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BRIEF COMMUNICATIONS
Gait Alterations in Healthy
Carriers of the LRRK2 G2019S
Mutation
Anat Mirelman, PhD,1,6 Tanya Gurevich, MD,1,3,4
Nir Giladi, MD,1,3,4 Anat Bar-Shira, PhD,5
Avi Orr-Urtreger, MD, PhD,4,5
and Jeffrey M. Hausdorff, PhD1,2,6
To test for an association between the LRRK2-G2019S
mutation and gait, we studied 52 first-degree relatives
of patients with Parkinson’s disease (PD) who carry this
mutation. An accelerometer quantified gait during usualwalking, fast-walking, and dual-tasking. Noncarriers (n ¼
27) and carriers (n ¼ 25) were similar with respect to age,
gender, height, and gait speed during all conditions.
During dual-tasking and fast-walking, gait variability
and the amplitude of the dominant peak of the
accelerometer signal were significantly altered among
the carriers. These findings support the possibility of
previously unidentified, presymptomatic motor changes
among relatives who have an increased risk of
developing PD.
ANN NEUROL 2011;69:193–197
T
he leucine-rich repeat kinase 2 (LRRK2) is an important genetic determinant of Parkinson’s disease (PD).
The autosomal dominant G2019S mutation in exon 41
is associated with an increased frequency of PD in Ashkenazi Jews,1 in whom rates approach as high as 26% in
familial and 14% in apparently sporadic PD. Penetrance
is incomplete and age-dependent, with age-specific estimates ranging from 15% at age 50–60 years to 21% to
85% at age 70 years.2–4 The asymptomatic first-degree
relatives of Ashkenazi Jewish PD patients who carry the
LRRK2 G2019S mutation, of whom about 50% may
carry the G2019S mutation, clearly represent a population at increased risk of developing PD.
Gait disturbances play a major role in the motor
manifestation of PD. Alterations in the gait pattern frequently observed in patients with PD include decreased
stride length and increased stride-to-stride variability.5–8
Changes in gait speed and variability can already be
detected in recently diagnosed, de novo patients, even
before any visible or symptomatic gait disturbances are
reported.5,9,10
PD is known for its long prediagnostic phase.11
Efforts to identify early biomarkers and predictors of PD
have identified disturbances in smell, sleep, autonomic
function, and affect to help clarify the pathogenesis of
the disease and inform the future development of novel
therapies.11 The possibility that subtle gait alterations are
also present in the prediagnostic phase of PD has never
been tested. To further elucidate the role of the G2019S
mutation in the development of PD, we investigated a
group of first-degree relatives of Ashkenazi PD patients
who are carriers of the G2019S mutation in the LRRK2
gene to test the hypothesis that challenging the central
gait network with a demanding paradigm will uncover
abnormalities in gait and unmask compensatory mechanisms12 among asymptomatic subjects.
Patients and Methods
Subjects
We recruited 52 healthy first-degree relatives of Ashkenazi PD
patients; all of these patients were carriers of the LRRK2G2019S mutation. The subjects were between 37 and 87 years
of age and were in good health; they were free of medical complaints (eg, dementia, unstable cardiovascular disease, rheumatologic disease, and orthopedic disease), acute illness, or pain.
Subjects were excluded if they had a diagnosis of PD, even
early, de novo PD, a history of stroke or neurological disorders,
major depression, dementia, or psychiatric diagnosis. The study
was approved by both the Internal Review Board of the Tel Aviv
Sourasky Medical Center and the National Israeli Committee for
Human Research, and all subjects signed informed written consent.
Procedures
Subjects underwent a thorough clinical, neurological and cognitive exam including the Unified Parkinson’s Disease Rating
Scale (UPDRS) motor (part III) and assessment of gait. A
small, lightweight accelerometer (DynaPort; McRoberts, The
Hague, Netherlands)13 was worn on the lower back during all
gait measurements to quantify walking. Subjects were asked to
walk along a 20-m-long, well-lit corridor for 1 minute, under
the following conditions: (1) preferred, usual-walking speed; (2)
From the 1Movement Disorders Unit, Department of Neurology, and
5
Genetics Institute, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel,
Departments of 2Physical Therapy and 3Neurology and 4Sackler Faculty of
Medicine, Tel-Aviv University, Tel-Aviv, Israel and 6Harvard Medical
School, Boston, MA.
Address correspondence to Dr Mirelman, Laboratory for Gait Analysis
and Neurodynamics, Movement Disorders Unit, Department of
Neurology, Tel Aviv Sourasky Medical Center, 6 Weizmann Street, Tel
Aviv 64239, Israel. E-mail: anatmi@tasmc.health.gov.il
Received May 26, 2010, and in revised form Jun 22, 2010. Accepted for
publication Jul 16, 2010.
View this article online at wileyonlinelibrary.com DOI: 10.1002/ana.22165
C 2011 American Neurological Association
V
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FIGURE 1: Gait variability measures in all 3 gait conditions. (A) Stride-time variability as measured during usual walking was
not significantly different between the groups, but during the more challenging conditions of fast-walking and dualtasking, carriers demonstrated significantly larger (worse) variability, as compared to the noncarriers. (B) Spectral analysis.
The amplitude of the dominant frequency is presented in all 3 gait conditions. Consistent with the stride time variability
findings in A, the amplitude of the peak was lower in the carriers, compared to the noncarriers, during fast and dual task
walking. Amplitude units are presented as power per radians per second (PRS). Means and standard errors are presented.
dual-task condition, defined as performing a secondary task while
walking (subtracting serial 7s); and (3) fast-speed walking.
Average stride time, gait speed, stride length, and stride
time variability were evaluated. Stride time variability was determined by calculating the magnitude of the stride-to-stride fluctuations of the stride time, normalized to each subject’s mean
stride time (coefficient of variation ¼ 100 standard deviation/mean). Data collected by the accelerometer was also used
for spectral analysis of the calibrated acceleration signal in the
locomotion band (0.5–3.0Hz). The peak amplitude of the dominant frequency in the anterior-posterior direction was extracted
from the raw signal; a sharper and narrower peak reflects a more
consistent, rhythmic, and healthier gait pattern, ie, reduced gait
variability and lower stride-to-stride fluctuations.
Total genomic DNA was isolated from peripheral blood
leukocytes and the 6055G_A (G2019S) mutation in exon 41 of
the LRRK2 gene was determined as previously described.4 All
testing was performed in 1 session by 1 investigator who did
not know the genetic status of participants until the end of the
gait assessment. This information was also not disclosed to the
participants.
Statistical Analysis
Dependent variables (eg, gait speed, stride-time variability) were
checked for normality and homoscedasticity (within groups).
Differences between groups (carriers of the LRRK2-G2019S
mutation vs noncarriers) were tested using repeated measures
analysis of variance (group X gait condition). When differences
between groups were significant (p < 0.05), a post hoc analysis
was performed using a Student t test for further evaluation.
Potential covariates (eg, age, gender, height, etc.) were evaluated
and compared between groups. Scatter plots and nonparametric
comparisons confirmed that any significant findings were not
skewed by extreme values.. Statistical analyses were performed
using SPSS version 16. To reduce the likelihood of false-positive
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results, given the multiple outcome measures, a Bonferroni correction was used and significance was assumed at the 0.025 level.
Results
Twenty-five subjects were identified as carriers of the
LRRK2-G2019S mutation. Carriers and noncarriers were
similar with respect to age, gender, height, body mass
index, scores on the UPDRS, and cognitive function, as
well as on measures of mood, autonomic function, smell,
and presence of sleep disorders (Table). Gait speed, stride
time, and stride length were similar between groups under
all walking conditions (see Table). In contrast, stride time
variability differed across the 2 groups (p ¼ 0.009). Posthoc analysis showed that it was significantly higher (worse)
during the dual-task walking condition among the carriers
(1.82 6 1.04%), as compared to the noncarriers (1.26 6
0.48%; p ¼ 0.018). Differences were also observed during
fast walking (carriers: 1.33 6 0.58%; noncarriers: 1.05 6
0.31%; p ¼ 0.03) (Fig 1A). Spectral analysis results also
revealed similar differences between the 2 groups (p ¼
0.010). The amplitude of the dominant frequency mode
was smaller among the carriers, compared to noncarriers, in
both the fast (p ¼ 0.005) and dual-task (p ¼ 0.03) conditions
(see Fig 1B). Subgroup analyses reveled that even in the subset of subjects 60 years old (noncarriers: n ¼ 21, carriers:
n ¼ 20), in which age-associated changes were not likely to
play any role, the 2 subgroups were well-matched, yet similar
increases in stride-time variability were observed among the
carriers during the fast (p ¼ 0.019) and dual-task walking
conditions (p ¼ 0.012). Figure 2 shows examples of the raw
accelerometer signal and the measures that were extracted to
quantify gait variability in a carrier and noncarrier, illustrating
the differences seen in the 2 groups.
Volume 69, No. 1
Mirelman et al: Gait Alterations in Healthy Carriers of the LRRK2 G2019S Mutation
FIGURE 2: Examples of raw signals extracted from data collected during the fast walk of (A) 1 subject in the noncarrier group
and (B) 1 subject from the carrier group. Top figures demonstrate acceleration signals, the middle figures represent the
change in stride time (in seconds) during the walk, and the bottom figures demonstrate the amplitude of the dominant
frequency during the fast-gait condition presented as power per radians per second (PRS).
