BRIEF COMMUNICATIONS
of MS, experimental autoimmune encephalomyelitis
Interferon-g Secretion by
Peripheral Blood T-Cell
Subsets in Multiple Sclerosis:
Correlation with Disease
Phase and Interferon-b
Therapy
Burkhard Becher, PhD, Paul S. Giacomini, BSc,
Daniel Pelletier, MD, Ellie McCrea, BSc,
Alexandre Prat, MD, and Jack P. Antel, MD
Interferon-g (IFN-g) is implicated as a participant in the
immune effector and regulatory mechanisms considered
to mediate the pathogenesis of multiple sclerosis (MS).
We have used an intracellular cytokine staining tech-
nique to demonstrate that the proportion of ex vivo pe-
ripheral blood CD4 and CD8 T-cell subsets expressing
IFN-g is increased in secondary progressing (SP) MS
patients, whereas the values in untreated relapsing-
remitting (RR) MS patients are reduced compared with
those of controls. Patients treated with interferon-b
(IFN-b) have an even more significant reduction in the
percentage of IFN-g–secreting cells. The finding that the
number of IFN-g–expressing CD8 cells is increased in
SPMS patients, a group with reduced functional suppres-
sor activity, and is most significantly reduced by IFN-b
therapy, which increases suppressor activity, indicates
that IFN-g secretion by CD8 T cells and functional sup-
pressor defects attributed to this cell subset in MS can be
dissociated.
Becher B, Giacomini PS, Pelletier D, McCrea E,
Prat A, Antel JP. Interferon-g secretion by
peripheral blood T-cell subsets in multiple
sclerosis: correlation with disease phase
and interferon-b therapy.
Ann Neurol 1999;45:247–250
Multiple sclerosis (MS) is considered to be an
immune-mediated disorder that results in multifocal
sites of demyelination within the central nervous sys-
tem. The disease most frequently manifests an initial
relapsing-remitting (RR) course which subsequently
evolves into a progressive phase. In the animal model
From the Neuroimmunology Unit, Montreal Neurological Institute,
McGill University, Montreal, Quebec, Canada.
Received Jul 6, 1998, and in revised form Sep 1. Accepted for pub-
lication Sep 1, 1998.
Address correspondence to Dr Becher, Neuroimmunology Unit,
Montreal Neurological Institute, McGill University, 3801 Univer-
sity Street, Montreal, Quebec H3A 2B4, Canada.
Copyright © 1999 by the American Neurological Association
(EAE), interferon-g (IFN-g)–producing Th1 myelin-
reactive CD4 T cells are used to adoptively transfer the
disease, whereas Th2 cells are not encephalitogenic.1,2
IFN-g has been directly implicated as participating in
the MS disease process based on the observation that
when given to MS patients, systemic IFN-g increases
the frequency of clinical relapse.3 In EAE, however,
disease severity can be increased either by systemic ad-
ministration of antagonistic IFN-g antibodies or by de-
letion of the genes encoding IFN-g.4,5 These results
suggest that IFN-g can also have a protective role in
autoimmune disease. Th1 cells are now recognized to
have relevant autoimmunity properties other than cy-
tokine (IFN-g) production, including those related to
chemokine responses and migration.6,7 Thus, whether
the disease-inducing capacity of Th1 CD4 T cells is
directly related to IFN-g production remains to be
resolved.
IFN-g can be produced by other lymphoid cells, in-
cluding CD8 T cells and natural killer cells.8,9 As re-
gards CD8 T cells, Balashov and colleagues10 reported
reduced production of IFN-g by CD8 T cells of pro-
gressive MS patients in an autologous mixed lympho-
cyte reaction assay system and correlated this finding
with reduced suppressor function. Suppressor function
mediated by activated mononuclear cells has also been
found to be reduced in MS.11,16 We subsequently ob-
served, however, that this functional defect could be
detected using CD8 T-cell lines from MS patients.12
Interferon-b (IFN-b), the initial approved therapy for
MS, augments mitogen-induced suppressor function.13
The purpose of the current study was to combine
intracellular cytokine staining with T-cell subset immu-
nostaining to evaluate expression of IFN-g in both
CD4 T cells and CD8 T cells derived from peripheral
blood of untreated MS patients with either RR or pro-
gressive forms of disease or from MS patients treated
with IFN-b.
Patients and Methods
Patients
MS patients were selected on the basis of having secondary
progressing (SP) disease (10 women, 1 man; mean age, 47 6
9 years; mean disease duration, 11.0 6 2.4 years; mean
EDSS, 5.5 6 0.4) or RR disease. The latter were further
divided into those who were untreated (11 women, 1 man;
mean age, 40 6 4 years; mean disease duration, 10 6 4
years; mean EDSS, 2.5 6 0.3) or were receiving IFNb-1b (4
women, 1 man; mean age, 41 6 4 years; mean disease du-
ration, 7 6 4 years; mean EDSS, 2.7 6 0.8). All had con-
firmatory magnetic resonance imaging abnormalities consis-
tent with MS. None of the patients were having acute
relapses, and none had received corticosteroids in the last 3
months. The mean age of subjects in the healthy control
group was 29 years.
247
Methods
Peripheral blood-derived mononuclear cells (PBMCs) were
treated for 4 hours with PMA (25 ng/ml), ionomycin (1 mg/
ml), and Brefeldin A (10 mg/ml) (all from Sigma, Missis-
sauga, Ontario, Canada). The cells were then extensively
washed with phosphate-buffered saline and immunostained
for 30 minutes with either anti-CD8 monoclonal antibody
(mAb) conjugated to phycoerithrine (PE) or IgG1-IgG2a iso-
type control conjugated to fluorosein-isothyocyanate (FITC)
and PE, respectively (Becton Dickinson, Mississauga, On-
tario, Canada). The cells were washed and fixed overnight in
4% PFA. The cell membrane was permeabilized with HBSS
buffer containing 0.2% saponin (Sigma) and 2% fetal calf
serum and was stained for 30 minutes with anti–IFN-g
mAbs conjugated to FITC (Becton Dickinson). Cells were
analyzed using a FacsScan and Lysis II software (Becton
Dickinson). The data were analyzed using WinMDI software
(Scripps, La Jolla, CA). Statistical analysis comparing the dif-
ferent MS groups with controls was by single-factor ANOVA
and Dunnet posttest (PrismII).
Results
Expression of IFN-g–Secreting T Cells in MS
Patient-Derived Lymphocytes
In initial experiments using ex vivo PBMCs, we found
that virtually all cells gated as lymphocytes were either
CD8 or CD4 cells but only rarely were natural killer
cells (CD561, CD161). Upon activation with PMA
and ionomycin, the levels of CD4 expression de-
creased, limiting the capacity to perform dual-color
Fig 1. Interferon-g (IFN-g) production by CD8 T cells from
multiple sclerosis patients. Representative cytofluorometric anal-
ysis of peripheral blood mononuclear cells that were activated
for 4 hours in the presence of PMA (25 ng/ml), ionomycin
(1 mg/ml), and Brefeldin A (10 mg/ml) and then were
stained for CD8 (ordinate) and IFN-g (abscissa).
248 Annals of Neurology Vol 45 No 2 February 1999
flow cytometry using anti-CD4 mAbs. Figure 1 shows
a representative FACS result of PBMCs derived from
an SPMS patient when stimulated for 4 hours and
stained for CD8 and IFN-g. We further documented
that the intracellular cytokine staining technique could
detect shifts in T-cell cytokine polarization by showing
that the proportion of IFN-g–secreting lymphocytes
was significantly increased when cultured for 18 hours
with anti-CD3 mAbs in the presence of recombinant
interleukin-12 (IL-12) or reduced if cultured with
anti–IL-12 mAbs plus IL-4 (data not shown).
Figure 2 compares the MS patient subgroups and
controls with regard to the percentage of IFN-g–
secreting cells in the overall lymphocyte population.
Values were increased in the untreated SPMS group
compared with the control group and even more so
compared with the RRMS group. The values in the
RRMS group were reduced compared with the con-
trols. The SPMS group has a wider range of values
than the other groups. The mean percentage of
IFN-g1 cells was significantly lower in the IFN-b–
treated group compared with all other groups.
When results are analyzed in terms of T-cell subsets,
there were no significant differences between groups
with regard to CD4:CD8 ratios (data not shown). As
shown in Figure 3, the mean percentage of CD4 T
cells expressing IFN-g is significantly less than in the
CD8 population ( p , 0.05). The proportion of CD4
T cells expressing IFN-g was significantly increased in
the SPMS patients compared with the RRMS patients
( p , 0.05). For CD8 T cells, the percentage of IFN-
g–expressing cells was significantly increased in the
Fig 2. Comparison of percentage of interferon-g (IFN-g)–
secreting T cells from multiple sclerosis (MS) patient subgroups
and controls. Cytofluorometric analysis of IFN-g secreting
within the total lymphocyte population derived from the fol-
lowing donor groups: ctrl 5 control, RR 5 nontreated
relapsing-remitting (RR) MS, SP 5 secondary progressive MS,
IFN-b 5 RRMS treated with interferon-b. Data indicate
results from individual donors plus the mean 6 SEM for each
subgroup. # p , 0.05, *p , 0.01 (compared with control
values).

