DNA analysis of Huntington s disease

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1 DNA analysis of Huntington s disease Five years of experience in Germany, Austria, and Switzerland F. Laccone, MD; U. Engel, MD; E. Holinski Feder, MD; M. Weigell Weber, MD; K. Marczinek, PhD; D. Nolte, MD; D.J. Morris Rosendahl, MD; C. Zühlke, PhD; K. Fuchs, PhD; H. Weirich Schwaiger, MD; G. Schlüter, MD; G. von Beust, MD; A.M.M. Vieira Saecker; B.H.F. Weber, PhD; and O. Riess, MD Article abstract Objective: To review the direct DNA testing for Huntington s disease (HD) in Germany, Switzerland, and Austria from 1993 to 1997, and to analyze the population with regard to age structure, gender, and family history. Methods: Twelve laboratories (nine in Germany, two in Austria, and one in Switzerland) recorded data pertaining to repeat number, gender, age at molecular diagnosis, and family history of probands. The molecular test was categorized as either diagnostic (for symptomatic individuals), presymptomatic (for individuals at risk), and prenatal (for pregnancies at risk). Results: A total of 3,090 HD patients, 992 individuals at risk, and 24 fetuses were investigated using DNA analysis. The clinical diagnosis was confirmed in 65.6% of patients. A total of 38.5% of individuals at risk inherited an expanded CAG repeat. The female-to-male ratio showed a distinct predominance of women both in the diagnostic and presymptomatic groups. Of the fetuses tested, six were carriers of an expanded CAG repeat. Two pregnancies were interrupted; one pregnancy was not. No information about the parents decision was obtained for the remaining three pregnancies. Conclusions: Approximately 20% of the estimated 10,000 HD patients living in Germany, Switzerland, and Austria have been identified by DNA analysis (total population, approximately 100 million; incidence of HD, 1:10,000). Assuming a ratio of HD patients to individuals at risk of 1:3, approximately 30,000 individuals are, in principle, eligible for a presymptomatic test. Less than 3 to 4% of individuals at risk have requested a presymptomatic test. This shows that the assumed enormous request of predictive testing has not occurred. More surprisingly, prenatal diagnoses were found to be rare. Key words: DNA analysis Huntington s disease Germany Switzerland Austria. NEUROLOGY 1999;53: The discovery of the Huntington s disease (HD) gene (IT15) and of the underlying mutation in 1993 led rapidly to the development of molecular tests capable of confirming the diagnosis in patients and identifying carriers among individuals at risk. 1 To cope with the technical problems and ethical issues raised by the molecular testing, and to develop identical strategies of the different laboratories offering the test to the community, a consortium of clinicians, human geneticists, patient support groups, and all laboratories offering DNA analysis for HD was founded in Germany, Switzerland, and Austria in HD belongs to the continuously expanding group of CAG repeat disorders, including at least eight neurodegenerative diseases. 2 Ranges of nonexpanded and expanded CAG repeats have been defined in HD by molecular testing: Individuals carrying alleles with as many as 35 CAG repeats harbor no risk of developing HD, whereas individuals with 40 or more CAG repeats are affected or will develop HD during their lifetime. The range of 27 to 35 repeats is referred to as mutable normal allele, although there is no general agreement on the exact limits. 3,4 Furthermore, alleles with 36 to 39 repeats have been found both in affected as well as in nonaffected individuals. 5 Therefore, it has been suggested that these alleles have reduced penetrance and have to be considered carefully with respect to diagnostic significance and presymptomatic value. 5 However, meiotic instability of alleles with 27 to 35 repeats is a widely accepted phenomenon, with consequences and implications for patients and individuals at risk that have been discussed recently. 6 Despite these difficulties regarding the clinical and prognostic implications of alleles harboring 27 to 39 CAG repeats, molecular genetic testing has spread quickly and has proved to be a reliable diagnostic tool for neurologists, psychiatrists, and geneticists. The worldwide testing of more than 2,800 individuals 7 has demonstrated the specificity and sensitivity of the test. In fact, CAG From the Institute of Human Genetics (Drs. Laccone, Engel, and von Beust), University of Göttingen, Germany; the Department for Paediatric Genetics (Dr. Holinski Feder), Children s Hospital, Munich, Germany; the Institute of Medical Genetics (Dr. Weigell Weber), University of Zürich, Switzerland; the Institute of Medical Genetics (Dr. Marczinek), Charité University Hospital, Berlin, Germany; the Institute of Human Genetics (Dr. Nolte), University of Giessen, Germany; the Institute of Human Genetics (Dr. Morris Rosendahl), University of Freiburg, Germany; the Institute of Human Genetics (Dr. Zühlke), University of Lübeck, Germany; the Department of General Psychiatry, University Hospital for Psychiatry (Dr. Fuchs), Vienna, Austria; the Institute of Medical Biology and Human Genetics (Dr. Weirich Schwaiger), Innsbruck, Austria; the Laboratory of Professor Seelig (Dr. Schlüter), Karlsruhe, Germany; the Institute of Human Genetics (Dr. Weber), Biocenter, Wuerzburg, Germany; and Molecular Human Genetics (Dr. Riess and A.M.M. Vieira Saecker), Ruhr-University, Bochum, Germany. Received December 14, Accepted in final form April 3, Address correspondence and reprint requests to Dr. F. Laccone, Institute of Human Genetics, Gosslerstrasse 12d, Goettingen, Germany. Copyright 1999 by the American Academy of Neurology 801

2 Table 1 Summary of diagnostic and presymptomatic DNA tests for HD in Germany, Switzerland, and Austria, Test 39 CAG repeats; HD confirmed CAG repeats; mutable normal allele CAG repeats; reduced penetrance 27 CAG repeats; HD not confirmed Total Diagnostic 2, ,090 Presymptomatic Total 2, ,511 4,082 HD Huntington s disease. expansion in the IT15 gene has been found only in HD patients and not in patients affected with other neurologic or psychiatric conditions. We report the results of data collected retrospectively from the molecular testing of 3,090 affected patients, 992 individuals at risk, and 24 fetuses performed nationwide in 12 laboratories in Germany, Switzerland, and Austria. Methods. Twelve laboratories of the Consortium for DNA Analysis of HD provided their data from the molecular testing for HD conducted from 1993 to For each individual tested, the age at molecular diagnosis, CAG repeat number, gender, and family history were provided. Family history was defined as positive (FHP) if relatives with movement disorders, cognitive impairment, or psychiatric symptoms were known, and as family history uncertain (FHU) if relatives with movement or psychiatric conditions could not be excluded. Family history was considered negative (FHN) if there were no relatives with a neurodegenerative disease or psychiatric symptoms. Data were taken mostly from patients files and not from direct interviews. Molecular testing and test availability were carried out according to international guidelines. 8 CAG repeat sizes were determined after PCR amplification of genomic DNA from peripheral white blood cells. In general, the CAG repeat was amplified by the method described by Warner et al. 9 or Riess et al. 10 with slight modifications. We considered 40 or more CAG repeats to be pathologic alleles, whereas alleles with 26 or less CAG repeats were considered to be not expanded. The range of 27 to 35 repeats comprises the mutable normal alleles, and alleles with 35 to 39 repeats were considered to have reduced penetrance. A retrospective distinction between data obtained with PCR, including the polymorphic CCG located at the 3 end of the CAG repeat, 10 and PCR amplification of the CAG repeat alone 9 was not possible for all subjects retrospectively. However, all alleles in the range of 30 to 39 CAG repeats were reassessed in one laboratory (O.R.) excluding the flanking CCG repeats. 9 In case of detection of only one allele with at least two independent PCR methods, Southern blot analysis was carried out to confirm homozygosity of the identified allele. 802 NEUROLOGY 53 September (1 of 2) 1999 Results. Diagnostic testing. Data from the molecular analysis of 3,090 individuals with suspected HD were collected from 12 laboratories (2,825 individuals from Germany, 199 patients from Switzerland, and 66 patients from Austria). Testing was generally requested by neurologists, psychiatrists, or genetic counselors. The diagnosis of HD was confirmed in 65.6% of all patients referred for differential diagnosis. We found HD alleles with more than 39 CAG repeats in 2,028 individuals. A total of 971 individuals (31.4%) had alleles with less than 27 CAG repeats, 55 individuals (1.8%) had mutable normal alleles, and 36 individuals (1.2%) carried alleles in the reduced penetrance range (table 1). Only one allele was detected by PCR in 140 patients (4.5%) who were considered to be homozygous for the nonexpanded allele. The range of expansion was 40 to more than 100 CAG repeats with a mean of 45.1 CAG repeats (SD, 4.6) and a median of 44 CAG repeats (figure 1). The nonexpanded alleles ( 27 CAG repeats) in HD patients ranged from 4 to 26 CAG repeats with a mean of CAG repeats. The most frequent expansion in our cohort was 43 CAG repeats (15% of the expanded alleles), and the most common nonexpanded allele had 17 repeats (22.2% of all alleles that were not expanded). Thirty-six patients harbor an allele in the reduced penetrance range and 55 patients carry a mutable normal allele. Twenty-three HD patients with more than 39 CAG repeats had a second allele in the mutable normal allele/ reduced penetrance range (31 through 39). Two patients of the latter group had two alleles with 39 and 43 CAG repeats, and 39 and 62 CAG repeats respectively. The distribution of expanded alleles is depicted in figure 1. Our results show an inverse correlation between repeat number and age at molecular diagnosis (r 0.67), which is comparable with the correlation between repeat number of expanded alleles and age at onset of HD. 11 Overall, 75% of the patients were examined between age 34 and 70 years, and 50% were between 41 and 62 years. Considering exclusively the patients with expanded alleles, 75% were examined between age 36 and 67 years, and 50% were examined between age 43 and 60 years, whereas patients without expansion had a broader age of distribution (75% were between 29 to 72 years and 50% of the patients were between 39 to 66 years). Seventeen patients were found who had HD before age 20 (juvenile cases), and represent 0.9% of all patients with an expansion. A total of 135 patients (6.6% of all patients with an expansion mutation) had a late-onset form of HD (age at diagnosis, 70 years of age or older). Molecular testing confirmed the diagnosis in 24.6% of the juvenile group, in 69.1% of patients with the classic form of HD, and in 43.4% of the late-onset patients. The sex ratio of the whole group shows a significant predominance of women (ratio of women to men, 1.4; p 0.02). The mean age of the female and male groups are 52.2 years and 50.0 years respectively. Considering the group of patients without expanded alleles, we found a significant difference between men and women with respect to the age at diagnosis. The mean age of this group was 53.1 years for women and 48.9 years for men (t 3.7, df 917, p 0.001). In contrast, the group of HD patients

