Download A NÀLISI GENÈTICA I MOLECULAR DE LES MIGRANYES HEREDITÀRIES

ANÀLISI GENÈTICA I MOLECULAR
DE LES MIGRANYES HEREDITÀRIES
memòria presentada per:
Ester Cuenca León
Per optar al grau de:
Doctora per la Universitat de Barcelona
bienni 2000-2002
Aquest treball ha estat realitzat sota la direcció del Dr. Alfons Macaya Ruiz i el
Dr. Bru Cormand Rifà, al Laboratori de Neurologia Infantil i Psiquiatria Genètica de la
Unitat de Neurologia Infantil de l’Hospital Universitari Vall d’Hebron i al Departament
de Genètica de la Universitat de Barcelona.
BARCELONA
Dr. Alfons Macaya Ruiz
Dr. Bru Cormand Rifà
Ester Cuenca León
RESULTATS
CAPÍTOL 1
FAMILIAL HEMIPLEGIC MIGRAINE: LINKAGE TO CHROMOSOME
14q32 IN A SPANISH KINDRED
E. Cuenca-León, R. Corominas, M. Montfort, J. Artigas, M. Roig, M. Bayés,
B. Cormand, A. Macaya.
ARTICLE 1
NEUROLOGY - SOTMÈS
Capítol 1 – Article 1
RESULTATS
RESUM
Migranya hemiplègica familiar: lligament a 14q32 en una família espanyola.
Objectius: Localització del gen responsable de la malaltia en una família extensa amb
migranya hemiplègica familiar (FHM), migranya amb aura (MA) i migranya sense aura (MO).
Mètodes: Es van obtenir mostres d’ADN de 20 familiars. Els pacients es van classificar en les
variants específiques de migranya seguint els criteris de l’ICHD-II. Després d’excloure el
lligament als loci de migranya prèviament descrits, es va dur a terme una anàlisi de lligament a
escala genòmica mitjançant polimorfismes d’un sol nucleòtid (SNPs) amb una densitat de
0,62cM.
Resultats: Dels 13 individus afectats, sis presentaven FHM com a fenotip migranyós
predominant, dos mostraven MA i tres MO. S’ha identificat un nou locus per a la malatia en un
segment de 4,15 Mb a la regió cromosòmica 14q32, amb un valor màxim de LOD score
paramètric de 3,1 a l’anàlisi de dos punts i de 3,8 a l’anàlisi multipuntual. Aquesta regió
genòmica no correspon al locus 14q21-22 prèviament descrit. Hi ha diversos gens candidats en
aquesta regió. L’anàlisi de seqüència d’un d’ells, el gen SLC24A4, que codifica una proteïna
intercanviadora de sodi/calci/potassi, no va permetre la identificació de cap mutació
responsable del fenotip en els pacients.
Conclusions: La identificació d’un nou locus genètic de FHM reforça el seu caràcter
monogènic i insinua una major heterogeneïtat genètica de la que se sospitava prèviament.
Tot i que, a més del nostre, s’han descrit diversos loci de susceptibilitat a migranya a la regió
14q, no hem pogut identificar encara el gen responsable del fenotip a la nostra família.
71
Capítol 1 – Article 1
RESULTATS
Familial Hemiplegic Migraine: Linkage to chromosome 14q32 in a large
Spanish kindred
E. Cuenca-León BS, R. Corominas BS, M. Montfort PhD, J. Artigas MD, M. Roig MD,
M. Bayés PhD, B. Cormand PhD, A. Macaya MD.
From the Grup de Recerca en Neurologia Infantil i Psiquiatria Genètica, Hospital
Universitari Vall d’Hebron, Barcelona (E.C-L., R.C., M.R., A.M.), Corporació Sanitària
Parc Taulí, Sabadell (J.A.), Genes and Disease Program, Center for Genomic
Regulation (CRG), UPF, Barcelona (M.M., M.B.) CIBER Epidemiología y Salud
Pública, Instituto de Salud Carlos III (CRG), Barcelona (M.M., M.B.), Centro Nacional
de Genotipado (CeGen), Barcelona (M.M., M.B.), Departament de Genètica, Facultat
de Biologia, Universitat de Barcelona (B.C.), CIBER Enfermedades Raras, Instituto de
Salud Carlos III, Barcelona (B.C.), Institut de Biomedicina de la Universitat de
Barcelona (IBUB) (B.C.), Barcelona, Spain
Supported by grants of Ministerio de Educación y Ciencia SAF 2003/04704, SAF200613893-C02-01 and Fundació La Marató de TV3 061330, Spain. E.C.-L. is funded by
Ministerio de Educación y Ciencia and R.C. by Institut de Recerca Vall d’Hebron, Spain
Supplemental Data
Correspondence:
Alfons Macaya, MD
Grup de Recerca en Neurologia Infantil i Psiquiatria Genètica
Hospital Universitari Vall d’Hebron
Pg. Vall d’Hebron 119-129, 08035 Barcelona, Spain
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Lligament genètic
Tel. +34 93 4894334
Fax: +34 93 2746837
Email: [email protected]
Abstract word count: 199
Manuscript word count: 1994
Title character count: 84
Disclosure: The authors report no conflicts of interest
Statistical Analysis conducted by Ester Cuenca BS, Grup de Recerca en Neurologia
Infantil i Psiquiatria Genètica, Hospital Universitari Vall d’Hebron, Barcelona, and Bru
Cormand PhD,
Departament de Genètica, Facultat de Biologia, Universitat de
Barcelona, Spain
Supplemental data: Supplemental Table: CuencaET1, E-Table 1, Supplemental
Figure: CuencaEF1, E-Figure 1.
Search terms: All Genetics [91], Genetic linkage [94], Migraine [101]
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Capítol 1 – Article 1
Abstract
Objective: To map the disease-causing gene in a large Spanish kindred with familial
hemiplegic migraine (FHM), migraine with aura (MA) and migraine without aura (MO).
Methods: DNA samples from 20 family members were obtained. Patients were
classified according to ICHD-II criteria for specific migraine subtypes. After ruling out
linkage to known migraine genetic loci, a single nucleotide polymorphism (SNP)-based,
0.62 cM density genomewide scan was performed.
Results: In 13 affected subjects, FHM was the prevailing migraine phenotype in six,
MA in four and MO in three. Linkage analysis revealed a disease locus in a 4.15 Mb
region on 14q32, with a maximum two-point LOD score of 3.1 and a multipoint
parametric LOD score of 3.8. This genomic region does not overlap with reported
migraine loci on 14q21-22. Several candidate genes map to this region. Sequence
analysis of one of them, SLC24A4, encoding a potassium-dependent sodium/calcium
exchanger, failed to show disease-causing mutations in our patients.
Conclusions: The finding of a new genetic locus in FHM underscores its monogenic
character and hints to greater genetic heterogeneity than previously suspected. While
several genes conferring increased susceptibility to migraine seem to reside on 14q,
the
underlying
disease-causing
gene
in
our
family
remains
unidentified.
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RESULTATS
Lligament genètic
Introduction
Familial hemiplegic migraine (FHM) is a rare subtype of migraine with aura (MA) with
autosomal dominant inheritance. In combination with sporadic hemiplegic migraine
(SHM), the condition has a prevalence of 0.01%.1 Three FHM genes have been
identified, its dysfunction resulting in increased synaptic glutamate and a lower
threshold for cortical spreading depression, the mechanism underlying migraine aura.
2,3
In FHM1, mutations in the CACNA1A gene on chromosome 19p13.13, encoding the
α subunit of the neuronal P/Q-type calcium channel (CACNA1A), were first reported in
five unrelated FHM families.4 To date, at least 21 CACNA1A mutations have been
reported.5 FHM2 is caused by mutations in the ATP1A2 gene on chromosome 1q23.2;
over 30 FHM2 mutations have been identified.6-8 Only three FHM3 mutations have
been described in the SCN1A gene, which encodes the α subunit of the neuronal
voltage-gated type I sodium channel.9-11 Mutations in these three genes account for just
50-70% of published cases of FHM. A recent population-based study from Denmark
indicated greater locus heterogeneity than previously assumed.12
In migraine families, previous genomewide linkage scans have detected loci for MA on
4q24 , 11q24,13,14 for migraine without aura (MO) or MA on 6p12-2115 and for MO on
4q21 and 14q21.2-22.3.16,17 A locus on 9q21-q22 has been linked to familial
occipitotemporal lobe epilepsy in a pedigree showing MA co-occurrence in five out of
ten affected members.18
Two studies using latent class analysis of migraine symptoms identified a locus on
5q21 for the cluster photophobia – phonophobia19 and putative loci on 3q29 and 18p11
for the severe migraine phenotype.20
We performed a genomewide scan in a Spanish FHM multigenerational family and
obtained conclusive linkage to a novel single genetic locus on chromosome 14q32.1232.13.
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RESULTATS
Methods
Patients: A multigenerational FHM family from Catalunya, North-Eastern Spain, was
assessed. All participants were directly interviewed by one of the authors (AM);
migraine clinical diagnosis was established according to the ICHD-II criteria from the
IHS.21 For genetic analysis, any member meeting IHS criteria for a type of migraine
headache was given an affected status.
Samples: DNA was extracted from peripheral blood of 20 available family members
using the QIAamp DNA Blood Maxi Kit (Hilden, GE). Written informed consent from
participants and approval from the local Ethics Committee were obtained according to
the guidelines of the Helsinki Declaration.
DNA analysis: Linkage between the migraine phenotype in our family and each one of
six previously reported migraine genetic loci on 1q21-23,22 1q31-32,23 4q24,13 6p12.2p21.1,15 14q21.2-22.317 and 19p.1324 was assessed. These loci were covered with 20
microsatellite markers mainly from the MD-10 Linkage Mapping Set v2.5 (Applied
Biosystems, Foster City, CA) and genotypes resolved by polyacrylamide gel
electrophoresis and silver staining following standard methods. Next, samples were
genotyped with the SNP-based Linkage IVb Gold Panel (Illumina, San Diego, CA)
comprising 6,008 SNP markers evenly distributed across the genome. Each sample
was genotyped in four highly multiplexed assays following the manufacturer’s
recommendations. The Illumina’s BeadArray Reader was used to analyze fluorescence
signals, and the Illumina’s BeadStudio GenoTyping Module v.2.1.10 to normalize raw
data, perform clustering and generate genotype calls.
The coding regions of the SLC24A4 gene were PCR-amplified, purified and sequenced
(ABI PRISM 3700 DNA Analyzer, Applied Biosystems, Foster City, CA). See E-Table 1
for primer sequences and product sizes.
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Statistical analysis:
The simulation program SLINK25-27 was used to compute the maximum expected
pairwise LOD score (Z) in our pedigree, assuming an autosomal dominant model of
inheritance with a penetrance (p) of 0.95, a phenocopy rate (f) of 0.01, a disease allele
frequency (q) of 0.001 and a marker heterozygosity of 0.5 over 1000 replicates. The
family was estimated to give a maximum two-point LOD score (Zmax) of 4.04 at a
recombination fraction (θ) of 0.00 from the disease gene.
The evaluation of previously reported migraine loci was performed by multipoint
parametric linkage analysis between microsatellite markers and the disease phenotype
using the LINKMAP software from the LINKAGE package28 with p = 0.95, f = 0.01 and
q = 0.001 under a dominant model.
The genomewide scan for linkage between SNP markers and migraine was performed
by multipoint parametric linkage analysis, assuming 0.8 ≤ p ≤ 1.0, 0.01 ≤ f ≤ 0.10 and q
= 0.001 under dominance. Exponential multipoint non-parametric linkage (NPL)
analysis was also performed to detect increased allele sharing among affected
individuals, without assumption of any inheritance model. Both calculations were
computed with MERLIN29 on a split pedigree to circumvent program constraints.
Finally, the critical disease interval was studied in more detail in the full pedigree by
both two-point linkage analysis using the MLINK program, and also by multipoint
parametric linkage analysis using LINKMAP and the “sliding window” method to avoid
loss of information. Several values of penetrance and phenocopy rate were assumed
under dominance. Both programs are implemented in the LINKAGE package.28 To
define the boundaries of the critical interval, haplotypes were constructed with
MERLIN29 by minimizing the number of recombination events.
For the genomewide screen, the SNP allele frequencies were considered to be equal,
while for the analysis of the critical interval on chromosome 14, Caucasoid allele
frequencies from the Central European (CEU) HapMap database were used
(www.hapmap.org).
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Capítol 1 – Article 1
Results
A three-generation pedigree with 13 affected and 7 healthy individuals was analyzed
(Figure 1). The 16-year-old proband (III.12) was the youngest FHM patient, with
episodes starting at age 12. She had five relatives diagnosed with FHM, including her
mother and two siblings, four with the main diagnosis of MA and three with MO. The
pedigree’s detailed clinical data are shown in Table 1; all subjects fulfilled ICHD-II
criteria regarding number and duration (4-72 hours) of episodes. Clinical diagnosis in
family members with MA ranged from typical visual aura with migraine headache in
individual II.10 to visual aura with non-migraine headache in II.12 and typical aura
without ensuing headache in III.16.
Patient II.13 had simultaneous bilateral
paresthesia, the occasional feature of FHM and of basilar-type migraine, occurring as
the single aura manifestation. Most patients presented in childhood or early puberty
and, overtime, five patients displayed more than one subtype of migraine. Interictal
neurological examination was normal in all cases; specifically, no cerebellar signs were
recorded. Brain MRI in the proband and in patients II.3 and II.13, were normal. No
patient or non-migraneur in the pedigree displayed ataxia, seizures or any other
paroxysmal neurological sign.
Multipoint analysis encompassing six loci previously linked to migraine allowed
exclusion of linkage to five of them (Z < -2.0) in our pedigree and displayed negative
scores (Z < -0.6) at the 4q24 locus (E-Figure 1).
