A duplication at chromosome 11q12.2-11q12.3 is associated with spinocerebellar ataxia type 20.
ABSTRACT Spinocerebellar ataxia type 20 (SCA20) has been linked to chromosome 11q12, but the underlying genetic defect has yet to be identified. We applied single-nucleotide polymorphism genotyping to detect structural alterations in the genomic DNA of patients with SCA20. We found a 260 kb duplication within the previously linked SCA20 region, which was confirmed by quantitative polymerase chain reaction and fiber fluorescence in situ hybridization, the latter also showing its direct orientation. The duplication spans 10 known and 2 unknown genes, and is present in all affected individuals in the single reported SCA20 pedigree. While the mechanism whereby this duplication may be pathogenic remains to be established, we speculate that the critical gene within the duplicated segment may be DAGLA, the product of which is normally present at the base of Purkinje cell dendritic spines and contributes to the modulation of parallel fiber-Purkinje cell synapses.
- SourceAvailable from: umn.edu
Article: Trinucleotide repeat disorders.[show abstract] [hide abstract]
ABSTRACT: The discovery that expansion of unstable repeats can cause a variety of neurological disorders has changed the landscape of disease-oriented research for several forms of mental retardation, Huntington disease, inherited ataxias, and muscular dystrophy. The dynamic nature of these mutations provided an explanation for the variable phenotype expressivity within a family. Beyond diagnosis and genetic counseling, the benefits from studying these disorders have been noted in both neurobiology and cell biology. Examples include insight about the role of translational control in synaptic plasticity, the role of RNA processing in the integrity of muscle and neuronal function, the importance of Fe-S-containing enzymes for cellular energy, and the dramatic effects of altering protein conformations on neuronal function and survival. It is exciting that within a span of 15 years, pathogenesis studies of this class of disorders are beginning to reveal pathways that are potential therapeutic targets.Annual Review of Neuroscience 02/2007; 30:575-621. · 20.61 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Dominantly inherited diseases are generally caused by mutations resulting in gain of function protein alterations. However, a CTG expansion located in the 3' untranslated portion of a kinase gene was found to cause myotonic dystrophy type 1, a multisystemic dominantly inherited disorder. The recent discovery that an untranslated CCTG expansion causes the same constellation of clinical features in myotonic dystrophy type 2 (DM2), along with other recent discoveries on DM1 pathogenesis, have led to the understanding that both DM1 and DM2 mutations are pathogenic at the RNA level. These findings indicate the existence of a new category of disease wherein repeat expansions in RNA alter cellular function. Pathogenic repeat expansions in RNA may also be involved in spinocerebellar ataxia types 8, 10 and 12, and Huntington's disease-like type 2.Current Opinion in Genetics & Development 07/2002; 12(3):266-71. · 7.47 Impact Factor
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ABSTRACT: Autosomal dominant cerebellar ataxias are hereditary neurodegenerative disorders that are known as spinocerebellar ataxias (SCA) in genetic nomenclature. In the pregenomic era, ataxias were some of the most poorly understood neurological disorders; the unravelling of their molecular basis enabled precise diagnosis in vivo and explained many clinical phenomena such as anticipation and variable phenotypes even within one family. However, the discovery of many ataxia genes and loci in the past decade threatens to cause more confusion than optimism among clinicians. Therefore, the provision of guidance for genetic testing according to clinical findings and frequencies of SCA subtypes in different ethnic groups is a major challenge. The identification of ataxia genes raises hope that essential pathogenetic mechanisms causing SCA will become more and more apparent. Elucidation of the pathogenesis of SCA hopefully will enable the development of rational therapies for this group of disorders, which currently can only be treated symptomatically.The Lancet Neurology 06/2004; 3(5):291-304. · 23.92 Impact Factor
A duplication at chromosome 11q12.2–11q12.3 is
associated with spinocerebellar ataxia type 20
Melanie A. Knight1,?, Dena Hernandez2, Scott J. Diede3,4, Hans G. Dauwerse5, Ian Rafferty2,
Joyce van de Leemput2, Susan M. Forrest6, R.J. McKinlay Gardner7,8, Elsdon Storey7,9,
Gert-Jan B. van Ommen5, Stephen J. Tapscott4, Kenneth H. Fischbeck1
and Andrew B. Singleton2
1Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health,
Bethesda, MD, USA,2Molecular Genetics Unit, National Institute in Aging, National Institutes of Health, Bethesda,
MD, USA,3Department of Pediatrics, University of Washington, Seattle, WA, USA,4Division of Human Biology, Fred
Hutchinson Cancer Research Center, Seattle, WA, USA,5Department of Human and Clinical Genetics, Leiden
University Medical Center, Leiden, The Netherlands,6Australian Genome Research Facility, Walter and Eliza Hall
Institute of Medical Research, Melbourne, Australia,7Genetic Health Services Victoria, Melbourne, Australia,
8Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, Australia and9Department of Medicine
(Neurosciences), Alfred Hospital Campus of Monash University, Melbourne, Australia
Received June 26, 2008; Revised and Accepted September 2, 2008
Spinocerebellar ataxia type 20 (SCA20) has been linked to chromosome 11q12, but the underlying genetic
defect has yet to be identified. We applied single-nucleotide polymorphism genotyping to detect structural
alterations in the genomic DNA of patients with SCA20. We found a 260 kb duplication within the previously
linked SCA20 region, which was confirmed by quantitative polymerase chain reaction and fiber fluorescence
in situ hybridization, the latter also showing its direct orientation. The duplication spans 10 known and 2
unknown genes, and is present in all affected individuals in the single reported SCA20 pedigree. While the
mechanism whereby this duplication may be pathogenic remains to be established, we speculate that the criti-
cal gene within the duplicated segment may be DAGLA, the product of which is normally present at the base of
Purkinje cell dendritic spines and contributes to the modulation of parallel fiber-Purkinje cell synapses.
