Molecular analysis of the spinal muscular atrophy and neuronal apoptosis inhibitory protein genes in Saudi patients with spinal muscular atrophy.

Abstract

Spinal muscular atrophy (SMA) is a common, often fatal, autosomal recessive disease leading to progressive muscle wasting and paralysis as a result of degeneration of anterior horn cells of the spinal cord. The prevalence of SMA cases in the Kingdom of Saudi Arabia (KSA) is much higher than the European and North American population. Deletions or mutations in 2 genes, telomeric form of the survival motor neuron (SMN1) and the neuronal apoptosis inhibitory protein (NAIP), are known to be associated with SMA. The aim of this study is to examine the deletions or interruptions of the SMN1 and NAIP genes in Saudi patients.
The study included 121 Saudi SMA patients [type I (60 patients); type II (26 patients); and type III (35 patients)]. The deletions or interruptions of the SMN1 and NAIP genes were detected by using polymerase chain reaction. The study was carried out at the King Fahad National Guard Hospital, Riyadh, KSA between 2000 and 2002.
The homozygous deletions of exons 7 and 8 of the SMN1 gene were found in 94% and 87% of the patients. Exon 5 of the NAIP gene was deleted in 70%, but its deletion was more frequent in SMA type I (93%) as compared to type II (54%) and type III (43%). Seven patients with SMA diagnosis did not show any of the above homozygous deletions. All 230 control subjects had at least one copy of both SMN1 and NAIP genes, as expected.
Our results demonstrate that the deletion rate (94%) of the SMN1 gene in Saudi SMA patients is similar, irrespective of types, compared with patients of other ethnic groups. We also show that the incidence of NAIP deletion is higher in the more severe SMA cases and the dual deletion of the SMN1 and NAIP genes are more common in Saudi SMA type I patients compared with patients of other ethnic groups.

Full text

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Results:
SMN1 gene were found in 94% and 87% of the patients.
Exon 5 of the NAIP gene was deleted in 70%, but its deletion
was more frequent in SMA type I (93%) as compared to type
II (54%) and type III (43%). Seven patients with SMA
diagnosis did not show any of the above homozygous
deletions. All 230 control subjects had at least one copy of
both SMN1 and NAIP genes, as expected.
The homozygous deletions of exons 7 and 8 of the
Conclusion:
(94%) of the SMN1 gene in Saudi SMA patients is similar,
irrespective of types, compared with patients of other ethnic
groups. We also show that the incidence of NAIP deletion is
higher in the more severe SMA cases and the dual deletion of
the SMN1 and NAIP genes are more common in Saudi SMA
type I patients compared with patients of other ethnic groups.
Our results demonstrate that the deletion rate
Saudi Med J 2003; Vol. 24 (10): 1052-1054
roximal spinal muscular atrophy (SMA) is a
neuromuscular disorder, which results in the loss of
motor neurons in the spinal cord and lower brainstem
leading to symmetrical progressive paralysis with
muscle atrophy. Spinal muscular atrophy is classified
into 3 clinical groups based on age at onset, achievement
P
ABSTRACT
Molecular analysis of the spinal muscular
atrophy and neuronal apoptosis inhibitory
protein genes in Saudi patients with spinal
muscular atrophy
Mohammed Al-Jumah, MD, Ramanath Majumdar, PhD, Saad Al-Rajeh, MD, Adnan Awada, MD,
Enrique Chaves-Carballo, MD, Mustafa Salih, MD, Saad Al-Shahwan, MD, Khalid Al-Subiey, MD, Shifa Al-Uthaim, BSc.
of developmental milestones and lifespan.1 Type I
SMA, known as Werdnig-Hoffmann disease (the most
severe form of SMA), is characterized by severe
generalized muscle weakness and hypotonia with onset
between birth and 6 months. Death from respiratory
failure usually occurs within the first 2 years. In type II
Objective:
often fatal, autosomal recessive disease leading to progressive
muscle wasting and paralysis as a result of degeneration of
anterior horn cells of the spinal cord. The prevalence of SMA
cases in the Kingdom of Saudi Arabia (KSA) is much higher
than the European and North American population. Deletions
or mutations in 2 genes, telomeric form of the survival motor
neuron (SMN1) and the neuronal apoptosis inhibitory protein
(NAIP), are known to be associated with SMA. The aim of
this study is to examine the deletions or interruptions of the
SMN1 and NAIP genes in Saudi patients.
Spinal muscular atrophy (SMA) is a common,
Methods:
I (60 patients); type II (26 patients); and type III (35 patients)].
The deletions or interruptions of the SMN1 and NAIP genes
were detected by using polymerase chain reaction. The study
was carried out at the King Fahad National Guard Hospital,
Riyadh, KSA between 2000 and 2002.
The study included 121 Saudi SMA patients [type
From the Neurogenetics Laboratory & Neurology Section (Al-Jumah, Majumdar, Awada, Al-Subiey, Al-Uthaim), King Fahad National Guard Hospital,
Neurology & Neuropediatric Division (Al-Rajeh, Salih) King Saud University, Department of Neurosciences (Chaves- Carballo), King Faisal Specialist Hospital
& Research Centre and the Department of Pediatrics (Al-Shahwan), Armed Forces Hospital, Riyadh, Kingdom of Saudi Arabia.
Received 12th May 2003. Accepted for publication in final form 15th July 2003.
Address correspondence and reprint request to: Dr. Mohammed Al-Jumah, Neurogenetics Laboratory (Mail code 2216), King Fahad National Guard Hospital,
PO Box 22490, Riyadh 11426, Kingdom of Saudi Arabia. Tel. +966 (1) 2520088 Ext. 6665. Fax. +966 (1) 2520088 Ext. 6662. E-mail: jumahm@ngha.med.sa
1052
Articles

