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Improvement in the Molecular Diagnosis of Machado-Joseph Disease
Patrícia Maciel, PhD;
Maria do Carmo Costa, BSc;
Anabela Ferro, BSc;
Marylène Rousseau, BSc;
Cláudia Sofia Santos, BSc;
Claudia Gaspar, PhD;
José Barros, MD;
Guy A. Rouleau, MD, PhD;
Paula Coutinho, MD, PhD;
Jorge Sequeiros, MD, PhD
Arch Neurol. 2001;58:1821-1827.
ABSTRACT
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Background Direct detection of the gene mutation
allows for the confirmation of the clinical diagnosis of Machado-Joseph
disease (MJD), the most frequent cause of autosomal dominant
spinocerebellar ataxia worldwide.
Objective To address the main difficulties in our national MJD
predictive testing program. The first was the emergence of intermediate
alleles, for which it is not yet possible to determine whether they
will cause disease. The second was the issue of homoallelism, ie,
homozygosity for 2 normal alleles with exactly the same
(CAG)n length, which occurs in about 10% of all test
results.
Methods A large pedigree with 1 affected patient carrying a 71
and a 51 CAG repeat and 2 asymptomatic relatives carrying the 51 CAG
repeat and normal-size alleles underwent clinical and molecular
studies. Intragenic haplotypes for these alleles were determined. A
representative sample of the healthy population in the region was
obtained to assess the distribution of the normal (CAG)n
length. We established the genotype for 4 intragenic polymorphisms in
the gene for MJD (MJD1) in 21 homoallelic individuals, to
distinguish their 2 normal chromosomes. In addition, we developed a new
Southern blot method to completely exclude cases of nonamplification of
expanded alleles in the homoallelic individuals.
Results The study of the family in which the 51 CAG repeat was
found suggests that the allele is apparently not associated with
disease. These intermediate alleles were not present in a large sample
of the healthy population from the same region. Intragenic
polymorphisms allowed distinction of the 2 different normal alleles in
all cases of homoallelism. The absence of an expanded allele was also
confirmed by Southern blot.
Conclusions We propose an improved protocol for molecular
testing for MJD. These strategies, developed to overcome the practical
difficulties mostly in the presymptomatic and prenatal diagnosis of
MJD, should prove useful for other polyglutamine-related disorders.
INTRODUCTION
MACHADO-JOSEPH disease (MJD) is a progressive, adult-onset, neurodegenerative disorder
transmitted in an autosomal dominant manner that affects the central
nervous system. Its manifestations include cerebellar ataxia and
progressive external ophthalmoplegia, associated in a
variable degree with pyramidal signs, extrapyramidal signs (dystonia or
rigidity), amyotrophy, and peripheral neuropathy.1 The gene
associated with this disease, MJD1, was cloned in 1994, and
the causative mutation was shown to be the expansion of a
(CAG)n tract within its coding region.2 This
tract contains 12 to 44 triplets in healthy individuals and from 61 to
87 in patients, as shown in numerous studies of populations of diverse
ethnic origin.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22
Machado-Joseph disease is highly pleomorphic in its clinical
presentation, and that led to the definition of the following 3
subphenotypes23: type 2, the most common, characterized by
cerebellar ataxia, ophthalmoplegia, pyramidal signs, and onset in
midadulthood (mean age, 40.5 years); type 1, the
most severe form, characterized by an earlier onset (mean age, 24.3
years), marked spasticity, and dystonic features in addition to the
cerebellar ataxia and ophthalmoplegia; and type 3, corresponding to the
mildest form, characterized by a later onset (mean age, 46.8 years )
and marked peripheral amyotrophies in addition to the main signs. A
fourth (rare) subphenotype characterized by the presence of
parkinsonian features, was also proposed.24 This variable
clinical presentation is partly explained by the length of the expanded
allele. The inverse correlation observed between age at onset and
(CAG)n length, however, is not sufficient to be
applicable in presymptomatic testing.3, 25, 26
There is, at present, no effective treatment for MJD. Although
the identification of the causative gene has increased our
understanding of the pathogenesis of MJD, the detailed pathways that
lead to neurodegeneration are still not clear, hampering the
development of new therapies. Therefore,
predictive testing and genetic counseling are still the only means to
diminish the impact of the disease in affected families.