3 Figure 1. Distribution of expanded alleles in patients with Huntington s disease. was homogeneous with no remarkable differences regarding gender, CAG repeat number, or age at diagnosis. Family history. In 1,527 of 2,028 patients with an expansion it was possible to retrieve information on the family history (table 2). In FHP patients, HD was confirmed in 946 of 1,017 (93.0%) patients. HD was confirmed in 445 of 833 patients (53.7%) with FHU, and in 136 of 435 patients (31.3%) with FHN. Within the group of HD patients, 62.0% had FHP, 29.1% had FHU, and 8.9% had FHN. The mean age at diagnosis in the three groups was FHP, 48.9 years; FHU, 53.9 years; and FHN, 57.1 years, and the mean CAG expansion was FHP, 45.6 CAG repeats; FHU, 44.7 CAG repeats; and FHN, 43.8 CAG repeats, which is in agreement with the inverse correlation of age at diagnosis (figure 2). The difference in the age at onset between the three groups carrying a CAG repeat expansion is significant by t-test analysis: FHP versus FHU patients: t 7.1, df 852, p ; FHP versus FHN patients: t 7.3, df 182, p ; and FHU versus FHN patients: t 2.5, df 235, p Considering the groups of patients without expansion, the statistical analysis is significant for the FHP patients versus the FHU patients (t 3.6, df 61, p 0.001) and versus the FHN patients (t 3.8, df 64, p 0.001), whereas there is no significance between the group of patients with FHU and FHN (t 0.5, df 602, p 0.6). Investigating the size of the expanded alleles we also found a significant difference between the three groups (FHP versus FHU: t 3.7, df 900, p ; FHP versus FHN: t 5.9, df 231, p ; and FHU versus FHN: t 2.5, df 319, p 0.05). Presymptomatic and prenatal diagnosis. A total of 992 individuals at risk requested presymptomatic testing (Germany, 886; Switzerland, 89; and Austria, 17). A total of 370 individuals (37.3%) were found to be carriers of alleles of more than 39 CAG repeats, and were therefore considered to have a high risk of developing HD. Fifty-two individuals have a mutable normal allele, and 30 individuals carry an allele in the reduced penetrance range. A total of 540 individuals have alleles in the nonexpanded range less than 27 CAG repeats (see table 1). The mean age of the whole group was 34.8 years. However, approximately 50% of the population at risk requested molecular testing between 23 and 33 years, with a clear predominance of women to men (ratio, 1.37). The remaining individuals at risk show a women-to-men ratio of We carried out 24 prenatal diagnoses. Fourteen parents were carriers of a CAG expansion for a total of 15 pregnancies. One woman requested a prenatal diagnosis in three subsequent pregnancies. The result of the first prenatal molecular test identified her as a carrier of the HD mutation. Nine parents had a 50% risk of being a carrier. The resulting average of the a priori risk for the fetuses was approximately 40%. Six fetuses were identified as carriers. The difference between the observed and expected number of carrier fetuses is not relevant ( 2 1.5; p 0.2). One Table 2 Distribution of the affected patients in relation to their family history Type of family history 39 CAG repeats; HD confirmed CAG repeats; mutable normal allele/ reduced penetrance 27 CAG repeats; HD not confirmed Total Positive ,017 Uncertain Negative Total 1, ,285 HD Huntington s disease. September (1 of 2) 1999 NEUROLOGY

4 Figure 2. Chart illustrating the cumulative frequency of identified patients grouped in relation to the family history in each age group. The x-axis represents the upper limit of each age group. The y-axis is the cumulative frequency of detected patients in percent. family history positive; y family history uncertain; family history negative. pregnancy continued; two subsequent pregnancies in the same woman, who herself carried the expansion, were terminated. No further information was available for the remaining three pregnancies. Discussion. Estimating the population of HD patients to be approximately 10,000 in Germany, Austria, and Switzerland, approximately 20% of the HD patients have been tested for the CAG repeat expansion of the HD gene. The population investigated also contained patients for whom HD was not considered to be the primary cause for the condition but was requested to be excluded (exclusion analysis). This could explain the lower rate of confirmation of diagnosis in our population compared with that of previously published studies. 11 In 2,285 patients it was possible to correlate the presence of an expanded allele with the family history. A total of 93% of the patients with FHP carried a CAG expansion, which highlights the significance of a pedigree analysis for the diagnosis. The rate of patients having a CAG expansion decreased to 53.4% in the FHU group and decreased to 31.2% in the FHN group. We identified two patients with two expanded alleles. Unfortunately we do not have enough clinical data to describe their phenotype exhaustively. However, the age at diagnosis for the patient harboring 39 and 43 CAG repeats was 55 years, and for the patient having 39 and 62 CAG repeats was 22 years. This fits perfectly with the mean age of the patients carrying 43 repeats (mean age, 55.8 years; n 304) and 62 repeats (mean age, 22.6 years; n 5), and it might suggest that the phenotype is determined predominantly by the larger expanded allele. The confirmation of the diagnosis in FHN patients is the lowest described until now. This is probably due to the composition of our group, which was not selected clinically and is therefore quite heterogeneous. However, the identification of the CAG repeat expansion in approximately one-third of the FHN 804 NEUROLOGY 53 September (1 of 2) 1999 group clearly demonstrates that the diagnosis of HD has to be considered for clinically affected individuals even in the absence of a positive family history. It remains to be determined which factors in particular contribute to such a high rate of HD within the FHN group. Nonpaternity, which is thought to be as high as 10% in high industrialized populations, new mutations, or early death of progenitors before manifestation of the disease are adding to this number. It can be hypothesized that alleles in the reduced penetrance/intermediate range progress to the range of full penetrance through moderate meiotic expansions. Alternatively, alleles in the low pathologic range (39 through 41 CAG repeats) may become fully penetrating relative to age. It is possible that parental generations were not affected because they did not reach the age of full penetrance of the alleles. Both hypotheses might explain the lowest average level of CAG repeats and the highest mean age, which was characteristic for our group of FHN HD patients. Analogous characteristics have been found in a group of 36 patients with FHN. 12 Our assessment cannot address the question of the new mutation rate in HD. In this respect it is interesting, however, that patients with FHN make up 8.9% of the whole group. This frequency was proposed prior to direct molecular testing as a realistic percentage of new mutations in HD. 13 Several reports have confirmed the existence of new mutations in HD. 4,6,14,15 Except for some cases, in which the analysis of the parental CAG demonstrated the presence of fresh mutations definitively, 16,17 most cases of recently reported new mutations 4,6,14,15 are biased by the lack of information regarding parental DNA or by the lack of exclusion of slight symptoms of HD in the parents. In our opinion the issue of the new mutation rate of HD cannot be cleared completely. As a matter of fact, considering the demographic profile of patients with FHN, it is practically impossible to analyze the DNA

5 of the patients parents in most cases to discriminate between cases caused by new mutations and cases of pseudo new mutations due to the early death of the carrier parent. However, our data are likely to portray the realistic probability of finding a CAG expansion in an unselected population of patients with FHN. Because details of the symptoms of these patients were not always available, it was not possible to define the phenotype in the patients with FHN. Additional studies characterizing the phenotype of patients who are carriers of the HD mutation and of patients who are clinically considered as having HD but do not have a CAG expansion would be worthwhile. In the juvenile-onset group, 17 probands (25%) were found to carry an expanded HD allele, which was much lower than in the classic or late-onset groups. This stresses the rarity of juvenile forms, although in our group they may have been underestimated because we did not consider age at onset, but rather age at diagnosis. Furthermore, this indicates the difficulty of an accurate selection of symptoms considered as first signs of manifestation of HD for patients in this group. Of the 13 juvenile cases with FHP who were tested, only 6 had an expansion mutation. The confirmation of the diagnosis in this group was much lower (46.2%) than that observed for the whole group of FHP patients. It can be hypothesized that nonspecific symptoms have been attributed mistakenly to HD in the presence FHP or even that molecular testing is requested under the pretense of a differential diagnosis to obtain a presymptomatic test for children. There is sometimes a conflict between the parents wish to test their children and the refusal of the testing laboratory according to the guidelines set for DNA analysis. 8 In fact, we identified a supposedly affected child who was 7 years old with 17 and 42 CAG repeats. Despite repeated requests, it was not possible to obtain detailed documentation attesting to the clinical state of the child. A similar situation has been described recently in a child carrying an allele with 43 CAG repeats with EEG changes and autism, who was tested at age 11 years. 18 In both cases the family history was positive. It is unlikely that these symptoms are early manifestations of the disease due to the low pathologic range of the repeat expansion. These two examples stress the necessity for the insistence on obtaining clinical documentation on children referred for an HD diagnostic test, particularly if the family history is positive. The demand for presymptomatic testing has been low, considering that only 3% of the eligible population decided to be tested. The relatively little interest in the test has already been observed, and our data confirm this attitude of the population at risk. 19,20 The predicted demand for presymptomatic testing after the cloning of the gene was not apparent. In our countries, the number of requests for a presymptomatic test plateaued during the last 2 years (approximately 160 requests per year). We do not expect a dramatic change in the demand for predictive testing in the future unless a decisive therapy for delaying the onset of the disease is found. Women are, in general, more motivated in requesting the test, particularly from 23 to 33 years of age, during which time the majority of women make decisions regarding family planning. This prevalence of women is not related to the female-to-male ratio in the general population, because there are slightly more men than women in this age group. 21 We found that 38.5% of individuals at risk are carriers of an expanded CAG repeat. This percentage of carriers is definitively different from the expected 50%. Most probably this is due to the presence of probands with a 25% or less a priori risk in our group rather than due to a selective force acting on individuals at risk. Alternatively, it is possible that individuals at risk with moderate symptoms did not request the molecular test. Prenatal diagnoses were a rare request. Unfortunately, we do not know the total number of pregnancies at risk during which parents at risk for HD had the option of a prenatal diagnosis to draw any conclusions. Acknowledgment The authors thank Prof. W. Engel, as initiator of the project, for his continuous encouragement, incitement, and critical advice. They also thank Profs. J.T. Epplen, U. Müller, and E. Schwinger for their support; and Profs. R. Witkowski, A. Schinzel, and B. Zoll, as well as Dr. R. Maiwald for critical review of the manuscript. Furthermore they thank Drs. H.N. Aschauer, D. Emmerich, and L. Pfeiffer, who collected data on numerous patients. They are grateful to all members of the consortium and to the patients support organization and are very obliged to the patients families. References 1. The Huntington s Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington s disease chromosomes. Cell 1993;72: Koeppen AH. The hereditary ataxias. J Neuropathol Exp Neurol 1998;57: ACMG/ASHG Statement. Laboratory guidelines for Huntington disease genetic testing. The American College of Medical Genetics/American Society of Human Genetics. Huntington Disease Genetic Testing Working Group. Am J Hum Genet 1998;62: Goldberg YP, Kremer B, Andrew SE, et al. Molecular analysis of new mutations for Huntington s disease: intermediate alleles and sex of origin effects. Nat Genet 1993;5: McNeil SM, Novelletto A, Srinidhi J, et al. Reduced penetrance of the Huntington s disease mutation. Hum Mol Genet 1997;6: Goldberg YP, McMurray CT, Zeisler J, et al. Increased instability of intermediate alleles in families with sporadic Huntington disease compared to similar sized intermediate alleles in the general population. Hum Mol Genet 1995;4: Nance MA. Huntington disease another chapter rewritten. Am J Hum Genet 1996;59:1 8. Editorial. 8. International Huntington Association (IHA) and the World Federation of Neurology (WFN) Research Group on Huntington s Chorea. Guidelines for the molecular genetics predictive test in Huntington s disease. Neurology 1994;44: Warner JP, Barron LH, Brock DJ. A new polymerase chain reaction (PCR) assay for the trinucleotide repeat that is unstable and expanded on Huntington s disease chromosomes. Mol Cell Probes 1993;7: Riess O, Noerremoelle A, Soerensen SA, Epplen JT. Improved PCR conditions for the stretch of (CAG)n repeats causing September (1 of 2) 1999 NEUROLOGY

