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Magnetic resonance imaging demonstrates differential atrophy of pontine base and tegmentum in Machado–Joseph disease
Journal of the Neurological Sciences 215 (2003) 45 – 50 www.elsevier.com/locate/jns
Magnetic resonance imaging demonstrates differential atrophy of pontine base and tegmentum in Machado–Joseph disease Toshihiro Yoshizawa *, Masahiko Watanabe, Kentaro Frusho, Shinichi Shoji Department of Neurology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba City, Ibaraki 305-8575, Japan Received 12 February 2003; received in revised form 3 June 2003; accepted 6 June 2003
Abstract The pons is one of the brain areas demonstrating selective degeneration in Machado – Joseph disease (MJD), which is caused by the expansion of a polyglutamine stretch in the protein called ataxin-3. Although the resultant pontine atrophy is readily recognized by magnetic resonance imaging (MRI), the features and natural process of atrophy are not fully understood. To characterize them, we analyzed the midsagittal images of the pons obtained by MRI. We found a difference in atrophy between the pontine base and tegmentum. The reduced size of the pontine tegmentum was prominent early after the onset of clinical symptoms. No overlap was seen in the range of the area of pontine tegmentum between MJD and controls. The quotient of atrophy of the pontine tegmentum divided by age correlated well with the CAG repeat number. In contrast, the area of the pontine base correlated negatively with disease duration. Particularly, the size of the pontine base remained in the range of controls for a relatively long time after the onset of symptoms. These results suggest that the atrophic process is not uniform in the pons in MJD and that the different patterns of atrophy may be derived from the differential vulnerability in pontine structures. D 2003 Elsevier B.V. All rights reserved. Keywords: Machado – Joseph disease; Magnetic resonance imaging; Atrophy; Pontine base; Pontine tegmentum; CAG repeat
1. Introduction Machado –Joseph disease (MJD) is an autosomal dominant spinocerebellar ataxia caused by the expansion of CAG repeats in the MJD1 gene [1]. The protein coded by the MJD1 gene, ataxin-3, possesses a polyglutamine motif in its structure [1]. An increased number of CAG repeats results in the expansion of the polyglutamine stretch in ataxin-3, which demonstrates a novel toxic function in susceptible cells [2– 4]. In MJD pathology, selective brain areas including the dentate nucleus of the cerebellum, the nucleus dorsalis of Clarke in the spinal cord, cranial motor nerve nuclei, pontine nuclei, and substantia nigra are reported to show degeneration [5– 7]. Among the heterogeneous pathological changes in MJD patients, modest pontine atrophy is a part of condition [5– 7]. Since pontine atrophy is readily recognized by magnetic resonance imaging (MRI), evaluation of the size of the pons on MRI provides useful information for
diagnosing MJD. Several studies have analyzed the size of the pons in MJD on MRI [8– 10] and described the effects of the CAG repeat size and age on pontine atrophy [9,10]. However, the features and natural process of atrophy have not been fully clarified. To characterize the pontine atrophy in MJD, we analyzed the size of the pontine base and tegmentum using MRI. We found a difference in atrophy between the pontine base and tegmentum. The reduced size of the pontine tegmentum was prominent from early after the onset of clinical symptoms. The quotient of atrophy of the pontine tegmentum divided by age correlated well with the CAG repeat number. In contrast, the area of the pontine base correlated negatively with disease duration. Our MRI results suggest a difference in susceptibility within the pontine structures in MJD.
