 |
|
A Novel Mutation in the Notch3 Gene in an Italian Family With Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy
Genetic and Magnetic Resonance Spectroscopic Findings
Rosario L. Oliveri, MD, MSc;
Maria Muglia, PhD;
Nicole De Stefano, MD, PhD;
Rosalucia Mazzei, PhD;
Angelo Labate, MD;
Francesca L. Conforti, PhD;
Allessandra Patitucci, PhD;
Anna L. Gabriele, PhD;
Giuseppe Tagarelli, PhD;
Angela Magariello, PhD;
Mario Zappia, MD;
Antonio Gambardella, MD;
Antonio Federico, MD;
Aldo Quattrone, MD
Arch Neurol. 2001;58:1418-1422.
ABSTRACT
| |
Background Cerebral autosomal dominant arteriopathy with subcortical infarcts and
leukoencephalopathy (CADASIL) is a hereditary syndrome caused by mutations
of the Notch3 gene, usually localized to exons 3
and 4.
Objectives To report a novel pathogenetic mutation occurring in exon 6 of the Notch3 gene, a location not previously recognized in patients
with CADASIL, and to report the results of magnetic resonance spectroscopy
in CADASIL.
Methods Mutation analysis of the Notch3 gene was performed
in 2 patients belonging to a large kindred manifesting CADASIL, as well as
in 7 clinically unaffected members of the family and 200 control chromosomes.
Proton magnetic resonance spectroscopy was used to estimate metabolite resonance
intensities in the 2 affected subjects.
Results Sequence analysis of the Notch3 gene showed
a new missense mutation CGC TGC in codon 332 of exon 6, resulting in
the replacement of an arginine residue with a cysteine. This mutation was
never observed in the 7 unaffected members of the family and the 200 control
chromosomes examined. Proton magnetic resonance spectroscopy showed a diffuse
decrease in cerebral N-acetylaspartate, indicating
the presence of widespread axonal damage.
Conclusions Our findings emphasize the role of direct DNA sequence analysis for
the diagnosis of CADASIL. Moreover, the results of proton magnetic resonance
spectroscopy suggest that widespread axonal damage may be an early finding
of the disease.
INTRODUCTION
CEREBRAL autosomal dominant arteriopathy with subcortical infarcts and
leukoencephalopathy (CADASIL) is an adult-onset hereditary syndrome characterized
by recurrent transient ischemic attacks and strokes.1
Mutations within the Notch3 gene (chromosome 19p13.1)
have been identified as the underlying genetic defect associated with CADASIL.2 The genetic diagnosis can be established by linkage
analysis, or by direct sequencing of the Notch3 gene
demonstrating missense mutations involving the loss or gain of a cysteine
residue.2, 3 Most of these mutations
have been localized to exons 3 and 4 in previous studies.2, 4
Herein we report a novel mutation within exon 6 of the Notch3 gene, identified in a kindred from southern Italy with CADASIL.
Clinical and neuroimaging data are also presented for affected individuals.
SUBJECTS AND METHODS
FAMILY
The simplified pedigree of the family is shown in Figure 1. Data on the medical histories of 8 previously deceased
subjects were ascertained through medical records and detailed accounts from
the older members of the family at the time of the study. Family members younger
than 18 years were not included in the study. Two living affected members
(III:16 and III:17) were studied in detail. The phenotype of CADASIL in all
other deceased subjects with the disease was quite uniform and characterized
by migraine with and without aura and recurrent strokes developing from the
third to fourth decades of life, progressing to a pseudobulbar palsy, spastic
quadriparesis, and dementia. Death occurred in the fifth to sixth decades.
|
|
|
|
Figure 1. Simplified pedigree of the southern
Italian kindred with cerebral autosomal dominant arteriopathy with subcortical
infarcts and leukoencephalopathy. Squares represent males; circles, females.
Filled symbols represent affected individuals. A diagonal line through the
symbol represents a deceased person.
|
|
|
Subject III:16
The proband was a 42-year-old, right-handed man. At age 37 years, he
experienced a sudden onset of right hemiparesis, which resolved partially
in 1 month. One year later, he manifested diplopia, which disappeared in 10
days. At age 41 years, he experienced another acute attack characterized by
right hemiparesis and dysarthria, followed by partial recovery. A few months
later, he noted a worsening of both dysarthria and hemiparesis and reported
to our department for hospitalization.
Neurologic examination showed right hemiparesis, dysarthria, diffuse
brisk deep tendon reflexes, right-sided Babinski sign, and bilateral dysmetria.
