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Spinal muscular atrophy: Molecular genetics and diagnostics

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Spinal muscular atrophy:

molecular genetics and diagnostics
Shuji Ogino† and Robert B Wilson

Spinal muscular atrophy is one of the most common autosomal recessive diseases,
affecting approximately one in 10,000 live births and with a carrier frequency of
approximately one in 50. Spinal muscular atrophy is caused by a deficiency of the
ubiquitous protein survival of motor neuron (SMN), which is encoded by the SMN genes,
SMN1 and SMN2. Due to a single nucleotide polymorphism (840C>T), SMN2 produces less
full-length transcript than SMN1 and cannot entirely prevent neuronal cell death at
CONTENTS physiologic gene dosages. The 38-kDa SMN protein comprises 294 amino acids and is
involved in the biogenesis of uridine-rich small nuclear ribonucleoproteins, facilitating their
Clinical features
cytoplasmic assembly into the spliceosome. Various animal models have been developed
Molecular genetics to study the pathogenesis of spinal muscular atrophy, as well as to test novel therapeutics.
Molecular diagnostics Common PCR-restriction fragment length polymorphism assays can detect the
Genetic counseling & homozygous absence of SMN1 in approximately 94% of patients with clinically typical
risk assessment spinal muscular atrophy. SMN gene dosage analysis can determine the copy number of
Potential therapeutic SMN1 to detect carriers and patients heterozygous for the absence of SMN1. Due to the
approaches genetic complexity and the high carrier frequency, accurate risk assessment and genetic
Conclusion counseling are particularly important. Comprehensive SMA genetic testing, combined with
appropriate genetic counseling and risk assessment, provides the most complete
Expert opinion
evaluation of patients and their families at this time. New technologies, such as monosomal
Five-year view
analysis techniques, may be widely available in the future.
Key issues
Expert Rev. Mol. Diagn. 4(1), 15–29 (2004)
References
Affiliations Clinical features may also be affected in SMA Type I patients
Typical features [4]. Type II SMA (intermediate; OMIM
Spinal muscular atrophy (SMA) is one of the number 253550) is characterized by the onset
most common autosomal recessive diseases, of proximal muscle weakness before
affecting approximately one in 10,000 live 18 months of age, the ability to sit but not to
births and with a carrier frequency of approxi- walk unaided and survival beyond 4 years of
mately one in 50 [1]. SMA is characterized by age. Type III SMA (Kugelberg–Welander dis-
symmetric proximal muscle weakness second- ease; OMIM number 253400) is characterized
†
ary to degeneration of the anterior horn cells by the onset of proximal muscle weakness
Author for correspondence
Department of Pathology,
of the spinal cord. SMA is clinically sub- after the age of 2 years, the ability to walk
Brigham and Women’s Hospital, divided into three types based on age of onset independently until the disease progresses
75 Francis Street, Amory 3rd Floor, and clinical severity, according to established and survival into adulthood. It was proposed
Boston, MA 02115, USA diagnostic criteria [2,3]. Type I SMA (Werdnig– that Type III SMA be subdivided into
Tel.: +1 617 632 3978 Hoffmann disease; Online Mendelian Inheri- Type IIIa and Type IIIb, depending on age of
Fax: +1 617 632 5762
sogino@partners.org
tance in Man [OMIM] database number onset (Type IIIa before 3 years and Type IIIb
253300) is characterized by the onset of after 3 years) [5]. The probabilities of being
KEYWORDS: severe muscle weakness and hypotonia in the ambulatory at 10, 20 and 40 years after onset
carrier, conversion, deletion,
gene dosage, genetic counseling, first few months of life and the inability to sit were 73, 44 and 34%, respectively, in patients
genetic testing, risk assessment, or walk. Fatal respiratory failure usually occurs with SMA Type IIIa, and 97, 89 and 67%,
SMN, SMN1, SMN2
before the age of 2 years. Sensory neurons respectively, in patients with SMA Type IIIb [5].

