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Association of a monoamine oxidase B allele with Parkinson's disease.

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Association of a Monoamine
Oxldase B Allele with Parlunson’s Disease
Janice H. Kurth, PhD, Matthias C. Kurth, MD, PhD, Shirley E. Poduslo, PhD, and John D. Schwankhaus, MD
Monoamine oxidase B (MAO-B) is implicated in the cause of Parkinson’s disease (PD) because of its role in metabolizing
and forming H,02 during dopamine metabolism. Althe neurotoxin l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine,
tered MAO-B activity has been observed in PD platelets. Polymerase chain reaction was used to amplify a portion of
the MAO-B gene. Polymerase chain reaction products were screened with restriction enzymes to identify fragments
useful for single-stranded conformational polymorphism analysis. A single-stranded conformational polymorphism
was identified in an MAO-B polymerase chain reaction product after Hae 111 digestion. One hundred twenty-one
control individuals were allelotyped with frequencies of 0.45 and 0.55 for alleles 1 and 2, respectively. Frequencies of
0.62 and 0.38 (1 and 2, respectively) were observed in a population of 46 patients with PD. The presence of MAO-B
allele 1 is associated with a relative risk for PD of 2.03-fold (confidence interval, 1.44-2.61;p < 0.02).For comparison,
a monoamine oxidase A polymorphism was used to determine allelic frequencies in these same populations and no
statistically significant differences were found. These results suggest that an inherited variant of MAO-B may be
involved in a genetic predisposition for PD.
Kurth JH, Kurth MC, Poduslo SE, Schwankhaus JD. Association of a monoamine
oxidase B allele with Parkinson’s disease. Ann Neurol 1993;33:368-372
The cause of Parkinson’s disease (PD) is uncertain, and
likely includes genetic C1-61 and environmental 17111 factors. The genetics of PD poses many technical
problems, including a strong environmental component, a late age of onset, a somewhat uncertain diagnosis, and the possibility that multiple causes may lead to
a common pathology. Gene polymorphisms have been
successfully used to identify alleles associated with diseases of complex cause such as multiple sclerosis, coronary heart disease, and diabetes, among others 1121,
and were used to address the genetics of PD here.
Monoamine oxidase B (MAO-B) was chosen as a
candidate gene for P D by consideration of the biochemistry, pathophysiology, and clinical manifestations
of the disease. MAO-B produces H,O, through the
oxidative metabolism of dopamine, the principal neurotransmitter produced by the substantia nigra neurons
that are lost in PD {13]. Patients with PD have different platelet MAO-B activity than control individuals
{14, 151. Furthermore, MAO-B produces l-methyl4-phenylpyridinium, the active neurotoxin from 1methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP)
[8}. Inhibition of MAO-B with selegiline blocks
METP-induced neuronal degeneration in primates
[ 10) and reduces the rate of PD progression in humans
{16-181. Variant MAO-B activity could therefore be
involved in PD. We hypothesized that MAO-B may
be a candidate enzyme involved in the pathogenesis of
P D and that unique MAO-B gene alleles might be
detected that occur with a higher frequency in a population of patients with PD. In contrast, monoamine
oxidase A (MAO-A) is involved in serotonin metabolism {I31 and is not known to be involved in the pathogenesis of PD. We hypothesized that MAO-A allelic
frequencies would not differ among patients with PD
and control subjects.
An MAO-B gene polymorphism was identified using polymerase chain reaction (PCR) El91 and singlestranded conformational polymorphism (SSCP) {20,
211. MAO-B allelic frequencies were determined in a
population of control subjects and patients with PD.
Statistically significant differences in frequencies were
observed between the two populations. MAO-A allelic
frequencies { 2 2 ) within these populations were determined and no differences were found.
From the Department of Neurology, Tarbox Parkinson’s Disease
Institute, Texas Tech Health Sciences Center, School of Medicine,
Lubbock, TX.
Address correspondence to Dr Kurth, Department of Neurology,
Tarbox Parkinson’s Disease Institute, Texas Tech Health Sciences
Center, School of Medicine, Lubbock, TX 79430.
