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DNA length polymorphism 5 to the myelin basic protein gene is associated with multiple sclerosis.

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DNA Length Polymorphsm 5' to the
Myelin Basic Protein Gene Is Associated
with Multiple Sclerosis
Kevin B. Boylan, MD,"§ Naoki Takahashi, PhD,'l# Donald W. Paty, MD,§ Adele D. Sadovnick, PhD,"
Marc Diamond, BA," Leroy E. Hood, MD, PhD,T and Stanley B. Prusiner, MD"?
A site of DNA polymorphism linked to the myelin basic protein gene, identified as restriction fragment length
polymorphism, was analyzed in a population-based study comparing patients with clinically definite multiple sclerosis
(MS) and population-matched control subjects. A 0.9-kilobase (kb) genomic DNA fragment (EcoG) encompassing the
first exon of the human myelin basic protein gene, located on the long arm of chromosome 18, identified ten alleles
arising from a region of DNA, 1.5 kb 5' to the myelin basic protein gene first exon coding region. Produced by RsaI
digests and ranging in length from 2.05 to 2.15 kb, these alleles vary in size by up to 100 base pairs due to insertion or
deletion, or both, from a 1-kb length of repetitive DNA. Allele frequencies among 65 patients with MS were compared
with those of 63 control subjects. Chi square for these data was significant ( p < 0.001), largely due to a preponderance
in the patients with MS of alleles in the 2.14- to 2.15-kb range. Comparison of the numbers of patients with MS and
control subjects bearing specific alleles showed that 45% of the patients carried at least one allele of 2.14 to 2.15 kb as
opposed to 19% of control subjects ( p < 0.005). These data, while preliminary, suggest that patients with MS differ
from population-matched control subjects with respect to DNA polymorphism linked to the myelin basic protein
gene. Although no pathogenic relationship between this polymorphism and MS can be presupposed, this finding raises
the possibility that the myelin basic protein gene or some other myelin basic protein-linked locus may be a factor in
susceptibility to MS.
Boylan KB, Takahashi N, Paty DW, Sadovnick AD, Diamond M, Hood LE, Prusiner SB.
DNA length polymorphism 5' to the myelin basic protein gene is associated
with multiple sclerosis. Ann Neurol 1990;27:291-297
Genetic and environmental factors appear to interact
in the development of multiple sclerosis (MS); however, the precise cause remains unknown 11, 2). Data
suggesting a genetic component of MS pathogenesis
have emerged over the last several decades. Epidemiological studies show that MS is a disease principally of
northern European Caucasians f3). First-degree relatives of patients with MS show an increased incidence
of MS, with empirical recurrence risks of 30 to 50
times those of the general population f4). In addition,
the concordance rate of MS is much higher in monozygotic twins than in dizygotic twins f5l.
Population studies have revealed association of
HLA antigens A3, B7, and DR2 with MS, but segregation and sib pair analyses have not consistently
shown HLA linkage f6-8). Population data suggesting
an association between immunoglobulin (Gm) marker
and MS have also appeared; however, this locus is less
well studied in MS than is HLA, and no linkage data
have been published 19).
Statistical modeling of genetic factors in MS indicates that familial occurrence is best explained by a
model involving a genetic locus or loci in addition to
HLA [lo, 11). The mode of inheritance of risk factors
for MS remains uncertain, and the specific role of
HLA-linked factors is unresolved [l 1). Identification
of non-HLA-linked loci influencing the development
of MS would clarify the present understanding of inherited susceptibility to MS.
Autoimmunity to myelin basic protein (MBP) has
been considered as a factor in MS pathogenesis, based
on studies of experimental allergic encephalomyelitis
From the Departments of *Neurology and ?Biochemistry and Biophysics, and the $Division of Medical Genetics, University of
California,
Francisco, CA; the $Departments of Neurology and
"Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; and the "Division of Biology, California Institute of Technology, Pasadena, CA.
'Present address: Department of Biochemistry, University of Tokyo, Faculty of Medicine, Tokyo, Japan.
