Dopamine D5 receptor gene polymorphism and the risk of levodopa-induced motor fluctuations in patients with Parkinson's disease

Neuroscience Letters 308 (2001) 21±24

www.elsevier.com/locate/neulet

Dopamine D5 receptor gene polymorphism and the risk of levodopainduced motor ¯uctuations in patients with Parkinson's disease Jian Wang*, Zhuo-Lin Liu, Biao Chen Department of Neurology, The First Af®liated Hospital, Sun Yat-Sen University of Medical Sciences, Guangzhou 510080, China Received 25 April 2001; received in revised form 28 May 2001; accepted 29 May 2001

Abstract Motor ¯uctuations are the most common complication of levodopa therapy for Parkinson's disease (PD). Genetic factors could play a role in determining the occurrence of motor ¯uctuations. To investigate whether dopamine receptor D5 (DRD5) T978C polymorphism is associated with the risk of developing motor ¯uctuations in PD, we studied this polymorphism in a case-control study of 120 subjects with sporadic PD and 110 control subjects. We found that the overall allelic and genotypic frequencies did not differ signi®cantly between patients with PD and control subjects (all P . 0:7), and between motor ¯uctuators (n ˆ 50) and non-motor ¯uctuators (n ˆ 50) (all P . 0:8). It suggests that DRD5 T978C polymorphism is not associated with the susceptibility to PD, nor with the risk of developing motor ¯uctuations in PD. Therefore, other polymorphisms that alter the expression of the dopamine receptors should be further studied. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Parkinson's disease; Dopamine D5 receptor; Gene polymorphism; Motor ¯uctuations

After more than 30 years, levodopa is still considered the `gold standard' of anti-parkinsonian treatment, yet despite its symptomatic effectiveness, approximately 50% of patients develop motor ¯uctuations after 5 years of treatment [10]. The pathophysiological basis of such ¯uctuations is poorly understood, although nigrostriatal degeneration with consequent reduced dopamine storage capacity [12] and postsynaptic receptor function alteration [2] have been proposed. Although early initiation and longer duration of levodopa therapy are recognized risk factors [4], since a substantial proportion of patients with Parkinson's disease (PD) never develop motor ¯uctuations, there may be important pharmacogenetic factors that could contribute to the individual risk for the development of motor ¯uctuations. The dopamine receptors D5 (DRD5) are localized in the substantia nigra-pars compacta, hypothalamus, striatum, cerebral cortex, nucleus accumbens and olfactory tubercle. The presence of DRD5 in the areas of dopamine pathways suggests that this receptor may participate actively in dopaminergic neurotransmission [3,8]. The DRD1 and DRD2 are * Corresponding author. Institute of Genetics, School of Life Science, Fudan University, 220 Handan Road, Shanghai 200433, P.R. China. Tel.: 186-21-65643717; fax: 186-2165643250. E-mail address: [email protected] (J. Wang).

primarily involved in the dopaminergic response to exogenous levodopa in patients with PD [6,18]. Due to the similarities in structure and function, the DRD5 with higher af®nity for dopamine than D1 [17] may also be involved in the pharmacologic treatment of PD, and polymorphism of the gene coding for this receptor could be implicated in the genetic susceptibility to the complications of long-term levodopa use. A common exon polymorphic T ) C transition was identi®ed at base pair 978 of DRD5 gene [16]. Using a polymerase chain reaction (PCR) allele-speci®c ampli®cation, we investigated whether this polymorphism in the DRD5 gene was associated with an increased risk for developing motor ¯uctuations in Chinese. One hundred and twenty PD patients (75 men and 45 women) were recruited from the outpatient clinic of Neurology at the First Af®liated Hospital of the Sun Yat-sen University of Medical Sciences, Guangzhou, P.R. China. All PD patients were diagnosed as having probable PD by using the criteria of Gelb et al. [5]. The age at onset and examination of all PD patients was 54.2 ^ 9.5 years (range from 30 to 82 years) and 60.8 ^ 10.0 years (range from 34 to 86 years), respectively. All PD patients had sporadic PD and those with a history of familial PD (i.e. an affected ®rstdegree relative) were excluded. One hundred and ten healthy unrelated control subjects, who were volunteers or

