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An allelic association study of monoamine oxidase B in parkinson's disease.

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served in unaffected tissues in patients with the 3243
mutation { 121. These observations would support the
view that factors in addition to tissue distribution and
proportion of mutation are important in determining
tissue-specific dysfunction and therefore phenotype.
The precise nature of these factors remains to be determined; possibilities include tissue-specific differences
in expression of nuclear-encoded respiratory chain subunits, and perhaps the efficiency with which they can
be upregulated, or differences in the distribution of
mtDNA defects, perhaps allowing complementation to
occur in some tissues but not others.
The mean proportion of mutant mtDNA in the tissues of our patient containing dividing populations of
cells was less than that in tissues containing predominantly nondividing populations (61.796 vs 89%, p <
.05, Mann-Whitney U test). It is possible that mutant
mtDNA is selected against in dividing cell populations,
as has been proposed for mtDNA deletions that are
rarely detected in blood {2]. Selection against point
mutations may also occur, as they are always present
in lower amounts in blood compared to muscle. Poulton and Morten { l 5 ] suggested that the proportion of
mutant (3243) mtDNA in blood declines with age.
In conclusion, we defined an unusual mitochondrial
encephalomyopathy phenotype at a molecular genetic
level and demonstrated that the mutation has a widespread tissue distribution. From a clinical point of view
these observations suggest that a mtDNA disease
should be considered in the differential diagnosis of
progressive dementia with chorea.
Financial support from the Medical Research Council of Great Britain and the European Economic Community is gratefully acknowledged.
References
1. Petty RKH,Harding AE, Morgan-Hughes JA. The clinical features of mitochondrial myopathy. Brain 1986;109:915-938
2. Holt IJ, Harding AE, Cooper JM, et al. Mitochondrial myopathies: clinical and biochemical features of 30 patients with major
deletions of muscle mitochondrial DNA. Ann Neurol 1989;26:
699-708
3. Moraes CT, DiMauro S, Zeviani M, et al. Mitochondrial DNA
deletions in progressive external ophthalmoplegia and KearnsSayre syndrome. N Engl J Med 1989;320:1293-1299
4. DiMauro S, Moraes CT. Mitochondrial encephalomyopathies.
Arch Neurol 1993;50:1197-1208
5. Truong DD, Harding AE, Scaravilli F, et al. Movement disorders in mitochondrial myopathies: a study of nine cases with
two autopsy studies. Mov Disord 1990;5:109-117
6. Morgan-Hughes JA, Hayes DJ, Cooper M, Clark JB. Mitochondrial myopathies: deficiencies localized to complex I and complex 111 of the mitochondrial respiratory chain. Biochem Soc
Trans 198S;13:648-650
7. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain
termination inhibitors. Proc Natl Acad Sci USA 1977;74:54635467
8. Anderson S, Bankier AT, Barrel1 BG, et al. Sequence and orga-
9.
10.
11.
12.
13.
14.
15.
nisation of the human mitochondrial genome. Nature 1981;290:
457-465
Holt IJ, Hdrding AE, Petty RKH, Morgan-Hughes JA. A new
mitochondrial disease associated with mitochondrial DNA heteroplasmy. Am J Hum Genet 1990;46:428-433
Sprinzl M, Hartmann, Weber J , et al. Compilation of tRNA
sequences and sequences of tRNA genes. Nucleic Acids Res
1989;17(suppl):rl-r 172
Sweeney MG, Bundey S, Brockington M, et al. Mitochondrial
myopathy associated with sudden death in young adults and a
novel mutation in the mitochondrial DNA leucine transfer
RNA (UUR) gene. Q J Med 1993;86:709-713
Shiraiwa N , Ishii A, Iwamoto H , et al. Content of mutant mitochondrial DNA and organ dysfunction in a patient with a
MELAS subgroup of mitochondrial encephalomyopathies. J
Neurol Sci 1993;120:174-179
Sweeney MG, Hammans SR, Duchen LW, et al. Mitochondrial
DNA mutation underlying Leigh’s syndrome: clinical, pathological, biochemical and genetic study of a patient presenting with
progressive myoclonic epilepsy. J Neurol Sci 1994;121:57-65
Boulet L, Karparti G, Shoubridge EA. Distribution and threshold expression of the tRNA Lys mutation in skeletal muscle
of patients with myoclonic epilepsy and ragged red fibers
(MERRF). Am J Hum Genet 1992;51:1187-1200
Poulton J, Morten K. Noninvasive diagnosis of the MELAS
syndrome from blood DNA. Ann Neurol 1993;34:116 (Letter)
An Allelic Association
Studv of Monoamine
oxidase B in
Parkinson’s Disease
S. L. Ho, MRCP,” A. L. Kapadi,? D. B. Ramsden, PhD,t
and A. C. Williams, MD, FRCPX
A single-stranded conformational polymorphism in the
monoamine oxidase B gene was shown to be A or G, 36
bases upstream from the intron 13-exon 14 boundary.
An allelic association study revealed no statistically significant associations between this single-base polyrnorphisrn and Parkinson’s disease, unlike the results of a
previous study.