www.future-drugs.com © Future Drugs Ltd. All rights reserved. ISSN 1473-7159 15

Ogino & Wilson
Approximately 60% of SMA patients have Type I and the
remaining patients have Type II or Type III, with Type II
being more common [6].
Atypical features
Type IV (or so-called adult-type) SMA has been a controversial
entity in the literature, especially with regard to the age of
onset [7–9]. It has been defined by some as SMA with an age of
onset after 30 years [5]. Although Type III SMA is character-
ized by a rather variable age of onset spanning three decades,
few individuals with Type III SMA become symptomatic in
their twenties [5]. SMN1 was not homozygously absent in four
patients with SMA and an age of onset after 30 years, although
they fulfilled the diagnostic criteria of SMA [8]. In another
study, five patients with ages of onset of SMA ranging from 20
to 32 years did show homozygous absence of SMN1 [7]. It
remains to be determined whether Type IV patients who do
not lack both copies of SMN1 have small intragenic mutations
in SMN1 or have an SMN1-unrelated disease.
Rare examples of SMN1-related SMA with atypical clinical
features have been described. These features include extended
CNS involvement (i.e., cerebral atrophy, abnormal head cir-
cumference, pyramidal tract signs and cerebellar signs), con-
genital fiber type disproportion (Type 1 fiber atrophy and
Type 2 fiber hypertrophy), creatine kinase (CK) values greater
than 1000 U/l, congenital malformations of other organs (i.e.,
ventricular septal defects of the heart and urogenital malforma-
tions) and complex syndromes [10]. It remains to be determined
whether these rare examples include deletions of additional,
contiguous genes (i.e., represent contiguous gene syndromes);
are part of the spectrum of SMN1-related defects; or represent
coincidental, simultaneous occurrences of conditions unlinked
to chromosome 5q.
Arthrogryposis represents a group of heterogeneous syn-
dromes, all of which have joint contractures present at birth [11].
The major clinical form, arthrogryposis multiplex congenita
(AMC), is caused by decreased fetal movements in utero, due to
a large number of conditions, including neuropathies, myo-
pathies, connective tissue diseases and conditions that limit the
space within the uterus [12]. SMA with congenital bone frac-
tures (which is chromosome 5 unlinked) is a rare syndrome
characterized by floppiness in infancy and by multiple fractures
of the long bones [13]. The features of SMA with congenital
bone fractures overlap with those of SMA with congenital
arthrogryposis and bone fractures [10]. Six of 12 patients with
neurogenic AMC showed homozygous absence of SMN1 [14].
Two of four patients with arthrogryposis, not otherwise speci-
fied, showed homozygous absence of SMN1 [15]. By contrast,
two of two patients affected with SMA with congenital arthro-
gryposis and bone fractures did not show homozygous absence
of SMN1 [10], as well as only one of seven cases referred for
arthrogryposis in another study [16]. However, comparing these
data is problematic because of the heterogeneous etiologies for
arthrogryposis and the lack of uniform inclusion criteria in
these studies.
16
The authors previously reported the case of an infant with
homozygous absence of SMN1 who was suspected to have
sudden infant death syndrome (SIDS) [16]. Another report
described an infant who nearly suffered sudden death at the age
of 7 weeks due to diaphragmatic paralysis and was later diag-
nosed to have Type I SMA [17]. Diaphragmatic paralysis and
respiratory distress were reported as initial manifestations of
SMA in six infants [18–20]. However, the diagnosis of SMN1-
related SMA may not be appropriate in a setting of muscle weak-
ness that is predominantly distal and the status of SMN1 in these
infants was not determined. The authors speculate that the cause
of some clinically unexplained infant deaths may be SMA.
Strictly speaking, the diagnosis of SIDS is inappropriate if a
homozygous SMN1 mutation confirms the diagnosis of SMA.
More studies are necessary to draw a definitive conclusion on the
frequency of undiagnosed SMA being misdiagnosed as SIDS.
Molecular genetics
Disease gene SMN1 & its homolog SMN2
SMA of all types is associated with homozygous mutations in
the survival of motor neurone 1 gene (SMN1; OMIM
number 600354) [21]. SMN1 and its centromeric homolog,
SMN2 (OMIM number 601627), lie within the telomeric
and centromeric halves, respectively, of a large inverted repeat
in chromosome region 5q13 [21]. SMN consists of nine exons
(exons 1, 2a, 2b and 3–8), with the stop codon present near
the end of exon 7 [22]. The coding sequence of SMN2 differs
from that of SMN1 by a single nucleotide (840C>T) in
exon 7 (FIGURE 1) [22]. In contrast to SMN1, of which at least
one copy is indispensable for the normal survival of motor
neurons, SMN2 is dispensable since approximately 5–10% of
normal individuals lack both copies [21,23,24]. The failure of
SMN2 to compensate fully for the loss of SMN1 is due to the
sequence difference in exon 7. Although translationally silent,
the 840C>T transition in SMN2 has been reported to
decrease the activity of an exonic splicing enhancer so that less
full-length transcript is generated, leading to deficiency of
normal, stable SMN protein [25,26]. More recent evidence sug-
gests that the 840C>T transition in SMN2 activates an exonic
splicing silencer, which functions as a binding site for the
known repressor protein hnRNP A1 [27].
SMN protein & the SMN complex
SMN is a ubiquitously expressed 38-kDa protein consisting
of 294 amino acids. Most of the SMN protein in a normal
individual is derived from SMN1, as indicated in part by the
fact that there is only a very small amount of the SMN pro-
tein derived from SMN2 in affected individuals with a
homozygous absence of SMN1. SMA patients with a
homozygous absence of SMN1 always have at least one copy
of SMN2 and a homozygous deletion of the mouse SMN
gene causes embryonic lethality. Thus, the SMN protein is
essential for viability. The SMN protein protected neurons
in culture and in vivo from virus-induced apoptosis, whereas
disease-associated mutant forms of the protein were proapoptotic
Expert Rev. Mol. Diagn. 4(1), (2004)