6 Huntington s disease. Hum Mol Genet 1993;2:637 (erratum: Hum Mol Genet 1993;2:1523). 11. MacMillan JC, Snell RG, Tyler A, et al. Molecular analysis and clinical correlations of the Huntington s disease mutation. Lancet 1993;342: Nance MA, Westphal B, Nugent S. Diagnosis of patients presenting to a Huntington disease (HD) clinic without a family history of HD. Neurology 1996;47: Bundey S. New mutations in Huntington s chorea. J Med Genet 1983;20: Letter. 14. Nance MA. Genetic testing of children at risk for Huntington s disease. US Huntington Disease Genetic Testing Group. Neurology 1997;49: Myers RH, MacDonald ME, Koroshetz WJ, et al. De novo expansion of a (CAG)n repeat in sporadic Huntington s disease. Nat Genet 1993;5: Squitieri F, Andrew SE, Goldberg YP, et al. DNA haplotype analysis of Huntington disease reveals clues to the origins and mechanisms of CAG expansion and reasons for geographic variations of prevalence. Hum Mol Genet 1994;3: Bozza A, Malagu S, Calzolari E, Novelletto A, Pavoni M, del Senno L. Expansion of a (CAG)n repeat region in a sporadic case of HD. Acta Neurol Scand 1995;92: Sanchez A, Mila M, Castellvi Bel S, et al. Maternal transmission in sporadic Huntington s disease. J Neurol Neurosurg Psychiatry 1997;62: Codori AM, Hanson R, Brandt J. Self-selection in predictive testing for Huntington s disease. Am J Med Genet 1994;54: Craufurd D, Dodge A, Kerzin Storrar L, Harris R. Uptake of presymptomatic predictive testing for Huntington s disease. Lancet 1989;2: Statistisches Jahrbuch fuer die Bundesrepublik Deutschland Wiesbaden: Metzler-Poeschel. Clinical correlation of striatal 1 H MRS changes in Huntington s disease R. Sánchez Pernaute, MD; J.M. García Segura, PhD; A. del Barrio Alba, PhD; J. Viaño, MD; and J.G. de Yébenes, MD Article abstract Objective: To study the clinical significance of metabolic alterations as measured in vivo with proton MRS in the striatum of patients with Huntington s disease (HD). Methods: Localized, single-voxel MRS was performed on the basal ganglia of 10 HD patients (4 presymptomatic gene carriers and 6 akinetic patients) and 5 age-matched healthy individuals. Metabolite quantification was performed by referring the areas of the respective spectral peaks to that of water in the analyzed voxel. The spectroscopic findings were correlated with motor and cognitive performance in several specific tests and with the length of the CAG repeat expansion normalized for age. Results: N-acetylaspartate (NAA) and creatine were reduced markedly in both groups of patients, particularly in the advanced group (approximately 60%), but the decrease was also significant in presymptomatic patients (approximately 30%) whose motor and cognitive performances were within the normal range. Both metabolites correlated highly with the motor score of the Unified Huntington s Disease Rating Scale and with computed measurements of saccadic and tapping speed. Creatine reduction was also well correlated with performance in cognitive timed tasks and with the length of CAG expansion (r 0.81). Conclusion: The creatine signal appears to be an interesting marker for progression in HD and could be useful in assessing therapeutic outcome, particularly during the initial stages when most clinical indices are still within the normal range. Key words: Proton MRS Huntington s disease Striatum. NEUROLOGY 1999;53: Huntington s disease (HD) is a neurodegenerative disorder caused by an unstable CAG trinucleotide expansion in the Huntington gene (IT15). 1 Striatal projection neurons and some cortical neurons are damaged selectively, despite wide expression of the abnormal protein huntingtin. 2 To account for this selective vulnerability, energy metabolism failure and excitotoxicity have been considered as likely pathogenic mechanisms. 3 Support for this theory comes from experimental models that reproduce either the clinical picture or the pathologic features of HD in animals, 4,5 and from reports of mitochondrial defects in the caudate nucleus of HD patients. 6,7 In vivo proton MRS provides a noninvasive means of assessing metabolites in brain. Jenkins et al., 8 using this technique, described an elevation of lactate an indicator of energy metabolism impairment in the occipital cortex of HD patients. The same group 9 reported a favorable effect of coenzyme Q on cortical lactate levels, and more recently they reported 10 a positive correlation in symptomatic patients between CAG length and several spectroscopic ratios. Taylor From the Department of Neurology (Drs. Sánchez Pernaute, del Barrio Alba, and de Yébenes), Fundación Jiménez Díaz, Universidad Autónoma; the Neuroradiology Unit (Drs. García Segura and Viaño), Clínica N a S a del Rosario; and the Department of Biochemistry and Molecular Biology (Dr. García Segura), Facultad de Biología, Universidad Complutense, Madrid, Spain. Supported in part by a fellowship of the Conchita Rábago Foundation (R.S.-P.). Received October 6, Accepted in final form April 10, Address correspondence and reprint requests to Dr. R. Sánchez Pernaute, Laboratory of Molecular Medicine and Neuroscience, NINDS Building 36, Room 5W21 36, Convent Drive, Bethesda, MD ; pernaute@codon.nih.gov 806 Copyright 1999 by the American Academy of Neurology

7 Table 1 Sample characteristics Variable HD1 patients (initial) HD2 patients (akinetic) All HD patients Control subjects n Sex, M/F 4/0 2/4 6/4 3/2 Age, y; mean SD (range) 30 4 (23 34) 28 4 (22 32) (21 34) 23 3 (21 28);NS (CAG) n ; mean SD (range) (43 46) 55 7 (50 70) 52 8 (43 70) NA FCS; mean SD (range) 13 0 (13) 8 2 (5 12) (5 13) NA Approximate evolution, y (0 1) (4 6) (0 6) NA FCS Functional Capacity Score rated according to Shoulson and Fahn s scale 12 ;NS not significant (two-tailed t-test); NA not applicable. Robinson et al. 11 communicated significant alterations of the proton spectra in HD, including an increase in the glutamine-to-creatine ratio but no lactate increase. The increase of glutamine was interpreted as an indication of an excitotoxic mechanism. Apart from gaining insight into HD pathogenesis, the promise of proton spectroscopy would be to detect and to quantify metabolic dysfunction in relation to neurologic condition. With such a view, we investigated the correlation between the spectroscopic alterations of the basal ganglia of 10 HD patients and their performance on several motor and cognitive tasks. Methods. Patients. Ten HD patients regularly attending the Movement Disorders Unit of Fundación Jiménez Díaz (University Autónoma, Madrid) and five age-matched control subjects (table 1) agreed to participate in this study. The protocol was approved by the ethics committee of the Fundación Jiménez Díaz, Madrid, Spain. Choreic patients were excluded deliberately; thus we had either presymptomatic gene carriers or advanced akinetic patients. Accordingly, two groups were defined: initial HD1 and advanced HD2. The HD1 group comprised four individuals considered asymptomatic (although in two of them soft motor signs were already apparent) who had undergone genetic predictive testing according to recommended guidelines of the World Federation of Neurology 1 to 3 years before entering this study. The HD2 group included six akinetic, young patients. As shown in table 1, HD2 patients had longer CAG expansions ( 50) and earlier onset of the disease, and thus mean age was similar in both groups. None of the patients had ever received neuroleptic treatment. Molecular analysis of the CAG expansion was performed in all patients as described previously. 13 Neurologic assessment. Motor impairment was rated according to the Unified Huntington s Disease Rating Scale motor score. 14 In addition, horizontal saccadic speed (with fixed predictable stimulus) measured with video nystagmo-oculography, 15 and tapping speed on a computer keyboard (calculated for each hand as the time needed to make 40 pulsations) were computed. Four timed tests sensitive to prefrontal dysfunction were selected from the full battery proposed in the Core Assessment Program for Intracerebral Transplantation in Huntington s Disease protocol 16 : 1. Verbal fluency 17 : to measure the speed and ease of word generation for semantic (animals) and phonologic (p) categories 2. Trail-making tests A and B 18 : to measure the speed and flexibility of sequential shifting Figure 1. MR images depict the selected voxel for spectroscopic acquisition (above) and the resulting proton spectra in a representative case of each group (below). (A) Control subject. (B) Asymptomatic gene carrier (patient with initial Huntington s disease (HD1). (C) Akinetic patient (HD2). Marked atrophy of the striatum and reduction of metabolite signals can be observed in HD2 (C) and milder changes but already notable in HD1 (B). The thickness of the spectroscopic voxel was slightly larger than that of the depicted MRI slices, extending into the adjacent slice above and below. These two slices were examined to ensure the absence of CSF contamination in the volume of interest. Cho choline-containing compounds; Cr creatine; NAA N-acetylaspartate. September (1 of 2) 1999 NEUROLOGY