Results of routine blood examination and cerebrospinal fluid analysis were
unremarkable. Very-long-chain fatty acids, vitamin E, and arylsulfatase A
levels were within normal limits. Mutation screening for the mitochondrial
myopathy, encephalopathy, lactacidosis, and stroke syndrome was negative,
and no mutations were found in the factor V Leiden or prothrombin genes. Results
of electromyography and electroneurography were normal, as were multimodality
evoked potentials. Neuropsychological testing indicated diffuse cognitive
impairment, particularly evident for memory and executive functions.
Subject III:17
A 32-year-old, right-handed man, the brother of the proband, experienced
an acute episode characterized by left hemiparesis, together with bladder
and emotional incontinence, at 30 years of age. These symptoms partially resolved.
Neurologic examination showed mild left hemiparesis, diffuse brisk deep tendon
reflexes, left-sided Babinski sign, and pseudobulbar palsy. Results of routine
blood examination were unremarkable, and cerebrospinal fluid analysis gave
normal results. Very-long-chain fatty acids, vitamin E, and arylsulfatase
A levels were within normal limits. The results of neurophysiological and
neuropsychological testing were normal.
GENETIC ANALYSIS
Mutation analysis of the Notch3 gene of these
2 affected subjects was undertaken. Genetic analysis was also performed in
7 clinically unaffected members of the family, including members II:9, III:14,
and III:15. Genomic DNA was extracted from peripheral blood after informed
consent had been obtained. Polymerase chain reaction (PCR) was performed with
primers specific for exons of the Notch3 gene. Because
most previously described mutations occur in exons 3 and 4, these regions
were screened first. When no mutations were detected in these exons, we designed
primer pairs comprising the intron-exon boundaries and sequenced the remaining
exons. The PCR products were sequenced by means of forward primers (ABI Prism
Dye Terminator Cycle Sequencing Ready Reaction Kit; Perkin Elmer, Foster City,
Calif). To confirm the mutation, PCR products were digested with MvnI restriction enzyme. To control for polymorphisms at this site,
the Notch3 genes of 100 unrelated healthy white subjects
originating from the same geographic area as the affected patients were examined.
IMAGING
Magnetic resonance (MR) imaging and MR spectroscopy were used to estimate
metabolite resonance intensities in the 2 affected subjects. Combined brain
proton (1H)–MR imaging and MR spectroscopy was performed
with a scanner (Philips Gyroscan NT; Philips Medical Systems, Best, the Netherlands)
operating at 1.5 T. Conventional multislice spin-echo and fluid-attenuated
inversion recovery images were obtained in transverse planes parallel to the
anterior commissure–posterior commissure line. The fluid-attenuated
inversion recovery MR images were then used to position an intracranial volume
of interest (VOI) for spectroscopy parallel to the anterior commissure–posterior
commissure line and centered craniocaudally on the corpus callosum. The VOI
measured approximately 100 mm anteroposteriorly x 20 mm craniocaudally
x 90 mm left to right, thus including a large proportion of the brain
white matter. Two-dimensional spectroscopic images were obtained with a point-resolved
spectroscopy sequence for volume selection (repetition time, 2000 milliseconds;
echo time, 272 milliseconds; 250 x 250-mm field of view; 32 x
32 phase encoding steps; 1 signal average per step), as previously described.5 Raw data were then postprocessed as previously described.5 Metabolite resonance intensities of N-acetyl groups (mainly N-acetylaspartate
[NAA], a marker of axonal integrity), choline (mainly from choline-containing
phospholipids [Cho], reflecting myelin integrity), and creatine (Cr) were
determined automatically from peak areas relative to a spline-corrected baseline.6 As Cr is evenly distributed throughout the brain and
relatively refractory to change, metabolite resonance intensity values were
normalized to Cr resonance intensity. Metabolite ratios were considered abnormal
if they were more than 2 SDs outside the mean values derived from 20 normal
adult control subjects. In this study, metabolite ratios were expressed as
a global value, which was obtained by averaging the metabolite/Cr for all
the voxels in the spectroscopic VOI for each subject.
RESULTS
GENETIC ANALYSIS
Sequence analysis of exon 6 showed a new missense mutation CGCTGC
in codon 332, resulting in the replacement of an arginine residue with a cysteine
(Figure 2). The restriction enzyme MvnI recognizes the sequence CGCG, and the CT transition
causes the loss of an MvnI restriction site in the
mutant PCR product. The MvnI cleaves the wild-type
PCR product of 279 base pairs (bp) into 224-bp and 55-bp digestion fragments,
but does not cleave the corresponding region of the Notch3 gene in patients with CADASIL. We demonstrated the occurrence of this
mutation in the affected subjects of the family, in none of the 7 unaffected
members of the family, and in none of the 200 control chromosomes examined.
|
|
|
|
Figure 2. Notch3 exon sequence
analysis. Sequencing with forward primer shows a heterozygous missense mutation
CGCTGC in codon 332 of exon 6 in an affected individual (A, arrow),
but not in an unaffected member (B).
