C9ORF72 HEXANUCLEOTIDE REPEAT
EXPANSIONS IN PATIENTS WITH ALS FROM
THE CORIELL CELL REPOSITORY
Amyotrophic lateral sclerosis (ALS) is a neurologic
disorder, characterized by progressive degeneration
of both upper and lower motor neurons in the brain
active response binding protein 43 (TARDBP), fused in
sarcoma (FUS), and valosin containing protein (VCP)
genes as being causative of disease.1Recently, an expan-
sion of the noncoding GGGGCC hexanucleotide re-
was identified as an important novel genetic defect in
patients with ALS without or with frontotemporal
dementia (FTD-ALS).2,3Here we report the fre-
quency of this new mutation and its associated clini-
cal features in a cohort of patients obtained from the
Coriell Cell Repository.
Methods. We studied 617 patients with a diagnosis of
ALS (n ? 568), FTD-ALS (n ? 20), progressive
muscular atrophy (PMA; n ? 26), primary lateral
sclerosis (PLS; n ? 2), and progressive bulbar palsy
(PBP; n ? 1). DNA samples from patients were ob-
tained from the Coriell Institute for Medical Re-
search. Table 1 summarizes detailed demographic
and clinical information.
The presence or absence of an expanded hexanucle-
otide repeat was determined using our 2-step protocol.2
First, the hexanucleotide repeat was PCR amplified in
all samples using 1 fluorescently labeled primer fol-
lowed by fragment-length analysis on an ABI3730
DNA analyzer. Patients showing only a single peak on
the electropherogram, suggesting homozygosity in this
assay, were further analyzed using the repeat-primed
tern on the electropherogram was considered evidence
of a pathogenic repeat expansion.
Fisher exact tests were used to compare frequen-
cies of demographic and clinical features among
Results. Of the 617 samples that were analyzed, 73
(11.8%) were found to carry pathogenic GGGGCC
repeat expansions in C9ORF72 (table 1 and table e-1
on the Neurology®Web site at www.neurology.org).
Interestingly, a significantly higher mutation fre-
quency was observed within the FTD-ALS patient
group (9/20; 45.0%), compared to the group of pa-
tients with pure ALS (64/568; 11.3%) (p ? 0.0002).
Among familial cases (fALS), 37.1% (49/132)
showed a repeat expansion, while 4.9% (24/485) of
the sporadic cases (sALS) were positive. In the re-
mainder of our patient series, repeat-units ranged
from 2 to 28, which we considered normal in this
Average age at onset for expansion carriers was
56.8 ? 8.0 years (range 39–80) and males ac-
counted for 53.4% (n ? 39). Ethnicities of positive
cases were white (n ? 72; 98.6%) and African Amer-
ican (n ? 1; 1.4%). The mutation cases with known
site of onset were equally distributed among bulbar,
limb-upper, and limb-lower for the pure ALS cases
(20/20/23 respectively, 1 unknown), whereas the
FTD-ALS cases presented with bulbar, limb-upper,
or generalized onset (4/4/1, respectively). Overall,
bulbar presentation was somewhat more common in
C9ORF72 mutation carriers (24/73; 32.9%) com-
pared to non-C9ORF72 mutation carriers (107/544;
19.7%) (p ? 0.014).
Discussion. The frequency of repeat expansion carri-
ers in fALS (37.1%) reported in this study was highly
similar to that previously reported in a selected series
of European patients with ALS (38.1%) and slightly
higher than our published mutation frequency from
a US fALS series (23.5%). This may be due to the
ALS cases in our previous publication being collected
at 1 location (Mayo Clinic Florida), primarily from
incident patients, whereas Coriell samples were col-
lected at multiple centers throughout the United
States. The frequency of repeat expansion carriers in
sALS (4.9%) in this study was highly comparable to
our previously reported frequency of 4.1%.
Only limited clinical features of C9ORF72 muta-
tion carriers have thus far been described.2–5Clinical
data available on the patients we studied indicate
considerable clinical heterogeneity among mutation
carriers with onset ages ranging from 39 to 80 years,
with limb, bulbar, and generalized presentations at
disease onset. All mutation carriers presented with
upper and lower motor neuron features, with the
highest mutation frequency among FTD-ALS pa-
tients. Although most mutation carriers were white,
we also report the first African American patient with
a C9ORF72 repeat expansion.