Materials and Methods
D N A Samples
After obtaining informed consent approved by the local Institutional Review Board, blood samples were collected from
46 patients seen at the Tarbox Parkinson’s Disease Clinic,
Texas Tech Health Sciences Center. The following diagnostic
Received Jun 22, 1992, and in revised form Sep 24. Accepted for
publication Sep 28, 1992.
368 Copyright 0 1993 by the American Neurological Association
criteria based on a recently published consensus statement
1231 were used for inclusion: (1) presence of at least two of
the following signs: resting tremor, cogwheel rigidity, bradykinesia, and postural reflex impairment, at least one of which
was resting tremor or bradykinesia; (2) parkinsonism was not
due to other known neurological disease or to known drugs,
chemicals, or toxins; (3) absence of prominent oculomotor
palsy, cerebellar signs, vocal cord paresis, orthostatic hypotension, pyramidal signs, or amyotrophy; and (4) improvement with levodopa therapy. All patients (26 men and 20
women) were of European origin. The average age was 71
-+ 8.4 years. Average duration of symptoms was 7.0 5 3.7
years and time since diagnosis was 5.4 k 4.0 years. Eightynine percent of the patients had asymmetrical symptoms or
signs early in the course of their disease.
Genomic DNA (gDNA) was extracted from 4 ml of frozen blood using a modification of the method by Grimberg
and colleagues [24}, and resuspended in 10 mM Tris-CI (pH
7.4) and 1 mM EDTA (pH 8.0).
The control population consisted of 12 1 individuals of European origin (61 men and 60 women). All were unrelated
individuals from the Centre DEtude du Polymorphisme Humain (CEPH) families. Purified gDNA was obtained directly
from CEPH.
D N A Amplzjkation 6y Polymerase Chain Reaction
The MAO-B cDNA sequence 1251 and introdexon boundary data [26) were used to design PCR oligonucleotide primers that would anneal within 2 adjacent exons and amplify
across the intervening intron. Only the approximate sizes of
the 14 introns is known 1261. We chose primers that would
amplify across small introns to give PCR products <2 kb
in size. Potential annealing sites within the MAO-B cDNA
sequence were chosen that were -20 bp in length, -50%
guanine-cytosine content, and did not have stem-loop formation potential or complimentary 3' ends. Primer sequences were excluded if they were >70% similar to any
other human sequence in the GenBank data base. MAOBl
is at the 5' end of the fourth exon. MAOB2 is at the 3' end
of the fifth exon. This pair of primers was predicted to amplify across the fourth intron of >400 bp [26} to give a
product >600 bp in length. MAOB3 is near the 5' end of
exon 13, and MAOB4 is at the 3' end of exon 14. This
primer pair was predicted to amplify across the 13th exon of
-700 bp to get an amplification product of -860 bp [26].
The primers for a portion of the MAO-A gene (MAOAI
and MAOA2) were published by Hotamisligil and Breakefield 1221. These primers amplify a 488 bp fragment of the
MAO-A gene, which contains an EcoRV restriction site polymorphism. Primer sequences are given in Table 1. All oligonucleotides (unpurified) were synthesized by Operon Technologies (Alameda, CA).
Two hundred nanograms of gDNA were amplified in 25-pl
reactions containing 1.25 units of Taq DNA polymerase
(Promega, Madison, WI) in buffer provided by the manufacturer, 200 WM of each deoxynucleotide (dATP, dCTP,
dGTP, d'ITP; Pharmacia, Piscataway, NJ), and 1 pM of each
primer. To radioactively label MAO-B PCR products for
SSCP analysis, 0.15 c1.1 of [a-32P)dATP(NEN, Boston, MA)
was added to each reaction. Twenty-five cycles of amplification were performed in a programmable thermal controller
Table 1. Primer Sequences
Primer Name
Sequence (5'-3')
MAOAI [227
MAOA2 [22)
MAOB 1
MAOB2
MAOB3
MAOB4
GACC'ITGACTGCCAAGAT
CTI'C'ITClTCCAGAAGGCC
GCAA ATCATACCCCTTC AGG
AGCAGAGCTI'GTCC AGTAGC
GGATITACTlTGC AGGC ACC
C AGACTCTGGTTCTGACTGC
(MJ Research, Watertown, MA) as previously described [27].