in the same systems [28]. It remains unexplained why motor
neurons are more susceptible than other cell types to defi-
ciency of such a ubiquitously expressed protein. It is likely
that requirements for the SMN protein differ among various
cell types.
SMN is involved in the biogenesis of uridine-rich small
nuclear ribonucleoproteins (UsnRNPs) and facilitates cytoplas-
mic assembly of UsnRNPs into the spliceosome, a large
snRNA–protein complex that catalyzes pre-mRNA splicing
[29,30]. UsnRNP biogenesis by the SMN complex is well
described in a recent review [30]. In the nucleus, SMN appears
to be directly involved in pre-mRNA splicing. SMN associates
with multiple proteins to form a complex [30,31]. Core SMN-
interacting proteins in the complex include Gemin2, Gemin3,
Gemin4, Gemin5, Gemin6 and Gemin7. Substrates and sub-
stoichiometric components include Sm and Sm-like (LSm)
proteins B, D1, D2, D3, E, F and G; uridine-rich snRNAs
(UsnRNAs); fibrillarin; GAR1; coilin; profilin; ZPR1; OSF;
nucleolin; B23; hsc70; RNA helicase A; RNA polymerase II;
snurportin; importin β; galectin1/3; and heterogeneous nuclear
RNP-Q and RNP-R [29–31]. UsnRNP assembly is mediated by
the SMN complex, which subsequently facilitates hypermethy-
lation of the methyl-G cap of UsnRNAs [30]. The hypermethy-
lated cap (m3G-cap) structure is specifically bound by snurpor-
tin, which then interacts with the import receptor importin β
and, together with an as-yet-unknown import receptor that
recognizes the Sm core, mediates UsnRNA import [30]. After
each round of assembly, the SMN complex must be reloaded
Centromeric
SMN2
500 Kb
Large inverted duplication in 5q13
SMN
1 2a 2b
g C
a T
Essential difference
Figure 1. The survival of motor neuron gene (SMN) consists of 9 exons, with the stop codon near the end of exon 7. The coding sequence of SMN2 differs
from that of SMN1 by a single nucleotide (840C>T) in exon 7.
www.future-drugs.com
Spinal muscular atrophy
with fresh Sm proteins to regain its activity and this loading
reaction may be regulated by the PRMT5 complex, composed
of methylosome (plCln, WD45 and PRMT5 [JBP1]) and Sm
proteins [30,31]. Sm proteins B, D1 and D3 are methylated by
methylosome before loading into the SMN complex [30].
SMN1 deletion & conversion mutations
Approximately 94% of individuals with clinically typical SMA
lack both copies of SMN1 exon 7 [32]. Loss of SMN1 can occur
by deletion (typically a large deletion that includes the whole
gene [33–35]) or by conversion to SMN2 [36]. Affected individuals
lacking both copies of SMN1 exon 7 but retaining at least one
copy of SMN1 exon 8 sequence have been described [21,36]. In
some of these individuals, exon 8 of SMN1 was found to be
physically juxtaposed to exon 7 of SMN2, consistent with an
SMN1-to-SMN2 gene conversion event involving the func-
tionally critical exon 7 sequence difference [36,37]. A large
majority of genes converted from SMN1 to SMN2 had SMN2
sequences in all of the polymorphic nucleotides except for the
SMN1 sequence in exon 8 and half of the hybrid
SMN1/SMN2 genes had a single Ag1-CA/C212 haplotype,
suggesting that these hybrid SMN alleles had a common origin
[37]. Minor variants of hybrid SMN genes have been described.
These include SMN2 intron 6 and exon 7 juxtaposed to
SMN1 intron 7 and exon 8 [37], SMN2 exon 7 flanked by
SMN1 intron 6 and exon 8 [38] and SMN1 sequences in all of
the polymorphic nucleotides except for the SMN2 sequence in
exon 8, the last of which was found in healthy individuals [37].
20 kb
Telomeric
SMN1
500 Kb
3 4 5 6 7 8
a a G
SMN1
SMN2
7 8
g g A
17
Ogino & Wilson
A number of hypotheses have been proposed for the generation
of these hybrid genes, including unequal recombination, intra-
chromosomal deletion and gene conversion [37]. At least one of
the SMN2 copies from mildly affected patients with SMA was
located in the telomeric position [39]. Taken together with the
increased number of SMN2 copies generally in mildly affected
patients with SMA, these data are consistent with gene conver-
sion from SMN1 to SMN2. The absence of SMN1 exon 7
sequence correlates with either a functional or a de facto
absence of the SMN1 gene.
At least one of the factors underlying the variable phenotype
of SMA is the SMN2 copy number. Individuals with Type III
SMA have, on average, more copies of SMN2 than individuals
with Type II or Type I SMA and likely express more full-length
SMN transcript derived from the extra copies of SMN2
[23,36,40,41]. It has been shown that 6.9, 73 and 20% of SMA
Type I patients had one, two and three copies of SMN2, respec-
tively. In contrast, 11, 82 and 7.3% of Type II patients and 3.9,
51 and 45% of Type III patients, had two, three and four copies
of SMN2, respectively [41].
SMN1 small intragenic mutations
The SMA in many of the approximate 6% of affected individ-
uals who do not lack both copies of SMN1 is not likely to be
related to SMN1 [40]. However, in a substantial number of the
SMA patients who do not lack both copies of SMN1, small
intragenic mutations (also referred to as subtle or nondeletion
mutations) in SMN1 have been identified (TABLE 1). These
intragenic mutations provide solid evidence that SMN1 is
indeed the SMA gene, since intragenic SMN1 mutations are
associated with the SMA phenotype regardless of the status of
NAIP or other genes. Commonly used nomenclature has not
been based on the nucleotide position starting from the initia-
tion ATG codon, which has led to some confusion. The use of
standard nomenclature based on the published sequence [22,37]
and published nomenclature guidelines [42,43] has been pro-
posed (TABLE 1) [40]. Use of correct nomenclature in the litera-
ture is essential since clinical diagnosis and decision making
are based on data in the literature regarding a specific muta-
tion detected in a consultand or affected family member. The
three most frequently reported small intragenic mutations are
the Y272C missense mutation (815A>G) [21,32,40,44], the
768_778dupTGCTGATGCTT frameshift mutation [32,45–48] and
the 399_402delAGAG frameshift mutation [48–50].
Wirth and coworkers found that 18 of 501 patients with
SMA – six of 265 Type I, six of 122 Type II and six of 114
Type III – with identifiable mutations in both chromosome 5s
had one small intragenic mutation and one deletion mutation
[40]. Thus, the proportion of disease alleles with small intragenic
mutations was highest in Type III SMA and lowest in Type I
SMA [40]. Among 525 patients with clinically typical SMA, 24
lacked identifiable mutations in both chromosome 5s and were
considered to have SMN1-unrelated disease [40]. Disease and
nondisease allele frequencies have a significant impact on risk
assessment in SMA genetic testing [1,6].
18
Some of these small intragenic mutations have been placed
into expression constructs and the effects on oligomerization
have been investigated. In vitro experiments showed that
G279V (836G>T) and Y272C (815A>G) caused a severe
impairment of oligomerization, while S262I (785G>T) and
T274I (821C>T) caused a mild impairment [51]. The degree of
impairment correlated with the severity of SMA associated with
the mutations in vivo [51]. A nonsense mutation, W102X
(305G>A), caused exon 3 skipping and thereby generated a
short form of SMN transcript lacking exon 3 [52]. Western blot
analysis showed a band at the size expected for SMN protein
lacking the amino acids encoded by exon 3. These mutations
were detected in three patients who had only two copies of
SMN2 but mild symptoms [52].
The frequency of small intragenic SMN1 mutations and/or
of SMA unlinked to chromosome 5q13 may be higher in cer-
tain non-European populations, such as black South African
patients [53], although data that do not support this hypothesis
also exist [54].
SMN polymorphisms
SMN polymorphisms have been reviewed elsewhere [32]. Most
polymorphisms are present in the promoter region and intron 6
[26]. The authors reported five unrelated individuals who have
two alleles that differ in length due to a single thymidine inser-
tion in a pre-existing polythymidine tract (8T) in SMN1
intron 6 (IVS6-24dupT or IVS6-24_IVS6-23insT) [16]. Two
were symptomatic (one with Type II SMA and the other with
Type III SMA) and the other three were asymptomatic. All five
of the samples contained the normal 8T SMN1 peak, in addi-
tion to the one-base-larger 9T SMN1 peak, and each peak
corresponded in dosage to one copy of SMN1 [16]. No indi-
viduals were found with only the 9T SMN1 peak without the
normal 8T SMN1 peak. The biological significance, if any, of
this single nucleotide insertion remains unknown.
De novo SMN1 mutations
SMN1 has a high rate of de novo deletion mutations. The high
rate of de novo mutations in SMN1 likely underlies the high
carrier frequency for SMA in the general population despite the
high genetic lethality of the disease. The large inverted repeat at
chromosome 5q13, as well as smaller repeat sequences around
the SMN1 and SMN2 loci, likely predispose to unequal crossing
over and other recombination events and therefore likely
underlie the high de novo mutation rate of SMN1. De novo
mutations were present in a total of 12 of 494 affected individ-
uals with a homozygous absence of SMN1 [6,55,56]. Among the
12 patients, there were nine paternal de novo deletion muta-
tions [6,55,56], one paternal de novo conversion mutation [55],
one maternal de novo deletion [55] and one maternal de novo
conversion mutation [40]. Thus, most reported de novo muta-
tions were paternal in origin. The reason why de novo muta-
tions of paternal origin are much more frequent than those of
maternal origin is unknown. Using the method of Wirth and
coworkers [56], paternal and maternal de novo mutation rates are
Expert Rev. Mol. Diagn. 4(1), (2004)
 Spinal muscular atrophy

Table 1. List of small intragenic SMN1 mutations (modified from [1]).