Ho SL, Kapadi AL, Ramsden DB, Williams AC.
An allelic association study of monoamine
oxidase B in Parkinson’s disease.
Ann Neurol 1995;37:403-405
Kurth and associates 111 identified a single-stranded
conformational polymorphism (SSCP) in intron 13 of
From the Departments of “Clinical Neurology and ?Medicine, University of Birmingham, Queen Elizabeth Hospital, Birmingham, UK.
Received Jun 14, 1994, and in revised form Sep 13. Accepted for
publication Nov 3, 1994.
Address correspondence to Dr Ho, Department of Neurology, University of Birmingham, Queen Elizabeth Hospital, Birmingham B15
2TH, UK.
Copyright 0 1995 by the American Neurological Association 403
the monoamine oxidase B (MAO-B) gene and claimed
that o n e allele occurred with a significantly higher frequency in their parkinsonian population compared
with t h e control group. We have sequenced intron 13
of t h e g e n e and elucidated the exact nature of t h e polymorphism. We then carried o u t an allelic association
study in a n attempt to substantiate t h e claim of Kurth
and associates [ l f .
Materials and Methods
Part I-Identification of the Polymorphism
A section of D N A containing MAO-B intron 13 was amplified from genomic D N A by polymerase chain reaction (PCR)
as described by Kurth and associates 111. PCR products of
the approximate anticipated size (800 bp) were purified
(Sephaglas BandPrep kit, Pharmacia Biotech Europe, Milton
Keynes, UK), cloned into TA cloning vector (Invitrogen version 1.3, R & D Systems Europe Ltd, Abingdon, UK), and
the inserts of positive bacterial colonies sequenced using an
automated D N A sequencer (model 373A, version 1.2, Applied Biosystems Inc, Warrington, UK). The above procedure was repeated using D N A from a total of 11white males.
Sequencing was repeated until each base was identified unambiguously. The 11 sequences were then compared with
each other to identify any polymorphic bases.
Part 11-Allelic Association Study
ALLELE-SPECIFIC POLYMERASE CHAIN
REACTION
ASSAY.
Downstream allele-specific primers ( 5 ‘-CACTGGCAAATAGCAAAAG T or C-3’) were used with a common upstream primer, 5’-GGATITACTITGCAGGCACC-3’, in
an allele-specific PCR assay. The PCR conditions were optimized such that a product of 663 bp was produced with the
appropriate allele-specific primer when using genomic D N A
of the 11 males as template, which had been characterized
previously by sequencing. Negative control (i.e., without any
D N A template) PCR reactions were carried out with every
set of amplifications to ensure that no false-positive reactions
occurred. The optimal conditions for allele-specific PCR
were as follows: genomic D N A (0.1-0.4 kg), d N T P (2.5
pM), Mg2+ (1 pM), and using “hot-start’’ PCR at 99°C for
5 minutes before adding Taq polymerase (2.5 units, Bioline,
UK) followed by 92°C for 45 seconds, 65°C for 1 minute,
72°C for 1 minute for 30 cycles, and an extension time of 5
minutes at 72°C for the final cycle.
Szcbjects
Allele-specific PCR assay was carried out on four different
white populations (all U K residents) as follows:
Idiopathic Parkinson’s disease (IPD) group (76 males, 36
females; age range, 39-83 yr; mean, 64.8 2 9.7 yr): Subjects
were unrelated with no family history of P D in their firstdegree relatives. IPD was diagnosed in the presence of three
of the following features: resting tremor, rigidity, bradykinesia, postural instability, and gait disturbance as assessed by
two neurologists. Patients with atypical features or parkinsonism due to any other neurological diseases, chemicals, or
404 Annals of Neurology Vol 37 NO 3 March 1995
toxins were excluded. All patients improved with levodopa
therapy.
Normal control group (37 males, 23 females; age range
10.1 yr): Subjects were healthy,
40-84 yr; mean, 60.3
unrelated volunteers. None had any significant past medical
or family history of PD.
Disease control group (11 males, 17 females; age range,
48-84 yr; mean, 62.2 t 9.8 yr): These subjects had vario-us
neurological diseases (9 had dementia, 8 had motor neuron
disease, 4 had Huntington’s chorea, 2 had progressive suprabulbar palsy, 2 had essential tremor, 1 each of Pick’s disease, Down’s syndrome, Hallervorden-Spatz disease).
Familial Parkinson’s disease group (index patients; 5 males,
7 females; age range, 32-77 yr; mean, 59.6 2 14.9 yr):
These subjects were index patients of families with P D (families had at least 2 members from different generations with
PD).
*
Statistical Analysis
Comparisons of the allelic frequencies were performed using
x 2 test (Minitab Statistical Package, Clecom, UK).
Results
Part I-Identification of the Polymorphism
The sequence of the initial PCR product (740 bp) is
recorded in EMBLlGenbank (accession no. 22907 l ) ,
intron 13 contained within this product being only 590
bp. HaeIII restriction cleaved t h e PCR product into
three fragments (66, 180, and 494 bp, respectively).