0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 97 1- 1

22

J. Wang et al. / Neuroscience Letters 308 (2001) 21±24

spouses of the patients, were recruited from the same hospital. Control subjects were matched for age, gender, ethnic origin, and area of residence. A medical examination was performed to identify control subjects in good health. The motor ¯uctuations were de®ned as having the obvious `wearing-off' effect or `on-off' response. Motor ¯uctuations were recorded as present or absent by the examining neurologist on the occasion of the last visit, which included an oral acute levodopa test [20]. Brie¯y, each levodopa test consisted of a 6-h observation by a trained neurologist, after the morning administration of a standard dose of 250 mg of levodopa plus 25 mg of carbidopa after an overnight without any drug. The clinical evaluation was made by using the Uni®ed Parkinson's Disease Rating Scale (UPDRS)-Subscale III (motor examination) at baseline, and at 30, 60, 90, 120, 240, and 360 min after the levodopa administration. The following clinical characteristics, as measured during the last acute oral levodopa test, were available for all the patients: age, sex, age at onset of the disease, duration of levodopa therapy, duration of disease, and mean daily dosage of levodopa. The severity of the disease was measured by UPDRS-Subscale III and the modi®ed Hoehn±Yahr scale; the degree of motor ¯uctuations was evaluated by UPDRS-Subscale IVB (clinical ¯uctuations); cognitive functions were evaluated by the Mini-Mental State Examination (MMSE) and by the Hamilton depression scale. All these evaluations were determined at baseline during the same visit. All subjects recruited to the study gave fully informed consent and the study protocol was approved by the ethical committee of the Sun Yat-sen University of Medical Sciences. High-molecular-weight genomic DNA was extracted from whole blood leukocytes using standard method. The DRD5 gene T978C polymorphism was genotyped by PCR

allele-speci®c ampli®cation [6]. We performed two ampli®cation reactions for each sample: T-speci®c reaction and C-speci®c reaction and simultaneously, a heterozygous sample was used as a positive control, and two-distilled water was used as a negative control when performing the PCR ampli®cation reactions every time. Among the whole cohort of 120 PD subjects, cases with motor ¯uctuations and non-motor ¯uctuations were selected and individually matched. A total sample of 50 pairs with a similar duration of treatment with levodopa (^10%) were available for the analysis. The characteristics of the patients were compared according to speci®c data distributions using the unpaired Student's t-test, or the Mann±Whitney rank sum test. The frequencies of the alleles and genotypes among case patients and control subjects (in the total PD samples) and among motor ¯uctuators and non-motor ¯uctuators (in the matched PD samples) were determined and compared using the chi-square test. P , 0:05 was taken as the level of signi®cance. The clinical characteristics of the 50 pairs of the PD patients with motor ¯uctuations and non-motor ¯uctuations matched for treatment duration are listed in Table 1. Both patients and control subjects were in Hardy±Weinberg equilibrium (all P . 0:05). The overall allelic and genotypic frequencies did not differ signi®cantly between patients with PD and control subjects (all P . 0:7), and between motor ¯uctuators and non-motor ¯uctuators (all P . 0:8) (see Table 2). To our knowledge, this is the ®rst study on DRD5 T978C polymorphism in patients with PD. Our data showed that DRD5 T978C polymorphism is not associated with the susceptibility to PD, nor with the risk of developing motor ¯uctuations in PD. Genetic in¯uence has increasingly been recognized as an important risk factor for PD [1]. During the last few years several studies have focused on polymorphisms of genes

Table 1 Clinical details of patients with PD with motor ¯uctuations and non-motor ¯uctuations matched for duration of levodopa treatment a Characteristic

Motor ¯uctuators (n ˆ 50)

Non-motor ¯uctuators (n ˆ 50)

P value

Age (years) Sex Age at onset of PD (years) Duration of PD (months) Duration of levodopa treatment, (months) Levodopa dosage (mg/day) UPDRS Subscale III Subscale IVB Hoehn±Yahr score Mini mental state examination Hamilton scale

63.8 ^ 9.0 30 M/20 F 51.8 ^ 9.6 78 ^ 46 65 (60Ð180) 500 (125±1250)

64.6 ^ 9.2 31 M/19 F 54.8 ^ 9.0 74 ^ 46.5 65 (60±198) 375 (125±1000)