Standard nomenclature Single amino Other nomenclature Site of Type of Type of Unrelated Ref.
of nucleotide change acid change used in the literature mutation mutation SMA patients

5C>G A2G 38C>G Exon 1 Missense II, III 3 [46]

22_23insA (or 22dupA) Exon 1 Frameshift I or II 1 [114]

43C>T Q15X 78C>T Exon 1 Nonsense I, III 2 [40]

81_81+1insG (or 81dupG) Q27insG Exon 1 Frameshift II 2 [115]

91_92insT (or 91dupT) 124insT Exon 2a Frameshift I 1 [40]

208_209insGTGT 241-242in4 Exon 2b Frameshift III 1 [40]

305G>A W102X Exon 3 Nonsense II, III 2 [52]

399_402delAGAG 430del4 Exon 3 Frameshift I, II, III 11 [48–50]

400G>A E134K 433G>A Exon 3 Missense I 1 [32,47,52]

439_443delGAAGT 425del5, 472del5 Exon 3 Frameshift I 2 [52,116]

509_510delGT 542delGT Exon 4 Frameshift I, II, III 3 [46,91]

558delA 591delA Exon 4 Frameshift II 1 [40]

585_586insT (or 585dupT) 618insT Exon 4 Frameshift I 1 [32,47]

IVS4_IVS6del Intron 4–6 Deletion I 2 [40]

683T>A L228X Exon 5 Nonsense I or II 1 [114]

734C>T P245L 767C>T Exon 6 Missense III 1 [44]

740_741insC (or 740dupC) 773insC Exon 6 Frameshift III 1 [48]

768_778dupTGCTGATGCTT 813ins/dup11, Exon 6 Frameshift I, II 8 [32,

(or 770_780dupCTGATGCTTTG) 800ins11 45–48]

785G>T S262I 818G>T Exon 6 Missense III 2 [23,117]

815A>G Y272C 848A>G Exon 6 Missense I, II, III 9 [21,32,

40,44]

821C>T T274I 854C>T Exon 6 Missense II, III 4 [40,46,

117]

823G>A G275S Exon 6 Missense III 1 [115]

834+2T>G (or IVS6+2T>G) c.867+2T>G Intron 6 Splice site I 1 [48]

IVS6-18_IVS6-12delCCTTTAT c.868-11del7 Intron 6 Splice site I 1 [21,32]

835G>T G279C 868G>T Exon 7 Missense II, III 2 [118]

836G>T G279V 869G>T Exon 7 Missense I 2 [119]

IVS7+4_IVS7+7delAGTC c.922+3del4 Intron 7 Splice site II 1 [21,32]

IVS7+6T>G c.922+6T>G Intron 7 Splice site III 1 [40]

EX8del§ Exon 8 Deletion II, III 2 [120]

§
Exact extent of deletion not determined. One patient revealed homozygous deletion of NAIP exon 5, and the other patient did not reveal homozygous deletion of NAIP exon 5.
SMA: Spinal muscular atrophy.

www.future-drugs.com 19
Ogino & Wilson
calculated as 2.11 × 10-4 and 4.15 × 10-5, respectively. However,
these figures are based on very small numbers of patients who
had de novo mutations. Further studies are necessary to calcu-
late de novo mutation rates more accurately. The possibility of a
de novo mutation is highly relevant to genetic risk assessments
in some clinical scenarios [1].
SMN1 mosaicism
Campbell and coworkers described possible maternal somatic
or germline mosaicism for an SMN1 mutation [57]. Germline
mosaicism may result from a de novo mutation if it occurs early
in embryonic gonadal development. De novo mutations are
usually associated with a very low recurrence risk. However, in
the setting of germline mosaicism, recurrence risk is unpredict-
able unless SMN gene dosage analysis is performed on both
gametes and peripheral blood. The authors have encountered a
possible case of germline mosaicism for SMN1 in which the
SMN1 signal from a sperm sample was approximately 80% of
that from the same individual’s peripheral blood, demonstrated
repeatedly by SMN gene dosage analysis [58,59].
Candidate modifying genes for SMA
SMN2 is a well-established, disease-modifying gene for SMA
and has been shown to decrease the severity of SMA in a dose-
dependent manner [23,40,41,58]. The copy number of SMN2 also
correlates with longer survival of individuals affected with
Type I SMA [41]. However, the SMN2 copy number in Type I,
II and III SMA overlaps. In addition, rare families have been
reported in which different degrees of disease severity were
present in affected individuals with identical SMN1 and SMN2
copy numbers [40,41]. Thus, although SMN2 copy number
apparently explains much of the phenotypic variation in
SMA, there must be other disease-modifying factors. Indeed,
a small intragenic SMN2 mutation (859G>C; G287R) [41,60]
and other SMN2 mutations or polymorphisms may underlie
the discrepancy between SMN2 copy number and SMA type.
The neuronal apoptosis inhibitory protein (NAIP) gene was a
candidate disease gene for SMA because it was found to be
deleted in approximately two-thirds of Type I SMA chromo-
somes and because of its homology with baculoviral apoptosis
inhibitory proteins [61]. With the confirmation of SMN1 as the
disease gene for SMA [21], NAIP has been hypothesized to be a
disease-modifying gene. Homozygous deletions of NAIP are
found more often in Type I SMA than in the other two types
[60,62–64]. However, homozygous deletions of NAIP may simply
reflect larger chromosomal deletions involving both SMN1 and
SMN2. Further studies are necessary to confirm or rule out a
role of NAIP in the pathogenesis of SMA. H4F5 (SERF1) is
closer to SMN1 on chromosome 5 than any other known gene.
By microsatellite analysis, homozygous deletions of H4F5 were
found in approximately 90% of cases of Type I SMA [65].
However, as with NAIP, homozygous deletions of H4F5 may
simply reflect larger chromosomal deletions involving both
SMN1 and SMN2. Further studies are necessary to confirm or
rule out a role of H4F5 in the pathogenesis of SMA. Htra2-β1,
20
a nonessential splicing factor, was found to modify the alterna-
tive splicing of SMN2 and to regulate the inclusion of SMN
exon 7 to produce full-length transcripts from SMN2 [66].
However, phenotypic variations between discordant siblings
did not correlate with polymorphic variants or differences in
expression of Htra2-β1 [67]. Likewise, phenotypic variations
between siblings with identical 5q13 haplotypes did not corre-
late with mutations or differences in expression of Gemin2
(SIP1) [68]. It remains a possibility that SMA phenotypes are
modified by genes responsible for other neuromuscular disorders,
including IGHMBP2, HEXA, SCO2 and others [1]. It is also
possible that some of the other components of the SMN and
PRMT5 complexes and other components of the spliceosome
may be modifiers for SMA.
Animal models & orthologs of SMN
SMN gene duplication occurred more than 5 million years ago,
before the separation of human and chimpanzee lineages; how-
ever, the SMN2 gene sequence per se appeared for the first time
only in Homo sapiens, whereas the chimpanzee has duplicated
SMN but no SMN2 [69]. A knockout of murine SMN resulted
in massive apoptosis and embryonic lethality, indicating that
the SMN protein is essential for cell survival [70]. To construct
mouse models of SMA, it was necessary either to retain some
amount of SMN protein in the mouse embryo by introducing
the human SMN2 gene [71–73], or to delete murine SMN
exon 7 in neurons using cre-lox-mediated recombination [74].
Human SMN2 rescued mice from embryonic lethality and
alleviated symptoms resembling human SMA in a dose-
dependent manner [71,73]. Deletion of murine SMN exon 7 in
skeletal muscle by cre-lox-mediated recombination led to
severe muscular dystrophy, suggesting that skeletal muscle may
also be a primary site of degeneration in SMA [75]. Other animal
models of human SMA include Drosophila with a point muta-
tion in SMN [76], and zebrafish with SMN protein levels
decreased by the use of an antisense morpholino oligonucleo-
tide [77]. These animal models will be useful for studies on the
pathogenesis of SMA, as well as for the development and testing
of novel therapeutics.
Molecular diagnostics
Linkage analysis
Localization of the SMA critical region allowed for linkage
analysis, the first genetic test for SMA. From centromeric to
telomeric, informative linkage markers that have been
reported are as follows: D5S679, D5S680, D5S125, D5S681,
D5S435, D5S629, D5S823, D5S1556/D5F150 (intra-
genic/SMN1 promoter region), D5S149 (intragenic/SMN1
promoter region), D5S557, D5S610, D5S351, 5´-MAP1B,
3´-MAP1B, D5S112, D5S127 and D5S539 [78]. Even after
identification of the disease gene [21], linkage analysis remains
an important tool in certain clinical scenarios [16,58]. Multi-
copy markers, such as C212 and C272, have also been used in
linkage analysis [33,79,80], although the interpretation of results
requires considerable skill.
Expert Rev. Mol. Diagn. 4(1), (2004)

