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Dr Pupak Derakhshandeh, PhD

Ass Prof of Medical Science of Tehran University

SMA SMA

SPINAL MUSCULAR SPINAL MUSCULAR ATROPHYATROPHY

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Mutation Detection &Mutation Detection &PNDPND

--ThalassemiaThalassemia// --ThalassemiaThalassemia••HaemophiliaHaemophilia (A / B)(A / B)••HbDHbD, G, E, S, G, E, S••DMD/BMDDMD/BMD••SMA(ISMA(I--III)III)••CF; CF; ……

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DELETION OF SMN AND NAIP GENES IN IRANIAN PATIENTS

WITH SPINAL MUSCULAR ATROPHY

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SMAGene: SMN (Survival Motor Neuron)Motor Neuron; Anterior Horn; Spinal CordSecond most common fatal autosomal recessive disorder after CFSecond most common pediatric neuromuscular disorder after DMDIncidence : 1 in 6000-10000 live birthsCarrier frequency : 1 in 40-60

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CLASSIFICATIONSMA TYPE I (Werdnig-Hoffmann)

SMA TYPE II (Classic)

SMA TYPE III (Kugelberg-Welander)

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SMA TYPE ISevere form of SMA

Onset : first 6 months

Death : < 2 year

Never raising the head or sitting

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SMA TYPE IILess 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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GENETICS1990: 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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DeletionsUp 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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188 bp

164 bp

188 bp

123 bp

65 bp

Exon 7, DraI

Exon 8, DdeI

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SMN Deletion AnalysisSMN Deletion Analysis

SMNt Exon 7 is deleted in affected child

Derakhshandeh-Peykar, et al. Annal Acad Med, 2007Derakhshandeh-Peykar, et al. Annal Acad Med, 2007(Depart Med Gen & NRCGEB)

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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

Derakhshandeh-Peykar, et al. Annal Acad Med, 2007Derakhshandeh-Peykar, et al. Annal Acad Med, 2007(Depart Med Gen & NRCGEB)

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The objective of this study was to genetically characterize the childhood onset

spinal muscular atrophy in Iran.

SMN NAIPDeletion of exon 7 &

8Deletion of exon 5 &

6

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