Direct detection of the MJD mutation allows the confirmation of the
clinical diagnosis, which can be useful given the potential clinical
overlap among the different forms of spinocerebellar
ataxia.27 Another immediate application of the molecular
test was the possibility of presymptomatic diagnosis, in the context of
genetic counseling programs. This is of utmost importance, particularly
in areas of high prevalence of disease, such as the Azorean islands
(1:3700 in São Miguel; 1:120 in Flores), where the disease is
considered a public health problem, and some areas of mainland Portugal
(1:1000 in a small area of the Tagus River
Valley).28, 29 In this study, we address the main
difficulties encountered in the context of the Portuguese Predictive
Testing and Genetic Counseling Program for Machado-Joseph Disease. The
first was the emergence of alleles of a size between the known normal
and pathogenic ranges (intermediate alleles), for which it is not
possible at this point to determine whether they will cause the
disease. The second was the issue of
homoallelism, ie, the homozygosity for 2 normal
alleles with exactly the same (CAG)n length. The strategies
developed to overcome these difficulties are presented herein and may
prove useful to other polyglutamine-related disorders.
SUBJECTS, MATERIALS, AND METHODS
SUBJECTS
A family from the Tagus River Valley (family MJD75) was
identified during an ongoing survey of inherited ataxias in
Portugal.29 Results of molecular testing showed the proband
and his 45-year-old affected son to carry an expanded allele at the
MJD1 gene; however, the son also carried another allele of
intermediate size, never previously described in the affected or the
healthy population (Figure
1). To clarify the possible
association of this allele with the presence or absence of disease, an
important issue for genetic counseling of the family (including the 2
asymptomatic siblings), we examined additional members of the family,
including the proband's spouse and 12 of her relatives (pedigree
MJD75b) (Figure 2).
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Figure 1.
Analysis of (CAG)n length in a pedigree with Machado-Joseph
disease (families MJD75 and MJD75b). N indicates normal
alleles; E, expanded alleles; and I, intermediate alleles. Family
members designated on the right side are depicted in Figure 2.
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Figure 2.
Pedigree with Machado-Joseph disease (family MJD75b). Other
than the proband of family MJD75 (IV:7) (arrow) and his son (V:4), only
1 of the members of the maternal side of the family available for study
carried an expanded allele (III:11). CAG repeats are
indicated for all members undergoing molecular diagnostic procedure.
Individuals represented by barred symbols presented neurologic symptoms
not associated with MJD and were not carriers of the mutation. The
allele with 51 CAG repeats was stable on 2 transmissions and not
associated with the disease on its own. Squares indicate male members;
circles, female members; and slash, deceased.
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To determine the (CAG)n size in normal chromosomes
and look for intermediate alleles, we tested anonymous Guthrie cards
(from the national program for phenylketonuria screening); 20 samples
were collected at random from 16 villages in the region, totaling 320
chromosomes tested.
During a 2-year period, the molecular diagnosis of MJD was obtained at
our laboratory for 149 patients referred by a neurologist or another
physician, and for 55 at-risk family members after indication by a
medical geneticist. Of these 204 subjects, 21 were shown to be
homoallelic and underwent studies for intragenic polymorphisms and
Southern blot analysis as described herein.
MOLECULAR CHARACTERIZATION OF THE CAG REPETITIVE TRACT AND
INTRAGENIC POLYMORPHISMS IN THE MJD1 GENE
Blood samples were obtained after informed consent from all
individuals in the familial study, and genomic DNA was extracted from
lymphocytes, as described elsewhere.30 The DNA was
extracted from the filter paper blots using the resin Chelex (Bio-Rad,
Hercules, Calif) in a final concentration of 0.8%.