Received for publication
28, 1989,
in revised form
29,
Accepted for publication Aug 30, 1989,
Address correspondence to Dr. Prusiner, Department of Neurology
HSE-781, University of California, San Francisco, CA 94143-0518.
Copyright 0 1990 by the American Neurological Association
291
(EAE) and investigation of immunity to MBP in patients with MS 112-161. The immune response in MS
demyelination remains poorly understood, however,
and many questions in this area remain unresolved
115, 17-21). Overall, it appears that MBP autoimmunity occurs in at least a subset of patients with MS
in the setting of a more uniform and extensive immune
system dysregulation.
Parallels between EAE and MS suggest that MBP
polymorphism could result in enhanced MBP autoantigenicity, thereby contributing to autoimmune CNS
demyelination 122, 231. In a more recently developed
model for MS, viral proteins bearing structural similarity to myelin components are hypothesized to initiate
autoimmune demyelination 1131. The primary immune response against the virus becomes directed
against myelin, owing to cross-reactivity between viral
and myelin epitopes. Under this hypothesis, termed
molecakzr mimicry 1241, MBP mutation that produced
structural homology between MBP and viral proteins
could facilitate development of autoimmune demyelination.
The forms and frequency of MBP protein and structural gene polymorphism both within and among
species are unknown. Only one MBP structural gene
polymorphism has been reported 1221. Because this
variant (a conservative amino acid substitution) has
been found in only one of a small number of persons
(both patients with MS and subjects without MS) analyzed at the amino acid level, its population frequency
is unknown.
Association of specific restriction fragment length
polymorphisms (RFLPs) with structural gene mutations is well known in a number of genetic diseases,
based on population studies 125-271. RFLPs linked to
the MBP gene have been identified using several
cloned genomic and cDNA probes derived from the
MBP gene, located on the long arm of chromosome
18 128, 271. However, no data regarding MBP-linked
U P S among patients with MS have been reported.
We previously reported a region of highly repetitive
DNA located 5‘ to the human MBP gene first exon
1271. DNA length polymorphism, identified as RFLPs
at this site, is due to insertion or deletion, or both, of
DNA from the chromosome. Eleven alleles have been
identified, showing stable inheritance as simple mendelian traits. A 0.7-kilobase (kb) human genomic probe
(EcoG) encompassing the first exon of the MBP gene
identifies polymorphisms arising from this region of
repetitive DNA. Sequence analysis reveals that the
repetitive region consists of approximately 1.0 kb of
the tandemly repeated tetramer [TGGA), located
within 1.5 kb 5’ to the probe site 1301. Here we report
the use of RFLPs generated by the MBP 5’ repetitive
site to compare patients with MS and control subjects
in a population-based study.
292 Annals of Neurology Vol 27 N o 3 March 1770
Methods
Subjects
All subjects who provided blood samples for this study were
enrolled after they gave informed consent, as approved by
the appropriate institutional review board.
Patients were identified through the MS clinic at the
Health Sciences Center Hospital of the University of British
Columbia, Vancouver. The patients with MS all had clinically
definite MS as determined by established criteria (either
chronic progressive or relapsing-remitting type) [3 1). All
were unrelated Caucasians of northern European extraction.
There were 65 patients with MS (45 [67%1 women), with an
age range of 23 to 65 years (average, 36.1 years). Control
subjects (also unrelated) were either healthy individuals or
persons with nondemyelinating neurological diseases. They
were thought to be free of MS according to findings on
history and examination. All were Caucasians of northern
European extraction and had no known family history of MS.
We chose control subjects who were 50 years or older to
reduce the chance of including in the control group persons
likely to develop MS later [321. There were 63 control subjects (27 [46%] women), with an age range of 50 to 99 years
(average, 67.6 years).
D N A lsolation
DNA was prepared from peripheral blood leukocytes (2050 ml of whole blood) using standard techniques, as previously described E291.