PCR-single-strand conformation polymorphism
PCR-single-strand conformation polymorphism (SSCP) analysis
has been used to detect the homozygous absence of SMN1
exon 7 and exon 8 [21,81]. However, although PCR-SSCP analysis
can detect small intragenic mutations in an amplified segment,
inconsequential polymorphisms are also detected. Using PCR-
SSCP analysis, Wang and coworkers found that 4% of asymp-
tomatic family members of patients with SMA had polymor-
phisms in SMN1 exon 7 that mimicked the homozygous
absence of SMN1 [82]. With the development of PCR-restriction
fragment length polymorphism (RFLP) assays [83], PCR-SSCP
has largely been replaced in clinical laboratories. PCR-SSCP
may still be used to screen for small intragenic mutations in
affected individuals who do not lack both copies of SMN1.
PCR-RFLP
The most commonly used method to genetically confirm the
diagnosis of SMA is a qualitative PCR-RFLP assay (also known
as the SMN1 deletion assay) to detect the homozygous absence
of SMN1 [83]. The PCR-RFLP assay takes advantage of the base
differences in exons 7 and 8 to distinguish SMN1 from SMN2.
The restriction endonuclease DraI digests only the SMN2 exon 7
PCR products because of a restriction site generated by a mis-
matched reverse primer [83]. Another restriction enzyme that
can be utilized is Hinf I [40]. In the Hinf I method, internal con-
trol restriction sites are introduced in both the SMN1 and the
SMN2 PCR products to ensure complete Hinf I digestion [40].
Wirth reviewed the frequencies of SMA patients without a
homozygous absence of SMN1 exon 8 among SMA patients with
a homozygous absence of SMN1 exon 7 [32]. The frequencies
were 21 of 400 (5.3%) for Type I, 19 of 241 (7.9%) for Type II,
26 of 185 (14%) for Type III and 79 of 1054 (7.5%) for all
types combined. These data are consistent with the tendency of
patients with Type III SMA to have more copies of SMN2
exon 7 juxtaposed to SMN1 exon 8 due to gene conversion.
Combining the data of Wirth with the data of Mailman and
coworkers (29 of 261 for all types combined) [84] and Ogino
and coworkers (16 of 126 for all types combined) [16], a total of
124 of 1441 (8.6%) patients with SMA and a homozygous
absence of SMN1 exon 7 had at least one copy of SMN1 exon 8.
In clinical practice, patients referred for SMA genetic testing
do not always have clinically typical features of SMA. In one
study, 349 of 610 (57%) patients tested did not have a
homozygous absence of SMN1 exon 7 [84]. In another study, a
total of 217 of 430 (50%) patients did not have a homozygous
absence of SMN1 exon 7 [16]. Among those patients in which
the SMA type was specified, this comprised 34 of 94 Type I
patients (36%), five of 40 Type II patients (12%) and 42 of 62
Type III patients (68%) [16]. Therefore, the clinical diagnosis of
SMA can be problematic and genetic testing is often ordered to
exclude SMN1-related SMA because of atypical clinical fea-
tures, or to confirm SMN1-related SMA. The predictive value
of a presumptive clinical diagnosis of Type II SMA was consid-
erably higher (35 of 40) than that of the other SMA types [16],
probably due to the fewer conditions that mimic Type II SMA.
www.future-drugs.com
Spinal muscular atrophy
Rapid PCR-denaturing high performance liquid chromatog-
raphy (DHPLC) methods to detect the homozygous absence of
SMN1 exon 7 have been described [85,86].
Quantitative SMN gene dosage analyses
Since qualitative PCR-RFLP cannot distinguish carriers with
one copy of SMN1 from normal individuals with two copies of
SMN1, a quantitative approach (i.e., SMN gene dosage analysis)
is necessary to identify SMA carriers. The rationale for a quan-
titative competitive PCR method using a genomic reference
sequence, along with internal control sequences for both the
target and the reference sequences, has been described [59].
McAndrew and coworkers developed the first highly precise
assay to determine copy numbers of SMN1 and SMN2 using
this method [23]. The assay utilizes quantitative competitive
PCR-RFLP analysis, with CFTR exon 4 as a two-copy genomic
reference. To correct for variation in amplification efficiency,
the assay includes SMN and CFTR internal standard sequences.
Each internal standard has the same primer binding sites as its
genomic counterpart and a small internal deletion, which
allows the internal standard PCR products and the genomic
PCR products to be separated and quantified independently
[23]. The copy number of SMN1 per cell (or more precisely per
diploid genome) is determined by coamplification of SMN1,
SMN2, the SMN internal standard, CFTR and the CFTR inter-
nal standard; DraI digestion of SMN2; and quantification of
each PCR product [23,58].
A variety of highly precise SMN gene dosage analyses have
since been developed, including a nonradioactive modification
using fluorescence-labeled primers and an automated sequencer
[40,58,87], combined quantitative competitive PCR with quanti-
tative primer extension [24] and real-time quantitative PCR
[41,88]. Various features of quantitative competitive PCR-RFLP
and real-time quantitative PCR have been compared [89].
Heteroduplex formation may affect quantitative PCR-RFLP
analyses [59]. Heteroduplexes between SMN1 and SMN2 PCR
products cannot be digested by DraI, increasing the SMN1 signal
and decreasing the SMN2 signal [59]. In addition to heteroduplex
formation, an unexpected and reproducible PCR bias between
SMN1 and SMN2 sequences has been found (i.e., amplifica-
tion efficiency for SMN2 was only approximately 80% that of
SMN1) [90]. One should be aware of heteroduplex formation
and/or PCR bias in any quantitative competitive PCR.
SMN gene dosage analysis can be used for detection of the
heterozygous absence of SMN1 in patients with SMA who do
not lack both copies of SMN1 [40,44,91]. However, SMN gene
dosage analysis for this purpose should be used with caution
and should be limited to patients for whom SMN1-related
SMA is strongly suspected (i.e., clinically typical cases). This is
because the high frequency of SMA carriers with one copy of
SMN1 in the general population would otherwise lead to an
unacceptably low positive predictive value [6,16].
It has been shown that occasional individuals have three or
four copies of SMN1, implying the presence of occasional
chromosome 5s with two copies of SMN1 [6,23,40,41]. These
21
Ogino & Wilson
chromosomes can mask a deletion or conversion mutation of
SMN1 on the other chromosome 5, causing false-negative
results in SMN gene dosage analysis (i.e., carriers with two
SMN1 copies on one chromosome 5 and a deletion/conversion
mutation on the other chromosome 5 cannot be distinguished
by dosage analysis alone from normal individuals). Two SMN1
copies on a single chromosome may be due to gene conversion
from SMN2 to SMN1, or alternatively due to remaining dupli-
cated SMN1 genes without conversion to SMN2 [92]. The dis-
tinction between two and three copies of SMN1 is sometimes
important in families with relatives suspected to have the 2 + 0
SMN1 genotype [1].
SMN1 small intragenic mutation analysis
Analysis for the detection of small intragenic mutations is not
currently offered routinely in the clinical laboratory setting
because it is typically performed by DNA sequencing and is
therefore too labor intensive. A multiplex, allele-specific PCR
assay to detect the seven most common small intragenic muta-
tions has been described [84]. For more information regarding
assays to detect small intragenic mutations, the laboratory of
H Scheffer (Groningen, The Netherlands), or the laboratory of
B Wirth (Bonn, Germany) may be contacted [78]. Assays to
detect small intragenic mutations in SMN1 may become a part
of routine practice in the future if sensitive and cost-effective
methods to detect such mutations are developed.
Prenatal & preimplantation genetic testing
Prenatal testing to detect the homozygous absence of SMN1
can be performed on chorionic villous sampling (CVS) speci-
mens or amniotic fluids. To avoid false-negatives due to mater-
nal contamination, the amount of maternal DNA relative to
fetal DNA should be determined, for example by quantitative
PCR of polymorphic microsatellite or SNP markers. The
authors have used a method that was developed to quantify
engraftment after bone marrow transplantation [93]. Although
CVS specimens are more often contaminated with maternal
DNA than amniotic fluid specimens, such contamination is
rarely a problem if CVS specimens are handled properly. In
each SMN PCR-RFLP assay, a 10%-contamination control is
also run that contains a mixture of DNA lacking SMN1 (90%)
and DNA that contains SMN1 (10%) [1]. If the SMN1 band of
a fetal sample with less than 5% maternal contamination is
much brighter than that of the 10%-contamination control,
the SMN1 band of the fetal sample is much more likely to
derive from the fetal DNA than from contaminated maternal
DNA. However, interpretation of such results is still carried out
with caution since the standard PCR-RFLP assay is at most
only semiquantitative.
Prenatal testing can be performed on circulating fetal cells
in maternal blood [94]. However, the issue of persistent fetal
cells from the prior pregnancy is a cause for concern, although
a recent study found no evidence that there is interference of
persistent fetal cells from prior pregnancies with prenatal
diagnosis [95].
22
Preimplantation genetic testing in general has been reviewed
[96–98].PCR-RFLP-based preimplantation genetic testing to
detect the homozygous absence of SMN1 exon 7 has also been
described [99–103].
Monosomal analysis
Yan and coworkers developed a monosomal analysis method
involving the separation of single human chromosomes of
homologous pairs by fusing human cells with mouse cells and
culturing with selection [104]. This method is useful for analyzing
oncogenic mutations, which may be masked by the presence of
wild type alleles. It can also be applied to the detection of muta-
tions in hereditary diseases. Mailman and coworkers used this
method to identify an SMN1 deletion carrier who had two
copies of SMN1 by dosage analysis [105]. Monosomal analysis
obviates the need to perform extensive linkage and dosage
analyses to distinguish carriers with two copies of SMN1 (the
2 + 0 SMN1 genotype) from noncarriers (with the normal 1 + 1
genotype) who passed a de novo deletion mutation to an
affected child [58].
Genetic counseling & risk assessment
Both genetic counseling and risk assessment are integral
components of genetic testing and are particularly impor-
tant for SMA families because of the high carrier frequency
(1 in 50) and the complexity of SMA genetics [1]: small
intragenic mutations are undetectable by the standard PCR-
RFLP and SMN gene dosage analyses; the presence of two
normal copies of SMN1 on one chromosome 5 can mask a
deletion mutation on the other chromosome 5 in SMN gene
dosage analyses; and the paternal de novo mutation rate is
relatively high.
It should be noted that genetic risk assessments are esti-
mates. They are based on published data from a finite number
of cases in specific populations. It should also be noted that
additional family information, especially additional genetic
information, often dramatically improves the accuracy of
genetic risk assessments. Thus, genetic risk assessment is often
an ongoing process and genetic testing on additional family
members should be performed whenever indicated by the
initial results [1].
SMN1 allele & genotype frequencies
For the purpose of SMA risk assessment, normal and disease
SMN1 allele frequencies in the general population and for
each SMA type have been estimated [1]. Most disease SMN1
alleles are deletions/conversions of at least exon 7 of SMN1
(0-copy alleles). These alleles are usually detectable by dosage
analysis. A normal chromosome 5 usually has one copy of
SMN1 (1-copy allele). However, two copies of SMN1 are often
present on the same chromosome 5, for which the term 2-copy
allele is used. Disease alleles with small intragenic mutations in
SMN1 are referred to as 1D alleles (standing for 1-copy-disease);
1D alleles are indistinguishable from normal 1-copy alleles by
dosage analysis.
Expert Rev. Mol. Diagn. 4(1), (2004)
 Spinal muscular atrophy