The most common cause of the autosomal dominant cerebellar
ataxias is simple sequence repeat expansion (SCAs 1–3, 6–8,
10, 12, 17) (1–3). Missense mutations have also been identified
in six SCAs (the Japanese 16q-linked SCA, SCAs 5, 11, 14, 15,
27) (4–9), and more recently genomic deletion at ITPR1 has
been associated with SCA15 and SCA16 (9,10), which can
now therefore be recognized as the same condition.
ful method for detecting chromosomal duplications and deletions
to discover the mutational mechanism of SCA15 (9).
Spinocerebellar ataxia type 20 (SCA20) is a dominantly
inherited cerebellar ataxia that is clinically distinct from the
other SCAs. Notably, dysphonia is present together with dys-
arthria; and palatal tremor (‘palatal myoclonus’) is typical. In
the eye movements, saccades are hypermetric, and there is no
nystagmus. A unique neuroradiological finding is a progress-
ive calcification of the dentate nucleus of the cerebellum,
which likely precedes the onset of clinical manifestations.
The age of onset of the disease ranges from 19 to 64 years
Genetic linkage was found to the pericentromeric region of
chromosome 11 (11). Since this region overlapped the SCA5
disease locus, locus homogeneity was considered, although
?To whom correspondence should be addressed at: Medical Genetics Branch, Section on Molecular Neurogenetics, National Human Genome Research
Institute, National Institutes of Health, Building 35, Room 1A105, 35 Convent Drive, MSC 3708, Bethesda, MD 20894-3708, USA. Tel: þ1
3014510902; Fax: þ1 3014026438; Email: firstname.lastname@example.org and Melanie_A_Knight@hotmail.com.
# 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is
Human Molecular Genetics, 2008, Vol. 17, No. 24
Advance Access published on September 18, 2008
the SCA5 and SCA20 clinical pictures differ considerably; but
subsequent discovery of the SCA5 gene, b-III spectrin
(SPTBN2) (6), allowed the demonstration that the two diseases
are genetically distinct (13).
The SNP microarray genotyping identified a structural vari-
ation in two affected SCA20 individuals, in comparison to
two unaffected family members. The profile obtained from
the log R ratio and the B allele frequencies indicated a
which is within the region to which SCA20 had previously
been linked (11) (Fig. 1). The duplication is defined by the
SNPs rs4963307 and rs10897193, which are 260 kb apart.
We examined 62 contiguous SNPs across the duplicated
region in two affected family members (GD1907 and
GD1918); using the B allele frequency metric, we were able
to assign the two affected family members a genotype
at each of these SNPs of A/A/A (n ¼ 16), A/A/B (n ¼ 24),
A/B/B (n ¼ 20) and B/B/B (n ¼ 48; we were unable to confi-
dently call 16 genotypes). At 32 of these SNPs, one of the
affected family members was called either A/A/B or A/B/B;
in no instance did we observe an A/A/B genotype in one indi-
vidual and an A/B/B genotype in the other. These data are con-
sistent with the notion that the duplication originally occurred
by intrachromosomal duplication. A search of repeats in NCBI
Genome Viewer revealed a large number of repeats at the
flanking ends of the duplicated region, but none at both ends
that were identical. The duplicated region contains 10
known and 2 unknown genes (Fig. 2). Quantitative real-time
polymerase chain reaction (PCR) was performed on eight
different genes to evaluate the extent of the duplication and
to assess the SCA20 family members. As expected, this analy-
Figure 2. A schematic diagram taken from the UCSC Genome Browser,
Human March 2006 (v174) Freeze of chromosome 11 showing the ten
genes within the SCA20 duplicated critical region and the two SNPs that
delineate the duplication, rs4963307 and rs10897193. The schematic
diagram also illustrates where the BACs lie in relation to the SNPs on chromo-
Figure 1. Infinium HumanHap550 SNP genotyping chips. (A) The top two panels show the results from one affected family member and the bottom two panels
show the results from an unaffected family member. The horizontal band in each panel represents heterozygous signal from two-allele SNP markers distributed
along chromosome 11. The arrow indicates a duplicated region that was shared by the two affected family members. (B) A higher magnification view showing
the duplication more clearly (circled in red).