2. Subjects and methods 2.1. MRI subjects
* Corresponding author. Tel.: +81-29-853-3224; fax: +81-29-8533273/3224. E-mail address: [email protected] (T. Yoshizawa). 0022-510X/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0022-510X(03)00185-0
Thirteen MJD patients (4 men and 9 women) from 11 pedigrees admitted to Tsukuba University Hospital from
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1990 to 2002 were recruited in this study. All MJD patients underwent MRI examinations when first admitted. Six underwent additional MRI during follow-up. Five underwent MRI twice, and one underwent MRI three times over 9 years. A total of 20 scannings of MRI from 13 MJD patients was used for analysis. The mean age at scanning F standard error of MJD patients was 45.5 F 3.3 years (range 19– 72). The mean duration of illness at scanning F standard error was 11.9 F 6.5 years (range 2 –25). The diagnosis of MJD was made by clinical findings and confirmed by molecular analysis of the MJD1 gene using a polymerase chain reaction [1]. The CAG repeat number of the expanded allele in the MJD1 gene ranged from 65 to 98 (mean F standard deviation: 77 F 7). Seventeen control subjects (6 men and 11 women) were chosen from patients undergoing MRI at Tsukuba University Hospital for diagnostic purposes. None had brain abnormalities. The mean age at scanning F standard error of controls was 49.2 F 3.8 years (range 17– 72). 2.2. Magnetic resonance imaging and analysis Magnetic resonance imaging was done using a machine at a 1.5-T magnetic field (Gyroscan, Philips, DA Best, The Netherlands, or Signa, GE, Milwaukee, WI, USA). Sagittal T1-weighted images (repetition time, 460 ms; echo time, 15 ms) were obtained at a slice thickness of 5 mm with a 1-mm gap. The acquisition matrix contained 205 256 pixels. Midsagittal images demonstrating the cerebral aqueduct clearly were scanned with scales and analyzed using NIH Image computer software (Version 1.6, National Institute of Mental Health, USA). Areas of the pontine base (Fig. 1a) and pontine tegmentum (Fig. 1b) were measured. Since we recognized the low intensity line demarcating the pontine base on MRI, the area ventral to this line was first deter-
mined as pontine base. The pontine tegmentum was the area dorsal to the pontine base. The upper margin of the pontine tegmentum was determined by a line drawn perpendicular to the longitudinal axis of the pons at the junction of the midbrain and pontine base. The lower margin was determined by a line drawn perpendicular to the longitudinal axis of the pons at the pontomedullary junction. In each MJD case, the area of the pontine base or tegmentum was subtracted from the mean value obtained from controls. This value was defined as the atrophy of the pontine base or tegmentum. Each measure of atrophy was divided by age at scanning. Then, the quotients were plotted against CAG repeat numbers. 2.3. Clinical grading of MJD Severity of MJD at scanning was evaluated by clinical grading based on the Japanese annual report of the research committee for ataxic diseases [11], as follows: grade 1: needed no assistance in walking; grade 2: needed assistance only in standing, turning, or stepping up or down; grade 3: needed assistance in walking; grade 4: unable to walk, but able to stand with assistance; grade 5: bedridden. 2.4. Statistical analysis Statistical analysis was done with StatView 4.0 computer software (Abacus Concept, Berkeley, CA, USA). To compare the mean of the area of the pontine base or tegmentum between MJD and controls, we used Mann –Whitney’s Utest. To determine the significance of the correlation coefficient, a Fisher’s r to z transformation was done on the correlation. The partial correlation of two variables with respect to the third was also calculated. We used Mann – Whitney’s U-test to compare the area of the pontine base or tegmentum among individual clinical grades.
3. Results
Fig. 1. Schematic representation of pontine areas measured in this study. (a) Pontine base, (b) pontine tegmentum.
Prior to evaluating midsagittal MRI in MJD, we examined the influence of aging on areas of the pontine base and tegmentum in controls. Age at scanning did not significantly correlate with either the size of the pontine base (r = 0.417, p = 0.0967) or tegmentum (r = 0.306, p = 0.237), suggesting that aging itself did not influence the size of the pontine base or tegmentum in control subjects. Next, we determined mean areas of the pontine base and tegmentum in MJD and controls. The mean area F standard deviation (S.D.) of the pontine base was 3.82 F 0.86 cm2 in MJD and 4.65 F 0.82 cm2 in controls. The mean area F S.D. of the pontine tegmentum was 1.22 F 0.07 cm2 in MJD and 2.52 F 0.43 cm2 in controls. Both sizes were significantly smaller in MJD than in controls ( p = 0.0055 in the pontine base; p < 0.0001 in the pontine tegmentum). The size reduction observed in MJD was more prominent in the pontine
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tegmentum (17.8% of reduction in the mean area of the pontine base compared to controls; 51.8% of reduction in the mean area of the pontine tegmentum compared to controls). When we plotted data from MJD and controls separately on the univariate plot, a considerable overlap was observed in the range of the area of the pontine base between these two groups (Fig. 2a). In contrast, almost no overlap was seen in the range of the area of the pontine tegmentum (Fig. 2b), suggesting a possible difference in the nature of atrophy between these two areas. Our patients suffered from MJD for 2 to 25 years, enabling us to explore the influence of disease duration on the size of the pontine base and tegmentum. Disease duration correlated negatively with the area of the pontine
Fig. 2. Univariate plots of areas of the pontine base and tegmentum in controls and MJD. (a) Pontine base, (b) pontine tegmentum. Broken lines indicate mean values of individual groups. Shaded areas above and below broken lines denote ranges of mean F standard deviation. The horizontal rendition was arbitrary. Between MJD and controls, a considerable overlap is observed in the range of the area of pontine base. Note that no overlap is seen in the range of the area of pontine tegmentum.