Fig 3. Comparison of percentage of interferon-g (IFN-g)–
secreting CD8 and CD4 T cells within each subset. Cytoflu-
orometric analysis of IFN-g–secreting CD8 and CD4 T cells
derived from the following donor groups: ctrl 5 control,
RR 5 nontreated relapsing-remitting (RR) MS, SP 5
secondary progressive MS, IFN-b 5 RRMS treated with
interferon-b. Patients are the same as in Figure 2. Data are
presented as mean 6 SEM percentage of either CD8 or CD4
IFN-g–secreting T cells. # p , 0.05, *p , 0.01 (compared
with control values).
SPMS patients compared with the control group ( p ,
0.01), whereas values in the RRMS group were de-
creased compared with the control group ( p , 0.05).
In the IFN-b–treated group, the percentage of IFN-g1
cells was significantly reduced ( p , 0.01) in both
T-cell subsets compared with both untreated patients
and controls.
Discussion
In this study, we have used intracellular cytokine stain-
ing to assess IFN-g cytokine secretion by CD4 and
CD8 T-cell subsets contained in peripheral blood of
MS patients and have correlated the results with clini-
cal disease phenotype and effects of IFN-b therapy.
The intracellular cytokine staining technique used in
this study assessed the original ex vivo population in
that no significant cell proliferation is required and is
well suited to identify the cell source of the cytokine.
Our data indicating significant differences in IFN-g
expression in lymphocyte populations derived from MS
patients in the SP compared with the RR phase of the
disease could reflect on the potential contribution of
this cytokine to the susceptibility and course of MS.
The increased proportion of IFN-g–expressing lym-
phocytes detected by intracellular cytokine staining in
SPMS patients is consistent with reports of increased
IFN-g production by mitogen, and anti-CD3 antibod-
ies stimulated PBMCs in vitro in this population.14,15
The increases could reflect the effects of the Th1 po-
larizing cytokine IL-12, whose production by mono-
Brief Communication: Becher et al: Altered IFN-g Secretion in MS
cytes is increased in this patient group.15 Whether the
increased production of IFN-g underlies the transition
of MS from the RR phase to the progressive phase re-
mains speculative. The wider range of values found in
the SPMS group may reflect a heterogeneity of disease
within this group.
Our findings regarding reduced IFN-g expression in
RRMS patients compared with healthy controls are in
accordance with previous studies using short-term acti-
vation of MS PBMCs.16 A number of studies have
now reported reduced IFN-g in short-term cultures of
mitogen or anti-CD3–activated mononuclear cells de-
rived from RRMS patients.17,18 Whether the reduced
IFN-g levels in the RRMS patients reflect a suscepti-
bility factor analogous to the effect of depleting sys-
temic IFN-g in the EAE model remains unanswered.4,5
Among its multiple actions, IFN-g has been impli-
cated as being the molecular mediator of CD8 T-cell
functional suppressor activity.10 As regards SPMS
patients, functional suppressor defects have been demon-
strated using multiple assay systems.10,12 Our current re-
sults of increased IFN-g production by CD8 T cells
from such patients would not suggest a correlation be-
tween these suppressor functions and IFN-g production.
Our observation that IFN-b therapy downregulates
IFN-g expression in both CD4 and CD8 T cells is
consistent with that of Crucian and co-workers,19 who
applied a similar intracellular cytokine staining proto-
col to unfractionated PBMCs derived from IFN-b–
treated MS patients.19 Similar results have been found
in in vitro assays dependent on cell proliferation.20 –22
Our finding that IFN-b reduced IFN-g expression in
CD8 T cells together with the report that IFN-b ther-
apy augmented mitogen-induced suppressor activity13
would further support the postulate that defects in
IFN-g production by CD8 T cells and suppressor
function are not directly linked. The combined intra-
cellular cytokine/cell subset detection assay can be ex-
tended to analysis of multiple cell subsets and cytokines
so as to permitmore detailed analysis of the apparently
complex immunoregulatory networks that participate
in the MS disease process.
Dr Becher has a fellowship from the German Academic Exchange
Service (DAAD-HSPIII). This work was supported by a grant from
the Multiple Sclerosis Society of Canada and the MRC.
References
1. Ando DG, Clayton J, Kono D, et al. Encephalitic T cells in the
B10.PL model of experimental allergic encephalomyelitis (EAE)
are of the Th-1 lymphokine subtype. Cell Immunol 1989;124:
132–143
2. Racke MK, Bonomo A, Scott DE, et al. Cytokine-induced im-
mune deviation as a therapy for inflammatory autoimmune dis-
ease. J Exp Med 1994;180:1961–1966
3. Panitch HS, Hirsch RL, Schindler J, Johnson KP. Treatment of
multiple sclerosis with gamma interferon: exacerbations associ-
249
 ated with activation of the immune system. Neurology 1987;
37:1097–1102
4. Heremans H, Dillen C, Groenen M, et al. Chronic relapsing
experimental autoimmune encephalomyelitis (CREAE) in mice:
enhancement by monoclonal antibodies against interferon-
gamma. Eur J Immunol 1996;26:2393–2398
5. Krakowski M, Owens T. Interferon-gamma confers resistance
to experimental autoimmune encephalomyelitis. Eur J Immunol
1996;26:1641–1646
6. Austrup F, Vestweber D, Borges E, et al. P- and E-selectin me-
diate recruitment of T-helper-1 but not T-helper-2 cells into
7.
inflamed tissues. Nature 1997;385:81– 83
Bonecchi R, Bianchi G, Bordignon PP, et al. Fifferential ex-
pression of chemokine receptors and chemotactic responsiveness
of type 1 T helper cells (Th1s) and Th2s. J Exp Med 1998;
187:129 –134
8. Zhang B, Yamamura T, Kondo T, et al. Regulation of experi-
mental autoimmune encephalomyelitis by natural killer (NK)
cells. J Exp Med 1997;186:1677–1687
9. Koh DR, Fung-Leung WP, Ho A, et al. Less mortality but
more relapses in experimental autoimmune encephalomyelitis in
CD81/2 mice. Science 1992;256:1210 –1213
10. Balashov KE, Khoury SJ, Hafler DA, Weiner HL. Inhibition of
T cell responses by activated human CD81 T cells is mediated
by interferon-gamma and is defective in chronic progressive
multiple sclerosis. J Clin Invest 1995;95:2711–2719
11. Bania MB, Antel JP, Reder AT, et al. Suppressor and cytolytic
cell function in multiple sclerosis: effects of cyclosporin A and
interleukin-2. J Clin Invest 1986;78:582–586
12. Antel JP, Bania MB, Reder A, Cashman N. Activated suppres-
sor cell dysfunction in progressive multiple sclerosis. J Immunol
1986;137:137–141
13. Noronha A, Toscas A, Jensen MA. Interferon beta augments
suppressor cell function in multiple sclerosis. Ann Neurol 1990;
27:207–210
14. Noronha A, Toscas A, Jensen MA. Interferon beta decreases T
cell activation and interferon gamma production in multiple
sclerosis. J Neuroimmunol 1993;46:145–153
15. Balashov KE, Smith DR, Khoury SJ, et al. Increased interleukin
12 production in progressive multiple sclerosis: induction by
activated CD41 T cells via CD40 ligand. Proc Natl Acad Sci
USA 1997;94:599 – 603
16. Neighbour PA, Miller AE, Bloom BR. Absence of virus-
induced lymphocyte suppression and interferon production in
multiple sclerosis. Neurology 1981;31:561–566
17. Crucian B, Dunne P, Friedman H, et al. Alterations in periph-
eral blood mononuclear cell cytokine production in response to
phytohemagglutinin in multiple sclerosis patients. Clin Diagn
Lab Immunol 1995;2:766 –769
18. Brod SA, Khan M, Bright J, et al. Decreased CD3-mediated
interferon-gamma production in relapsing-remitting multiple
sclerosis. Ann Neurol 1995;37:546 –549
19. Crucian B, Dunne P, Friedman H, et al. Detection of altered T
helper 1 and T helper 2 cytokine production by peripheral blood
mononuclear cells in patients with multiple sclerosis utilizing in-
tracellular cytokine detection bu flow cytometry and surface
marker analysis. Clin Diagn Lab Immunol 1996;3:411– 416
20. Arnason BG. Interferon beta in multiple sclerosis. Clin Immu-
nol Immunopathol 1996;81:1–11
21. Petereit HF, Bamborschke S, Esse AD, Heiss WD. Interferon
gamma–producing blood lymphocytes are decreased by inter-
feron beta therapy in patients with multiple sclerosis. Mult
Scler 1997;3:180 –183
22. Bongioanni MR, Durelli L, Ferrero B, et al. Systemic high-dose
recombinant alpha-2a-interferon therapy modulates lymphokine
production in multiple sclerosis. J Neurol Sci 1996;143:91–99
250 Copyright © 1999 by the American Neurological Association
An Association between
Autosomal Dominant
Cerebral Cavernomas and a
Distinctive Hyperkeratotic
Cutaneous Vascular
Malformation in 4 Families
Pierre Labauge, MD,* Odile Enjolras, MD,†
Jean-Jacques Bonerandi, MD,‡ Sophie Laberge, MD,*
Michel Dandurand, MD,§ Jean-Marie Joujoux, MD,i
and Elisabeth Tournier-Lasserve, MD*¶
Cerebral cavernomas (CCMs) are vascular malformations
that may be inherited as an autosomal dominant condi-
tion for which a gene, CCM1, was mapped to chromo-
some 7. Poorly defined cutaneous malformations were
sometimes described in association with CCMs. During a
national survey, 57 French CCM families were studied.
Co-occurrence of CCMs and a distinctive cutaneous vas-
cular malformation was observed in 4 families. Ten in-
dividuals belonging to these families showed similar hy-
perkeratotic cutaneous capillary venous malformations
(HCCVMs). In 3 families, the histology showed ortho-
keratosis and hyperkeratosis as well as dilated capillaries in
the dermis extending to the hypodermis and confirmed the
diagnosis of HCCVM. Genetic analysis strongly supports
linkage of these families to the CCM1 locus on chromo-
some 7. The HCCVM seems to be a peculiar cutaneous
vascular malformation associated with CCMs. These data
strongly suggest that HCCVMs and CCMs in these fami-
lies are due to the same genetic abnormality.
Labauge P, Enjolras O, Bonerandi J-J, Laberge S,
Dandurand M, Joujoux J-M, Tournier-Lasserve E.
An association between autosomal dominant cerebral
cavernomas and a distinctive hyperkeratotic
cutaneous vascular malformation in 4 families.
Ann Neurol 1999;45:250–254
Cavernomas are vascular malformations mainly located
in the central nervous system. They are characterized
by abnormally enlarged capillary cavities without inter-
From *INSERM U25 Faculté de Médecine Necker, Paris, †Consul-
tation des Angiomes, Hôpital Lariboisière, Paris, ‡Service de Der-
matologie, CHU Timone, Marseille, and §Service de Dermatolo-
gie and iService d’Anatomopathologie, CHU Caremeau, Nîmes,
¶Hôpital Lariboisière, Paris, France.
Received Jul 16, 1998, and in revised form Sep 14. Accepted for
publication Sep 16, 1998.
Address correspondence to Dr Tournier-Lasserve, INSERM U25
Faculté de Médicine Necker, 156 Rue de Vaugirard, 75730 Paris
Cedex 15, France.
Fig 1. Pedigree structure of the 4 families. Affected individuals are represented by filled symbols, unaffected individuals by empty
symbols, and individuals having an unknown status by symbols with a question mark. Individuals designated by an asterisk bear a
hyperkeratotic cutaneous capillary venous malformation (HCCVM). Alleles of the polymorphic markers spanning the CCM1 interval
on chromosome 7 (D7S2410, D7S2409, D7S1813, D7S1789, M65B, D7S646, D7S558, D7S689) that segregate within the
families are shown for each individual. Filled boxes indicate the affected haplotypes cosegregating with the disease. Empty boxes in-
dicate haplotypes unlinked to the disease. All cerebral cavernoma (CCM) and HCCVM patients share the affected haplotype segre-
gating with the cavernous angioma phenotype in their family. Two unaffected individuals (I-15, III-4) were carrying the affected
haplotype, which is most likely a consequence of the established incomplete penetrance of CCMs.

vening brain parenchyma.1 Large autopsy and mag-
netic resonance imaging (MRI) series evaluated their
frequency at 0.5% in the general population.2 They
may be inherited as an autosomal dominant condition
designated as familial cerebral cavernomas (CCMs).
CCMs have been studied extensively in the Hispanic
population in the United States, in which more than
50% of cavernomas are familial.3 An as yet unidenti-
fied gene mapping on chromosome 7, CCM1 4, has
been shown to be involved in all CCM families be-
longing to this ethnic group.5,6 Few non-Hispanic
families have been studied so far, with some of them
being linked to chromosome 7 and some unlinked,
demonstrating the implication of at least one other
gene.7 In some rare sporadic and familial cases of
CCM, angiomatosis may affect other organs such as
retina and/or skin.8
During a recent survey of 57 French familial cerebral
cavernomas families, including 173 CCM patients,9 we
identified 10 cases of localized peculiar cutaneous hy-
perkeratotic vascular malformations segregating in 4
families, including at least 2 cases of these cutaneous
vascular lesions in each family. Cerebral MRI per-
formed in 8 of the patients with skin lesions showed
CCMs in all cases. The cosegregation of these types of
cutaneous and neurological vascular malformations has
not been reported before. It is unlikely to have oc-
curred by chance, as these distinctive cutaneous lesions
are uncommon and, to our knowledge, not familial.
Genetic linkage analysis with chromosome 7 markers

Brief Communication: Labauge et al: Association between CCMs and HCCVMs 251
 tologist (O.E.), and pathological samples were examined by a
pathologist (J-J.B.). Eight polymorphic microsatellite markers
spanning the interval containing the CCM1 locus were se-
lected for linkage analysis.9 Two-point linkage analysis was
performed (data not shown but available on request).

Fig 2. Axial T2-weighted magnetic resonance image of the
brain of an affected patient (Patient II-4). Mixed hypersignals
and hyposignals surrounded by a rim of hyposignal located
in the right external capsule are strongly suggestive of a
cavernoma.
spanning the CCM1 interval strongly suggests that all 4
families are linked to chromosome 7.
Materials and Methods
A total of 57 French CCM families were studied during a
survey conducted throughout all 28 French neurosurgery
centers from November 1996 to June 1997.9 The “affected”
diagnosis was based on the presence of typical histological or
MRI-detected lesions (n 5 173 subjects). The “unaffected”
diagnosis was retained based on the normality of MRI (n 5
89). In addition to detailed neurological and neuroimaging
investigations, specific inquiries on skin lesions were con-
ducted. All skin lesions were examined by a certified derma-
Results
Of the overall 57 identified families, 4 were character-
ized by the association of CCMs and cutaneous lesions
(Fig 1). Within these 4 families, 8 of 21 individuals
who had CCMs detected by MRI (Fig 2) also had cu-
taneous lesions (Table). Two additional family mem-
bers (Patients I-23 and IV-9) in whom MRI was not
performed because of young age (Patient I-23) and re-
fusal (Patient IV-9), respectively, had cutaneous
changes. These lesions were congenital, single, and lo-
calized on the lower limbs in all patients, except 1 in
whom the lesion was located on the left arm (Patient
III-6). No extension was noticed since childhood. Sur-
gical removal was performed in 3 patients (Patients
I-18, I-23, and IV-9) followed by local recurrence in 1
(Patient IV-9). Lesions were crimson-colored tiny pap-
ules (3–10 mm) or larger macules (20 – 65 mm) with a
hyperkeratotic epidermis. Bluish discoloration of the
skin or several tortuous vessels radiating outward from
the superficial lesion led us to consider clinically the
diagnosis of associated subcutaneous venous malforma-
tion (Fig 3). Histological confirmation was available in
4 patients (Patients I-18, I-23, II-4, IV-9) belonging to
3 families (Families I, II, IV). The histology revealed
orthokeratosis and hyperkeratosis, abundant dilated
capillaries, and blood-filled spaces in papillary and re-
ticular dermis extending to the hypodermis in all 4 pa-
tients (Fig 4). These vascular lesions can be described
as hyperkeratotic cutaneous capillary venous malforma-
tions (HCCVMs). None of the 89 clinically healthy
individuals having a normal MRI scan had any
HCCVMs.
Haplotypes obtained with eight microsatellite mark-
ers spanning the CCM1 locus on chromosome 7 are

Table. Cutaneous Findings of HCCVMs and CCMs

HCCVM Skin Pathological

Patients Age (yr) CCM MRI Location Size (mm) Confirmation

I-4 60 1 Left calf 10 3 5 2

I-12 29 1 Left calf 835 2
I-18 34 1 Buttock 334 1
I-23 7 NA Left calf 10 3 10 1
II-4 60 1 Right thigh 65 3 60 1
II-11 34 1 Left thigh 20 3 10 2
III-3 26 1 Left leg 534 2
III-6 31 1 Left arm 432 2
IV-2 78 1 Left thigh 20 3 15 2
IV-9 28 NA Right thigh 10 3 5 1
CCM 5 cerebral cavernoma; HCCVM 5 hyperkeratotic cutaneous capillary-venous malformation; NA 5 not available.