In our study a characteristic stutter amplification
pattern by repeat-primed PCR was considered evi-
dence for pathogenicity; however, future Southern
blot analyses will be required to determine the mu-
tant allele length in each individual patient. Future
studies should also clarify the minimal length of a
pathogenic repeat expansion.
The reporting of C9ORF72 mutation status of this
munity. Acquisition of available lymphoblast cell lines
derived from this cohort of mutated patients will allow
accurate repeat sizing, genotype–phenotype correla-
tions, and a source of mutant cells for in vitro studies.
Nicola J. Rutherford,
Matt C. Baker, BSc
Thomas B. Kryston, MS
Patricia E. Brown, MS
Kevin Boylan, MD
Zbigniew K. Wszolek,
Rosa Rademakers, PhD
Neurology 79July 31, 2012
Our findings confirm that C9ORF72 GGGGCC Download full-text
hexanucleotide repeat expansions are a major cause
of ALS and FTD-ALS.
From the Departments of Neuroscience (N.J.R., M.D.-H., M.C.B.,
T.B.K., P.E.B., R.R.) and Neurology (K.B., Z.K.W.), Mayo Clinic,
Jacksonville, FL; and Department of Neurology (C.L.-H.), University
of California San Francisco, San Francisco.
Author contributions: Nicola Rutherford: drafting/revising the man-
uscript for content, analysis or interpretation of data, Acquisition of
data. Mariely DeJesus-Hernandez, Matt Baker, Thomas Kryston,
and Patricia Brown: analysis or interpretation of data, acquisition
of data. Drs. Boylan, Wszolek, and Lomen-Hoerth: drafting/revis-
ing the manuscript for content, including medical writing for con-
tent, contribution of vital reagents/tools/patents, obtaining funding.
Dr. Rademakers: drafting/revising the manuscript for content, in-
cluding medical writing for content, study concept and design, anal-
ysis or interpretation of data, study supervision, obtaining funding.
Acknowledgment: This study used ALS samples from the NINDS
Human Genetics Resource Center DNA and Cell Line Repository
Study funding: Supported by NIH grant R01AG26251, the ALS
Association, and the ALS Therapy Alliance.
N. Rutherford, M. DeJesus-Hernandez, M. Baker, T. Kryston, P.
Brown, and C. Lomen-Hoerth report no disclosures. K. Boylan re-
ceives research support from the ALS Association, Biogen Idec, Neu-
raltus Pharmaceuticals, Cytokinetics Inc, and Mayo Foundation,
and has received research support from Avanir Pharmaceuticals and
Synapse Biomedical. Z. Wszolek is supported by the NIH/NINDS
NS057567, 1RC2NS070276, P50NS072187-01S2, Mayo Clinic
Florida Research Committee CR program, and the Dystonia Medical
Research Foundation. R. Rademakers receives research support from the
NIH (R01 NS065782, R01AG02651, and P50 AG16574), the ALS
association, the ALS Therapy Alliance, CurePSP, and the Consortium
for Frontotemporal Degeneration Research. Dr. Rademakers further re-
ceived honoraria for lectures or educational activities not funded by
industry and serves on the medical advisory board of the Association
for Frontotemporal Degeneration. Go to Neurology.org for full
Correspondence& reprint requests
Copyright © 2012 by AAN Enterprises, Inc.
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panded GGGGCC hexanucleotide repeat in noncoding
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Table 1 Clinical characteristics in patients with and without C9ORF72
hexanucleotide repeat expansions
Average onset age, y
55.0 (19–88)54.8 (19–88) 56.8 (39–80)
Site of symptom onset
ALS and FTD/dementia
No family history of ALS
Abbreviations: ALS ? amyotrophic lateral sclerosis; FTD ? frontotemporal dementia;
PBP ? progressive bulbar palsy; PLS ? primary lateral sclerosis; PMA ? progressive mus-
aIncludes 2 Native Americans and 2 Pacific Islanders.
Neurology 79 July 31, 2012