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base (r = 0.757, p < 0.0001). Most area data during the first 13 years of duration was distributed above the value of the control mean 1 S.D. (10/13 cases). In contrast, all area data after 14 years of duration was below the value of the control mean 1 S.D. (7/7 cases). These results indicated that disease duration is an important factor influencing the progression of atrophy of the pontine base in MJD. The atrophic process may not linearly develop in the pontine base. The size of the pontine base in MJD appeared to remain in the range of controls for a relatively long time after the onset of clinical symptoms. Disease duration also correlated negatively with the area of the pontine tegmentum to a lesser extent (r = 0.464, p = 0.0386). All data for the pontine tegmentum except 1 was distributed below the value of the control mean 2 S.D. (19/20 cases). This was quite different from that observed in the pontine base. Atrophy may already be present in the pontine tegmentum at the onset of clinical symptoms. Structures in the pontine tegmentum are likely to be more susceptible to pathological processes in MJD. In addition to the above analysis, we studied the influence of age at scanning on the size of the pontine base and tegmentum in MJD. Although we have seen no significant effect of aging in controls, a weakly negative correlation between age at scanning and the area of the pontine base was observed in MJD (r = 0.422, p = 0.050). No significant correlation was seen between age at scanning and the area of the pontine tegmentum (r = 0.034, p = 0.889). Since the weakly negative correlation between age at scanning and the area in the pontine base of MJD could be derived from the influence of disease duration, the partial correlation between age and the area with respect to duration was calculated. This value was 0.025. In contrast, the partial correlation between disease duration and the area with respect to age was 0.685. These results appear to show that the negative correlation between duration and the area in the pontine base was more significant compared to that between age at scanning and the area. To examine the relationship between disease duration and pontine atrophy more clearly, we focused our analysis on data obtained from six MJD patients who underwent MRI more than twice over several years. One underwent MRI three times during the 2 to 11 years of duration, and five underwent MRI twice during 10 to 21 years. Area data on the pontine base and tegmentum of individual patients was plotted against disease duration (Fig. 3a and b). The size of the pontine base and tegmentum of these six patients progressively decreased in the course of MJD regardless of age at first MRI. These results again showed that the size of pontine structures was significantly affected by disease duration, with a longer duration gave rise to greater reduction of the pontine base and tegmentum. In addition to the above data, we did several additional analyses. The influence of clinical severity on the size of pontine structures was examined. Significant differences were detected in the size of the pontine base ( p = 0.033,
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against CAG repeat numbers (Fig. 4). The size of pontine base of two MJD patients was almost same as the control mean. Therefore, the atrophy and calculated quotients of these patients were almost zero (Fig. 4a). Three MJD patients had larger pontine bases compared to the control mean; hence, the atrophy and quotients were negative figures (Fig. 4a). No correlation was seen between the CAG repeat number and the quotient of atrophy of the pontine base divided by age (r = 0.189, p = 0.429, Fig. 4a). In contrast, the tegmental area in MJD was always smaller than in average controls, resulting positive figures of the atrophy and quotients (Fig. 4b). A strong correlation was
Fig. 3. Sequential MRI analyses demonstrate the progressive reduction of the areas of the pontine base and tegmentum. (a) Pontine base, (b) pontine tegmentum. Symbols indicate individual MJD patients. The age at MRI scanning is shown in figures next to each symbol. The size of the pontine base and tegmentum progressively decreased in the course of MJD regardless of age at first MRI. A longer duration gave rise to greater reduction of the pontine base and tegmentum.
Mann – Whitney’s U-test) and tegmentum ( p = 0.011, Mann –Whitney’s U-test) only between grades 1 and 4. We also examined the correlation between the CAG repeat size of the expanded allele and the area of pontine structures. Although a strong correlation was seen between age at onset and the expanded repeat size (r = 0.880, p < 0.0001), no correlation was detected between the area itself and the expanded repeat size. A previous study reported that the CAG repeat number correlated with the quotient of the degree of atrophy divided by age at examination in MJD [10]. We did a similar analysis with our data sets, in which the atrophy determined by subtracting each measure of area from the control mean was divided by age at MRI. These quotients were plotted
Fig. 4. Correlation between the CAG repeat number and the quotient of pontine base atrophy divided by age or the quotient of pontine tegmentum atrophy divided by age. (a) No correlation was observed between the CAG repeat number and the quotient of pontine base atrophy divided by age. (b) A strong correlation was present between the CAG repeat number and the quotient of pontine tegmentum atrophy divided by age. The correlation coefficient is 0.839, highly significant ( p < 0.0001). The equation for the simple regression line through points is CAG = 62.6 + 446.8 (atrophy/age at scanning).
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seen between the CAG repeat number and the quotient of atrophy of the pontine tegmentum divided by age (r = 0.839, p < 0.0001, Fig. 4b).