8 Table 2 Neurologic scores and their correlation with CAG, creatine, and NAA in Huntington s disease (HD) Control subjects, n 5 HD1 patients, n 4 HD2 patients, n 6 All HD patients, n 10,r(p) Variable Mean (SD) Range Mean (SD) Range Mean (SD) Range CAG* Creatine NAA MMS score 29.8 (0.4) (0.8) c (4) NS NS NS TMA, sec 30 (8) (3) (64) NS NS NS TMB, sec 48 (18) (9.5) ,a (48) ( 0.05) NS NS SDMT score 52 (7.5) (10) ,a (6) ( 0.005) 0.8 (0.005) NS VF(p) 16 (3) (8) ,b (2) (0.01) 0.66 (0.05) NS VF(c) 22 (5) (7) ,b (3.6) ( 0.05) 0.72 (0.01) 0.74 ( 0.01) S word 123 (19) (3) ,a (21) ( 0.01) 0.76 ( 0.01) NS S color 79 (18) (4.8) ,b (12) (0.01) 0.78 ( 0.01) NS S interf 50 (14) (2) ,c (13) 0 33 NS 0.76 ( 0.01) 0.76 ( 0.01) UHDRS score NA 4.5 (6) ,a (13) ( 0.005) 0.83 ( 0.005) 0.73 ( 0.01) Hsacc, deg/sec 385 (52) (51) ,a (21) ( 0.005) 0.85 ( 0.005) 0.74 ( 0.01) Tapping, sec 5.2 (0.3) (0.5) ,c (9) NS NS 0.85 ( 0.005) Significant differences between advanced and initial patients are marked with numbers, and significant differences between patients and control subjects are marked with letters. 1,a p 0.001; 2,b p 0.01; 3,c p 0.05, analysis of variance followed by Bonferroni s correction. * Significant correlation coefficients between neurologic scores and CAG expansion corrected for age (CAG n 35.5) years of age, creatine, and N-acetylaspartate (NAA) are shown in the last column (control subjects were not included in correlation analyses). HD2, n 3 (three patients were unable to complete the test in 6 minutes). HD2, n 5 (one patient was daltonic). Mean of left and right hands. HD1 patients with initial stage of HD; HD2 patients with akinetic HD; MMS Folstein s mini mental status; TMA Trail Making Test A; TMB Trail Making Test B; SDMT Symbol Digit Modalities Test; VF(p) phonologic verbal fluency; VF(c) categoric verbal fluency; S word Stroop Test, word reading; S color Stroop Test, color naming; S interf Stroop test, interference; UHDRS Unified Huntington s Disease Rating Scale; NS not significant; Hsacc speed of horizontal saccadic movements. 3. Stroop test 19 : to rate the ability to inhibit automatic response and the capacity of shifting performance to changing demands 4. Symbol Digit Modalities Test 20 : to rate attention and visuomotor coordination with time limitation (90 seconds) Folstein s Mini-Mental State Examination 21 was also included as a global indicator of intellectual function. MRI and MRS. Acquisition. MRI and MRS were performed on a 1.5-T Signa Advantage unit (General Electric Medical Systems, Milwaukee, WI) using the standard quadrature head coil as both the transmitter and the receiver. MR studies and clinical evaluation were performed within the same month. T2-weighted axial images of the basal nuclei were obtained with a fast spin echo sequence with a repetition time (TR) of 4,400 msec, an echo time (TE) of 119 msec, and a field of view of 22 cm. The slice orientation was parallel to the orbitomeatal line. The axial plane that cut through the mid-axial section of the caudate nucleus was chosen to measure the bicaudate ratio (BCR) as described. 22,23 Axial images were used to prescribe regions of interest (ROIs) on the left striatum from which proton spectra were subsequently acquired. Care was taken to include in the ROI as much caudate nucleus as compatible with the absence of contamination from the adjacent ventricular horn (figure 1). Mean volume of the ROI was 4.54 cm 3. Atrophy of the caudate nucleus forced us to define slightly smaller 808 NEUROLOGY 53 September (1 of 2) 1999 volumes in advanced patients (mean volume, 3.27 cm 3 )to minimize contribution from other structures. Proton spectra were acquired with the PROBE procedure of the General Electric spectroscopic package (release 5.6, Milwaukee, WI), working in a double-echo spin mode (probe p) and using a TR of 1,500 msec and a TE of 288 msec. 24 In all subjects, prescan automatic optimization resulted in both 6 Hz or less of full width at half height of the unsuppressed water peak and percentages of water suppression higher than 98%. A total of 256 or 512 acquisitions were accumulated, depending on ROI dimensions. In addition, the probe procedure accumulated automatically 16 nonwater-suppressed signals that were of use as an internal reference (discussed later). Data processing. Acquired signals consisting of two frames of raw data (nonwater suppressed and water suppressed) were transferred to a Sun workstation (SPARC station 2, SunMicrosystems, Mountain View, CA) and processed with the spectral analysis software from General Electric (SA/GE version 3.0, Milwaukee, WI). Processing of water-suppressed data consisted of eddy current correction 25 (using the nonwater-suppressed peak as a reference), apodization (exponential multiplication, line broadening, 3.5 Hz; followed by hemigaussian multiplication line broadening, 10 Hz), zero filling from 2 to 8 K data, fast Fourier transformation, zero-order phase correction, and frequency assignment of 304 Hz (4.7 ppm) to the residual water peak. No baseline correction was required except

9 Figure 2. Individual values of metabolites and bicaudate ratio (BCR) measurements. Metabolite values are ratios to water signal ( 10 3 ). NAA N-acetylaspartate; HD1 patient with initial Huntington s disease (HD); HD2 patient with akinetic HD. for one HD1 patient in whom a linear tilt was performed. Processing of nonwater-suppressed data was performed in the same way, excluding the eddy current correction step. The resulting spectra were analyzed by the fitting routine included in the SA/GE package (Marquardt Levenberg algorithm), obtaining for each patient the series of gaussian curves that best fit either the metabolic peaks at 2.0 ppm (N-acetylaspartate), 3.0 ppm (creatine/ phosphocreatine), and 3.2 ppm (choline-containing compounds) in the water-suppressed spectrum, or the peak from water in the corresponding nonwater-suppressed spectrum. For each ROI, the areas of the curves fitted to the metabolite peaks were referred to that of the respective water peak, which was used as an internal reference. 26 Statistics. Statistical analyses were performed with the aid of the Statistical Package for Social Sciences (SPSS; release 7.5 for Windows, SPSS Inc., Chicago, IL). Comparisons among groups (control subjects, HD1, and HD2) were performed using analysis of variance followed by Bonferroni s multiple comparison post hoc test. Excluding control subjects, bivariate correlations were calculated between spectroscopic data, BCR, and clinical scores. The product of triplets in excess (CAG n 35.5) years of age was used 27 to correlate neurologic and spectroscopic data with the severity of the genetic defect. Results. Neurologic data. The results of the motor evaluation and cognitive performance are summarized in table 2. The scores of the HD1 group were not significantly different from control subjects. On the other hand, the HD2 patients had a poor performance on every test. Considering all HD patients, most clinical scores showed a significant decline correlated with the length of the expansion corrected for age (CAG n 35.5) years of age. Significant correlation coefficients between neurologic scores and CAG are presented in table 2. Spectroscopic data. Representative images and spectra for each group are shown in figure 1, illustrating the atrophy of the striatum and the reduction of metabolite signals in the HD2 group (see figure 1C), and similar al- Table 3 Spectroscopic and bicaudate ratio (BCR) statistics Control subjects (n 5) HD1 patients (n 4) HD2 patients (n 6) Variable Mean (SD) Range Mean (SD) Range Mean (SD) Range NAA 6.9 (0.7) b (0.3) ,a (1.1) Creatine 3.53 (0.6) c (0.3) ,a (0.3) Choline 3.76 (0.6) (0.4) c (1.1) BCR (0.005) (0.017) ,b (0.04) Significant differences between advanced and initial patients are marked with numbers, and between patients and control subjects are marked with letters: 1 p 0.01; 2 p 0.05; a p 0.001; b p 0.01; c p 0.05; ANOVA, followed by Bonferroni s correction. HD1 patients with initial Huntington s disease (HD); HD2 patients with akinetic HD; NAA N-acetylaspartate. September (1 of 2) 1999 NEUROLOGY

10 though milder changes in the HD1 group (see figure 1B). The results of spectroscopic metabolite quantification and BCR measurements are represented in figure 2, and the statistics are summarized in table 3. NAA was decreased significantly in all HD patients (see figure 2). Mean reduction was 36% for the HD1 group and 66% for the HD2 group. NAA levels correlated with caudate atrophy as measured by BCR (r 0.73, p 0.01). NAA decrease was well correlated with motor scores but not with cognitive scores (see table 2) or CAG length (r 0.45, p 0.06). Creatine levels were reduced significantly in HD patients, although individual values showed some overlap (see figure 2). The HD1 group showed a mean decrease of 30% and the HD2 group exhibited a mean decrease of approximately 60% of control levels. The decrease of creatine showed significant associations with all clinical scores excluding the mean computed tapping speed, the Trail Making Test, and mini mental status. Significant correlations are shown in table 2. Creatine correlated strongly with the length of the CAG expansion normalized for age (r 0.81, p 0.005). With regard to the choline signal, only the HD2 group showed a significant decrease (50%) because the scatter of the data was considerable (see figure 2). No clear association with clinical or genetic indices was found for this metabolite. BCR was grossly enlarged in the HD2 group ( 138% of control subjects). The increase in the initial group (HD1) was also evident ( 64% of control subjects), even if it did not reach statistical significance (mean difference, ; p 0.1). As mentioned earlier, BCR correlated inversely with NAA but not with creatine (r 0.5) or choline. The relationship with clinical scores was not significant. Discussion. We observed a remarkable reduction of both NAA and creatine in the basal ganglia of HD patients (see figure 2). Interestingly, metabolite changes were already significant in the group of patients without relevant neurologic impairment (HD1 patients; see tables 2 and 3). Moreover, the changes in creatine levels correlated significantly with several motor and cognitive neurologic scores (see table 2) and with the length of CAG expansion. Although increased CSF content in the localized volumes cannot be ruled out completely, it does not seem to represent a major contribution to the observed reductions in spectral peak intensities because relevant CSF contamination would affect all metabolites in parallel, which was not the case (see figure 2). In addition, the absence of obvious partial volume effects from ventricles was made certain in every study by visual inspection of all MRI slices covered by the spectroscopic voxel (see figure 1). Lastly, the values reported in table 3 for the different metabolites correspond with the ratios between their respective signal intensities and that of the water from the same voxel (see the discussion of data processing in Methods). In this way, the calculated metabolite-to-water ratios can be compared among the different subjects by taking the water signal 810 NEUROLOGY 53 September (1 of 2) 1999 as a valid internal reference. To assess metabolite changes it is mandatory to perform absolute quantification because using peak ratios can yield ambiguous results. For such a purpose the use of the water signal as an internal standard has been proposed, 25 but calculating absolute metabolite concentrations in this way could result in some error because the concentration of water standard is not known precisely. 28 In addition, because it is impractical to collect fully relaxed spectra, correction for T1 and T2 relaxation times are necessary. To our knowledge no striatal metabolite relaxation times have been reported. This led us to refer the intensity of metabolite signals to that of water, obtaining ratios that have an absolute value in themselves (although they still require the above-mentioned correction for striatal T1 and T2 to translate them into concentrations). The decrease in NAA the neuronal marker in the proton spectra correlated inversely to the BCR, a good index of caudate atrophy in HD patients. 22 Atrophy of the caudate nucleus as measured by MRI volumetry has been demonstrated to occur before clinical onset In this study we found a significant reduction of NAA in the initial group (see table 3), but this metabolite showed no significant relationship to either the repeat length by age or by cognitive performance. A similar lack of association between caudate/basal ganglia atrophy and several neuropsychological scores has been reported. 32,33 The available information from recent clinical, pathologic, and experimental studies supports a major role of the CAG expansion in the progression of the disease. 27,34-37 Accordingly, most clinical scores measured in this study appeared associated significantly with the length of the expansion by age. These tests measure processing speed, which is considered a reliable indicator of disease severity and progression. The creatine peak proceeds from the combined contribution of creatine plus phosphocreatine. Switching between both forms from dephosphorylated to phosphorylated creatine and vice versa constitutes an efficient way of reserving and obtaining energy as required. Several lines of evidence point to energy impairment as a pathogenic mechanism of neuronal death in HD. 3 The early and progressive reduction of creatine/phosphocreatine levels associated with clinical and molecular evolutive indices (see figure 2 and table 2) supports the theory of energy impairment underlying striatal dysfunction. This has also been argued as an explanation for the increased levels of lactate found in proton spectra of HD patients However, we did not find increased lactate contribution in any spectrum acquired from either the basal ganglia (this study) or the occipital lobe (unpublished data) in HD patients. Jenkins et al has repeatedly reported increased lactate levels in HD patients, but in their recent study 10 they acknowledge that lactate levels are found to fall within the normal range in some patients with clinical HD (p 1359). 10 They report