D N A Polymorphism Analysis
RFLP studies were performed using the Southern transfer
technique, as previously described in detail 129, 331, except
as will be noted. For detailed polymorphism analysis,
genomic DNA was probed with the cloned genomic DNA
probe, EcoG (Fig 1).This is a 900-base pair (bp) fragment
encompassing the coding region for the first 58 amino acids
of human MBP. This sequence represents the first exon of
the human MBP gene by comparison with the fully sequenced mouse MBP gene 1341. The EcoG probe encodes a
single copy sequence mapped to the distal long arm of
chromosome 18 by in situ hybridization [351.
Restriction enzyme digests of genomic DNA were carried
out as described previously [271. The enzymes used for
RFLP analysis were EcoRI and RraI;relevant restriction sites
are shown in Figure 1. DNA polymorphism arising in the 5’
repetitive region is detected with RsaI and EcoG owing to the
generation of a restriction fragment covering the entire repetitive region, which also encompasses the 5’ half of the
probe side (see Fig 1). Detailed comparison of MBP polymorphism in the 5’ repetitive region in the control group
and the group with MS was made by Southern transfer analysis of DNA digested with &a1 and probed with EcoG.
To confirm that polymorphic fragments seen with EcoG
were in fact produced by repetitive region alleles as opposed
to DNA structural variation of some other type, DNA of all
subjects was analyzed with a second genomic probe in separate experiments. This fragment, Eco2.1, is the 2.1-kb EcoRI
genomic fragment immediately 5’ to EcoG and encompasses
the entire repetitive site (see Fig 1). This fragment has been
fully sequenced and is reported in detail elsewhere [331. In
5'
3'
REPETITIVE DNA [TGGA].
o,
ECOG PROBE SITE
kb
U
..
...
.
I
POLYMORPHIC 2 05 to 2.1 5 kb
Fig 1. Map ofthe region encompassing the first exon of the human myelin basic protein (MBP) gene, including the adjacent 5
repetitive sequence. The EcoG probe site is shown by the open
box; its 5' and 3' ends are marked by EcoRI sites. The region
within the probe site encoding thefirst 58 amino acids ofMBP
is hatched. The region of repetitive D N A sequence {TGGA), is
indicated ly the shaded box. The Eco2.1 probe is represented &y
the fragment extending from the 5' border of the EcoG probe
(EcoRI site) to the left-most EcoRl site. The EcoG probe detects
the Rsal fragments shown ly the open bars. The 3' 1.6836
fragment was seen in all subjects tested. The 5' polymorphic
fragment varied from 2.05 t o 2.15 kb, owing to length variation
in the repetitive D N A region. Restriction sites from the 3'
EcoRI site upstream to the 5' EcoRl site to the repetitive region
(on the extreme kft of the diagram) were determinedly D N A
sequencing. The Rsal site marking the 3' margin of the 1.68-kb
invariant fragment was localized through double restriction enzyme digests. E = EcoRI site; R = Rsal site.
EcoRI digests of genomic DNA for a given subject, the
Ec02.1 probe should yield polymorphic bands in an identical
pattern to those seen with the EcoG probe in RsaI digests,
because both probes recognize the same repetitive polymorphic site.
For RraYEcoG RFLP analysis, electrophoresis gels were
1.5% agarose, 13 X 30 cm or 20 X 30 cm run at 1.2 to 1.5
Vkm. Band migration artifacts were controlled for by transfer of an invariant 1.68-kb RsaI fragment in each sample in
the experiments. Resolving power of the gel system used was
such that bands differing in size by 30 bp or more were
clearly separated. Standards were a collection of radioactively
labeled pBR322 fragments transferred to the filters with the
sample DNA.Restriction fragment sizes were averaged from
at least two separate experiments. The individual gels contained DNA samples entirely from control subjects or from
patients with MS.
Statistical Methods
The null hypothesis of this study was that there was no difference in REXP allele frequencies at the 5' repetitive site
between the group with MS and the control group. Comparison of the two populations was performed by chi-square
analysis {36]. Comparison of allele frequencies for specificsize alleles or discrete-size ranges was by chi-square analysis
in 2 x 2 tables with continuity correction 1363.