For the MAO-B amplifications, each cycle consisted of 0.5
minutes at 94"C, followed by 1 minute at 55"C, and 2 minutes at 72°C. The MAO-A amplification conditions were similar except that the samples were incubated at 94°C for 0.75
minutes.
Agarose Gel Electrophoresis and Restriction
Enzyme Digestion
Each pair of the MAO-B PCR primers was used to amplify
gDNA from 6 individuals. The products were screened for
digestion with a panel of 6 restriction enzymes ( A h I, Hae
111, HznP1, Hpa 11, Rsa I, BstNI; NEB, Beverly, MA). Each
20-p.1 reaction contained 10 p1 of unpurified PCR product.
The restricted products were electrophoresed in agarose to
identify fragments of desirable size for SSCP analysis. Those
enzymes that produced such products were used to digest
radioactively labeled MAO-B PCR product to search for
SSCPS.
Ten microliters of unpurified MAO-A PCR products was
digested with EcoRV (NEB 1. Products were electrophoresed
in agarose and visualized with ethidium bromide to detect
bands of 488 andlor 456 bp as previously described [22f.
Acrylamide Gel Electrophoresis and Single-Stranded
Conformational Polymorphism Analysis
Radioactively labeled and restricted MAO-B PCR products
were electrophoresed through a standard sequencing gel
(6.5% acrylamide/7 M urea) to detect small size differences.
For SSCP analysis, a modification of the method developed
by Orita and co-workers 120) as previously described 12 1J
was used. These same radioactively labeled samples were run
at 15 W for 28 hours at room temperature on a 10% glyceroV10% polyacrylamide gel to evaluate for polymorphisms.
Statistical Analysis
x2 analysis was used to compare the allelic frequencies of
MAO-A and MAO-B alleles in the patients with Parkinson's
disease and the control population. Relative risk was calculated according to Emery 112). All means are expressed with
standard deviations.
Results
Monoamine Oxidase B Polymorphism and
Allelic Frequencies
Primers were successfully designed that amplified portions of the MAO-B gene as predicted. Oligonucleotide sequences are given in Table 1.
Kurth et al: MAO-B in Parkinson's Disease 369
Fig I. Monoamine oxidase B (MAO-B) amplificationproduct.
The -860 bp products from ampl$cation of human genomic
DNA using oligonucleotideprimers MAOB3 and MAOB4 are
shown in lanes 2 to 5. Lanes 1 and 6 contain molecular weight
markers. with sizes given in base pairs to the left.
The MAOBl and MAOB2 primer pair amplified a
fragment of -1.3 kb, showing that the intron between
the fourth and fifth exon is -1.1 kb, which is significantly greater than the 400-bp minimum size that was
previously published 1263. When these PCR products
were screened with restriction enzymes, 3 (Rsa I, A h
I, Hae 111) from the panel of 6 were found to cut the
product into smaller fragments ranging up to -600 bp.
When subjected to both denaturing polyacrylamide gel
electrophoresis and SSCP analysis, no polymorphism
was observed among 6 individuals.
The MAOB3 and MAOB4 primers amplified an
-860 bp fragment as was predicted (Fig 1). When
these PCR products were screened with the panel of
restriction enzymes, Hae 111, HinPI, and BstNI were
found to produce fragments between -150 and -600
bp. When these products from 6 individuals were subjected to denaturing gradient gel electrophoresis, no
polymorphism was observed. However, when subjected to SSCP analysis, a polymorphic shift was observed in one of the Hae 111 digestion products. Two
bands, defined as MAO-B allele 1 and allele 2, were
observed as shown in Figure 2. Two informative CEPH
families were analyzed for this polymorphism to confirm X-linked inheritance.
The gDNA of the population of patients with Parkinson’s disease and control subjects was analyzed to
determine the MAO-B allelic frequencies shown in Table 2. Comparison of allelic frequencies in a population
of patients with P D with control subjects demonstrated
a statistically significant difference (x2 = 5.67, p <
0.02). The calculated relative risk for PD when allele
1 is present is 2.03, with a confidence interval of 1.44
to 2.61 (p < 0.02).