11 normal levels in all occipital and in five of eight striatal spectra obtained in presymptomatic gene carriers (none of them on psychoactive drugs). On the other hand, of 31 patients they include data from 8 patients on neuroleptics and 7 patients on antidepressants. Thus one factor that might be relevant is that our patients had never received neuroleptics and were not taking any psychoactive drug at the time of the study. In another study Taylor Robinson et al. 11 found no abnormalities in lactate in either the striatum, thalamus, or the temporal or occipital cortex. Their patients were also drug free for more than 3 months at the time of the study. Another interesting result from the study by Jenkins et al. 10 is a significant correlation between the length of the CAG expansion and NAA and lactate levels measured as ratios to creatine in symptomatic HD patients. There is a major methodological difference preventing further comparisons between both studies, which is the fact that they use creatine as a reference when, according to our results, creatine is affected profoundly in HD and the changes in this metabolite might account for significant variations in the ratios. Nevertheless, despite apparent discrepancies, both studies share a common conclusion: MRS in HD can detect metabolic abnormalities that correlate with the severity of the molecular CAG defect and that point to an alteration of energy metabolism. Very recently a detailed biochemical study in HD brains 38 has confirmed a selective deficit of aconitase activity (a sensitive marker of excitotoxic cell damage) in the striatum (8 to 27% of normal) and cortex (52%), further supporting the role of energy failure and excitotoxicity in the pathogenesis of this disorder. Considering the major spectroscopic alterations reported herein, the decrease of neuronal density (NAA), and the impairment of energy metabolism (creatine), proton spectroscopy appears to be a valuable tool for obtaining both structural and functional information from the basal ganglia in HD patients. As in other studies combining functional and volumetric measurements, 39,40 atrophy and metabolic dysfunction seem closely related but might progress independently because metabolic markers appear better correlated with neurologic condition. We propose that the creatine signal might become a useful outcome measure, particularly in early HD stages when other indices are normal or only slightly impaired, making the assessment of progression or recovery very difficult. Acknowledgment The authors thank the patients for their participation in this study and the technical staff of the MR unit for their assistance. We also thank Vicenta Sánchez Bernardos, who did the tapping registration; Francisco Esperón, who performed the oculographic measurements; and Pedro García Ruíz, for sharing these data with us. R.S.P. gratefully acknowledges a fellowship of the Conchita Rábago Foundation. References 1. Huntington s Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is unstable in Huntington s disease chromosomes. Cell 1993;72: Sharp AH, Ross CA. Neurobiology of HD. Neurobiol Dis 1996; 3: Young AB. Impairment of energy metabolism and excitotoxic cell death in Huntington s disease. Rev Neurol (Paris) 1997; 153: Brouillet E, Hantraye P, Ferrante RJ, et al. Chronic mitochondrial energy impairment produces selective striatal degeneration and abnormal choreiform movements in primates. Proc Natl Acad Sci USA 1995;92: Burns LH, Pakzaban P, Deacon TW, et al. Selective putaminal excitotoxic lesions in non-human primates model the movement disorder of Huntington disease. Neuroscience 1995;64: Gu M, Gash MT, Mann VM, Javoy Agid F, Cooper JM, Schapira AHV. Mitochondrial defect in Huntington s disease caudate nucleus. Ann Neurol 1996;39: Brownne SE, Bowling AC, MacGarvey U, et al. Oxidative damage and metabolic dysfunction in Huntington s disease: selective vulnerability of the basal ganglia. Ann Neurol 1997; 41: Jenkins BG, Koroshetz WJ, Beal MF, Rosen BR. Evidence for an energy metabolism defect in Huntington s disease using localized proton spectroscopy. Neurology 1993;43: Koroshetz WJ, Jenkins BG, Rosen BR, Beal MF. Energy metabolism defects in Huntington s disease and effects of coenzyme Q10. Ann Neurol 1997;41: Jenkins BG, Rosas HD, Chen Y-CI, et al. 1H NMR spectroscopy studies of Huntington s disease. Correlations with CAG repeat numbers. Neurology 1998;50: Taylor Robinson SD, Weeks RA, et al. Proton magnetic resonance spectroscopy in Huntington s disease: evidence in favour of the excitotoxic theory? Mov Disord 1996;11: Shoulson I, Fahn S. Huntington s disease: clinical care and evaluation. Neurology 1979;29: Benítez J, Fernández E, García Ruíz PJ, Ramos C, García Yébenes J. Trinucleotide (CAG) repeat expansion in chromosomes of Spanish patients with Huntington s disease. Hum Genet 1994;94: Huntington Study Group. Unified Huntington s Disease Rating Scale: reliability and consistency. Mov Disord 1996;11: Vitte E, Semont A. Assessment of vestibular function by videonystagmoscopy. J Vestib Res 1995;5: Quinn N, Brown R, Craufurd D, et al. Core Assessment Program for Intracerebral Transplantation in Huntington s Disease (CAPIT-HD). Mov Disord 1996;11: Benton AL, Hamsher K de S. Multilingual aphasia examination. Iowa City, IA: University of Iowa Press, Reitan RM. Validity of the Trail Making Test as an indicator of organic brain damage. Percept Mot Skills 1958;8: Stroop JR. Studies of interference in serial verbal reactions. J Exp Psychol 1935;18: Smith A. The Symbol Digit Modalities Test: a neuropsychological test for economic screening of learning and other cerebral disorders. Learn Disord 1968;3: Folstein MF, Folstein SE, MacHugh PR. Mini mental state: a practical guide for grading the mental state of the patient for the clinician. J Psychiatr Res 1975;12: Barr A, Heinze W, Dobben G, Valvassori G, Sugar O. Bicaudate index in computerized tomography of Huntington s disease and cerebral atrophy. Neurology 1978;28: Aylward EH, Schwartz J, Machlin S, Pearlson G. Bicaudate ratio as a measure of caudate volume on MR images. AJNR Am J Neuroradiol 1991;12: Webb PG, Sailasuta N, Kohler SJ, Raidy T, Moats RA, Hurd RE. Automated single-voxel proton MRS: technical development and multisite verification. Magn Reson Med 1994;31: Klose U. In vivo proton spectroscopy in the presence of eddy currents. Magn Reson Med 1990;14: Christiansen P, Henriksen O, Stubgaard M, Gideon P, Larsson HBW. In vivo quantification of brain metabolites by 1H- September (1 of 2) 1999 NEUROLOGY

12 MRS using water as an internal standard. Magn Reson Imaging 1993;2: Penney JB, Vonsattel JP, MacDonald ME, Gusella JF, Myers RH. CAG repeat number governs the development rate of pathology in Huntington s disease. Ann Neurol 1997;41: Ende G, Laxer KD, Knowlton RC, Matson GB, Schuff N, Weiner MW. Temporal lobe epilepsy: bilateral hippocampal metabolite changes revealed at proton MR spectroscopic imaging. Radiology 1997;202: Aylward EH, Brandt J, Codori A, Mangus R, Barta PE, Harris G. Reduced basal ganglia volume associated with the gene for Huntington s disease in asymptomatic at-risk persons. Neurology 1994;44: Aylward EH, Codori AM, Barta PE, Pearlson GD, Harris GJ, Brandt J. Basal ganglia volume and proximity to onset in presymptomatic Huntington disease. Arch Neurol 1996;53: Aylward EH, Li Q, Stine OC, et al. Longitudinal change in basal ganglia volume in patients with Huntington s disease. Neurology 1997;48: Harris GJ, Aylward EH, Peyser CE, et al. Single photon emission computed tomographic blood flow and magnetic resonance volume imaging of basal ganglia in Huntington s disease. Arch Neurol 1996;53: Davies SW, Turmaine M, Cozens BA, et al. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 1997; 90: Becher MW, Kotzuk JA, Sharp AH, et al. Intranuclear neuronal inclusions in Huntington s disease and dentatorubro and pallidoluysian atrophy: correlation between the density of inclusions and IT15 CAG triplet repeat length. Neurobiol Dis 1998;4: Duyao M, Ambrose C, Myers R, et al. Trinucleotide repeat length instability and age at onset in Huntington s disease. Nat Genet 1993;4: Furtado S, Suchowersky O, Rewcastle NB, Graham L, Klimek ML, Garber A. Relationship between trinucleotide repeat and neuropathological changes in Huntington s disease. Ann Neurol 1996;39: Antonini A, Leenders KL, Eidelberg D. [ 11 C]Raclopride PET studies of the Huntington s disease rate of progression: relevance of the trinucleotide repeat length. Ann Neurol 1998;43: Tabrizi SJ, Cleeter MWJ, Xuereb J, Taanman J-W, Cooper JM, Schapira AHV. Biochemical abnormalities and excitotoxicity in Huntington s disease brain. Ann Neurol 1999;45: Grafton ST, Mazziotta JC, Pahl JJ, et al. Serial changes of cerebral glucose metabolism and caudate size in persons at risk for Huntington s disease. Arch Neurol 1992;49: Bäckman L, Robins Wahlin TB, Lundin A, Ginovart N, Farde L. Cognitive deficits in Huntington s disease are predicted by dopaminergic PET markers and brain volumes. Brain 1997; 120: Neuro Images Figure. (A) Sagittal T2-weighted imaging of the cervical cord. (B) Axial gadolinium-enhanced T1-weighted imaging of the C 2 cord level. (C) Magnetic resonance angiography of the neck. (D) The left vertebral arteriogram. Arteriovenous malformation of the cervical cord Ken Ikeda, MD, PhD, Yasuo Iwasaki, MD, Masao Kinoshita, MD, Tokyo, Japan A 53-year-old woman who had a head injury 2 years previously developed muscle weakness and dysesthesia in the lower extremities. These motor and sensory deficits spread to the upper extremities and trunk rapidly, leading to quadriplegia and respiratory failure. Neurologic examination showed absent muscle stretch reflexes in the four extremities and hypalgesia below C 4 levels. Her deep sensations, including vibration and position sense, were normal. T2-weighted images revealed hyperintensity signal areas and antero-marginal hypointensity signal areas in the medulla oblongata and C 1 4 cord (A). Gadoliniumenhanced T1-weighted images disclosed abnormal enhancement in the left extramedullary regions of the C 1 3 cord (B, arrow). Magnetic resonance angiography (C, arrow) and arteriogram of the left vertebral artery (D, arrows) demonstrated arteriovenous malformation of the C 1 3 cord level. 812 NEUROLOGY 53 September (1 of 2) 1999

