Transcript Document

Mutation Detection &
PND
-Thalassemia/ -Thalassemia
• Haemophilia (A / B)
• HbD, G, E, S
• DMD/BMD
• SMA(I-III)
• CF; …
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SMA
SPINAL MUSCULAR
ATROPHY
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DELETION OF SMN AND
NAIP GENES IN IRANIAN
PATIENTS WITH SPINAL
MUSCULAR ATROPHY
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SMA
Gene: SMN (Survival Motor Neuron)
Motor Neuron; Anterior Horn; Spinal
Cord
Second most common fatal autosomal
recessive disorder after CF
Second most common pediatric
neuromuscular disorder after DMD
Incidence : 1 in 6000-10000 live births
Carrier frequency : 1 in 40-60
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CLASSIFICATION
SMA TYPE I (Werdnig-Hoffmann)
SMA TYPE II (Classic)
SMA TYPE III (Kugelberg-Welander)
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SMA TYPE I
Sever form of SMA
Onset : first 6 months
Death : < 2 year
Never raising the head or sitting
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SMA TYPE II
Less sever
Clinical appearing : < 18 months
Able to sit unaid
Death : about 9 years
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SMA TYPE III
Mildest form of SMA
Onset : > 18 months
Walking without aid
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OTHER
CLASSIFICATION
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DIAGNOSIS
EMG
Muscle Biopsy
Genetic Testing/PND
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GENETICS
1990: The three types of SMA were
mapped to 5q13
The SMA locus contain two inverted
copies of a 500kb element
The two copies named telomeric and
centromeric
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GENETIC MAP
Three candidate genes named SMN
(Survival Motor Neuron), NAIP (Neuronal
Apoptosis Inhibitory Protein) and P44
were identified in this locus
Up to 95% of SMA patients (SMNI-III) are
homozygously deleted for two exons (7&8)
of both telomeric copy of SMN gene
(SMNt)
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Deletions
Up to 5% of SMA patients have
frameshift mutations, gene conversions
and point mutation
Exons 5 and 6 of NAIPt gene are
deleted in approximately 50% of type
I SMA and 18% of types II and III SMA
P44t is lacked or intrrupted in 73% of
SMA type I patients and 7% in types II
and III
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The summery of normal alleles (N) , mutant
alleles (M) and deletion types (D) of SMN
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MOLECULAR
DIAGNOSIS & PND
PCR-SSCP or PCR-RFLP of SMN
gene enables confirmation of a
suspected clinical diagnosis of SMA
or prenatal diagnosis
These two techniques based on
nucleotide differences of both exon 7
and exon 8 of telomeric and
centromeric copy of SMN
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Deletion Analysis of
SMN gene
Exones 7 and 8 of
SMN gene were
amplified and cut
by Dra I and Dde I ,
respectively. (only
centromeric copy
is cutted)
Absence of SMNt
exone(s) 7 (and 8)
confirm diagnosis
of SMA
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Exon 7, DraI
188 bp
164 bp
Exon 8, DdeI
188 bp
123 bp
65 bp
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SMN Deletion Analysis
Derakhshande et al.
(Farhud Genetic Lab/ NRCGEB)
SMNt Exon 7
is deleted in
affected child
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NAIP Gene Deletion
Analysis
Exones 5 & 6 of NAIP gene were amplified
with exon 13 which was the internal control
Absence of exon
5 and exon 6 ( which
only exist within the
telomeric functional
copy of NAIP) was
detected in ~50% of
type I SMA and 18%
of types II and III SMA
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NAIP Deletion analysis
Derakhshande et al.
(Farhud Genetic Lab/ NRCGEB)
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The objective of this study was to genetically
characterize the childhood onset spinal muscular
atrophy in Iran.
The objective of this study was to genetically
characterize the childhood onset spinal muscular
atrophy in Iran.
SMN
NAIP
Deletion of exon 7 & Deletion of exon 5 &
8
6
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SMA type I (n=70)
70(100%)
61(87%)
SMA type II (n=3)
2(66%)
1(33%)
SMA type III (n=2)
1(50%)
0(0%)
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Various deletion haplotypes were constructed by using
genotypes of SMN and NAIP genes.
Haplotype A, which has the deletions of all two involved genes,
were deleted in approximately 83% of type I and II SMA but not
in type III and was found predominantly in the severe group
with an early onset at less than 6 month of age.
we report Thirty four our experiences for prenatal diagnosis
Haplotypes (%)
A
B
C
SMA type I
(n=70)
87
100
0
SMA type II
(n=3)
33
66
33
SMA type III
(n=2)
0
50
50
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These studies suggested that the frequency
of gene deletions of SMN1 and NAIP gene
is a few higher than previous reports. It is
may be due to high rate of consanguine
marriage by Iranian Muslims (96 % in this
families). Thus, the conformation of SMA
related gene deletion will also be a useful
tool for the pre and postnatal diagnostic. In
addition to common PCR methods for SMN
exon 7 and 8 and NAIP exons 4 and 5, we
also conducted multiplex PCR of exon 5, 6
and 13 of the NAIP telomere in one
reaction.
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Derakhshandeh
Esmaiili
Rahmani
Babrzadeh
Taeb
Attaran
Sajedifar
Farhud
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