Download Machado-joseph

Conceição Bettencourt
Portugaliӕ Genetica 2010
Conceição Bettencourt
Introduction
• Machado-Joseph Disease (MJD) or Spinocerebellar
ataxia type 3 (SCA3)
– Autosomal dominant neurodegenerative disorder
– Onset
• Mean: ~40 yr (range: 4-70 yr)
• First symptoms: ataxia (92.4%) or diplopia (7.6%)
– Multisystem
• Cerebellar, oculomotor, pyramidal, extrapyramidal
and peripheral motor systems
Portugaliӕ Genetica 2010
Conceição Bettencourt
Introduction
– Complex and pleomorphic phenotype
• 3 main clinical types (in common: cerebellar ataxia and EPO)
Portugaliӕ Genetica 2010
Type 1
• Onset ~24 yr
• Pyramidal and extrapyramidal signs
Type 2
• Onset ~40 yr
• Cerebellar ataxia and EPO
Type 3
• Onset ~47 yr
• Peripheral alterations
Occasionally
in the same
family
Conceição Bettencourt
Introduction
– MJD locus was mapped to 14q32.1
– CAG repeat expansion at the exon 10 of the ATXN3 gene
Wild-Type
12-44 CAGs
Intermediate
45-56 CAGs
Expanded
61-87 CAGs
Rarely reported
– Incomplete genotype-phenotype correlation
Expanded
(CAG)n size
Explaining only
~50-70% of
onset variance
Portugaliӕ Genetica 2010
Conceição Bettencourt
Introduction
– ATXN3 gene encodes for ataxin-3
• Expansion of (CAG)n tract
Elongation of
polyglutamine tract
Ubiquitous
expression
Not explaining
Specificity of
neuropathology
Portugaliӕ Genetica 2010
Ichikawa et al. (2001)
J Hum Genet 46:413-422
Conceição Bettencourt
Introduction
• Large number of alternative
splicing variants
several isoforms
of ataxin-3
Bettencourt et al. (2009) Neurogenetics
• Although ubiquitously expressed
Differential levels of ataxin-3 (allele/isoform)
may have modifying effects
Portugaliӕ Genetica 2010
Conceição Bettencourt
Introduction
• Mechanisms
of
ATXN3
translational regulation
– 5’ regulatory regions
transcriptional
and
allele-specific manner
• ATXN3 core promoter region is directly upstream
(between -291 and the transcription start site)
• cis-regulatory elements in 5’-UTR (between -89 and -1)
– Genomic structure of ATXN3 5’ flanking region already
described
Extent of variation
is unexplored
Portugaliӕ Genetica 2010
Conceição Bettencourt
Objectives
• Analyze the extent of genetic variation upstream of
the ATXN3 start codon
– 5’ regulatory regions
• Core promoter
• 5’-UTR
• Possibly influence gene
expression levels
• May be associated with
variance in MJD’s onset
Portugaliӕ Genetica 2010
Conceição Bettencourt
Subjects and Methods
– 30 unrelated MJD patients
• Sample
– 59 controls
• Amplification and Sequencing (ATXN3)
Core promoter + 5’-UTR
– PCR: fragment of 816bp (primers P-1aF and P-1R)
– Sequencing: fragment of ~480bp (primers P-1aF and P-1aR)
Size of (CAG)n tract
– Determined according to Bettencourt et al. (2008) J Hum Genet 53:333–339
Portugaliӕ Genetica 2010
Conceição Bettencourt
Subjects and Methods
• Statistical analysis
– Genotypic and allelic frequencies (1 SNP in 5’UTR)
– Conformity with the HWE (1 SNP in 5’UTR)
– Linear regression analysis (MJD patients)
• Onset
– (CAG)n in expanded ATXN3 alleles
– (CAG)n in wild-type ATXN3 alleles
– Genotypes of 1 SNP in 5’UTR
Portugaliӕ Genetica 2010
Conceição Bettencourt
Results and Discussion
• Sequence analysis
Core promoter region
no polymorphisms (178 chr)
Conserved
Portugaliӕ Genetica 2010
5’-UTR
1 SNP (c.-31C>T)
Conceição Bettencourt
Results and Discussion
• Sequence analysis
Core promoter region
5’-UTR
1 SNP (c.-31C>T)
c.-31C>T
C seems to be the ancestral
Portugaliӕ Genetica 2010
Conceição Bettencourt
Results and Discussion
• Sequence analysis
Core promoter region
5’-UTR
1 SNP (c.-31C>T)
Samples
MJD patients (N=30)
Frequency (%)
Genotypes
C/C
93.3 (28)
C/T
6.7 (2)
T/T
0.0 (0)
Alleles
C
96.7
T
3.3
Controls (N=59)
78.0 (46)
18.6 (11)
3.4 (2)
87.3
12.7
Not statistically significant
Portugaliӕ Genetica 2010
HWE
Conceição Bettencourt
Results and Discussion
• Sequence analysis
Core promoter region
5’-UTR
1 SNP (c.-31C>T)
Schmitt et al. (2003) Gene 314: 81–88
CMYB binding site
Portugaliӕ Genetica 2010
Putative regulatory role
Conceição Bettencourt
Results and Discussion
• Genotype-phenotype correlations
– (CAG)n size in expanded alleles
Onset = 251.1 – 2.9CAGexp
r2 = 0.761
– (CAG)n size in wild-type alleles
– C.–31C>T genotypes
Portugaliӕ Genetica 2010
No significant effects
in onset variance
Conceição Bettencourt
Conclusions
• ATXN3 core promoter region was shown to be highly
conserved
• One SNP (c.-31C>T) was found in the 5’-UTR of both
MJD patients and controls
– Notwithstanding the putative functional role of this SNP in
the ATXN3 transcriptional and/or translational regulation
No significant effects in the MJD’s age at onset
Portugaliӕ Genetica 2010
Conceição Bettencourt
Acknowledgements
• University of the Azores
– Manuela Lima
– Mafalda Raposo
– Francisca Silva
• Universitat Autònoma de Barcelona
– Cristina Santos
• University of Minho
– Patrícia Maciel
• SEEBMO
– Jácome Bruges-Armas
• GAIN
– João Vasconcelos
– Teresa Kay
Governo dos Açores
Portugaliӕ Genetica 2010