Results
DNA extracted from peripheral blood leukocytes was
digested with RraI and EcoRI, and analyzed by the
method of Southern using the EcoG and Eco2.1 probes,
respectively.
RraI digests produced one or two bands in the 2.05to 2.15-kb range, and a single band of 1.68 kb. Exam-
INVARIANT1 68 kb
21152066
-
bP
1922
-
17251646
-
1425
-
Fig 2. Example of polymorphism detected with the EcoG probe
in Rsal digests of D N A from patients with MS.DNA was isolated and digested with Rsal, and the digests were fractionated by agarose gel electrophoresis as described in Methods. The
D N A was transferred to nitrocellulose and probed with EcoG,
and the bands were imaged by autoradiography as described.
Samples from the control group and the group with MS were
electrophoresed separately. The D N A size markers indicated
were restriction enzymefragments of pBR322, radioactively
labeled and transfmed to nitrocellulosealongside the sample
bands. The two most closely spaced standard bands differin size
Sy 49 bp. The EcoG probe hybridizes a 1.68-kb band in all
subjects. This fragment represents the 3' half of the probe and
additional downstreamjanking D N A (see Fig 1). The 5' repetitive region gives rise to multiallelic D N A length variation
upstream of the probe site. This polymorphism is seen as size
variation in the Rsal fragment encompassing this region and the
adjacent 5' half of the EcoG probe (see Fig 1). Persons with two
bands in the 2.1-kb range are considered heterozygous for the 5'
fragments detected by the probe. Subjects with one band in this
region were assumed to be homozygous for the same-sizefragment.
Gel resolution was such that bands in the 2.1-kb region differing by approximately 30 bp or more were clearb resolved (note
Subject 032).
ples of RsaI digests of MS patients' DNA hybridized
with EcoG are shown in Figure 2. DNA size markers
closely spaced over the fragment size range of interest
(as indicated in Fig 2) facilitated precise measurement
of fragment sizes. The presence of one or two bands in
the 2.05- to 2.15-kb range in all subjects is due to
multiallelic size variation in restriction fragments extending 5' to the EcoG probe site (see Fig 1). A subject
possesses two bands if his or her respective chromosomes 18 have DNA length polymorphism in the
5 repetitive region. In EcoRI digests of all samples, the
Boylan et al: MBP Gene Polymorphism and MS 293
Table I. Polymorphic Rsd Fragments Hybridizing the Myelin
Basic Protein Gene First-Exon Probe EcoG in Human DNA
from Control Subjects and Patients with MS”
Subject
No.
5’ Fragment
Size (kb)
Subject
No.
5’ Fragment
Size (kb)
Control Subjects
00 1
002
003
004
005
006
007
008
009
010
01 1
012
013
014
015
016
017
018
019
020
02 1
022
02 3
024
02 5
026
027
028
029
030
03 1
032
00 1
002
003
004
005
006
007
008
009
010
01 1
012
013
014
015
016
2.06
033
2.06
034
2.14
035
2.14
036
2.14, 2.06
037
2.07
038
2.06
039
040
2.13, 2.05
04 1
2.05
2.14
042
2.06
04 3
044
2.13, 2.05
2.14, 2.10
04 5
2.14
046
2.14, 2.06
04 7
048
2.14
2.06
049
050
2.13
05 1
2.14
2.13
052
2.13
053
2.13
054
2.05
055
056
2.13
2.13, 2.09
057
058
2.13, 2.06
2.13, 2.05
059
060
2.13, 2.05
06 1
2.13
062
2.12
2.14, 2.05
063
2.12, 2.06
Patients with MS
2.14, 2.06
034
2.15, 2.06
035
2.14
036
2.14
037
2.14
038
2.13
039
2.13
040
2.09, 2.05
04 1
2.14, 2.06
042
2.14
04 3
2.14, 2.06
044
2.13, 2.10
04 5
2.14
046
2.14
047
2.14
048
2.06
049
2.13, 2.05
2.12, 2.05
2.13, 2.05
2.13, 2.05
2.09, 2.05
2.13, 2.05
2.13, 2.05
2.05
2.05
2.13
2.13, 2.06
2.13, 2.06
2.13
2.13
2.14, 2.06
2.14, 2.05
2.13
2.14, 2.06
2.12, 2.05
2.13
2.13
2.09, 2.05
2.13
2.13
2.13, 2.05
2.13
2.09, 2.05
2.13, 2.05
2.13, 2.05
2.13
2.06
2.13, 2.06
2.13, 2.05
2.14, 2.07
2.06
2.14, 2.07
2.14, 2.06
2.14, 2.06
2.14, 2.07
2.11, 2.06
2.05
2.13, 2.06
2.14, 2.06
2.13, 2.06
2.13, 2.06
2.05
2.12, 2.06
294 Annals of Neurology Vol 27 No 3 March 1990
Table 1. Continued
Subject
No.
