Neurogenetics (2006) 7: 269276

DOI 10.1007/s10048-006-0051-3
SHORT COMMUNI CATION
Oronzo Scarciolla
.
Liborio Stuppia
.
Maria Vittoria De Angelis
.
Stefania Murru
.
Chiara Palka
.
Rossella Giuliani
.
Marta Pace
.
Antonio Di Muzio
.
Isabella Torrente
.
Annunziata Morella
.
Paola Grammatico
.
Manlio Giacanelli
.
Maria Cristina Rosatelli
.
Antonino Uncini
.
Bruno Dallapiccola
Spinal muscular atrophy genotyping by gene dosage using
multiple ligation-dependent probe amplification
Received: 31 May 2006 / Accepted: 7 June 2006 / Published online: 22 July 2006
# Springer-Verlag 2006
Abstract Spinal muscular atrophy (SMA) is an autosomal
recessive disease characterized by degeneration of the
anterior horn cells of the spinal cord, causing symmetric
proximal muscle weakness. SMA is classified in three
clinical types, SMA I, SMA II, and SMA III, based on the
severity of the symptoms and the age of onset. About 95%
of SMA cases are caused by homozygous deletion of the
survival motor neuron 1 (SMN1) gene (5q13), or its
conversion to SMN2. The molecular diagnosis of this
disease is usually carried out by a polymerase chain
reactionrestriction fragment length polymorphism ap-
proach able to evidence the absence of both SMN1 copies.
However, this approach is not able to identify heterozygous
healthy carriers, which show a very high frequency in
general population (1:50). We used the multiple ligation-
dependent probe amplification (MLPA) approach for the
molecular diagnosis of SMA in 19 affected patient and in
57 individuals at risk to become healthy carriers. This
analysis detected the absence of the homozygous SMN1 in
all the investigated cases, and allowed to discriminate
between SMN1 deletion and conversion to SMN2 on the
basis of the size showed by the peaks specific for the
different genes mapped within the SMA critical region.
Moreover, MLPA analysis evidenced a condition of the
absence of the heterozygous SMN1 in 33 out of the 57
relatives of the affected patients, demonstrating the
usefulness of this approach in the identification of healthy
carriers. Thus, the MLPA technique represents an easy, low
cost, and high throughput system in the molecular diag-
nosis of SMA, both in affected patients and in healthy
carriers.
Keywords Spinal muscular atrophy (SMA)
.
MLPA
.
SMN1
.
SMN2
Introduction
Spinal muscular atrophy (SMA) is a genetic disease
characterized by symmetric proximal muscle weakness
due to a degeneration of the anterior horn cells of the spinal
cord. Based on the severity of the symptoms and age of
onset, SMA is classified into three clinical types. Type I
O. Scarciolla
.
L. Stuppia (*)
.
R. Giuliani
Dipartimento di Scienze Biomediche, Sezione di Genetica
Medica, Universit G. dAnnunzio,
Via dei Vestini 35,
Chieti-Pescara, 66013, Italy
e-mail: stuppia@unich.it
Tel.: +39-0871-3554131
Fax: +39-0871-3554135
O. Scarciolla
.
L. Stuppia
.
M. V. De Angelis
.
M. Pace
.
A. Di Muzio
.
A. Uncini
Aging Research Center, CESI,
G. dAnnunzio, University Foundation,
Chieti-Pescara, Italy
O. Scarciolla
Dipartimento di Scienze Cliniche e Bioimmagini,
G. dAnnunzio, University,
Chieti-Pescara, Italy
L. Stuppia
ITOI-CNR, Unit of Bologna, c/o IOR,
Bologna, Italy
M. V. De Angelis
.
M. Pace
.
A. Di Muzio
.
A. Uncini
Department of Oncology, Neurosciences and Interuniversitary
Institute of Myology, G. dAnnunzio, University Foundation,
Chieti-Pescara, Italy
S. Murru
.
M. C. Rosatelli
Instituto di Clinica e Biologia dellEta Evolutiva,
Universit degli Studi,
Cagliari, Italy
C. Palka
.
I. Torrente
.
A. Morella
.
B. Dallapiccola
IRCCS-CSS, San Giovanni Rotondo and CSS-Mendel Institute,
Rome, Italy
P. Grammatico
.
B. Dallapiccola
Genetica Medica Universit La Sapienza,
Azienda Ospedaliera S. Camillo-Forlanini,
Rome, Italy
M. Giacanelli
Neurology, S. Camillo-Forlanini Hospital,
Rome, Italy
[WerdnigHoffman disease, Online Mendelian Inheritance
in Man (OMIM) 253300], the most severe form, is
characterized by muscle weakness and hypotonia within
the first days/months of life, resulting in death before the
age of 2 years. Type II (Dubowitz disease, OMIM 253550)
is characterized by proximal muscle weakness with onset
before the age of 18 months, inability to walk, and survival
beyond 4 years. Type III (KugelbergWelander disease,
OMIM 253400) shows proximal muscle weakness after the
age of 18 months with survival to the adulthood [12].
Based on age of onset, type III can be subclassified as type
IIIa (onset before the age of 3 years) and type IIIb (onset
after 3 years) [23]. SMA is inherited as an autosomal
recessive trait with a prevalence of about 1 in 10,000
newborns and a carrier frequency of 1 in 50 [13]. The three
SMA types result from homozygous mutations of the
survival motor neuron 1 (SMN1) gene, mapped to
chromosome 5q13 [10]. This region also contains SMN2,
which, compared to SMN1, has a few nucleotide changes,
of which only one is functional (840C>T in exon 7) [3].