252 Annals of Neurology Vol 45 No 2 February 1999


Fig 3. Aspect of skin lesion of the right thigh (Patient II-4).
Note the underlying tortuous bluish vessels. The white area
was left by a previous partial treatment (radiotherapy and
cryotherapy).
shown in Figure 1. Detailed linkage analysis and LOD
score values of these families are not shown but are
available on request. In each family, a distinct haplo-
type was shared by all CCM patients, and the 8 indi-
viduals bearing HCCVMs were carrying the CCM
haplotype segregating in their family. Patient I-23, who
did not undergo MRI, shared the CCM haplotype seg-
regating in his affected relatives. The affected haplo-
types segregating with the CCM and HCCVM pheno-
types were distinct in the 4 families. Two-point and
Fig 4. Cutaneous biopsy findings (Patient II-4). Hyperkeratosis and acanthosis associated with dilated vessels in the upper dermis
(A) and capillaries and veins extending into the hypodermis (B) (hematoxylin-eosin and safran; magnification 3 100 before 47%
reduction).
Brief Communication: Labauge et al: Association between CCMs and HCCVMs
multipoint linkage as well as HOMOG analysis
showed that Families I, II, and IV had a greater than
90% probability of being linked. No definite conclu-
sion could be made for Family III (58% probability)
due to its small size.
Discussion
Skin lesions in our patients consisted of more or less
prominent, plaque-like, irregularly shaped, crimson-
colored vascular malformations. These circumscribed
lesions were strikingly similar in all patients. According
to the current classification of superficial vascular ab-
normalities accepted by the International Society for
the Study of Vascular Anomalies,10 they can be defined
as capillary and venous malformations. All the cutane-
ous lesions of our patients were vascular malformations
of predominantly capillary type with a venular compo-
nent in association with an overlying hyperkeratotic
epidermis. No cellular hyperplasia was present, and the
vascular proliferation invaded not only the dermis but
also the hypodermis.
Several arguments strongly suggest that the occur-
rence of HCCVMs and CCMs in several members of
the families reported here is not fortuitous, including
the facts that (1) HCCVM is a rare vascular lesion that
253
was observed in 8 of 173 individuals with CCMs and
in none of the 89 healthy family members, and (2) in
each case, an HCCVM was observed in at least 2
members of the affected families, with 1 family (Family
IV) including 4 cases.
There are few reports about the association between
CCMs and cutaneous lesions. The types of these cuta-
neous lesions are quite heterogeneous and were mainly
described as bluish nodules,11,12 cherry angiomas,13
and capillary vascular anomalies.14 Filling-Katz and
colleagues15 reported 1 family suffering from terminal
transverse limb defect, CCMs, and skin lesions called
cavernous “angiomas.” In 1 patient, several CCMs
were associated with eruptive multiple angiokerato-
mas.16 Histological data are available in only one of
these case and family reports.16 All of these reports
concerned patients bearing multiple CCMs11,12,16 or
belonging to a family with multiple cases of caverno-
mas.13–15 To our knowledge, CCMs have never been
associated with HCCVMs. In addition, this is the first
report on familial cases of this hyperkeratotic vascular
malformation.
Interestingly, all 9 genetically studied HCCVM pa-
tients share the affected haplotype present in their
CCM-affected relatives. These data strongly suggest
that the occurrence of the vascular cutaneous lesions in
these patients is due to the same genetic abnormality
causing CCMs, although with a lower penetrance. As
we did observe these HCCVMs only in a subset of the
FCC families linked to chromosome 7, one may sug-
gest that these 4 families may harbor a specific CCM1
mutation. This fact will be tested only when CCM1
gene identification is achieved.
In conclusion, we focus on HCCVMs, a cutaneous
vascular hallmark for the development of CCMs in at-
risk family members.
Dr Laberge is the recipient of a grant from the Fonds de Recherche
en Santé du Québec (FRSQ). Dr Labauge benefitted from a poste
d’accueil INSERM during the initial part of the study and is now
supported by the Collège des Enseignants de Neurologie. This work
was supported by INSERM, Ministère de l’Enseignement Supérieur
et de la Recherche (MESR, ACCSV 1995).
We are indebted to the families included in this study for their
participation; to Drs A. L. Benabid, A. Carriere, P. Freger, B.
George, J. Guillemette, D. Hache, J.-P. Lejeune, J.-P. Machayeki, J.
Petit, B. Silhouette, and A. Turcarelli for referring medical observa-
tions; and to Dr M. Wassef and Prof J.-F. Pellissier for pathological
advice. We also thank Profs J.-C. Piette and E. Roullet for excellent
critical reading of this manuscript.
References
1. Russel DS, Rubenstein LJ. Pathology of tumors of the nervous
system. 5th ed. Baltimore: Williams & Wilkins, 1989:730 –736
2. Otten P, Pizzolato GP, Rilliet B, Berney J. A propos de 131 cas
d’angiomes caverneux (cavernomes) du SNC, repérés par
254 Annals of Neurology Vol 45 No 2 February 1999
l’analyse rétrospective de 24,535 autopsies. Neurochirurgie
1989;35:82– 83
3. Rigamonti D, Hadley MN, Drayer BP, et al. Cerebral cavern-
ous malformations. Incidence and familial occurrence. N Engl
J Med 1988;319:343–347
4. Johnson EW, Lyer LM, Rich SS, et al. Refined localization of
the cerebral cavernous malformation gene (CCM1) to a 4-cM
interval of chromosome 7q contained in a well-defined YAC
contig. Genome Res 1995;5:368 –380
5. Dubovsky J, Zabramski JM, Kurth J, et al. A gene responsible
for cavernous malformations of the brain maps to chromosome
7. Hum Mol Genet 1995;4:453– 458
6. Gûnel M, Awad IA, Finberg K, et al. A founder mutation as a
cause of cerebral cavernous malformation in Hispanic Ameri-
cans. N Engl J Med 1996;334:946 –951
7. Gûnel M, Awad IA, Finberg K, et al. Genetic heterogeneity of
inherited cerebral cavernous malformation. Neurosurgery 1996;
38:1265–1271
8. Schwartz AC, Weaver RG, Bloomfield R, Tyler ME. Cavernous
hemangioma of the retina, cutaneous angiomas and intracranial
vascular lesion by computed tomography and nuclear magnetic
resonance imaging. Am J Ophthalmol 1984;98:483– 487
9. Labauge P, Laberge S, Brunereau L, et al. Clinical and genetic
study of 57 French familial cavernomas pedigrees. Neurology
1998;50:A441 (Abstract)
10. Enjolras O, Mulliken JB. Vascular tumors and vascular malfor-
mations. Adv Dermatol 1998;13:375– 423
11. Wood MW, White RJ, Kernohan KW. Cavernous hemangio-
matosis involving the brain, spinal cord, heart, skin and kid-
ney: report of a case. Staff Meet Mayo Clinic 1957;32:
249 –254
12. Bartolomei F, Lemarquis P, Alicherif A, et al. Angiomatose cav-
ernomateuse systématisée avec localisations multiples cérébrales
et cutanées. Rev Neurol (Paris) 1992;148:568 –570
13. Gass JD. Cavernous hemangioma of the retina: a neuro-oculo-
cutaneous syndrome. Am J Ophthalmol 1971;71:799 – 814
14. Goldberg RE, Pheasant TR, Shields JA. Cavernous hemangi-
oma of the retina: a four generation pedigree with neurocuta-
neous manifestations and an example of bilateral retinal in-
volvement. Arch Ophthalmol 1979;97:2321–2324
15. Filling-Katz MR, Levin SW, Patronas NJ, Katz NNK. Termi-
nal transverse limb defects associated with familial cavernous
angiomatosis. Am J Med Genet 1992;42:346 –351
16. Ostlere L, Hart Y, Misch KJ. Cutaneous and cerebral haeman-
giomas associated with eruptive angiokeratomas. Br J Dermatol
1996;135:98 –101

Epilepsia Partialis Continua:
A New Manifestation of
Anti-Hu–Associated
Paraneoplastic
Encephalomyelitis
Yaël Bellaı̈che Shavit, MD,* Francesc Graus, MD,‡
Alphonse Probst, MD,† Ramon Rene, MD,§
and Andreas J. Steck, MD*
We report on 3 anti-Hu–positive patients who presented
with clinical and electroencephalographic (EEG) features
of epilepsia partialis continua (EPC). Two of the patients
had an associated small cell carcinoma. Magnetic reso-
nance imaging (MRI) disclosed a hyperintense nonen-
hancing focal lesion in T2-weighted images in the senso-
rimotor area in 2 patients. Histopathological analysis of
the lesion revealed inflammatory infiltrates and neuronal
cell loss. In the patient who had a postmortem study,
these neuropathological changes were not observed in
other areas of the nervous system. This study emphasizes
that the possibility of an anti-Hu–associated paraneoplas-
tic disorder must be considered in patients with cortical
encephalitis presenting with EPC when a brain tumor
can be excluded.
Shavit YB, Graus F, Probst A, Rene R, Steck AJ.
Epilepsia partialis continua: a new manifestation of
anti-Hu–associated paraneoplastic encephalomyelitis.
Ann Neurol 1999;45:255–258
Anti-Hu–associated paraneoplastic encephalomyelitis
and sensory neuronopathy (PEM/SN) are two often
concomitant syndromes associated with small cell lung
carcinoma (SCLC) in nearly 80% of patients. Neuro-
logical dysfunction is usually multifocal, including sen-
sory neuronopathy, motor neuron dysfunction, brain-
stem encephalopathy, limbic encephalitis, autonomic
neuropathy, and cerebellar degeneration.1–3 Although
the inflammatory infiltrates and neuronal loss charac-
teristic of PEM/SN may be observed in multiple areas
of the cerebral cortex in postmortem studies, no symp-
toms suggestive of a particular area of the cerebral cor-
tex other than limbic encephalitis have been reported
From the Departments of *Neurology and †Pathology, University
Hospital, Basel, Switzerland, ‡Department of Neurology, Hospital
Clı́nic i Provincial, University of Barcelona, and §Department of
Neurology, C.S.U. Bellvitge, L’Hospetalet, Barcelona, Spain.
Received May 21, 1998, and in revised form Sep 28, 1998. Ac-
cepted for publication Sep 28, 1998.
Address correspondence to Prof Steck, Neurologische Klinik, Kan-
tonsspital Basel, Petersgraben 4, CH-4031 Basel, Switzerland.
Copyright © 1999 by the American Neurological Association
in patients with Hu-associated PEM/SN. Seizures are
rarely the presenting symptom of PEM/SN and occur
almost always in the setting of limbic encephalitis.2
We describe 3 patients with focal sensorimotor en-
cephalitis presenting with epilepsia partialis continua
(EPC), which represents a new clinical picture in the
spectrum of anti-Hu–associated neurological disorders.
Case Reports
Patient 1
A 56-year-old woman with a history of SCLC diagnosed in
June 1995 went into complete remission after chemotherapy.
In September 1996, she presented with dysesthesia of the left
trunk from T6 to T10 and involuntary clonic muscular
twitching of the left leg. A neurological examination showed
left facial weakness, increased tendon reflexes on the left side,
negative Babinski reflex, slight paresis, and distal hypesthesia
of the left extremities. A stimulus-sensitive myoclonic move-
ment disorder of the left arm and leg could be observed.
Magnetic resonance imaging (MRI) of the head showed a
nonenhancing lesion in the right postcentral area in T2-
weighted images (Fig 1A). The electroencephalogram (EEG)
demonstrated periodic epileptiform discharges in the right
parietal area, which were sometimes synchronous with an ob-
served myoclonus of the left hand. A cerebrospinal fluid
(CSF) examination, including oligoclonal IgG bands, was
negative. There was no evidence for recurrence of her previ-
ous cancer on chest computed tomography (CT), including
liver and adrenal glands, or on spinal and pelvic radiography.
Tumor markers were normal, except for b2-microglobulin,
which was slightly elevated (3.7 mg/L).
The patient underwent a stereotactic biopsy of the right
postcentral lesion. A histopathological examination revealed a
subacute cortical encephalitis with a few perivascular B-cell
infiltrates, a larger number of T lymphocytes diffusely infil-
trating the cortex, and neuronal cell loss. There was evidence
for microglia activation and proliferation as well as for astro-
cytic gliosis (Fig 2). Intracytoplasmic IgG reactivity was
found in some neurons (not shown). Despite intravenous
immunoglobulin treatment and plasmapheresis, she experi-
enced a relentless progression of her left hemiparesis until she
became unable to walk. The motor seizures involving the left
face and the left upper and lower extremities became almost
constant but were finally partially controlled with a combi-
nation of anti-epileptic drugs. On MRI 3 months later, the
patient showed an enlargement of the nonenhancing lesion
to the right precentral area (see Fig 1B). She subsequently
received cyclophosphamide under which the lesion regressed,
the EPC disappeared, and the left hemiparesis notably im-
proved. Nevertheless, at her last follow-up examination 2
years after the onset of neurological symptoms, the hemipa-
resis had again increased with subsequent extension of the
lesion, although the SCLC is still in remission.
Patient 2
A 54-year-old man was admitted to the emergency room due
to a generalized tonic-clonic seizure. The patient recovered
completely within a few hours. Routine laboratory analysis
and a CT scan of the head were normal. Over the ensuing 3
days, the patient noticed progressive difficulty in evoking
255