TABLE: Subject Characteristics
Non-Carriers (n 5 27)
Carriers (n 5 25)
p
Age, yr (range)
50.08 6 8.05 (39–80)
53.56 6 11.81 (37–87)
0.23
Gender, number (% female)
14 (51%)
11 (44%)
0.39
UPDRS Part III–motor
2.43 6 2.72
3.64 6 3.81
0.29
Height (cm)
167.12 6 11.13
168.31 6 9.62
0.76
Weight (kg)
73.27 6 11.81
79.96 6 13.80
0.07a
BMI (kg/m2)
25.89 6 3.74
27.36 6 3.70
0.16
Cognitive function (MoCA)
26.34 6 2.58
26.81 6 2.42
0.67
Depressive symptoms (GDS)
4.63 6 5.83
3.68 6 2.72
0.46
Autonomic dysfunction (SCOPA-AUT)
6.18 6 7.03
5.68 6 4.20
0.13a
Sleep disturbances (% affected)b
0%
12%
0.06a
Smell (UPSIT)
27.85 6 6.17
28.72 6 4.12
0.56
Usual walking (m/sec)
1.37 6 0.19
1.36 6 0.17
0.96
Fast walking (m/sec)
1.61 6 0.21
1.69 6 0.18
0.86
Dual task (m/sec)
1.21 6 0.18
1.19 6 0.16
0.91
Usual walking (sec)
1.06 6 0.07
1.05 6 0.09
0.81
Fast walking (sec)
0.95 6 0.06
0.94 6 0.10
0.78
Dual task (sec)
1.11 6 0.09
1.10 6 0.11
0.94
146.42 6 13.71
145.09 6 15.60
0.89
Gait speed
Stride time
Stride length: usual walking (cm)
These potential confounders, including the UPDRS motor scores, were not correlated with gait variability (p > 0.101). None of
the subjects had resting tremor or had other findings that might suggest the presence of early PD.
b
Sleep disturbances assessed using the REM Sleep Behavior Disorder Questionnaire.
GDS ¼ Geriatric Depression Scale; MoCA ¼ Montreal Cognitive Assessment; SCOPA-AUT ¼ Scales for Outcomes in
Parkinson’s Disease-Autonomic Questionnaire; UPSIT ¼ University of Pennsylvania Smell Identification Test.
a
January 2011
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Discussion
To our knowledge, this is the first report of changes in
motor performance among healthy asymptomatic carriers
of the LRRK2-G2019S mutation. Although results of
both groups were within normative ranges, gait variability of the carriers was worse than in the noncarriers in all
conditions, especially during more challenging tasks.
Stride-time variability has been shown to be a
highly sensitive measure of gait consistency and stability,
especially under challenging conditions.7,14,15,19 In PD,
the ability to regulate stride-to-stride fluctuations
decreases and gait variability increases during dual tasking
as this task requires the recruitment of additional attentional resources.12,16,20 The poorer performance of the
LRRK2-G2019S mutation carriers may be consistent,
therefore with subtle abnormalities in the central gait
network as manifested during the challenging conditions,
thus demonstrating decreased compensatory reserve.
There are at least 2 competing explanations for the
present findings. It may be possible that among the carriers,
a population that is at increased risk for developing PD, the
subtle changes observed in gait variability could reflect early
preclinical motor alterations, ie, an early manifestation of
subtle alterations to the central gait network. This intriguing
possibility can only be confirmed by long-term, prospective
follow-up of these subjects. Another possibility is that the
gait network of the LRRK2-G2019S mutation carriers is
simply different, unrelated to the future development of
PD. Compensatory mechanisms may be sufficient during
usual-walking, but not during more challenging conditions.
The deficits observed in these first-degree relatives might
therefore reflect, at least in part, an endophenotypic marker
and not an early biomarker of PD.
The present observation is supported by 2 recent
functional magnetic resonance imaging studies that tested
upper extremity movements in asymptomatic carriers of
mutations in the PARKIN or PINK1 genes. A stronger
increase in movement-related activity in the right rostral
cingulate motor area and left dorsal premotor cortex and
increased compensatory recruitment of the rostral supplementary motor area during movements was observed
compared to noncarriers.17,18 While these studies did not
examine gait, the findings are consistent with the possibility of changes to the neural networks involved in
motor control and perhaps in recruitment of compensatory reserves among asymptomatic mutation carriers,
which may be independent of a diseased state.
The present, preliminary results should be interpreted cautiously. Still, these novel findings have implications for understanding the genotype-phenotype relation
of the LRRK-G2019S mutation. The findings also support the intriguing possibility that gait dynamics during
196
challenging conditions may serve as a new, sensitive biological marker of presymptomatic PD. Larger scale, longitudinal studies are, however, needed to assess whether
the gait changes observed are predictive of PD.
Acknowledgments
This research was supported by grants from the Tel Aviv
Sourasky Medical Center Grant of Excellence, Khan Foundation, Israel Science Foundation Heritage Legacy, and the
Michael J. Fox Foundation for Parkinson’s Research.
We thank the participants and staff of this project for
their invaluable contributions: Shiran Levy, Avner Thaler,
Hertzel Shabtai, Yakov Balash, Ariellla Hilel, Meirav
Kedmi, Ziv Gan-Or, Aner Weiss, and Leor Gruendlinger.
Potential Conflicts of Interest
Nothing to report.
References
1.
Ozelius LJ, Senthil G, Saunders-Pullman R, et al. LRRK2 G2019S
as a cause of Parkinson’s disease in Ashkenazi Jews. N Engl J
Med 2006;354:424–425.
2.
Goldwurm S, Zini M, Mariani L, et al. Evaluation of LRRK2 G2019S
penetrance: relevance for genetic counseling in Parkinson disease.
Neurology 2007;68:1141–1143.
3.
Kachergus J, Mata IF, Hulihan M, et al. Identification of a novel
LRRK2 mutation linked to autosomal dominant parkinsonism: evidence of a common founder across European populations. Am J
Hum Genet 2005;76:672–680.
4.
Orr-Urtreger A, Shifrin C, Rozovski U, et al. The LRRK2 G2019S
mutation in Ashkenazi Jews with Parkinson disease: is there a gender effect? Neurology 2007;69:1595–1602.
5.
Baltadjieva R, Giladi N, Gruendlinger L, et al. Marked alterations
in the gait timing and rhythmicity of patients with de novo Parkinson’s disease. Eur J Neurosci 2006;24:1815–1820.
6.
Blin O, Ferrandez AM, Serratrice G. Quantitative analysis of gait in
Parkinson patients: increased variability of stride length. J Neurol
Sci 1990;98:91–97.
7.
Hausdorff JM, Cudkowicz ME, Firtion R, et al. Gait variability and
basal ganglia disorders: stride-to-stride variations of gait cycle
timing in Parkinson’s disease and Huntington’s disease. Mov Disord 1998;13:428–437.
8.
Hausdorff JM. Gait dynamics in Parkinson’s disease: common and
distinct behavior among stride length, gait variability, and fractallike scaling. Chaos 2009;19:026113
9.
Carpinella I, Crenna P, Calabrese E, et al. Locomotor function in
the early stage of Parkinson’s disease. IEEE Trans Neural Syst
Rehabil Eng 2007;15:543–551.
10.
Frenkel-Toledo S, Giladi N, Peretz C, et al. Effect of gait speed on
gait rhythmicity in Parkinson’s disease: variability of stride time and
swing time respond differently. J Neuroeng Rehabil 2005;2:23.
11.
Wolters EC, Francot C, Bergmans P, et al. Preclinical (premotor)
Parkinson’s disease. J Neurol 2000;247(suppl 2):II103–II109.
12.
Bezard E, Gross CE. Compensatory mechanisms in experimental
and human parkinsonism: towards a dynamic approach. Prog
Neurobiol 1998;55:93–116.
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13.
Zijlstra W, Hof AL. Assessment of spatio-temporal gait parameters from
trunk accelerations during human walking. Gait Posture 2003;18:1–10.
14.
Rao AK, Muratori L, Louis ED, et al. Spectrum of gait impairments
in presymptomatic and symptomatic Huntington’s disease. Mov
Disord 2008;23:1100–1107.
15.
Verghese J, Holtzer R, Lipton RB, et al. Quantitative gait markers
and incident fall risk in older adults. J Gerontol A Biol Sci Med Sci
2009;64:896–901.
16.
Yogev-Seligmann G, Hausdorff JM, Giladi N. The role of executive
function and attention in gait. Mov Disord 2007;23:329–342.
17.
Buhmann C, Binkofski F, Klein C, et al. Motor reorganization in
asymptomatic carriers of a single mutant Parkin allele: a human model
for presymptomatic parkinsonism. Brain 2005;128:2281–2290.
18.
van Nuenen BF, Weiss MM, Bloem BR, et al. Heterozygous carriers of a Parkin or PINK1 mutation share a common functional
endophenotype. Neurology 2009;72:1041–1047.
19.
Herman T, Mirelman A, Giladi N, et al. Executive control deficits
as a prodrome to falls in healthy older adults: A prospective study
linking thinking, walking, and falling. J Gerontol A Biol Sci Med
Sci 2010 (ePub ahead of print).
20.
Yogev G, Giladi N, Peretz C, et al. Dual tasking, gait rhythmicity,
and Parkinson’s disease: which aspects of gait are attention
demanding? Eur J Neurosci 2005;22:1248–1256.
Motor Nerve Biopsy: Clinical
Usefulness and
Histopathological Criteria
Nilo Riva, MD,1 Sandro Iannaccone, MD,2
Massimo Corbo, MD,3 Chiara Casellato, MD,4
Barbara Sferrazza, MD,2 Alberto Lazzerini, MD,5
Marina Scarlato, MD, PhD,1 Federica Cerri, MD,1
Stefano C. Previtali, MD, PhD,1
Eduardo Nobile-Orazio, MD, PhD,4
Giancarlo Comi, MD,1,6 and Angelo Quattrini, MD1
Early differential diagnosis of motor neuropathies (MN)
and lower motor neuron diseases (LMND) is important,
as prognosis and therapeutic approaches are different.
We evaluated the diagnostic contribution of the biopsy
of the motor branch of the obturator nerve and gracilis
muscle in 21 consecutive patients in which, after proper
clinical and neurophysiological studies, the differential
diagnosis was still open. At baseline, motor biopsy was
performed; diagnostic confirmation was obtained by
2-year clinical follow-up. Our results support the
usefulness of this diagnostic procedure for selected
cases of MN and LMND.