Amplification of the CAG repeat–containing fragment in the
MJD1 gene was performed by means of polymerase chain reaction
(PCR) analysis, using previously described conditions,2 and
the size of the PCR products was determined by denaturing 6%
polyacrylamide gel electrophoresis in parallel with an M13 sequence
ladder, visualized by means of autoradiography. In every reaction, we
used as a positive control genomic DNA from a patient with an
expanded allele containing 86 CAG repeats, the largest we have ever
amplified by means of PCR.
The intragenic polymorphisms
C987GG/G987GG and
TAA1118/TAC1118 (single
nucleotide substitutions at positions indicated in each codon) were
detected by means of allele-specific PCR.31 The
polymorphism A669TG/G669TG
was detected by means of single-strand conformational polymorphism
analysis.31 The intragenic polymorphism
C1178/A1178
(single nucleotide substitution at position 1178 in the 3' noncoding region of
the gene) was detected by means of allele-specific PCR, using
primers MJD8 (5'-GATTACAAATTTACTTAAGAG-3') or MJD9
(5'-GATTACAAATTTACTTAAGAT-3') combined with MJD6
(5'-GACAGAGTTCACATCCATGTG-3'). The allele-specific PCR was
performed in the same conditions used for amplification of the CAG
repeat, except for the annealing step, which was performed at 52°C
for 30 seconds. The PCR products were analyzed in a denaturing 6%
polyacrylamide gel and visualized by means of autoradiography. The DNA
sequencing was performed using the primer MJD52a
(5'-CCAGTGACTACTTTGATTCGT-3') to determine the genotype for the
C987GG/G987GG polymorphism
in a few individuals used as controls in the allele-specific PCR.
Sequencing with primer MJD6 was used to determine genotype for
polymorphisms TAA1118/TAC1118 and
C1178/A1178 for
the same purpose. Reactions were performed using 5 µL of DNA and a
cycle-sequencing kit (Thermo Sequenase; USB, Cleveland, Ohio) following
the manufacturer's instructions.
Another approach was also used to confirm homoallelism. A
186–base pair (bp) fragment was generated by means of PCR, with
primers MJD1 (5'-TGGCCATGATAGGTTATTTTGTGA-3') and MJD2
(5'-GGAAAATACATTGTTTCACGAATCAAA-3'). This fragment was
purified from agarose gel using an extraction kit (QIAEX II;
QIAGEN, Valencia, Calif) and labeled with phosphorus 32
(32P) using a labeling kit (Prime-It II Random Primer;
Stratagene, Cedar Creek, Tex), to be used as a probe in a Southern
blot. The genomic DNA (40 µg) was digested with the restriction
enzyme AluI, and the fragments were separated in a 1% agarose
gel (Nusieve 3:1 agarose; FMC BioProducts, Rockland, Me) and blotted in
a nylon membrane (Hybond H-N+; Amersham Pharmacia Biotech,
Buckinghamshire, England) that was hybridized with the probe at 65°C.
This results in bands of approximately 600 bp for normal alleles and
bands larger than 733 bp for expanded alleles. These were visualized by
means of autoradiography.
Figure 3 represents the
location of all primers and restriction sites used in the molecular
diagnostic procedure.
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Figure 3.
Position of primers and restriction sites used in the molecular
diagnostic procedures. MJD indicates Machado-Joseph disease
(MJD); ASP, allele-specific polymerase chain reaction
analysis.