Complete, updated haplotype and genotype frequencies in
the general population and for each SMA type are shown in
TABLE 2. The frequencies for a given SMA type should be used
only when the substantial risk of a 1D haplotype is present due
to an index patient who is affected with clinically typical SMA
of the given type [1,6].
In-depth and detailed methods for risk calculations have
been illustrated utilizing a variety of examples [1].
Potential therapeutic approaches
There is currently no effective treatment or cure for SMA.
However, potential therapies are an active area of investiga-
tion and new therapeutic approaches will likely emerge in the
near future. These include: to increase full-length mRNA
from SMN2 by altering the splicing pattern of SMN2 exon 7
in vivo, either through the activation of transacting factors or
by using antisense oligonucleotides [106]; to activate the over-
all level of SMN transcription; to stabilize the SMN protein;
to repair degenerated motor neurons; to replace degenerated
motor neurons by stem cell therapy [107]; and to target modi-
fying factors other than SMN2 [108]. To date, aclarubicin
[109], sodium butyrate [110] and valproic acid [111] have been
shown to increase the level of full-length transcripts from
SMN2 and thus are promising potential therapeutic agents for
SMA. High-throughput drug screening to identify compounds
that upregulate full-length SMN protein is underway [108].
Potential gene therapy strategies for SMA are also under inves-
tigation; for example, adenovirus-mediated gene delivery [112]
and cardiotrophin-1 gene transfer [113]. Further elucidation of
the molecular genetics and pathogenesis of SMA should facili-
tate development of effective treatments and potentially a cure
for SMA.
Conclusion
Since the discovery of the SMA disease gene, SMN1, consid-
erable progress in our understanding of the biochemical basis
for SMA and in genetic testing for SMA have been made. At
this time, comprehensive SMA testing comprising qualitative
PCR-RFLP, SMN gene dosage analysis and linkage analysis, in
combination with appropriate genetic counseling and risk
assessment, offers the most complete evaluation of SMA
patients and their families. The accuracy of risk assessments for
SMA will increase as more data on the genetics of SMA emerge.
In addition, new technologies, such as monosomal analysis,
may be widely available in the near future.
Expert opinion
Published guidelines state that genetic testing laboratories
should perform SMN gene dosage analysis for SMA carrier
testing on SMA family members only when the index patient