Fig 1. (A) T2-weighted axial magnetic resonance imaging
(MRI) performed during the initial course of the disease dis-
closed high signals located in the right postcentral area (TR 5
2,500). (B) Head MRI performed 9 months later. T2-
weighted images show an extension of the hyperintense lesion
to the precentral area.
names, with dysarthria and frequent focal-clonic seizures in-
volving the muscles of the right side of the face.
General physical and neurological examinations were nor-
mal, except for severe dysarthria, mild facial paresis of central
origin, increased deep tendon reflexes, and extensor plantar re-
sponse in the right side. A second CT scan of the head, repeat
MRI; three CSF examinations; routine laboratory analysis; se-
rologies for herpesvirus, human immunodeficiency virus, bor-
relia, and syphilis; testing of somatosensory evoked potentials;
and four-vessel angiography were all normal or negative. Sev-
eral EEGs demonstrated periodic lateralized epileptiform dis-
charges in the left parasagittal frontoparietal region.
The clinical course of the patient was characterized by fo-
cal clonic seizures involving the right side of the face and
256 Annals of Neurology Vol 45 No 2 February 1999
right hand, which became almost continuous and unrespon-
sive to treatment. The patient remained collaborative and
oriented throughout the clinical course. He died due to as-
piration pneumonia 30 days after the onset of the neurolog-
ical disorder.
The autopsy study did not show any tumor. The macro-
scopic examination of the brain was normal. The micro-
scopic study of multiple areas of the brain was normal, except
for the presence of perivascular and intraparenchymatous
inflammatory infiltrates, gliosis, and neuronal cell loss in the
left motor strip. Immunohistochemical studies demonstrated
microglia proliferation. Most of the inflammatory cells were
T cells, with occasional perivascular B-cell lymphocytes.
Patient 3
A 43-year-old man noticed involuntary repetitive movements
of the tongue that interfered with his speech and deglutition
in April 1997. The movements were almost continuous and
persisted during sleep. The patient was evaluated elsewhere,
and head MRI and a CSF examination were normal. He was
discharged with a diagnosis of lingual myoclonus and treated
with clonazepam, which reduced the movements. Over the
ensuing months, the patient noticed additional left perioral
twitching and occasional clonic movements of the first two
fingers of the left hand.
The patient was admitted for a severe hematemesis in No-
vember 1997. A general physical examination was unreveal-
ing. A neurological examination was normal, except for fre-
quent twitching of the tongue and platysma of both sides,
which was more prominent on the left side. There were oc-
casional clonic jerks of the left side of the face. The twitching
interfered with voluntary movements of the tongue, which
were slowed. Deep tendon reflexes were abolished in the legs.
Pallesthesia was diminished in the left foot. Routine labora-
tory analysis and a CSF examination were normal. The EEG
demonstrated periodic epileptiform discharges in the right
frontoparietal area, and MRI of the head showed a nonen-
hancing lesion in the inferior portion of the right Rolandic
area (Fig 3). The electromyographic examination was nor-
mal, except for absent sensory potential in the left sural and
superficial peroneal nerves. Upper gastrointestinal endoscopy
and a CT scan of the abdomen disclosed a large tumor in the
cardia of the stomach with enlarged perigastric lymph nodes.
A biopsy of the tumor confirmed a small cell tumor. After
three cycles with carboplatin and etopiside, the tumor
showed partial remission. The patient’s movements greatly
improved, and MRI revealed a complete remission of the
brain lesion.
Immunological Studies
Serum and CSF anti-Hu antibodies were detected on frozen
sections of normal human cortex and confirmed by immu-
noblotting of human neuronal nuclei extracts and recombi-
nant HuD as described in detail previously.4 Anti-Hu anti-
bodies were detected in the serum of the 3 patients and in
the two available CSF samples. Anti-Hu antibodies remained
positive at serum dilutions greater than 1:10,000, a feature
that is almost always associated with paraneoplastic neurolog-
ical disorders.4
Fig 2. Biopsy of the cerebral cortex of Patient 1. (a) The cerebral cortex is diffusely infiltrated by lymphocytes. The cell infiltrates
surrounding the vessel mainly consist of B lymphocytes. (b) The cerebral cortex shows hypertrophic astrocytes (arrowheads), loose neu-
ropil texture, and loss of neurons. (c) The patient’s biopsy was incubated with pan–T-cell antibody CD3. The cortex is diffusely
infiltrated by T cells, with most of them bearing CD8 surface antigen (not shown). (d) Immunostaining with antibody CD68
shows diffuse infiltration of the cerebral cortex by activated microglia/macrophages. (All hematoxylin and eosin; original magnifica-
tion: a 3 40, b 3 200, c 3 80, d 3 100.).

Fig 3. T2-weighted transverse magnetic resonance imaging of
Patient 3. The arrow shows the hyperintense lesion in the
right frontoparietal area.
Discussion
These 3 patients demonstrate a previously unreported
clinical presentation of PEM/SN associated with
anti-Hu antibodies. The clinical, EEG, and histopatho-
logical findings in our patients fulfill the criteria of the
EPC syndrome.5 In addition, MRI disclosed a focal
nonenhancing lesion in the sensorimotor area as de-
scribed in patients with EPC of other etiologies.6 EPC
is usually caused by structural focal lesions (cerebrovas-
cular disorders, trauma, or tumors), metabolic disor-
ders, or presumed encephalitis, but in some patients,
the cause is unknown.5,6 The present study shows that
the possibility of a paraneoplastic origin must be in-
cluded in the differential diagnosis. The absence of a tu-
mor in the autopsy of Patient 2 does not rule out this
possibility. The autopsy was done without the clinical
diagnosis of a paraneoplastic syndrome, and a small tu-
mor can easily be missed in the postmortem analysis if a
direct search for an occult tumor is not considered.7 The
possibility of a brain metastasis was reasonably excluded
by the brain biopsy in Patient 1 and by a complete au-
topsy in Patient 2, which did not disclose malignant
cells in the brain. Furthermore, the clinical course of
Patient 1, who survived for 2 years after the onset of
manifestations, is not typical for a brain metastasis.

Brief Communication: Shavit et al: EPC in Anti-Hu–Associated PEM 257

 Pathological and immunohistochemical abnormali-
ties in the brain tissue of patients with PEM/SN are
usually widespread, with certain areas being more af-
fected than others.8 This may be explained by the
ubiquitous expression of Hu antigens in the nervous
system. The remarkable findings in our patients are the
exquisitely focal clinical, paraclinical, and, to a certain
extent, pathological features. Why some patients with
PEM/SN and anti-Hu antibodies demonstrate a focal
neurological disorder throughout the clinical course of
the disease is an unsettled issue. Patients with anti-
Hu–associated PEM/SN sometimes build an immune
response against other neural antigens, and they may
demonstrate other autoantibodies such as anti-CV29 or
antiamphiphysin.10 Our patients could have other
autoantibodies that would be relevant to the seizure
disorder. Patients with Rasmussen’s encephalitis, a pro-
gressive disorder characterized by intractable focal sei-
zures and inflammatory neuropathology, demonstrate
antibodies against subunit 3 of the glutamic receptor
(GLuR3). Animals immunized with GLuR3 develop
anti-GLuR3 antibodies and clinical and pathological
features similar to those of Rasmussen’s encephalitis.11
Recently, antibodies against GLuR1, GLuR4, and
GluR5/6 were reported in 6 of 7 patients with para-
neoplastic cerebellar degeneration.12 Although we did
not detect GLuR antibodies in our 3 patients, no an-
tibodies against any subunit of GLuR were found in a
large number of patients with paraneoplastic neurolog-
ical disorders, including anti-Hu–associated PEM/SN
(J. Dalmau, personal communication, 1997).13
Whether the tissue distribution of the products of
the different genes of the Hu family correlates with the
diverse neurological syndromes of PEM/SN is still a
matter requiring further research.14 Studies by Manley
and colleagues15 could not find any correlation be-
tween antibody reactivity with any of the Hu antigens
and the type of neurological symptoms. Furthermore,
analysis of IgG subclass distribution did not reveal any
correlation between anti-Hu isotype and specific brain
area or clinical features.16 Finally, the focal nature of
the symptoms could be explained by the circumscribed
expression of major histocompatibility complex pro-
teins, which eventually leads to the demonstration of
Hu antigens. This could induce a T-cell–mediated pro-
cess either as a direct cytotoxic reaction with neuronal
cells or as the activation of T and B cells.17
In conclusion, the present study extends our knowl-
edge of the clinical spectrum of PEM/SN and demon-
strates that the possibility of a paraneoplastic origin must
be considered in the differential diagnosis of EPC.
We thank Prof E. W. Radü for providing the MR images of Patient
1 and Dr H. R. Stöckli for referring Patient 1. We are grateful to V.
Bruce for revising the manuscript.
258 Annals of Neurology Vol 45 No 2 February 1999
References
1. Graus F, Ribalta T, Campo E, et al. Immunohistochemical
analysis of the immune reaction in the nervous system in para-
neoplastic encephalomyelitis. Neurology 1990;40:219 –222
2. Dalmau J, Graus F, Rosenblum MK, Posner JB. Anti-Hu–
associated paraneoplastic encephalomyelitis/sensory neuronopa-
thy. A clinical study of 71 patients. Medicine 1992;71:59 –72
3. Henson RA, Urich H. Encephalomyelitis with carcinoma. In:
Cancer of the nervous system. Oxford, UK: Henson Blackwell
Scientific Publications, 1982:314 –345
4. Graus F, Dalmau J, Reñé R, et al. Anti-Hu antibodies in pa-
tients with small cell lung cancer: association with complete re-
sponse to therapy and improved survival. J Clin Oncol 1997;
15:2866 –2872
5. Thomas JE, Reagan TJ, Klass DW. Epilepsia partialis continua.
A review of 32 cases. Arch Neurol 1977;34:266 –275
6. Singh BM, Strobos RJ. Epilepsia partialis continua associated
with nonketotic hyperglycemia: clinical and biochemical profile
of 21 patients. Ann Neurol 1980;8:155–160
7. Anderson NE, Budde-Steffen C, Wiley RG, et al. A variant of
the anti-Purkinje cell antibody in a patient with paraneoplastic
cerebellar degeneration. Neurology 1988;38:1018 –1026
8. Dalmau J, Furneaux HM, Rosenblum MK, et al. Detection of
the anti-Hu antibody in specific regions of the nervous system
and tumor from patients with paraneoplastic encephalomyelitis/
sensory neuronopathy. Neurology 1991;41:1757–1764
9. Honnorat J, Antoine JC, Derrington E, et al. Antibodies to a
subpopulation of glial cells and a 66-kDa developmental pro-
tein in patients with paraneoplastic neurological syndromes.
J Neurol Neurosurg Psychiatry 1996;61:270 –278
10. Dropcho EJ. Anti-amphiphysin antibodies with small-cell lung
carcinoma and paraneoplastic encephalomyelitis. Ann Neurol
1996;39:659 – 667
11. Rogers SW, Andrews PI, Gahring LC, et al. Autoantibodies to
glutamate receptor GLuR3 in Rasmussen’s encephalitis. Science
1994;265:648 – 652
12. Gahring LC, Twyman RE, Greenlee JE, Rogers SW. Autoan-
tibodies to neuronal glutamate receptors in patients with para-
neoplastic neurodegenerative syndrome enhance receptor activa-
tion. Molecular Medicine 1995;1:245–253
13. Degenhardt A, Hoard R, Duvoisin RM, et al. Glutamate re-
ceptor antibodies and paraneoplastic neurologic disorders. Neu-
rology 1996;46:A410 (Abstract)
14. Darnell RB. Onconeural antigens and the paraneoplastic neu-
rologic disorders: at the intersection of cancer, immunity, and
the brain. Proc Natl Acad Sci USA 1996;93:4529 – 4536
15. Manley GT, Smitt PS, Dalmau J, Posner JB. Hu antigens: re-
activity with Hu antibodies, tumor expression, and major im-
munogenic sites. Ann Neurol 1995;38:102–110
16. Jean WC, Dalmau J, Ho A, Posner JB. Analysis of the IgG
subclass distribution and inflammatory infiltrates in patients
with anti-Hu–associated paraneoplastic encephalomyelitis. Neu-
rology 1994;44:140 –147
17. Dalmau J, Graus F, Cheung NV, et al. Major histocompatibil-
ity proteins, anti-Hu antibodies, and paraneoplastic encephalo-
myelitis in neuroblastoma and small cell lung cancer. Cancer
1995;75:99 –109

