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Chromosomal translocation t(18;21)(q23;q22.1) indicates novel susceptibility loci for frontotemporal dementia with ALS

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Chromosomal Translocation
t(18;21)(q23;q22.1) Indicates
Novel Susceptibility Loci for
Frontotemporal Dementia
with ALS
Johannes Prudlo, MD,1 Burkhard Alber, MD,2
Vera M. Kalscheuer, PhD,3 Klaus Roemer, PhD,4
Thomas Martin, MD,5 Joern Dullinger, MD,6
Helmut Sittinger, MD,7 Stephan Niemann, MD,8
Peter Heutink, PhD,9 Albert C. Ludolph, MD,2
Hilger H. Ropers, MD,3 Klaus Zang, MD,5
and Thomas Meyer, MD6
A chromosomal translocation t(18;21)(q23;q22) is reported in a patient with frontotemporal dementia (FTD)
and amyotrophic lateral sclerosis (ALS). We exclude the
physical involvement and silencing of the ALS-linked
gene for copper/zinc superoxide dismutase (SOD1) on
chromosome 21q22.1. The breakpoints are assigned to
sequences flanked by the markers ATA1H06, D18S462,
D21S1915, and D21S1898. These critical regions may
contain susceptibility loci for FTD associated with ALS.
Ann Neurol 2004;55:134 –138
Two to 5% of amyotrophic lateral sclerosis (ALS) cases
concurrently occur with frontotemporal dementia
(FTD), a neurodegenerative disorder with personality
changes and language dysfunction related to a primary
progressive frontotemporal lobar atrophy.1– 4 For the
familial form of FTD/ALS, a first genetic locus has
been identified on chromosome 9q21-q22.5 Approximately 10% of ALS patients have inherited the disease.
Ten to 20% of these patients have been linked to a
locus on chromosome 21q22 and are caused by muta-
From the 1Department of Neurology, University Hospital, Homburg/Saar; 2Department of Neurology, University of Ulm, Ulm;
3
Max Planck Institute for Molecular Genetics, Berlin; 4Department
of Virology, University Hospital; 5Department of Human Genetics,
University Hospital, Homburg/Saar; 6Department of Neurology,
Charité University Hospital, Humboldt University, Berlin; 7Department of Psychiatry, University Hospital, Homburg/Saar; 8Department of Human Genetics, Justus-Liebig-University, Giessen, Germany; and 9Department of Clinical Genetics, Erasmus University,
Rotterdam, The Netherlands.
Received Mar 24, 2003, and in revised form Jun 23 and Sep 25.
Accepted for publication Sep 25, 2003.
Address correspondence to Dr. Johannes Prudlo, University Hospital, Department of Neurology, Kirrberger Strasse, 66421 Homburg/
Saar, Germany. E-mail: nejpru@uniklinik-saarland.de
134
tions in the gene encoding copper/zinc superoxide dismutase (SOD1).6 Recently, we found constitutional
chromosomal abnormalities in several apparently sporadic ALS patients, all affecting distinct chromosomal
loci.7 We proposed that the chromosome rearrangements may represent a genomic risk factor for apparently sporadic ALS. Among these patients we identified
a man aged 64 years with FTD and ALS carrying a
balanced chromosomal translocation t(18;21)(q23;
q22). The cytogenetic breakpoint on chromosome 21
localized to the region 21q22 which harbors the ALSassociated SOD1 gene. Here, the physical involvement
and the silencing of the SOD1 gene by the chromosomal rearrangement was excluded. This suggests the
presence of genetic susceptibility loci for FTD and ALS
in previously undescribed genomic regions, one of
them in the proximity of the SOD1 locus.
Case Report
The patient presented with a slowly progressive behavioral and affective disorder. Furthermore, there was a
prominent cognitive dysfunction such as impairment
of comprehension, perseveration, and bradyphrenia indicating prominent frontal executive deficits. Immediate free recall, attention, and calculation also were disturbed as shown by digit span subtest of the Wechsler
Memory Scale (WMS) and logical memory subtest of
the WMS. More complex tests (eg, verbal fluency,
Trail Making Test) were not applicable. The diagnosis
of FTD was established according to the Lund and
Manchester criteria.8
Motor symptoms became evident within several
months after the onset of dementia, consisting of gradually progressive bulbar palsy, weakness, wasting, and
fasciculations. On examination, he showed a severe
lower and upper motor syndrome that fulfilled the
clinical criteria for the diagnosis of definite ALS.9 Furthermore, we observed a selective loss of upward vertical saccades, which is found in some ALS patients.10
Magnetic resonance imaging showed moderate midbrain atrophy as indicated by a decreased midsagittal
anterioposterior diameter of the midbrain (Fig 1A).11
Upon further inspection with 18F-fluorodeoxyglucose
positron emission tomography (PET), we found a significant decrease in glucose metabolism within the
frontal and temporal lobes (see Fig 1B–D). Finally, examination by 123I-IBZM-SPECT showed an asymmetric and reduced striatal postsynaptic D2-receptor density, reflecting an extrapyramidal involvement, which
has been described in ALS previously (see Fig 1E).3
After a total clinical course of 2.5 years, death resulted
from recurrent aspiration pneumonia and respiratory
insufficiency. Consent for an autopsy was not given.
