Original Paper
Received: August 26, 2002
Eur Neurol 2003;50:64–68
DOI: 10.1159/000072500
HLA Typing and Parkinson’s Disease
J.B. Lampe a G. Gossrau a B. Herting a A. Kempe a U. Sommer a
M. Füssel b M. Weber d R. Koch c H. Reichmann a
a Department of Neurology, b Institute of Immunology, c Department of Medical Informatics and Biometrics, and
d Department of Internal Medicine, University of Technology, Dresden, Germany
Key Words
Neurogenetics W Parkinson’s disease W HLA W
Neurodegenerative disorder W Linkage disequilibrium
Abstract
Idiopathic Parkinson’s disease (IPD) is a neurodegener-
ative disorder of unknown aetiology. Several antigens
have been associated with IPD using serological
methods. We systematically analysed HLA class I and II
alleles in 45 German Caucasian IPD patients using se-
quence-specific oligonucleotides and sequence-specific
primer technology. Applying Bonferroni adjusted p
values, we demonstrate a statistically significant in-
crease of the DQB1*06 allele (p = 0.002) in IPD which
may indicate an association between IPD and the im-
mune system. Alternatively, HLA alleles might be in
linkage disequilibrium with genes located next to the
HLA locus.
Copyright © 2003 S. Karger AG, Basel
Introduction
Several genes have been identified which are responsi-
ble for autosomal-dominant (PARK1, PARK3, PARK4,
PARK5) or autosomal-recessive (PARK2, PARK6) forms
of Parkinson’s disease (PD) [1]. However, most cases of
© 2003 S. Karger AG, Basel
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Accepted: February 25, 2003
this neurodegenerative disease are idiopathic Parkinson’s
disease (IPD) without any known cause. Nevertheless,
various observations may point to an involvement of the
immune system in the aetiology of this disorder. For
example, postmortem analysis of the substantia nigra in
IPD patients revealed the presence of activated microglia
[2]. Additionally, a possible association between IPD and
certain HLA antigens has been suggested in several stud-
ies, since the frequency of various antigens was increased
in patients with IPD and in patients dying from post-
encephalitic PD [3, 4]. In contrast to former investigators,
we systematically analysed both HLA class I and class II
alleles in a series of 45 German IPD patients using
sequence-specific oligonucleotides (SSO) and sequence-
specific primer (SSP)-PCR. We identified a statistically
significant increase in the DQB1*06 allele. The reason for
this increase remains unclear.
Patients and Methods
Patients included in this study were admitted as in-patients or
out-patients to our neurological clinic specialised in the treatment of
movement disorders. All patients were of German Caucasian origin.
The clinical diagnosis of each case was made by neurologists experi-
enced in movement disorders (B.H., H.R.) and followed the clinical
criteria for probable or possible IPD [5]. The clinical diagnosis of
probable and possible IPD is based on the identification of some
combination of the cardinal motor signs (bradykinesia, rigidity,
tremor, postural instability). For the diagnosis of definite IPD both
Dr. J.B. Lampe
Schering AG, Clinical Development CNS
Sellerstrasse 31, DE–13342 Berlin (Germany)
Tel. +49 30 468 16691, Fax +49 30 468 16648
E-Mail johannes.lampe@schering.de
clinical criteria for possible or probable IPD and neuropathologic Table 1. Clinical data of IPD patients (n = 45)
confirmation are required. Furthermore a subset of additional clini-
cal features was applied (asymmetrical onset, no atypical clinical fea- Men 29
tures for IPD, no possible aetiology for another parkinsonian syn- Women 16
drome) [6, 7]. Average age, years 61.5 (range 28–81)
The severity of parkinsonism was assessed by Hoehn and Yahr Average duration of disease, years 8.5 (range 0.5–33)1
staging. Genomic DNA was isolated from peripheral blood lympho-
Hoehn and Yahr staging2
cytes using commercial kits (Puregene DNA Isolierungskit, Diagno-
Stage I 2
stik GmbH Biozym, Hess. Oldendorf, Germany; QIA-amp mini
Stage I.5 2
DNA Kit; QIAGEN GmbH, Hilden, Germany).
Stage II 11
To determine HLA class I and II allele frequencies, we applied
Stage II.5 9
(i) SSP-PCR and (ii) SSO. Commercial SSP (HLA-A, HLA-B, HLA-
Stage III 9
DQ, HLA-C medium resolution; all BAG, Lich, Germany) and SSO