5’ Fragment
Size (kb)
017
018
019
020
02 1
022
02 3
024
02 5
026
02 7
028
029
030
03 1
032
033
2.12,
2.13,
2.10,
2.15
2.14,
2.14,
2.13
2.14
2.09,
2.14,
2.15,
2.11,
2.14,
2.14,
2.14,
2.14,
2.15,
Subject
No.
Patients with MS
2.05
050
2.06
05 1
2.05
052
05 3
2.07
054
2.10
055
056
05 7
2.05
058
2.06
059
2.07
060
2.07
06 1
2.06
062
2.07
063
2.06
064
2.11
065
2.11
5’ Fragment
Size (kb)
2.13, 2.06
2.13
2.14
2.13
2.05
2.13, 2.09
2.13, 2.05
2.13
2.12, 2.05
2.05
2.05
2.13
2.12
2.13
2.12, 2.05
2.10. 2.05
“The EroG probe detects fragments that include the 3‘- and 5’flanking regions of the myelin basic protein gene first exon. Only the
5’4anking fragment is polymorphic in these subjects, and is tabulated above. The 3’-flanking fragment was 1.68 kb in all subjects
(see Fig 2) and is not included. Subjects for whom a single 5’ fragment size was seen were assumed to be homozygous for similar-size
fragments.
Ec02.1 probe yielded the same number of polymorphic
bands (one or two), showing the same relative size
difference, as did the EcoG probe in RruI digests (data
not shown).
&I fragments differing in length by 30 bp or more
were clearly resolved (note Subject 032 in Fig 2).
However, smaller size variations in the 10- to 20-bp
range were also reproducibly observed between subjects. Fragment sizes were measured in duplicate experiments under similar electrophoretic conditions.
For the polymorphic fragments (2.05- to 2.15-kb size
range), RsuI fragment sizes in control subjects and patients were k 8 bp. Fifty-nine (94%) of 63 control
samples were k 5 bp or less. Fifty-eight (89%) of 65
MS samples were 5 bp or less. Fragment sizes were
rounded to the nearest 10 bp because reproducible
measurements were generally within this size range.
RsaI fragment sizes for the 63 control subjects and
65 patients with MS were measured and are presented
in Table 1. Subjects with one band are considered
homozygous for length polymorphism at the 5’ repetitive site, and those with two bands are considered
heterozygous. From the foregoing it is apparent that
heterozygotes bearing alleles that differ in size by
much less than 30 bp would necessarily be considered
homozygotes, owing to incomplete resolution of the
alleles in the gels. Therefore, while 29 control subjects
*
(46%) and 39 patients with MS (60%) were found to
be heterozygous (not significant p > 0.05), these numbers probably underestimate the degree of heterozygosity.
The data in Table 1 are presented as a histogram in
Figure 3. The allele frequency distributions are similar
in being bimodal and skewed. This is analogous to the
&a1 allele distribution noted previously in a smallscale general population study {29]. In Table 2 the
distribution of allele frequencies (from Fig 3) is presented with the distribution of control subjects or patients with MS possessing at least one of a specific-size
allele. Overall, the absolute allele frequencies paralleled the proportion of control subjects or patients
with MS bearing a given allele. Chi-square analysis of
the allele and subject distributions was performed to
determine whether the control group and group with
MS differed with respect to allele frequencies or with
respect to the numbers of subjects carrying specific
alleles. Data for the ten alleles were analyzed for the
allele and subject distributions in a 5 x 2 table. The
columns were constructed for the fragment size ranges
of 2.05 to 2.06 kb, 2.07 to 2.09 kb, 2.10 to 2.11 kb,
2.12 to 2.13 kb, and 2.14 to 2.15 kb. Chi squares for
the allele and subject distributions were significant
(20.35,p < 0.001, and 15.68, p < 0.005, respectively).