13 Bilineal transmission in Tourette syndrome Philip A. Hanna, MD; Fatima N. Janjua, MD; Charles F. Contant, PhD; and Joseph Jankovic, MD Article abstract Objective: To investigate the frequency and pattern of bilineal transmission in families of patients with Tourette syndrome (TS) compared with normal control subjects. Methods: The study population consisted of two groups: 1) consecutive patients with TS with both parents (51 family sets; 153 individuals), and 2) normal control subjects randomly selected from public schools (20 family sets; 60 individuals). All patients with TS, normal control subjects, and their parents were evaluated for evidence of TS and associated features. Structured interviews and detailed questionnaires designed to assess tics, obsessive-compulsive behavior (OCB), and attention deficit disorder (ADD) were administered to all people in both groups. Results: In addition to tics, 43 (84.3%) patients with TS had ADD, 33 (64.7%) had OCB, and 31 (60.8%) had both ADD and OCB. In 42 (82.4%) of the 51 patients, at least one parent exhibited features of TS; unilineal transmission (only one parent with tics, OCB, or ADD) was present in 29 (56.9%) TS families and an additional 13 (25.5%) TS families manifested evidence of bilineal transmission (both parents affected). More fathers than mothers of patients with TS had tics (31.4% versus 15.7%), whereas more mothers had OCB than did fathers (33.3% versus 15.7%). Features of ADD were equally distributed among fathers (35.3%) and mothers (33.3%) of patients with TS. Eight of 1,142 (0.7%) children in the general school population had some evidence of TS. One of 40 parents of the normal control subjects (2.5%) had symptoms of ADD, but none of the parents of normal control subjects manifested any features of TS or OCB. Conclusions: This study found evidence for bilineal transmission in one fourth of TS families. Features of the TS spectrum were rare in normal control families. Evidence of tics, OCB, and ADD should be investigated in both parents of patients with TS. Key words: Tourette syndrome Genetics Tics. NEUROLOGY 1999;53: Tourette syndrome (TS) is a childhood-onset, neurobehavioral disorder characterized by motor and phonic tics, frequently accompanied by features such as obsessive-compulsive behavior (OCB) and attention deficit disorder (ADD). 1-7 Several studies have documented increased frequency of family history of tics and comorbid features in patients with TS Family 11 and twin studies 12,13 have provided additional evidence for a strong genetic component in TS. Genetic models described in early family studies included single major locus models and mixed models. 17,18 Segregation analyses of large pedigrees have supported an autosomal dominant pattern and intermediate models of inheritance. 22 Obsessivecompulsive disorder (OCD), but not ADD, has been thought to be genetically related to TS. 23 Walkup et al. 24 suggested a major locus in combination with a multifactorial background. A multicenter sib pair study recently identified at least two TS-related gene markers on chromosomes 4q and 8p. 25 Several studies have provided increasingly compelling evidence for bilineal transmission in TS (both parents of patients with TS with tics, OCB, ADD, or a combination of these features). Comings et al. 25 reviewed 170 TS pedigrees and found a history of tics on both the maternal and paternal sides in 8.2% of families studied. Behaviors including OCB, attention deficit hyperactivity disorder (ADHD), panic attacks, or drug or alcohol abuse were present on both maternal and paternal sides in 34.7% of patients with TS. Kurlan et al. 26 reported bilineal transmission of tics in 15% of 39 families with consecutively evaluated patients with TS. When tics or OCB were included, bilineal transmission was noted in 41% of these families. Other studies have reported bilineal transmission in families of children with TS. 27,28 Before it can be concluded that the higher frequency of TS and associated symptoms in both parents is meaningful, it is necessary to compare the parents of patients with TS with those of agematched, normal control subjects. Our study provides new evidence that the pattern of inheritance of TS is complex and that it is in part determined by transmission of some or all features of TS from both parents to the patient. Methods. To investigate the frequency of bilineal transmission in TS, we studied patients with TS and their parents and normal control subjects and their parents. The study was approved by the Baylor Institutional Review Board for Human Research. The first group consisted of consecutive patients diagnosed with TS according to the TS Classification Study Group criteria, 29 along with both parents. There was a total of 51 family sets (patient with TS and both parents), for a total of 153 people. From the Parkinson s Disease Center and Movement Disorders Clinic (Drs. Hanna, Janjua, and Jankovic), Department of Neurology; and the Department of Neurosurgery (Dr. Contant), Baylor College of Medicine, Houston, TX. Received January 1, Accepted in final form April 10, Address correspondence and reprint requests to Dr. Joseph Jankovic, Department of Neurology, Director of Parkinson s Disease Center and Movement Disorders Clinic, Baylor College of Medicine, 6550 Fannin St. #1801, Houston, TX Copyright 1999 by the American Academy of Neurology 813

14 The second group served as normal control subjects and consisted of students in the second, fifth, and eighth grades at schools in the Houston Independent School District (HISD). The schools in HISD, the largest school district in the city and the fourth-largest urban school district in the nation, were selected to represent a broad socioeconomic distribution. During observations, a physician skilled in recognizing tics and other features of TS (FNJ) sat in such a position in the classroom as to see most of the students faces. The children were not aware, however, of the reason why the observer was in the classroom. The observing physician spent approximately 1 hour in each classroom. All students were numbered in order and all even-numbered students were invited to participate as randomly selected normal control subjects. Although 77 families initially agreed to participate, 33 full family sets (child, father, and mother) completed the questionnaires. Seven families (21 people) were excluded from the control group because the subject exceeded the threshold for ADD based on a score of 14 on the modified ADD scale. 30 Six additional families (18 people) were excluded because the subject had either definite TS or TS by history according to the TS Classification Study Group criteria. 29 The remaining 20 completed family sets (60 people) were used as normal control subjects. Questionnaires designed to assess symptoms of tics, OCB, and ADD were administered by structured interviews to each of three members of a family set (patient with TS or control subject, mother, and father). Children who did not have access to both biologic parents were excluded. A parent was allowed to answer certain questionnaire items for the child if the child appeared to have difficulties understanding the question. Families were asked to complete the questionnaires in the presence of a study physician, and control parents were personally evaluated. During the interview session, the physician reviewed the survey questions in detail and answered any of the subject s questions. Furthermore, the physician clarified the language of the questionnaire if the participant was not familiar with the terminology. Spouses were asked to verify each other s self-evaluations. These structured interviews were conducted in a similar manner for clinic families. The content of the questionnaires included the following: 1. Tourette Syndrome Rating Scale 2. ADD Inventory (modified) Obsessive-Compulsive Disorder (OCD) Inventory (modified) TS Global Disability Rating Scale 5. MOVES (Motor tic, Obsession and compulsion, Vocal tic Evaluation Scale) Survey Y-BOCS (Yale-Brown Obsessive-Compulsive Scale) Symptom Checklist 7. Y-BOCS Scale 33 Subjects were classified as having tics if they fulfilled the criteria for definite TS 29 : multiple motor and one or more vocal tics present at some time during the illness, although not necessarily concurrently; duration of tics 1 year; and onset of tics before 21 years of age. OCB was classified qualitatively based on a score of 10 (of 40) on the Y-BOCS scale, similar to the methodology used by Kurlan et al. 26 Subjects were considered to have features of ADD if they scored 14 (of 70) on the modified ADD inventory NEUROLOGY 53 September (1 of 2) 1999 Figure 1. Percentage of parents (of patients with Tourette syndrome [TS] and of normal control subjects) with symptoms of tics, obsessive-compulsive behavior (OCB), or attention deficit disorder (ADD). Statistical analyses were performed to compare certain parameters (as described for the following groups): fathers (TS families versus control subjects), mothers (TS families versus control subjects), children (patients with TS versus control subjects), and fathers versus mothers of TS families. We also compared the number of fathers (TS families versus control subjects) with regard to tics, OCB, and ADD. Similar analyses were performed for mothers. In addition, we compared the number of patients with TS versus control subjects with one versus both parents exhibiting tics, OCB, or ADD (unilineal versus bilateral transmission). The percentage of parents with each of the TS spectrum symptoms (tics, OCB, or ADD) was further subdivided (figures 1 and 2). Other parameters included scores on the following rating instruments: OCD inventory, ADD inventory, TS Global Disability Rating Scale, MOVES, Y-BOCS current and past symptoms, and Y-BOCS scale. Categorical data were analyzed using contingency tables. The two-sided Fisher s exact test was calculated for each table. Medians of ordinal and scale variables were compared using the Kruskal-Wallis test. Results. A total of 1,142 students were observed in second-, fifth-, and eighth-grade classrooms. Five students were observed to have tics in the classroom; three of these five students fulfilled diagnostic criteria for TS (multiple motor and at least one vocal tic for period of 1 year) and completed the questionnaires. These three students were excluded from the normal control group. One of these students carried a prior diagnosis of TS. The two remaining students did not wish to participate in the study. Three additional students who participated had a history of multiple motor and at least one vocal tic for period of 1 year; although tics were not personally observed by the

15 Figure 2. Percentage of patients with Tourette syndrome (TS) and of normal control subjects in whom one or both parents have tics, obsessive-compulsive behavior (OCB), or attention deficit disorder (ADD). investigator, these three students were excluded from the normal control group. A total of six family sets (18 people) who completed questionnaires were excluded for the aforementioned reasons. Thus, the prevalence of observed tics in our school population was 5 of 1,142 (0.4%), and evidence of TS was found in 8 of 1,142 (0.7%) in the general school population. We found an 84% male preponderance in our population of patients with TS (51 people: 43 boys, 8 girls), compared with a 55% female preponderance in normal control subjects (20 people: 9 boys, 11 girls; p 0.002). The patients with TS were older (mean, 13.7 years; range, 6 to 22 years) compared with normal control subjects (mean, 10.2 years; range, 7 to 14 years; p 0.003). Table 1 summarizes the features of tics, OCB, and ADD in patients with TS and their parents as well as in the normal control subjects. The median values are depicted for the subcategories of OCD inventory (from a total score of 70), ADD inventory (of 70), TS Global Disability Rating Scale (10% fully disabling tics to 100% no tics), MOVES (of 60), Y-BOCS current and past symptoms (each of 74), and Y-BOCS scale (of 40). There is no significant difference in these features between fathers and mothers of patients with TS, but, as expected, there are significant differences in the occurrence of these features between the TS families and normal control families. Specifically, compared with fathers of normal control subjects, fathers of patients with TS had more tics( p 0.003), and ADD ( p 0.008) and a trend toward more severe OCB ( p 0.095). In contrast to mothers of normal control subjects, mothers of patients with TS had more OCB ( p 0.002) and ADD ( p 0.002), but the higher frequency of tics did not reach statistical significance. There were significant differences between patients with TS versus control subjects (children) with regard to tics, OCB, and ADD. Parents of patients with TS had a significantly higher frequency of TS and associated disorders than those of normal control subjects: 24 (23.5%) versus 0 (0%) parents Table 1 Characteristics of controls and patients TS families, n 51 Control families, n 20 p Values Characteristic Fathers (A) Mothers (B) Children (C) Fathers (D) Mothers (E) Children (F) Avs D Bvs E Cvs F Avs B Tics, n (%) 16 (31.4) 8 (15.7) 51 (100) 0 (0) 0 (0) 0 (0) OCB, n (%) 8 (15.7) 17 (33.3) 33 (64.7) 0 (0) 0 (0) 0 (0) ADD, n (%) 18 (35.3) 17 (33.3) 43 (84.3) 1 (5) 0 (0) 0 (0) OCD inventory ADD inventory TS disability, % MOVES Y-BOCS current Y-BOCS past Y-BOCS scale TS Tourette syndrome; OCB obsessive-compulsive behavior; ADD attention deficit disorder; MOVES Motor tic, Obsession and compulsion, Vocal tic Evaluation Scale; Y-BOCS Yale-Brown Obsessive-Compulsive Scale. September (1 of 2) 1999 NEUROLOGY

