Review Of Literature 3

CHAPTER 3
REVIEW OF LITERATURE

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3. REVIEW OF LITERATURE
3.1 Epidemiology.

 Andrea et al. in 1994 presented the most extensive epidemiological

data on chronic forms of spinal muscular atrophy in childhood (CSMA)
in West-Thtiringen in Germany. The incidence of CSMA was calculated
to be 1 in 9,420 live births. The prevalence was 1,624 in 100,000 of the
general population (Andrea et al. 1994).

3.2 Molecular Analysis.

 Bussaglia et al. in 1995 presented a genetic analysis of 54 unrelated

Spanish SMA Families that has revealed a 4-basepair (bp) deletion
(AGAG) in exon 3 of SMN in four unrelated patients. This deletion,
which results in a frame shift and a premature stop codon, occurs on
the same haplotype background, suggesting that a single mutational
event is involved in the four families. The other patients showed either
deletions of the SMN gene (49/54) or a gene conversion event
changing SMN exon 7 into its highly homologous copy (cBCD541,
1/54). This observation gives strong support to the view that mutations
of the SMN gene are responsible for the SMA phenotype as it is the
first frame shift mutation reported in SMA (Bussaglia et al. 1995).

 Chang et al. in 1997 assayed deletions of two candidate genes for

spinal muscular atrophy (SMA), the survival motor neuron (SMN) and
neuronal apoptosis inhibitory protein (NAIP) genes, in 101 patients
from 86 Chinese SMA families. Deletions of exon 7 and 8 of the
telomeric SMN gene were detected in 100%, 78.6%, 96.6%, and
16.7%, in type I, II, III, and adult-onset SMA patients, respectively.
Deletion of exon 7 only was found in eight type II and one type III
patient. One type II patient did not have a deletion of either exon 7 or 8.
The prevalence of deletions of exon 5 and 6 of the NAIP gene were

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22.5% and 2.4% in type I and II SMA Patients, respectively. They also
examined four polymorphisms of SMN genes and found that there
were only two, SMN-2 and CBCD541-2, in Chinese subjects. They
analyzed the ratio of the telomeric to centromeric portion (T/C ratio) of
the SMN gene after enzyme digestion to differentiate carriers, normals,
and SMA patients. They found the T/C ratio of exon 7 of the SMN gene
differed significantly among the three groups, and may be used for
carrier analysis. An asymptomatic individual with homozygous deletion
of exon 7 and 8 of the SMN gene showed no difference in
microsatellite markers in the SMA-related 5q11.2–5q13.3 (Chang et al.
1997).

 Glotov et al. in 2001 carried out Analysis of Deletions in SMN1, SMN2,

and NAIP Genes in Spinal Muscular Atrophy Patients from the
Northwestern Region of Russia. They used Polymerase chain reaction
with subsequent SSCP (single-strand DNA conformational
polymorphism) and restriction (Bsel I restriction endonuclease)
analyses to type the DNA samples of affected individuals and their
relatives from 23 Russian families with high risk of spinal muscular
atrophy (SMA) residing in the northwestern region of Russia. Deletions
of exon 7 of the SMN1 gene were found in 96% of the individuals
examined. The frequency of homozygous deletion of exon 7 and 8 of
the SMN1 gene was 65%. The frequency of homozygous isolated
deletion of the SMN1 gene exon 7 among the SMA patients was 4.3%.
Homozygous deletion of exon 5 of the NAIP gene was found in 22% of
SMA patients. In SMA patients, a total of seven deletion types involving
the SMN1, NAIP, and SMN2 genes were detected. Deletion of exon 7
and 8 of the SMN1 gene was the most common mutation associated
with SMA in patients from the northwestern Russia (Glotov et al. 2001).