Epstein-Barr Virus in
Monitoring the Response
to Therapy of Acquired
Immunodeficiency
Syndrome–Related Primary
Central Nervous
System Lymphoma
Andrea Antinori, MD,* Antonella Cingolani, MD,*
Andrea De Luca, MD,* Gianluca Gaidano, MD, PhD,‡
Adriana Ammassari, MD,* Luigi M. Larocca, MD,†
and Luigi Ortona, MD*
To evaluate the value of Epstein-Barr virus DNA (EBV-
DNA) assay in cerebrospinal fluid (CSF) for monitoring
the response to treatment in acquired immunodeficiency
syndrome–related primary central nervous system lym-
phoma (AIDS-PCNSL), 9 human immunodeficiency
virus–infected patients with biopsy-proven AIDS-PCNSL
who underwent multimodal therapy were investigated for
EBV-DNA detection in CSF by semiquantitative nested
polymerase chain reaction (PCR). Tumoral tissue expres-
sion of bcl-6 oncogene protein and of EBV-encoded la-
tent membrane protein (LMP-1) was also investigated.
The 2 patients who had a response to chemotherapy
showed a reduction of mean EBV-DNA concentration
values after chemotherapy and displayed a large non-
cleaved morphology and a BCL-61/LMP-12 phenotype.
Conversely, the 4 patients with progressive disease after
chemotherapy showed increasing mean values of EBV-
DNA and displayed an immunoblastic morphology and a
BCL-6 2/LMP-11 phenotype. No significant changes
were observed for patients with stable disease. EBV-DNA
burden reduction was significantly associated with pro-
longed survival. These results suggest that EBV-DNA
monitoring might be helpful in predicting response to
chemotherapy and in segregating distinct biological and
prognostic categories of AIDS-PCNSL.
Antinori A, Cingolani A, De Luca A, Gaidano G,
Ammassari A, Larocca LM, Ortona L. Epstein-
Barr virus in monitoring the response to therapy
of acquired immunodeficiency syndrome–related
primary central nervous system lymphoma.
Ann Neurol 1999;5:259 –261
From the Departments of *Infectious Diseases and †Pathology,
Catholic University, Rome, and ‡Division of Internal Medicine,
Department of Medical Sciences, University of Torino at Novara,
Novara, Italy.
Received Aug 18, 1998, and in revised form Oct 5, 1998. Accepted
for publication Oct 5, 1998.
Address correspondence to Dr Antinori, Department of Infectious
Diseases, Catholic University, L.go A. Gemelli 8 — 00168 Rome,
Italy.
Copyright © 1999 by the American Neurological Association
Epstein-Barr virus (EBV) infection is associated with
virtually all acquired immunodeficiency syndrome–
related primary central nervous system lymphoma
(AIDS-PCNSL),1 and EBV-DNA is detectable in the
cerebrospinal fluid (CSF) of most patients with the dis-
ease.2– 4 For these reasons, EBV-DNA detection in
CSF has been proposed as a valid tool for the rapid
selection of patients to undergo brain biopsy.5 Re-
cently, our group reported that detection of EBV-DNA
in CSF may allow a minimally invasive diagnosis of
AIDS-PCNSL.6,7 At present, it is noteworthy to assess
the value of this assay for monitoring tumor response
to treatment.7 Here, we report the results of a pilot
study performed to assess the relationship between
EBV load in CSF, response to therapy, and survival of
patients with AIDS-PCNSL who received multimodal
therapy.
Materials and Methods
Nine consecutive patients with biopsy-proven AIDS-PCNSL
who underwent multimodal therapy between January 1995
and April 1998 were included in the study. All pathological
specimens were classified according to the working formu-
lation for non-Hodgkin’s lymphoma8 and to a revised
European-American classification of lymphoid neoplasms.9
EBV-encoded small RNAs were detected by in situ hybrid-
ization. All cases were investigated for protein expression of
the BCL-6 proto-oncogene and of EBV-encoded latent
membrane protein-1 (LMP-1). The BCL-6 protein was de-
tected by the PG-B6 monoclonal antibody directed against
the amino terminal portion of the human bcl-6 gene product
(obtained from B. Falini, Institute of Hematology, Univer-
sity of Perugia, Perugia, Italy). The LMP-1 antigen was de-
tected using a pool of four anti-LMP-1 monoclonal antibod-
ies (CS-1– 4, Dakopatts, Glostrup, Denmark). Subsequently,
after incubation with primary antibodies, an avidin-biotin
peroxidase complex method was employed using tyramide
amplification (Dako Catalyzed Amplification System, Dako-
patts) and diaminobenzidine as a chromogen. A positive re-
sult was defined as 10% or greater positive neoplastic cells.10
Eight patients received multimodal therapy with high-dose
intravenous methotrexate (MTX), oral procarbazine, and in-
trathecal MTX followed by whole-brain irradiation to 30
Gy. The last patient (Patient 9 in Table) was treated with a
combination of high-dose intravenous MTX, high-dose
zidovudine, and intrathecal MTX followed by whole-brain
irradiation to 50 Gy. The response to chemotherapy as well
as that to radiation therapy was determined by computed
tomographic scan or magnetic resonance imaging according
to reported criteria.11
EBV-DNA amplification was performed in CSF samples
at baseline and after the end of the chemotherapy phase us-
ing a nested polymerase chain reaction (PCR) technique ex-
ploiting primers derived from the EBNA1 gene.3 The detec-
tion limit of the nested PCR assay was 1,000 EBV-DNA
copies per milliliter as defined by serial dilution experiments.
EBV-DNA load in CSF was measured semiquantitatively by
serial testing of twofold dilutions of each sample. Sample di-
lutions were prepared and run in triplicate; the assay was
259
Table. Host’s CD4 Count, Tumor Histology, and Expression of BCL-6, EBV-Encoded Small RNAs, and LMP-1 as Well as
Viral Load in CSF and Response to Therapy of Patients Included in the Study

Baseline Changes in Response to

CD4 EBV-Encoded EBV-DNA at EBV-DNA after Response to Multimodal
Case (per mL) REAL WF bcl-6a Small RNAb LMP-1a Diagnosisc Chemotherapyc Chemotherapy Therapy

1 5 DLCL LNCCL 1 1 2 5.0 21.3 PR CR

2 5 DLCL LNCCL 1 1 2 4.7 21.4 PR PR
3 20 DLCL LNCCL/IBPL 1 1 1 5.3 0.0 SD PR
4 4 DLCL IBPL 2 1 1 3.3 1.4 SD PR
5 4 DLCL IBPL 2 1 1 3.7 20.4 SD PR
6 13 DLCL IBPL 2 1 1 3.0 1.3 PD PD
7 8 DLCL IBPL 2 1 1 3.3 0.7 PD SD
8 29 DLCL IBPL 2 1 1 4.0 1.3 PD NE
9 41 DLCL IBPL 2 1 1 4.7 1.0 PD PR
a
Analyzed by immunohistochemistry.
b
Analyzed by RNA in situ hybridization.
c
Epstein-Barr virus DNA is expressed as log copies per milliliter in cerebrospinal fluid.
LMP-1 5 latent membrane protein; REAL 5 revised European-American lymphoma classification; WF 5 working formulation for non-Hodgkin’s
lymphoma; DLCL 5 diffuse large B-cell lymphoma; LNCCL 5 large noncleaved-cell lymphoma; IBPL 5 immunoblastic plasmacytoid lymphoma;
LNCCL/IBPL 5 diffuse large-cell lymphoma with an admixture of centroblasts and immunoblasts; CR 5 complete response; PR 5 partial response;
SD 5 stable disease; PD 5 progressive disease; NE 5 not evaluable due to early death before starting radiotherapy.

considered positive only if at least two of the triplicate PCR

tests for each dilution scored positive. Changes of the EBV-
DNA concentration in CSF between the baseline value and
the value after chemotherapy were expressed as log copies per
milliliter. Differences between mean values of EBV-DNA
concentration at baseline in different response groups were
analyzed by one-way ANOVA. Mean values of EBV-DNA
concentration at baseline and after chemotherapy were com-
pared by paired t test. Survival probability was estimated by
the Kaplan-Meier method, and differences between groups
according to the reduction of EBV-DNA log copies were cal-
culated using the Mantel-Cox test.

Results
The Table summarizes the host’s CD4 count, tumor
histology, and expression of BCL-6, EBV-encoded
small RNAs, and LMP-1 as well as the viral load in
CSF and the response to therapy of all patients in-
cluded in
the study.
Changes of EBV-DNA burden correlated with re-
sponse to chemotherapy. In fact, in patients who re-
sponded to chemotherapy (Cases 1 and 2), the mean
value of the EBV-DNA load was 4.85 log copies per
milliliter at baseline (range, 4.70 –5.0) and 3.50 log
copies per milliliter after therapy (range, 3.3–3.7) ( p 5
0.022, paired t test). In patients who progressed (Cases
6 –9), the mean values were 3.75 (range, 3.0 – 4.7) and
4.82 (range, 4.0 –5.7) log copies per milliliter, respec-
tively ( p 5 0.0049). In patients with stable disease
(Cases 3–5), the mean values were 4.10 (range, 3.3–
5.3) and 4.43 (range, 3.3–5.3) log copies per milliliter,
respectively ( p 5 0.60) (Fig). Six of 9 patients had a
final response to multimodal therapy, but only those
patients whose EBV-DNA concentration in CSF was
reduced after chemotherapy showed a response to the
Fig. Epstein-Barr virus DNA log copies per milliliter expressed
as mean and range values at baseline and after chemotherapy
in patients with acquired immunodeficiency syndrome–related
primary central nervous system lymphoma according to the
grade of response to therapy. Statistical significance was as-
sessed by paired t test.
chemotherapy phase as well. A final assessment of
EBV-DNA burden after radiotherapy was not per-
formed in most patients (see Table).
Unlike longitudinally observed changes, absolute val-
ues of EBV-DNA at baseline did not predict a response
to therapy. In fact, no significant differences for mean
EBV-DNA concentrations were detected at baseline in
the three response groups of patients (4.85, 4.10, and
3.75 log copies per milliliter for responders, stable pa-
tients, and progressive patients, respectively; F value at
ANOVA 5 1.205; p 5 0.36).
The correlation with response to chemotherapy re-
sulted in an effect on patients’ survival. In fact, the
median length of survival was 243 days in those with a