16 Table 2 Unilineal versus bilineal transmission: occurrence of clinical features in parents of patients with Tourette syndrome Characteristic Fathers Mothers One parent Both parents Tics* 16 (31.4) 8 (15.7) 16 (31.4) 4 (7.8) OCB* 8 (15.7) 17 (33.3) 21 (41.2) 2 (3.9) ADD* 18 (35.3) 17 (33.3) 23 (45.1) 6 (11.8) Tics only 6 (11.8) 2 (3.9) 8 (15.7) 0 (0) OCB only 2 (3.9) 7 (13.7) 9 (17.6) 0 (0) ADD only 8 (15.7) 6 (11.8) 12 (23.5) 1 (2.0) Tics OCB 2 (3.9) 1 (2.0) 3 (5.9) 0 (0) Tics ADD 6 (11.8) 2 (3.9) 6 (11.8) 1 (2.0) OCB ADD 2 (3.9) 6 (11.8) 7 (13.7) 0 (0) Tics OCB ADD 2 (3.9) 3 (5.9) 4 (7.8) 0 (0) Any combination of tics, OCB, ADD 28 (54.9) 27 (52.9) 29 (56.9) 13 (25.5) Values are n (%). * Total number of parents manifesting these features (with or without additional features). OCB obsessive-compulsive behavior; ADD attention deficit disorder. had tics; 25 (24.5%) versus 0 (0%) had OCB, and 35 (34.3%) versus 1 (2.5%) had ADD (figure 1). Of the patients with TS, 56.9% had only one parent with either tics, OCB, or ADD (unilineal transmission), whereas only 5% of normal control subjects had one parent with any of these three features ( p 0.001); 25.5% of patients with TS had both parents with either tics, OCB, or ADD (bilateral transmission) versus 0% of control subjects ( p 0.014; figure 2). Thus, 82.4% of patients with TS had at least one parent with tics, OCB, or ADD. Table 2 shows the number (and percentage) of patients with TS with one (unilineal) or both (bilineal) parents having various combinations of tics, OCB, and ADD. This table also delineates the features of TS in fathers and mothers of patients with TS. There were no statistically significant differences between patients with TS from bilineal families versus patients with TS from either unilineal or sporadic (neither parent with tics, OCB, or ADD) families with regard to the following scores: ADD inventory, Y-BOCS scale, TS Global Disability Rating Scale, and OCD inventory. Discussion. We found evidence for bilineal transmission (both parents of patients with TS with tics, OCB, ADD, or a combination of these features) in 25.5% of patients with TS (0% of normal control subjects), and unilineal transmission (only one parent with tics, OCB, ADD or a combination) in 56.9% of patients with TS (5.0% of normal control subjects). Thus, 82.4% of our patients with TS had at least one parent with tics, OCB, or ADD. Furthermore, 39 of 102 (38.2%) TS parents had evidence of two or more of the TS spectrum traits of tics, OCB, or ADD. Our findings of bilineal transmission are supported by other studies. Walkup et al. 24 performed complex segregation analysis on family study data in 52 independently evaluated children and adolescents with TS and their 154 first-degree relatives. Their data suggested a major locus in combination with a multifactorial background. Although none of the 816 NEUROLOGY 53 September (1 of 2) 1999 families had both parents affected with TS, 19% of families had both parents affected with the broader TS phenotype, including TS, chronic tic disorder, or OCD. Comings et al. 25 reviewed 170 TS pedigrees and found a history of tics on both the maternal and paternal sides in 8.2% of families studied, whereas OCB, ADHD, panic attacks, and drug or alcohol abuse were present on both maternal and paternal sides in 34.7% of patients with TS. Kurlan et al. 26 reported bilineal transmission from 15% (tics) in 39 families of consecutively evaluated patients with TS to 41% (tics or OCB) in high-density families, depending on the defined TS phenotype. These authors did not include ADD in their analyses of bilineal transmission. None of these studies compared their TS populations with normal control subjects. Unlike Kurlan et al., 26 we included ADD in the TS spectrum for analysis of transmission patterns. We were very conservative in diagnosing tics, restricting inclusion to people with TS according to the TS Classification Study Group criteria. 29 We found that more fathers of patients with TS had tics (31.4% versus 15.7%) than did mothers of patients with TS ( p 0.101), and mothers of patients with TS manifested more OCB by history (33.3% versus 15.7%) than fathers of patients with TS ( p 0.064), but this difference was not significant. Symptoms of ADD were rather equally distributed among fathers and mothers of patients with TS. Most (60.8%) of our patients with TS had all three features: tics, OCB, and ADD. In contrast to most other population genetic studies, we examined the frequency of TS and associated disorders not only in our TS population, but in normal control subjects (and their parents) randomly selected from a local school district. A few previous studies have addressed the frequency of TS symptoms in general and in special education populations Furthermore, comparative analyses of TS families

17 versus normal control families with regard to transmission of tics, OCB, and ADD have not been previously performed. We found a prevalence rate of observed tics in 0.4% and definite TS or TS by history 29 in 0.7% of the school population. The prevalence rates of TS have been estimated to vary widely between 1/83, /100,000, 38 and 1/10, In one study of 3,034 students in three schools in Los Angeles observed over a 2-year period, the frequency of TS was found to be 1/95 boys and 1/759 girls. 36 The marked variability in the reported prevalence rates is due to a number of reasons, including the use of different criteria, different populations and ascertainment methods, and other methodologic differences. Assortative mating (in which couples marry based on similar physical or psychological traits) in contrast to random mating may play a role in observed cases of bilineal transmission. McMahon et al. 27 studied 175 members of a large, four-generation, TS family and 16 spouses who had married into this family. Of interest, they found evidence of TS in 36% of the family members and in 31% of the married-in spouses (with some form of tic found in 67% and 44%, respectively). Based on multivariate analysis, tics were more severe in the offspring of parents both of whom were affected with tics. They suggested the possibility of assortative mating in TS families in contrast to nonassortative (random) mating, which is presumed in the general population, but they provided no explanation for this peculiar occurrence. Certain studies, such as those by Hasstedt et al., 22,39 further explored the effect of assortative mating in the transmission of TS. When assortative mating was assumed in analyzing the family data, an intermediate model of inheritance for TS was determined, whereas assuming random mating resulted in a dominant mode of inheritance. Thus, assortative mating can result in ascertainment bias, particularly in large, high-density pedigrees, and this must be taken into account in selection of the most appropriate model of inheritance. 24 If TS is transmitted in an autosomal dominant pattern, a homozygote patient (more likely a product of a bilineal transmission) may not necessarily be more affected than his or her parents or patients with TS from unilineal or sporadic families. This may at least in part explain why we did not find significant differences between patients with TS from bilineal families versus patients with TS from either unilineal or sporadic (neither parent with tics, OCB, or ADD) families with regard to the following scores: ADD inventory, Y-BOCS scale, TS Global Disability Rating Scale, and OCD inventory. Although autosomal dominant transmission is characterized by a unilineal pattern of inheritance, bilineal transmission seen in our population of TS suggests a polygenic or recessive inheritance. 40 Although we did not demonstrate increased severity of TS in patients who were products of bilineal transmission (perhaps because of sample size), other studies suggested that bilineally transmitted tics 26,27 and OCB 28 were more severe. This would imply a semidominant semi-recessive 17 or polygenic inheritance. 41 The frequency of bilineality of tics, OCB, and ADD in our control families cannot be determined because of the small number of affected people identified in our study. This study provides evidence in support of bilineal transmission in a subset of patients with TS. It is possible that the parents without apparent tics, OCB, or ADD represent gene carriers, but do not exhibit these features because of low penetrance. Alternatively, TS may be a heterogeneous condition, in which bilineal transmission is one form of genetic transmission. Because of the rigid criteria for the diagnosis of tics, OCB, and ADD used in this study, we believe that the frequency of bilineal transmission found in our TS population represents a rather conservative estimate of the true frequency of bilineal transmission in TS. Several limitations of this study should be noted. Although the classroom observer was not identified as a physician, some control subjects may have suppressed tics if they knew that they were being observed. Also, there was no information on whether the control subjects may have been on medication that suppressed or exacerbated tics. These factors could result in underreporting of the actual number of children with tics. Self-reporting questionnaires may be adversely affected by denial of the responder, difficulty of parents recalling childhood symptoms (particularly tics), and lack of insight into one s symptomatology. In our experience, parents of children with TS minimize or deny symptoms in themselves, unless pressed, possibly because of concern that they may have contributed in some way (genetically) to their child s condition. Another potential source of bias may be the possibility that people with OCB (i.e., affected people) may be more inclined to participate in a study. In addition, we evaluated only the biologic parents of the children of interest and no other relatives on either maternal or paternal sides. We experienced difficulty enrolling control subjects, particularly the older children, for participation in the study; hence, our sample size is rather small and the control population is on the average 3 years younger than the TS population. Finally, we recognize that our clinic is a university-based tertiary referral center and thus our patients with TS may be more affected than patients with TS in the general population. Despite these limitations, our findings draw attention to the importance of diligently investigating both parents of patients with TS for evidence of tics, OCB, or ADD, because bilineal transmission, found in one fourth of TS families, is more prevalent than has been hereto appreciated. Furthermore, using structured interviews in family sets of normal control subjects from a single school district, we found relatively high prevalence (0.4 to 0.7%) of TS features in the general population. September (1 of 2) 1999 NEUROLOGY