3848 Human Molecular Genetics, 2008, Vol. 17, No. 24
sis indicated that the duplication co-segregated with the
disease haplotype in the SCA20 family (Figs 3 and 4).
Based on examination of copy number metrics of the Human-
Hap550 BeadChip, none of the 1129 control samples showed a
duplication in the same region.
To determine the orientation of the large genomic repeat,
we used Genome-wide Analysis of Palindrome Forma-
tion (GAPF), a procedure that enriches for palindromes
(i.e. inverted repeats) in genomic DNA (14). This approach
is based on a relatively simple and efficient method to
make ‘snap-back DNA’ from palindromic sequences by
intra-molecular base-pairing, followed by elimination of
non-palindromic single-strand DNA using S1 nuclease. This
procedure has previously been used to show that DNA inverted
repeats are non-randomly distributed and enriched in cancer
cells (14). To analyze the SCA20 region in high-resolution
in an affected individual, we modified the GAPF assay to
use genomic tiling arrays, with probes spaced every 10 bp
along the SCA20 region of the chromosome. We did not
detect a GAPF-positive signal in the genomic area known to
Figure 3. Gene dosage analysis of the SCA20 duplicated region. Results are the mean of three replicates done in triplicate experiments and are expressed as
22DDCt+SD. (A) Gene dosage results obtained for the entire SCA20 duplicated region comparing an unaffected family member to an affected family
member. The genes outside the duplication are SYT7 and INCENP. (B) A representative gene dosage result for the entire SCA20 pedigree that was tested
for one of the duplicated genes, DAGLA exon 5.
Human Molecular Genetics, 2008, Vol. 17, No. 24 3849
be duplicated, indicating that the duplication is unlikely to be
an inverted repeat (data not shown), but presumably direct.
To confirm that the duplication is direct, we used fiber fluor-
escence in situ hybridization (FISH) with three BAC clones,
RP11-467L20, RP11-61G16 and RP11-810P12, in different
combinations. Most informative was the co-hybridization of
RP11-467L20 in red and RP11-61G16 in green. Both clones
map for the largest part within the duplication (Fig. 5);
RP11-467L20 spans the proximal breakpoint, and RP11-61G16
spans the distal breakpoint of the duplication. The alternating
red–green–red–green pattern of signal sequence is consistent
with the duplication being of direct orientation (Fig. 5).
We have demonstrated by quantitative real-time PCR that all
the affected individuals in the single known SCA20 family
have a duplication on chromosome 11q12.2–11q12.3, within
the previous linked SCA20 region (11). The orientation is
direct: that is to say, the DNA of the duplicated segment
runs in the same direction as the original segment. Since the
duplication lay well within the linkage region, it was expected
that all affected individuals with the disease haplotype would
also carry the duplication; but it was useful to corroborate this.
As no controls of similar ethnicity were found with this dupli-
cation, it is unlikely that this alteration is a common copy
number variant (CNV). However, since many CNVs are
rare, it is nonetheless possible that the observed duplication
is a rare ‘private’, neutral (non-pathogenic) CNV. A pointer
against this possibility is that benign CNVs do not generally
affect multiple transcripts, but are more likely to involve
only a single gene, or to be localized to intergenic regions
of the genome. The balance of evidence thus strongly suggests
that the observed duplication is truly pathogenic, and is the
cause of SCA20 in this family. As SCA20 is the first SCA
to implicate a CNV, it remains to be seen if CNVs have a
role in other dominant or sporadic forms of ataxia.
Further evidence that could be adduced were there to be
known individuals, whose phenotype was recorded, and carry-
ing a chromosomal duplication encompassing this region.
However, we could find only one such duplication case in
the cytogenetic literature (15,16), a 6-year-old child, who
had a more severe neurological deficit, presumably due to
the broader effects of his relatively large (11q11–11q13.3)
duplication. A CT brain scan was reported as normal; but
the inferred absence of dentate calcification cannot, at this
young age, exclude SCA20.