To determine whether the difference between the
control group and the group with MS could be localized within the allele and subject distributions, subsections of the respective distributions were compared.
Significant differences between the control group and
the group with MS occurred only in the 2.13- to 2.15kb range in both the allele and human subjects distributions (see Table 2).
Twenty-nine (45%) of 65 patients with MS possessed at least one allele in the 2.14- to 2.15-kb range,
compared with 12 (19%) of 63 control subjects ( p <
0.005). Ten patients with MS were homozygous for
2.14- or 2.15-kb alleles, versus 5 in the control group
( p < 0.10). Nineteen patients with MS had a single
2.14- to 2.15-kb allele, while only 7 control persons
were heterozygotes with an allele in this range ( p <
0.10). Thirty-two (51%) of 63 control subjects had at
least one 2.13-kb allele, while 18 (29%) of 65 patients
with MS had an allele of this size ( p < 0.025).
Discussion
The patients with MS and the control subjects in this
study were unrelated but were drawn from the same
background population in order to reduce the chance
of introducing population stratification artifacts. The
control subjects were 50 years or older to decrease the
possibility that individuals with occult MS would be
included. All subjects were from the Vancouver, British Columbia area. MS prevalence in this well-studied
population is approximately 100 per lo5 persons, mak-
50-I
a
.
2.05
.
.
.
.
2.15
2.10
RESTRICTION FRAGMENT SIZE (kb)
A
m
1
r
11
C
40 34
30 -
t
8U
W
m
5
3
z
20
-
10-
2.05
2.10
2.15
RESTRICTION FRAGMENT SIZE (kb)
Fig 3. Frequency distribution of polymorphic Rsal fragments fm
63 controI subjects (A)and 65 patients with multiple sclerosis
(B).The data are fmm Tabk 1. Each restrictionfragment aIIeIe
corresponds to a fragment of one of the two chromosomes 18 that
are present per diploid ceII. The frequency of each size fragment is
shown as the number of chromosome 18 variants counted. Subjects with a single Rsal band in Tabk I were assumed to 6e
homozygous for the same-size fragment and were counted twice.
The number of chromosomes 18 countedfor the various fragment
sizes is indicated b-~the number above each column.
ing the likelihood of having included one person with
occult MS in the control group less than
Although RFLP fragment sizes could be estimated
with relative precision, the designation of fragment
sizes, particularly in subjects with a single band (“homozygotes”), is arbitrary. Presentation of the various
allele sizes as shown appeared justified by the reproducibility of the size measurements. In all likelihood,
the true number of alleles is larger than the ten indicated. There are probably small differences in size
between some alleles designated as being the same
length that were unrecognized due to limits in resolving power of the electrophoresis system used. The
purpose of the RFLP analysis was not to assign specificsize repetitive region alleles to the control or MS populations, but to determine qualitatively whether any
Boylan et al: MBP Gene Polymorphism and MS 295
Table 2. Numbers of Rsd Polymorphic Alleles and Numbers of Subjects C a v i n g at Least One Allele ofthe Sizes Noted
No. of Subjects"
(Homozygous or Heterozygous)
Allele Frequency
(No. of Chromosomes)
ha1
Fragment Size
(kb)
Control Subjects
(n = 126)
2.05
2.06
2.07
26
20
2
19
22
7
1
2.08
0
2.12
5
1
0
7
0
4
0
2.09
2.10
2.13
2.14
48
17
2.15
0
2.11
Patients with
MS
(n = 130)
3
4
6
26b
Control Subjects
(n = 63)
Patients with
MS
(n = 65)
22
14
14
20
7
0
5
1
4
0
4
5
5
32
3
18'
12
0
"Number of subjects with at least one allele of the size noted. Homozygotes are counted only once.