Table 2. Updated SMN1 allele and genotype frequencies.

SMN1 allele Designation General Type I§ Type II§ Type III§
population
0-copy (disease) a 9.88 × 10-3 9.95 × 10-3 9.86 × 10-3 9.76 × 10-3
1-copy (normal) b 9.50 × 10-1 9.50 × 10-1 9.50 × 10-1 9.50 × 10-1
2-copy (normal) c 4.04 × 10-2 4.04 × 10-2 4.04 × 10-2 4.04 × 10-2
1D-copy (disease) d 1.81 × 10-4 1.14 × 10-4 2.08 × 10-4 3.06 × 10-4
Status Genotype§§ Copy Designation General Type I Type II Type III
number population
Noncarrier 2+2 4 c2 1.63 × 10-3 1.63 × 10-3 1.63 × 10-3 1.63 × 10-3
2+1 3 2bc 7.67 × 10-2 7.67 × 10-2 7.67 × 10-2 7.67 × 10-2
1+1 2 b2 9.02 × 10-1 9.02 × 10-1 9.02 × 10-1 9.02 × 10-1

Carrier 2 + 1D 3 2cd 1.46 × 10-5 9.20 × 10-6 1.68 × 10-5 2.47 × 10-5
2+0 2 2ac 7.98 × 10-4 8.04 × 10-4 7.96 × 10-4 7.88 × 10-4
1 + 1D 2 2bd 3.43 × 10-4 2.16 × 10-4 3.95 × 10-4 5.82 × 10-4
1+0 1 2ab 1.88 × 10-2 1.89 × 10-2 1.87 × 10-2 1.85 × 10-2

Affected 1D + 1D 2 d2 3.27 × 10-8 1.30 × 10-8 4.32 × 10-8 9.38 × 10-8

1D + 0 1 2ad 3.57 × 10-6 2.27 × 10-6 4.10 × 10-6 5.98 × 10-6
0+0 0 a2 9.77 × 10-5 9.90 × 10-5 9.72 × 10-5 9.52 × 10-5
§
Relative allele frequencies for each spinal muscular atrophy (SMA) type, which should be used when the risk of a 1D allele is present due to an index case affected with
clinically typical SMA of a given type.
§§
Genotype is indicated by (SMN1 allele on one chromosome 5) + (SMN1 allele on the other chromosome 5).

www.future-drugs.com 23
Ogino & Wilson
has been found to lack both copies of SMN1 [78]. When the
index patient with clinically typical SMA is unavailable for
testing, SMN gene dosage analysis can be performed on the
parents to confirm the presence of deletion/conversion muta-
tions. In the authors experience, however, a deceased index
patient, as well as his or her parents, are occasionally unavailable
for genetic testing when an unaffected relative is referred for
genetic testing. After careful genetic counseling and informed
consent, SMN gene dosage analysis can be offered in such
cases. One reason is that unaffected relatives are more than
occasionally found to have one copy of SMN1, which is of
course readily interpretable. Had testing of such individuals
been denied, important information for family planning would
have been unavailable. Another reason is that when the index
patient and his or her parents are unavailable for testing, other
relatives of the patient are sometimes available and are found to
have only one copy of SMN1.
As previously shown [1], accurate risk assessment in such
cases is possible even when negative results are obtained. One
of the aims of this previous study was to provide versatile
methods for calculating genetic risk in complicated clinical
scenarios, even when genetic information on the index case or
the parents of the index case is limited. SMN gene dosage
analysis and carrier risk assessment are appropriate provided
that all available information is taken into account and pro-
vided that the limitations are explained clearly to those being
tested. One of the limitations is the dependence of the risk
assessment on whether the disease in the affected family mem-
ber is SMN1-related. In general, of course, the more individuals
tested, and the more genetic information that is available, the
more accurate the genetic risk assessment becomes. This prin-
ciple holds true even in the absence of genetic information on
the index case or the parents.
With regard to SMA genetic testing for affected individuals
who do not lack both copies of SMN1, SMN gene dosage
analysis for the detection of heterozygous SMN1 deletion/
conversion should be used with caution and should be
restricted to patients with a high index of clinical suspicion, in
order to ensure a high positive predictive value. This is because
the frequency of 1-copy carriers in the general population
(~2%) is comparable with the fraction of SMA patients who
have a small intragenic mutation in one SMN1 allele (~3.5%
for all SMA types [40]) [1]. Provided that this limitation is fully
understood, SMN gene dosage can be offered in such circum-
stances. Although not a routine practice at this time, the SMN1
alleles without gene deletion/conversion mutations can be
interrogated for small intragenic mutations.
Linkage analysis in combination with SMN gene dosage
analysis is recommended when small intragenic mutations are
present in families, or when two copies of SMN1 are found in
the parent of an affected child who lacks both copies of SMN1
[16,58]. In the former case, linkage analysis can be used to track
the mutation through the family. In the latter case, linkage
analysis can detect crossover events associated with de novo
SMN1 deletions, helping to distinguish the 1 + 1 SMN1
24
genotype (with a low recurrence risk) from the 2 + 0 genotype
(with a high recurrence risk of 1 in 4) [58]. The monosomal
analysis method in combination with dosage analysis, though
less extensively evaluated as yet, can also be used to distinguish
the 1 + 1 genotype from the 2 + 0 genotype [105].
Five-year view
Elucidation of SMA pathogenesis and the precise function of
the SMN protein are highly active areas of investigation. High-
throughput drug screening for SMA is in progress [108]. There is
every reason to believe that in 5 years, our understanding of
SMA pathogenesis will have advanced considerably and
improved therapies may be available. Data on SMA genetics
will also accumulate, serving to improve genetic risk assessment
and our understanding of the molecular evolution of the SMN1
gene and its homolog SMN2. Disease modifying genes and
their polymorphisms will likely be further elucidated. Large
population registries and databases for population-based studies
should aid in testing various hypotheses concerning modifying
factors for SMA.
Diagnostic testing techniques will likely continue to improve,
particularly in terms of cost effectiveness. Monosomal analysis
is a promising technology that obviates the necessity for extensive
dosage analysis and linkage analysis to distinguish the 2 + 0
genotype from the 1 + 1 genotype. Assays to detect various
small intragenic SMN1 mutations may improve and be per-
formed routinely for patients who do not lack both copies of
SMN1. As SMA modifiers are identified, molecular testing for
their presence will likely emerge. Lastly, genetic testing for drug
sensitivity and resistance may be needed in the future as new
pharmaceutical agents become available for SMA patients.
Acknowledgements
We thank the following individuals for their support of various
aspects of our work: DGB Leonard, V Van Deerlin, H Rennert,
TW Prior, PE McAndrew, C Turino, T Smith, K-L Chen, H-J
Dong, S Gao, WJ Ewens, M Loda and CS Fuchs.
Information resources
• GeneTests
www.genetests.org
(Viewed December 2003)
• GeneCards for SMN1
http://bioinfo.weizmann.ac.il/cards-bin/carddisp?SMN1
(Viewed December 2003)
• NiceProt View of Swiss-Prot: SMN, accession number Q16637
http://us.expasy.org/cgi-bin/niceprot.pl?Q16637
(Viewed December 2003)
• Spinal Muscular Atrophy Net
www.affari.com/smanet
(Viewed December 2003)
• Families of Spinal Muscular Atrophy (FSMA)
www.fsma.org
(Viewed December 2003)
Expert Rev. Mol. Diagn. 4(1), (2004)
 Spinal muscular atrophy