Stage III.5 3
kits (RELI SSO-Kit, Dynal Biotech GmbH, Hamburg, Germany)
Stage IV 6
were used in 45 IPD patients. In unclear cases both SSP-PCR and
Stage IV.5 1
SSO were applied. For the distinction of DRB1*15/16 we used a fur-
Stage V 1
ther test kit (Olerup SSP AG, Saltsjöbaden, Sweden). All tests were
performed following the instructions of the manufacturers. 1
The SSP technique uses oligonucleotide sequences as primers Data in 1 patient is missing.
2 Number of patients.
complementary to known nucleotide sequences. Therefore, only
primers whose sequences are completely complementary to a known
target sequence will be amplified. In contrast, non-complementary
primers will not be converted into PCR products. Accordingly, HLA
typing is performed by using a set of different PCR primers specific
for different HLA alleles. ty of the disease in each patient was assigned to the stages
In principle, SSO PCR uses oligonucleotide probes to identify of Hoehn and Yahr. In detail, two patients were classified
specific sequences which in turn allow the identification of different
as Hoehn and Yahr stage I, two as stage I.5, eleven as stage
alleles. In HLA analysis the sequence for a whole HLA region is
amplified during PCR using biotinylated primer pairs for the target II, nine as stage II.5, nine as stage III, three as stage III.5,
region. The denatured biotinylated PCR products are then hybrid- six as stage IV, one as stage IV.5, and one as stage V. In 1
ised to oligonucleotide probes immobilised on a nylon-membrane. patient the clinical data concerning the staging of Hoehn
Finally, a streptavidin-horseradish peroxidase (SA-HRP) conjugate and Yahr is missing; 17 patients had a predominantly aki-
is added. This SA-HRP conjugate binds to the biotin-labelled probe
netic-rigid syndrome, 10 had parkinsonian tremor as the
which itself is bound to the membrane. This is visualised by colour
formation by adding H2O2 and tetramethylbenzidine. Therefore, the leading symptom, in 17 patients akinesis, rigor and trem-
oligonucleotide probes will identify the presence of specific se- or were equivalent and 1 patient suffered from severe
quences of HLA alleles. motor fluctuations and dyskinesias.
For PCR amplification a PE Thermocycler 2400 (Perkin Elmer, Using both SSO and SSP technology, we were able to
Applied Biosystems, Foster City, Calif., USA) and Thermocycler
identify 77 allele specificities in HLA-A, 205 in HLA-B,
‘Personal’ (Biometra, Göttingen, Germany) were used.
Our results were compared with the data of normal controls from 66 in HLA-C, 128 in HLA-DRB1, and 31 in HLA-DQB1.
the general German population for HLA-A, -B, -DR, and -DQ [8] These alleles were referred to different allele groups which
and HLA-C [9]. Contrasts between allele frequencies in patients and correspond to serologically defined antigens. Bonferroni
controls were evaluated using the chi-square test. In the case of low- adjusted p values are presented for all antigens within one
filled cells, the Fisher exact test was performed for a two-tailed test of
HLA class I or II locus: 22 in HLA-A, 51 in HLA-B, 16 in
the difference. Unadjusted p values are presented. Moreover, Bon-
ferroni adjustment was performed over all allele groups correspond- HLA-C; 15 in HLA-DRB, and 5 HLA-DQ. Results were
ing to serologically defined antigens within one HLA class I or II compared with a published normal control population
locus. Bonferroni adjusted p values were used to avoid an alpha infla- [8, 9].
tion when chaining statistical tests. Even after Bonferroni adjustment, there was a statisti-
cally significant increase in the frequency of the
DQB1*06 allele (table 2). We found the DQB1*06 allele
Results in 35.56% of patients (32/90) vs. 20.13% in the general
population (p unadjusted = 0.0003; p adjusted = 0.002).
Altogether 45 patients (29 men and 16 women) partici- The unadjusted p value indicates statistical significance
pated in this study (table 1). The average age was 61.5 in several further cases. However, the increase in those
years (range 28–81). The average duration of disease at cases did not remain statistically significant when calcu-
the time of study was 8.5 years (range 0.5–33). The severi- lating Bonferroni adjusted p values (table 2): A*25, B*42,