The nature of the genes within the segment may support the
proposition of pathogenicity of the duplication, and a gene nor-
mally expressedincerebellumcould beimportant tothis disease
mechanism. Of the 10 annotated genes within the 260 kb dupli-
cated interval, diacylglycerol lipase a subunit (DAGLA) (17), a
neural stemcell-deriveddendriteregulator, isthe mostattractive
in this respect. The remaining genes, listed following, have little
support for candidacy: hypothetical protein LOC745, chromo-
some 11 open reading frame 9 (C11orf9) (18), hypothetical
protein LOC746, chromosome 11 open reading frame 10
(C11orf10) (19), flap structure-specific endonuclease 1 (FEN1)
Figure 4. The SCA20 family pedigree showing the individuals who carry the duplication that is segregating with the disease. Individuals in the family who do
not carry the disease haplotype do not have the duplication (Normal). Three individuals were not tested as indicated because their DNA was unavailable.
Figure 5. Fiber FISH analysis on EBV-transformed lymphocytes of patient
00101063 from the family. The DNA fibers were hybridized with two
BACs which span each end of the breakpoint, namely, RP11-467L20 visual-
ized in red and RP11-61G16 visualized in green. In the lower part of the
image the signals on one fiber are shown. The upper part of the figure
shows only the RP11-467L20 (red) signal on this fiber; the middle part of
the figure shows only the RP11-61G16 (green) signal on the fiber. The alter-
nating green-overlap-red-green-overlap-red pattern is apparent. It is important
to note that the red and green signals in the centre are not overlapping: this
indicates a direct tandem duplication.
3850 Human Molecular Genetics, 2008, Vol. 17, No. 24
(20), fatty acid desaturase 1 (FADS1), fatty acid desaturase 2
(FADS2), fatty acid desaturase 3 (FADS3) (21), RAB3A-
interacting protein (rabin3)-like 1 (RAB3IL1) (22), bestrophin-1
(vitelliform macular dystrophy protein 2) (BEST1) (23), ferritin,
heavy polypeptide 1 (FTH1) (24) and an otherwise undefined
predicted gene (UCSC Genome Browser, Human Mar. 06
DAGLA is interesting as a SCA20 candidate because of its
known function and its association with the ataxia interactome
(DAG) to 2-arachidonoyl-glycerol (2-AG), an endocannabinoid
with an important role in retrograde trans-synaptic suppression
of synaptic transmitter release (26). From mouse studies, the
gene is highly expressed in two particular neuronal classes of
the brain, cerebellar Purkinje cells, where its protein product is
present in the dendritic field, and pyramidal cells of the CA1
region of the hippocampus. The dendritic field of the Purkinje
cell receives input from the parallel fibers of the granule cells,
and this input is modulated at the synaptic junction; DAGLA
spines near the anatomical site of this synapse. It is not clear
why an incorrect amount (150%) of DAGLA production (if the
duplication does indeed causethis) should lead to a gradual com-
promise of cerebellar function; but the observation in SCA15
provides a notable parallel, in which an incorrect amount (50%)
of another factor, ITPR1, expressed at high level in the Purkinje
cell, is associated with a quite similar very slowly progressive
pure cerebellar degenerative phenotype (9).
The only known disease-associated gene within the region
is BEST1, which is mutated in Best macular dystrophy
(BMD) (MIM: 153700), also known as vitelliform macular
dystrophy type 2. The pathogenesis is due to abnormal
accumulation of lipofuscin within and beneath the retinal
pigment epithelium cells. BEST1 forms calcium-sensitive
chloride channels, and may conduct other physiologically
significant anions such as bicarbonate (27). No obvious
connection to the phenotype of SCA20 is evident.
expression, and present no obvious features that would impli-
cate them in the mechanism of SCA20. C11orf9, whose func-
tion is unknown, is not highly expressed in the cerebellum,
although it is expressed in other regions of the brain, such
as the brainstem and basal ganglia (18). FEN1 is an endonu-
clease that cleaves the 50end overhanging flap structure that
is generated by displacement synthesis when DNA polymerase
encounters the 50end of a downstream Okazaki fragment.