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SMN1 gene deletions in SMA patients ... Al-Jumah et al

www.smj.org.saSaudi Med J 2003; Vol. 24 (10) 1053
fulfilled diagnostic criteria for proximal SMA defined
by the International SMA Consortium.1 The patients
were classified into SMA type I (60 patients), type II (25
patients) and type III (35 patients). One hundred and ten
healthy family members,
individuals and 30 patients with other neuromuscular
diseases were used as controls. The local ethics
committee reviewed the study and an informed consent
was obtained from the parents of the patients. The study
was carried out at the King Fahad National Guard
Hospital, Riyadh, Kingdom of Saudi Arabia between
2000 and 2002. Genomic DNA was extracted from 2-5
ml of peripheral blood collected from each individual
following the method by QIAampTM DNA blood midi kit
according to manufacturer (QIAGEN, Germany)
protocol.
Deletion analysis of the survival motor neuron gene.
Deletions in the SMN1 gene were identified by
restriction site analysis after amplification of exon 7 and
exon 8.5 This method allows exons 7 and 8 of SMN1 to
be distinguished from the corresponding exons of
SMN2. The polymerase chain reaction (PCR) protocol
is similar to the method described previously.5 The
amplification was carried out with a hot start for 7
minutes at 94oC and subsequently with 35 cycles
(denaturation for one minute at 94oC, annealing for one
minute at 55oC [for exon 7] or one minute at 57oC [for
exon 8], and extension for one minute at 72oC). The
amplified products are further digested with either Dra1
(for exon 7) or Dde1 (for exon 8) for 4 hours at 37oC.
The digested products are run on 2% agarose gel and
subsequently visualized under UV light.
Deletion analysis of the neuronal apoptosis
inhibitory protein gene.
Neuronal apoptosis inhibitory
protein gene analysis was performed by PCR
amplification of exon 5 and exon 13 following the
method described earlier.8 All reactions were carried out
with a hot start for 5 minute at 94oC and subsequently
with 35 cycles (denaturation for one minute at 94oC,
annealing for one minute at 57oC, and extension for one
90 unrelated normal
Table 1 - Percentage distribution of SMN1 (survival motor neuron) and
NAIP (neuronal apoptosis inhibitory protein) deletions in
different groups.
SMA Type
(n of patients)
Type I 60
Type II 26
Type III 35
Total 121
SMN1 exon 7
n of patients
had deletion
n (%)
57
25
32
(95)
(96)
(91)
114 (94)
SMN1 exon 8
n of patients
had deletion
n (%)
52
22
31
(87)
(85)
(88)
105(87)
NAIP exon 5
n of patients
had deletion
n (%)
56
14
15
(93)
(54)
(43)
85 (70)
Table 2 - Percentage of consanguinity and family history.
SMA Type
(n of patients)
Type I 60
Type II 26
Type III 35
Total 121
Parents related
(%)
(69)
(55)
(52)
(59)