4. Discussion We found that the pontine tegmentum responded differently to atrophy than the pontine base in MJD. A previous study of MRI analysis indicated that the reduced size of the pontine tegmentum was more striking than the size reduction of the pontine base in MJD [8,9], which was confirmed by our investigation. In addition, we found that the size reduction of the pontine tegmentum was prominent from early after the onset of clinical symptoms and gradually worsened as disease progressed. In contrast, the size of the pontine base in MJD appeared to remain in the range of controls for several years after the onset of clinical symptoms. Due to the subsequent progressive decrease, the size of the pontine base reached the level of overt atrophy more than 15 years after the onset of symptoms, suggesting a difference between the pontine base and tegmentum in the background of atrophy. Structures in the pontine tegmentum appear more susceptible to the pathological process in MJD. Atrophic changes in the pontine tegmentum may start before the onset of clinical symptoms of MJD. We detected a strong correlation between the CAG repeat number and the quotient of atrophy of the pontine tegmentum divided by age. From the nearly straight-line correlation between these two factors, the following equation was introduced: CAG repeat number = 62.6 + 446.8 (atrophy of tegmentum/age). This data suggested that the longer CAG repeat length is associated with a faster rate of atrophy of pontine tegmentum. The association between the CAG repeat number and a faster rate of striatal pathology was also described in Huntington’s disease [12,13]. The length of CAG repeat may influence the rate of pathological changes in the polyglutamine diseases in general. In contrast, our present analysis did not demonstrate a significant correlation between the CAG repeat number and the quotient of atrophy divided by age in the pontine base of MJD. Rather, the area of the pontine base correlated well with disease duration, suggesting a difference in the atrophic process between the pontine base and tegmentum. Our data favored the presence of the latent phase during which the size of pontine base remains in the range of controls. There were two discrepancies in our analysis of pontine base atrophy in MJD compared to previous studies. One concerned the correlation between the area of the pontine base and disease duration. A previous paper found no significant correlation between these two factors in MJD [9]. Our data demonstrated a strong significant correlation between these two variables. Although it was not clear from where the difference between ours and the previous result derived, the distribution of data on disease duration may affect the consequence. When we calculated the correlation
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coefficient between these two variables using 13 data points in which the duration was under 14 years, we did not observe a significant value (r = 0.282, p = 0.3597). As discussed above, there may be a latent phase of pontine base atrophy even after the onset of MJD, which is followed by a progressive decrease in size of the pontine base. The presence of a latent phase may affect calculation of the correlation coefficient. In addition to the above, our sequential analysis of the pontine base on MRI in six MJD patients showed that the size of the pontine base decreased during disease progression, indicating that disease duration is an important factor influencing the size of the pontine base in MJD. The other discrepancy concerned the correlation between the CAG repeat number and the quotient of atrophy of the pontine base divided by age. Although a previous paper reported a significant correlation [10], we did not observe one. According to previous data, the correlation between the CAG repeat number and the quotient of atrophy of the pontine tegmentum was much greater than that between the CAG repeat number and the quotient of atrophy of the pontine base [10]. We also observed a strong correlation between the CAG repeat number and the quotient of atrophy of the pontine tegmentum, so the difference observed in the atrophy of the pontine base may be due to variations in cases. From our present study, it can be assumed that the process of atrophy in MJD differed in the pontine base compared to the tegmentum. The susceptibility to death induced by the expression of mutant ataxin-3 varies among individual cell types, even among neurons [14]. Therefore, the region-specific variations in the atrophic process in MJD may be due to the difference in susceptibility.
References [1] Kawaguchi Y, Okamoto T, Taniwaki M, Aizawa M, Inoue M, Katayama S, et al. CAG expansions in a novel gene for Machado – Joseph disease at chromosome 14q32.1. Nat Genet 1994;8:221 – 8. [2] Ikeda H, Yamaguchi M, Sugai S, Aze Y, Narumiya S, Kakizuka A. Expanded polyglutamine in the Machado – Joseph disease protein induces cell death in vitro and in vivo. Nat Genet 1996;13:196 – 202. [3] Paulson HL, Perez MK, Trottier Y, Trajanowski JQ, Subramony SH, Das SS, et al. Intranuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3. Neuron 1997;19:333 – 44. [4] Yoshizawa T, Yamagishi Y, Koseki N, Goto J, Yoshida H, Shibasaki F, et al. Cell cycle arrest enhances the in vitro cellular toxicity of the truncated Machado – Joseph disease gene product with an expanded polyglutamine stretch. Hum Mol Genet 2000;9:69 – 78. [5] Yuasa T, Ohama E, Harayama H, Yamada M, Kawase Y, Wakabayashi M, et al. Joseph’s disease: clinical and pathologic studies in a Japanese family. Ann Neurol 1986;19:152 – 7. [6] Du¨rr A, Stevanin G, Cancel G, Duyckaerts C, Abbas N, Didierjean O, et al. Spinocerebellar ataxia 3 and Machado – Joseph disease: clinical, molecular, and neuropathological features. Ann Neurol 1996;39: 490 – 9. [7] Iwabuchi K, Tsuchiya K, Uchihara T, Yagishitra S. Autosomal dominant spinocerebellar degenerations. Clinical, pathological, and genetic correlations. Rev Neurol (Paris) 1999;155:255 – 70.