Monoamine Oxidase A Allelic Frequencies
The MAO-A PCR primers and subsequent EcoRV digestion that detect an MAO-A polymorphism [22f
were successfully used. The panels of PD and control
DNAs were analyzed to determine the MAO-A allelic
370 Annals of Neurology
Vol 3 3
No 4
April 1993
Fig 2. Single-stranded conformational polymorphism of the human monoamine oxiduse B (MAO-B) gene. The arrows point to
each of the positions of the MAO-B alleles. Lane 1 contains
product from a heterozygous female with a copy of both alleles.
Lanes 3 and 5 contain amplificationproduct.! from individuals
either homozygous or hemizygous for allele 1 . Lanes 2 and 4 contain products from individuals either homozygous or hemizygous
for allele 2.
Table 2. Monoamine Oxidase B Allelic Frequencies
Control (n = 177)
Parkinson’s disease (n = 64)
Allele 1
Allele 2
0.45
0.62
0.55
0.38
~~~~~~
x2 =
n =
5.67, df = 1, p < 0.02.
number of chromosomes analyzed
Table 3. Monoamine Oxidase A Allelic Frequencies
~
Control (n = 181)
Parkinson’s disease (n = 66)
x2 =
n
=
Allele 1
Allele 2
0.71
0.65
0.29
0.35
1.13, df = 1, p = 0.29.
number of chromosomes analyzed.
frequencies in each population given in Table 3. No
statistically significant difference was noted in the allelic frequencies in our population of patients with PD
and control subjects (x2 = 1.13, = 0.20).
Discussion
MAO-B allelic frequencies for a newly identified polymorphism in patients with PD and a control population
differ significantly. This result supports the role of
MAO-B in the pathogenesis of PD, as a functional
difference must be very closely linked to the intron
polymorphism (i.e., within the gene) to see an association among unrelated individuals. The presence of allele l increases the risk for developing PD 2.03-fold
compared with the risk without the allele. In contrast,
allelic frequencies for an MAO-A DNA polymorphism
in the same group of patients with P D did not differ
significantly from the control group and therefore is
not associated with PD in our population. However,
this finding does not necessarily exclude the MAO-A
locus from playing a role in the cause of PD because
a polymorphism different from the one evaluated here
may show some disease association.
The MAO-B gene is on the X chromosome, suggesting that PD should affect men more frequently
than women. Indeed, an increased incidence and prevalence for PD in men has been reported in two population-based studies 128, 291. The MAO-B gene may be
partially responsible for this. That only a small difference is found is likely due to the effect of other unidentified genes and environmental factors that may be involved in the development of PD.
The choice of the control population is extremely
important in associative studies. The CEPH samples
are a well defined, easily available group of D N A samples from individuals of European origin. The use of
unrelated CEPH samples to determine allelic frequencies has become relatively standard in the genetics literature. The disadvantage of using this population is the
lack of clinical and pathological information concerning
these individuals. However, pathological studies of
brains in normal subjects of 51 to 100 years of age
suggests that up to 9% of individuals may develop
Lewy bodies without necessarily developing clinical
features of PD {30, 31), so that up to 9% (10 individuals) of the control sample may develop subclinical Lewy
body pathology and be considered preclinical individuals with PD. Though this could alter the observed
allelic frequencies and relative risk values slightly, our
conclusion would remain unchanged.
The identification of an allele of MAO-B that is associated with an increased risk for the development of
PD confirms that the search for a genetic predisposition for a complex multifactorial disease like P D can
be undertaken using this approach. Identification of
additional candidate genes through an understanding
of the mechanisms of neuronal cell death may permit
the development of genetic markers that can predict a
predisposition to PD. Ultimately, a number of such
markers may identify individuals at risk for PD, and
allow intervention in the disease process before the
disabling symptoms occur.
After completion of this work, we became aware
that another polymorphism within the MAO-B gene
has recently been identified by Konradi and associates
{32], which is different from the polymorphism reported here.
This work was supported by the Tarbox Parkinson’s Disease Institute
at the Texas Tech Health Sciences Center.
We thank Dr Xandra 0. Breakefield for sharing her MAO-B polymorphism with us before publication, and Drs Arthur W. Coquelin,
Judith E. Hogg, and William H. Lyness for their careful review of
the manuscript, and Julia E. Treland for help in compiling the clinical
data base.
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