 Tooko et al. in 2002 analyzed SMN genes in 32 SMA patients and

found that the SMN1 gene was deleted in 30 of 32 patients (94
%), irrespective of clinical type. The NAIP gene was deleted in 6

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patients and its deletion rate was higher in type I patients than that in
type II or III. Further, in type I patients lacking the NAIP gene,
deterioration in their respiratory function was more rapid than in those
type I patients retaining the NAIP gene. Since complete p44t
deletion was observed in only 3 patients, the correlation between the
p44t deletion and severity of SMA remained ambiguous. They
concluded that the NAIP deletion was closely related to the clinical
severity of SMA and was a predictive marker of SMA prognosis, while
the SMN1 deletion did not correlate with clinical severity (Tooko et al.
2002).

 Khanh et al. in 2002 carried out molecular genetic analyses of five

Vietnamese Patients with Spinal Muscular Atrophy. In this preliminary
study, they analyzed five Vietnamese SMA patients and found that
SMN1 gene exons 7 and 8 were completely absent in one of them,
a 6-month-old girl with hypotonic muscles. Thus, homozygous
deletion of the gene can be a cause of SMA in Vietnam, although
other genetic abnormalities should be considered as etiological factors
in many cases. In conclusion, they identified a homozygous deletion of
the SMN1 gene in a Vietnamese SMA patient. Since the number of the
patients analyzed in this study was very limited, it is too early to
determine whether homozygous deletion of the gene is not a main
cause of SMA in Vietnam (Khanh et al. 2002).

 Bouhouche et al. in 2003 conducted deletion analysis of SMN and a

neighboring gene, NAIP (neuronal apoptosis inhibiting protein). Among
54 SMA patients types I–IV), all of Moroccan origin, Exon 7 of the
SMN1 gene was homozygously absent in 100% of type I, 90% of type
II, 74% of type III and 80% of type IV SMA patients. Deletion of SMN1
exon 8 was detected in 100% of type I, 53% of type II, 53% of type III
and 80% of type IV patients. NAIP exon 5 was homozygously deleted
in 67% of type I, 32% of type II, 5% of type III and 20% of type IV SMA
Patients. Thirty control individuals who were studied had normal SMN1

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and NAIP genes. Their results showed a high incidence of SMN1 gene
deletion in adult-onset SMA patients indicating that SMN1 is the
autosomal recessive adult SMA-causing gene. While NAIP was
commonly deleted in SMA, this was unlikely to affect disease severity;
it was deleted in two adult SMA patients with mild phenotypes
(Bouhouche et al. 2003).

 Shafeghati et al. in 2004 carried out a molecular diagnosis of Iranian

patients with Spinal Muscular Atrophy. They investigated the presence
of the survival motor neuron gene in 47 Iranian families, including 60
patients by polymerase chain reaction amplification of exon 7 and 8 in
affected individuals and parents of patients for carrier testing. They
also performed prenatal testing on 15 pregnant mothers. Mutation
detection in the 22 live patients showed that in the 21 cases, both
alleles were deleted. In 1 case only one of the mutations was detected,
therefore the other must have been a point mutation. In 34 families,
both of the parents were carriers, that is, they carried only one copy of
the normal SMN gene. In 9 of the couples only one mutation was
detected, therefore in the other one, it should have been a point
mutation that was not diagnosed. Molecular testing of 15 fetuses by
prenatal diagnostic procedures showed that 4 of the fetuses were
normal, 3 fetuses were affected and carried both of the mutations, five
fetuses were carriers (they carried one of the mutations), and the other
2 were carriers or healthy, but only one case might have been carrier
or affected. They conclude that SMA is a very common disease in the
Iranian population, due to the high frequency of consanguineous
marriages (Shafeghati et al. 2004).

 Chan et al. in 2004 reported Carrier incidence for spinal muscular

atrophy in southern Chinese. They used A real time quantitative PCR
(QPCR) method using TaqMan technology to assess the copy number
of the two survival motor neuron genes SMN1 and SMN2) on
chromosome 5q13.Analysis of 569 normal southern Chinese

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individuals revealed a carrier incidence of 1.6%, similar to that found in

the western society. Study of 42 obligatory carriers showed a (2+0)
genotype in two (4.8 %). In 27 SMA patients with homozygous deletion
of the SMN1 gene, the number of SMN2 gene correlated with disease
phenotype, with 68% of type II and III patients carrying three or more
SMN2 genes, whilst the incidence of three or more SMN2 genes in the
normal population was 1.57% (Chan et al. 2004).