FEN1 also possesses 50to 30exonuclease activity on nicked
or gapped double-stranded DNA, and exhibits RNase H
activity (20). Not much is known about C11orf10 (19),
except that it is located immediately upstream of the FEN1
gene, but in the reverse orientation, with the 50ends overlap-
ping. FTH1 stores iron in a soluble, non-toxic, readily avail-
able form and is important for iron homeostasis. FTH1 has
ferroxidase activity. Iron is taken up in the ferrous form and
deposited as ferric hydroxides after oxidation. Defects in fer-
ritin proteins are associated with several neurodegenerative
diseases. FADS1, FADS2 and FADS3 are fatty acid desa-
turases. Desaturase enzymes regulate desaturation of fatty
acids through the introduction of double bonds between
defined carbons of the fatty acyl chain. This cluster of fatty
nothave high cerebellar
acid desaturases is thought to have arisen evolutionarily
from gene duplication, based on the similar exon/intron of
its constituent genes (21). RAB3IL1 (22) is a Ras-like
GTPase that regulates synaptic vesicle exocytosis. RAB3IL1
is a physiologic guanine nucleotide exchange factor for
If indeed the duplication is the cause of SCA20, then
its discovery has enabled us to place SCA20 among other
neurological diseases caused by chromosomal duplication
(Charcot–Marie–Tooth disease type 1A being the classic
example). Whether an extra copy of the DAGLA gene is suffi-
cient, of itself, to determine the SCA20 phenotype, or whether
involvement of other genes within or near the duplicated
region is necessary for the disease, awaits clarification. Identi-
fication of additional SCA20 families, or other adults with
interstitial cytogenetic duplication involving proximal 11q,
who have had neurological evaluation and brain imaging,
would be very useful in this regard. In the absence of such sup-
portive evidence from other families, animal models with
overexpression of DAGLA and other candidate genes in the
segment may be needed to confirm the mechanism of the
MATERIALS AND METHODS
Patient samples were collected as previously described (11).
Structural alterations in genomic DNA were sought by SNP
analysis. Two affected and two unaffected individuals from
the original SCA20 pedigree were genotyped as per the man-
ufacturer’s instructions, using the Infinium HumanHap550
SNP genotyping chips, which contain 555, 352 unique SNPs
(Illumina Inc, San Diego, CA, USA). Data were collected on
an Illumina BeadStation scanner, and genotypes generated
from the genotyping module (v2.3.25, Illumina Inc). Signal
intensities (log R ratio) and SNP B allele frequencies were
assessed via the visualization tool in the BeadStudio
package (Genome viewer), as outlined in Simon-Sanchez
et al. (28). Allele frequencies (‘theta values’) were obtained
for individual SNPs, and corrected for cluster position.
These data then give the log R ratio, which is the log2ratio
of the observed normalized R value for each SNP, divided
by the expected normalized R value for that SNP’s theta value.
Scores close to 1, 0.5 and 0, indicate B allele homozygosity,
heterozygosity and A allele homozygosity, respectively, while
deviations from these values, in contiguous SNPs, indicate a
change in the copy number at that locus. An increase in
copy number (.1) denotes a likely duplication, and a decrease
(,1), a deletion.
Quantitative real-time PCR analysis
Quantitative real-time PCR for eight different genes was used
to assess gene dosage and to determine the extent of the dupli-
cation in all available family members (Fig. 4). Twenty-two
samples were studied with 13 probes across the region
Human Molecular Genetics, 2008, Vol. 17, No. 24 3851
(Figs 3 and 4). Quantitative duplex PCR of genomic DNA was
performed on the ABI Prism 7900 Sequence Detection
System. b-globin was co-amplified as an internal control,
using the following primers: 50-TGGGCAACCCTAAGG
TGAAG-30(b-Globin F, Exon 2) and 50- GTGAGCCAG
GCCATCACTAAA-30(b-Globin R, Exon 2), and a probe
GCAAGAAAGTGCTCGGTGC-30. The probes for each
region examined were labeled with VIC. Specific primers
and probes (obtainable upon request) were designed using
the Primer Express Program for TaqMan MGB probes
(Applied Biosystems, Foster City, CA, USA). All primers
were purchased from Integrated DNA Technologies (Inte-
grated DNA Technologies, Coralville, IA, USA). Each PCR
reaction was performed in a total reaction volume of 15 ml
containing 25 ng genomic DNA, TaqMan Universal PCR
Master Mix (Applied Biosystems), 900 nM primers and
250 nM probes. The PCR cycling conditions were standard,
958C for 10 min, then 958C for 15 s and 608C for 1 min for
40 cycles. The plates for PCR each contained the genomic
DNA samples, control DNA and a no-template water control
in triplicate, and the experiments were performed in triplicate.
The quantification of each amplicon was determined as the
cycle at which the PCR amplification was in log phase in
fluorescent signal (Ct), relative to b-globin as the internal
control. The dosage of each region relative to b-globin was
normalized to the mean of the unrelated controls using the
22DDCtmethod (29). Values of 0.8–1.2 were assumed to be
normal, and values between 1.3 and 1.7 were considered
indicative of heterozygous duplication.
Genome-wide analysis of palindrome formation
In order to determine whether the SCA20 amplicon is in the
form of a large inverted repeat, the GAPF procedure was per-
formed as described previously (30), with modifications.
Genomic DNA (1.5 mg) was treated with either KpnI or SbfI
to enrich for DNA close to palindromic centers, or with no
enzyme. The restriction enzymes were then heat-inactivated.