The results of linkage to 5,627 autosomal SNPs are shown in Figure 2; sex-linked
inheritance was ruled out in this pedigree. The average genotype call rate was 99.58
(±1.52) % after excluding 36 SNPs (0.64%) with genotype calls <80%. Evidence of
linkage to chromosome 14q32.12-32.13 was found. The highest two-point LOD score
value was obtained with marker rs882023 (Zmax = 3.11 at θ = 0.00), with several close
markers preserving positive LOD scores, under the assumption of 95% penetrance and
1% phenocopy rate (Table 2). In line was the multipoint parametric linkage analysis
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which provided strong evidence of linkage between markers rs972905 and rs1054195
(Z > 3), with a maximum LOD score of 3.83 at markers rs742893 and rs1054195
(Figure 3). No other region in the whole genome surpassed a LOD score value of 1
and, indeed, 95.7% of the genome was ruled out to hold the causative gene (Z < -2).
Exponential NPL analysis reached its highest value in the same region of chromosome
14 (Z = 2.71, p = 0.0002).
The limits of the disease-causing haplotype, spanning 4.15 Mb, were set by a proximal
recombination between rs755102 and rs972905 in III.16, a MA patient, and a distal
recombination between rs1054195 and rs1007813 in III.10, a FHM patient. It was
shared by all the affected members while the unaffected members carried a different
haplotype (Figure 1). The only exception was individual III.14, a 12 year-old boy that
either was presymptomatic at the time of the study or displayed incomplete penetrance
of the disease phenotype.
The SLC24A4 gene, encoding a potassium-dependent sodium/calcium exchanger
(NCKX4) is located within the critical interval, around 1 Mb distal from rs755102.
Sequence analysis of all exons and intronic flanking regions of the gene was carried
out in affected individuals, but no potential disease-causing mutations were found.
Discussion
Whole-genome linkage analysis on a single multigenerational dominant pedigree
revealed a novel FHM locus on 14q32, although the underlying gene remains to be
identified. The prevailing phenotype of the affected members in this family was FHM,
without associated episodic or progressive ataxia; the seven cases of MA or MO were
considered as affected in the linkage calculations, since occurrence of non-hemiplegic
migraine within FHM pedigrees is well acknowledged. Indeed, co-occurrence of MO
and MA is observed in some FHM patients during their lifetime, both in our family and
in other reports.12 In addition, mutations in the CACNA1A gene are often expressed as
MA30,31 or MO,32 although certainly FHM1 does not appear to be a major susceptibility
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locus for non-hemiplegic migraine.14,33,34 That dissimilar migraine phenotypes may
share the same molecular defect is also illustrated by some FHM2 pedigrees, where
mutations in the ATP1A2 gene have been reported in MA or MO individuals.35,36
All the affected members of the pedigree shared a common haplotype spanning 4.15
Mb, regardless of their specific migraine phenotype (FHM, MA, MO). Only one
asymptomatic individual (III.14) was a carrier of the disease haplotype, and the
question remains whether he will develop migraine symptoms in the future. Using the
stringent phenotype FHM-only, the haplotypes segregating with the disease defined a
wider disease-harboring interval of about 28 Mb (between rs1015023 and rs1007813)
that included the 4.15 Mb region defined when patients with FHM or MA, or patients
with any of the three migraine phenotypes, were considered. A recombination in
individual III.10, an FHM individual, defined the distal limit of the smaller critical region,
whereas III.16, a MA patient, defined the proximal border.
The newly identified locus shows no overlap with a previous one described on
chromosome 14q21 in a large Italian MO family.17 Considering the disease-associated
haplotypes in each family, the two loci are more than 30 Mb apart. A latent class
analysis in families from Australia20 found suggestive linkage of migraine symptoms to
a locus on 14q22 (Z = 2.06, p = 0.002), in close proximity (< 5cM) to the critical region
in the above mentioned Italian family. Again, the locus does not overlap with the one
described here, although it is conceivable that a cluster of genes conferring increased
susceptibility to migraine may reside in this region on chromosome 14q.
The UCSC Human Genome Browser database (genome.ucsc.edu, NCBI Build 36.1)
lists 47 genes within the critical disease interval. One of them, SLC24A4 [OMIM
609840], encoding a multi-pass membrane protein for ion exchange, was screened on
the basis of its genomic position, expression profile and function. Even though we
failed to detect putative disease-causing mutations, the possibility remains of changes
outside the coding region or that may have gone undetected by PCR and direct
sequencing. Also in the region is the ATXN3 gene [OMIM 607047] encoding ataxin 3,
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whose expansion at a (CAG)n repeat is responsible for Machado - Joseph disease
(SCA3).37 FHM-causing mutations in this gene would match the pattern associated with
CACNA1A mutations, which cause both FHM1 and SCA6. Other candidate genes are
FBLN5 [OMIM 604580], encoding fibulin5, a calcium binding protein expressed in
arterial vasculature and ITPK1 [OMIM 601838], encoding inositol 1,3,4-triphosphate
kinase, which indirectly regulates plasma membrane Ca2+-activated chloride channels.
Linkage to a single locus in our family adds to the existing evidence that FHM is usually
inherited as a monogenic defect and that genetic heterogeneity in FHM appears to be
greater than previously suspected. Further studies are warranted to ascertain the
relevance of this locus in other large migraine families. Identification of the disease
gene in the locus described here may lead to a better understanding of the complex
molecular mechanisms involved in this condition.
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FIGURE LEGENDS
Figure 1. Spanish migraine pedigree showing haplotypes for 20 SNP markers on
chromosome 14q24.1 - 14q32.31. The haplotypes segregating with the disease
phenotype are boxed. The names and order of the markers are depicted in the inset,
with the genetic distances between them indicated in cM. FHM= familial hemiplegic
migraine, MA= migraine with aura, MO= migraine without aura.
Figure 2. Whole-genome parametric multipoint linkage analysis between the disease
phenotype and 5,627 autosomal SNPs from the Linkage IVb Gold Panel (Illumina, San
Diego, CA). The -2 and +3 LOD score thresholds are indicated with horizontal lines
within the graph. The arrow shows the linked area on chromosome 14q32.
Figure 3. Parametric multipoint linkage analysis between the disease phenotype and
12 SNP markers on chromosome 14q31-q32. The marker names are indicated on the
top of the graph. The LOD scores, on the y-axis, were calculated with the LINKMAP
software assuming different penetrance (p) and phenocopy (f) values, which are
indicated in % in the inset. The -2 and +3 LOD score thresholds are indicated with
horizontal lines within the graph. On the x-axis, genetic distances in cM from the 14p
telomere, as defined in the Linkage IVb Gold Panel (Illumina, San Diego, CA) and in
the deCODE Genetics recombination map (www.decode.com).
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Table 1. Patients clinical features.
Individual
Sex
Age at onset (years)
Unilateral pain
Pulsating pain
Aggravation by physical activity
Pain intensity
Nausea
Vomiting
Photophobia
Phonophobia
Highest attack frequency
Visual disturbances
Language disturbances
Sensory / Motor deficit
Ataxia
Vertigo
Clinical diagnosis (ICHD-II code)
I.2
II.2
II.3
II.8
II.10
II.12
II.13
III.3
III.5
III.10
III.11
III.12
III.16
F
14
Yes
Yes
Yes
NA
No
No
Yes
Yes
F
28
Yes
Yes
Yes
NA
No
No
Yes
Yes
F
13
No
Yes
Yes
NA
No
No
Yes
Yes
F
12
Yes
Yes
Yes
NA
No
No
Yes
Yes
F
10
No
Yes
Yes
Disabling
Yes
Yes
Yes
Yes
M
16
No
Yes
No
Disabling
No
No
No
Yes
F
12
Yes
Yes
Yes
Disabling
Yes
No
Yes
Yes
F
5
Yes
Yes
Yes
NA
No
No
Yes
Yes
M
12
Yes
Yes
Yes
NA
No
No
Yes
Yes
M
10
No
No
Yes
NA
Yes
Yes
No
Yes
F
12
Yes
No
Yes
NA
Yes
Yes
Yes
Yes
F
7
No
No
No
Moderate
No
No
No
No
2/month
2/week
5 FHM/year,
2 MO/month
2/year
1/month
2/year
2/month
F
5
Yes
Yes
NA
NA
Yes
Yes
No
Yes
3 FHM
overall, 1
MO/week
1/month
3/year
6/year
2/month
2/month
No
No
No
No
No
No
No
No
No
No
yes
yes
Yes/Yes
No
No
yes
yes
Yes/Yes
No
No
yes
No
No
No
No
yes
No
No
No
No
No
No
Yes/No
No
No
yes
no
Yes/Yes
No
No
No
No
No
No
No
yes
yes
Yes/Yes
No
No
yes
yes
Yes/Yes
No
No
Yes
Yes
Yes/Yes
No
No
Yes
No
No
No
Yes
MO (1.1)
MO (1.1)
FHM (1.2.4)
MO (1.1)
FHM (1.2.4)
MA (1.2.1)
MO (1.1)
MA (1.2.1),
MO (1.1)
MA (1.2.2)
MA (1.2.1),
MO (1.1)
FHM (1.2.4)
MO(1.1)
MO (1.1)
FHM (1.2.4)
FHM (1.2.4)
FHM (1.2.4)
MA (1.2.3)
NA= not available; FHM= familial hemiplegic migraine, MA= migraine with aura, MO= migraine without aura
RESULTATS
87
Position
Marker
cM
Mb
LOD score at θ =
0.00
0.01
0.05
0.10
0.20
0.30
0.40
Zmax
θmax
rs1999916
84.31
85.53
-2.12
-0.75
0.15
0.53
0.69
0.54
0.26
0.69
0.19
rs719572
87.25
88.45
-2.40
-0.76
-0.01
0.29
0.46
0.38
0.17
0.46
0.21
rs972905
94.46
91.27
1.74
1.71
1.61
1.47
1.18
0.84
0.45
1.74
0.00
rs882023
96.43
92.60
3.11
3.06
2.84
2.56
1.94
1.26
0.53
3.11
0.00
rs1004958
99.39
94.11
1.78
1.75
1.63
1.46
1.11
0.71
0.27
1.78
0.00
rs742893
99.69
94.22
1.01
1.05
1.14
1.15
1.01
0.76
0.42
1.15
0.08
rs1054195
101.33
94.72
1.78
1.75
1.62
1.46
1.11
0.71
0.27
1.78
0.00
rs1007813
102.71
95.08
1.22
1.25
1.32
1.31
1.15
0.86
0.48
1.33
0.07
rs2369522
104.11
95.81
1.94
1.92
1.80
1.64
1.31
0.93
0.50
1.94
0.00
rs1159799
109.62
98.32
0.94
0.93
0.91
0.86
0.72
0.54
0.30
0.94
0.00
rs941731
112.31
98.96
-1.77
-1.28
-0.64
-0.36
-0.22
-0.23
-0.14
0.00
0.50
rs1007904
117.78
101.03
-2.99
-1.40
-0.72
-0.45
-0.20
-0.08
-0.02
0.00
0.50
RESULTATS
88
Table 2. Two-point LOD scores between migraine (FHM, MA, MO) and chromosome 14q31-q32 markers.
Lligament genètic
Capítol 1 – Article 1
RESULTATS
Figure 1
89
Lligament genètic
Figure 2
RESULTATS
90
Capítol 1 – Article 1
RESULTATS
91
RESULTATS
Lligament genètic
Figure 3
(Z)
92
RESULTATS
Capítol 1 – Article 1
E-Table 1
93
MIGRANYA AMB AURA: LLIGAMENT A 14q EN UNA
FAMILIA AUTOSÒMICA DOMINANT EXTENSA
ANNEX A L’ARTICLE 1
Capítol 1 – Annex a l’article 1
RESULTATS
RESUM
Migranya amb aura: lligament a 14q en una família autosòmica dominant
extensa
Amb l’objectiu final d’identificar el gen responsable d’una forma freqüent de migranya en
una família autosòmica dominant extensa, s’ha realitzat un cribratge genòmic per anàlisi de
lligament. La família té 30 individus en quatre generacions, set d’ells afectats de migranya
amb aura (MA) i set amb migranya sense aura (MO). La identificació d’un haplotip compartit
pels membres afectats de la família ha permès localitzar el gen de la malaltia a una regió de
19,45Mb a 14q24.3-34.2. L’interval de 4,15Mb identificat prèviament a 14q32.12-32.13 en una
altra família amb individus amb FHM, MA i MO (Article 1), està inclòs en aquesta regió. Es va
realitzar l’anàlisi mutacional del gen SLC24A4, un candidat posicional i funcional que codifica
una proteïna intercanviadora de sodi/calci/potassi, en dos individus afectats de la família i
portadors de l’haplotip de risc per la malaltia i no es va detectar cap canvi potencialment
patogènic.
El fet que la regió de lligament a migranya en aquesta família inclogui un locus prèviament
descrit lligat a un fenotip més greu de la mateixa malaltia suggereix una causa genètica
comú en variants diferents de migranya. Seria interessant reclutar altres famílies
migranyoses amb herència autosòmica dominant per intentar identificar lligaments al
mateix locus que permetin acotar encara més la regió genòmica crítica.
97
RESULTATS
Capítol 1 – Annex a l’article 1
MIGRANYA AMB AURA: LLIGAMENT A 14q EN UNA FAMÍLIA
AUTOSÒMICA DOMINANT EXTENSA
Consideracions generals
Donat que es desconeix la base genètica de les formes comuns de migranya i que vam poder
identificar una família de 30 individus que inclou membres MA i MO en què la migranya
segrega aparentment de forma autosòmica dominant (figura 1), ens vam plantejar dur a
terme un estudi de lligament a escala genòmica. El fet de poder utilitzar una única família de
grans dimensions permet evitar els problemes potencials d’heterogeneïtat genètica inherents
al fenotip migranyós.