Key issues

• Qualitative PCR-restriction fragment length polymorphism is used to detect the homozygous absence of SMN1 exon 7, which is
observed in approximately 94% of clinically typical spinal muscular atrophy (SMA) patients. It is a reasonably sensitive assay, but
cannot detect patients with small intragenic SMN1 mutations.
• Quantitative SMN gene dosage analysis can determine the copy number of SMN1 and SMN2 and is used for SMA carrier testing.
However, it cannot detect a carrier with two SMN1 copies on one chromosome 5 and a deletion/conversion mutation on the other
chromosome 5 (the 2 + 0 SMN1 genotype) or a carrier with a small intragenic mutation. SMN gene dosage analysis can also detect
SMA patients who do not lack both copies of SMN1 exon 7, although it should be used with caution because of a potentially low
positive predictive value if the index of clinical suspicion is low.
• SMN1 has an exceptionally high paternal de novo mutation rate (ν = 2.11 × 10-4).
• Linkage analysis or monosomal analysis can be used in combination with SMN gene dosage analysis in certain clinical scenarios to
distinguish a carrier with the 2 + 0 genotype from a 1 + 1 normal individual who has passed a de novo mutation to the index case.
• Due to the complexity of SMA genetics and the high carrier frequency (1 in 50), genetic counseling and risk assessment are essential
components of SMA genetic testing. Accurate genetic risk assessment is possible if all available family and genetic information is
taken into account. Testing of additional family members may dramatically improve the accuracy of genetic risk calculations;
therefore, testing of additional family members should be suggested and offered when appropriate.

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Sci. 96, 6307–6311 (1999).
• Demonstrated that the translationally silent
single nucleotide polymorphism at position
840 in SMN2 exon 7 is responsible for the
inefficient splicing of the SMN2 transcript.
26 Monani UR, Lorson CL, Parsons DW et al.
A single nucleotide difference that alters
splicing patterns distinguishes the SMA
gene SMN1 from the copy gene SMN2.
Hum. Mol. Genet. 8(7), 1177–1183 (1999).
• Demonstrated that the translationally silent
single nucleotide polymorphism at position
840 in SMN2 exon 7 is responsible for the
inefficient splicing of the SMN2 transcript.
27 Kashima T, Manley JL. A negative element
in SMN2 exon 7 inhibits splicing in spinal
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• Demonstrated that the translationally
silent single nucleotide polymorphism at
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• Excellent overview of SMA.
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• Excellent review of the function of SMN.
31 Meister G, Eggert C, Fischer U.
SMN-mediated assembly of RNPs: a
complex story. Trends Cell Biol. 12(10),
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• Excellent review of the function of SMN.
26
32 Wirth B. An update of the mutation
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• First comprehensive review of SMN1
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33 Wirth B, Hahnen E, Morgan K et al.
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•• Only comprehensive study investigating
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45 Parsons DW, McAndrew PE, Monani UR
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•• Largest study investigating the frequency
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66 Hofmann Y, Lorson CL, Stamm S,
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• Investigated the molecular evolution of
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70 Schrank B, Gotz R, Gunnersen JM et al.
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• Established a mouse model of SMA.
Spinal muscular atrophy
72 Monani UR, Coovert DD, Burghes AH.
Animal models of spinal muscular atrophy.
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73 Hsieh-Li HM, Chang JG, Jong YJ et al.
A mouse model for spinal muscular atrophy.
Nature Genet. 24(1), 66–70 (2000).
• Established a mouse model of SMA.
74 Frugier T, Tiziano FD, Cifuentes-Diaz C
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76 Chan YB, Miguel-Aliaga I, Franks C et al.
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Best practice guidelines for molecular
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79 Melki J, Lefebvre S, Burglen L et al.
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27
Ogino & Wilson
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91 Parsons DW, McAndrew PE, Allinson PS
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92 Ogino S, Gao S, Leonard DG, Paessler M,
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•• Describes methods to detect carriers with
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• Excellent overview of SMA.
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117 Hahnen E, Schonling J,
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 Spinal muscular atrophy

119 Talbot K, Ponting CP, Theodosiou AM
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Affiliations • Robert B Wilson, MD, PhD
• Shuji Ogino, MD, PhD Associate Professor
Associate Pathologist Department of Pathology and
Department of Pathology, Laboratory Medicine,
Brigham and Women’s Hospital University of Pennsylvania Medical Center,
Philadelphia, PA 19104, USA
Staff Research Scientist
Tel.: +1 215 898 0606
Department of Medical Oncology,
Dana–Farber Cancer Institute Fax: +1 215 573 8944
wilsonr@mail.med.upenn.edu
Harvard Medical School,
Boston, MA 02115, USA
Tel.: +1 617 632 3978
Fax: +1 617 632 5762
sogino@partners.org

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