 Smith et al. in 2007 studied 422 individuals to identify SMA carriers.

This cohort included 117 parents of children homozygously deleted for
SMN1 (94% were carriers and 6% had two copies of SMN1; of these
individuals, two in seven had the ‘2+0’ genotype, two in seven were
normal but had children carrying a de novo deletion and three in seven
were unresolved), 158 individuals with a significant family history of
SMA (47% had one copy, 49% had two copies and 4% had three
copies of SMN1) and 146 individuals with no family history of SMA
(90% had two copies, 2% had one copy and 8% had three copies of
SMN1). The SMA carrier frequency in the Australian population
appears to be 1/49 and the frequency of two-copy SMN1 alleles and de
novo deletion mutations are both at least 1.7%. A multimodal approach
involving quantitative analysis, linkage analysis and genetic risk
assessment (GRA), facilitates the resolution of SMA carrier status in
individuals with a family history as well as individuals of the general
population (Smith et al. 2007).

 Swaminathan et al. in 2008 performed Deletion analysis of spinal

muscular atrophy in southern Indian population to determine the
molecular genetics of SMN1 and NAIP genes in SMA from southern
India. In the this study, 37 patients from the Neuromuscular disorders
clinic of National Institute of Mental Health and Neurosciences were
assayed for the deletions in the SMN1 and NAIP genes using PCR-
RFLP methods. Among the SMA Type I patients, 43% showed
deletions of SMN1 and NAIP. In patients Type II SMA, 57% showed

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deletions of the SMN1 exons. Thus, Deletions were found to occur in

47.8% of the Type I and II patients. Lower sensitivity of gene deletion
study in clinically suspected SMA needs further study as clinical
diagnosis of SMA is not gold standard (Swaminathan et al. 2008).

 Liang et al. in 2009 analyzed the deletion of SMN1 and NAIP genes in
southern Chinese children with SMA. They used polymerase chain
reaction (PCR) Combined with restriction fragment length
polymorphism (RFLP) to detect the deletion of both exon 7 and exon 8
of SMN1 and exon 5 of NAIP in 62 southern Chinese children with
strongly suspected clinical symptoms of SMA. All the 32 SMA1 patients
and 76% (13/17) of SMA2 patients showed homozygous deletions for
exon 7 and exon 8, and all the 13 SMA3 patients showed single
deletion of SMN1 exon 7 along with 24% (4/17) of SMA2 patients.
Eleven out of 32 (34%) SMA1 patients showed NAIP deletion, and
none of SMA2 and SMA3 patients was found to have NAIP deletion.
The findings of homozygous deletions of exon 7 and/or exon 8 of
SMN1 gene confirmed the diagnosis of SMA, and suggested that the
deletion of SMN1 exon 7 was a major cause of SMA in southern
Chinese children, and that the NAIP gene may be a modifying factor for
disease severity of SMA1 (Liang et al. 2009).

 Alias et al. in 2009 studied the molecular pathology of SMA in 745

unrelated Spanish patients using PCR-RFLP, SMN gene dosage
analysis, linkage studies, long-range PCR and direct sequencing. The
systematic approach allowed them to complete genetic testing and risk
assessment in 736 SMA patients (98.8%). Females were more
frequently affected by the acute form of the disease (type I), whereas
chronic forms (type II–III) predominated in males (p < 0.008). Absence
of the SMN1 gene was detected in 671 patients (90%), and hybrid
SMN1–SMN2 genes were observed in 37 cases (5%). They detected
13 small mutations in 28 patients (3.8%), four of which were previously