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[8] Murata Y, Yamaguchi S, Kawakami H, Imon Y, Maruyama H, Sakai T, et al. Characteristic magnetic resonance imaging findings in Machado – Joseph disease. Arch Neurol 1998;55:33 – 7. [9] Onodera O, Idezuka J, Igarashi S, Takiyama Y, Endo K, Takano H, et al. Progressive atrophy of cerebellum and brainstem as a function of age and size of the expanded CAG repeats in the MJD1 gene in Machado – Joseph disease. Ann Neurol 1998;43:288 – 96. [10] Abe Y, Tanaka F, Matsumoto M, Doyu M, Hirayama M, Kachi T, et al. CAG repeat number correlates with the rate of brainstem and cerebellar atrophy in Machado – Joseph disease. Neurology 1998;51: 882 – 4. [11] Hirayama K, Takayanagi T, Yanagisawa N. Ataxic diseases (spinocer-
ebellar degenerations and Shy-Drager syndrome): epidemiologic study. Annual report of research committee for ataxic diseases; 1990. [12] Furtado S, Suchowersky O, Rewcastle B, Graham L, Klimek ML, Garber A. Relationship between trinucleotide repeats and neuropathological changes in Huntington’s disease. Ann Neurol 1996;39:132 – 6. [13] Penney JB, Vonsattel JP, MacDonald ME, Gusella JF, Myers RH. CAG repeat number governs the development rate of pathology in Huntington’s disease 1997;41:689 – 92. [14] Yoshizawa T, Yoshida H, Shoji S. Differential susceptibility of cultured cell lines to aggregate formation and cell death produced by the truncated Machado – Joseph disease gene product with an expanded polyglutamine stretch. Brain Res Bull 2001;56:349 – 52.
Magnetic resonance imaging demonstrates differential atrophy of pontine base and tegmentum in Machado–Joseph disease Toshihiro Yoshizawa *, Masahiko Watanabe, Kentaro Frusho, Shinichi Shoji Department of Neurology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba City, Ibaraki 305-8575, Japan Received 12 February 2003; received in revised form 3 June 2003; accepted 6 June 2003
Abstract The pons is one of the brain areas demonstrating selective degeneration in Machado – Joseph disease (MJD), which is caused by the expansion of a polyglutamine stretch in the protein called ataxin-3. Although the resultant pontine atrophy is readily recognized by magnetic resonance imaging (MRI), the features and natural process of atrophy are not fully understood. To characterize them, we analyzed the midsagittal images of the pons obtained by MRI. We found a difference in atrophy between the pontine base and tegmentum. The reduced size of the pontine tegmentum was prominent early after the onset of clinical symptoms. No overlap was seen in the range of the area of pontine tegmentum between MJD and controls. The quotient of atrophy of the pontine tegmentum divided by age correlated well with the CAG repeat number. In contrast, the area of the pontine base correlated negatively with disease duration. Particularly, the size of the pontine base remained in the range of controls for a relatively long time after the onset of symptoms. These results suggest that the atrophic process is not uniform in the pons in MJD and that the different patterns of atrophy may be derived from the differential vulnerability in pontine structures. D 2003 Elsevier B.V. All rights reserved. Keywords: Machado – Joseph disease; Magnetic resonance imaging; Atrophy; Pontine base; Pontine tegmentum; CAG repeat
1. Introduction Machado –Joseph disease (MJD) is an autosomal dominant spinocerebellar ataxia caused by the expansion of CAG repeats in the MJD1 gene [1]. The protein coded by the MJD1 gene, ataxin-3, possesses a polyglutamine motif in its structure [1]. An increased number of CAG repeats results in the expansion of the polyglutamine stretch in ataxin-3, which demonstrates a novel toxic function in susceptible cells [2– 4]. In MJD pathology, selective brain areas including the dentate nucleus of the cerebellum, the nucleus dorsalis of Clarke in the spinal cord, cranial motor nerve nuclei, pontine nuclei, and substantia nigra are reported to show degeneration [5– 7]. Among the heterogeneous pathological changes in MJD patients, modest pontine atrophy is a part of condition [5– 7]. Since pontine atrophy is readily recognized by magnetic resonance imaging (MRI), evaluation of the size of the pons on MRI provides useful information for
diagnosing MJD. Several studies have analyzed the size of the pons in MJD on MRI [8– 10] and described the effects of the CAG repeat size and age on pontine atrophy [9,10]. However, the features and natural process of atrophy have not been fully clarified. To characterize the pontine atrophy in MJD, we analyzed the size of the pontine base and tegmentum using MRI. We found a difference in atrophy between the pontine base and tegmentum. The reduced size of the pontine tegmentum was prominent from early after the onset of clinical symptoms. The quotient of atrophy of the pontine tegmentum divided by age correlated well with the CAG repeat number. In contrast, the area of the pontine base correlated negatively with disease duration. Our MRI results suggest a difference in susceptibility within the pontine structures in MJD.