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RESULTS
INTERMEDIATE ALLELES IN A FAMILY WITH MJD
A family with the MJD mutation was identified, where the index case was
a 69-year-old man (IV:7 in Figure 2). Age at onset was 45
years with gait imbalance, followed some years later by diplopia,
dysarthria, and dysphagia. He used a wheelchair since 54 years of
age, due to severe cerebellar ataxia. He had a complete
limitation of upward movements of his eyes and partial limitation
of lateral movements, without lid retraction or nystagmus. A
bilateral facial palsy with atrophy and fasciculations was evident, as
well as a moderate, generalized muscle weakness and atrophy of the
limbs with areflexia. Except for a brisk jaw jerk, no corticospinal
signs were present, and no dystonia. The clinical picture corresponded
to a classic subtype 3.
The proband's oldest son (V:4, born in 1952) was also affected, with
difficulty walking since 32 years of age, followed 2 years later by
dysarthria and diplopia. At 45 years of age, with 13 years of evolution
of the disease, he still had an independent gait but was unable to
work. He showed a moderate cerebellar ataxia, more marked in the gait
and in the lower limbs. A coarse nystagmus was present. Vertical eye
movements were limited, and some contraction fasciculations of the face
were observed. Muscular strength was normal. All deep-tendon reflexes
were exaggerated, but plantar reflexes were normal, and there was no
spasticity. This clinical picture corresponded to a subtype 2. The
other son (V:5) and the daughter (V:6) of the proband had normal
results of neurologic examinations at 35 and 32 years of age,
respectively; the proband's wife (IV:8) had normal results of
neurologic examination at 67 years of age.
The proband (IV:7) carried an expanded allele with 68 CAG
repeats (Figure 1 and Figure 2). His oldest son (V:4) carried an
expanded allele of 71 repeats, and, in addition, an allele with 51
repeats (Figure 1 and Figure 2). The unaffected 35-year-old son
(V:5) carried the paternal normal allele with 21 repeats and the
intermediate allele with 51 repeats, whereas the 32-year-old unaffected
daughter (V:6) carried 2 normal alleles (21 and 23 CAGs).
Their unaffected mother (IV:8) had a normal allele (23 repeats) and the
intermediate allele of 51 repeats.
The length of the (CAG)n in 12 other members on
the maternal side of the family is shown in Figure 2; all were thought,
owing to history, not to have MJD. On examination, all but 4 were
considered healthy. Two sisters had juvenile parkinsonism (IV:1 and
IV:2), and 1 male member was bedridden due to spinal cord injury
(III:12); all 3 had 2 normal alleles. One female member (III:11),
however, who was bedridden after a stroke,
had an expanded allele with 63 CAG repeats. She
was aged 85 years at the time of examination and had been in good
health until 78 years of age, when she had a stroke with right-sided
hemiparesis and speech difficulties. Since then, she had been unable to
walk without assistance. For 3 years, her difficulties walking and
speaking worsened. Dysphagia was noticed for the first time
at the time of observation. She presented with cerebellar ataxia and
limitation of upward gaze. There was a moderate atrophy of the hands
and legs, with generalized fasciculations, contrasting with muscular
weakness only on the right side (probably due to her previous
stroke). In conclusion, along with mental deterioration,
advanced age, and her history of diabetes (probably associated with the
occurrence of stroke and the peripheral neuropathy), cerebellar ataxia
combined with anterior horn signs could have been developing for
3 to 4 years, corresponding to a very-late-onset type 3 MJD.
HAPLOTYPES ASSOCIATED WITH INTERMEDIATE AND EXPANDED ALLELES IN FAMILY MJD75b
The intragenic markers A669TG/G669TG,
C987GG/G987GG,
TAA1118/TAC1118, and
C1178/A1178 were used to define the haplotypes
associated with the intermediate and expanded alleles in the family
under study. The 51 CAG repeat coming from the unaffected mother (IV:8)
was associated with the same haplotype as the expanded allele coming
from the affected father (IV:7), GGCA. The expanded allele present in
the 85-year-old relative (III:11) was also associated with haplotype
GGCA.