To make snap-back DNA, genomic DNA was then boiled in
50 ml of water with 100 mM NaCl for 7 min and then placed
in an ice-water bath to cool quickly. After snap-back
treatment, DNA was treated with S1 nuclease (Invitrogen,
Carlsbad, CA, USA) and amplified by ligation-mediated
PCR. 7.5 mg DNA was labeled with biotin (GeneChip WT
Double-stranded Target Labeling Kit, Affymetrix, Santa
Clara, CA, USA) and hybridized to a Human Tiling 2.0R F
array (Affymetrix). A comparison of GAPF profiles between
affected and unaffected individuals was done with Tiling Analy-
sis Software (Affymetrix, Version 1.1). The probe intensities
were normalized using quantile normalization plus scaling,
and bandwidth was set at 250 bp. Results were visualized with
the Integrated Genome Browser (Affymetrix, Version 4.56).
Fiber FISH analysis
To confirm the GAPF interpretation of the duplication orien-
tation, we applied fiber FISH, according to the methodology
described previously (31,32), with some adaptations. In short,
the cells of EBV-transformed cell line were suspended in water
to a concentration of 1–5 ? 105cells/ml (5). Approximately
100 ml of cell suspension was pipetted onto a Repel-Silane (GE
Healthcare)-coated slide, spread out over the entire surface of
the slide, and quickly dried using a hair-dryer. Two 50 ml
drops of 0.5% SDS, 50 mM EDTA, 0.2 M Tris–HCl, pH 7.0,
were pipetted onto a 24 ? 60-mm coverslip. The slide with the
side containing the cells facing down was then lowered on
top of the coverslip. The slide was turned upside-down and
the coverslip was very carefully slid off. Again, the slide was
dried using a hair-dryer, and then incubated for 5 min in metha-
used directly in the denaturation and hybridization procedures.
FISH was performed as described previously (33).
Analysis of control and population samples
for duplication at SCA20
We analyzed the disease locus for copy number alteration in
1129 samples, previously typed by us using the Infinium
HumanHap550 GeneChip product. These comprised 644
samples from neurologically normal subjects deposited at
the National Institute of Neurological Disorders and Stroke
Neurogenetics Repository (http://ccr.coriell.org/ninds/, unpub-
lished data) and 485 samples from 29 world populations (34).
The research was funded in part by the Intramural Research
Programs of the National Institute on Aging (project number
1 Z01 AG000949-02) and the National Institute of Neurologi-
cal Disorders and Stroke, both of the National Institutes of
Health, Department of Health and Human Services, USA.
M.A.K. was supported by a NINDS Competitive Postdoctoral
Fellowship. The National Institute on Aging project number
associated with this work is Z01 AG000957-05. S.J.D. was
supported by a Pediatric Oncology Research Training
Program, National Institutes of Health 2T32CA009351-29.
The authors wish to thank the family members for their
cooperation with this study. This study used samples from
the NINDS Human Genetics Resource Center DNA and Cell
Line Repository (http://ccr.coriell.org/ninds).
Conflict of Interest statement. None declared.
1. Orr, H.T. and Zoghbi, H.Y. (2007) Trinucleotide repeat disorders. Annu.
Rev. Neurosci., 30, 575–621.
2. Ranum, L.P. and Day, J.W. (2002) Dominantly inherited, non-coding
microsatellite expansion disorders. Curr. Opin. Genet. Dev., 12, 266–271.
3. Scho ¨ls, L., Bauer, P., Schmidt, T., Schulte, T. and Riess, O. (2004)
Autosomal dominant cerebellar ataxias: clinical features, genetics, and
pathogenesis. Lancet Neurol., 3, 291–304.
4. Houlden, H., Johnson, J., Gardner-Thorpe, C., Lashley, T., Hernandez, D.,
Worth, P., Singleton, A.B., Hilton, D.A., Holton, J., Revesz, T. et al.
(2007) Mutations in TTBK2, encoding a kinase implicated in tau
phosphorylation, segregate with spinocerebellar ataxia type 11. Nat.
Genet., 39, 1434–1436.
3852Human Molecular Genetics, 2008, Vol. 17, No. 24
5. Waters, M.F., Minassian, N.A., Stevanin, G., Figueroa, K.P., Bannister,
J.P., Nolte, D., Mock, A.F., Evidente, V.G., Fee, D.B., Muller, U. et al.
(2006) Mutations in voltage-gated potassium channel KCNC3 cause
degenerative and developmental central nervous system phenotypes. Nat.
Genet., 38, 447–451.