First degree
(%)
(56)
(35)
(40)
(44)
Positive family
history (%)
(67)
(75)
(61)
(68)
SMA - spinal muscular atrophy, SMN1 - survival motor neuron,
NAIP - neuronal apoptosis inhibitory protein
SMA - spinal muscular atrophy
(intermediate form) and type III (Kugelberg-Welander
disease), patients learn to walk, but suffer from proximal
muscle weakness with onset after the age of 2. The
candidate region for SMA locus has been mapped to
chromosome 5q11.2-5q13.3 by linkage analysis.2,3 Two
candidate genes are known to be involved in SMA. The
survival motor neuron gene (SMN) is present in 2 highly
homologous copies, telomeric SMN (SMN1) and
centromeric SMN (SMN2) within the SMN region.4
Both copies are composed of 9 exons, which encode
identical amino acid sequences. The 2 genes differ in
their exons by only 2 base pairs, one in exon 7 and one
in exon 8, which allows distinction of SMN1 from
SMN2 by single strand conformation polymorphism
analysis (SSCP) and restriction site assay.4,5 Independent
of the clinical severity, homozygous deletions or
interruptions of the SMN1 gene that affect exon 7 or
exons 7 and 8 were found in 90-98% of SMA patients in
many ethnic groups.4-7 The SMN2 gene is not
interrupted in 95% normal and SMA chromosomes.7
Thus, there is a strong correlation between deletion of
the SMN1 gene and SMA and this deletion has been
suggested to be the most reliable means of diagnosis of
the disease. Other candidate gene is the neuronal
apoptosis inhibitory protein gene (NAIP), which
contains more than 16 exons.8 The NAIP gene shows
homozygous deletions in 45-67% of type I and 20-42%
of type II and type III patients.6-9 Spinal muscular
atrophy is reported to have an overall worldwide
incidence of one in 10,000 live births; however, due to
the prevailing custom of consanguineous marriage,
incidence, prevalence, and carrier frequency of SMA in
Saudi population are much higher than the European and
the North American population.10 In this report we
present gene deletions analysis in 121 SMA cases
belonging to 97 families.
Methods.
collection.
patients. All patients were evaluated by neurologists and
Patient
The study included 121 SMA Saudi
selection and blood

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SMN1 gene deletions in SMA patients ... Al-Jumah et al

1054 Saudi Med J 2003; Vol. 24 (10)www.smj.org.sa
minute at 72oC). The PCR products were analyzed for
the presence and absence of exon 5 and exon 13.
Amplification of NAIP exon 13 served as control for the
presence of the NAIP gene.
Results.
gene was absent or interrupted in 57 out of 60 SMA type
I patients, 25 out of 26 type II patients and 32 out of 35
type-III patients. Exon 8 of the SMN1 gene was absent
or interrupted in 105 out of 121 patients (87%). Seventy
percent of the patients showed homozygous deletion of
NAIP exon 5 although the deletions of NAIP exon 5
were more common in type I SMA patients (93%). All
control subjects (n=230) had at least one copy of both
SMN1 and NAIP genes, as expected. Positive family
history of SMA in the siblings or other first-degree
relatives was seen in 82 out of 121 SMA patients (Table
2). Fifty-nine percent of the SMA patients were born of
consanguineous marriages, but the parents who are first
degree cousins were more common in SMA type I (56%)
as shown in Table 2.
As shown in Table 1, exon 7 of the SMN1
Discussion.
deletion of exons 7 of the SMN1 gene in 94% of Saudi
SMA patients with severe or milder form, which is in
agreement with previous reports dealing with different
ethnic groups.4,6,7,9,11-13 Neuronal apoptosis inhibitory
protein gene exon 5 was mostly deleted in type I
patients. We also demonstrated that most patients who
lacked the NAIP exon 5 also lacked SMN1 exons 7 and
8. Seven of our SMA patients (3 each from SMA type I
and III and one from type II) did not show homozygosity
for a deletion in the SMN1 gene in exon 7. These
patients have been diagnosed based on the International
SMA Consortium criteria. Several studies have reported
that a small minority of SMA patients lack homozygous
SMN1 deletions.4,7,11 For some of the SMA patients,
smaller mutations in the SMN gene have been
described.4,14 In some SMA patients gene conversion
and rearrangement events leading to formation of hybrid
SMN1 or SMN2 genes have been reported.13,15 Studies
are underway to determine, which of the above events
are playing a role in causing the disease in minority of
Saudi patients without homozygous deletion of SMN1.
Our results are in agreement with the general
consensus that there is no correlation between genotype
and phenotypic expression of the disease. The data also
suggest that the incidence of NAIP deletion is higher in
the more severe SMA cases, and the dual deletion of the
SMN1 and NAIP genes are more common in the Saudi
SMA type I patients compared with patients of other
ethnic groups.7-9 Akutusu et al16 have suggested that
NAIP deletion may be a prognostic indicator of SMA.
Presence of positive family history is more common in
Saudi SMA patients comparing with other ethnic groups.
Furthermore, although consanguinity in Saudi SMA
parents is similar to the Saudi general population, first
degree consanguinity related to SMA type I cases is
higher than the general population (56% versus 35%).
Our study showed homozygous
Acknowledgment. This study was supported by King Fahad
National Guard Hospital and King Abdul Aziz City for Science and
Technology (KACST project AT 18-03), Riyadh, Kingdom of Saudi
Arabia. We also thank Ms. Nouf Fetaini for her technical assistance.
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