HLA and Parkinson’s Disease Eur Neurol 2003;50:64–68 65

Table 2. HLA alleles increased in IPD
Allele Allele frequency in Allele frequency in p value1 Bonferroni
patients
(corresponding antigen) PD patients normal controls adjusted
[8, 9] p value2

A*25 (A25 (10)) 6/90 (6.67%) 792/29,670 0.02 n.s.

(2.67%)
B*42 (B42) 1/90 (1.11%) 6/29,670 0.02 (F) n.s.
(0.02%)
DRB1*04 (DR4) 20/90 (22.22%) 3,632/29,670 0.004 n.s.
(12.24%)
DRB1*15 (DR15) 19/90 (21.11%) 3,461/29,670 0.005 n.s.
(11.66%)
DQB1*02 (DQ2) 10/90 (11.11%) 2,521/12,700 0.04 n.s.
(19.85%)
DQB1*06 (DQ6) 32/90 (35.56%) 2,556/12,700 0.0003 0.002
(20.13%)

1 Chi-square test or two-tailed Fisher exact test (F), significant at global alpha = 0.05; n.s. =
not significant.
2 p values are adjusted for all corresponding antigens within one HLA class I or II locus (22
in HLA-A; 51 in HLA-B; 16 in HLA-C; 15 in HLA-DRB, and 5 HLA-DQ).

DRB1*04, DRB1*15, DQB1*02. Additionally, we calcu-
lated p values for antigen frequencies deduced from the
analysis of allele frequencies in all s-IBM patients (data
not shown). However, we did not find any additional sta-
tistical significant increase when applying Bonferroni ad-
justment. Parts of the preceding results were presented at
the 127th annual meeting of the American Neurological
Association [10].
Discussion
Viruses have been discussed as causative in the aetiolo-
gy of various neurodegenerative disorders [11]. Epidemi-
ological studies suggest that encephalitis lethargica (von
Economo encephalitis) and post-encephalitic PD cases
were due to the pandemic influenza in 1918 [12]. Enceph-
alitis lethargica is a meningoencephalitis, clinically char-
acterised by fever, somnolence, and oculomotor paresis
[13]. Therefore, the increase in HLA-B14 in post-enceph-
alitic PD in one study is of special interest [4]. However,
this increase is discussed controversially [14].
Several studies have analysed HLA antigen frequen-
cies in IPD. Emile et al. [3] reported a possible association
of the HLA-B17 and -B18 antigen with IPD. One pre-
vious study has reported an increased frequency of A2
and A28 antigens in a study of 39 patients [15]. However,
these reports did not correct the p value for the number of
antigens tested. While calculating adjusted p values, var-
ious investigators did not find any significant deviation in
HLA types of IPD patients in comparison to normal con-
trols [16–18].
In all studies dealing with the analysis of HLA antigens
in IPD to date, detailed information concerning the clini-
cal criteria for the diagnosis of IPD is lacking. However, a
critical point of genetic association studies in neurodegen-
erative disorders is the reliability of the clinical diagnosis
of the respective disease. The clinical diagnosis of IPD in
our study was based on the clinical criteria for probable
and possible IPD [5–7]. Additionally, the diagnosis was
made by neurologists specialised in movement disorders.
Recently, the correlation between the clinical diagnosis of
IPD and the pathologically confirmed diagnosis was esti-
mated by Hughes et al. [7] to reach 90%. Moreover, when
the diagnosis of IPD is made by neurologists specialised
in movement disorders it seems to be more accurate than
when made by a non-specialised neurologist [7].
Several studies have considered a possible association
between HLA class I and II alleles and neurodegenerative
diseases. For example, the HLA-A2 allele was identified
as a potential risk factor in the development of sporadic
Alzheimer’s disease in most studies which addressed this
question [19–22]. More recently, the DQ7 antigen was
identified as a possible resistance factor for the develop-
ment of the new variant of Creutzfeldt-Jakob disease
(vCJD) [23]. Our own analysis of HLA class I and HLA
class II alleles in a group of 20 sCJD patients did not dem-
onstrate any increase in the frequency of HLA class I or II