© 2003 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
Fig 1. Brain imaging. (A) Moderate midbrain atrophy indicated by a decreased midsagittal anterioposterior diameter (normal
⬎17mm) on axial T2-weighted MRI as depicted in the scheme. (B–D) PET images of 18F-fluorodeoxyglucose metabolism. Markedly reduced tracer uptake in both frontal and temporal lobes, axial (B) and midline sagittal plane (C), paramedian right temporal
lobe (D). (E) 123I-IBZM-SPECT: reduced and asymmetric striatal postsynaptic D2-receptor density.
Cytogenetic Analysis
Cytogenetic analysis was conducted on G-banded chromosomes of cultured peripheral blood lymphocytes.
Fluorescence in situ hybridization (FISH) analysis of
mega-yeast artificial chromosome (YAC) clones from a
genomewide YAC panel (YAC/BAC FISH mapping resource at the Max Planck Institute for Molecular Genetics, Berlin) were used to narrow the breakpoint on
the chromosomes 21 and 18. The YACs are derived
from the Centre d’Etude du Polymorphisme Humain
YAC library and YAC descriptions refer to this library.
SOD1 Gene Analysis
To investigate whether the translocation breakpoint occurred within the SOD1 gene, we performed a Southern blot analysis (Fig 2A). Standard methods were used
for probe labeling, hybridization, washing, and autoradiography.
SOD1 exons 1 to 5 (complete cDNA sequence) in-
cluding flanking intronic sequences were amplified by
polymerase chain reaction (PCR). This was followed by
direct sequencing of both strands of PCR products on
an ABI-Prism 3100 DNA sequencer.
Reverse Transcription Polymerase Chain
Reaction Analysis
For semiquantitative PCR of the SOD1 transcript, we
conducted a reverse transcription (RT) and subsequent
PCR amplification of two overlapping fragments of the
SOD1 cDNA. The primer design and sequences are
given in Figure 2C. RT-PCR was performed on equal
amounts of lymphocyte total RNA (500ng) of three
controls, the patient and the son of the index patient,
who carried the same chromosomal abnormality. The
semiquantitative RT-PCR was repeated thrice. Contamination was excluded with a blank PCR probe. Five
microliters of PCR products was removed after 28, 30,
32, 34, 36, 38, and 40 cycles, respectively. Images were
Prudlo et al: Susceptibility Loci for FTD/ALS
135
Fig 2. Analysis of the SOD1 gene locus. (A) Restriction sites of four DNA endonucleases ( BamHI, SacI, EcoRI, XbaI) in relation
to the SOD1 gene used for the digestion of the genomic DNA. (B) Southern blot of genomic DNA of the patient (P) and three
controls (C1, C2, C3) restricted with the endonucleases BamHI, SacI, EcoRI, XbaI (lanes 1– 4) hybridized with a radioactive
probe of a 610bp fragment of the SOD1 complementary DNA (GenBank Accession number E00882). (C) Schematic presentation
of a multiplex polymerase chain reaction (PCR) of the complete complementary DNA (primers F1-R1) and two overlapping fragments
of the SOD1 transcript (5⬘-fragment: primers F1-R2; 3⬘-fragment: primers F2-R1). Primers used: SOD1-F1 (5⬘-CTGCAGCGTCTGGGGTTT-3⬘), SOD1-R1 (5⬘-CAGTGTTTAATGTTTATCAGGAT-3⬘), SOD1-F2 (5⬘-CAGTGAAGGTGTGGGGAA-3⬘),
SOD1-R2 (5⬘-GTCCATTACTTTCCTTCTGC-3⬘). (D) Competitive PCR amplification of SOD1 mRNA in the patient (P) and
a normal control. The SOD1 gene dosage effect is defined by the expression of a distinct PCR product (eg, product 1) in relation
to the other PCR products (eg, products 2 and 3) of the same patient. M ⫽ marker (100bp).
captured by ultraviolet translumination (GelDoc1000;
Bio-Rad, Richmond, CA). Quantitative expression of
SOD1 transcript was determined at cycle 38 during
linear regression of amplification.
Results
Karyotype Analysis
The karyotype analysis of the index patient has been
reported showing an apparently balanced translocation
t(18;21)(q23;q22) (Fig 3).7 In addition, the index patient’s 37-year-old son who was clinically unaffected
was found to carry the same constitutional chromosomal rearrangement. The mother and two sisters of
the patient were karyotypically normal.