2. Subjects and methods 2.1. MRI subjects
* Corresponding author. Tel.: +81-29-853-3224; fax: +81-29-8533273/3224. E-mail address: [email protected] (T. Yoshizawa). 0022-510X/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0022-510X(03)00185-0
Thirteen MJD patients (4 men and 9 women) from 11 pedigrees admitted to Tsukuba University Hospital from
46
T. Yoshizawa et al. / Journal of the Neurological Sciences 215 (2003) 45–50
1990 to 2002 were recruited in this study. All MJD patients underwent MRI examinations when first admitted. Six underwent additional MRI during follow-up. Five underwent MRI twice, and one underwent MRI three times over 9 years. A total of 20 scannings of MRI from 13 MJD patients was used for analysis. The mean age at scanning F standard error of MJD patients was 45.5 F 3.3 years (range 19– 72). The mean duration of illness at scanning F standard error was 11.9 F 6.5 years (range 2 –25). The diagnosis of MJD was made by clinical findings and confirmed by molecular analysis of the MJD1 gene using a polymerase chain reaction [1]. The CAG repeat number of the expanded allele in the MJD1 gene ranged from 65 to 98 (mean F standard deviation: 77 F 7). Seventeen control subjects (6 men and 11 women) were chosen from patients undergoing MRI at Tsukuba University Hospital for diagnostic purposes. None had brain abnormalities. The mean age at scanning F standard error of controls was 49.2 F 3.8 years (range 17– 72). 2.2. Magnetic resonance imaging and analysis Magnetic resonance imaging was done using a machine at a 1.5-T magnetic field (Gyroscan, Philips, DA Best, The Netherlands, or Signa, GE, Milwaukee, WI, USA). Sagittal T1-weighted images (repetition time, 460 ms; echo time, 15 ms) were obtained at a slice thickness of 5 mm with a 1-mm gap. The acquisition matrix contained 205 256 pixels. Midsagittal images demonstrating the cerebral aqueduct clearly were scanned with scales and analyzed using NIH Image computer software (Version 1.6, National Institute of Mental Health, USA). Areas of the pontine base (Fig. 1a) and pontine tegmentum (Fig. 1b) were measured. Since we recognized the low intensity line demarcating the pontine base on MRI, the area ventral to this line was first deter-
mined as pontine base. The pontine tegmentum was the area dorsal to the pontine base. The upper margin of the pontine tegmentum was determined by a line drawn perpendicular to the longitudinal axis of the pons at the junction of the midbrain and pontine base. The lower margin was determined by a line drawn perpendicular to the longitudinal axis of the pons at the pontomedullary junction. In each MJD case, the area of the pontine base or tegmentum was subtracted from the mean value obtained from controls. This value was defined as the atrophy of the pontine base or tegmentum. Each measure of atrophy was divided by age at scanning. Then, the quotients were plotted against CAG repeat numbers. 2.3. Clinical grading of MJD Severity of MJD at scanning was evaluated by clinical grading based on the Japanese annual report of the research committee for ataxic diseases [11], as follows: grade 1: needed no assistance in walking; grade 2: needed assistance only in standing, turning, or stepping up or down; grade 3: needed assistance in walking; grade 4: unable to walk, but able to stand with assistance; grade 5: bedridden. 2.4. Statistical analysis Statistical analysis was done with StatView 4.0 computer software (Abacus Concept, Berkeley, CA, USA). To compare the mean of the area of the pontine base or tegmentum between MJD and controls, we used Mann –Whitney’s Utest. To determine the significance of the correlation coefficient, a Fisher’s r to z transformation was done on the correlation. The partial correlation of two variables with respect to the third was also calculated. We used Mann – Whitney’s U-test to compare the area of the pontine base or tegmentum among individual clinical grades.
3. Results
Fig. 1. Schematic representation of pontine areas measured in this study. (a) Pontine base, (b) pontine tegmentum.