I.1
II.1
III.1
III.1
1
6
2
3
1
2
3
4
IV.1
III.2
3
6
1
4
1
1
3
3
1
3
5
5
4
4
1
3
IV.2
4
1
6
4
3
2
1
4
III.4
III.5
2
8
5
3
3
4
1
3
1
6
3
6
1
1
1
4
2
5
6
2
1
1
1
2
IV.4
IV.5
IV.6
IV.7
IV.8
1
6
2
3
1
2
3
4
4
1
6
4
3
2
1
4
4
1
6
4
3
2
1
4
4
6
7
3
2
4
3
1
4
6
7
3
2
4
3
1
4
6
7
3
2
4
3
1
2
5
6
2
1
1
1
2
II.3
4
6
7
3
2
4
3
1
IV.3
3
6
1
4
1
1
3
4
II.2
III.3
4
1
6
4
3
2
1
4
2
5
6
2
1
1
1
2
2
5
5
3
3
4
1
3
2
8
5
3
3
4
1
2
IV.9
2
8
5
3
3
4
1
3
I.2
III.6
1
7
4
5
1
4
1
3
IV.10
IV.11
1
6
3
6
1
1
1
3
1
7
4
5
1
1
1
4
3
6
1
4
1
1
3
2
III.7
3
6
1
4
1
1
3
3
5
6
4
3
1
1
1
2
IV.12
1
6
3
6
1
1
1
3
5
6
4
3
1
2
1
3
1
9
2
1
2
2
3
1
IV.13
1
7
4
5
1
4
1
3
3
6
1
4
1
1
3
3
IV.14
5
6
6
2
1
1
2
2
III.8
5
6
4
3
1
2
1
2
5
6
6
2
1
1
2
2
IV.15
III.9
2
4
3
4
1
3
5
3
3
6
1
4
1
1
3
3
IV.16
IV.17
IV.18
1
9
6
2
1
1
3
1
5
6
6
2
1
1
2
1
5
6
6
2
1
1
2
2
3
6
1
4
1
1
3
3
3
6
1
4
1
1
3
3
5
6
4
3
1
2
1
2
3
6
4
3
1
2
1
2
III.10
6
5
3
3
3
1
3
2
5
6
4
3
1
2
1
2
IV.19
IV.20
3
6
1
4
1
1
3
1
5
6
4
3
1
2
1
2
2
5
3
3
3
1
3
2
2
4
3
4
1
3
5
3
II.4
III.11
III.12
3
6
1
4
1
1
3
1
2
2
8
6
1
4
1
1
IV.21
3
6
1
4
1
1
3
1
IV.22
IV.23
2
4
3
4
1
3
5
3
2
1
4
3
1
4
2
3
IV.24
2
2
8
6
1
4
1
1
GRUP EXCLÒS
V.1
V.2
3 2
6 1
1 6
4 1
1 1
1 4
3 4
2 2
V.3
D14S59
D14S1037
D14S67
D14S68
D14S256
D14S1044
D14S280
D14S65
5,33
3,2
0,1
0,53
3,46
5,12
12,3
: MA, HOME
: MO, DONA
Figura 1. Estructura de la família estudiada amb els haplotips de 8 marcadors corresponents al locus del
cromosoma 14. L’haplotip de risc per desenvolupar la malaltia es representa enquadrat. A la caixa s’especifica
l’ordre dels marcadors i les distàncies entre ells en cM segons el mapa Marshfield
(research.marshfieldclinic.org/genetics). La caixa amb línia discontínua emmarca els individus que, tot i
genotipats, s’han exclòs de l’anàlisi de lligament.
99
V.4
4
6
1
4
1
1
3
3
RESULTATS
Lligament genètic
Pacients
La família consta de 30 individus disponibles per a l’estudi, 14 d’ells afectats de migranya
que compleixen els criteris ICHD-II de la IHS per MA (7 casos) o per MO (7 casos) (taula 1).
L’individu índex (V.2) presentà des dels 10 dies de vida i amb una freqüència d’un cop al mes,
episodis de disfunció neurològica aguda (hipotonia, postració) que en arribar als 3 anys
d’edat, el pacient pot descriure com a dolor cefàlic intens unilateral que empitjora amb
l’activitat física. Aquest individu, que encara està en edat pediàtrica, manifesta molt
probablement una forma clínica precursora de migranya, i encara que no compleix els
criteris ICHD-II, s’ha considerat com a afectat per a les anàlisis de lligament. L’individu III.7,
afectat de MO, prové de fora de la família i es diferencia del tipus de cefalea present en la
resta del pedigrí per l’inici tardà dels episodis, a l’edat de 30 anys. Aquest individu i els seus 5
fills (IV.14, IV.15, IV.16, IV.17 i IV.18) han estat exclosos dels càlculs de lligament, ja que,
tenint en compte l’elevat grau d’heterogeneïtat genètica de la malaltia, és possible que
l’individu III.7 transmeti a algun dels seus fills un al·lel mutant en un gen diferent al que és
responsable de la patologia a la resta de la família.
Avaluació de loci prèviament descrits mitjançant anàlisi de lligament genètic
L’anàlisi haplotípica de loci prèviament lligats a migranya mitjançant polimorfismes de tipus
microsatèl·lit va permetre excloure en aquesta família bona part dels 6 loci coneguts a l’inici
d’aquest estudi. L’anàlisi de lligament multipuntual en 11 individus afectats de la família (taula
1), assumint una herència autosòmica dominant amb p=90% i f=10%, exclogué clarament els
loci 4q24, 14q21-23 i gran part de 1q31-32 com a responsables del fenotip migranyós, i
suggerí, sense assolir significació estadística, que els loci 6p12.2-p21.1 i 1q21-23 tampoc
estaven implicats (figura 2). El sisè locus, a 19p13, conté el gen CACNA1A i es va excloure
mitjançant anàlisi de lligament de dos punts utilitzant el marcador intragènic D19S1150,
situat a l’intró 7 del gen CACNA1A.
100
Individu
Sexe
Edat d'inici dels símptomes (anys)
>5 episodis
4 a 72h
Dolor unilateral
Dolor pulsatiu
Empitjorament amb l'activitat física
intensitat del dolor
Nàusees
Vòmits
Fotofòbia
Fonofòbia
Freqüència màxima dels episodis
Alteracions visuals
Alteracions del llenguatge
Alteracions sensorials/ motores
Atàxia
Vertigen
Diagnòstic clínic (codi ICHD-II)
III.2
F
10
Sí
Sí
No
Sí
Sí
incapacitant
ND
ND
Sí
Sí
10/any
Sí
No
No
No
No
MA (1.2.1)
III.3
M
20
Sí
Sí
No
Sí
Sí
incapacitant
No
No
Sí
Sí
1-2/setm
No
No
No
No
No
MO (1.1)
III.8
F
10
Sí
Sí
Sí
Sí
Sí
incapacitant
Sí
Sí
Sí
Sí
3/setm
No
No
No
No
Sí
MO (1.1)
III.10
F
10
Sí
Sí
No
Sí
Sí
incapacitant
Sí
ND
Sí
Sí
1/mes
Sí
No
No
No
No
MA (1.2.1)
IV.3
M
20
Sí
Sí
No
Sí
Sí
moderada
No
No
Sí
Sí
4/any
Sí
No
No
No
Sí
MA (1.2.1)
IV.5
F
16
Sí
Sí
Sí
Sí
Sí
incapacitant
Sí
No
Sí
Sí
1/setm
No
No
No
No
No
MO (1.1)
IV.10
F
10
Sí
Sí
Sí
Sí
Sí
incapacitant
No
No
Sí
Sí
4/mes
Sí
No
Sí
No
No
MA (1.2.1)
IV.13
F
5
Sí
Sí
Sí
Sí
Sí
moderada
Sí
Sí
Sí
Sí
1/mes
No
No
No
No
No
MO (1.1)
IV.19
M
12
Sí
Sí
No
Sí
Sí
moderat
No
No
Sí
Sí
2-3/setm
Sí
No
No
No
No
MA (1.2.1)
IV.24
F
7
Sí
Sí
No
Sí
Sí
incapacitant
No
No
Sí
ND
1/setm
No
No
No
No
Sí
MO (1.1)
V.2
F
10 dies
Sí
Sí
Sí
?
Sí
incapacitant
?
no
?
?
1/mes
?
?
Capítol 1 – Annex a l’article 1
Taula 1. Simptomatologia clínica dels pacients inclosos en el cribratge a escala genòmica.
NO IHS
ND: no disponible, MA: migranya amb aura, MO: migranya sense aura
RESULTATS
101
RESULTATS
Lligament genètic
M1 1q31- 32
M1 1q21-23
M1 4q24
M1 14q21-22
M1 6p12-22
M1 f 10% - p 90%
Distància en cM
0
5
10
15
20
25
LOD score (Z)
0
-1
-2
-3
-4
Figura 2. Representació gràfica dels valors de LOD score multipuntuals obtinguts per a cinc dels sis loci
autosòmics de migranya descrits a la literatura a l’inici de l’estudi. El sisè locus, 19p13, conté el gen
CACNA1A i es va excloure utilitzant el marcador intragènic D19S1150. Els valors de LOD score s’han
calculat mitjançant el programa GENEHUNTER i considerant p= 90% i f= 10%. L’ordre i distàncies entre
marcadors es corresponen amb el mapa Marshfield (research.marshfieldclinic.org/genetics).
Anàlisi de lligament genètic a escala genòmica
Es va dur a terme un cribratge de tot el genoma en 11 dels 18 individus afectats de migranya
de la família (5 MA i 6 MO) utilitzant 400 marcadors de tipus microsatèl·lit del Linkage
Mapping Set v2.5-MD10 (Applied Biosystems) amb una densitat mitjana d’1 marcador/9 cM.
Dels 18 individus afectats de la família, 7 no es van genotipar o no es van considerar a l’anàlisi
de lligament per diverses raons: els individus II.2, II.4 i III.11 ja eren morts i no hi havia mostra
biològica disponible, l’individu III.7, també afectat, no és descendent de la parella fundadora
de la genealogia (I.1 i I.2), i els individus IV.14, IV.17 i IV.18 són fills de III.7. Es va fer una anàlisi
de lligament paramètric multipuntual considerant una herència autosòmica dominant amb
p=90% i f=10%, i es va obtenir un únic valor de LOD score per sobre de 1,5 a la regió
cromosòmica 14q24-32. Uns altres 4 loci van presentar Z>1: 1p31, 3p25, 12p12 i 15qtel (figura
3a). En paral·lel, es va dur a terme una anàlisi de lligament no paramètrica (sense considerar
un model d’herència concret) de tipus exponencial i s’obtingué un valor màxim de LOD score a
la mateixa regió del cromosoma 14 (Z= 1,62, p= 0,003) (figura 3b).
102
Capítol 1 – Annex a l’article 1
RESULTATS
Definició i acotament de la regió crítica
A continuació es va aprofundir en l’anàlisi dels 5 loci que presentaven LOD scores superiors a 1
genotipant nous marcadors de tipus mirosatèl·lit i afegint 14 individus addicionals de la
família, tots ells sans. El LOD score obtingut en aquestes noves anàlisis va davallar respecte a
l’anàlisi inicial a quatre de les regions potencialment candidates a contenir el gen responsable
a
b
Figura 3. Anàlisi de lligament multipuntual paramètric (a) i no paramètric (b) entre el fenotip MA i/o MO i 400
marcadors microsatèl·lits de la col·lecció de mitjana densitat ABI PRISM Linkage Mapping Set v2.5 MD10 Panel
d’Applied Biosystems (Foster City, CA). Les fletxes indiquen el punt amb el valor màxim de LOD score a la regió
cromosòmica 14q24.3-34.
de la malaltia: 1p31, 3p25, 12p12 i 15qtel. Només en el cas de la regió 14q24-32 es va obtenir
un LOD score màxim superior al de l’anàlisi inicial, amb Zmax = 2,12 a θ = 0,00 pel marcador
D14S67, i valors positius pels marcadors més propers (taula 2). Això va confirmar aquesta
regió genòmica com la de major probabilitat de contenir el gen de risc per a la malaltia en
aquesta família.
103
RESULTATS
Lligament genètic
Taula 2. Valors de LOD de 2 punts calculats amb el programa MLINK, considerant f=10 i p=90 i tots els individus
genotipats excepte el grup exclòs (25 individus).
Posició
Marcador
cM
Mb
D14S261
D14S283
D14S275
D14S70
D14S288
D14S276
D14S63
D14S258
D14S74
D14S59
D14S1037
D14S67
D14S68
D14S256
D14S1044
D14S280
D14S65
D14S985
D14S292
6,46
13,89
28,01
40,11
47,51
56,36
69,18
76,28
87,36
87,36
92,69
95,89
95,89
96,42
99,88
105,00
117,30
126,61
134,30
19,91
21,76
25,77
33,53
43,17
54,75
63,62
69,55
77,63
77,14
84,27
87,46
87,70
88,28
89,04
91,15
96,59
100,27
103,67
LOD score a θ=
0,00
0,01
0,05
0,10
0,20
0,30
0,40
Zmax
θmax
1,368
-0,321
-1,290
-1,394
-0,344
-1,851
-1,672
-1,168
-0,179
0,497
-0,137
2,122
1,477
0,710
1,410
0,468
-2,168
-0,353
-1,540
1,330
-0,230
-1,072
-1,261
-0,224
-1,670
-1,483
-1,065
-0,173
0,599
-0,127
2,088
1,482
0,675
1,389
0,483
-2,033
-0,350
-1,412
1,177
-0,014
-0,643
-0,878
0,076
-1,158
-0,959
-0,776
-0,147
0,822
-0,089
1,929
1,456
0,538
1,281
0,506
-1,596
-0,326
-1,044
0,990
0,098
-0,377
-0,580
0,251
-0,768
-0,570
-0,550
-0,112
0,906
-0,048
1,691
1,352
0,379
1,111
0,482
-1,165
-0,275
-0,747
0,635
0,121
-0,109
-0,264
0,322
-0,339
-0,169
-0,281
-0,050
0,807
0,008
1,150
1,015
0,149
0,716
0,364
-0,552
-0,159
-0,375
0,324
0,046
-0,009
-0,125
0,235
-0,129
-0,007
-0,130
-0,008
0,529
0,028
0,621
0,609
0,054
0,340
0,230
-0,206
-0,066
-0,155
0,098
-0,020
0,006
-0,056
0,106
-0,031
0,035
-0,041
0,006
0,198
0,020
0,216
0,230
0,020
0,090
0,108
-0,042
-0,011
-0,035
1,368
0,121
0,006
-0,056
0,322
-0,031
0,035
-0,041
0,006
0,906
0,028
2,122
1,477
0,710
1,410
0,506
-0,042
-0,011
-0,035
0,00
0,20
0,40
0,40
0,20
0,40
0,40
0,40
0,40
0,10
0,30
0,00
0,00
0,00
0,00
0,05
0,40
0,40
0,40
El LOD score multipuntual per aquesta regió enriquida en individus i marcadors va assolir un
valor màxim Zmax de 2,5, sobre el marcador D14S67 (figura 4).