ANN NEUROL 2011;69:197–201
(ALS) is the most common form, involving both lower
motor neurons (LMN) and upper motor neurons
(UMN), whose biological, psychological, and social
impacts are devastating.1 ALS diagnosis is generally
fairly simple,2 but may be less certain in patients presenting with sporadic progressive disease of LMN.
These patients were diagnosed as ‘‘suspected ALS’’
according to the 1994 El-Escorial criteria but this category no longer exists in the 2000 revised criteria.3 The
term lower MND (LMND) is more appropriately used
to indicate this heterogeneous group of diseases, which
includes progressive muscular atrophy (PMA). A substantial proportion of PMA patients develop ALS or
have an ‘‘ALS-like’’ disease course.4 Notably, the
reported percentage of misdiagnosis is 19% for PMA,5
and up to 10% for ALS (1% rediagnosed as neuropathy).6–8 Therefore, in some cases, only follow-up can
lead to a certain diagnosis.
Motor neuropathies (MN) are an heterogeneous
group of diseases primarily affecting the motor nerves.
In most MN cases the absence of UMN signs and
demyelinating features at nerve conduction studies
lead to a straightforward diagnosis. However, demyelinating features may not always be demonstrated and
purely axonal electrophysiologic findings are found
in selected cases, some responding to intravenous
immunoglobulin therapy.9–11
Early differential diagnosis between LMND and
MN is important, as prognosis and therapeutic
approach are different; moreover, current and future
therapies might be more effective in the first stages of
disease.12
The morphological aspects of the motor branch of
the obturator nerve have been shown to differ in patients
with a definite diagnosis of MN or MND; however, the
From the 1 Department of Neurology, INSPE and Division of
Neuroscience, San Raffaele Scientific Institute, Milan, Italy; 2Department
of Clinical Neurosciences, San Raffaele Turro Hospital, Milan, Italy;
3
NEuroMuscular Omnicentre (NEMO), Niguarda Ca Granda Hospital,
Milan, Italy; 42 Neurology, IRCCS Istituto Clinico Humanitas, Milano
University, Milan, Italy; 5Hand Surgery and Microsurgery Unit, IRCCS
Istituto Clinico Humanitas, Milan, Italy; 6Università Vita e Salute San
Raffaele Milan, Milan, Italy.
Address correspondence to Dr Riva, Department of Neurology, INSPE
and Division of Neuroscience, San Raffaele Scientific Institute, via
Olgettina 48, 20132 Milan, Italy. E-mail: riva.nilo@hsr.it
Received Feb 3, 2010, and in revised form May 27, 2010. Accepted for
publication May 28, 2010.
M
otor neuron disease (MND) indicates a group of
neurological disorders characterized by degeneration of motor neurons. Amyotrophic lateral sclerosis
January 2011
View this article online at wileyonlinelibrary.com. DOI: 10.1002/
ana.22110
Additional Supporting Information can be found in the online version of
this article.
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clinical value and potential diagnostic contribution of
this investigation in the early stages of disease are still
unknown and are therefore the focus of our study.13
Patients and Methods
Patients
We studied 21 consecutive patients over about 900 screened
(neuropathies 630; MND 270). Patients provided
informed consent to the study, approved by the local ethic
committee. We included patients presenting with sporadic,
recent-onset LMN syndrome in which, after extended clinical,
neurophysiological and hematochemical examination, a conclusive diagnosis could not be reached. Neurophysiological findings were consistent with pure motor axonal neuropathy/neuronopathy at limbs. Follow-up neurological examination was
performed at 6, 12, 18, and 24 months (see Supplementary
Material and Supplementary Table S1 and S3).
Neuropathology
All patients underwent biopsy of the motor branch of the obturator nerve and gracilis muscle.
Light and electron microscope examinations were perfomed.14,15 Nerve morphometric analysis included fiber density
and g-ratio (axonal/fiber diameter); the regeneration parameter
was calculated as the number of regenerating clusters per mm2
(cluster density [CD]) and as the ratio of clusters to fibers.16
Nerve and muscle morphological examination were performed by 3 blinded independent examiners. Inter-reader agreement was evaluated with Cohen’s kappa index.
Criteria for nerve biopsy analysis:
1. Signs of myelin pathology: nerve fiber demyelination/remyelination, onion bulbs.
2. Signs of axonal pathology: reduction of myelinated fibers,
signs of active axonal degeneration.
3. Pathological signs suggesting a known cause of neuropathy.15
4. Nerve regeneration parameter. The only previous study on the
obturator nerve found a mean CD of 19.2/mm2 in patients
with a definite clinical diagnosis of MND (standard deviation
(SD)¼ 8.4).13 CD <27.6/mm2(mean þ SD) was chosen as a
supportive criterion for LMND (CD > 27.6/mm2 for MN).
Based on these findings, patients were divided into 2
groups: 1) Group I, suspected LMND; and 2) Group II, suspected MN.
Follow-Up: Clinical Diagnosis
MND or peripheral neuropathy/MN diagnosis was performed
according to standard criteria.3,5,17
Statistical Analysis and Development of
Neuropathological Diagnostic Criteria
Statistical analysis of morphometric data was performed after
clinical diagnostic confirmation at 2 years of follow-up using
SPSS software (Chicago, IL); for group comparisons, the
198
Mann-Whitney U-test was applied (statistical significance
threshold: p < 0.05%).
We propose neuropathological criteria for motor nerve
biopsy interpretation. CD (and cluster/fiber ratio) reference
intervals have been defined as follows:
• Upper CD limit for LMND: mean LMND-CD þ SD;
• Lower CD limit for MN: mean MN-CD 1SD.
(CD obtained from compound descriptive statistic analysis including morphometric data from our previous study performed in patients with a definite clinical diagnosis at
biopsy).13
Results
Baseline: Histopathological Diagnosis
Inter-reader agreement was 100%(Cohen’s kappa).
Twelve patients (1–12; mean age: 51.8 years, range:
43–62 years) were classified in Group I (suspected
LMND). Morphological examination showed reduction
of myelinated fibers, sometimes associated with signs of
active axonal degeneration, poor/no signs of nerve regeneration, and no signs of demyelination/remyelination
and/or inflammatory cell infiltration. Notably, the reduction in fiber density tended to be a focal/multifocal distribution among and within fascicles (Fig 1).
Eight patients (14–21; mean age: 55.6 years; range:
45–65 years) were classified in Group II (suspected
MN). Morphological examination showed a reduction of
myelinated fibers uniformly distributed within and
between the fascicles; in 6 patients (16–21) the diagnosis
was supported by high CD, in 3 patients (19, 20, and 21)
it was associated with signs of demyelination/remyelination. In 2 patients (14 and 15) nerve biopsy showed lowmoderate signs of nerve regeneration, associated in one
(14) with amyloid deposits on Congo-red staining, and in
the other (15) with sign of demyelination/remyelination.
Muscle biopsy did not help in differentiating MN
from LMND, in all patients showing small angulated denervated fibers, variable degree of type grouping, type II
fiber hypertrophy in 2 patients (10 and 12) and fiber I
hypertrophy in 3 (14, 17, and 18).
In 1 patient (13) nerve and muscle biopsy showed
normal pathological findings. This patient did not have
clinical/neurophysiological abnormalities of lower limbs
at time of biopsy.
Differences in clinical characteristics between group
I and II were unremarkable.
Follow-Up: Clinical Diagnosis
Two years of clinical follow-up confirmed the baseline
histopathological diagnosis in all patients (see Supplementary Table S1 and S2).
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FIGURE 1: Representative neuropathological cases. Transverse semi-thin sections of biopsy of motor nerve from case 1 (A: MND)
and 19 (B: MN). Focal decreased density of myelinated nerve fibers (A*) is evident. In A, axonal degeneration is present at higher
magnification (arrows). (B) Mild reduction of large myelin nerve fibers is present in representative sections from patients with
definite diagnosis of MN. There are many clusters of small myelinated fibers (arrowheads) indicating axonal regeneration. In
addition a thinly myelinated nerve fiber (arrow), indicating remyelination, and poliglucosan bodies inclusions (white arrows) are
present. Bar: 50 lm; high magnification: 15 lm. MN 5 motor neuropathies; MND 5 motor neuron diseases.
Group I: in 8 patients (1, 3, 4, 5, 8, 10, 11, and 12)
the final diagnosis was ALS. Four patients (2, 6, 7, and 9)
developed diffuse LMN signs and a rapidly progressive disease leading to respiratory failure: LMND was diagnosed.
Group II: in 5 patients (14, 15, 18, 19, and 21) a
final diagnosis of motor-sensory axonal neuropathy was
made, while in 3 patients (16, 17, and 20) the final diagnosis was axonal MN. The patient with amyloid neuropathy died within 1 year.
Patient 13 developed ALS.
We obtained an overall sensibility for disease detection of 0.95 (95% confidence interval: 0.74–0.99). No
patient complained of adverse symptoms related to biopsy.
Morphometric Analysis and Neuropathological
Diagnostic Criteria
Morphometric studies showed increased CD in MN
patients nerves (p < 0.001), a border-line reduction of gratio in LMND (p < 0.05) but no group differences in
myelinated fiber density (Table 1).
The proposed pathological diagnostic criteria
should be applied in the appropriate clinical context
(namely, recent-onset LMN syndromes)(Table 2; Supplementary Table S4 and S5).
Signs of axonal pathology and low CD should lead
to suspicion of LMND, which is also suggested, in our
experience, by focal fiber loss. Signs of demyelination/
remyelination and/or high CD support a pathological diJanuary 2011
agnosis of MN; rarely, specific findings can be demonstrated, such as pathologic deposits or axonal inclusions.