6. Ikeda, Y., Dick, K.A., Weatherspoon, M.R., Gincel, D., Armbrust, K.R.,
Dalton, J.C., Stevanin, G., Durr, A., Zuhlke, C., Burk, K. et al. (2006)
Spectrin mutations cause spinocerebellar ataxia type 5. Nat. Genet., 38,
7. Chen, D.H., Brkanac, Z., Verlinde, C.L., Tan, X.-J., Bylenok, L., Nochlin,
D., Matsushita, M., Lipe, H., Wolff, J., Fernandez, M. et al. (2003)
Missense mutations in the regulatory domain of PKC gamma: a new
mechanism for dominant nonepisodic cerebellar ataxia. Am. J. Hum.
Genet., 72, 839–849.
8. van Swieten, J.C., Brusse, E., de Graaf, B.M., Krieger, E., van de Graaf,
R., de Koning, I., Maat-Kievit, A., Leegwater, P., Dooijes, D., Oostra,
B.A. and Heutink, P. (2003) A mutation in the fibroblast growth factor 14
gene is associated with autosomal dominant cerebellar ataxia. Am. J. Hum.
Genet., 72, 191–199.
9. van de Leemput, J., Chandran, J., Knight, M.A., Holtzclaw, L.A., Scholz,
S., Cookson, M.R., Houlden, H., Gwinn-Hardy, K., Fung, H.C., Lin, X.
et al. (2007) Deletion at ITPR1 underlies ataxia in mice and
spinocerebellar ataxia 15 in humans. PLoS Genet., 3, 1076–1082.
10. Iwaki, A., Kawano, Y., Miura, S., Shibata, H., Matsuse, D., Li, W.,
Furuya, H., Ohyagi, Y., Taniwaki, T., Kira, J. and Fukumaki, Y. (2008)
Heterozygous deletion of ITPR1, but not SUMF1, in spinocerebellar
ataxia type 16. J. Med. Genet., 45, 32–35.
11. Knight, M.A., Gardner, R.J.M., Bahlo, M., Matsuura, T., Dixon, J.A.,
Forrest, S.M. and Storey, E. (2004) Dominantly inherited ataxia and
dysphonia with dentate calcification: spinocerebellar ataxia type 20.
Brain, 127, 1172–1181.
12. Storey, E., Knight, M.A., Forrest, S.M. and Gardner, R.J.M. (2005)
Spinocerebellar ataxia type 20. Cerebellum, 4, 55–57.
13. Lorenzo, D.N., Forrest, S.M., Ikeda, Y., Dick, K.A., Ranum, L.P. and
Knight, M.A. (2006) Spinocerebellar ataxia type 20 is genetically distinct
from spinocerebellar ataxia type 5. Neurology, 67, 2084–2085.
14. Tanaka, H., Bergstrom, D.A., Yao, M.C. and Tapscott, S.J. (2005)
Widespread and nonrandom distribution of DNA palindromes in cancer
cells provides a structural platform for subsequent gene amplification.
Nat. Genet., 37, 320–327.
15. Jehee, F.S., Bertola, D.R., Yelavarthi, K.K., Krepischi-Santos, A.C., Kim,
C., Vianna-Morgante, A.M., Vermeesch, J.R. and Passos-Bueno, M.R.
(2007) An 11q11–q13.3 duplication, including FGF3 and FGF4 genes, in
a patient with syndromic multiple craniosynostoses. Am. J. Med. Genet.,
16. Zarate, Y.A., Kogan, J.M., Schorry, E.K., Smolarek, T.A. and Hopkin,
R.J. (2007) A new case of de novo 11q duplication in a patient with
normal development and intelligence and review of the literature.
Am. J. Med. Genet., 143A, 265–270.
17. Bisogno, T., Howell, F., Williams, G., Minassi, A., Cascio, M.G., Ligresti,
A., Matias, I., Schiano-Moriello, A., Paul, P., Williams, E.J. et al. (2003)
Cloning of the first sn1-DAG lipases points to the spatial and temporal
regulation of endocannabinoid signaling in the brain. J. Cell Biol., 163,
18. Sto ¨hr, H., Marquardt, A., White, K. and Weber, B.H. (2000) cDNA
cloning and genomic structure of a novel gene (C11orf9) localized to
chromosome 11q12–.q13.1 which encodes a highly conserved, potential
membrane-associated protein. Cytogenet. Cell Genet., 88, 211–216.
19. Adachi, N., Karanjawala, Z.E., Matsuzaki, Y., Koyama, H. and Lieber,
M.R. (2002) Two overlapping divergent transcription units in the human
genome: the FEN1/C11orf10 locus. OMICS: J. Integr. Biol., 6, 273–279.
20. Hiraoka, L.R., Harrington, J.J., Gerhard, D.S., Lieber, M.R. and Hsieh,
C.L. (1995) Sequence of human FEN-1, a structure-specific endonuclease,
and chromosomal localization of the gene (FEN1) in mouse and human.
Genomics, 25, 220–225.