' p < 0.005.
' p < 0.025.
' p C 0.05.
significant difference exists between the RFLP pattern
in the two groups.
In terms of absolute allele frequency, the group with
MS differed significantly from the control population
(see Fig 3 and Table 2). Categorization of individuals in
the group with MS and the control group by carriage
of specific-size alleles revealed similar differences (see
Table 2). Analysis showed that the variation was
largely derived from differences in the 2.13- to 2.15kb fragment range. This difference between patients
with MS and control subjects remained consistent for
persons considered homozygotic and heterozygotic for
2.14- or 2.15-kb RsaI bands; these alleles were more
common in the patients with MS. The MS population
differed from the control population with respect to
alleles in the 2.09- to 2.11-kb range as well, but the
numbers of alleles and subjects seen with these RFLPs
were small and of unclear significance.
A feature of the RFLP data, made clear by Figure
3, is seen in the appearance of the two major peaks of
the allele distribution in patients with MS. Both of
these peaks are slightly broader in the MS group than
in the control subjects, principally because of an increased number of alleles in the 2.06- to 2.07-kb and
2.14- to 2.15-kb regions of the respective peaks. These
variations are largely the bases for the significant differences in allele and subject distribution found between
the control group and the MS group.
It may be argued that this divergence in allele frequency represents a systematic error in the measurement of fragment sizes in the control or MS samples.
The differences in allele sizes are small, and in the
absence of having D N A sequence data for all of the
alleles this must be considered as a possible explana296 Annals of Neurology Vol 27 N o 3 March 1990
tion for the data. However, given the reproducibility
of the allele sizes (the variation being _t 5 bp for
approximately 90% of samples) as obtained in independent experiments, we believe that the fragment
sizes are accurate as presented.
Based on the present data, no pathogenetic relationship between polymorphism at the MBP 5' site and
MS can be supposed. It is unknown whether the 5'
repetitive region has any influence on the expression
of the MBP gene, or if RFLPs at this site should be
considered MBP gene alleles, as opposed to alleles of
the repetitive region in and of itself. Although it is
undetermined whether the repetitive region is part of
the MBP structural gene, RFLPs generated at this site
are tightly linked to the MBP gene and thus are capable of serving as markers for MBP structural gene variants. These data suggest that patients with MS differ
from control subjects without MS in an MS high-risk
population with respect to MBP gene-linked RnPs.
Whether this finding represents true association of one
or more specific MBP genotypes with MS is unproved.
Our data may represent RFLP association due to linkage to a locus other than MBP, or may simply represent population stratification of repetitive region
RFL.Ps in the MS population and have no specific relationship to MBP or MS.
Since this study was begun, in addition to the 5'
repetitive region, at least four RFLPs produced by gain
or loss of a restriction site have been found for the
human MBP gene as detected by an MBP cDNA
probe (128); K. B. Boylan, J. W. Rose, S. B.
Prusiner, unpublished observation, 1987). More certain demonstration of RFL.Ps associated with MS could
be obtained by haplotype determination of multiple
RFLPs in patients with MS. This approach applied to
families who are multiply affected by MS would allow
a more convincing analysis of possible MBP gene linkage to MS.
Dr. Boylan was a recipient of a National Multiple Sclerosis Society
fellowship (FG 668-A). This work was supported in part by a research grant from the National Institutes of Health (NS 14069) as
well as a gift from the Folger Foundation.
We thank Mrs Rochelle Farquhar of the Multiple Sclerosis Clinic of
the University of British Columbia Health Sciences Center Hospital
for her invaluable assistance in procuring blood samples for this
study.
Presented in part at the 112th Annual Meeting of the American
Neurological Association, San Francisco, CA, October 18-2 1,
1987, and published in abstract form (Ann Neurol 1987;22:139).
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Boylan e t al: MBP G e n e Polymorphism and MS 297
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