Figura 4. Anàlisi de lligament paramètric entre el fenotip MA i/o MO i els 12 marcadors de la regió 14q2432. Els LOD score s’han calculat amb el programa LINKMAP assumint f=10 i p=90.
La construcció dels haplotips de la regió 14q24-32 (figura 1) revelen que l’haplotip que segrega
amb la migranya en aquesta família té una extensió de 19,45 Mb. La regió crítica està
104
Capítol 1 – Annex a l’article 1
RESULTATS
delimitada a l’extrem proximal per una recombinació entre els marcadors D14S59 i D14S1037
en l’individu afectat de MO, IV.24 i per una recombinació entre els marcadors D14S280 i
D14S65 en els individus III.10, IV.3 i IV.10, tots ells amb fenotip MA, a la part distal.
Tots els individus afectats de la família són portadors de l’haplotip de risc a excepció dels
individus III.3 i la seva filla IV.5, que tenen MO. Entre els individus sans hi ha dos individus
portadors de l’haplotip de risc, el III.6 que és portador obligat, i l’individu IV.21. El “grup
exclòs” (l’individu III.7 i els seus fills) s’ha genotipat en l’última fase del estudi per estudiar els
haplotips presents, però no s’ha tingut en compte per a les anàlisis de lligament.
El gen l’SLC24A4, que codifica un intercanviador de calci i sodi dependent de potassi, i que
està situat a l’interval crític compartit per les dues famílies (aquesta i la de l’article 1), es va
escollir com el millor gen candidat funcional d’entre els candidats posicionals. L’anàlisi
mutacional de les regions codificants i intròniques flanquejants del gen SLC24A4 a l’individu
III.10, portador de l’haplotip que cosegrega amb la malaltia, no va revelar cap variant
potencialment patogènica.
105
CAPÍTOL 2
GENETIC
27 SPANISH
HEMIPLEGIC MIGRAINE, BASILAR-TYPE
CHILDHOOD PERIODIC SYNDROMES.
ANALYSIS OF
PATIENTS WITH
MIGRAINE AND
E Cuenca-León, R Corominas, N Fernàndez-Castillo, V Volpini, M del Toro,
M Roig, A Macaya, B Cormand
ARTICLE 2
CEPHALALGIA: ACCEPTAT
Capítol 2 – Article 2
RESULTATS
RESUM
Anàlis genètica de 27 pacients espanyols amb migranya hemiplègica, migranya de tipus
basilar i síndromes periòdiques de la infància.
La migranya hemiplègica familiar (FHM) és un tipus rar de migranya amb aura. Fins ara s’han
descrit mutacions en tres gens diferents en pacients amb FHM: CACNA1A (subunitat α1A del
canal de calci neuronal de tipus P/Q, FHM1), ATP1A2 (ATPasa transportadora de Na+ i Ca2+,
FHM2) i SCN1A (subunitat α1A del canal de Na+ neuronal de tipus I, FHM3). Hem analitzat
aquests gens en 27 pacients espanyols amb migranya hemiplègica (HM), migranya de tipus
basilar (BM) o síndromes periòdiques de la infància (CPS). Tot i que encara no hem demostrat
la seva patogenicitat a nivell funcional, s’han identificat dues noves variants en individus amb
HM, p.Val581Met i p.Tyr1245Cys, i un canvi prèviament descrit, p.Cys1534Ser, en el gen
CACNA1A. Cal destacar que la variant p.Tyr1245Cys s’ha identificat en un pacient amb un
fenotip canviant en funció de l’edat que comença com a torticoli paroxístic benigne del lactant
(BPT) i evoluciona a vertigen paroxístic benigne de l’adolescència (BPV) per acabar finalment
en HM. Aquest és el primer exemple en què es descriu una alteració genètica en un cas de
CPS. El fet que el cribratge molecular permeti identificar mutacions potencialment
patogèniques només en un 15% dels pacients amb HM constata l’existència d’heterogeneïtat
genètica a les formes de migranya presumiblement monogèniques.
109
Capítol 2- Article 2
RESULTATS
Genetic analysis of 27 Spanish patients with hemiplegic migraine, basilar-type
migraine and childhood periodic syndromes
E Cuenca-León1, R Corominas1, N Fernàndez-Castillo2,3, V Volpini4, M del Toro1, M
Roig1, A Macaya1, B Cormand2,3,5
1
Grup de Recerca en Neurologia Infantil i Psiquiatria Genètica, Hospital Universitari
Vall d’Hebron, Barcelona, Catalonia, Spain
2
Departament de Genètica, Universitat de Barcelona, Barcelona, Catalonia, Spain
3
Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER),
Barcelona, Catalonia, Spain
4
Center for Molecular Genetic Diagnosis - IDIBELL, l’Hospitalet de Llobregat,
Barcelona, Catalonia, Spain
5
Institut de Biomedicina de la Universitat de Barcelona (IBUB), Catalonia, Spain
Correspondence should be addressed to:
Bru Cormand, PhD
Associate Profesor of Genetics
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona
Av. Diagonal 645, edifici annex, 3ª planta
08028 Barcelona, SPAIN
Phone: 34-934021013
Fax: 34-934034420
email: [email protected]
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Anàlisi mutacional
ABSTRACT
Familial Hemiplegic Migraine (FHM) is a rare type of migraine with aura. Mutations in
three genes have been described in FHM patients: CACNA1A (FHM1), ATP1A2
(FHM2) and SCN1A (FHM3). We screened 27 Spanish patients with hemiplegic
migraine (HM), basilar-type migraine (BM), or childhood periodic syndromes (CPS) for
mutations in these three genes. We identified two novel CACNA1A variants,
p.Val581Met and p.Tyr1245Cys, and a previously annotated change, p.Cys1534Ser, in
individuals with HM, although they have not been proven to be pathogenic as yet.
Interestingly, p.Tyr1245Cys was detected in a patient displaying a changing, agespecific phenotype that began as benign paroxysmal torticollis of infancy (BPT),
evolving into benign paroxysmal vertigo of childhood (BPV) and later becoming HM.
This is the first instance where a specific gene alteration is described in a subject
affected with CPS. The fact that the molecular screen identified potential pathogenic
mutations in less than 15% of our HM patients further stresses the genetic
heterogeneity underlying the presumably monogenic forms of migraine.
Keywords: mutation analysis, CACNA1A, hemiplegic migraine, childhood periodic
syndromes, basilar-type migraine
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Capítol 2- Article 2
INTRODUCTION
Familial Hemiplegic Migraine (FHM) [MIM# 141599] and Sporadic Hemiplegic Migraine
(SHM) are rare types of migraine with aura. The deciphering of the first two molecular
defects underlying the condition has led to its being classified into subtypes. In FHM1,
missense mutations in the CACNA1A gene on chromosome 19p13.13, encoding the α
subunit of the neuronal P/Q-type calcium channel (CACNA1A), were first reported in
five unrelated FHM families (1). To date, at least 18 different CACNA1A missense
mutations have been reported in FHM families (2). Most of these mutations recur very
infrequently, with the exception of p.Thr666Met, found in 20 families worldwide and
p.Arg583Gln, reported in six families (3). About half of the FHM families show
additional signs of progressive cerebellar ataxia (4) or, less frequently, epileptic
seizures (5), cognitive dysfunction (6) or migraine coma (7).
Mutations in the CACNA1A gene also cause episodic ataxia type 2 (EA-2)
[MIM#108500], and spinocerebellar ataxia type 6 (SCA6) [MIM#183086]. Most of the
EA-2-related CACNA1A mutations are nonsense, splice site, small deletions or
insertions which disrupt the open reading frame and result in a truncated protein, while
SCA6 is usually caused by expansion of an unstable CAG repeat on the C-terminal
region of the protein (8). However, missense mutations have also been identified in
SCA6 and EA-2.
Conversely, FHM2 is linked to mutations in the ATP1A2 gene on chromosome
1q23.2. More than 30 FHM2 missense mutations have been identified in this gene so
far (2, 3, 9); but also in other phenotypes including alternating hemiplegia of childhood
(10), migraine with aura (MA),
migraine without aura (MO) (11) and basilar-type
migraine (BM) (12).
Recently, a third gene has been associated with FHM. The same mutation,
p.Gln1489Lys, was identified in three independent FHM families, now classified as
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Anàlisi mutacional
FHM3, in the SCN1A gene, which encodes the α subunit of the neuronal voltage-gated
type I sodium channel (13).
Some clinical syndromes that are precursors of migraine in young patients,
including cyclical vomiting (CV), abdominal migraine and benign paroxysmal vertigo in
childhood (BPV), have recently been reclassified by the IHS as Childhood Periodic
Syndromes (CPS). Although these syndromes have been known for decades (14, 15),
their suggested relationship with migraine remains a matter of debate, as is the case
for another possible subtype of migraine precursor, benign paroxysmal torticollis of
infancy (BPT). In addition, there is very little genetic evidence to date linking familial
migraine and CPS (16).
Here we report the clinical manifestations of a series of 27 Spanish patients
diagnosed with hemiplegic migraine (HM), BM or CPS and the results of a molecular
analysis of the three known FHM genes that allowed the identification of three
CACNA1A potential pathogenic mutations.
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Capítol 2- Article 2
RESULTATS
MATERIALS AND METHODS
Subjects
This study includes 27 unrelated Spanish patients referred to Vall d’Hebron University
Hospital, Barcelona, during the period 1999-2004 with diagnoses of HM, BM or CPS,
the migraine phenotypes previously associated with mutations in the CACNA1A,
ATP1A2 and SCN1A genes. CPS presented as BPV or BPT (the latter being
considered as a possible CPS by virtue of its present classification and encoded A1.3.5
in the International Classification of Headache Disorders (ICHD-II) appendix (17)).
Twenty-two patients were directly interviewed and examined by one of the authors
(AM) and information about the remaining 5 cases was obtained from referring
physicians and completed through telephone interview. Positive family history in first or
second degree relatives was elicited from the patients or from their parents in case of
paediatric patients. In kindreds with positive family history, at least one affected relative
was directly interviewed; the diagnosis of FHM was established upon the presence of
two or more cases of migraine with aura including motor weakness. The diagnosis of
CPS was based on the presence of recurrent torticollis, vertigo, vomiting and/or
behavioural changes in neurologically and audiologically intact children, and was later
confirmed in all cases following the guidelines given by the IHS in the ICHD-II (17). The
clinical diagnosis of migraine types was initially established in accordance with the
ICHD-I criteria (18) and later confirmed according to ICHD-II. For genetic studies we
recruited 64 unrelated control individuals from the Blood Extraction Unit of the Vall
d’Hebron University Hospital. They were Spanish, Caucasoid and lacked any history of
recurrent or disabling headache, as did their first-degree relatives.
Samples
Blood venous samples were obtained from 27 probands and from 64 unrelated nonmigraineurs. Genomic DNA was isolated using the QIAamp DNA Blood Maxi Kit
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RESULTATS
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(QIAGEN, Hilden, GE) after each subject had provided written informed consent for
DNA analysis and the local ethics committee had approved the study, which follows the
guidelines of the Helsinki Declaration.
DNA and Mutation analysis
The 47 exons of the CACNA1A gene, the 23 exons of the ATP1A2 gene and exon 23
of the SCN1A gene and their corresponding exon/intron junctions, including splice sites
and branch points, were PCR-amplified and sequenced in 42, 16 and 1 independent
PCR products, respectively. The primers were designed using Primer3 software
(frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi)
(19).
Primer
sequences
are
available as supplementary material (Tables S1 and S2). The details of the PCR
procedures are available from the authors upon request. The PCR products were
purified and sequenced (ABI PRISM 3700 DNA analyzer, Applied Biosystems, Foster
City, CA). The identified changes were confirmed by digestion of the corresponding
PCR product with the appropriate restriction enzyme: NspI (p.Val581Met), ItaI
(p.Tyr1245Cys) and HpyCH4V (p.Cys1534Ser).
Sixty-four unrelated healthy Spanish individuals were screened for the presence
of the gene variants identified in the patients by either restriction enzyme cleavage of
PCR products or Single-Strand Conformation Polymorphism (SSCP) analysis (20)
The potential disease-causing mutations and the polymorphic variants identified
in the CACNA1A gene were named according to HGVS guidelines (www.hgvs.org),
using RefSeq accession number NM_023035 as a cDNA reference sequence, with
nucleotide 283, the A of the ATG initiation codon, corresponding to +1, and
NP_075461 as the protein reference sequence. Both sequences correspond to the
CACNA1A transcript variant 2. The ATP1A2 reference sequences were NM_000702
for the cDNA and NP_000693 for the protein.
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Capítol 2- Article 2
RESULTATS
RESULTS
Clinical data
The main clinical features of the 27 patients are shown in Table 1. Episodes of HM
were documented in 21 patients, irrespective of the mode of onset. Age at onset
ranges from 15 days to 2 months in BPT (n=4), from 11 months to 6 years in BPV
(n=7) and from 8 to 18 years in HM (n=21). In the single case presenting as BM, onset
was in childhood.