21. Marquardt, A., Sto ¨hr, H., White, K. and Weber, B.H.F. (2000) cDNA
cloning, genomic structure, and chromosomal localization of three
members of the human fatty acid desaturase family. Genomics, 66,
22. Luo, H.R., Saiardi, A., Nagata, E., Ye, K., Yu, H., Jung, T.S., Luo, X.,
Jain, S., Sawa, A. and Snyder, S.H. (2001) GRAB: a physiologic guanine
nucleotide exchange factor for Rab3A, which interacts with inositol
hexakisphosphate kinase. Neuron, 31, 439–451.
23. Sto ¨hr, H., Marquardt, A., Rivera, A., Cooper, P.R., Nowak, N.J., Shows,
T.B., Gerhard, D.S. and Weber, B.H. (1998) A gene map of the Best’s
vitelliform macular dystrophy region in chromosome 11q12–q13.1.
Genome Res., 8, 48–56.
24. Hentze, M.W., Keim, S., Papadopoulos, P., O’Brien, S., Modi, W.,
Drysdale, J., Leonard, W.J., Harford, J.B. and Klausner, R.D. (1986)
Cloning, characterization, expression, and chromosomal localization of a
human ferritin heavy-chain gene. Proc. Natl Acad. Sci. USA, 83,
25. Lim, J., Hao, T., Shaw, C., Patel, A.J., Szabo ´, G., Rual, J.F., Fisk, C.J., Li,
N., Smolyar, A., Hill, D.E. et al. (2006) A protein–protein interaction
network for human inherited ataxias and disorders of Purkinje cell
degeneration. Cell, 125, 801–814.
26. Yoshida, T., Fukaya, M., Uchigashima, M., Miura, E., Kamiya, H., Kano,
M. and Watanabe, M. (2006) Localization of diacylglycerol lipase-alpha
around postsynaptic spine suggests close proximity between production
site of an endocannabinoid, 2-arachidonoyl-glycerol, and presynaptic
cannabinoid CB1 receptor. J. Neurosci., 26, 4740–4751.
27. Sun, H., Tsunenari, T., Yau, K.W. and Nathans, J. (2002) The vitelliform
macular dystrophy protein defines a new family of chloride channels.
Proc. Natl Acad. Sci. USA, 99, 4008–4013.
28. Simon-Sanchez, J., Scholz, S., Fung, H.C., Matarin, M., Hernandez, D.,
Gibbs, J.R., Britton, A., de Vrieze, F.W., Peckham, E., Gwinn-Hardy, K.
et al. (2007) Genome-wide SNP assay reveals structural genomic
variation, extended homozygosity and cell-line induced alterations in
normal individuals. Hum. Mol. Genet., 16, 1–14.
29. Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene
expression data using real-time quantitative PCR and the 2(-DD C(T))
method. Methods, 25, 402–408.
30. Tanaka, H., Cao, Y., Bergstrom, D.A., Kooperberg, C., Tapscott, S.J. and
Yao, M.C. (2007) Intrastrand annealing leads to the formation of a large
DNA palindrome and determines the boundaries of genomic amplification
in human cancer. Mol. Cell. Biol., 27, 1993–2002.
31. Datson, N.A., Semina, E., van Staalduinen, A.A., Dauwerse, H.G.,
Meershoek, E.J., Heus, J.J., Frants, R.R., den Dunnen, J.T., Murray, J.C.
and van Ommen, G.J. (1996) Closing in on the Rieger syndrome gene on
4q25: mapping translocation breakpoints within a 50-kb region.
Am. J. Hum. Genet., 59, 1297–1305.
32. Giles, R.H., Petrij, F., Dauwerse, H.G., den Hollander, A.I., Lushnikova,
T., van Ommen, G.J., Goodman, R.H., Deaven, L.L., Doggett, N.A.,
Peters, D.J. and Breuning, M.H. (1997) Construction of a 1.2-Mb contig
surrounding, and molecular analysis of, the human CREB-binding protein
(CBP/CREBBP) gene on chromosome 16p13.3. Genomics, 42, 96–114.
33. Dauwerse, J.G., Jumelet, E.A., Wessels, J.W., Saris, J.J., Hagemeijer, A.,
Beverstock, G.C., van Ommen, G.J. and Breuning, M.H. (1992) Extensive
cross-homology between the long and the short arm of chromosome 16
may explain leukemic inversions and translocations. Blood, 79,
34. Jakobsson, M., Scholz, S.W., Scheet, P., Gibbs, J.R., VanLiere, J.M.,
Fung, H.C., Szpiech, Z.A., Degnan, J.H., Wang, K., Guerreiro, R. et al.
(2008) Genotype, haplotype and copy-number variation in worldwide
human populations. Nature, 451, 998–1003.
Human Molecular Genetics, 2008, Vol. 17, No. 243853