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identified in other populations (c.91dupT; c.770_780dup11;

p.Tyr272Cys and p.Thr274Ile), while five mutations were found to date
only in Spanish patients (c.399_402delAGAG, p.Ile116Phe,
p.Gln136Glu, c.740dupC and c.834+2T>G). The c.399_402delAGAG
mutation accounted for 1.9% of all Spanish SMA Patients. They
discovered four novel mutations: c.312dupA, c.411delT, p.Trp190X and
p.Met263Thr. Their results confirmed that most SMA cases were due
to large genetic rearrangements in the repetitive region of the SMA
locus, resulting in absence-dysfunction of the SMN1 gene. Four
prevalent changes in exon 3 and 6 (c.399_402delAGAG;
c.770_780dup11; p.Tyr272Cys; p.Thr274Ile) accounted for almost 70%
of our patients with these subtle mutations (Alias et al. 2009)

3.3 Novel Mutations

 Wang et al. in 1998 reported a novel G279C mutation with a G to T

transversion on exon 7 (nucleotide position 868) of SMNT. Another
missense mutation was on position 869. The fact that two mutations on
the same codon both result in SMA suggest a functional significance of
this amino acid within the SMN protein (Wang et al. 1998).

 Parsons et al. in 1998 reported a child with clinical findings consistent

with Werdnig-Hoffmann disease (spinal muscular atrophy type I) who
was found not to have the homozygous absence of the survival motor
neuron SMNT) gene observed in -95% of spinal muscular atrophy
patients. A quantitative PCR based dosage assay for SMNT copy
number showed that this patient possessed a single copy of the SMNT
gene. Heteroduplex and sequence analysis of the remaining copy of
SMNT showed a 2 base pair deletion within exon 4 which produces a
frame shift and premature termination of the deduced SMNT protein.
This protocol of initial SMNT gene dosage analysis followed by
mutation detection allows identification of SMA compound

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heterozygote (patients lacking one copy of SMNT and having another

mutation in their other copy), thereby increasing the sensitivity of SMA
molecular diagnosis (Parsons et al. 1998).

 Leigh et al. in 2001 reported two novel mutations in three cases of

spinal muscular atrophy (SMA), including two distant cousins who
followed an unexpectedly severe course. Diagnosis was confirmed by
reduced SMN protein and full-length SMN mRNA levels. Sequencing of
the non-deleted SMN1 gene revealed a single G insertion at the end of
exon 1 in the two cousins and a novel G275S exon 6 mis-sense
mutations in the milder case (Leigh et al. 2001).

 Arkblad et al. in 2006 reported a previously unreported non-sense

mutation in SMN1 causes spinal muscular atrophy. Using MLPA in
clinical diagnostics they have identified six patients having only one
copy of SMN1 each and a clinical suspicion of SMA. The SMN-genes
of these patients have been sequenced and two were found to have
point mutations in their remaining SMN1. One patient had the known
point mutation SMN1 c.815A > G (Tyr272Cys), which is one of the
more commonly detected point mutations associated with SMA. The
MLPA analysis showed 1 SMN1, 3 SMN2, 1 BIRC1 (NAIP), 3 yNAIP,
and 3 GTF2H2. One patient with SMA type 1 had a new non-sense
mutation SMN1 c.831T > G (Tyr277Stop). Long range nested PCR
confirmed that the mutation was located in the SMN1. It was shown
that the full-length SMN mRNA in this patient was almost only from the
SMN2 copies. This might be due to non-sense mediated degradation of
the mRNA from the mutated SMN1. The MLPA result showed 1 SMN1,
2 SMN2, 1 BIRC1 (NAIP), 2 yNAIP, and 2GTF2H2. The father of this
patient also carried this mutation. They found point mutations in two of
six patients with one copy of SMN1 and a clinical suspicion of SMA
(Arkblad et al. 2006).