Eight patients presented as CPS. The main symptom of CPS, torticollis or
vertigo, was often associated with pallor, hypotonia, irritability, anxiety, vomiting or
crying during the attacks. Of note, three of the four patients that had their onset in early
infancy with episodes of BPT, subsequently developed BPV and one of them has
finally evolved into the HM phenotype. The fourth BPT patient developed HM at age 8
years. Among patients who had their onset in childhood with episodes of BPV (n=4),
one has developed HM, one BM and two continue to display BPV at the ages of five
and six years, the latter with accompanying headache. All BPV patients had normal
EEG and audiometric testing; clinical screening of vestibular function in school-aged
children was also normal.
The remaining 19 patients presented with more typical “adult” phenotypes,
including FHM (n=7), SHM (n=7), MA (n=4) and BM (n=1). All patients with MA went on
to develop typical HM attacks. No patient had concomitant ataxia.
Interictally, all 27 patients had normal neurological examinations and all had normal
brain MRI.
Overall, family history of migraine was present in 23/27 cases, including 18/21
of those developing HM, 2/2 with BM and 3/4 of the younger patients with CPS that
have not developed migraine. In 12 families, the affected relatives fulfilled the ICHD-II
criteria (17) for FHM.
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No other signs suggestive of neuronal channelopathy were noted, except for
infantile convulsions that were reported in case 6 and in one of his relatives and
epilepsy in three other unrelated subjects belonging to the families of cases 14, 16 and
27.
Genetic analysis
Two new and one previously cited gene variants were identified in three unrelated
individuals (cases 1, 9 and 27) after extensive sequencing of the CACNA1A gene in
the 27 probands (Table 2). In the remaining 24 patients not bearing CACNA1A
alterations, a sequence analysis of the ATP1A2 gene and exon 23 of the SCN1A gene
did not reveal any putative pathogenic change.
In a patient presenting the age-specific sequential phenotypes of BPT, BPV and
FHM (case 1) a change from A to G at cDNA nucleotide 3734 in exon 22 was
identified, prompting the new gene variant p.Tyr1245Cys, located at domain III,
segment 1 (DIII-S1) of Cav2.1. A patient with pure FHM (case 9) was found to harbour
the p.Val581Met variant, encoded in exon 13. This was brought about by a G to A
substitution at cDNA nucleotide 1741 leading to the substitution of a methionine for a
highly conserved valine located at domain II, segment 4 (DII-S4) of the protein. Finally,
in another FHM patient (case 27), a change from G to C at cDNA nucleotide 4601 in
exon 29 produced the p.Cys1534Ser variant which lies in the intracellular loop between
domains DIII and DIV (Fig. 1 and Table 2) . The pedigrees of patients 1, 9 and 27 are
shown in Fig. 2.
The presence of these gene variants was confirmed by a restriction analysis of
the corresponding PCR products and they were not present in 64 unrelated Spanish
non-migraineurs. The residues that were replaced are highly conserved in evolution in
paralogous human α1 subunits of Cav2 and Cav1 channels (CACNA1A, B, E, D, F, C
and S) as well as in orthologous CACNA1A subunits of cattle (Bos taurus), mouse
(Mus musculus), rat (Rattus norvegicus), rabbit (Oryctolagus cuniculus), zebrafish
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Capítol 2- Article 2
RESULTATS
(Danio rerio) and fruit fly (Drosophila melanogaster) (Fig. 3). The conservation is only
lost for residue p.Tyr1245 in the human CACNA1D, F, C and S protein (present in Cav1
channels), but not in CACNA1B and E (Cav2 channels), evolutionary and functionally
closer to CACNA1A (21).
No potential pathogenic mutations were found in the coding region of the
CACNA1A gene in the remaining 24 probands, some of which bear polymorphic
variants with non-apparent pathogenic effect (Table S3). To our knowledge, four of
these changes, c.400-28C>T (intron 2), c.5253-101C>T (intron 34), c.5944-52C>T
(intron 40) and c.6840G>A or p.Pro2280Pro (exon 47) are described here for the first
time, the latter being located in the coding region of the CACNA1A isoform 2. All these
changes were found in several patients and also in a subset of 64 Spanish healthy
controls in which minor allele frequency (MAF) ranged from 0.008 to 0.211. We also
found three previously described non-synonymous variants located in exons 19
(rs16022C>G or p.Asp918Glu and rs16023T>A or p.Val993Glu) and 20 (rs16027A>G
or p.Ser1105Gly), which were present in several patients and in the general population
(Table S3). Three previously described polymorphic variants (rs16041C>T in intron 35,
rs16006A>G in exon 6 and c.579G>A in exon 4) were found only in one patient each.
Their population frequencies had been studied previously by other authors in several
panels of individuals and were found to range between 0.02 and 0.04. One of them,
c.579G>A (p.Thr193Thr), was not found in a screening of 64 Spanish non-migraineurs
performed by us, although it was present in a set of 50 randomly collected Dutch
individuals with a MAF of 0.02 (1).
The 24 individuals not bearing potential pathogenic CACNA1A mutations did
not show ATP1A2 changes either. Yet, several polymorphic variants were once again
identified, including two novel changes in the 5’-UTR region of the gene (c.1-48C>G
and c.1-42G>C), both present only in patient 11, and two previously described
polymorphic variants (c.749-43G>C in intron 7 and rs2070701G>A in the 3’-UTR
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region), also found in only one patient each (Table S3). The presence of the
p.Gln1489Lys SCNA1A missense mutation was ruled out in these 24 individuals.
DISCUSSION
We report two novel and one previously cited potential pathogenic mutations in
the CACNA1A gene in a set of 27 unrelated, predominantly familial, Spanish
cases with different migraine variants (HM, BM, CPS). To our knowledge, this is
the first molecular analysis of the CACNA1A, ATP1A2 and SCN1A genes to
have been conducted in Spanish patients.
Molecular genetics of HM
In our series, the ratio of three cases bearing CACNA1A changes out of 11 pure
FHM cases (27.3 %) is roughly in line with figures reported elsewhere (e.g. 4/12
(22) or 6/42 (3)), but stands in contrast to the widely accepted notion that
mutations in the CACNA1A gene account for more than half of FHM cases,
reviewed in (2). However, a more detailed review of the literature reveals that
these high percentages were only recorded when mutational screenings
included FHM families selected on the basis of their putative linkage to the
CACNA1A locus on 19p13 or on that of the occurrence of concomitant
cerebellar ataxia. Thus, 15 mutations were found in 16 FHM families with
cerebellar signs and positive linkage to CACNA1A (22) and 5 out of 6 FHM
families with previous positive linkage, two of them with cerebellar signs (1).
The proportion of patients with CACNA1A mutations is much lower in the case
of SHM, and always below 5% in the absence of cerebellar signs (1, 23-25). In
our study we did not find any CACNA1A mutation among the 10 SHM cases
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Capítol 2- Article 2
RESULTATS
without cerebellar signs, despite the fact that seven of them displayed familial
history of (non-hemiplegic) migraine.
It is of note that no ATP1A2 changes were found in our series. This is in
contrast with previous studies that report percentages of 42% (11/26) (26), 17%
(1/6) (27) and 7% (3/42) (3) for ATP1A2 mutations in FHM patients without
mutations in the CACNA1A gene. However, no ATP1A2 mutations were found
in a study including 19 FHM and 7 BM patients (25). Mutations in SCN1A, the
only other gene linked to FHM and involved in several forms of epilepsy, would
appear to be as a rather unusual cause of FHM. Indeed, more than 160
mutations have been identified in SCN1A (HGMD, www.hgmd.cf.ac.uk) (28)
compared to just one in FHM (13). Finally, at least one other FHM locus has
been mapped to 1q31, but the underlying gene awaits identification (29).
The fact that only 3 CACNA1A potential pathogenic mutations have been
identified in 27 patients with HM, BM or CPS after an exhaustive screening of
CACNA1A and ATP1A2 may be explained by the high level of genetic
heterogeneity in this group of paroxysmal conditions. Nevertheless, it is also
possible that a low number of mutations have remained unidentified in the
genes studied, including changes in introns or in the regulatory regions, or
gross alterations that may be undetectable by PCR.
Childhood Periodic Syndromes and CACNA1A
In this report we describe eight patients that presented with a CPS
phenotype in infancy or childhood. The various CPS phenotypes have generally
been considered paediatric equivalents of migraine and three of them, BPV, CV
and abdominal migraine, are recognized by the IHS as migraine equivalents in
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infancy or early childhood. A fourth phenotype, BPT, awaits validation as CPS
or migraine precursor. BPT might very well be the earliest manifestation of
migraine in life. In fact, our patient 1 presented with torticollis episodes during
the neonatal period, similar to two cases reported elsewhere (30, 31). Our
patient was found to bear the p.Tyr1245Cys variant in the CACNA1A gene and
his clinical course reflects the changing, age-specific phenotypes associated
with CACNA1A dysfunction, i.e. BPT, BPV and FHM. This sequence in the
leading clinical manifestations, which may at times overlap, was seen in three of
our four BPT patients and has also been suggested by other authors (32-35).
Our results provide evidence of a common genetic background for some forms
of CPS and FHM. A previous study (16) reported a patient with BPT featuring
interictal ataxia and who belonged to one kindred with FHM and ataxia linked to
a CACNA1A mutation. However, the authors did not disclose the genotype of
the probandus. BPV, in turn, is the most common cause of childhood vertigo
without ear disease or hearing loss (36). Classic BPV begins between 1 and 4
years of age and further evolution towards MA is observed in more than 50% of
cases (37, 38). Among our seven BPV cases, three have developed classical
migraine phenotypes by age 10: FHM in two cases and BM in one. The
remaining four patients are younger than 7 years; two of them complain of
recurrent headaches.
Description of identified CACNA1A missense variants
Several findings point to a causative role of the three missense changes
identified
122
in
the
CACNA1A
gene,
p.Val581Met,
p.Tyr1245Cys
and
Capítol 2- Article 2
RESULTATS
p.Cys1534Ser, although the eventual demonstration of their pathogenicity in
FHM will require functional studies.
The p.Val581Met variant (patient 9) involves a residue located within the
transmembrane S4 segment of the DII domain (DII-S4), and the change is
predicted to shorten a α helix, as determined by the PSIPRED software
(www.psipred.net) (39). This segment probably represents the voltage sensor of
the channel, in which most mutations have been described. All the mutations
reported to date in S4 segments, except for the mutation reported here, are
substitutions of a conserved arginine (192, 195, 583, 1347, 1661, 1664 and
1667) and the majority of them have been associated with the combination of
hemiplegic migraine and episodic ataxia or cerebellar signs.
The p.Tyr1245Cys variant (patient 1) affects the transmembrane DIII-S1
segment. No other missense/nonsense changes have previously been
described in the S1 segment of any domain of the α1A subunit of the CaV2.1
channel in any patient with FHM. This may argue against its involvement in the
disease, although the total number of FHM mutations that have been described
in the gene so far (around 20) is still small.
The third gene variant identified, p.Cys1534Ser (patient 27), was
described in a previous report in three relatives with FHM, two of them with
concomitant episodic ataxia (40). However, the presence of this change in the
general population was not assessed, and its putative structural/functional
relevance not discussed. Prediction of the protein secondary structure indicates
that the amino acid change produces a shortening of a α helix which might have
functional consequences for the channel. The mutation involves a residue
located in the intracellular loop between domains DIII and DIV, where no other
123
RESULTATS
Anàlisi mutacional
mutations have been described in FHM to our knowledge. Interestingly, the
CACNA1A protein is structurally very similar to SCN1A, and the only FHM
mutation (p.Gln1489Lys) described so far in the latter is, like Cys1534Ser, also
located in the DIII-DIV linker.
Other evidence supports the relation of these three missense variants
with the disease phenotype: First, the residues involved are highly conserved in
evolution, both at the intraspecific (human CACNA1A, B, C and E subunits) and
interspecific levels (CACNA1A subunit of human, cattle, mouse, rat, rabbit,
zebrafish and fruit fly), indicating functional/structural relevance (Fig. 3).
Second, we did not detect the presence of these variants in a screening of 64
healthy Spanish individuals in which migraine had been specifically excluded,
and the changes are not present in the public SNP databases, indicating that
they are not polymorphic variants. And third, no other molecular alterations
were identified within the gene after the analysis of the whole coding region and
the exon-intron boundaries, including splice sites and branch points.
Unfortunately, family members were unavailable for cosegregation analysis
between the disease phenotype and the identified variants, which may had
provided additional clues about their putative causative role.
Conclusions
Mutational analysis of ion channel genes is often expensive and timeconsuming, but it is conceivable that some clinical clues might help guiding
molecular diagnosis in HM. Thus, episodic or permanent cerebellar ataxia or
trauma-induced episodes should prompt investigation of CACNA1A mutations
(FHM1). Alternating hemiplegia or BM may suggest a defect in ATP1A2
124
Capítol 2- Article 2
RESULTATS
(FHM2). Concomitant epilepsy suggests mutations in SCN1A (FHM3), although
absence epilepsy has been described in FHM1 and benign familial infantile
convulsions in FHM2.
On the basis of the present results reported here and previous molecular
genetic studies, we hypothesize that, while the list of FHM-causative genes is
expected to grow, a substantial number of cases may follow more complex
patterns of inheritance, similar in this respect to other migraine variants.
ACKNOWLEDGEMENTS
The authors wish to thank all the patients and family members for their cooperation and
J Artigas, M Pineda, A Rodríguez, and M Galván for patient referral. JM Fernández is
acknowledged for helpful suggestions in the preparation of the manuscript and M
Ribasés for technical assistance. This study was supported by the Spanish Ministry of
Education and Science (SAF-2000/197 and SAF-2003/04704), Red Española de
Ataxias, Fondo de Investigación Sanitaria, Spain (G03/056 and PI052129), Instituto de
Salud Carlos III, Spain (PI061073, PI050996) and AGAUR (2005SGR00848). E.C-L.
was a recipient of a FPI scholarship of the Ministerio de Ciencia y Tecnología, Spain.
R.C. was funded by the Institut de Recerca Vall d’Hebron.