Discussion
As main result of this study, motor nerve pathologic examination was helpful for early differential diagnosis of
LMN syndromes. At morphometric examination, CD
was the best parameter for differentiating MN from
MND patients and was also a prognostic factor,
independently from the diagnosis. As expected from neurophysiological inclusion criteria, neuropathological examination showed scant signs of demyelination/remyelination in MN. These results are consistent with a
previous study, which was performed, unlike the present
study, on patients with a definite diagnosis of MND or
MN.13 We observed a tendency toward a relatively focal/
patchy fiber loss in MND motor nerves. However, this
observation was not included as a diagnostic criterion for
2 reasons. First, this observation has never been reported
before. This might be explained by the different timing
of motor nerve biopsy, performed in this study at an
early stage, while in previous reports in patients at an
advanced stage of disease or postmortem.13,18,19 Second,
a varying degree of focal/multifocal fiber loss is seen in a
variety of neuropathies, including vasculitis or demyelinating neuropathies and has recently been described in
biopsies of upper limb nerves obtained at sites of conduction block from long-lasting cases of MMN.20
199
<0.001
<0.001
0.02
Inclusion Criteria
31.8 (20.8)
13.6 (8.8)
1. Recent onset lower motor neuron syndrome
0.54 (0.04)
4358 (1093)
21
2. Informative biopsy
Pathologic Diagnostic Criteria
1. Signs of axonal pathology
172.4 (83.4)
<0.001
43.0 (20.4)
208.4 (53.4)
<0.001
23.4 (17.4)
145.3 (94.6)
b. Intermediate regeneration: cluster density
between 22.4 and 42.2mm2(or cluster/fiber
ratio between 0.52% and 0.89%)
c. High regeneration cluster density > 42.2 mm2
(or cluster/fiber ratio > 0.89%)
3. Signs of demyelination/remyelination, pathologic
deposits, or other potential causes of neuropathy
Pathologic Diagnostic
Pathologic Diagnostic
Categories
Criteria Required
Values are mean (SD).
MN ¼ motor neuropathy; MND ¼ motor neuron disease; NS ¼ not significant; SD ¼ standard deviation.
Cluster/fiber ratio (%)
a. Low regeneration: cluster density < 22.4mm2
(or cluster/fiber ratio < 0.52%)
a
75.4 (33.2)
<0.001
<0.001
9.4 (6.7)
a
62.5 (33.7)
Cluster density (mm )
G ratio
a
Fiber density (mm )
92.7 (25.8)
19.2 (8.4)
0.58 (0.03)
0.03
0.02
2 a
0.60 (0.02)
0.57 (0.03)
0.56 (0.03)
0.51 (0.04)
4761 (1502)
NS
4788 (1291)
4684 (1714)
NS
4350 (933)
12
9
Patients (n)
2 a
4819 (1443)
25
9
6
Diagnosis
MND
MN
Diagnosis
MND
p
MN
Corbo et al.13
Present Study
TABLE 1: Results of Morphometric Studies and Comparison with Previous Literature Results
200
TABLE 2: Proposed Neuropathological Diagnostic
Criteria for Motor Nerve Biopsy
2. Regeneration parameter
p
MN
Diagnosis
MND
p
NS
of Neurology
Present Study 1 Corbo et al.15
ANNALS
Definite MN
1 þ 2b þ 3
1 þ 2c þ 3
2b þ 3
2c þ 3
3
Probable MN
1þ2c
Possible MN (LMND
not excluded)
1 þ 2b
Probable LMND
1 þ 2a
In this study, muscle biopsy, considered by the ElEscorial criteria as a possible diagnostic investigation for
ALS, did not help in differentiating MN from MND.3
Type grouping percentage was similar, in spite of the
marked increase in CD observed in MN patients (see Table
2). Different re-enervation mechanisms in fact take place in
MN and LMND.14 Collateral re-enervation through
sprouting of surviving distal motor fibers can be observed
both in MN and MND, underlying type grouping formation on muscle pathological inspection and increased motor
unit potential amplitude on needle examination, while cluster formation underlies nerve regeneration, requires a vital
LMN, and is more prominent in MN.
We avoided the introduction of the pathologic
diagnostic category ‘‘definite LMND’’ because for a pathologic confirmative diagnosis, an extensive central nervous system examination, including motor neuron cell
bodies, should be performed.3 LMND remains,
Volume 69, No. 1
Riva et al: Motor Nerve Biopsy
therefore, a diagnosis of exclusion because a disease morphological marker in the peripheral nerve is still lacking.
However, a definite diagnosis of MN is possible.
The biopsy of the motor branch of the obturator nerve
should be considered as a potential diagnostic tool for early
differential diagnosis of selected cases of LMND and MN.
Acknowledgments
This research was supported by Istituto Superiore di Sanità e Fondo per gli investimenti della Ricerca di Base TissueNet (to A.Q.) and by MoH RF-FSR-2007-637144 to
S.I.
Potential Conflict of Interest
Nothing to report.
References
1.
Carus R. Motor neurone disease: a demeaning illness. Br Med J
1980;280:455–456.
2.
de Carvalho M, Dengler R, Eisen A, et al. Electrodiagnostic criteria
for diagnosis of ALS. Clin Neurophysiol 2008,119:497–503.
3.
Brooks BR, Miller RG, Swash M, Munsat TL. World Federation of
Neurology Research Group on Motor Neuron Diseases. El Escorial
revisited: revised criteria for the diagnosis of amyotrophic lateral
sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord
2000;1:293–300.
4.
Van den Berg-Vos RM, Visser J, Kalmijn S, et al. A long-term prospective study of the natural course of sporadic adult-onset lower
motor neuron syndromes. Arch Neurol 2009;66:751–757.
5.
Visser J, van den Berg-Vos RM, Franssen H, et al. Mimic syndromes in sporadic cases of progressive spinal muscular atrophy.
Neurology 2002;58:1593–1596.
6.
Scottish Motor Neuron Disease Research Group. The Scottish
Motor Neuron Disease Register: a prospective study of adult
onset motor neuron disease in Scotland. Methodology, demography and clinical features of incident cases in 1989. J Neurol Neurosurg Psychiatry 1992;55:536–541.
7.
Davenport RJ, Swingler RJ, Chancellor AM, Warlow CP. Avoiding
false positive diagnoses in motor neuron disease: lessons from
the Scottish Motor Neuron Disease Register. J Neurol Neurosurg
Psychiatry 1996;60:147–151.
8.
Traynor BJ, Codd MB, Corr B, et al. Clinical features of amyotrophic lateral sclerosis according to the El Escorial and Airlie House
diagnostic criteria. Arch Neurol 2000;57:1171–1176.
9.
Van Asseldonk JT, Franssen H, Van den Berg-Vos RM, et al. Multifocal motor neuropathy. Lancet Neurol 2005;4:309–319.
10.
Katz JS, Barohn RJ, Kojan S, et al. Axonal multifocal motor neuropathy without conduction block or other features of demyelination. Neurology 2002;58:615–620.
11.
Delmont E, Azulay JP, Giorgi R, et al. Multifocal motor neuropathy
with and without conduction block: a single entity? Neurology
2006;67:592–596.
12.
Shook SJ, Pioro EP. Racing against the clock: recognizing, differentiating, diagnosing, and referring the amyotrophic lateral sclerosis patient. Ann Neurol 2009;65:S10–S16.
13.
Corbo M, Abouzahr MK, Latov N, et al. Motor nerve biopsy studies in motor neuropathy and motor neuron disease. Muscle Nerve
1997;20:15–21.
January 2011
14.
Dubowitz V, Sewry CA. Neurogenic disorders. In: Dubowitz V,
Sewry CA, eds. Muscle biopsy. A practical approach. 3th ed. Philadelphia: Elsevier Saunders, 2007:275–292.
15.
Dick PJ, Dyck PJB, Engelstad J. Pathologic alterations of nerves.
In: Dyck PJ, Thomas PK, eds. Peripheral neuropathy. 4th ed. Philadelphia: Elsevier Saunders, 2005:733–830.
16.
Previtali SC, Malaguti MC, Riva N, et al. The extracellular matrix
affects axonal regeneration in peripheral neuropathies. Neurology
2008;71:322–331.
17.
Grant IA, Benstead TJ. Differential diagnosis of polineuropathy.
In: Dyck PJ, Thomas PK, eds. Peripheral neuropathy. 4th ed. Philadelphia: Elsevier Saunders. 2005:1163–1180.
18.
Atsumi T. The ultrastructure of intramuscular nerves in amyotrophic lateral sclerosis. Acta Neuropathol 1981;55:193–198.
19.
Mitsumoto H, Chad DA, Pioro EP. Neuropathology. In: Amyotrophic
lateral sclerosis. Philadelphia: FA Davis Company, 1998:179–196.
20.
Taylor BV, Dyck PJ, Engelstad J, et al. Multifocal motor neuropathy: pathologic alterations at the site of conduction block. J Neuropathol Exp Neurol 2004;63:129–137.
Ictal Very Low Frequency
Oscillation in Human
Epilepsy Patients
Liankun Ren, MD,1,2 Kiyohito Terada, MD,2
Koichi Baba, MD,3 Naotaka Usui, MD,3
Shuichi Umeoka, MD,3 Keiko Usui, MD,2
Kazumi Matsuda, MD,3 Takayasu Tottori, MD,3
Fumihiro Nakamura, MD,4 Tadahiro Mihara, MD,3
and Yushi Inoue, MD4
Using intracranial electroencephalographic recordings,
we identified a distinct brain activity in 3 patients with
refractory epilepsy characterized by very early
occurrence from 8 minutes 10 seconds to 22 minutes 40
seconds prior to clinical seizure onset, periodical
appearance of slow negative baseline shift, long
interpeak interval of 40 to 120 seconds, and
disappearance after clinical seizure. We named this
activity ‘‘very low frequency oscillation’’ (VLFO), which
reflected a dynamic process during the preictal state.
From the 1Department of Neurology, China-Japan Friendship Hospital,
Beijing, China; and Departments of 2Neurology, 3Neurosurgery, and
4
Psychiatry, National Epilepsy Center, Shizuoka Institute of Epilepsy and
Neurological Disorders, Aoi-ku, Shizuoka, Japan.