66 Eur Neurol 2003;50:64–68 Lampe/Gossrau/Herting/Kempe/Sommer/

Füssel/Weber/Koch/Reichmann
alleles in German sporadic Creutzfeldt-Jakob disease
(sCJD) patients, which is in accordance with the results of
Jackson et al. [23] for sCJD patients [Lampe et al., unpub-
lished results].
We identified a statistically significant increase in
HLA-DQB1*06 in IPD which may indicate an involve-
ment of the immune system in the pathogenesis of IPD.
MPTP-treated mice – which serve as a model for Parkin-
son’s disease – reveal several changes typical of inflamma-
tion, including transient microglial reaction, an increase
in MHC class I and II expression, and CD4+ and CD8+
lymphocytic infiltrates [24, 25]. Additionally, several
genes are expressed typically found in oxidative stress and
inflammation [26]. Interestingly, elevation of HLA class I
B44 has been reported in neuroleptica-induced Parkin-
son’s syndrome [27] and HLA-DR4 was more prevalent
in patients suffering from tardive dyskinesia [28]. There-
fore, it is tempting to speculate that similar pathogenetic
mechanisms may underlie those drug-associated cases of
Parkinson’s syndrome and IPD cases. Moreover, inflam-
mation may play an important neuroprotective role in
neurodegenerative diseases [29].
An alternative explanation for the increase in HLA-
DQB1*06 in IPD may be given by the phenomenon of
linkage disequilibrium, which leads us to consider the oth-
er factors that are found along with HLA class I and II
alleles. According to mendelian inheritance, the frequen-
cy of alleles at one locus does not influence the frequency
of alleles at another locus. Nevertheless, in HLA genetics
some HLA class I, II, and III alleles are inherited together
more often than could be expected [30].
Several genes have been located in the central region of
the MHC, including the tumour necrosis factor (TNF)
alpha and beta gene pair [31], the second and fourth com-
ponents of complement (C2 and C4, respectively), factor
B (Bf) [32], and three heat shock protein-70 (HSP70)
genes [33]. It is also worth noting that the HSP70–1 alleles
are in linkage disequilibrium with different HLA markers
[34]. Likewise, elevated HSP70 mRNA levels in mononu-
clear blood cells have been described in CJD patients [35]
and the receptor for advanced glycation end products
(RAGE) gene has been located 150 kb centromeric to the
C4 gene. This is of special interest, since RAGE has been
implicated in the aetiology of Alzheimer’s disease [36,
37]. Moreover, the results of one study suggest that a poly-
morphism within the promoter region of the TNF-alpha
gene may serve as a risk factor for the development of
Alzheimer’s disease and vascular dementia [38].
The statistically significant increase of a HLA allele in
IPD patients is of special interest: It may indicate that
(i) typing of HLA is helpful in epidemiological studies of
IPD and (ii) certain HLA haplotypes or other genes that
are in linkage disequilibrium with HLA loci contribute to
the aetiology or pathogenesis of IPD. Further studies will
be needed to determine whether the disturbances de-
scribed above constitute a primary or a secondary phe-
nomenon.
Acknowledgement
This work was supported in part by a grant from the Deutsche
Parkinson-Vereinigung to J.B.L. and H.R.