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3.4 Gene conversion

 Van der Steege et al. in 1996 reported a one of the possible cause of
spinal muscular atrophy that gene conversion involving SMN gene. The
large majority of SMA patients show homozygous deletions of at least
exon 7 and 8 of the SMN gene. A minority of patients show absence of
SMN exon 7 but retention of exon 8. This was explained by results of
analysis of 13 such patients providing evidence for apparent gene-
conversion events between SMN and the centromeric copy gene.
Instead of applying a separate analysis for absence or presence of
SMN exon 7 and 8, they used a contiguous PCR from intron 6 to exon
8. In every case they found a chimeric gene with a fusion of exon 7 of
the copy gene and exon 8 of SMN and absence of a normal SMN
gene. Similar events, including the fusion counterpart, were observed
in a group of controls, although in the presence of a normal SMN gene.
Chimeric genes as the result of fusions of parts of SMN and cBCD541
apparently are far from rare and may partly explain the frequently
observed SMN deletions in SMA patients (Van der Steege et al. 1996).

3.5 SMN2 copy number, NAIP gene deletion and severity

of SMA.

 Mohd Shamshudin et al. in 2009 investigated the potential association

between the number of copies of SMN2 and the deletion in the NAIP
gene with the clinical severity of SMA in patients of Malaysian origin.
Forty-two SMA patients (14 of type I, 20 type II, and 8 type III) carrying
deletions of the SMN1 gene were enrolled in this study. SMN2 copy
number was determined by fluorescence-based quantitative
polymerase chain reaction assay. Twenty-nine percent of type I
patients carried one copy of SMN2, while the remaining 71% carried
two copies. Among the type II and type III SMA Patients, 29% of cases
carried two copies of the gene, while 71% carried three or four copies

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of SMN2. Deletion analysis of NAIP showed that 50% of type I SMA

patients had a homozygous deletion of exon 5 of this gene and that
only 10% of type II SMA Cases carried a homozygous deletion, while
all type III patients carried intact copies of the NAIP gene. They
concluded that there exists a close relationship between SMN2 copy
number and SMA disease severity, suggesting that the determination
of SMN2 copy number may be a good predictor of SMA disease type.
Furthermore, NAIP gene deletion was found to be associated with SMA
severity (Mohd Shamshudin et al. 2009).

3.6 Microsatellite Markers in SMA.

 Morrison et al. in 1993 described the isolation of two new microsatellite

markers (EF1/2a and EF13/14) near locus D5S125, which lies 2 cm
distal to D5S6. They showed by linkage analysis and the study of the
recombinants in 55 SMA pedigrees that the disease lies in the 4-cM
interval between EF1/2a and D5S112. Fluorescence in situ analysis of
cosmids from D5S6, EF1/2a and D5S112 confirms the genetic order
and relative distance of markers. The microsatellites EF1/2a and
EF13/14 are the first highly polymorphic PCR-based proximal markers
in SMA to be described, and will be of value in prenatal prediction of
the disorder (Morrison et al. 1993).

3.7 Mitochondrial DNA and SMA

 Alexandra et al. in 2003 analyzed the amount of mitochondrial DNA

(mtDNA) in skeletal muscle of 20 unrelated children with genetically
proven SMA and 31 controls to gain a better understanding of low
energy supply. Quantitative Southern blot analysis revealed a severe
and homogeneous decrease in the content of muscle mtDNA in
relation to nuclear DNA in SMA patients (90.3±7.8%), whereas by
immunofluorescence no decrease in the number of mitochondria was

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detected. In addition, a two- to three fold reduction of the nuclear-

encoded complex II (succinate dehydrogenase) activity was detected in
SMA muscle tissue. Western blot analysis showed a significant
reduction of both mitochondrial- and nuclear-encoded cytochrome c
oxidase subunits. Their results indicate that mtDNA depletion in SMA is
a consequence of severe atrophy, and has to be differentiated by
measurement of complex II from an isolated reduction of mtDNA as
found in patients with mitochondriocytopathies and the so called
mtDNA depletion syndrome (Alexandra et al. 2003).

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