125
RESULTATS
Anàlisi mutacional
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Dichgans M, Freilinger T, Eckstein G, Babini E, Lorenz-Depiereux B, Biskup S, et
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SUPPLEMANTARY MATERIAL REFERENCES
1
Ophoff RA, Terwindt GM, Vergouwe MN, van Eijk R, Oefner PJ, Hoffman SM, et
al. Familial hemiplegic migraine and episodic ataxia type-2 are caused by
mutations in the Ca2+ channel gene CACNL1A4. Cell 1996;87:543-52.
129
RESULTATS
2
Anàlisi mutacional
Riant F, De Fusco M, Aridon P, Ducros A, Ploton C, Marchelli F, et al. ATP1A2
mutations in 11 families with familial hemiplegic migraine. Hum Mutat
2005;26:281.
130
Capítol 2- Article 2
RESULTATS
FIGURE LEGENDS
Figure 1. Schematic representation of the human CACNA1A gene (top), the three
potential pathogenic mutations identified in this study with the corresponding sequence
electropherograms (middle) and the encoded protein (bottom). Previously reported
FHM mutations are indicated with numbers on the protein. cDNA and protein sequence
numbering are according to HGVS guidelines (www.hgvs.org), using NM_023035 as a
reference sequence for the cDNA and NP_0755461 for the protein.
Figure 2. Structure of pedigrees of patients with identified gene variants. Affected
individuals are denoted by solid symbols. Clinical characteristics are indicated below
each individual (HM: migraine with hemiplegic aura, MA: migraine with aura; MO:
migraine without aura). The CACNA1A variant carrier status is indicated below each
index patient.
Figure 3. Protein alignment performed with clustalW (www.ebi.ac.uk/clustalw). The
human CACNA1A Val581 and Cys1534 residues are conserved in all the human calcium
channel α1 subunits studied (CACNA1B, E, D, F, C and S) whereas Tyr1245 is
conserved only in the evolutionary closer subunits (CACNA1B and E). All three
positions are conserved in the orthologous CACNA1A proteins of several organisms.
Non-conserved amino acids are indicated in grey. In brackets, the RefSeq code of
each protein. ZEFI: Zebrafish, Danio rerio. DROME: Fruit fly, Drosophila melanogaster.
131
RESULTATS
Figure 1
Figure 2
132
Anàlisi mutacional
Capítol 2- Article 2
RESULTATS
Figure 3
133
RESULTATS
134
Anàlisi mutacional
Capítol 2- Article 2
RESULTATS
135
CRIBRATGE MUTACIONAL DEL GEN CACNA1A EN
INDIVIDUS AMB A TÀXIA EPISÒDICA DE TIPUS 2
ANNEX A L’ARTICLE 2
Capítol 2- Annex a l’article 2
RESULTATS
RESUM
Cribratge mutacional del gen CACNA1A en individus amb atàxia episòdica de tipus 2 (EA2)
L’atàxia episòdica de tipus 2 (EA2) és un tipus rar de canalopatia autosòmica dominant que es
manifesta amb episodis de desequilibri i incoordinació. Fins ara s’han identificat més de 50
mutacions responsables del fenotip EA2 en el gen CACNA1A. Aquest treball se centra en
l’anàlisi mutacional d’aquest gen en 27 pacients espanyols que manifesten EA2 d’inici tardà
associada a símptomes acompanyants o a altres trastorns paroxístics. S’han identificat 4
canvis potencialment patogènics: p.Gly638Asp i p.Pro1011Ala en pacients afectats d’EA2
d’inici tardà, i p.Arg583Gln (prèviament descrita) i p.Thr501Met en dos individus que
presenten EA2 i migranya amb aura (MA). La baixa proporció de mutacions identificades en el
gen CACNA1A suggereix que aquest gen no és la causa principal de la EA2 i, en canvi, sembla
que juga un paper important quan EA2 es presenta en combinació amb MA.
139
Capítol 2 – Annex a l’article 2
RESULTATS
CRIBRATGE MUTACIONAL DEL GEN CACNA1A EN INDIVIDUS AMB
ATÀXICA EPISÒDICA DE TIPUS 2 (EA2)
Pacients
Gràcies a la pertinença a la la Red Española de Ataxias (REA) es van poder identificar i reclutar
35 individus amb sospita d’EA2 i en funció de les proves diagnòstiques realitzades i la
disponibilitat de mostres se’n van seleccionar 27 per a l’estudi mutacional del gen CACNA1A,
realitzat en el marc d’aquesta Tesi. Tots els pacients, a excepció de l’individu 3 que debuta
amb 16 anys, tenen la particularitat que presenten una variant d’EA2 d’inici tardà, amb els
primers episodis entre els 30 i els 65 anys. La major part dels pacients presenten símptomes
acompanyants durant els episodis com nistagmus o vertigen, o bé tenen associats trastorns
paroxístics d’altres tipus, com migranya o més rarament epilèpsia. En alguns pacients, la
sensació d’inestabilitat i el vertigen són fins i tot més prominents que la pròpia atàxia, el signe
objectiu que pot documentar el metge. Així, aquests pacients es trobarien nosològicament
entre el diagnòstic d’EA2 i el d’atàxia episòdica hereditària vestíbulo-cerebel·losa o EA4. Els
pacients EA2 poden presentar història d’atàxia crònica progressiva o alteracions
oculomotores interictals, a part dels episodis aguts d’atàxia (taula 1). En dos familiars del cas
índex 1 s’ha documentat atròfia cerebel·losa (CA) mitjançant proves de neuroimatge.
Individu
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
EA2,
EA2,
EA2,
EA2,
EA2,
EA2,
EA2,
EA2
EA2
EA2
EA2,
EA2,
EA2
EA2
EA2,
EA2,
EA2,
EA2,
EA2,
EA2
EA2
EA2
EA2,
EA2,
EA2
EA2,
EA2
Característiques clíniques
PCA, disàrtria, MA
FHM
vertigen
vertigen, nistagmus
PCA
PCA
PCA
Taula 1. Característiques
clíniques dels pacients EA2.
EA2= Atàxia episòdica de
tipus 2, PCA= Atàxia
cerebel·losa progressiva,
MA= migranya amb aura,
FHM= migranya hemiplègica
familiar, MO= migranya
sense aura.
vertigen
vertigen
nistagmus
nistagmus
nistagmus
vertigen
PCA
migranya, epilèpsia
disàrtria
vertigen, torticoli paroxístic, convulsions febrils
141
RESULTATS
Anàlisi mutacional
Els casos esporàdics són rars, de manera que gairebé tots els individus estudiats presenten
història familiar positiva d’atàxia episòdica o progressiva. En el cas del pacient 1, que prové
d’una família extensa amb recurrència d’atàxia episòdica i/o progressiva, l’estudi es va fer
extensiu a tots els membres disponibles de la genealogia (figura 1).
I.1
2
I.2
3
1
p.Arg583Gln/+
II.1
II.2
2 1
+/+
1 1
+/+
III.1
II.3
3 2
p.Arg583Gln/+
2
+/+
II.4
II.5
2 2
+/+
2 1
+/+
III.2
1 3
p.Arg583Gln/+
II.6
3 2
p.Arg583Gln/+
II.7
3 2
p.Arg583Gln/+
EA2
CA
MA
HM
Figura 1. Representació esquemàtica de la família del pacient 1 amb la mutació identificada al gen CACNA1A. Els
al·lels del marcador microsatèl·lit D19S1150 estan representats en rectangles. L’al·lel que cosegrega amb la malaltia
s’indica en negre. L’individu índex II.3 és el pacient 1 de la taula 1. EA2= atàxia episòdica de tipus 2; CA= atròfia
cerebel·losa; MA= migranya amb aura; HM= migranya hemiplègica.
Exclusió d'atàxies espinocerebel·loses (SCAs)
Com que alguns pacients presenten atàxia cerebelosa progressiva o van desenvolupar els
primers símptomes de la malaltia episòdica a edats molt avançades, es va considerar oportú
determinar el número de repeticions CAG en els gens responsables de les SCA de tipus 1, 2, 3,
6, 7, 8 i 12 i en el gen DRPLA. Tots els individus presentaven un nombre de repeticions dins el
rang de la ‘normalitat’. Aquesta feina fou realitzada pel grup del Dr. Víctor Volpini a l’Institut
de Recerca Oncològica (IRO).
142
RESULTATS
Capítol 2 – Annex a l’article 2
Lligament al locus CACNA1A a la família del pacient 1 (II.3)
Amb la genotipació de tots els individus disponibles de la família del pacient 1 pel marcador
D19S1150, situat a l’intró 7 del gen CACNA1A, es va poder observar cosegregació del fenotip
atàxic amb l’al·lel 3 del polimorfisme (figura 1). En fer càlculs de lligament genètic entre el
marcador i la malaltia amb el programa MLINK (Terwilliger and Ott, 1994), es va obtenir un
LOD score de dos punts de 1,87 considerant una penetració del fenotip del 90% i una taxa de
fenocòpies del 5%. Les simulacions realitzades amb el programa SLINK (Ott, 1989; Weeks et
al., 1990) mostren que aquest valor coincideix amb el valor màxim de LOD score esperat tenint
en compte l’estructura de la família i la informativitat del marcador utilitzat. Aquesta feina es
va realitzar en col·laboració amb el grup del Dr. Víctor Volpini de l’Institut de Recerca
Oncològica (IRO).
Anàlisi mutacional del gen CACNA1A
L’anàlisi exhaustiu dels 47 exons del gen CACNA1A i de les regions intròniques flanquejants en
els 27 pacients (inclòs el pacient 1) amb EA2 i altres símptomes acompanyants mitjançant
seqüenciació de productes de PCR ha permès identificar quatre mutacions de canvi de sentit:
dues mutacions noves (p.Thr501Met i p.Gly638Asp), una mutació ja descrita prèviament
(p.Arg583Gln) i una aparent mutació (p.Pro1011Ala) curiosament descrita a les bases de dades
com a polimorfisme (rs28413664), tot i que no s’ofereixen dades de freqüències al·lèliques en
cap població (taula 2).
Taula 2. Pacients i mutacions en el gen CACNA1A
Mutació
Pacient
cDNA
Proteïna
Posició de la mutació
Canvi de
codó
Exó
Domini de la proteïna
Mètode de
detecció
Fenotip
Pacient 2
c.1502C>T
p.Thr501Met
ACG>ATG
11
Transmembrana S1, DII
SSCP
EA, FHM
Pacient 1
c.1748G>A p.Arg583Gln
CGA>CAA
13
Segment transmembrana S4, DII
Enzim -Ban II
PCA, MA
Pacient 13 c.1913G>A p.Gly638Asp
GGC>GAC
14
Segment extracelular S5-S6, DII
Enzim -Ita I i SSCP
EA
Pacient 14 c.3031C>G p.Pro1011Ala
CCA>GCA
19
Llaç citoplasmàtic DII-DIII
Enzim +Pst I
EA
La numeració del cDNA i de la seqüència proteica segueix les pautes de la Human Genome Variation Society
(www.hgvs.org). Per la numeració del cDNA s’ha considerat com a posició +1 el nucleòtid A del codó d’iniciació
ATG (que correspon al nucleòtid 283 de la seqüència de referència NM_023035). La numeració de la proteïna s’ha
realitzat en base a la seqüència de referència NP_0755461. El símbol ‘+’ o ‘-’ indica que el canvi crea o destrueix una
diana per l’enzim de restricció corresponent. EA= Atàxia episòdica de tipus 2. PCA= Atàxia cerebel·losa
progressiva. FHM= Migranya hemiplègica familiar. MA= Migranya amb aura.
143
RESULTATS
Anàlisi mutacional
Mutació p.Thr501Met:
Situada a l’exó 11 del gen CACNA1A, aquesta mutació s’ha identificat en un pacient amb
atàxia episòdica i migranya hemiplègica familiar. El canvi provoca una substitució de
l’aminoàcid treonina, polar, per l’aminoàcid metionina, no polar, en el segment
transmembrana S1 del segon domini de la proteïna, S1DII (figura 2). La posició mutada està
molt conservada tan a nivell interespecífic com intraespecífic (figura 3), i el canvi no està
present en 128 cromosomes d’individus no migranyosos analitzats.
D19S1150
Figura 2. Representació esquemàtica del gen CACNA1A (a dalt), les quatre mutacions identificades en aquest
estudi amb els corresponents electroferogrames (al mig) i la proteïna codificada (a baix). Les mutacions s’han
anomenat seguint les pautes de la HGVS (www.hgvs.org), utilitzant com a referència la seqüència
NM_023035 pel cDNA i la NP_0755461 per la proteïna.
Mutació p.Gly638Asp:
Es va detectar en un pacient amb EA2 i està situada en el llaç extracel·lular entre els segments
S5 i S6 del segon domini de la proteïna, S5-S6DII (figura 2). La substitució nucleotídica
produeix un canvi de l’aminoàcid polar glicina per l’àcid aspàrtic, carregat negativament.
Aquesta mutació tampoc no es va trobar en 64 individus control. Aquesta posició presenta
una alta homologia entre les diferents espècies analitzades i també està conservada en altres
canals de calci humans (figura 3).