References

1 Gasser T: Genetics of Parkinson’s disease. J
Neurol 2001;248:833–840.
2 McGeer PL, Itagaki S, Boyes BE, McGeer EG:
Reactive microglia are positive for HLA-DR in
the substantia nigra of Parkinson’s and Alz-
heimer’s disease brains. Neurology 1988;38:
1285–1291.
3 Emile J, Truelle JL, Pouplard A, Hurez D:
Association of Parkinson’s disease with HLA-
B17 and B18 antigens. Nouv Presse Méd 1977;
6:4144.
4 Elizan TS, Terasaki PI, Yahr MD: HLA-B14
antigen and postencephalitic Parkinson’s dis-
ease: Their association in an American-Jewish
ethnic group. Arch Neurol 1980;37:542–544.
5 Gelb DJ, Oliver E, Gilman S: Diagnostic crite-
ria for Parkinson disease. Arch Neurol 1999;
56:33–39.
6 Calne DB, Snow BJ, Lee C: Criteria for diag-
nosing Parkinson’s disease. Ann Neurol 1992;
32(suppl):S125–S127.
7 Hughes AJ, Daniel SE, Ben-Shlomo Y, Lees
AJ: The accuracy of diagnosis of parkinsonian
syndromes in a specialist movement disorder
service. Brain 2002;125:861–870.
8 Mueller CR, Goldmann SF, Wegener S: ‘Ger-
man Normal’ in HLA 1998. Larosa, American
Society for Histocompatibility and Immunoge-
netics, 1997.
9 Eichler H, Richter E, Schwartz K, Woelpel A,
Goldmann SF: ‘Caucasian German Normal’ in
HLA 1998; in Gjestson DW, Terasaki PI (eds):
Larosa, Kans., American Society for Histocom-
patibility and Immunogenetics, 1997, pp 150–
151.
10 Gossrau G, Herting B, Sommer U, Füssel M,
Koch R, Reichmann H, Lampe JB: HLA and
Parkinson’s disease. Ann Neurol Suppl 2002:
S57.
11 Adams RD, Victor M: Viral infections of the
nervous system; in Adams RD, Victor M, Rop-
per AH (eds): Principles of Neurology, ed 6.
New York, McGraw-Hill, 1997, pp 742–776.
12 Ravenholt RT, Foege WH: 1918 influenza, en-
cephalitis lethargica, parkinsonism. Lancet
1982;ii:860–864.
13 Esiri MM, Kennedy PGE: Viral diseases; in
Graham DI, Lantos PL (eds): Greenfield’s
Neuropathology, ed 6. London, Arnold and
Oxford University Press, 1997, pp 3–64.
14 Lees AJ, Stern GM, Compston DA: Histocom-
patibility antigens and post-encephalitic par-
kinsonism. J Neurol Neurosurg Psychiatry
1982;45:1060–1061.