LENGTH OF THE (CAG)n TRACT IN THE MJD1 GENE IN A CONTROL POPULATION
The distribution of (CAG)n tract length in 302 chromosomes
of control individuals born in the same district where the individual
with the 51 CAG repeat originated is shown in Figure 4. The largest allele found was 37 CAG
repeats. No intermediate alleles were found in this sample.
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Figure 4.
Distribution of (CAG)n length in a sample of the healthy
population (n = 320) obtained from the region of origin
of family MJD75. No intermediate alleles were found.
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APPARENT HOMOALLELISM OF THE MJD1 GENE IN MEMBERS OF MJD FAMILIES
We have performed a total of 204 diagnostic and predictive tests.
According to our results, 107 family members carried an expansion, 79
had 2 normal alleles of different size, and 21 (10.3%) were apparently
homoallelic, ie, had 2 normal alleles with the same (CAG)n
size. When these 21 individuals underwent typing for 3 intragenic
polymorphisms of the MJD1 gene to try to distinguish the 2
normal chromosomes (Figure 5),
in 2 cases (9.5%) the distinction was possible using polymorphism
C987GG/G987GG;
in 4 (19.0%), using A1178/C1178; and in 18
(85.7%), using TAA1118/TAC1118. In
combination, these 3 intragenic polymorphisms allowed for the
distinction of both normal alleles in all 21 cases of homoallelism.
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Figure 5.
Frequency of genotypes for 3 intragenic polymorphisms in the gene
for Machado-Joseph disease among the 21 homoallelic individuals
identified in the context of our diagnostic and predictive testing
program. The
TAA1118/TAC1118
polymorphism has the highest frequency of heterozygosity, but only the
combination of all 3 polymorphisms allowed for the distinction of 2
different normal alleles in all cases. Polymorphisms are described in
the "Subjects, Materials, and Methods" section.
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Results of Southern blot analysis also confirmed that none of these
individuals carried an expanded allele (an example is shown in
Figure 6).
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Figure 6.
Southern blot analysis of the (CAG)n-containing segment of
the gene for Machado-Joseph disease. bp indicates base pair.
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COMMENT
The molecular diagnosis of MJD is currently based on the determination
of the (CAG)n length in the MJD1 gene. If someone
carries alleles with more than 60 CAG repeats, one can predict with
some certainty that if the person lives long enough, the disease will
develop in this individual, although it is not possible to establish at
what age symptoms will begin. Below this limit, there are reports of 3
patients with alleles containing 56, 55, and 54 CAG
repeats,32, 33, 34 making the last one the shortest CAG repeat
known to date in patients.
Precision of size determination has its limitations;
although we can apply more accurate methods using densitometry to
select the peak with the largest area
and using a cloned, sequenced allele as control,
one still has to consider that somatic mosaicism exists, with
differences in (CAG)n length between lymphocytes (where
length is usually measured) and central nervous system cells, as well
as among subpopulations of lymphocytes. An error of ±1 CAG repeat is
considered acceptable. In addition, (CAG)n size on its own
is not useful as a more precise predictor of outcome. Although the
variability in clinical presentation is partly explained by the length
of the expanded allele, this inverse correlation is incomplete and not
applicable for prediction of age at onset or clinical
presentation.3, 25, 26 For that reason, the sizes of the
alleles are not usually communicated to the consultant. We believe it
is important, however, to determine and to keep an accurate record of
the sizes of normal and expanded alleles, since the molecular diagnosis
of MJD is still in a research phase. In the future, knowledge of other
factors affecting disease onset and progression, eg, environmental
agents or modifier genes, may contribute to a more accurate prediction.
To illustrate this point, we describe herein an MJD1 allele with
a repeat length not previously encountered. Although studies in several
different populations had shown a wide gap between the normal (12-44
CAG repeats) and the disease (61-87 CAG repeats) range, these limits
are expected to change with the increasing size of our sample. The
present identification of a formerly undescribed "intermediate"
allele containing 51 CAG repeats was the source of a potential problem
for genetic counseling, since we are not able to predict whether or not
the disease will develop in an individual carrying this allele.