|
|
|
IMAGING
The fluid-attenuated inversion recovery and T2-weighted MR images showed
diffuse hyperintense lesions in white matter of both subjects. The 1H-MR spectroscopy showed diffuse decreases in brain NAA/Cr (NAA/Cr =
2.58 and 2.68 in subjects III:1 and III:7, respectively, and mean NAA/Cr =
3.05 ± 0.19 in normal controls), whereas values of Cho/Cr were within
normal limits and lactate signals were not found (Figure 3).
|
|
|
|
Figure 3. Proton brain magnetic resonance
imaging and magnetic resonance spectroscopy in a normal subject (A) and a
patient (III:3) with cerebral autosomal dominant arteriopathy with subcortical
infarcts and leukoencephalopathy (CADASIL) (B). Conventional transverse magnetic
resonance image of the patient with CADASIL shows diffuse white matter abnormalities
(B, top). Proton spectra from representative voxels located in the deep white
matter (black square) of a normal control subject (A, bottom) and the patient
with CADASIL (B, bottom) show a significant decrease of N-acetylaspartate
(NAA) in the latter. Cho indicates choline-containing phospholipids; Cr, creatine.
|
|
|
COMMENT
Herein we describe a novel pathogenetic mutation occurring in the exon
6 of Notch3 gene, a location not previously recognized
in patients with CADASIL. Previously identified mutations in the Notch3 gene occur mainly in exons 3 and 4, although mutations have
been reported in other exons.7, 8
Similar to previously reported mutational modifications in other exons of Notch3,4, 7
this mutation is located at the 5' end of the gene and is characterized
by the gain of a cysteine residue. It has been proposed that these modifications
to the primary sequence affect the folding of Notch3
or induce inappropriate disulfide bonding between Notch3 and other cysteine-containing proteins.4
Our finding further supports the notion that mutational analysis is
a valuable tool for this complex disease, both in confirming the diagnosis
of CADASIL and, as in our case, in finding novel mutations.9
In the past few years, there has been increasing evidence that the clinical
picture of CADASIL is highly heterogeneous, ranging from severe dementia to
migraine with aura, associated with white matter abnormalities.4, 8, 10
Moreover, it has recently emerged that sporadic cases also exist, as mutation
in the Notch3 gene may occur de novo.9
In addition, although electron microscopy of skin biopsy specimens is considered
a useful screening method for the diagnosis of CADASIL,11
with the finding of the characteristic granular, electron-dense, osmiophilic
material attached to vascular smooth muscle cells, there is now evidence that
skin biopsy specimens may occasionally be negative.12
All of these findings account for the difficulty in identifying patients with
CADASIL. Thus, a mutational screening for the Notch3
gene should be performed in patients who present with the aforementioned clinical
features, regardless of the family history. A final molecular diagnosis of
CADASIL, in fact, has important implications for the patient and for the family.
The Notch3 gene, however, contains 33 coding exons
and, therefore, sequencing the whole gene is time-consuming and expensive.
Thus, as also suggested by other authors,4
a rational testing strategy is the following: the first step consists of direct
sequencing analysis of exons 3 and 4, where mutations are located in most
cases. If no mutation is found, screening of other exons that contain more
rare mutations should be pursued. If no mutation is found by the second step,
direct sequencing of the remaining exons may be performed as the last step.
Single-voxel 1H-MR spectroscopy and MR spectroscopic imaging
results have been reported by Desmond et al13
in 4 patients with CADASIL with variable disease duration. Elevated lactate
levels in the right frontal lobe of 1 patient and normal findings in 3 patients
were observed in that study.13 It is difficult
to compare the results of the previous study with those reported here, as
Desmond and coworkers used different spectroscopic techniques (single-voxel
and multivoxels) and placed their VOI for spectroscopy in different brain
regions for each patient. In the present study, with the use of a VOI always
localized to include most of the cerebral white matter (Figure 3), 1H-MR spectroscopy of our patients showed
a diffuse decrease in cerebral NAA/Cr. This suggests that marked axonal damage
can occur in the deep white matter of patients with CADASIL, even relatively
early in the course of the disease. In contrast, we did not find changes in
Cho/Cr or increases in lactate in the brains of these patients, suggesting
that neither active myelin breakdown nor substantial inflammation or mitochondrial
impairment should be expected in patients with CADASIL, at least far from
clinically relevant attacks.
In conclusion, our findings emphasize the role of direct DNA sequence
analysis for the diagnosis of CADASIL. Moreover, the results of 1H-MR
spectroscopy indicate that widespread axonal damage might be an early finding
of the disease.
AUTHOR INFORMATION
Accepted for publication February 21, 2001.