Prior to evaluating midsagittal MRI in MJD, we examined the influence of aging on areas of the pontine base and tegmentum in controls. Age at scanning did not significantly correlate with either the size of the pontine base (r = 0.417, p = 0.0967) or tegmentum (r = 0.306, p = 0.237), suggesting that aging itself did not influence the size of the pontine base or tegmentum in control subjects. Next, we determined mean areas of the pontine base and tegmentum in MJD and controls. The mean area F standard deviation (S.D.) of the pontine base was 3.82 F 0.86 cm2 in MJD and 4.65 F 0.82 cm2 in controls. The mean area F S.D. of the pontine tegmentum was 1.22 F 0.07 cm2 in MJD and 2.52 F 0.43 cm2 in controls. Both sizes were significantly smaller in MJD than in controls ( p = 0.0055 in the pontine base; p < 0.0001 in the pontine tegmentum). The size reduction observed in MJD was more prominent in the pontine
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tegmentum (17.8% of reduction in the mean area of the pontine base compared to controls; 51.8% of reduction in the mean area of the pontine tegmentum compared to controls). When we plotted data from MJD and controls separately on the univariate plot, a considerable overlap was observed in the range of the area of the pontine base between these two groups (Fig. 2a). In contrast, almost no overlap was seen in the range of the area of the pontine tegmentum (Fig. 2b), suggesting a possible difference in the nature of atrophy between these two areas. Our patients suffered from MJD for 2 to 25 years, enabling us to explore the influence of disease duration on the size of the pontine base and tegmentum. Disease duration correlated negatively with the area of the pontine
Fig. 2. Univariate plots of areas of the pontine base and tegmentum in controls and MJD. (a) Pontine base, (b) pontine tegmentum. Broken lines indicate mean values of individual groups. Shaded areas above and below broken lines denote ranges of mean F standard deviation. The horizontal rendition was arbitrary. Between MJD and controls, a considerable overlap is observed in the range of the area of pontine base. Note that no overlap is seen in the range of the area of pontine tegmentum.
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base (r = 0.757, p < 0.0001). Most area data during the first 13 years of duration was distributed above the value of the control mean 1 S.D. (10/13 cases). In contrast, all area data after 14 years of duration was below the value of the control mean 1 S.D. (7/7 cases). These results indicated that disease duration is an important factor influencing the progression of atrophy of the pontine base in MJD. The atrophic process may not linearly develop in the pontine base. The size of the pontine base in MJD appeared to remain in the range of controls for a relatively long time after the onset of clinical symptoms. Disease duration also correlated negatively with the area of the pontine tegmentum to a lesser extent (r = 0.464, p = 0.0386). All data for the pontine tegmentum except 1 was distributed below the value of the control mean 2 S.D. (19/20 cases). This was quite different from that observed in the pontine base. Atrophy may already be present in the pontine tegmentum at the onset of clinical symptoms. Structures in the pontine tegmentum are likely to be more susceptible to pathological processes in MJD. In addition to the above analysis, we studied the influence of age at scanning on the size of the pontine base and tegmentum in MJD. Although we have seen no significant effect of aging in controls, a weakly negative correlation between age at scanning and the area of the pontine base was observed in MJD (r = 0.422, p = 0.050). No significant correlation was seen between age at scanning and the area of the pontine tegmentum (r = 0.034, p = 0.889). Since the weakly negative correlation between age at scanning and the area in the pontine base of MJD could be derived from the influence of disease duration, the partial correlation between age and the area with respect to duration was calculated. This value was 0.025. In contrast, the partial correlation between disease duration and the area with respect to age was 0.685. These results appear to show that the negative correlation between duration and the area in the pontine base was more significant compared to that between age at scanning and the area. To examine the relationship between disease duration and pontine atrophy more clearly, we focused our analysis on data obtained from six MJD patients who underwent MRI more than twice over several years. One underwent MRI three times during the 2 to 11 years of duration, and five underwent MRI twice during 10 to 21 years. Area data on the pontine base and tegmentum of individual patients was plotted against disease duration (Fig. 3a and b). The size of the pontine base and tegmentum of these six patients progressively decreased in the course of MJD regardless of age at first MRI. These results again showed that the size of pontine structures was significantly affected by disease duration, with a longer duration gave rise to greater reduction of the pontine base and tegmentum. In addition to the above data, we did several additional analyses. The influence of clinical severity on the size of pontine structures was examined. Significant differences were detected in the size of the pontine base ( p = 0.033,
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against CAG repeat numbers (Fig. 4). The size of pontine base of two MJD patients was almost same as the control mean. Therefore, the atrophy and calculated quotients of these patients were almost zero (Fig. 4a). Three MJD patients had larger pontine bases compared to the control mean; hence, the atrophy and quotients were negative figures (Fig. 4a). No correlation was seen between the CAG repeat number and the quotient of atrophy of the pontine base divided by age (r = 0.189, p = 0.429, Fig. 4a). In contrast, the tegmental area in MJD was always smaller than in average controls, resulting positive figures of the atrophy and quotients (Fig. 4b). A strong correlation was
Fig. 3. Sequential MRI analyses demonstrate the progressive reduction of the areas of the pontine base and tegmentum. (a) Pontine base, (b) pontine tegmentum. Symbols indicate individual MJD patients. The age at MRI scanning is shown in figures next to each symbol. The size of the pontine base and tegmentum progressively decreased in the course of MJD regardless of age at first MRI. A longer duration gave rise to greater reduction of the pontine base and tegmentum.