144
RESULTATS
Capítol 2 – Annex a l’article 2
Mutació p.Pro1011Ala:
Situada a l’exó 19 del gen CACNA1A, aquest canvi es va identificar en un pacient, amb episodis
d’atàxia de dies de durada que es resolen espontàniament. Aquesta substitució, absent en 128
cromosomes control, provoca el canvi d’una prolina per una alanina que està en el segment
citoplasmàtic d’unió entre els dominis II i III on s’han identificat regions d’interacció amb
d’altres proteïnes reguladores del canal (figura 2). Aquesta posició està conservada en els gens
ortòlegs de vaca, ratolí, rata i conill però no ho està a nivell intraespecífic quan es comparen
els diferents canals de calci humans (figura 3).
p.Thr501Met
p.Arg583Gln
p.Gly638Asp
p.Pro1011Ala
O00555|CACNA1A_HOME
Q00975|CACNA1B_HOME
Q15878|CACNA1E_HOME
Q01668|CACNA1D_HOME
O60840|CACNA1F_HOME
Q13936|CACNA1C_HOME
Q13698|CACNA1S_HOME
WTVLSLVALNTLCVAIVHYNQ
WVVLCVVALNTLCVAMVHYNQ
WIVLSLVALNTACVAIVHHNQ
WLVIVLVFLNTLTISSEHYNQ
WAVLLLVFLNTLTIASEHHGQ
WLVIFLVFLNTLTIASEHYNQ
WLVILIVALNTLSIASEHHNQ
PGTSFGISVLRALRLLRIFKV
PGSSFGISVLRALRLLRIFKV
PGTSFGISVLRALRLLRIFKI
IMSPLGISVFRCVRLLRIFKV
AMQPLGISVLRCVRLLRIFKV
IMSPLGISVLRCVRLLRIFKI
AMTPLGISVLRCIRLLRIFKI
FALLGMQLFGGQFNFDEGTPFALLGMQLFGGQFNFQDETPFALLGMQLFGGRFNFNDGTPFSLLGMQLFGGKFNFDETQTK
FSLLGMQLFGGKFNFDQTHTK
FSLLGMQLFGGKFNFDEMQTR
FALLGMQLFGGRYDFEDTEVR
ERRRRHRHGAPATYEGDARRE
EPARRHRARHKAQPAHEAVEK
QDLRRTNSLMVSGLAGGL
---------------------------------------------------------------------------------
O00555|CACNA1A_ HOME
Q1ADE8|Q1ADE8_VACA
P97445|CACNA1A_RATOLÍ
P54282|CACNA1A_RATA
P27884|CACNA1A_CONILL
P91645|CACNA1A_DROME
WTVLSLVALNTLCVAIVHYNQ
WTVLSLVALNTLCVAIVHYNQ
WTVLSLVALNTLCVAIVHYNQ
WTVLSLVALNTLWLAIVHYNQ
WTVLSLVALNTLCVAIVHYNQ
WFVIVLVFLNTVCVAVEHYGQ
PGTSFGISVLRALRLLRIFKV
PGTSFGISVLRALRLLRIFKV
PGTSFGISVLRALRLLRIFKV
PGTSFGISVLRALRLLRIFKV
PGTSFGISVLRALRLLRIFKV
–GGSFGLSVLRALRLLRIFKV
FALLGMQLFGGQFNFDEGTPP
FALLGMQLFGGQFNFDEGTPP
FALLGMQLFGGQFNFDEGTPP
FALLGMQLFGGQFNFDEGTPP
FALLGMQLFGGQFNFDEGTPP
FALLGMQLFGGQFNLPGGTPE
ERRRRHRHGAPATYEGDARRE
ERRRRHRHGPPPAYDADMRRE
ERKRRHRHGPP-------AHD
ERKRRHRHGPP-------AHD
ERRRRHRHGPPPAYDPDARRD
---------------------
Figura 3. Alineament proteic realitzat amb el programa ClustalW (www.ebi.ac.uk/clustalw). Els residus Thr501,
Arg583 i Gly638 de la proteïna CACNA1A humana estan conservats en totes les subunitats α1 estudiades (CACNA1B,
E, D, F, C i S), però no així l’aminoàcid Pro1011, el més C-terminal dels canvis identificats. Les quatre posicions estan
conservades a la proteïna ortòloga de diversos organismes. La posició mutada es destaca en negre i les regions no
conservades, en gris. Davant de cada seqüència protèica hi ha el codi SwissProt. HOME: Homo sapiens, VACA: Bos
taurus, RATOLÍ: Mus musculus, RATA: Rattus norvegicus, CONILL: Oryctolagus cuniculus, DROME: Drosophila
melanogaster.
Mutació p.Arg583Gln:
És una mutació ja descrita prèviament, situada a l’exó 13 del gen CACNA1A en el pacient 1
(figura 2), que correspon al cas índex (II.3) d’una família amb migranya i atàxia (figura 1). Es va
estudiar la presència/absència del canvi identificat a la resta d’individus de la família i es va
detectar en tots els individus afectats i portadors de l’al·lel 3 del marcador intragènic
D19S1150 i en cap dels individus sans (figura 1). La mutació cosegrega amb tots els fenotips
d’atàxia de la família, que inclouen EA2 en els individus més joves - un d’ells és portador de la
mutació (III.2) i l’altre no va ser analitzat per falta de consentiment (III.1)-, atàxia i atròfia
cerebel·losa, que és present en dos individus portadors de la mutació (I.2 i II.6) i atàxia, atròfia
cerebel·losa i MA (individu II.3) o HM (individu II.7). Aquesta mutació provoca el canvi de
l’aminoàcid arginina, amb càrrega positiva, per una glutamina, un residu polar no carregat, en
el segment S4 del segon domini de la proteïna, S4DII (figura 2), situat en el sensor de voltatge
del canal. Aquesta posició està molt conservada tant a nivell interespecífic com intraespecífic
145
RESULTATS
Anàlisi mutacional
(figura 3). Aquesta és la segona mutació més prevalent identificada fins ara al gen CACNA1A,
amb un mínim de 7 casos familiars i un cas esporàdic (Battistini et al., 1999; Ducros et al.,
2001; Terwindt et al., 2001; Alonso et al., 2003; Thomsen et al., 2007).
146
CAPÍTOL 3
EPISODIC
SPONTANEOUS
HYPOTHERMIA
WITH
HYPERHIDROSIS IN A PATIENT WITH A NOVEL CACNA1A
VARIANT: EVIDENCE FOR A NEW MIGRAINE PRECURSOR?
E. Cuenca-León, R. Corominas, B. Cormand and A. Macaya
ARTICLE 3
EN PREPARACIÓ
Capítol 3- Article 3
RESULTATS
RESUM
Hipotèrmia episòdica espontània amb hiperhidrosi en un pacient amb una nova
variant al gen CACNA1A: un nou precursor de migranya?
Es va identificar una pacient de 7 mesos d’edat que presentava episodis periòdics d’hipotèrmia
amb hiperhidrosi i absència de tremolors. Als sis anys d’edat els episodis d’hipotèrmia
persistien i va començar a tenir cefalees recurrents. També es va documentar apnea
obstructiva del son. La pacient té història familiar de migranya i epilèpsia. L’anàlisi mutacional
del gen CACNA1A va revelar una mutació a l’exó 20. Es tracta d’una transversió de C a G a la
posició 3638 del cDNA (c.3638C>G), que dóna com a resultat la substitució d’un residu de
prolina per un d’alanina (p.Pro1138Ala) en el bucle intracel·lular situat entre els dominis DII i
DIII de la proteïna.
Tot i que encara no s’han estudiat les conseqüències funcionals, aquest canvi podria posar de
manifest un mecanisme fisiopatològic comú entre la hipotèrmia episòdica espontània amb
hiperhidrosi (ESHH) i la migranya. Així, la ESHH podría situar-se com a una nova variant de
síndromes periòdiques de la infància (CPS). El defecte en la termoregulació podria ser
conseqüència d’una disfunció hipotalàmica, fet que relacionaria la ESHH amb altres cefalees
primàries, principalment amb la cefalea acuminada o en clusters.
149
Capítol 3- Article 3
RESULTATS
EPISODIC SPONTANEOUS HYPOTHERMIA WITH HYPERHIDROSIS IN A
PATIENT WITH A NOVEL CACNA1A VARIANT: EVIDENCE FOR A NEW
MIGRAINE PRECURSOR?
E. Cuenca-León, R. Corominas, B. Cormand1 and A. Macaya
Grup de Recerca Neurologia Infantil i Psiquiatria Genètica, Hospital Universitari
Vall d’Hebron, 1Departament de Genètica, Facultat de Biologia, Universitat de
Barcelona , CIBER Enfermedades Raras, Instituto de Salud Carlos III, Institut
de Biomedicina de la Universitat de Barcelona (IBUB), Barcelona, Spain
Correspondence:
Alfons Macaya, MD
Grup de Recerca Neurologia Infantil i Psiquiatria Genètica
Hospital Universitari Vall d’Hebron
Pg. Vall d’Hebron 119-129, 08035 Barcelona, Spain
Tel. +34 93 4894334
Fax: +34 93 2746837
Email: [email protected]
151
RESULTATS
Hipotèrmia: CPS?
ABSTRACT
A young girl presented at the age of 7 months with periodic episodes of
hypothermia with absent shivering and hyperhidrosis. Her family history was
notable for the presence of both migraine and partial epilepsy. At age 6 years
the hypothermia episodes persisted and she had developed recurrent
headaches. Obstructive sleep apnea was documented. A mutation in exon 20 of
the CACNA1A gene was detected. A C to G transversion at cDNA nucleotide
3638 resulted in a replacement of a proline for an alanine residue,
p.Pro1138Ala, in the intracellular loop between the domains DII and DIII of the
protein. The functional consequences of this change are unclear, but it raises
the issue of a common pathophysiology of episodic spontaneous hypothermia
with hyperhidrosis (ESHH) and migraine. This finding may therefore support the
inclusion of ESHH as a new variant of childhood periodic syndrome.
Thermoregulation failure might also result from hypothalamic dysfunction; in this
respect the condition might also relate to other primary headaches, most
notably cluster headache.
152
RESULTATS
Capítol 3- Article 3
INTRODUCTION
Hypothermia
is
defined
as
core
body
temperature
below
35ºC.
Nonenvironmental hypothermia occurs infrequently in childhood; some well
known acquired causes are brain tumors, sepsis, endocrinologic disturbances
and drug intoxications. It has been also described in adults with brain injury (1),
subarachnoidal hemorrhage (2), limbic encephalitis (3) or multiple sclerosis (3).
Some rare congenital syndromes may present with episodic hypothermia,
including agenesis of the corpus callosum (Shapiro syndrome) and semilobar
holoprosencephaly (3, 4). The majority of instances of pediatric episodic
hypothermia are, however, considered idiopathic and most commonly referred
to as episodic spontaneous hypothermia with hyperhidrosis (ESHH). The
profuse sweating that characterizes this condition and the typical absence of
shivering during the hypothermia, point to a lowered body temperature set point
in ESHH. In the aggregate, the diverse causes of episodic hypothermia are
thought to result from a disorder of thermoregulation, most likely arising from
periodic hypothalamic dysfunction. Episodic brainstem dysregulation, but also of
its interconnected hypothalamic areas (4), has been also proposed to play a
pivotal role in the generation of the migraine attack, another paroxysmal
disorder. The character of the premonitory symptoms of the migraine attack, its
circadian rhythmicity and the dependence on hormonal fluctuations, are all
clues towards a transient hypothalamic dysfunction in the initiation of migraine
(5). Indeed, some clinical observations (6) led to postulate that ESHH may
represent a novel variant of childhood periodic syndromes (CPS), the ICHD-IIdefined category encompassing the phenotypes considered as pediatric
153
RESULTATS
Hipotèrmia: CPS?
precursors of migraine (7). To our knowledge, there have been no studies
addressing the molecular basis of ESHH.
PATIENTS AND METHODS
Patients
A 17 month-old girl presented with a clinical history of recurrent episodes of
hypothermia (core temperature between 34 and 35ºC) pallor and profuse
diaphoresis. The first episode occurred at the age of seven months. According
to her parents, during the episode the patient was unable to remain seated and
appeared much less active. In subsequent episodes, once the patient attained
independent walking, some degree of instability became evident. While the
patient was hypothermic, there was a conspicuous absence of shivering.
Episodes lasted typically from a few minutes to 1 hour, after which mild
somnolence was sometimes noted, and their frequency was between one and
four per month during the first four years of life. They were more common at
night, during sleep, and were rarely recorded during daytime.
The family history was notable for a nine year-old brother had been diagnosed
with focal idiopathic epilepsy at age 5. He received carbamazepine for three
years and remained seizure-free after discontinuation of the drug. Both of the
patient parents had a history of recurrent headaches. The patient’s father had
headaches fulfilling the criteria for migraine without aura, while the mother had a
relatively recent onset of tension-type headaches. However, telephone
interviews revealed that the maternal great-grandmother, the grandmother, her
sister and a daughter of the latter (Figure 1), all complained of migraine without
154
Capítol 3- Article 3
RESULTATS
aura and occasional bouts of vertigo. No other relative had other paroxysmal
neurological events, including epilepsy or episodic ataxia.
Interictally, the patient’s physical examination was unremarkable. Mental status,
cranial nerve function, muscle strength, sensory examination, gait and
coordination were normal. There were brisk tendon reflexes and flexor plantar
responses bilaterally. Two hypochromic maculae were noted over the abdomen
and left lower limb. Her psychomotor development was judged adequate
overtime.
Laboratory exams included blood sugar at time of hypothermia episodes and
serum lactate, ammonia and amino acids, urine organic acids and EEG soon
after the episodes. A polysomnogram at age five years showed an ApneaHypopnea index of 16.1, consistent with sleep apnea syndrome. The concurrent
EEG recording showed physiological theta hypnagogic arousals but no
epileptiform discharges or asymmetries.
A brain MRI at the age of 23 months revealed some periventricular white matter
hypomyelination (terminal myelination) but was otherwise normal.
By age six, the patient continues to show a normal exam and is attending
normal school. The frequency of the episodes of hypothermia has diminished to
less than one per month. Since age five, the patient complains, as a separate
type of paroxysms, from headache attacks. The headache, lasting about 2
hours, is of moderate-severe intensity and predominates over the occipital
region, although the age of the patient hampers the formal assessment of the
IHS criteria.
155
RESULTATS
Hipotèrmia: CPS?
Samples
Peripheral blood venous samples were obtained from the proband and five
family members, as well as from 100 healthy Spanish individuals, in whom
paroxysmal neurological diseases were specifically ruled out, that were used as
control population. Genomic DNA was isolated from leucocytes using a
standard salting-out procedure (8). The study was approved by the local IRB
and each subject gave informed consent for DNA analysis.
DNA and Mutation analysis
The CACNA1A gene was screened for mutations since the proband phenotype,
an unusual episodic brain disorder, and the family history of paroxysmal
neurological signs, including migraine, raised the suspicion of
a neuronal
channelopathy.