18 Acknowledgment The authors thank Tetsuo Ashizawa, MD for his review of the manuscript, and Christine Hunter, RN and Jennifer Beach, RN for their technical assistance. References 1. Jankovic J. Phenomenology and classification of tics. Neurol Clin 1997;15: Feigin A, Clarke H. Tourette syndrome: update and review of the literature. Neurologist 1998;4: Shapiro AK, Shapiro E. Evaluation of the reported association of obsessive-compulsive symptoms or disorder with Tourette s disorder. Compr Psychiatry 1992;33: Zametkin AJ, Ernst M. Problems in the management of attention-deficit-hyperactivity disorder N Engl J Med 1999; 340: Cohen DJ, Leckman JF. Developmental psychopathology and neurobiology of Tourette s syndrome. J Am Acad Child Adolesc Psychiatry 1994;33: Singer HS. Neurobiological issues in Tourette syndrome. Brain Dev 1994;16: Hyde TM, Weinberger DR. Tourette s syndrome: a model neuropsychiatric disorder [clinical conference]. JAMA 1995;273: Eldridge R, Sweet R, Lake R, Ziegler M, Shapiro AK. Gilles de la Tourette s syndrome: clinical, genetic, psychologic, and biochemical aspects in 21 selected families. Neurology 1977;27: Pauls DL, Leckman JF, Cohen DJ. Familial relationship between Gilles de la Tourette s syndrome, attention deficit disorder, learning disabilities, speech disorders, and stuttering. J Am Acad Child Adolesc Psychiatry 1993;32: LaBuda MC, Pauls DL. Gilles de la Tourette syndrome. In: Conneally PM, ed. Molecular basis of neurology. Boston: Blackwell Scientific, 1993: Pauls DL, Cohen DJ, Heimbuch R, Detlor J, Kidd KK. Familial pattern and transmission of Gilles de la Tourette syndrome and multiple tics. Arch Gen Psychiatry 1981;38: Price RA, Leckman JF, Pauls DL, Cohen DJ, Kidd KK. Gilles de la Tourette s syndrome: tics and central nervous system stimulants in twins and nontwins. Neurology 1986;36: Hyde TM, Aaronson BA, Randolph C, Rickler KC, Weinberger DR. Relationship of birth weight to the phenotypic expression of Gilles de la Tourette s syndrome in monozygotic twins. Neurology 1992;42: Baron M, Shapiro E, Shapiro A, Rainer JD. Genetic analysis of Tourette syndrome suggesting major gene effect. Am J Hum Genet 1981;33: Kidd KK, Pauls DL. Genetic hypotheses for Tourette syndrome. Adv Neurol 1982;35: Price RA, Pauls DL, Kruger SD, Caine ED. Family data support a dominant major gene for Tourette syndrome. Psychiatry Res 1988;24: Comings DE, Comings BG, Devor EJ, Cloninger CR. Detection of major gene for Gilles de la Tourette syndrome. Am J Hum Genet 1984;36: Devor EJ. Complex segregation analysis of Gilles de la Tourette syndrome: further evidence for a major locus mode of transmission. Am J Hum Genet 1984:36: Pauls DL, Pakstis AJ, Kurlan R, et al. Segregation and linkage analysis in Tourette syndrome. J Am Acad Child Adolesc Psychiatry 1990;29: Eapen V, Pauls DL, Robertson MM. Evidence for autosomal dominant transmission in Tourette s syndrome: United Kingdom cohort study. Br J Psychiatry 1993;162: Curtis D, Robertson MM, Gurling HM. Autosomal dominant gene transmission in a large kindred with Gilles de la Tourette syndrome. Br J Psychiatry 1992;160: Hasstedt SJ, Leppert M, Filloux F, van de Wetering BJ, Mc- Mahon WM. Intermediate inheritance of Tourette syndrome, assuming assortative mating. Am J Hum Genet 1995;57: Pauls DL, Leckman JF. The inheritance of Gilles de la Tourette s syndrome and associated behaviors. Evidence for autosomal dominant transmission. N Engl J Med 1986;315: Walkup JT, LaBuda MC, Singer HS, Brown J, Riddle MA, Hurko O. Family study and segregation analysis of Tourette syndrome: evidence for a mixed model of inheritance. Am J Hum Genet 1996;59: The Tourette Syndrome Association International Consortium on Genetics: a complete genome screen in sib-pairs affected with the Gilles de la Tourette syndrome. 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Obsessions and compulsions in Gilles de la Tourette s syndrome. Neurology 1986;36: Gaffney GR, Sieg K, Hellings J. The MOVES: a self-rating scale for Tourette s syndrome. J Child Adolesc Psychopharmacol 1994;4: Goodman WK, Price LH, Rasmussen SA, et al. The Yale- Brown Obsessive Compulsive Scale: I. development, use, and reliability. Arch Gen Psychiatry 1989;46: Eapen V, Robertson MM, Zetlin H, Kurlan R. Gilles de la Tourette s syndrome in special education schools: a United Kingdom study. J Neurol 1997;244: Kurlan R, Whitmore D, Irvine C, McDermott MP, Como PG. Tourette s syndrome in a special education population: a pilot study involving a single school district. Neurology 1994;44: Comings DE, Himes JA, Comings BG. An epidemiologic study of Tourette s syndrome in a single school district. J Clin Psychiatry 1990;51: Comings DE, Comings BG. A controlled study of Tourette syndrome: revisited. Am J Hum Genet 1988;43: Caine ED, McBride MC, Chiverton P, Bamford KA, Rediess S, Shiao J. 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19 Impairments of attention after cingulotomy R.A. Cohen, PhD; R.F. Kaplan, PhD; D.J. Moser, PhD; M.A. Jenkins, PhD; and H. Wilkinson, MD Article abstract Background: Outcome studies have generally not indicated significant cognitive disturbances after cingulotomy. There is now considerable evidence that the cingulate may play an important role in emotional behavior and attention. Objective: To characterize impairments of attention associated with bilateral lesions of the anterior cingulate cortex produced by cingulotomy. Methods: Twelve patients who underwent cingulotomy for treatment of intractable pain were administered tests of attention, executive functions, response intention and production, and a broad range of other neurocognitive functions before surgery and again 3 and 12 months after surgery. Data from this within-subjects repeated-measures design were analyzed by multivariate analysis of variance procedures. Results: After cingulotomy, patients initially had executive and attentional impairments. By 12 months, these had resolved into more circumscribed deficits, with greatest impairments on tasks requiring intention and spontaneous response production, and milder impairments of focused and sustained attention. Other aspects of attention and other cognitive functions were generally unaffected. Conclusion: The anterior cingulate cortex modulates response intention and focused attention. Key words: Impairment of attention Cingulotomy. NEUROLOGY 1999;53: Although the anterior cingulate cortex (ACC) has long been implicated in the experience and expression of human affect 1,2 and animal 3,4 and human behavior, 5,6 relatively few investigations have examined how ACC lesions affect other neurocognitive functions. Most early studies of cingulotomy found minimal long-term cognitive sequelae, and researchers concluded that cingulotomy is relatively safe and free of major adverse cognitive effects. 7,8 Despite strong evidence that intellectual, language, memory, and other core cognitive abilities are largely unaffected, it would be premature to conclude that cingulotomy has no neurocognitive consequences. The batteries used in early studies were typically designed to assess a broad range of cognitive domains, but not specifically to be sensitive to functions that might be associated with the ACC. Attention was assessed to a limited degree, and the relationship between changes in emotional experience and attentional functions was largely unexplored. Furthermore, few studies have examined cingulotomy patients longitudinally to determine how neurocognitive functions recover after surgery. We previously found mild attentional impairments among cingulotomy patients. 9 Deficits primarily involved focused and sustained attention, intention, and spontaneous response production. These findings augmented an emerging body of clinical 10 and experimental data indicating that the cingulate cortex is involved in the control of attention and behavioral activation. 2,11-13 Focal cingulate lesions have been shown to result in hemi-spatial neglect, 14 as well as a frontal-type syndrome involving decreased activity, spontaneity, and range of affect. 15 Altered habituation of the autonomic orienting response also occurs after cingulotomy, 16 and among normal subjects, functional brain imaging reveals cingulate activation when tasks require focused attention and effortful responding In the current investigation, we studied 12 patients who underwent cingulotomy for intractable pain to determine the effects of ACC lesions on behavior. In light of what is now known about the functional neuroanatomy of the ACC, we hypothesized that this region plays a central role in attentional processes involving response intention and production, in addition to influencing emotional experience, and that patients who underwent cingulotomy would have impairments of these attentional functions. Based on previous studies, we anticipated that patients would perform normally across other neurocognitive domains (e.g., language, visual, motor, memory, and intellectual functioning). We also hypothesized that after cingulotomy, patients would have a pattern of recovery characterized by initial impairments of attention and executive functions, most of which would resolve over time, and specific impairments of intention and spontaneous responding would remain. Methods. Subjects. Twelve patients (aged 40 to 58 years) who underwent anterior bilateral cingulotomy for treatment of chronic, intractable pain were studied at the From the Department of Psychiatry and Human Behavior (Drs. Cohen, Moser, and Jenkins), Brown University School of Medicine, Providence, RI; Department of Psychiatry (Dr. Kaplan), University of Connecticut School of Medicine, Farmington, CT; and Department of Neurosurgery (Dr. Wilkinson), University of Massachusetts Medical Center, Worcester, MA. Received November 6, Accepted in final form April 10, Address correspondence and reprint requests to Dr. Ronald A. Cohen, Neuropsychology, The Miriam Hospital, Brown University School of Medicine, 164 Summit Ave., Providence, RI Copyright 1999 by the American Academy of Neurology 819

20 Figure. Axial CT showing bilateral anterior cingulate lesions in one patient after surgery. These surgical lesions are typical of those observed among patients in this study. Lesions were symmetric and well circumscribed to the ACC ( mm 3 ) for all patients. 820 NEUROLOGY 53 September (1 of 2) 1999 University of Massachusetts Medical Center (UMMC). All patients (9 men and 3 women) reported chronic pain secondary to noncerebral traumatic injury and had not responded to medical treatment for pain before their cingulotomy, including pharmacologic interventions, nerve blocks, transcutaneous nerve stimulation, or surgical procedures (e.g., laminectomy) directed at peripheral nerve relief. None had a history of neurocognitive or neuropsychiatric disorder before surgery. Medical and neurologic examinations conducted before surgery were normal in all patients, except for findings related to the specific peripheral mechanisms responsible for the pain syndrome. Two of the 12 patients had symptoms of depression attributed to the pain syndrome before surgery. Twenty patients with chronic pain served as control subjects for baseline comparison with the cingulotomy patients. Controls were recruited based on consecutive referral from the UMMC Pain Disorders Clinic and had chosen nonsurgical pain interventions offered by this clinic. They were included only if they had pain levels comparable with the ACC patients (Pain Index 6). Sex, age, and educational characteristics did not differ significantly between groups. Both cingulotomy patients and control subjects reported severe pain on a 10-point visual analog rating scale, in which 0 no pain and 10 severe, excruciating pain. Groups did not differ in reported pain levels at baseline (cingulotomy patients , controls ). Across groups, patients tended to have pain associated with neuromuscular injury involving the cervical, thoracic, or lumbar regions of the back and neck. For all patients, the chronicity of pain was greater than 2 years (cingulotomy patients years, controls years). Surgical procedure. For all cingulotomy patients, surgery was performed using stereotactic thermal probes, producing bilateral lesions of the ACC. Lesions were approximately 5 mm in diameter, slightly lateral of midline. Postsurgically, a neuroradiologist determined the location of each lesion and the extent to which lesions were confined to the cingulate region. Lesion volumes were quantified using CT images on which the lesion area was traced and digitized on successive 5-mm axial slices. Lesion volume was then computed by summing across the lesion areas (mm 2 ) and multiplying by 5 mm (figure). Assessment procedure. Cingulotomy patients underwent baseline neurocognitive assessment within 1 month before surgery. Control patients were administered an identical test battery as part of their Pain Clinic workup. During the inpatient postsurgical recovery period, cingulotomy patients underwent a brief structured mental status examination along with a test of verbal fluency (Animal Naming) before being sent home. Approximately 3 months after surgery (mean months), cingulotomy patients were reassessed on the same neurocognitive battery as was used at baseline, with the exception of the Wisconsin Card Sorting Test (WCST). A third full assessment was conducted during a follow-up session a minimum of 12 months postsurgically (mean months). All assessments were completed during individual sessions lasting approximately 6 hours per patient. Neurocognitive assessment. Patients and control subjects were evaluated using a battery of neurocognitive tests designed to measure the functional domains of language, visual integration, learning and memory, executive control, attention, motor control, and intellectual functioning, as well as several experimental measures of response intention, generation, and persistence. The neurocognitive battery consisted largely of standardized tests with well-established reliability and validity. 20 The tests used to assess each domain were as follows: Intellectual functioning Wechsler Adult Intelligence Scale Revised (WAIS-R); Language Boston Diagnostic Aphasia Exam (Auditory Comprehension, Commands, Repetitions), Boston Naming Test; Visuospatial Hooper Visual Organization Test, Judgment of Line Orientation, Complex Figure Test Copy (CFT-Copy); Learning and memory Wechsler Memory Scale (WMS), Auditory Verbal Learning Test, CFT (immediate and delayed recall); Motor Grooved Pegboard, Finger Tapping Test; Attention and executive functions WCST, Stroop Color-Word Interference Test (Stroop), Trail Making (A and B), Reciprocal Motor Programs, Rampart Figures, Verbal Fluency-Animal Naming, Controlled Oral Word Association Test, Letter Cancellation, Digit Span, Adaptive Rate Continuous Performance Test (ARCPT). 13 It should be noted that we used the WMS rather than its revision, because data collection began before the time that the Wechsler Memory Scale Revised was published. For the memory tests, alternate forms were used during follow-up assessments. In addition to the battery of standard neurocognitive tests, the following experimental measures were obtained: Spontaneous utterances. Given that we hypothesized a reduction in spontaneous verbal output after cingulotomy, a measure was developed for the present study to provide an index of spontaneous verbal response initiation and production. Standard verbal fluency tests have specific task demands and therefore do not measure spontaneous verbalization. Our task employed a behavioral event recording method and provided information about the frequency of verbal production without prompts or specific