Address correspondence to Dr Terada, Department of Neurology,
National Epilepsy Center, Shizuoka Institute of Epilepsy and
Neurological Disorders, 886 Urushiyama, Aoi-ku, Shizuoka, 420-8688
Japan. E-mail: kyht-terada@umin.net
Additional Supporting Information can be found in the online version of
this article.
Received Mar 14, 2010, and in revised form Jun 14, 2010. Accepted for
publication Jul 9, 2010.
View this article online at wileyonlinelibrary.com. DOI: 10.1002/
ana.22158
201
ANNALS
of Neurology
This observation may render new insight into
epileptogenesis and provide additional information
concerning the epileptogenic zone as well as prediction
of epileptic seizures.
ANN NEUROL 2011;69:201-206
minutes per epoch (30 times the routine EEG display). For
each patient, a 2-hour EEG window with 1 hour before and 1
hour after the clinical seizures, and 5-hour continuous interictal
EEG was evaluated.
Results
E
lectroencephalography (EEG) is essential for assessing
patients with epilepsy. Conventionally, EEG is usually
analyzed at a narrow frequency band ranging from 0.5Hz
to 70Hz. However, brain activities beyond the conventional frequency range, such as high-frequency oscillation
(HFO) ranging from 100Hz to 500Hz,1,2 very high frequency oscillation (VHFO)3 (over 1,000Hz), infraslow
brain activity,4 and ictal direct current shift (IDS)5 have
also been observed. In recent years, these unconventional
EEG findings have remained a topic of intensive investigation for their clinical significance.
We first report here a distinct ictal electrophysiological activity in the infraslow band, which was detected in
3 patients with refractory neocortical epilepsy by means
of subdural electrodes. It was characterized by very early
occurrence before the clinical seizure onsets, periodical
appearance of slow negative baseline shifts, gradual evolution in amplitude, frequency, and distribution, and disappearance soon after the clinical seizures. Since it was distinguishable from any previously known ictal EEG
patterns, we named it ‘‘ictal very low frequency oscillation’’ (VLFO) to highlight its unique neurophysiological
features.
Patients and Methods
We investigated 26 patients with intractable neocortical epilepsy
who underwent presurgical evaluation with subdural electrodes
because noninvasive investigations could not delineate their epileptogenic zone (EZ), between July 2004 and June 2009 at the
Shizuoka Institute of Epilepsy and Neurological Disorders. A
low-frequency filter of 0.016Hz, which was in accordance with
a time constant (TC) of 10 seconds was used during recording
in these patients.
We used a digital EEG machine (Neurofax, NihonKoden Corp., Tokyo, Japan). The invasive electrodes were
placed according to the clues indicated by noninvasive studies.
Each subdural electrode was 2.3mm in diameter, linearly
arrayed, made of platinum-iridium alloy, and with a center-tocenter electrode distance of 10mm (Ad-Tech Medical Instrument Corp., Racine, WI). Recording sessions started 1 week after the implantation of electrodes, and lasted for 2 weeks.
The parameters for the conventional analysis of EEG are
TC of 0.1 second, high frequency filter (HFF) of 70 Hz, and
10 seconds per epoch of EEG display. To pick up very slow
periodic electrical potentials, we evaluated the EEG with TC of
10 seconds and a highly compacted EEG display with 5
202
Among 26 patients, only 3 patients clearly demonstrated
VLFO (Figs 1–3, Supporting Fig S1D, and Supporting
Figs S2–S4). Patient 1 was an 18-year-old female, having
drug-resistant seizures including simple partial seizures
(SPS) manifesting focal motor symptoms and secondarily
generalized tonic-clonic seizures (sGTC) since 2 years of
age. Her brain magnetic resonance imaging (MRI)
showed increased signal intensity on the right parietal
cuneus in fluid attenuation inversion recovery (FLAIR)
imaging. The scalp EEG demonstrated interictal low-amplitude spikes or sharp waves over the right parietal area.
Ictal discharges started with low-amplitude slow waves
over the right occipital and posterior temporal areas.
Patient 2 was a 30-year-old female whose seizures
occurred at the age of 16 years. Her seizures, including
auras manifesting as complex auditory symptoms, complex partial seizures (CPS), and sometimes followed by
sGTC, were refractory to antiepilepsy drugs (AEDs). Her
brain computed tomography (CT) and MRI were normal. Scalp interictal EEG showed epileptiform discharges
in the left anterior temporal area, and ictal discharges
started with 6Hz, theta activities over the left anterior
temporal area, evolving into the surrounding areas.
Patient 3 was a 23-year-old male having seizures since 12
years of age. His refractory CPS started with the eyes
and head turning to the left side, sometimes followed by
sGTC. Surface interictal EEG showed intermittent spikes
over the right frontal, temporal, and parietal lobes
dependently, while diffuse ictal discharges were found
widely over the right hemisphere. His brain MRI showed
mild increased signal intensity in the right frontal inferior
sulcus in FLAIR imaging (see Fig 3).
During intracranial EEG monitoring, 9 SPS and 7
sGTC in Patient 1; 2 SPS, 4 CPS, and 2 sGTC in
Patient 2; and 4 CPS and 11 sGTC in Patient 3 were
captured and analyzed in total.
With highly compacted EEG display and long TC
of 10 seconds, very slow, irregular, low-amplitude baseline fluctuations were found during the interictal period.
However, there was no regular, periodic, or evolving pattern (see Supporting Fig S1B). In contrast, VLFO were
clearly identified only prior to CPS (8/8 seizures) and
sGTC (20/20 seizures) but not SPS (0/11 seizures) in
these 3 patients. The pattern of VLFO was homogenous,
but with variation of time duration and amplitude
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Ren et al: Ictal VLFO in Human
FIGURE 1: Ictal EEG changes during one sGTC with schematic location of electrodes in Patient 1. (A) Conventional ictal
discharges (a TC of 0.1 second, HFF 70Hz) started with low amplitude, followed by fast activities originating from C4, D4, and
H2 (black arrow and bold lines) 6 seconds before clinical onset (white arrow). (B) VLFO (a TC of 10 seconds, HFF 70 Hz, highly
compacted display) developed over C2, D6, and G2 (black dots), followed by gradual evolution in morphology, amplitude,
frequency, and wider distribution. A particularly well-organized periodic pattern developed with an interpeak interval from 40
to 60 seconds over C4, G1, G2, H1, and H2. The maximal amplitude became more than 3mV. The onset of VLFO (black arrow)
preceded clinical seizure onset (white arrow) by 18 minutes 10 seconds. Notably, positive IDS could be detected in the initial
stage of seizure onset on A1, A2, D4, and D5, channels in which VLFO was insignificant. The black triangle marks enhanced
interictal discharges, which were superimposed over VLFO. The open frame of black lines indicates the time window in A.
among seizures in individual patients. No subjective
complaints or objective symptoms were reported during
VLFO. The observation of the interictal discharges, conventional ictal EEG, VLFO, and cortical mapping are
summarized in Supporting Table S1.
VLFO in 3 patients shared common essential features: (1) The occurrence always preceded clinical seizure
onset and conventional ictal EEG onset by a period of
time ranging from 8 minutes 10 seconds to 22 minutes
40 seconds. Gradually, repetitive negative slow baseline
shifts developed from very limited areas involving a few
electrodes. (2) The morphology, amplitude, frequency,
and distribution of VLFO evolved progressively. Generally, VLFO became more regular and periodical. Interpeak intervals of 40–120 seconds and more than 3mV of
maximal amplitudes were detected. (3) The intensive
interictal discharges in Patient 1 and subclinical discharge
in Patient 2 were superimposed on the VLFO waveform
several minutes after VLFO occurrence. (4) The spatial
distributions of VLFO were in the vicinity of, or even
overlapped with the conventional ictal EEG onset and
IDS. (5) Finally, VLFO disappeared or significantly attenuated after cessation of the clinical seizure.
Patients 1 and 2 were not operated on because the
EZ could not be localized clearly and/or the eloquent
January 2011
areas overlapped with the possible EZ. Patient 3 underwent lesionectomy (see Supporting Fig 1C). The lesion
was confirmed as focal cortical dysplasia histologically.
His seizures reduced by about 50%, although the followup period is still less than 1 year.
Discussion
The current study revealed a distinct ictal brain activity
in the range of the infraslow band. Theoretically, if EEG
recording is carried out with direct current (DC) amplification or alternate current (AC) amplification, but with
very long TC, slow potentials can be evaluated.6 Nevertheless, significant artifacts in surface EEG, the routinelyused short TC, or unavailability of a DC amplifier made
it difficult or even impossible to observe DC shift. Intracranial recording, very long TC, and highly compacted
EEG display were necessary to identify VLFO. The
platinum-iridium alloy electrodes are optimal to minimize electrode potentials, which could distort slow
potential signals.7 It is essential to differentiate real activities from artifacts resulting from electrode placement,
patient movements, or other noise in the evaluation of
VLFO. Artifacts are readily detected based on irregularity
or bizarre morphology, long-lasting instability of the
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of Neurology
FIGURE 2: Ictal EEG changes during 1 CPS with schematic location of electrodes in Patient 2. (A) Conventional ictal discharges
(a TC of 0.1 seconds, HFF 70Hz) started with low-amplitude fast activity (black arrow and bold lines) 12 seconds before clinical
onset (white arrow) over the basal temporal and posterior lateral temporal lobes simultaneously. (B) VLFO (a TC of 10
seconds, HFF 70 Hz, highly compacted display) developed at LBP4 and D1 (black dots), evolving into a higher-amplitude and
well- organized periodic pattern with interpeak interval from 90 to 120 seconds. The maximal amplitude became more than
3mV. The onset of VLFO (black arrow) preceded clinical seizure onset (white arrow) by 22 minutes 40 seconds. The black
triangle indicates onset of subclinical discharges, which were superimposed over VLFO. The open frame of black lines indicates
the time window in A.