260 Annals of Neurology Vol 5 No 2 February 1999

log EBV-DNA reduction and 121 days in those with-
out a reduction. The 6-month probabilities of survival
were 0.67 and 0.20, respectively ( p 5 0.048, Mantel-
Cox test).
Discussion
The results of this study suggest a prognostic signifi-
cance of the changes in EBV-DNA viral burden in
CSF before and after chemotherapy for AIDS-PCNSL.
In particular, an increasing burden of EBV-DNA in
CSF may reflect an active proliferation of an EBV-
positive tumor cell population which is totally or par-
tially resistant to therapy. Conversely, a significant
decrease of viral load after chemotherapy may be asso-
ciated with response to therapy and prolonged survival.
AIDS-PCNSL cases showing a response to therapy
and a decrease in EBV-DNA viral load appear to dis-
play biological peculiarities when compared with cases
that are resistant to therapy and show no decrease in
EBV-DNA viral load (see Table). Previously, it has
been shown that the expression of BCL-6 and LMP-1
is mutually exclusive in AIDS-PCNSL and segregates
two distinct phenotypical categories of the disease, that
is, BCL-6 1/LMP-12 and BCL-6 2/LMP-11.10 AIDS-
PCNSL showing the BCL-61/LMP-12 phenotype re-
flects a germinal center stage of B-cell differentiation,
tends to display a large noncleaved cell morphology,
and as suggested by the present study, appears to be
more susceptible to currently available therapeutic
strategies (see Table).10 Conversely, BCL-6 2/LMP-11
AIDS-PCNSL reflects a postgerminal center stage of
B-cell differentiation, tends to display an immunoblas-
tic morphology, and appears to be more resistant to
therapy (see Table).10
This pilot study includes a limited number of closely
monitored patients. Nevertheless, this is one of the
largest series of AIDS-PCNSL cases treated with mul-
timodal therapy published to date. Overall, our results
prompt large-scale investigation aimed at defining
whether molecular monitoring of EBV-DNA viral load
in CSF may be a reliable tool for predicting the re-
sponse of AIDS-PCNSL to therapy and identifying
subgroups of patients with different clinical outcomes
and lengths of survival.
This work was supported by the Programma Nazionale di Ricerca
sull’AIDS—1997. Istituto Superiore di Sanità (Rome, Italy) grant
no. 30A.0.47.
Special thanks to Emanuela Vaccher from Aviano, Italy, for the ma-
jor contribution to the zidovudine/MTX chemotherapy protocol
and to Laura Gillini for invaluable technical assistance.
References
1. MacMahon EME, Glass JD, Hayward SD, et al. Epstein-Barr
virus in AIDS-related primary central nervous system lym-
phoma. Lancet 1991;338:969 –973
Brief Communication: Antinori et al: EBV-DNA Monitoring and Cerebral Lymphoma
2. Cinque P, Brytting M, Vago L, et al. Epstein-Barr virus DNA
in cerebrospinal fluid from patients with AIDS-related primary
lymphoma of the central nervous system. Lancet 1993;342:
398 – 401
3. De Luca A, Antinori A, Cingolani A, et al. Evaluation of CSF
EBV-DNA and IL-10 as markers for in vivo diagnosis of AIDS-
related primary central nervous system lymphoma. Br J Haema-
tol 1995;90:844 – 849
4. Arribas JR, Clifford DB, Fichtenbaum CJ, et al. Detection of
Epstein-Barr virus DNA in CSF for diagnosis of AIDS-related
central nervous system lymphoma. J Clin Microbiol 1995;33:
1580 –1583
5. Antinori A, Ammassari A, De Luca A, et al. Diagnosis of
AIDS-related focal brain lesions: a decision-making analysis
based on clinical and neuroradiologic characteristics combined
with polymerase chain reaction assays in CSF. Neurology 1997;
48:687– 694
6. Cingolani A, De Luca A, Larocca LM, et al. Minimally invasive
diagnosis of acquired immunodeficiency syndrome–related pri-
mary central nervous system lymphoma. J Natl Cancer Inst
1998;90:364 –369
7. Yarchoan R, Jaffe ES, Little R. Diagnosing central nervous sys-
tem lymphoma in the setting of AIDS: a step forward. J Natl
Cancer Inst 1998;90:346 –347
8. Non-Hodgkin’s Lymphoma Pathologic Classification Project.
National Cancer Institute sponsored study of classification of
non-Hodgkin’s lymphomas: summary and description of a
working formulation for clinical usage. Cancer 1982;49:2112–
2135
9. Harris NL, Jaffem ES, Stain H, et al. A revised European-
American classification of lymphoid neoplasms: a proposal from
the International Lymphoma Study Group. Blood 1994;84:
1361–1392
10. Larocca LM, Capello D, Rinelli A, et al. The molecular and
phenotypic profile of primary central nervous system lymphoma
identifies distinct categories of the diseases and is consistent
withhistogenetic derivation from germinal center–related
B-cells. Blood 1988;92:1011–1019
11. Forsyth PA, Yahalom J, DeAngelis LM. Combined-modality
therapy in the treatment of primary central nervous system lym-
phoma in AIDS. Neurology 1994;44:1473–1479
261

Genetic Locus Heterogeneity
in Lafora’s Progressive
Myoclonus Epilepsy
Berge A. Minassian, MD,*†‡§ Jesus Sainz, PhD,*†
Jose M. Serratosa, MD, PhD,*†i Manyee Gee, PhD,*†
Lise M. Sakamoto,*† Saeed Bohlega, MD,¶
Guy Geoffroy, MD,** Cathy Barr, PhD,§††
Steve W. Scherer, PhD,§ Uwamie Tomiyasu, MD,‡‡
Stirling Carpenter, MD,§§ Karen Wigg,††§§
A. V. Sanghvi,* and Antonio V. Delgado-Escueta, MD*†ii
In 1995, we mapped a gene for Lafora’s progressive my-
oclonus epilepsy in chromosome 6q23-25. In 1997 and
1998, we reduced the size of the locus to 300 kb, and an
international collaboration identified mutations in the
protein tyrosine phosphatase gene. Here, we examine for
heterogeneity through the admixture test in 22 families
and estimate the proportion of linked families to be 75 to
85%. Extremely low posterior probabilities of linkage
(Wi), exclusionary LOD scores, and haplotypes identify 4
families unlikely to be linked to chromosome 6q24.
Minassian BA, Sainz J, Serratosa JM, Gee M,
Sakamoto LM, Bohlega S, Geoffroy G, Barr C,
Scherer SW, Tomiyasu U, Carpenter S, Wigg K,
Sanghvi AV, Delgado-Escueta AV. Genetic locus
heterogeneity in Lafora’s progressive myoclonus
epilepsy. Ann Neurol 1999;5:262–265
In 1911, Lafora and Glueck1,2 showed characteristic
large basophilic periodic acid-Schiff (PAS)–positive in-
clusion bodies in neuronal perikarya of the central ner-
vous system of a young adult with a fatal form of pro-
gressive myoclonus epilepsy (PME). Almost half a
century later, Harriman and Millar3 and Schwarz and
Yanoff 4 showed that PAS-positive granular deposits
can be found in liver,5 heart, retina, peripheral nerve,
From the *Comprehensive Epilepsy Program, Department of Neu-
rology, and iiBrain Research Institute, University of California, Los
Angeles School of Medicine, and †Neurology and Research and
‡‡Pathology Services, West Los Angeles DVA Medical Center, Los
Angeles, CA; ‡Division of Neurology, Department of Pediatrics,
Bloorview Epilepsy Program, §Department of Genetics, Hospital for
Sick Children, and Departments of ††Psychiatry and §§Pathology,
The Toronto Hospital, University of Toronto, Toronto, Ontario,
and **Department of Neurology, Ste-Justine Hospital, University of
Montreal, Montreal, Canada; iEpilepsy Unit, Jimenez Diaz Foun-
dation, Madrid, Spain; and ¶Department of Medicine, King Faisal
Specialist Hospital, Riyadh, Saudi Arabia.
Received May 29, 1998, and in revised form Oct 8, 1998. Accepted
for publication Oct 9, 1998.
Address correspondence to Dr Delgado-Escueta, Comprehensive
Epilepsy Program, UCLA and West Los Angeles DVA Medical
Center, Building 500, Room 3405, 11301 Wilshire Boulevard, Los
Angeles, CA 90073.
262 Copyright © 1999 by the American Neurological Association
and skeletal muscle, and others showed similar inclu-
sion bodies in sweat glands and apocrine myoepithelial
cells.6 –9 In 1965, Schwarz and Yanoff4 described elec-
troencephalographic abnormalities consisting of back-
ground slowing and diffuse spike wave and polyspike
wave bursts. In 1977, Schwarz10 contended that the
characteristic clinical features of stimuli-sensitive myoc-
lonus (ie, absences, tonic-clonic seizures, rapid deterio-
ration in neurological function ending in dementia and
death by 30 years of age, and typical electroencephalo-
gram and pathology) together with its autosomal reces-
sive mode of inheritance warranted its separation from
other PMEs and its designation as a single homoge-
neous entity called Lafora’s disease (LD).
In 1995, our laboratories localized a gene for LD to
a 17-cM span of chromosome 6q23-25 between
D6S292 and D6S420.11 In 1997, we reduced the LD
region to less than 3 cM (1.2 Mb of physical distance)
between D6S1003 and D6S311.12 In 1998, homozy-
gosities and recombinations in 30 families further re-
duced the region to 300 kb, and an international col-
laborative effort isolated the LD gene and identified
mutation(s) in a gene encoding for a protein tyrosine
phosphatase.13,14
Here, we present the results of the HOMOG admix-
ture test in 22 LD families, their posterior probability
of linkage, and the exclusionary LOD scores and hap-
lotypes of 4 families. The present data suggest that ge-
netic locus heterogeneity exists even within this rare
but clinically homogeneous entity.
Methods
Family Material
We studied 39 patients with biopsy-proven LD who be-
longed to 26 unrelated families, 14 of which were inbred.
Families LD3, LD4, LD6, LD9, LD12, LD27, LD28 and
LD33 have 2 or more affected offspring. These eight multi-
plex pedigrees and the 14 consanguineous families were used
for linkage analyses. This study was approved by the Human
Subjects Protection Committees at the UCLA School of
Medicine and the West Los Angeles DVA Medical Center.
Each participating patient, or the responsible adult on behalf
of minors or deceased relatives, signed an informed consent
form before a blood sample was drawn.
DNA Analyses and Genotyping
DNA was extracted from 200 ml of peripheral blood using
the QIAamp Blood kit (Qiagen, Inc, Valencia, CA), and
highly polymorphic short tandem repeats or microsatellites
were typed using the method of Weber and May.15 We used
30 microsatellites in 6q23-25, including D6S314, D6S471,
D6S453, D6S308, D6S409, D6S1003, D6S1010,
D6S1049, D6S1703, D6S1042, D6S311, and D6S420. The
order of the markers had been firmly established in our po-
sitional cloning of the LD gene using high-resolution yeast
artificial chromosome and P1-derived artificial chromosome
mapping.13,14
Linkage Analyses
Model-dependent pairwise linkage analyses were performed
using the MLINK portion of the LINKAGE program pack-
age16 and GENEHUNTER,17 assuming a single autosomal
recessive gene with gene frequency 0.001 and 100% pen-
etrance.12 Unaffected family members under 20 years of
age were classified as unknown. The admixture test in the
HOMOG program was used to test for genetic heterogene-
ity. Haldane’s mapping function was used to convert recom-
bination fractions to map distances.18
Results
We observed extended areas of homozygosities, ranging
from 2.7 to 31 cM, in 15 offspring belonging to 10
families. Of these 10 families, 6 with homozygosities
were consanguineous, but 4 families (LD15, LD16,
LD17, LD20) with homozygosities did not report
consanguinity.
Of the 14 families reporting consanguinity, 4 (LD7,
LD27, LD28 @Quebec, Canada#, and LD25 @Saudi
Arabia#) did not show evidence of homozygosities
among the chromosome 6q23-25 markers. This made
us suspect heterogeneity. As a result, we performed
pairwise linkage analyses between LD in 22 consan-
guineous or multiplex families and chromosome 6q
markers D6S471, D6S310, and D6S311 under the as-
sumption of heterogeneity. LOD scores excluded link-
age to chromosome 6q23-25 markers in Families LD7,
LD25, LD27, and LD28. LOD scores for Families
LD6 and LD12 were in the positive range but were
not significant.
Next, we tested for homogeneity in the 22 multiplex
or consanguineous families using pairwise LOD scores
for D6S471, D6S310, and D6S311 (Table). H2 versus
H1 showed significance ( p 5 0.0009 for D6S311 and
p 5 0.004 for D6S310), supporting the hypothesis of
linkage with heterogeneity, and provided an estimated
a of 0.75 (95% CI, 0.05– 0.99) for D6S311, an esti-
mated a of 0.70 for D6S310, and an estimated a of
0.85 for D6S471 for the proportion of chromosome
6q–linked families among these 22 families. The
posterior probability of linkage (Wi) for D6S471,
Table. Tests of Linkage Homogeneity
D6S471
Marker Test (df ) x2 p x2
H2 versus H1 (1) 1.526 0.2167
H1 versus H0 (1) 36.123 ,0.0001
H2 versus H0 (2) 37.649 ,0.0001
The HOMOG test generates the likelihood of the data under each hypothesis, allowing the use of the likelihood ratio test. The
resulting 22 Ln (likelihood) is approximately distributed as a x2 with df equal to the difference in the number of parameters
estimated. Heterogeneity is suspected if the H1 versus H2 test is significant.
H0 5 the hypothesis of no linkage in any families; H1 5 the hypothesis of linkage in all families; H2 5 the hypothesis of
linkage in only a subset of families.
D6S310, and D6S311 favored a chromosome 6q locus
for all 22 families, except Families LD7, LD25, LD27,
and LD28. The Lafora progressive myoclonus epilepsy
traits of these latter 4 families are unlikely to be linked
to chromosome 6q24 because of their posterior proba-
bility of linkage; for example, the probability of linkage
for D6S311 was 0.0002 for Family LD7, 0.0002 for
Family LD25, and 0.0000 for Family LD27.
In addition, LOD scores (#2) were exclusionary for
many of the chromosome 6q23-25 microsatellites
(D6S314 –D6S420) in Families LD7, LD25, and
LD27. The fourth family (LD28) showed positive
LOD scores of 1.03 at u 5 0 for D6S420 and 1.54 at
u 5 0 for D6S1042. Nevertheless, because of the low
conditional probability of linkage and because a chain
of homozygous markers is not seen in the affected
members as shown by their haplotypes, it is unlikely
that the LD gene in this consanguineous family
(LD28) is located in the chromosome 6q23-25 region.
Haplotype Analyses
In almost all cases, consanguineous unions of first-
degree cousins resulting in rare autosomal recessive dis-
orders are due to transmission by the carrier parents of
two copies of the same mutation that they inherited
from their common grandparent.19 Similarly, geno-
types of microsatellite markers in the vicinity of the
gene are expected to be the same on both chromo-
somes carrying the mutated gene.
In Families LD7 and LD28 from Quebec and Fam-
ily LD25 from Saudi Arabia, the parents of the affected
individual are first-degree cousins. There is no chain of
homozygous markers in the 6q23-25 LD gene region.
Moreover, the affected individuals have the same hap-
lotypes for the LD region as unaffected siblings who are
past the age of possible onset of LD (.26 years old).
In Family LD27 from Quebec (Fig 1), the parents
are first-degree cousins (LD27-2, LD27-3, LD27-4,
LD27-5). There is no chain of homozygous markers in
the living affected individual (LD27-6). He and his
healthy 16-year-old sister (LD27-7) have the same hap-
D6S310 D6S311
p x2 p
8.115 0.004 11.092 0.0009
38.725 ,0.0001 38.683 ,0.0001
46.840 ,0.0001 49.775 ,0.0001
Brief Communication: Minassian et al: Lafora’s Disease 263