HLA and Parkinson’s Disease Eur Neurol 2003;50:64–68 67

15 Davidowitz S: HLA antigens in Parkinson’s
disease. Excerpta Med (Amst) 1977;126.
16 Leheny WA, Davidson DL, deVane P, House
AO, Lenman JA: HLA antigens in Parkinson’s
disease. Tissue Antigens 1983;21:260–261.
17 Takagi S, Shinohara Y, Tsuji K: Histocompati-
bility antigens in Parkinson’s disease. Acta
Neurol Scand 1982;66:590–593.
18 Reed E, Lewison A, Mayeaux R, Suciu-Foca N:
HLA antigens in Parkinson’s disease. Tissue
Antigens 1983;21:161–163.
19 Matsuyama SS, Jarvik LF, Miller B, McManus
DQ, Bird TD, Katzman R, Heston L, Norman
D, Small GW: Evidence for association of
HLA-A2 allele with onset age of Alzheimer’s
disease. Neurology 1997;49:512–518.
20 Ballerini C, Nacmias B, Rombola G, Marcon
G, Massacesi L, Sorbi S: HLA A2 allele is asso-
ciated with age at onset of Alzheimer’s disease.
Ann Neurol 1999;45:397–400.
21 Harris JM, Cumming AM, Craddock N, St
Clair D, Lendon CL: Human leucocyte anti-
gen-A2 increases risk of Alzheimer’s disease
but does not affect age of onset in a Scottish
population. Neurosci Lett 2000;294:37–40.
22 Zareparsi S, James DM, Kaye JA, Bird TD,
Schellenberg GD, Payami H: HLA-A2 homo-
zygosity but not heterozygosity is associated
with Alzheimer disease. Neurology 2002;58:
973–975.
23 Jackson GS, Beck JA, Navarrete C, Brown J,
Sutton PM, Contreras M, Collinge J: HLA-
DQ7 antigen and resistance to variant CJD.
Nature 2001;414:269–270.
24 Kohutnicka M, Lewandowska E, Kurkowska-
Jastrzebska I, Czlonkowski A, Czlonkowska A:
Microglial and astrocytic involvement in a mu-
rine model of Parkinson’s disease induced by
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP). Immunopharmacology 1998;39:167–
180.
68 Eur Neurol 2003;50:64–68
25 Kurkowska-Jastrzebska I, Wronska A, Kohut-
nicka M, Czlonkowski A, Czlonkowska A: The
inflammatory reaction following 1-methyl-4-
phenyl-1,2,3, 6-tetrahydropyridine intoxica-
tion in mouse. Exp Neurol 1999;156:50–61.
26 Grunblatt E, Mandel S, Maor G, Youdim MB:
Gene expression analysis in N-methyl-4-phe-
nyl-1,2,3,6-tetrahydropyridine mice model of
Parkinson’s disease using cDNA microarray:
Effect of R-apomorphine. J Neurochem 2001;
78:1–12.
27 Metzer WS, Newton JE, Steele RW, Claybrook
M, Paige SR, McMillan DE, Hays S: HLA anti-
gens in drug-induced parkinsonism. Mov Dis-
ord 1989;4:121–128.
28 Metzer WS, Newton JE, Steele RW, Claybrook
M, Paige SR, McMillan DE, Hays S: HLA anti-
gens in tardive dyskinesia. J Neuroimmunol
1990;26:179–181.
29 Hohlfeld R, Kerschensteiner M, Stadelmann
C, Lassmann H, Wekerle H: The neuroprotec-
tive effect of inflammation: Implications for
the therapy of multiple sclerosis. J Neuroim-
munol 2000;107:161–166.
30 Dausset J, Legrand L, Lepage V, Contu L, Mar-
celli-Barge A, Wildloecher I, Benajam A, Meo
T, Degos L: A haplotype study of HLA complex
with special reference to the HLA-DR series
and to Bf. C2 and glyoxalase I polymorphisms.
Tissue Antigens 1978;12:297–307.
31 Carroll MC, Katzman P, Alicot EM, Koller
BH, Geraghty DE, Orr HT, Strominger JL,
Spies T: Linkage map of the human major his-
tocompatibility complex including the tumor
necrosis factor genes. Proc Natl Acad Sci USA
1987;84:8535–8539.
32 Dunham I, Sargent CA, Trowsdale J, Campbell
RD: Molecular mapping of the human major
histocompatibility complex by pulsed-field gel
electrophoresis. Proc Natl Acad Sci USA 1987;
84:7237–7241.
33 Sargent CA, Dunham I, Trowsdale J, Campbell
RD: Human major histocompatibility complex
contains genes for the major heat shock protein
HSP70. Proc Natl Acad Sci USA 1989;86:
1968–1972.
34 Cascino I, D’Alfonso S, Cappello N, Giordano
M, Pugliese A, Awdeh Z, Alper CA, Richiardi
PM: Gametic association of HSP70–1 promot-
er region alleles and their inclusion in extended
HLA haplotypes. Tissue Antigens 1993;42:62–
66.
35 Shyu WC, Kao MC, Chou WY, Hsu YD, Soong
BW: Creutzfeldt-Jakob disease: Heat shock
protein 70 mRNA levels in mononuclear blood
cells and clinical study. J Neuol 2000;247:929–
934.
36 Yan SD, Chen X, Fu J, Chen M, Zhu H, Roher
A Roher A, Slattery T, Zhao L, Nagashima M,
Morser J, Migheli A, Nawroth P, Stern D,
Schmidt AM: RAGE and amyloid-beta peptide
neurotoxicity in Alzheimer’s disease. Nature
1996;382:685–691.
37 Prevost G, Fajardy I, Fontaine P, Danze PM,
Besmond C: Human RAGE GLY82SER di-
morphism and HLA class II DRB1-DQA1-
DQB1 haplotypes in type 1 diabetes. Eur J
Immunogenet 1999;26:343–348.
38 McCusker SM, Curran MD, Dynan KB,
McCullagh CD, Urquhart DD, Middleton D,
Patterson CC, McIlroy SP, Passmore AP: Asso-
ciation between polymorphism in regulatory
region of gene encoding tumour necrosis factor
alpha and risk of Alzheimer’s disease and vas-
cular dementia: A case-control study. Lancet
2001;357:436–439.
Lampe/Gossrau/Herting/Kempe/Sommer/
Füssel/Weber/Koch/Reichmann
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