FIGURE 3: Ictal EEG changes during one sGTC with schematic location of electrodes in Patient 3. (A) Conventional ictal EEG (a
TC of 0.1 seconds, HFF 70Hz) started with low-voltage fast activities regionally 10 seconds prior to clinical onset (white arrow)
in a start-attenuation-restart pattern (black arrow and bold lines). (B) VLFO (a TC of 10 seconds, HFF 70 Hz, highly compacted
display) developed at E3 and E6 (black dots), becoming higher, wider, and periodic, and disappeared with seizure cessation. A
particularly well-organized periodic pattern developed with an interpeak interval from 40 to 60 seconds over B5, E3, and E6.
Clearly, negative IDS could be identified in the initial stage of seizure onset over E5, H7, H8, L1, and L2, channels in which
VLFO was insignificant. VLFO onset (black arrow) preceded clinical seizure onset (white arrow) by 13 minutes 20 seconds. The
open frame of black lines indicates the time window in A. (C) MRI findings demonstrated the thickness of gyrus with mildly
increased signal in the right inferior frontal lobe (white arrow).
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Ren et al: Ictal VLFO in Human
baseline, lack of evolution pattern, or continuation after
the seizures.
Identification of VLFO would expand the brain activity spectrum, and it is different from any other previously known slow activities. The exclusive correlation
between VLFO and clinical seizure distinguish it from
interictal infraslow activity,4 which might represent the
slow, cyclic modulation of cortical gross excitability. Also,
it is easily distinguished from IDS, which occurred with
conventional EEG onset almost simultaneously and is
supposed to result from astrocytic depolarization due to
the increased extracellular potassium released from the vicinity of firing neurons,8 because of its very early appearance, periodic evolving pattern, and the discrepancy in
the distribution.
The observation of VLFO would also broaden
our understanding of epileptogenesis. Traditionally, epileptic physiology was neuron-centric. A 50-msec to
200-msec paroxysmal depolarization shift (PDS) was
considered a hallmark of epilepsy.9 However, epileptogenesis might be heterogeneous. The role of astrocytes
is especially interesting, for they not only can exhibit
excitability in the form of spontaneous calcium oscillation with an interval of up to several minutes,10,11 but
also have a complex interaction with neurons.12,13
Astrocytes are found to express similar ion channels
and receptors to neurons and release gliotransmitters,
including glutamate, to act on neurons, resulting in
synchronizing neuronal activity14,15 or triggering PDSlike events.16 Therefore, it is assumed that astrocytes
might initiate the VLFO, followed by activating neurons and driving epileptiform discharges, finally resulting in clinical seizure.
VLFO may be of clinical significance for identification of reliable biological markers for EZ, since currently
no single technique can point out the EZ perfectly.17
Since VLFO was only identified with seizure, and the
spatial distributions of interictal discharges, conventional
ictal EEG onset, IDS and VLFO were adjacent, or even
overlapped, VLFO has the potential to provide valuable
additional information to determine EZ despite the limited number of patients in this study. Furthermore,
VLFO provides evidence for the dynamic preictal state
that has long been debated. One of the most disabling
aspects of epilepsy is the unpredictability of seizures. If
reliable prediction is possible, epilepsy treatment will
improve dramatically.18 Although many efforts have been
made to extract preictal EEG changes from continuous
EEG since the 1970s,19 more recent studies could not
reproduce previously optimistic findings.20 Hence, the
relatively long time lag between occurrence of VLFO and
January 2011
clinical seizure makes VLFO potentially a potent parameter for predicting seizures.
Acknowledgments
This research was supported by the Japan Epilepsy Society and the Epilepsy Research Foundation of Japan (to
L.R.).
We thank Ms. Miyako Yamaguchi, and Ms. Mariko
Ishikawa for technical assistance.
Potential Conflicts of Interest
Nothing to report.
References
1.
Bragin A, Engel JJ, Wilson CL, et al. High-frequency oscillations in
human brain. Hippocampus 1999;9:137–142.
2.
Bragin A, Engel JJ, Wilson CL, et al. Hippocampal and entorhinal
cortex high-frequency oscillations (100–500 Hz) in human epileptic
brain and in kainic acid-treated rats with chronic seizures. Epilepsia 1999;40:127–137.
3.
Usui N, Terada K, Baba K, et al. Very high frequency oscillations
(over 1000 Hz) in human epilepsy. Clin Neurophysiol (in press).
DOI:10.1016/j.clinph.2010.04.018
4.
Vanhatalo S, Palva JM, Holmes MD, et al. Infraslow oscillations
modulate excitability and interictal epileptic activity in the human
cortex during sleep. Proc Natl Acad Sci USA 2004;101:5053–5057.
5.
Ikeda A, Terada K, Mikuni N, et al. Subdural recording of ictal DC
shifts in neocortical seizures in humans. Epilepsia 1996;37:662–674.
6.
Caspers H. Handbook of electroencephalography and clinical neurophysiology. Vol 10.Amsterdam: Elsevier, 1974:7–11.
7.
Tallgren P, Vanhatalo S, Kaila K, Voipio J. Evaluation of commercially available electrodes and gels for recording of slow EEG
potentials. Clin Neurophysiol 2005;116:799–806.
8.
Dietzel I, Heinemann U, Lux HD. Relations between slow extracellular potential changes, glial potassium buffering, and electrolyte
and cellular volume changes during neuronal hyperactivity in cat
brain. Glia 1989;2:25–44.
9.
Johnston D, Brown TH. Giant synaptic potential hypothesis for
epileptiform activity. Science 1981;211:294–297.
10.
Scemes E, Giaume C. Astrocyte calcium waves: what they are and
what they do. Glia 2006;54:716–725.
11.
Wolf F, Kirchhoff F. Neuroscience: imaging astrocyte activity. Science 2008;320:1597–1599.
12.
Fields RD. Stevens-Graham B. New insights into neuron-glia communication. Science 2002;298:556–562.
13.
Allen NJ, Barres BA. Neuroscience: glia-more than just brain glue.
Nature 2009;457:675–677.
14.
Angulo MC, Kozlov AS, Charpak S, Audinat E. Glutamate released
from glial cells synchronizes neuronal activity in the hippocampus.
J Neurosci 2004;24:6920–6927.
15.
Fellin T, Pascual O, Gobbo S, et al. Neuronal synchrony mediated
by astrocytic glutamate through activation of extrasynaptic NMDA
receptors. Neuron 2004;43:729–743.
16.
Tian GF, Azmi H, Takano T, et al. An astrocytic basis of epilepsy.
Nat Med 2005;11:973–981.
17.
Rosenow F, Lüders HO. Presurgical evaluation of epilepsy. Brain
2001;124:1683–1700.
205
ANNALS
of Neurology
18.
Theodore WH, Fisher RS. Brain stimulation for epilepsy. Lancet
Neurol 2004;3:111–118.
19.
Le Van Quyen M, Martinerie J, Navarro V et al. Anticipation of epileptic seizures from standard EEG recordings. Lancet 2001;357:
183–188.
20.
Mormann F, Andrzejak RG, Elger CE, Lehnertz K. Seizure prediction: the long and winding road. Brain 2007;130:314–333.
Large Genomic Deletions:
A Novel Cause of Ullrich
Congenital Muscular
Dystrophy
A. Reghan Foley, MD,1,2 Ying Hu, MS,1
Yaqun Zou, MD,1 Michele Yang, MD,1,3
Lı̄vija Medne, MS, CGC,1,4
Meganne Leach, MSN, CRNP,1 Laura K. Conlin, PhD,4
Nancy Spinner, PhD,5,6 Tamim H. Shaikh, PhD,6,3
Marni Falk, MD,4,6 Ann M. Neumeyer, MD,7
Laurie Bliss,7 Brian S. Tseng, MD, PhD,7
Thomas L. Winder, PhD, FACMG,8
and Carsten G. Bönnemann, MD1,6,9
Two mutational mechanisms are known to underlie
Ullrich congenital muscular dystrophy (UCMD):
heterozygous dominant negatively-acting mutations
and recessively-acting loss-of-function mutations. We
describe large genomic deletions on chromosome
21q22.3 as a novel type of mutation underlying
recessively inherited UCMD in 2 families. Clinically
unaffected parents carrying large genomic deletions of
COL6A1 and COL6A2 also provide conclusive evidence
that haploinsufficiency for COL6A1 and COL6A2 is not a
disease mechanism for Bethlem myopathy. Our findings
have important implications for the genetic evaluation
of patients with collagen VI–related myopathies as well
as for potential therapeutic interventions for this patient
population.
ANN NEUROL 2011;69:206–211
U
llrich congenital muscular dystrophy (UCMD; MIM
254090), Bethlem myopathy (BM; MIM 158810) and
phenotypes intermediate to UCMD and BM form a group
of congenital muscular dystrophies known as the collagen
VI–related myopathies.1 Underlying these conditions is a
decrease, absence, or dysfunction of the extracellular matrix
protein collagen VI. The collagen VI heterotrimeric monomer is composed of 3 alpha chains: a1(VI), a2(VI), and
a3(VI),2,3 each containing a short triple helical domain
flanked by globular domains. Assembly of collagen VI proceeds intracellularly with monomers aligning in an antiparallel fashion to form dimers, which then align laterally to
206
form tetramers. The tetramers are secreted and align extracellularly in an end-to-end fashion, forming beaded microfilaments as the final product of collagen VI assembly.4–6
Mutations in any of the 3 genes coding for the 3
collagen VI alpha chains, COL6A1, COL6A2, or COL6A3,
can affect the complex assembly and secretion of collagen VI, resulting in the phenotype of a collagen VI–
related myopathy. COL6A1 and COL6A2 are located
on chromosome 21q22.3 (Heiskanen and colleagues7)
and COL6A3 is located on chromosome 2q37 (Weil
and colleagues8). UCMD results from either recessive
or dominantly-acting mutations9,10 and is characterized by a combination of early-onset muscle weakness,
congenital contractures of the proximal joints, and
hyperlaxity of the distal joints.11 BM typically follows
autosomal dominant inheritance; however, autosomal
recessive inheritance has recently been described as
well.12,13 BM is characterized by slowly progressive
muscle weakness and joint contractures.14
Here we describe a novel type of mutation underlying recessively inherited UCMD by delineating large
genomic deletions on chromosome 21q22.3, resulting in
loss of COL6A2 or both COL6A1 and COL6A2, and
occurring in combination with a mutation in COL6A2
or a deletion of COL6A2 on the other allele to cause disease. We also conclusively demonstrate that haploinsufficiency for COL6A1 and COL6A2 is associated with
decreased collagen VI deposition but is not associated
with clinical neuromuscular disease.