lotypes. Although LD27-2 and his brother, LD27-4,
both have sons with LD, the haplotype they share
(boxed haplotype, Fig 1) is not transmitted to LD27-
2’s son, LD27-6. Similarly, the haplotype common to
Sisters LD27-3 and LD27-5 (shaded haplotypes, see
Fig 1) is not transmitted to LD27-6.
Biopsies
The biopsy slides of LD patients belonging to Families
LD7, LD25, LD27, and LD28 were carefully reana-
lyzed by two senior neuropathologists (U.T. and S.C.),
who reconfirmed the presence of Lafora inclusions.
Figure 2 shows an example of a skin biopsy of an af-
Fig 2. Example of Lafora inclusion body in a family member
(LD25-9) afflicted with the clinical disease but whose family
(LD25) does not genetically link to chromosome 6q24. Oval
inclusions in peripheral cells of an eccrine duct stain strongly
positive with periodic acid-Schiff–hematoxylin. Bar 5
20 mm.
264 Annals of Neurology Vol 5 No 2 February 1999
Fig 1. Haplotypes of Family LD27 suggest a
locus outside chromosome 6q23-25. An open
square means an unaffected male individual,
an open circle means an unaffected female
individual, and a filled symbol means an
affected individual. A double line indicates
a consanguineous union. A diagonal line
through a filled symbol signifies a deceased
individual. The vertical columns of numbers
indicate genotypes for each of the microsatel-
lites arranged in proper physical order from
centromere to telomere, according to a yeast
artificial chromosome and P1-derived artifi-
cial chromosome contig of chromosome
6q23-25.
fected individual from Family LD25 that revealed
prominent PAS-positive oval inclusions in peripheral
cells of eccrine ducts. Hepatocytes in liver biopsies
from the affected individuals of Family LD27 and au-
topsy brain tissue from a deceased member of Family
LD28 revealed numerous large Lafora bodies with a
dark core and paler peripheral zone.
Discussion
Three of the four families who do not show linkage to
chromosome 6q23-25 are from the French-Canadian
“isolate” of Quebec (LD7, LD27, LD28). None
showed homozygous 6q23-25 microsatellites, despite
being products of first-cousin marriages. Recombina-
tions could have eliminated homozygosities from
around the gene in affected individuals. Chances for
such strategically placed recombinations resulting in
elimination of homozygosities are more likely to occur
in families where the relationship is distant, however.
Recombinations eliminating homozygosities are least
likely to occur in offspring of first-degree cousins, be-
cause the number of generations separating the com-
mon grandparent from the affected individuals is only 2.
Nevertheless, the absence of homozygosities in first-
degree consanguineous families such as LD7, LD25,
LD27, and LD28 is not of itself absolute proof of ex-
clusion of the LD gene from the 6q23-25 region. It is
still possible, although highly unlikely, that strategic re-
combinations within the narrow LD gene region might
have occurred on either side of the gene even within
the 2 generations separating the founder haplotype
from the one seen in the affected individuals. This
could offer a possible explanation for the positive LOD
scores in Family LD28.
Taking all the data into account, including an a es-
timate of 75 to 85% of LD families linked to chromo-
some 6q23-25, low conditional probability of linkage
in 4 consanguineous families who lack homozygosities,
sharing of 6q23-25 haplotypes by affected and unaf-
fected individuals in these 4 families, and exclusionary
LOD scores in 3 of these 4 families, one has to accept
the possibility of another genetic locus in LD.
This study was supported by NIH grant NS21908 to Dr Delgado-
Escueta, the DVA West Los Angeles Medical Center, The Hospital
for Sick Children, Bloorview Children’s Hospital Foundation, and
the Lafora’s Disease Associations of Quebec (Odette Malenfant) and
Sweden (Vera Faludi).
We thank Dr Jacques Thibeault for providing the biopsies on Fam-
ily LD28.
References
1. Lafora GR. Uber das vorkommen amyloider korperchen im in-
nern der ganglienzellen; zugleich ein beitrag zum studium der
amyloiden substanz im nervensystem. Virchows Arch A Pathol
Anat Histopathol 1911;205:295–303
2. Lafora GR, Glueck B. Contribution to the histopathology and
pathogenesis of myoclonic epilepsy. Bull Gov Hosp Insane
1911;3:96
3. Harriman DGF, Millar JHD. Progressive familial myoclonic
epilepsy in three families: its clinical features and pathological
basis. Brain 1955;78:325–349
4. Schwarz GA, Yanoff M. Lafora’s disease. Distinct clinicopatho-
logic form of Unverricht’s syndrome. Arch Neurol 1965;12:
172–188
5. Nishimura RN, Ishak KG, Reddick R, et al. Lafora disease:
diagnosis by liver biopsy. Ann Neurol 1980;8:409 – 415
6. Carpenter S, Karpati G. Sweat gland duct cells in Lafora
disease: diagnosis by skin biopsy. Neurology 1981;31:1564 –
1568
7. Busard HLSM, Gobreels-Festen AAWM, Renih WU, et al. Ax-
illa skin biopsy; a reliable test for the diagnosis of Lafora’s dis-
ease. Ann Neurol 1987;21:599 – 601
8. Van Heycop Ten Ham MW. Lafora disease, a form of progres-
sive myoclonus epilepsy. Handbook of Clinical Neurology
1974;15:382– 422
9. Roger J, Pellissier JF, Bureau M, et al. Le diagnostic précoce de
la maladie de Lafora. Importance des manifestations paroxys-
tiques visuelles et intérêt de la biopsie cutanée. Rev Neurol
(Paris) 1983;139:115–124
10. Schwarz GA. Lafora’s disease: a disorder of carbohydrate me-
tabolism. In: Goldensohn ES, Appel SH, eds. Scientific ap-
proaches to clinical neurology. Philadelphia: Lea & Febiger,
1977:148 –159
11. Serratosa JM, Delgado-Escueta AV, Posada I, et al. The gene
for progressive myoclonus epilepsy of the Lafora type maps to
chromosome 6q. Hum Mol Genet 1995;4:1657–1663
12. Sainz J, Minassian BA, Serratosa JM, et al. Lafora’s progressive
myoclonus epilepsy: narrowing the 6q24 locus by recombinations
and homozygosities. Am J Hum Genet 1997;61:1205–1209
13. Minassian BA, Lee JR, Hherbrick JA, et al. Mutations in a gene
encoding a novel protein tyrosine phosphatase cause progressive
myoclonus epilepsy, Lafora type. Nat Genet 1998;20:171–174
14. Serratosa JM, Gomez-Garre P, Anta B, et al. A novel protein ty-
Copyright © 1999 by the American Neurological Association
rosine phosphatase gene is mutated in progressive myodonus epi-
lepsy of the Lafora type (EPM2). Hum Mol Genet 1999,8 (In
press)
15. Weber JL, May PE. Abundant class of human DNA polymor-
phisms which can be typed using the polymerase chain reac-
tion. Am J Hum Genet 1989;44:388 –396
16. Lathrop GM, Lalouel JM, Julier C, Ott J. Multilocus linkage
analysis in humans: detection of linkage and estimation of re-
combination. Am J Hum Genet 1985;37:482– 498
17. Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES. Parametric
and nonparametric linkage analysis: a unified multipoint ap-
proach. Am J Hum Genet 1996;58:1347–1363
18. Haldane JBS. The combination of linkage values and the cal-
culation of distances between the loci of linked factors. J Genet
1919;8:299 –309
19. Lander ES, Botstein D. Homozygosity mapping: a way to map
human recessive traits with the DNA of inbred children. Sci-
ence 1987;236:1567–1570
Three-Dimensional Tracking
of Axonal Projections in
the Brain by Magnetic
Resonance Imaging
Susumu Mori, PhD,* Barbara J. Crain, MD, PhD,†
V. P. Chacko, PhD,* and Peter C. M. van Zijl, PhD*
The relationship between brain structure and complex
behavior is governed by large-scale neurocognitive net-
works. The availability of a noninvasive technique that
can visualize the neuronal projections connecting the
functional centers should therefore provide new keys to
the understanding of brain function. By using high-
resolution three-dimensional diffusion magnetic reso-
nance imaging and a newly designed tracking approach,
we show that neuronal pathways in the rat brain can be
probed in situ. The results are validated through compar-
ison with known anatomical locations of such fibers.
Mori S, Crain BJ, Chacko VP, van Zijl PCM.
Three-dimensional tracking of axonal projections
in the brain by magnetic resonance imaging.
Ann Neurol 1999;45:265–269
The white matter fibers in the brain are essential in
linking functional regions. These neuronal projections
have been traced in experimental animals, using inva-
sive in vivo experiments,1 but comparable human data
From the *Department of Radiology, Division of MRI Research,
and †Department of Pathology, Johns Hopkins Medical School,
Baltimore, MD.
Received Aug 17, 1998, and in revised form Oct 21. Accepted for
publication Oct 23, 1998.
Address correspondence to Dr Mori, 217 Traylor, 720 Rutland Ave,
Baltimore, MD 21205.
265
are necessarily much more limited. The availability of a
noninvasive method for fiber tracking therefore would
have a tremendous impact on our understanding of
normal and abnormal brain function. Diffusion-
weighted magnetic resonance imaging (MRI) allows in
vivo mapping of the diffusional properties of brain wa-
ter, and has revealed a high degree of diffusional an-
isotropy (directionality) in white matter.2–11 Despite
many studies of this anisotropy and the production of
vector pictures showing fiber orientation within the
volume elements (voxels) of image planes, the actual
reconstruction of neuronal projections by tracking of
these vectors has never been accomplished. In the
present study, we demonstrate an approach called fiber
assignment by continuous tracking (FACT), which is
able to achieve such high-resolution three-dimensional
(3D) tracking of axonal projections. The results are
subsequently validated by in situ tracking of known fi-
ber tracts in a rat brain.

Materials and Methods

The principle of water-diffusion anisotropy is shown in Fig-
ure 1a. For a region where axons are aligned, water is re-
stricted in the direction perpendicular to the axons and dif-
fuses preferentially in a direction parallel to them. This
situation can be represented mathematically by a so-called
diffusion ellipsoid5–11 (see Fig 1b), characterized by diffusion
constants l1, l2, and l3 along its three orthogonal directions
and the (vector) direction of the longest axis (l1). For exam-
ple, l1 .. l2 5 l3 (anisotropic diffusion) suggests the ex-
istence of cylindrical structures preferentially aligned along
l1, whereas l1 5 l2 5 l3 (isotropic diffusion) suggests
sparse or unaligned axons. Although diffusion anisotropies
have been detected previously in white matter, until now it
has not been possible to translate these into neuronal trajec-
tories. This problem is related to difficulties in the postpro-
cessing reconstruction of 3D fiber structures from diffusion
tensor MRI data, where decisions have to be made concern-
ing the connections between voxels of the image. This is il-
lustrated in Figure 1c, where the fibers (long curved arrows)
are assumed to be confined to the two-dimensional (2D)
plane and the fiber direction within each voxel is indicated
by a straight open arrow. Starting from the voxel with an
asterisk, tracking should follow the bold curved arrow. The
most intuitive way to perform this tracking is by connecting
each voxel to the adjacent one toward which the fiber direc-
tion is pointing. However, when using this approach, the
tracking (indicated by the dotted voxels) often deviates from
the true fiber orientation, because the choice of direction is
limited to only eight angle ranges (26 in the case of 3D) (see
Fig 1c). This problem is avoided when tracking a continuous
rather than a discrete vector field (see Fig 1d). Here, tracking
is initiated from the center of a voxel and proceeds according
to the vector direction. At the point where the track leaves
Fig 1. Principle of fiber tracking. (a) Schematic view of re-
stricted water diffusion (solid sphere) in an environment with
strongly aligned fibers (depicted by bars). The diffusion proper-
ties can be fully described by an ellipsoid (b) with three prin-
cipal axes (l1, l2, and l3 ) of which the orientation of the
main axis represents the average fiber direction. Once this
direction is determined in each voxel, tracking can be per-
formed by using either a discrete (c) or a continuous (d) num-
ber field. The actual fibers are indicated by curved arrows,
and the average fiber directions in the voxel are displayed as
open arrows. The connected voxels resulting from tracking are
shaded, using dots. The discrete approach leads to deviation
from the actual fiber to be tracked (c), whereas the continuous
approach succeeds as indicated by the train of solid arrows
(d). A three-dimensional axonal projection can be tracked as
long as nearby vectors are strongly aligned (e). When vector
orientation becomes random, as judged quantitatively from
summation of the inner products of these vectors (Eq 1), the
tracking is ended ( f ).
the projection is judged based on the occurrence of sudden
transitions in the fiber orientation (see Fig 1e and f). The
severity of such a transition is quantified through a parame-
ter R, presenting the summation of the inner products of
nearby data points:
O O abs~n
s s

the voxel and enters the next, its direction is changed to that R5 l 1i z n l 1j!/s~s 2 1! (1)
of the neighbor. Due to the presence of continuous inter- i j
cepts, this tracking now connects the correct voxels and the
actual fiber (bold straight arrows in Fig 1d) can be assigned. where nl1 is the unit vector representing the longest princi-
We therefore dubbed this approach FACT. The end point of pal diffusion axis (l1) and s is the number of data points