From the Institute of Neurology, University Magna Græcia, Catanzaro,
Italy (Drs Oliveri, Labate, Zappia, Gambardella, and Quattrone); Institute
of Experimental Medicine and Biotechnology, National Research Council, Mangone,
Italy (Drs Oliveri, Muglia, Mazzei, Conforti, Patitucci, Gabriele, Tagarelli,
Magariello, Gambardella, and Quattrone); and Department of Neurological Sciences,
University of Siena, Siena, Italy (Drs De Stefano and Federico).
Dr
Oliveri died February 25, 2001.
Corresponding author and reprints: Aldo Quattrone, MD, Clinica Neurologica,
Policlinico Universitario Mater Domini, Via T. Campanella, 115, 88100 Catanzaro,
Italy (e-mail: neurol.unicz{at}interbusiness.it).
REFERENCES
| |
1. Hutchinson M, O'Riordan J, Javed M, et al. Familial hemiplegic migraine and autosomal dominant arteriopathy with
leukoencephalopathy. Ann Neurol. 1995;38:817-824.
FULL TEXT
|
ISI
| PUBMED
2. Joutel A, Corpechot C, Ducros A, et al. Notch3 mutations in CADASIL, a hereditary
adult-onset condition causing stroke and dementia. Nature. 1996;383:707-710.
FULL TEXT
| PUBMED
3. Tournier-Lasserve E, Joutel A, Melki J, et al. Cerebral autosomal dominant arteriopathy with subcortical infarcts
and leukoencephalopathy maps to chromosome 19q12. Nat Genet. 1993;3:256-259.
FULL TEXT
|
ISI
| PUBMED
4. Joutel A, Vahedi K, Corpechot C, et al. Strong clustering and stereotyped nature of the Notch3 mutations in CADASIL patients. Lancet. 1997;350:1511-1515.
FULL TEXT
|
ISI
| PUBMED
5. De Stefano N, Matthews PM, Antel JP, et al. Chemical pathology of acute demyelinating lesions and its correlation
with disability. Ann Neurol. 1995;38:901-909.
FULL TEXT
|
ISI
| PUBMED
6. Arnold DL, Matthews PM. Practical aspects of clinical applications of MRS in the brain. In: Young IR, Charles HC, eds. MR Spectroscopy:
Clinical Applications and Techniques. London, England: Martin Dunitz;
1996:139-159.
7. Kalimo H, Viitanen M, Amberla K, et al. CADASIL: hereditary disease of arteries causing brain infarcts and
dementia. Neuropathol Appl Neurobiol. 1999;25:257-265.
FULL TEXT
|
ISI
| PUBMED
8. Oberstein SA, Ferrari MD, Bakker E, et al. Diagnostic Notch3 sequence analysis in CADASIL:
three new mutations in Dutch patients. Neurology. 1999;52:1913-1915.
FREE FULL TEXT
9. Joutel A, Dodick DD, Parisi JE, Cecillon M, Tournier-Lasserve E, Bousser MG. De novo mutation in the Notch3 gene causing
CADASIL. Ann Neurol. 2000;47:388-391.
FULL TEXT
|
ISI
| PUBMED
10. Ceroni M, Poloni TE, Tonietti S, et al. Migraine with aura and white matter abnormalities: Notch3 mutation. Neurology. 2000;54:1869-1871.
FREE FULL TEXT
11. Mayer M, Straube A, Bruening R, et al. Muscle and skin biopsies are a sensitive diagnostic tool in the diagnosis
of CADASIL. J Neurol. 1999;246:526-532.
FULL TEXT
|
ISI
| PUBMED
12. Rubio A, Rifkin D, Powers JM, et al. Phenotypic variability of CADASIL and novel morphologic findings. Acta Neuropathol. 1997;94:247-254.
FULL TEXT
| PUBMED
13. Desmond DW, Moroney JT, Lynch T, et al. CADASIL in a North American family: clinical, pathologic, and radiologic
findings. Neurology. 1998;51:844-849.
FREE FULL TEXT
RELATED ARTICLE
Archives of Neurology Reader's Choice: Continuing Medical Education
Arch Neurol. 2001;58(9):1503-1504.
FULL TEXT
THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES
The spectrum of Notch3 mutations in 28 Italian CADASIL families
Dotti et al.
J. Neurol. Neurosurg. Psychiatry 2005;76:736-738.
ABSTRACT
| FULL TEXT
A novel Notch3 gene mutation not involving a cysteine residue in an Italian family with CADASIL
Mazzei et al.
Neurology 2004;63:561-564.
ABSTRACT
| FULL TEXT
Recurrent hemiplegia, normal MRI, and NOTCH3 mutation in a 14-year-old: Is this early CADASIL?
Golomb et al.
Neurology 2004;62:2331-2332.
FULL TEXT
|
