Ueno-Natsukari I, Okamoto H, et al. Phenotypic heterogeneity of an adult form of adrenoleukodystrophy in monozygotic twins. Ann Neurol 1994;36:912–915. 4. Korenke GC, Fuchs S, Krasemann E, et al. Cerebral adrenoleukodystrophy (ADL) in only one of monozygous twins with an identical ADL genotype. Ann Neurol 1996;40:254 –257. 5. Wilichowski E, Ohlenbusch A, Korenke GC, et al. Identical mitochondrial DNA in monozygotic twins with discordant adrenoleukodystrophy phenotype. Ann Neurol 1998;43:835– 836.