266 Annals of Neurology Vol 45 No 2 February 1999

referenced. When adjacent fibers are aligned strongly (see Fig
1e), the R value is large, while it becomes small (see Fig 1f)
in regions without continuity in fiber direction. Theoreti-
cally, there are many ways to modify this criterion. In the
present study, we calculated R values by using the four
neighboring voxels closest with respect to the continuous
tracking position, and a fiber was judged discontinued for
R , 0.8. The procedure of mapping the neuronal connec-
tions is started through the input of an arbitrary point in 3D
space, after which the extent of the axonal projections into
functional regions is traced in both the orthograde (forward)
and the retrograde (backward) directions.
Studies were performed on a 400 MHz GE Omega (9.4
T), using a field of view of 32 3 16 3 16 mm and 128 3
64 3 64 data points zero-filled to a final resolution of
256 3 128 3 128. Diffusion images were recorded by using
a spin-echo Stejskal-Tanner sequence (echo time [TE] 5 44
msec) and 10 gradient orientations. Gradient strength and
length were 16 G/cm and 5 msec, with a separation of 16
msec. For an additional image with low diffusion weighting,
a strength of 5 G/cm was used in the xyz direction. Diffusion
tensor elements at each voxel were determined by multivari-
ant least-square fitting weighted by signal-to-noise ratio.12
The tensors were diagonalized to obtain l1, l2, and l3
and the direction of l1. The brain sample was obtained from
an adult Sprague–Dawley rat and fixed on 5% formalin for
2 weeks.
Results
Figure 2a shows a 2D representation of the water-
diffusion measurements. Although existence of axonal
projections can be clearly appreciated from this 2D
vector picture, tracing of neuronal projections through
the brain requires a complete 3D analysis. After ex-
tending the diffusion MRI into 3D, we applied the
FACT analysis to track 10 different pathways in a
formalin-fixed rat brain (Fig 3; see Fig 2b). It can be
seen that our method successfully reconstructs the well-
known structure of these projections. To further illus-
trate the validity of our approach, the tracking of the
anterior commissure is also shown in a series of 2D
planes and compared with the corresponding rat brain
atlas from Paxinos and Watson13 (see Fig 3d–f). The U
shape of this structure can be easily appreciated from
the atlas and it can be clearly seen that our diffusion
result (dark blue) accurately traces this projection.
These 2D slices also show part of the tracking of ol-
factory tract (ol), middle forebrain bundle (mfb), and
fornix (f).
One interesting aspect of Figure 2a is the demon-
stration of aligned fibers in most areas of gray matter.
Such anisotropy of gray matter has been reported pre-
viously, using diffusion measurements in two spatial
orientations,14 and our results extend on this earlier
finding by showing the ordered fiber structures that
cause the anisotropy.
Brief Communication: Mori et al: 3D Brain Fiber Tracking
Discussion
Our results from using the FACT approach show that
it is now possible to track the 3D structure of axonal
projections by using the diffusion MRI technique. Al-
though this is very promising, some limitations should
be pointed out. First, MRI can only give information
on the average axonal orientation within a voxel.
Therefore, it is resolution dependent and it cannot dis-
tinguish among several very small projections that are
immediately adjacent to each other. Second, the track-
ing method is unable to distinguish between afferent
and efferent fibers. Further problems may occur if por-
tions of two pathways actually touch, because the pro-
gram may inadvertently switch pathways. For example,
the initial tracking of the optic tract was complicated
when reaching the level of the dorsal lateral geniculate,
where the algorithm began a partial tracking of adja-
cent fimbrial axons. Other limitations are functions of
the current algorithm. For example, one of the most
challenging problems is the tracking of branched axons
or axons leaving major pathways as they approach their
targets. Our program traces only one of the branches
in such a situation. An example is the tracking of the
lateral olfactory tract (see Fig 3) for which the anterior
portion is quite faithfully followed (see arrow in Fig 3d
and e), but where the fibers that are actually identified
in Figure 3f are small branches into olfactory and piri-
form cortex (indicated by dotted lines in the atlases of
Fig 3d–f). As a result, the lateral olfactory tract itself,
the location of which is still visible in the T2-weighted
MRI scan (see arrow in Fig 3f), is not labeled at this
level. Possible solutions to this problem depend on the
specific question. For instance, if the goal is to follow
various components of a pathway to their end points,
one might simply identify multiple points in a large
fiber bundle and track each component separately.
In summary, we introduced a 3D tracking method
for diffusion MRI that allows the successful identifica-
tion of axonal projections. Potential applications of this
method include the study of neuronal development
and of diseases affecting neuronal integrity. In the
present study, a total of 12 hours were needed for the
completion of the study. This time period can be easily
shortened by a factor of 8 to 32 by using multiple-
echo acquisition techniques.15–17 Combined with re-
cent methodological advances for the suppression of
motion-related artifacts,18 –20 we expect clinical appli-
cation of this fiber-reconstruction technique to be fea-
sible in the near future.
This research was funded in part by a grant from Johns Hopkins
School of Medicine, the American Federation of Aging Research,
and the Whitaker Foundation.
267
 Fig 2. Two-dimensional (2D) and three-dimensional (3D) track-
ing of prominent axonal projections. (a) 2D vector field presenta-
tion in the parietal lobe of a rat brain as localized from a section
of the T2-weighted magnetic resonance imaging scan. Because
axonal orientations are projected onto the 2D plane, axons per-
pendicular to the plane appear as dots. Rough boundaries for
regions of prominent fiber bundles are indicated by color lines
(yellow 5 corpus callosum [cc] and external capsule [ec];
green 5 fimbria [ fi]; and red 5 internal capsule [ ic]. Notice
the presence of preferential axonal orientation in the gray matter.
(b) 3D presentation of the fibers. Color coding is the same as in
(a) except for the blue color that shows axons tracked from the
splenium of corpus callosum into the external capsule. Some axons
within the fimbria are tracked into ventral hippocampal commis-
sure, and axons within the internal capsule are tracked into the
corpus callosum.

Fig 3. Three-dimensional (3D) projections and

two-dimensional (2D) validation of eight fiber
bundles in the rat brain. The results of the track-
ing are superimposed on 3D volume images, using
an oblique angle (a) or three orthogonal angles (b
and c). The vertical lines between (b) and (c)
indicate the positions of the 2D axial slices of
T2-weighted images on which the position of the
tracking is superimposed (d–f ). Images from the
rat brain atlas13 corresponding to the three slices
in (d–f ) are shown in the bottom row. Color
codes are as follows: green 5 fimbria; dark
blue 5 anterior commissure (ac); light blue 5
medial forebrain bundle (mfb); yellow 5 fornix
(f); white 5 stria terminalis; pink 5 stria med-
ullaris; red 5 optic tract; peach 5 lateral olfac-
tory tract (ol).

268 Annals of Neurology Vol 45 No 2 February 1999

References
1. Heimer L, Robards MJ. Neuroanatomical tract-tracing meth-
ods. New York: Plenum Press, 1981
2. Chenevert TL, Brunberg JA, Pipe JG. Anisotropic diffusion in
human white matter: demonstration with MR technique in
vivo. Radiology 1990;177:401– 405
3. Moseley ME, Cohen Y, Kudarczyk J, et al. Diffusion-weighted
MR imaging of anisotropic water diffusion in cat central ner-
vous system. Radiology 1990;176:439 – 445
4. Moonen CTW, Prkar J, de Vleeschouner MH, et al. Restricted
and anisotropic displacement of water in healthy cat brain and
in stroke studied by NMR diffusion imaging. Magn Reson
Med 1991;19:322–327
5. Basser PJ, Mattiello J, Le Bihan D. MR diffusion tensor spec-
troscopy and imaging. Biophys J 1994;66:259 –267
6. Hsu EW, Mori S. Analytical interpretations of NMR diffusion
measurements in an anisotropic medium and a simplified
method for determining fiber orientation. Magn Reson Med
1995;34:194 –200
7. Pierpaoli C, Jezzard P, Basser PJ, et al. Diffusion tensor MR
imaging of human brain. Radiology 1996;201:637– 648
8. Ulug AM, Bakht O, Bryan RN, van Zijl PCM. Mapping of
human brain fibers using diffusion tensor imaging. In: Proceed-
ings of the International Society for Magnetic Resonance in
Medicine. 1325 New York: 1996:1325
9. Nakada T, Matsuzawa H. Three-dimensional anisotropy con-
trast magnetic resonance imaging of the rat nervous system:
MR axonography. Neurosci Res 1995;22:389 –398
10. Makris N, Worth AJ, Sorensen AG, et al. Morphometry of in
vivo human white matter association pathways with diffusion
weighted magnetic resonance imaging. Ann Neurol 1997;42:
951–962
11. Conturo TE, McKinstry RC, Aronovitz JA, Neil JJ. Diffusion
MRI: precision, accuracy and flow effects. NMR Biomed 1995;
8:307–332
12. Basser PJ, Mattiello J, LeBihan D. Estimation of the effective
self-diffusion tensor from the NMR spin echo. J Magn Reson B
1994;103:247–254
13. Paxinos G, Watson C. The rat brain in stereotaxic coordinates,
4th ed. San Diego: Academic Press, 1998
14. Thornton JS, et al. Anisotropic water diffusion in white
and gray matter on the neonatal piglet brain before and after
transient hypoxia-ischaemia. Magn Res Imaging 1997;15:433–
440
15. Mansfield P. Multi-planar image formation using NMR spin-
echoes. J Phys 1977;C10:L55–L58
16. Hennig J, Nauerth A, Friedburg H. RARE imaging: a fast im-
aging method for clinical MR. Magn Reson Med 1986;3:823–
833
17. Beaulieu CF, Zhou X, Cofer GP, Johnson GA. Diffusion-
weighted MR microscopy with fast spin-echo. Magn Reson
Med 1993;30:201–206
Brief Communication: Mori et al: 3D Brain Fiber Tracking
18. Ordidge RJ, Helpern JA, Qing ZX, et al. Correction of mo-
tional artifacts in diffusion-weighted NMR images using navi-
gator echoes. Magn Reson Imaging 1994;12:455– 460
19. Anderson AW, Gore JC. Analysis and correction of motion ar-
tifacts in diffusion weighted imaging. Magn Reson Med 1994;
32:379 –387
20. Mori S, van Zijl PCM. A motion correction scheme by twin-
echo navigation for diffusion weighted magnetic resonance im-
aging with multiple RF echo acquisition. Magn Reson Med
1998;40:511–516
Correction
The abstract by Yamasaki and colleagues that appeared
in the September issue (Ann Neurol 1998;44:490 –
491) and the program of the American Neurological
Association’s 123rd Annual Meeting (pp 76 –77)
contained an error of copy editing that distorted
its meaning. The full text of the corrected abstract
appears here:
T193. A Protein Critical for Immune-Mediated
Demyelinating Disease
Kenji Yamasaki, G. D. Ghadge, and R. P. Roos; Chicago, IL
DA and other members of the TO subgroup of Theiler’s
murine encephalomyelitis virus persistently infect mice and
cause a chronic demyelinating disease that resembles multiple
sclerosis. Both diseases have a similar pathology, and in both
the immune system plays a role in pathogenesis. In contrast,
members of a GDVII subgroup produce an acute fatal en-
cephalomyelitis and do not persist. We previously reported
that demyelinating (but not nondemyelinating) strains have
an initiation codon for translation for a protein (L*) out of
frame with the viral polyprotein; mutant DA virus that does
not have the L* initiation codon fails to induce a persistent
infection or demyelination. These data suggest that L* is crit-
ically important for viral persistence and demyelination. To
further test the role of L* in demyelinating disease, we have
now engineered the GDVII viral genome to contain the L*
initiation codon and to synthesize L*. The efficiency of ex-
pression of L* in mutant GDVII depends on particular nu-
cleotides in an RNA stem loop structure near the L* initia-
tion codon. Mutant GDVII virus that synthesizes L* is no
longer neurovirulent. The presence of demyelination and vi-
rus persistence in survivors is under study.
Study supported by the National Multiple Sclerosis Society.
The publisher apologizes for the error.
269