Exons of the CACNA1A gene (NM_023035.1) and exon/intron junctions including
splice sites and branch points, were amplified from the index proband in a total
of 42 independent PCR products. Primers were designed using the Primer3
software (frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) (9). The general
PCR procedure was: 100ng of genomic DNA, 0.2mM dNTPs, 5pmol of each
primer, 0.75U AmpliTaq Gold polymerase (Applied Biosystems), 1XGold buffer
with 2mM MgCl2 in a final volume of 50µl. The mixes were subjected to 10 min
at 94ºC, 35 cycles of 1 min at 94ºC, 1 min at 53 to 62ºC (depending on the
specific PCR fragment) and 1 min at 72ºC, and a final extension step of 10 min
at 72ºC. Exons 19, 20, 36, 38, 41 and 44 were supplemented with 5% DMSO
and exons 1, 13+14, 45+46 and 47 needed a different PCR procedure. Primer
sequences and specific PCR conditions are available from the authors upon
request.
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Capítol 3- Article 3
RESULTATS
The PCR products were column-purified using the GFX PCR DNA and Gel
Band purification Kit (Amersham Pharmacia Biotech, Wien, Austria), sequenced
from the reverse PCR primer using the BigDye Terminator Cycle Sequencing
Kit v3.1 (Applied Biosystems Foster City, CA), purified with Sephadex G-50
(Amershan Biosciences Freiburg, Germany) using MultiScreen Plates (Millipore,
Billerica, MA) and run in an ABI PRISM 3700 DNA analyzer (Applied
Biosystems, Foster City, CA). Sequence chromatograms were inspected
manually and analysed with the SeqMan v3.6 software (DNASTAR Inc). When a
change was observed, a comparison with the reference sequence available at
UCSC Human Genome Browser (genome.ucsc.edu) was carried out and the
complementary strand from a new PCR product was sequenced for
confirmation. Digestion of the exon 20 PCR product with the FauI restriction
enzyme was performed as an additional detection method to confirm the
identified mutation (WT: 661bp, mutant allele: 431+230bp). The enzymatic
digestion was performed according to the manufacturer’s recommendations,
and the resulting products were separated by electrophoresis on a 1.5%
agarose gel and stained with ethidium bromide.
A group of 100 healthy unrelated Spanish individuals was screened for the
presence of the mutation identified in the index proband by FauI restriction of
the exon 20 PCR product.
The mutation identified in the CACNA1A gene was numbered using the
reference sequence NM_023035.1, with nucleotide 283 of that sequence, the A
of the ATG initiation codon, corresponding to +1, following the Human Genome
Variation Society (HGVS) recommendations.
157
RESULTATS
Hipotèrmia: CPS?
RESULTS
A new mutation was identified in the proband after extensive sequencing of the
CACNA1A gene (Figure 2). The index patient displays a novel variant in exon
20, consisting in a C to G transversion at cDNA nucleotide 3638 of the
reference sequence NM_023035.1 (c.3638C>G). The change results in a
replacement of a proline for an alanine residue, p.Pro1138Ala, in the
intracellular loop between the domains DII and DIII of the CACNA1A protein.
The presence of the change was confirmed by restriction analysis in the patient
and in two relatives: the mother and the maternal grandmother (Figure 2). The
mutation was not present in 200 chromosomes from unrelated healthy
individuals.
DISCUSSION
We reported a new case of infantile ESHH where a potentially disease-causing
mutation was found in the CACNA1A gene.
The etiology of ESHH is unknown. This rare condition has been speculatively
ascribed to defective thermoregulation arising from hypothalamic dysfunction.
Various observations of secondary episodic hypothermia, often associated with
acquired diencephalic lesions or congenital anomalies of corpus callosum,
thalamus or hypothalamus may lend support to this hypothesis, which also
claims that in ESHH a lowered body temperature set point would produce the
body to sweat profusely, in an attempt to maintain the temperature low (10).
The hypothalamus is believed to play an important role in primary headaches
and other episodic brain disorders such as narcolepsy; it is activated during the
initiation of the migraine attack (5) and several lines of evidence suggest a
158
Capítol 3- Article 3
RESULTATS
pivotal role of hypothalamus in cluster headache pathophysiology (11). It is thus
conceivable that episodic disordered thermoregulation might constitute a
migraine equivalent or be in some way related to the primary headaches arising
in the hypothalamic region. Some evidences seem to support the contention
that ESHH is a migraine variant, akin to other CPS. Four unrelated cases of
ESHH had concurrent manifestations of either migraine or CPS (cyclical
vomiting) and family history of migraine in three of them (6). Also the 5 year-old
girl reported by Greenberg and Rittichier (12) had a family history of migraine in
her father and panic attacks in several other relatives. Some of the clinical
symptoms accompanying hypothermia both in our patient and in those from
previous reports (6), such as droopiness or somnolence, are reminiscent of
those featured in CPS. Our patient has also developed recurrent headaches at
preschool age. Conversely, ESHH has also been proposed to represent a form
of diencephalic epilepsy (10). This would not thwart the notion of CACNA1A
mutation pathogenicity, since several generalized epilepsy phenotypes have
been linked to this gene (13, 14).
An alternative view would be that ESHH represents an early form of cluster
headache (CH) a severe form of primary headache which characteristically
does not present in childhood. Both entities have nocturnal presentation and
have been related to hypothalamic dysfunction. Polysomnograms have been
repeatedly obtained in CH and it is of note that obstructive sleep apnea has
been found in over 80% of patients (15). Our patient, who also had
predominantly nocturnal episodes, had significant obstructive sleep apnea.
If ESHH is indeed a CPS, this is only the third reported patient with a migraine
precursor exhibiting an apparently causative mutation in a migraine-related
159
RESULTATS
Hipotèrmia: CPS?
gene. Giffin et al. (16) reported a case of benign paroxysmal torticollis that,
although not subjected to genetic analysis, belonged into a FHM pedigree
carrying a missense mutation in the CACNA1A gene. We have recently
reported a mutation in a young infant presenting with BPTI, which later evolved
into BPV and then to migraine (17).
Regarding our molecular findings, the mechanism through which a mutation in a
calcium channel subunit should produce episodic hypothermia is far from
obvious. Interestingly, the hypothalamus is densely populated with neurons
expressing the Cav2.1 channel (www.genecards.org). Although CACNA1A
mutations are common in FHM (18) (17), none has been found in the single
series of patients with cluster headache which was subjected to mutational
screen (19).
The missense mutation p.Pro1138Ala is expected to produce a more benign
phenotype, such as ESHH, through a gain of function, as it happens with most
FHM analyzed mutations, rather than a severe phenotype, as is the case of
some EA2 with cerebellar progressive ataxia resulting from CACNA1A null
alleles (20).
Although the present results do not substantiate the pathogenicity of the
described change, some clues may suggest a causal relationship between the
mutation and the phenotype of the index patient:
First, the mutant alanine substitutes a proline, present in the normal protein, that
is structurally disruptive. Second, the change is located in the intracellular linker
between domains DII and DIII of the protein, close to the SNARE protein
binding site. The SNARE proteins mediate neurotransmitter vesicles fusion at
the presynaptic membrane, so abnormal interaction with the channel could
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Capítol 3- Article 3
RESULTATS
compromise neurotransmitter exocytosis (21). Third, the mutation is highly
conserved in evolution at interspecific level (mouse –Mus musculus-, rat -Rattus
norvegicus-, cattle -Bos taurus- and rabbit -Oryctolagus cuniculus- α1A
subunits). The position, is conserved in CACNA1B and not in CACNA1E, but
does not exist in the other paralogous human proteins, thus precluding further
comparisons (Figure 3). And fourth, the mutation was not detected in a
screening of 200 control chromosomes from non-migraneurs.
Finally, only in vitro functional studies showing altered electrophysiological
properties of the p.Pro1138Ala mutated channel will eventually allow
confirmation of causality of the described phenotype. Follow-up data on the
ESHH patients reported thus far is clearly warranted. Whether this intriguing
condition is an early form of migraine, of another primary headache, such as
CH, another channelopathy as epilepsy, or even a channelopathy on its own
right, remains to be elucidated.
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ACKNOWLEDGEMENTS
The authors wish to thank all the patients and family members for their cooperation
This study was supported by the Spanish Ministry of Education and Science (SAF2000/197 and SAF-2003/04704), Red Española de Ataxias, Fondo de Investigación
Sanitaria, Spain (G03/056 and PI052129), Instituto de Salud Carlos III, Spain
(PI061073, PI050996) and AGAUR (2005SGR00848). E.C-L. was a recipient of a FPI
scholarship of the Ministerio de Ciencia y Tecnología, Spain. R.C. was funded by the
Institut de Recerca Vall d’Hebron.
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RESULTATS
FIGURE LEGENDS
Figure 1. A three generation Spanish pedigree with episodic spontaneous
hypothermia (arrow, index case), migraine and vertigo attacks.
Figure 2. The index patient displays a novel variant in exon 2, consisting in a C
to G transversion at nucleotide 3638 of the reference cDNA sequence
NM_023035.1 with nucleotide A of the ATG initiation codon (nucleotide 283)
corresponding to +1. The change results in a replacement of a proline for an
alanine residue, p.Pro1138Ala. The presence of the change was confirmed by
restriction analysis of the PCR product of exon 20 in the patient and two
relatives. The FauI restriction enzyme generated band patterns of 661bp in the
WT allele and 431+230bp in the mutant allele. ND= non digested.
Figure 3. Protein alignment performed with clustalW (www.ebi.ac.uk/clustalw).
The human CACNA1A Pro1138 residue is conserved in the orthologous
CACNA1A proteins of several organisms, whereas it is conserved only in the
human calcium channel subunit CACNA1B, since the position is not present in
the other shorter CACNA1 human proteins. Non-conserved amino acids are
marked in grey. The RefSeq code of each protein is indicated. HUMAN: Homo
sapiens, CATTLE: Bos taurus, MOUSE: Mus musculus, RAT: Rattus
norvegicus, RABBIT: Oryctolagus cuniculus.
165
RESULTATS
Hipotèrmia: CPS?
I.1
I.2
II.1
II.2
II.3
III.1
II.4
III.2
II.5
III.3
III.4
Headache
IV.1
Migraine with aura
IV.2
Vertigo
Hypothermia
Migraine without aura
Figure 1
19p13.13
CACNA1A
c.3638C>G (p.Pro1138Ala)
-
650bp
500bp
400bp
300bp
200bp
ND
II.2
II.3
p.Pro1138Ala/+
III.2
III.1
+/+
p.Pro1138Ala/+
IV.1
+/+
Figure 2
166
IV.2
p.Pro1138Ala/+
1 Kb
RESULTATS
Capítol 3- Article 3
p.Pro1138Ala
O00555|CACNA1A_HUMAN
Q00975|CACNA1B_HUMAN
Q15878|CACNA1E_HUMAN
Q01668|CACNA1D_HUMAN
O60840|CACNA1F_HUMAN
Q13936|CACNA1C_HUMAN
Q13698|CACNA1S_HUMAN
NPGNPSNPGPPKTPENSLIVT
----------PEDADNQRNVT
--------TDKATTESTSVTV
---------------------------------------------------------------------------------
O00555|CACNA1A_HUMAN
P97445|CACNA1A_MOUSE
P54282|CACNA1A_RAT
Q1ADE8|CACNA1A_CATTLE
P27884|CACNA1A_RABBIT
NPGNPSNPGPPKTPENSLIVT
NPGNPSNPGPPKTPENSLIVT
NPGNPSNPGPPKTPENSLIVT
NPGNPSNPGPPKTPENSLIVT
NPGNPSNPGPPKTPENSLIVT
Figure 3
167
CAPÍTOL 4
A MUTATION IN THE FIRST INTRACELLULAR LINKER OF
CACNA1A MODIFIES P/Q CHANNEL REGULATION BY CAVβ
SUBUNITS, G PROTEINS AND SYNTAXIN-1A UNDER CONDITIONS
OF HIGH ELECTRICAL ACTIVITY.
Selma A. Serra, Ester Cuenca-León, Artur Llobet, Noelia Fernández-Castillo,
Roser Corominas, Jacqueline Fernandes, Miguel A. Valverde, Alfons Macaya,
Bru Cormand and José M. Fernández-Fernández.
ARTICLE 4
NATURE NEUROSCIENCE - EN 2A REVISIÓ
Capítol 4 – Article 4
RESULTATS
RESUM
Una mutació en el primer llaç intracel·lular de la proteïna CACNA1A modifica la
regulació del canal de tipus P/Q, la unió a sintaxina 1A i baixa l’eficiència de
secreció: rellevància pel fenotip clínic de la migranya.
Les mutacions responsables de migranya hemiplègica familiar (FHM) en el gen que codifica
la subunitat α1A del canal de Ca2+ de tipus P/Q (CACNA1A) estan situades a les regions del
porus o del sensor de voltatge i impliquen en general un guany de funció del canal. En aquest
treball es descriu una mutació situada al primer llaç intracel·lular (ILI-II) del canal CACNA1A
(α1A(A454T)) associada a la manca de símptomes sensitivo-motors en una família amb
migranya amb aura. El canal α1A(A454T) presenta alteracions en la regulació de la inactivació
de l’estat de repòs dependent de voltatge per part de les subunitats CaVβ. El canal α1A(A454T)
també mostra una inactivació accelerada després d’una despolarització facilitadora, mediada
pels dímers βγ de la proteïna G. A més, la mutació A454T impedeix la interacció de la sintaxina
1A amb el canal P/Q i per tant, aboleix la modulació del canal a través de la sintaxina 1A i,
conseqüentment, redueix l’eficàcia de secreció. Els nostres resultats suggereixen un nou
paper de l’ILI-II en la interacció entre el canal de tipus P/Q i la sintaxina 1A i indiquen que
variants en el gen CACNA1A poden, a vegades, no ser les responsables del fenotip sinó
participar com a modificadors d’aquest.
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p.Ala454Tyr: estudis funcionals
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