NS NS b NS NS NS c NS c

19 (6.0±34) 4.5 (3Ð6) 2.5 (1Ð5) 24.1 ^ 5.0 9 (2±22)

18.5 (4.5±33)

NS c

2.5 (1±4) 25 ^ 5.1 10 (2±23)

NS NS NS c

a

The values are the mean ^ SD, and range values are the median. All P values refer to the unpaired t-test unless speci®c otherwise. UPDRS-Subscale III, Uni®ed Parkinson's Disease Rating Scale, Motor Examination section; UPDRS-Subscale IVB, Uni®ed Parkinson's Disease Rating Scale, Clinical Fluctuations section; NS, not signi®cant. b x 2 test. c Mann±Whitney U test.

J. Wang et al. / Neuroscience Letters 308 (2001) 21±24

23

Table 2 Genotypic and allelic frequencies for the DRD5 T978C polymorphism in PD patients and controls, and in ¯uctuators and non-¯uctuators a Variable DRD5 T978C alleles T C Genotypes T-T T-C C-C

Patients (n ˆ 120)

Control subjects (n ˆ 110)

Motor ¯uctuators (n ˆ 50)

Non-motor ¯uctuators (n ˆ 50)

119 (0.50) 121 (0.50)

113 (0.51) 107 (0.49)

52 (0.52) 48 (0.48)

53 (0.53) 47 (0.47)

24 (0.20) 71 (0.59) 25 (0.21)

26 (0.24) 61 (0.55) 23 (0.21)

12 (0.24) 28 (0.56) 10 (0.20)

12 (0.24) 29 (0.58) 9 (0.18)

a Numbers shown refer to number of subjects in each category with frequencies shown in brackets.*No signi®cant differences were found in either genotypic or allelic distributions between groups of patients with PD and control subjects (all P . 0:7), and between ¯uctuators and non-¯uctuators (all P . 0:8).

encoding for proteins involved in the dopaminergic functions [9,14]. Association studies on DRD3 were all negative [7,11], whereas investigations on DRD2 polymorphisms have yielded contradictory results [7,11,13]. Using a large group of patients with PD we were unable to show an association between patients with PD and control subjects in DRD5 T978C polymorphism. It therefore seems that this polymorphism is not a susceptibility factor for PD. The roles of the DRD5 in the pathogenesis of PD are likely to be minor because the major changes are in the presynaptic nigrostriatal pathway. However, the presence of DRD5 in the areas of dopamine pathways suggests that this receptor may participate actively in dopaminergic neurotransmission [3,8]. Due to the similarities in structure and function with DRD1, the DRD5 with higher af®nity for dopamine than D1 [17] could also be involved in the dopaminergic response to exogenous levodopa in patients with PD. However, our data show that DRD5 T978C polymorphism does not contribute to the risk of developing motor ¯uctuations in PD which suggests that other polymorphic loci that alter the expression of the receptors in DRD5 [15] or in other dopamine receptors such as DRD2 [19] may be involved in the development of levodopa related motor ¯uctuations. It should be further studied. This work was supported in part by China Postdoctoral Science Foundation, National Natural Science Foundation of China (30000095) and 211 Engineering Research Grant (98119) from Sun Yat-sen University of Medical Sciences in China.

[1] Bandmann, O., Marsden, C.D. and Wood, N.W., Genetics aspects of Parkinson's disease, Mov. Disord., 13 (1998) 203±211. [2] Chase, T.N., Mouradian, M.M. and Engber, T.M., Motor response complications and the function of striatal efferent systems, Neurology, 43(Suppl. 6) (1993) 23±27. [3] Ciliax, B.J., Nash, N., Heilman, C., Sunahara, R., Hartney, A., Tiberi, M., Rye, D.B., Caron, M.G., Niznik, H.B. and Levey, A.I., Dopamine D(5) receptor immunolocalization in rat and monkey brain, Synapse, 37 (2000) 125±145.