136
Annals of Neurology
Vol 55
No 1
January 2004
Mapping of Chromosomal Breakpoints Using
Fluorescence In Situ Hybridization
FISH analysis on chromosome 18 with YAC 932B10
(118cM) identified signals on 18q23, der(18), and
der(21). Thus, the breakpoint is localized between YAC
STS markers ATA1H06 and D18S462 spanning a 1Mb
region. The breakpoint on chromosome 18 mapped at
least 17Mb distal from a recently reported gene locus of
familial ALS.12 The breakpoint region on 21q22 is
flanked by the proximal YAC 752D12 with STS
D21S1915 (29cM) and the distal YAC 901E1 with STS
D21S1898 (35cM). YAC 752D12 maps to chromosome 21 and to der(21), whereas the YAC 901E1 maps
to chromosome 21 and der(18) localizing the breakpoint
in a 6Mb region including the SOD1 gene.
Fig 3. Representative G-banded karyotype of the index patient. The cytogenetic analysis is showing an apparently balanced translocation in the 46,XY,t(18;21)(q23;q22.1) karyotype. The arrowheads indicate the breakpoint positions.
The SOD1 Gene Locus
The SOD1 probe detected the expected restriction pattern of one (BamHI, SacI, EcoRI) and two (XbaI)
bands, respectively (see Fig 2B, lanes 1– 4). Several
bands of other molecular weights were detected and
likely represent restriction products of known SOD1
pseudogenes. One of them is visible in the Figure 2B,
EcoRI. Importantly, no alteration of the DNA restriction pattern was observed in our patient, as compared
with three controls. Southern analysis did not indicate
a disruption of SOD1 locus. Furthermore, no mutation
was identified by sequence analysis of SOD1 exons 1 to
5 (complete cDNA sequence) including flanking intronic sequences in genomic DNA (data not shown).
Expression of SOD1 RNA
Amplification of the SOD1 cDNA and two overlapping fragments yielded PCR signals of 617, 468, and
149bp, respectively. However, no differences in signal
intensity were observed in our patient when compared
with the PCR signals of a nonneurological control (see
Fig 2D), and in the patient’s son (data not shown).
This result indicates that the SOD1 expression is not
silenced by the translocation. Moderately altered expression of the SOD1 transcript, however, could not be
excluded by the semiquantitative PCR method.
The Tau Gene
Mutation analysis of the complete coding region of the
tau gene including the exon/intron boundaries, performed on the patient’s genomic DNA by direct sequencing, failed to show any sequence alterations.
Discussion
Karyotype analysis of our index patient demonstrated a
chromosomal breakpoint on chromosome 21q22, the
cytogenetic region of the ALS-linked SOD1 gene.7
This observation raised the question whether the
genomic rearrangement has disrupted the SOD1 locus.6
We excluded a physical involvement of the SOD1
gene. Also, there was no evidence for a position effect
and silencing of the SOD1 gene. Furthermore, the
translocation breakpoint was narrowed to a 6Mb
genomic region on chromosome 21q22.1, flanked by
the markers D21S1915 and D21S1898. Given this
physical proximity to the SOD1 gene, it is conceivable
that the genomic rearrangement interacts in some way
with the SOD1 locus. This possibility is supported by
the report of a disease-modifying factor in close proximity but outside the SOD1 gene, which is shared by
recessive kindreds of the unique SOD1-D90A mutation.13
In a series of apparently sporadic ALS patients, we
previously identified a high rate of constitutional chromosomal abnormalities (⬎5 %).7 This frequency is significantly higher than the frequencies in the general
population, as reported in three different studies.14 –16
However, these historical controls are not optimal, and
a better control population would have been asymptomatic subjects studied in a blinded fashion during
the actual karyotyping of the patients. Frequencies of
chromosome abnormalities are mostly derived from
large-scale incidence studies in newborns or amniocen-
Prudlo et al: Susceptibility Loci for FTD/ALS
137
tesis, which show a prevalence of balanced rearrangements in 0.1 to 0.28%.14 –16
Family studies of ALS patients identified several
asymptomatic carriers of constitutional rearrangements,
including some of advanced age.7 These findings suggest that in conjunction with the chromosomal abnormalities, other genetic or external factors may be required to produce the clinical phenotype.16 This
observation contributes to the current concept that apparently sporadic FTD and ALS represent complex disorders in which modifying genes and environmental
agents can contribute to the risk of disease.
We examined genome databases for possible candidate genes in the cytogenetic regions of rearrangements
on both involved chromosomes. Several genes map
near the breakpoint regions on chromosome 21 (encoding ubiquitin specific protease, the glutamate receptor of the kainate subtype, claudin, synaptojanin, and
interferon receptor 2) and the candidate region on
chromosome 18 (encoding galanin receptor 1, zinc finger protein 236, myelin basic protein). The characterization of the breakpoint regions on chromosomes 21
and 18 using positional cloning will help to define critical sequences in the human genome which predispose
to FTD and ALS. It remains to be determined whether
the chromosomal rearrangements define sets of functionally significant genes. If this is the case, an understanding of the associated proteins and pathways may
provide further insight into the genetic risk of FTD
and ALS.
This work was supported by the VERUM Foundation, Munich (T.
M.) and the German Genome Programme (01KW99087, T. M.).
We thank H. Madle, C. Menzel, and U. Reuter for technical assistance. We are especially grateful to Drs Kirsch and Moellers for
providing the SPECT and PET images.
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