A single-base polymorphism (G, 7 o f 11 males; A,
4 of 11) was found in t h e 180-bp HaeIII fragment
(base number 644, EMBL/Genbank accession no.
229071), 36 bases upstream from the intron 13-exon
14 boundary.
Part 11-Allelic Association Study
The results of the population study are summarized in
the Table. The A:G ratio was 1.08 in IPD patients
compared with 0.73 in normal controls, 0 . 6 1 in disease
control, and 1.11 in index patients of families with PD.
Therefore, no statistically significant differences were
found w h e n frequencies were compared.
Discussion
The only difference found when the sequence of 11
male individuals were compared with each o t h e r was
that of single base (A o r G). This polymorphic base
was in t h e 180-bp HaeIII restriction fragment. This
site would fit with the SSCP identified by Kurth and
associates [ 11, because single-stranded polymorphic
shifts are not likely to be observed in fragments larger
than 450 bp. However, it was not possible to identify
which of t h e alleles in o u r study were equivalent t o
the SSCP bands reported earlier [11.
Kurth and associates [I} reported a significant difference in allelic frequencies of this SSCP in their parkin-
Distribution of Pohmorphic Bases and Monoamine Oxidase B Allelic Frequencies in Subyects
Polymorphic Bases
Allelic Frequencies
A
AIA
AIG
G
GIG
36
15
13
6
6
3
40
22
2
2
10
10
2
77
35
17
10
71
48
28
3
14
7
1
2
9
1.08
0.73
0.61
1.11
63
24
28
66
31
139
156
0.89
A
G
A:G Ratio
~~
IPD
Normal control
Disease control
Familial Parkinson’s disease
(index patients)
Total
IPD
=
9
9
idiopathic Parkinson’s disease.
sonian group compared with those of their control
population. They found that one allele was associated
with a relative risk for IPD of 2.03-fold. In contrast
we have not found any statistically significant differences in allelic frequencies in our population groups
and, therefore, have been unable to substantiate the
claim that this polymorphism in the MAO-B gene is
implicated in any genetic predisposition to develop
IPD. Further, the distance of the single-base polymorphism from recognized regulatory elements in the 5’
flanking region of the gene (>42 kb) 12, 31 suggests it
would be unlikely to influence gene transcription. Finally, there is still no direct evidence that any structural
variations in the MAO-B gene are responsible for the
wide variations in MAO-B activity seen in humans or
that implicates the gene in the genetic predisposition
to develop IPD.
We are grateful to Orion-Farmos Pharmaceuticals Ltd and The Sir
Jules Thorne Trust for their financial support.
References
1. Kurth JH, Kurth MC, Poduslo SE, SchwankhausJD. Association
of a monoamine oxidase B allele with Parkinson’s disease. Ann
Neurol 1993;33:368-372
2. Grimsby J, Chen K, Wang L-J, et al. Human monoamine oxidase
A and B genes exhibit identical exon-intron organisation. Proc
Natl Acad Sci USA 1991;88:3637-3641
3. Zhu Q-S, Grimsby J, Chen K, Shih JC. Promoter organisation
and activity of human monoamine oxidase gene (MAO) A and
B genes. J Neurosci 1992;12:4437-4446
The Gene for Hereditary
Progressive Dystonia with
Marked Diurnal Fluctuation
Maps to Chromosome 144
H. Tanaka, MD,” K. Endo, MD,* S. Tsuji, MD, PhD,*
T. G. Nygaard, MD,t D. E. Weeks, PhD,$
Y. Nomura, MD,§ and M. Segawa, MD§
Hereditary progressive dystonia with marked diurnal
fluctuation (HPD) is a childhood-onset, postural dystonia that is characterized by marked diurnal fluctuation
and a dramatic response to levodopa. Recently, the gene
for dopa-responsive dystonia (DRD),an autosomal dominant dystonia showing similarly marked response to
levodopa, has been mapped to chromosome 14q. Since
HPD and DRD share many clinical characteristics, we
have analyzed microsatellite polymorphisms in the region of the DRD locus and obtained a maximal lod score
of 2.0 at D14S52 without obligate recombination events
in the affected individuals. The results strongly suggest
that HPD and DRD are to be caused by mutations in
the same gene on the long arm of chromosome 14.
Tanaka H, Endo K, Tsuji S, Nygaard TG, Weeks
DE, Nomura Y, Segawa M. The gene for
hereditary progressive dystonia with marked
diurnal fluctuation maps to chromosome 14q.
Ann Neurol 1995;37:405-408
~
~
From the *Department of Neurology, Brain Research Institute, Niigata University, Niigata, and OSegawa Neurological Clinic for Children, Tokyo, Japan; tDepartment of Neurology, Columbia Presbyterian Medical Center, New York, NY; and $Department of Human
Genetics, University of Pittsburgh, Pittsburgh, PA.
Received Sep 12, 1994, and in revised form Nov 8. Accepted for
publication Nov 9, 1994.
Address correspondence to Dr Tanaka, Department of Neurology,
Brain Research Institute, Niigata University, 1 Asahimachi, Niigata
95 1, Japan.
Copyright 0 1995 by the American Neurological Association
405
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