[4] Denny, A.P. and Behari, M., Motor ¯uctuations in Parkinson's disease, J. Neurol. Sci., 165 (1999) 18±23. [5] Gelb, D.J., Oliver, E. and Gilman, S., Diagnostic criteria for Parkinson's disease, Arch. Neurol., 56 (1999) 33±39. [6] Gonzalez, A.M., Berciano, J., Figols, J., Pazos, A. and Pascual, J., Loss of dopamine uptake sites and dopamine D2 receptors in striatonigral degeneration, Brain Res., 852 (2000) 228±232. [7] Higuchi, S., Muramatsu, T., Arai, H., Hayashida, M., Sasaki, H. and Trojanowski, J.Q., Polymorphisms of dopamine receptor and transporter genes and Parkinson's disease, J. Neural. Transm., 10 (1995) 107±113. [8] Khan, Z.U., Gutierrez, A., Martin, R., Pena®el, A., Rivera, A. and de la Calle, A., Dopamine D5 receptors of rat and human brain, Neuroscience, 100 (2000) 689±699. [9] Mellick, G.D., Buchanan, D.D., McCann, S.J., James, K.M., Johnson, A.G., Davis, D.R., Liyou, N., Chan, D. and Le Couteur, D.G., Variations in the monoamine oxidase B (MAOB) gene are associated with Parkinson's disease, Mov. Disord., 14 (1999) 219±224. [10] Miyawaki, E., Lyons, K., Pahwa, R., Troster, A.I., Hubble, J., Smith, D., Busenbark, K., McGuire, D., Michalek, D. and Koller, W.C., Motor complications of chronic levodopa therapy in Parkinson's disease, Clinic. Neuropharmacol., 20 (1997) 523±530. [11] Nanko, S., Ueki, A., Hattori, M., Dai, X.Y., Sasaki, T., Fukuda, R., Ikeda, K. and Kazamatsuri, H., No allelic association between Parkinson's disease and dopamine D2,D3, D4 receptor gene polymorphism, Am. J. Med. Genet., 54 (1994) 361±364. [12] Nutt, J.G., Levodopa: rational and irrational pharmacology, Ann. Neurol., 36 (1994) 4±5. [13] Oliveri, R.L., Annesi, G., Zappia, M., Civitelli, D., De Marco, E.V., Pasqua, A.A., Annesi, F., Spadafora, P., Gambardella, A., Nicoletti, G., Branca, D., Caracciolo, M., Aguglia, U. and Quattrone, A., The dopamine D2 receptor gene is a susceptibility locus for Parkinson's disease, Mov. Disord., 15 (2000) 127±131. [14] PlanteÂ-Bordeneuve, V., Davis, M.B., Maraganore, D.M., Marsden, C.D. and Harding, A.E., Tyrosine hydroxylase gene polymorphisms in familial and sporadic Parkinson's disease, Mov. Disord., 9 (1994) 337±339. [15] Propping, P. and Nothen, M.M., Genetics variation of CNS receptors- a new perspective for pharmacogenetics, Pharmacogenetics, 5 (1995) 318±325. [16] Sommer, S.S., Sobell, J.L. and Heston, L.L., A common exonic polymorphism in the human D5 dopamine receptor gene, Hum. Gene, 92 (1993) 633±634. [17] Sunahara, R.K., Guan, H.C., O'Dowd, B.F., Seeman, P., Laur-

24

J. Wang et al. / Neuroscience Letters 308 (2001) 21±24

ier, L.G., Ng, G., George, S.R., Torchia, J., Van Tol, H.H. and Niznik, H.B., Cloning of the gene for a human dopamine D5 receptor with higher af®nity for dopamine than D1, Nature, 350 (1991) 614±619. [18] Turjanski, E., Lees, A.J. and Brooks, D.J., In vivo studies on striatal dopamine D1 and D2 site binding in L-dopa-treated Parkinson's disease patients with and without dyskinesias, Neurology, 49 (1997) 717±723.

[19] Wang, J., Liu, Z.L. and Chen, B., Association study of dopamine D2, D3 receptor gene polymorphisms with motor ¯uctuations in PD, Neurology, 56 (2001) 1757±1759. [20] Zappia, M., Montesanti, R., Colao, R., Branca, D., Nicoletti, G., Aguglia, U. and Quattrone, A., Short-term levodopa test assessed by MT accurately predicts dopaminergic responsiveness in Parkinson's diseases, Mov. Disord., 12 (1997) 103±106.