The study of the nuclear family in which this allele with 51 CAG
repeats was found suggested, however, that this allele might not be
pathogenic. First, the 67-year-old transmitting mother was still
unaffected. Second, the individual carrying this allele in addition to
a full expansion did not have a particularly severe clinical
presentation or juvenile onset, as could possibly be expected in a
homozygous patient.35, 36, 37 Third, this allele was stable on
at least 2 transmissions. The reduced number of cases with the allele
does not, however, allow us to establish this conclusion with
certainty. When we studied other living members of this family, no
other individuals carrying the allele with 51 CAG repeats were found.
We did, however, find an expanded allele with 63 CAG repeats in an
85-year-old woman (Figure 2) in whom the MJD phenotype had not been
detected previously, possibly only because of masking by the sequelae
of a previous stroke and a diabetic neuropathy.
The alleles with 51 and 63 CAG repeats share the same intragenic
haplotype (GGCA) and may have a common origin. We cannot determine,
however, whether the 51-CAG allele resulted from the contraction of a
previously expanded allele, or whether the ancestral allele was
of intermediate size and expanded only in 1 branch of the
family. However, the GGC haplotype (not including the polymorphism
C/A1178) is known to be the most common in the healthy
population in Portugal and corresponds to a small subgroup of the
Portuguese families with MJD, namely those originating from the island
of São Miguel and those in the Tagus River
Valley.38, 39
In a previous screening of a large control population from all
districts of Portugal (2000 chromosomes, 100 per district) (P.M., M.do
C.C., A.F., C.S.S., Laura Guimarães, Alda Sousa, PhD, and J.S.,
unpublished data, August 2001), we have found no alleles larger than 36
CAG repeats, suggesting that intermediate alleles must be quite
uncommon. After an additional control sample (320 chromosomes) from the
very district in the Tagus River Valley where these families originated
(where prevalence of MJD is 80 times that of the rest of the country)
underwent screening, no intermediate alleles were found.
In Huntington disease, nonpenetrance with intergenerational instability
for alleles with 29 to 35 CAG repeats and low penetrance for alleles
with 36 to 39 CAG repeats were described.40, 41 It is
possible that the same will occur for intermediate alleles in MJD, but
further studies are needed to clarify this question.
It was also suggested that smaller expansions could be associated with
unusual clinical presentations of MJD, such as autonomic dysfunction
(present in a patient with an allele with 56 CAG repeats, combined with
cerebellar ataxia),32 progressive proximal weakness and
sensory disturbances (in a patient with an allele with 54 CAG
repeats),34 or parkinsonian features42 (type 4
cases confirmed by results of molecular testing had average size
alleles, with 61 and 71 CAG repeats). Other presentations
that have been suggested for MJD include pure cerebellar ataxia and
spastic paraplegia phenotypes.43, 44 In this context, the 2
sisters from pedigree MJD75b with juvenile parkinsonism carried 2
normal alleles; in fact, the consanguinity of their parents may suggest
that another (possibly recessive) mutation might be the cause of their
disease.