Mann – Whitney’s U-test) and tegmentum ( p = 0.011, Mann –Whitney’s U-test) only between grades 1 and 4. We also examined the correlation between the CAG repeat size of the expanded allele and the area of pontine structures. Although a strong correlation was seen between age at onset and the expanded repeat size (r = 0.880, p < 0.0001), no correlation was detected between the area itself and the expanded repeat size. A previous study reported that the CAG repeat number correlated with the quotient of the degree of atrophy divided by age at examination in MJD [10]. We did a similar analysis with our data sets, in which the atrophy determined by subtracting each measure of area from the control mean was divided by age at MRI. These quotients were plotted
Fig. 4. Correlation between the CAG repeat number and the quotient of pontine base atrophy divided by age or the quotient of pontine tegmentum atrophy divided by age. (a) No correlation was observed between the CAG repeat number and the quotient of pontine base atrophy divided by age. (b) A strong correlation was present between the CAG repeat number and the quotient of pontine tegmentum atrophy divided by age. The correlation coefficient is 0.839, highly significant ( p < 0.0001). The equation for the simple regression line through points is CAG = 62.6 + 446.8 (atrophy/age at scanning).
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seen between the CAG repeat number and the quotient of atrophy of the pontine tegmentum divided by age (r = 0.839, p < 0.0001, Fig. 4b).
4. Discussion We found that the pontine tegmentum responded differently to atrophy than the pontine base in MJD. A previous study of MRI analysis indicated that the reduced size of the pontine tegmentum was more striking than the size reduction of the pontine base in MJD [8,9], which was confirmed by our investigation. In addition, we found that the size reduction of the pontine tegmentum was prominent from early after the onset of clinical symptoms and gradually worsened as disease progressed. In contrast, the size of the pontine base in MJD appeared to remain in the range of controls for several years after the onset of clinical symptoms. Due to the subsequent progressive decrease, the size of the pontine base reached the level of overt atrophy more than 15 years after the onset of symptoms, suggesting a difference between the pontine base and tegmentum in the background of atrophy. Structures in the pontine tegmentum appear more susceptible to the pathological process in MJD. Atrophic changes in the pontine tegmentum may start before the onset of clinical symptoms of MJD. We detected a strong correlation between the CAG repeat number and the quotient of atrophy of the pontine tegmentum divided by age. From the nearly straight-line correlation between these two factors, the following equation was introduced: CAG repeat number = 62.6 + 446.8 (atrophy of tegmentum/age). This data suggested that the longer CAG repeat length is associated with a faster rate of atrophy of pontine tegmentum. The association between the CAG repeat number and a faster rate of striatal pathology was also described in Huntington’s disease [12,13]. The length of CAG repeat may influence the rate of pathological changes in the polyglutamine diseases in general. In contrast, our present analysis did not demonstrate a significant correlation between the CAG repeat number and the quotient of atrophy divided by age in the pontine base of MJD. Rather, the area of the pontine base correlated well with disease duration, suggesting a difference in the atrophic process between the pontine base and tegmentum. Our data favored the presence of the latent phase during which the size of pontine base remains in the range of controls. There were two discrepancies in our analysis of pontine base atrophy in MJD compared to previous studies. One concerned the correlation between the area of the pontine base and disease duration. A previous paper found no significant correlation between these two factors in MJD [9]. Our data demonstrated a strong significant correlation between these two variables. Although it was not clear from where the difference between ours and the previous result derived, the distribution of data on disease duration may affect the consequence. When we calculated the correlation
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coefficient between these two variables using 13 data points in which the duration was under 14 years, we did not observe a significant value (r = 0.282, p = 0.3597). As discussed above, there may be a latent phase of pontine base atrophy even after the onset of MJD, which is followed by a progressive decrease in size of the pontine base. The presence of a latent phase may affect calculation of the correlation coefficient. In addition to the above, our sequential analysis of the pontine base on MRI in six MJD patients showed that the size of the pontine base decreased during disease progression, indicating that disease duration is an important factor influencing the size of the pontine base in MJD. The other discrepancy concerned the correlation between the CAG repeat number and the quotient of atrophy of the pontine base divided by age. Although a previous paper reported a significant correlation [10], we did not observe one. According to previous data, the correlation between the CAG repeat number and the quotient of atrophy of the pontine tegmentum was much greater than that between the CAG repeat number and the quotient of atrophy of the pontine base [10]. We also observed a strong correlation between the CAG repeat number and the quotient of atrophy of the pontine tegmentum, so the difference observed in the atrophy of the pontine base may be due to variations in cases. From our present study, it can be assumed that the process of atrophy in MJD differed in the pontine base compared to the tegmentum. The susceptibility to death induced by the expression of mutant ataxin-3 varies among individual cell types, even among neurons [14]. Therefore, the region-specific variations in the atrophic process in MJD may be due to the difference in susceptibility.
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