Patients and Methods
Clinical details were collected according to a protocol approved
by the institutional review board and are summarized in the
Table 1.
From the 1Division of Neurology, 4Division of Human Genetics, 5Division
of Pathology, The Children’s Hospital of Philadelphia, University of
Pennsylvania, Philadelphia, PA; 2Dubowitz Neuromuscular Centre,
University College London Institute of Child Health and Great Ormond
Street Hospital for Children, London, UK; 3Department of Pediatrics,
University of Colorado Denver, Aurora, CO; 6Department of Pediatrics,
University of Pennsylvania, Philadelphia, PA; 7 Department of
Neurology, Massachusetts General Hospital, Harvard University,
Boston, MA; 8Prevention Genetics, Marshfield, WI; 9Neuromuscular and
Neurogenetic Disorders of Childhood Section, Neurogenetics Branch,
National Institute of Neurological Disorders and Stroke/NIH,
Bethesda, MD.
Address correspondence to Dr Bönnemann, Neuromuscular and
Neurogenetic Disorders of Childhood Section, Neurogenetics Branch,
National Institute of Neurological Disorders and Stroke/NIH, Porter
Neuroscience Research Center, Building 35, Room 2A-116, MSC 35
Convent Drive, Bethesda, MD 20892-3705. E-mail: carsten.bonnemann@
nih.gov
Received Jun 23, 2010, and in revised form Sep 9, 2010. Accepted for
publication Sep 24, 2010.
View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.22283
Volume 69, No. 1
Foley et al: Novel Cause of UCMD
TABLE 1: Clinical Details
Patients
P1 (Ullrich)
P2 (Ullrich)
P3
Sex
Male
Male
Male
Congenital hypotonia
Y
Y
N
Congenital hip dislocation
N
Y
N
Congenital abnormal position of hands and/or feet
Y
Y
N
Congenital torticollis
N
Y
N
Age at evaluation (years)
3.25
3.25
1.17
Weakness
Y
Y
N
Proximal
Y (moderate)
Y (severe)
N
Distal
Y (moderate)
Y (severe)
N
Contractures
Y
Y
N
Elbows
N
N
N
Hips
þþ
þþ
N
Knees
þ
þ
N
Ankles
þ
þ
N
Distal joint hyperlaxity
Y
Y
(Y) (mild)
Scoliosis
N
N
N
Abnormal skin findings
N
N
N
Hypertrophic scars
N
N
N
Keratosis pilaris
N
N
N
Y
N
N
Age achieved
3
—
—
Age lost
—
—
—
Pulmonary compromise
N
Y (severe)
N
CK
1.5 normal
1.4 normal
Normal
Achieved independent ambulation
þ ¼ moderate; þþ ¼ severe; CK ¼ creatine kinase; N ¼ no; Y ¼ yes.
Genomic and Biochemical Analyses
Exonic sequencing of COL6A1, COL6A2, and COL6A3 was
performed at Prevention Genetics by extracting genomic DNA
from patient blood cells, followed by polymerase chain reaction
(PCR) amplification of individual exons and sequencing of
PCR products on an ABI 3130x1 capillary sequencer in the
forward and reverse directions.
Single nucleotide polymorphism (SNP) array analysis was
performed using an Illumina Quad 610 at the Center for
Applied Genomics at the Children’s Hospital of Philadelphia as
per described protocols.15,16
Dermal fibroblasts from skin biopsies obtained from
Patient 1, Patient 2, Patient 2’s mother, and Patient 2’s brother
were cultured, and mutational analysis by reverse transcription
(RT)-PCR and DNA sequencing was completed as described.10
January 2011
Immunostaining of fibroblasts and western blot analysis on
fibroblasts were performed using polyclonal and monoclonal
antibodies as described.10,17
Results
Patient 1 has a phenotype of moderately severe UCMD,
and Patient 2 has a phenotype of severe UCMD (see
Table). The parents of Patients 1 and 2 have no neuromuscular complaints and normal neuromuscular examinations.
The brother of Patient 2 has global developmental delays
and epilepsy of unclear etiology but no symptoms suggestive of a congenital muscular dystrophy. Patient 3 does not
have a phenotype consistent with UCMD or BM but was
evaluated for a clinical picture of global developmental
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FIGURE 1: Mapping of chromosome 21q22.3 showing the extent of the large genomic deletions as found by SNP genomic
array analysis in Patient 1 (P1), Patient 2 (P2), and Patient 3 (P3), as well as the splice mutation on the nondeleted allele in
Patient 1 (P1) as confirmed by cDNA sequencing (see Fig 2). The mapping of genes located within deletions was performed
using the UCSC Genome Browser (http://genome.ucsc.edu) with nucleotide sequence numbers based on the March 2006
human reference sequence (NCBI Build 36.1), as produced by the International Human Genome Sequencing Consortium
(http://genome.ucsc.edu/cgi-bin/hgGateway). (Chromosome 21 ideogram from http://www.genecards.org.)
delays and axial hypotonia. Patient 3’s father had a completely normal neuromuscular examination. Both Patient 3
and his father had muscle ultrasounds performed, which
revealed normal-appearing muscles.
Mutational Analysis
Genomic DNA sequencing revealed that Patient 1 was
heterozygous for an intronic nucleotide change (G>A) at
position c.1970–9 at the intron 25–exon 26 junction of
COL6A2 (Fig 1). On COL6A2 sequencing using complementary DNA (cDNA) extracted from Patient 1’s fibroblasts, we detected a 7-bp insertion resulting from the
use of a novel splice acceptor site in intron 25 created by
the mutation (Fig 2). This insertion causes a frameshift,
resulting in a premature stop codon (G656AfsX17). In
addition, SNP analysis revealed a 69kb genomic deletion
at 21q22.3 encompassing at least the first 18 exons of
the COL6A2 gene. Patient 1’s asymptomatic mother was
found to be heterozygous for the nucleotide change
208
(G>A) at c.1970–9 of COL6A2; his asymptomatic father
was found to be heterozygous for the 69kb deletion.
SNP-based genome array analysis performed on
Patient 2 revealed evidence of 2 deletions at 21q22.3: a
deletion of 1.61Mb encompassing the entire COL6A1
and COL6A2 genes plus surrounding genes on 1 allele
and a smaller deletion of 47kb encompassing the entire
COL6A2 gene on the other allele (see Fig 1). Patient 2’s
asymptomatic mother was found to be heterozygous for
the 1.61Mb deletion; his asymptomatic father was
found to be heterozygous for the 47kb deletion.
Genomic DNA sequencing of Patient 2’s mother and
father did not reveal any mutation in either COL6A1 or
COL6A2 in the nondeleted alleles. Patient 2’s brother
was found to be heterozygous for the 1.61Mb deletion
inclusive of COL6A1 and COL6A2. Dermal fibroblasts
cultured from skin biopsies of Patient 2, his mother,
and his brother were stained for collagen VI and
revealed a complete absence of collagen VI
Volume 69, No. 1
Foley et al: Novel Cause of UCMD
immunoreactivity in Patient 2’s fibroblasts and evidence
of a slightly reduced collagen VI matrix intensity in the
fibroblasts of Patient 2’s mother and brother (see Fig 2).
(Patient 2’s mother is clinically asymptomatic; his
brother has a history of global developmental delays
and epilepsy of unclear etiology.)
SNP-based genomic array analysis performed on
Patient 3 for developmental delays revealed a heterozygous
1.09Mb deletion encompassing the entire COL6A1 and
the COL6A2 genes as well as adjacent genes (see Fig 1).
The same deletion was detected in Patient 3’s asymptomatic father (see Fig 2).
FIGURE 2.
January 2011
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Discussion
Two principal mutational mechanisms are known to underlie classic Ullrich congenital muscular dystrophy: heterozygous
dominant negatively-acting mutations and recessively-acting
loss-of-function mutations. Recessive null mutations have
included nonsense mutations as well as exon-skipping mutations precluding assembly. Small heterozygous in-frame intragenic deletions in the COL6A1 gene exerting a dominant
negative effect have been described.10,18 The large genomic
deletions on chromosome 21 described here involving
COL6A2 or both COL6A1 and COL6A2 have not yet been
described and establish a novel type of mutation in the collagen VI–related myopathies. This finding also adds to the
complexity of genetic evaluations in the collagen VI–related
myopathies, as this type of mutation will not be detected by
single-exon amplification and sequencing (unless done quantitatively). In addition, a hemizygous change detected on the
nondeleted allele will seemingly appear homozygous, potentially obscuring the true genetic causation of the patient’s
disease.
Large genomic deletions in recessive disorders are
only pathogenic if they unmask a pathogenic mutation
on the second nondeleted allele. In severe UCMD patients
the mutation on the nondeleted allele will most likely be
another loss-of-function mutation, although the precise nature of the second mutation will influence the severity of
disease. It is notable that Patient 1 has acquired ambulation,
whereas Patient 2, with a complete deletion of both
COL6A2 and COL6A1 on the other allele, has not. Patient
1’s second mutation is an intronic mutation leading to an
3
erroneous splice acceptor, adding a frameshifting 7bp to the
transcript. Based on RT-PCR evidence in Patient 1’s fibroblasts (see Fig 2), there also appears to be some normally
spliced transcript from this allele, which likely is responsible
for ameliorating Patient 1’s phenotype to a degree. We and
others recently reported patients with classical Bethlem myopathy who were compound heterozygous for a functional
null mutation and a missense mutation in the COL6A2
gene.12,13 Thus, large genomic deletions of the COL6A1 and
COL6A2 loci in compound heterozygosity with a milder missense mutation would be predicted to result in a phenotype
consistent with BM and, therefore, would have to be considered in that patient population also.