Another important source of ambiguity in the molecular testing of MJD
was the relatively high frequency of apparent homoallelism (ie,
homozygosity for exactly the same size CAG repeat). Given the
highly polymorphic nature of repetitive tracts, it is not surprising
that most of the individuals with 2 normal alleles at the MJD1
locus (homozygous in the classic mendelian sense) have 2 alleles with
different size CAG repeats (heteroallelism). The exclusion of
the disease is very clear in these cases, and the interpretation of the
molecular test results does not raise major difficulties. Approximately
10% of cases of homoallelism for the normal allele were found,
however, in our diagnostic and predictive tests. Although PCR was
systematically repeated in every such case, and large expansions were
used as positive controls, it is impossible to completely exclude
nonamplification of an expanded allele, because of either extremely
large size or the presence of polymorphisms in the primer-annealing
regions, leading to false-negative results such as have been described
in Huntington disease.45 One case of homoallelism was found
in an instance of prenatal diagnosis, creating a particularly delicate
situation, given time constraints and the dependence of the parents'
decision to proceed with or to terminate the pregnancy on the status of
the fetus regarding the MJD mutation.46 In these cases, to
confirm that we are in the presence of cases of true homoallelism, our
proposed approach (Figure 7) is
(1) to confirm, whenever possible, that it was compatible with the
parents' genotypes, ie, that it was possible for the individual to
carry 2 alleles of the same size; (2) to check the population frequency
of that allele (this should be mentioned in the report); (3) to
determine the intragenic haplotypes in both chromosomes (the 2 normal
chromosomes carrying repetitive CAG tracts of the same length might be
associated with different haplotypes); and (4) to now use, in addition,
a molecular method not dependent on PCR, which has allowed us to
exclude retrospectively the presence of an expansion in the
MJD1 gene in all cases of homoallelism in study, thus
confirming the results obtained with the intragenic polymorphisms.
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Figure 7.
Flowchart for the molecular diagnosis of Machado-Joseph disease.
Polymorphisms are described in the "Subjects, Materials, and
Methods" section.
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CONCLUSIONS
We have tried to address the major difficulties found while
testing for a genetic disease largely still under study, such as MJD.
We suggest specific solutions for these problems, and reinforce the
need for permanent interaction between the diagnostic services and the
research process.
AUTHOR INFORMATION
Accepted for publication July 24, 2001.
The National Machado-Joseph Disease Predictive Testing and Genetic
Counseling Program was supported by grants PECS/P/SAU/50/95 and
PRAXIS/PSAU/C/SAU/84/96 from the Junta Nacional de
Investigação Científica e Tecnológica (Lisbon,
Portugal). Drs Maciel, Santos, and Gaspar were the recipients
of PhD scholarships from Praxis XXI (Fundação para a
Ciência e a Tecnológia [FCT], Ministério para a
Ciência e Tecnologia [Lisbon]). Other grants from FCT
and grants STRDA/C/SAU/277/92 and PECS/C/SAU/219/95 from the
Portuguese Health Administration (Lisbon) supported the survey of
inherited ataxias and spastic paraplegias in Portugal. Dr Rouleau is
supported by the Medical Research Council of Canada
(Montreal).
We would like to thank the families for their cooperation in this study
and Mário Silva, MD, and Sister Maria do Rosário for their
organizational support. We are also indebted to Rui Vaz Osório,
MD, and Laura Vilarinho, PhD, at Instituto de Genética
Médica Jacinto de Magalhães (Porto, Portugal), for
supplying us with anonymous Guthrie test cards for this study.
From the UnIGENe, Instituto de Biologia
Molecular e Celular (Drs Maciel, Gaspar, and Sequeiros and Mss Costa,
Ferro, and Santos), and the Deptartamento Estudos de
Populações, Instituto de Ciências Biomédicas de
Abel Salazar (Dr Sequeiros), Universidade do Porto, and Serviço
de Neurologia, Hospital de St António (Dr Barros), Porto,
Portugal; Instituto Superior de Ciências da Saúde-Norte,
Paredes, Portugal (Dr Maciel); Centre for Research in Neuroscience,
McGill University and the Montreal General Hospital Research Institute,
Montreal, Québec (Ms Rousseau and
Drs Gaspar and Rouleau); and
Serviço de Neurologia, Hospital de St Sebastião, Santa
Maria da Feira, Portugal (Dr Coutinho).
Corresponding author: Patrícia Maciel, PhD, UnIGENe,
IBMC, Universidade do Porto, 4150-180 Porto, Portugal.
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