The increased use of chromosomal microarray platforms including those based on SNPs leads to an increasing catalog of genomic deletion and duplication variants
of unknown significance (http://projects.tcag.ca/variation).
It remains to be seen whether deletions within and
around the COL6A1 and COL6A2 loci will be identified
more often in asymptomatic carriers as a copy number
variation (CNV), which would be benign but confer
carrier status for a collagen VI–related myopathy. It is to
be anticipated that additional deletions encompassing neuromuscular disease loci will be found with more widespread use of this molecular technology, and vice versa,
that chromosomal microarray platforms have a place in the
diagnostic repertoire for neuromuscular disorders.
Our observation that the carrier status in the
parents—for the large deletions found in Patients 1, 2,
and 3—are clinically asymptomatic is the most
FIGURE 2: (A) Immunocytochemistry for collagen VI on cultured dermal fibroblasts derived from (a) control, (b) Patient 1,
(c) Patient 2, (d) Patient 2’s brother, and (e) Patient 2’s mother, probed with a monoclonal anti-collagen VI antibody, to
demonstrate the effect of the mutations on collagen VI matrix deposition. There is decreased collagen VI expression in the
matrix of cells from (b) Patient 1, a complete absence of collagen VI expression in the cells of (c) Patient 2, and only slightly
decreased collagen VI expression in the matrix of cells from (d) Patient 2’s brother and (e) mother. This slight decrease is best
seen early after confluency is reached (shown here at 3 days after confluency) and less so as the matrix accumulates in longer
culture. Insets show DAPI nuclear staining to demonstrate equal cell density. Images were taken at 320 magnification. Bar 5
100lm. (B) Western blot for collagen VI a2 chain on fibroblast cell extracts to show effect of the mutations on cellular a2(VI)
synthesis. There is complete absence of a2(VI) in Patient 2. Patient 1, Patient 2’s mother, and Patient 2’s brother show
reduced amounts of the collagen VI a2 chain, although the quantification can only be viewed as approximate. Relative
quantification of a2(VI) performed by densitometry (ImageJ software; NIH), normalized against tubulin and calibrated with a
control sample. (C) Gel electrophoresis of RT-PCR product from RNA isolated from Patient 1’s fibroblasts amplified with
primers flanking exons 25 and 26 in COL6A2 demonstrating 2 amplification products, indicating an incomplete use of the
alternate splice site: a larger band corresponding to the 7bp insertion caused by the use of an alternate slice site, resulting in
a frameshift and premature stop codon and possibly leading to some nonsense-mediated decay and a smaller band
corresponding to normal use of the intron 25–exon26 splice site. This normal band could possibly also include an aberrantly
initiated but correctly spliced transcript from the deleted allele, as exons 25 and 26 are not included in the deletion.
Sequencing of the combined RT-PCR product demonstrates coamplification of both the normal sequence and of the transcript
with the 7bp insertion. (D) Clinical photographs of the father of Patient 3 who also carries the 1.09Mb deletion inclusive of
COL6A1 and COL6A2. There is no evidence of (f) long finger flexor contractures or (g) distal hyperlaxity as typically seen in
collagen VI–related myopathy patients and as would be expected if haploinsufficiency for COL6A1 and COL6A2 were a
mechanism for Bethlem myopathy. Neuromuscular examination revealed that he has normal strength. In this case SNP array
analysis was used as a diagnostic tool for nonspecific developmental delays in the child (Patient 3), and the 1.09Mb deletion
identified in the child was also identified in the father, where it was found to be of no clinical consequence.
210
Volume 69, No. 1
Foley et al: Novel Cause of UCMD
conclusive evidence yet that haploinsufficiency for collagen type VI is not a disease mechanism for Bethlem
myopathy, even though it is associated with a reduction
in the deposited collagen VI matrix evidenced by Patient
2’s mother’s cells in culture (see Fig 2). These findings
suggest that the COL6A1 mutation reported in the literature as causing haploinsufficiency as a mechanism for
BM must in fact have more complex consequences.19
This observation is also of great translational importance,
as therapeutic strategies directed at the elimination of a
dominant negatively-acting mutation are conceivable and
would create a functional state of haploinsufficiency,
which, as demonstrated here, would not be associated
with clinical manifestations of neuromuscular disease.
3.
Timpl R, Engel J. Type VI collagen. In: Mayne R, Burgeson R, eds.
Structure and function of collagen types. Orlando: Academic
Press, 1987:105–143.
4.
Chu ML, Conway D, Pan TC, et al. Amino acid sequence of the
triple-helical domain of human collagen type VI. J Biol Chem
1988;263:18601–18606.
5.
Chu ML, Pan TC, Conway D, et al. Sequence analysis of alpha
1(VI) and alpha 2(VI) chains of human type VI collagen reveals
internal triplication of globular domains similar to the A
domains of von Willebrand factor and two alpha 2(VI) chain variants that differ in the carboxy terminus. EMBO J 1989;8:
1939–1946.
6.
Chu ML, Zhang RZ, Pan TC, et al. Mosaic structure of globular
domains in the human type VI collagen alpha 3 chain: similarity to
von Willebrand factor, fibronectin, actin, salivary proteins and
aprotinin type protease inhibitors. EMBO J 1990;9:385–393.
7.
Heiskanen M, Saitta B, Palotie A, Chu ML. Head to tail organization of the human COL6A1 and COL6A2 genes by fiber-FISH.
Genomics 1995;29:801–803.
8.
Weil D, Mattei MG, Passage E, et al. Cloning and chromosomal
localization of human genes encoding the three chains of type VI
collagen. Am J Hum Genet 1988;42:435–445.
9.
Camacho Vanegas O, Bertini E, Zhang RZ, et al. Ullrich scleroatonic muscular dystrophy is caused by recessive mutations
in collagen type VI. Proc Natl Acad Sci USA 2001;98:
7516–7521.
10.
Pan TC, Zhang RZ, Sudano DG, et al. New molecular mechanism
for Ullrich congenital muscular dystrophy: a heterozygous in-frame
deletion in the COL6A1 gene causes a severe phenotype. Am J
Hum Genet 2003;73:355–369.
11.
Ullrich O. Congenital atonic-sclerotic muscular dystrophy: an
additional type of heredodegenerative disease of the neuromuscular system. Z Ges Neurol Psychiat 1930;126:171–201.
12.
Foley AR, Hu Y, Zou Y, et al. Autosomal recessive inheritance of
classic Bethlem myopathy. Neuromuscul Disord 2009;19:813–817.
13.
Gualandi F, Urciuolo A, Martoni E, et al. Autosomal recessive
Bethlem myopathy. Neurology 2009;73:1883–1891.
14.
Bethlem J, van Wijnaarden GK. Benign myopathy, with autosomal
dominant inheritance: a report on three pedigrees. Brain 1976;99:
91–100.
15.
Gunderson KL, Steemers FJ, Lee G, et al. A genome-wide scalable SNP genotyping assay using microarray technology. Nat
Genet 2005;37:549–554.
16.
Shaikh TH, Gai X, Perin JC, et al. High-resolution mapping and
analysis of copy number variations in the human genome: a data
resource for clinical and research applications. Genome Res 2009;
19:1682–1690.
17.
Pepe G, Bertini E, Giusti B, et al. A novel de novo mutation in the
triple helix of the COL6A3 gene in a two-generation Italian family
affected by Bethlem myopathy. A diagnostic approach in the
mutations’ screening of type VI collagen. Neuromuscul Disord
1999;9:264–271.
18.
Pepe G, Lucarini L, Zhang RZ, et al. COL6A1 genomic deletions in
Bethlem myopathy and Ullrich muscular dystrophy. Ann Neurol
2006;59:190–195.
19.
Lamande SR, Bateman JF, Hutchison W, et al. Reduced collagen
VI causes Bethlem myopathy: a heterozygous COL6A1 nonsense
mutation results in mRNA decay and functional haploinsufficiency.
Hum Mol Genet 1998;7:981–989.
Acknowledgments
This research was supported by grants from NIH/
NIAMS (R01AR051999 to C.G.B.) and from MDA USA
(MDA3896 to C.G.B.); NIH/NIGMS grant (GM081519
to T.H.S.).
We thank the patients and their families for their
participation.
A.R.F. is a MDC (UK) clinical research fellow
(MC3/1057/2).
Potential Conflicts of Interest
A.R.F., A.M.N., B.S.T., L.B., L.K.C., M.F., M.L., N.S.,
T.L.W., Y.H., Y.Z., L.M., and T.H.S. have none to report.
C.G.B. has received honoraria from Datamonitor Group,
London, for a telephone survey on therapeutic management in
CMD; has had travel/accommodations expenses covered or
reimbursed by Cure CMD, a not-for-profit patient advocacy
group, for participation in meetings; and is chair of the scientific
and medical advisory board of Cure CMD, from which he does
not draw any honoraria. M.Y. has had travel/accommodations
expenses covered or reimbursed by the Medical College of
Georgia for a Child Neurology Society Meeting.
References
1.
Bertini E, Pepe G. Collagen type VI and related disorders: Bethlem myopathy and Ullrich scleroatonic muscular dystrophy. Eur J
Paediatr Neurol 2002;6:193–198.
2.
Timpl R, Chu ML. Microfibrillar collagen type VI. In: Mecham RP,
ed. Extracellular matrix assembly and structure. Orlando: Academic
Press, 1994:207–242.
January 2011
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