SMN1 and SMN2 genes are located at the telomeric and
centromeric portions of a large inverted repeat. Despite the
high homology between these two genes, only SMN1 is
necessary for the survival of motor neurons. In about 95%
of patients, the pathogenic mutation consists of homozy-
gous functional absence of SMN1 gene due to deletion or
conversion to SMN2 [2]. The remaining cases are com-
pound heterozygotes for a deletion/conversion of one
SMN1 allele and a small intragenic mutation of the other
allele [22]. Loss of SMN2 is unrelated to the disease, and
deletion of both SMN2 genes occurs in about 510% of
unaffected individuals [7]. Nevertheless, SMN2 is con-
sidered a disease-modifying gene because its copy number
relates with the disease severity and survival of affected
patients [2, 5, 11, 22].
The standard molecular diagnosis of SMA is based on a
polymerase chain reactionrestriction fragment length
polymorphism (PCR-RFLP) test, which is suitable for
detecting homozygous SMN1 loss [19]. However, this
method does not detect heterozygous SMN1 loss, and
cannot be used for identifying healthy carriers, which can
be checked by linkage [21] or quantitative analysis of
SMN1 copy number. Several additional techniques were
proposed for the identification of SMA healthy carriers,
including LightCycler PCR [5], TaqMan Technology [1],
and denaturing high-performance liquid chromatography
[17]. More recently, the multiple ligation-dependent probe
amplification (MLPA) assay has proved to be an efficient
tool for detecting deletions and duplications of different
genes, such as BRCA1 [8, 9], CMT [16], and DMD [6].
This assay provides the unique ability to hybridize several
probes specific for the target region and control sequences
in a single experiment.
In this study, we report on the use of MLPA assay for
genotyping SMA on affected individuals and healthy
carriers.
Materials and methods
Patients
Seventy-six subjects entered this study (patients 176).
They included 19 patients (patients 119) in which the
diagnosis of SMAwas performed according to Munsta and
Davies [12]. Based on age onset and clinical features, 4
patients were classified as SMA I (patients 14), 2 as SMA
II (patients 5 and 6), 2 as SMA IIIa (patients 7 and 8), and
11 as SMA IIIb (patients 919). In all cases, the loss of both
SMN1 copies had been previously established by PCR-
RFLP [19]. The remaining 57 subjects were relatives of
these patients or individuals at risk for SMA, being first-
degree relatives of patients not included in the present
study (patients 2076). In 35 of these individuals, their
carrier status had been assessed previously by real-time
PCR. Ten healthy subjects (individuals showing two copies
each of SMN1 and SMN2 genes at real-time analysis) were
used as controls.
Real-time analysis
Real-time analysis was performed using TaqMan technol-
ogy (ABI Prism 7000) and the comparative method of
Ct [18]. TaqMan MGB probe and primers used in this
study were as previously described [1]. RNAse P was
employed as endogenous control gene. PCR was carried
out using an ABI Prism 7000 sequence detection system
and 96-well microamp optical plates (Applied Biosystems).
Each DNA sample was analyzed in triplicate and the mean
of triplicates was used in each calculation. The same DNA
sample of a carrier individual (one SMN1 gene copy) was
used as calibrator in all amplification reactions.
MLPA reaction
MLPA analysis [15] was carried out according to the
manufacturers recommendations (MRC-Holland, Amster-
dam, The Netherlands) using the SALSA Probe Mix 021.
This mix contains 16 probes specific for the SMA critical
region (5q12.2q13.3), namely, 1 probe each for exons 1,
4, 6, and 8 of SMN1 and SMN2 (probes SMN-D04, SMN-
D05, SMN-D06, and SMN-D03); 2 probes for the C-to-T
transition in exon 7 (SMN-D01a for SMN1 and D01b for
SMN2); 2 probes for the G-to-A transition in exon 8
(SMN-D07a for SMN1 and SMN-D07b for SMN2); 2
probes for BIRC1 (NAIP) gene (BIRC1-D01 specific for
both NAIP and NAIP genes, and BIRC1-D02 specific for
NAIP gene only); 3 probes for GTF2H2 gene; 1 probe
for N-cadherin-like-gene; 1 probe for CDH6 (K-cadherin)
gene; and 1 probe for RAD17 gene. In addition, the SALSA
Probe Mix 021 contains 21 control probes mapping to other
autosomes.
270
MLPA analysis
After MLPA treatment, samples were run on an ABI
PRISM 3100 Genetic Analyzer and analyzed using Gene
Mapper version 3.5 software. For each sample, relative
peak area (RPA) was calculated and compared to five
normal controls using the Coffalyzer version 2 software
(MRC-Holland). This software is able to calculate the RPA
for each probe within the same test and to compare each
RPA to those derived from five normal controls to obtain
the ratio peak area for each probe. In the Coffalyzer
analysis, a peak size indicates a normal copy number when
showing a 0.71.3 ratio compared to normal controls; a
deletion when showing a ratio <0.7; a gain when showing a
ratio >1.3; and an absence when showing a ratio equal to 0.
Based on this correlation, a normal peak indicates the
presence of two gene copies, except for probes SMN-D04,
D05, D06, and D03, which, when showing normal peaks,
indicate the presence of four SMN1+SMN2 gene copies,
and probe BIRC-D01, which indicates four NAIP+NAIP
gene copies.
Using this approach, normal SMN1 copy number (two
copies) was expected to produce normal peaks for the
probes SMN-D01a and D07a, while homozygous SMN1
loss (SMA-affected patients) was expected to cause the
absence of these peaks. In this latter case, analysis of SMN-
D04, D05, D06, and D03 probes, specific for both SMN1
and SMN2 genes, was used to distinguish between SMN1
deletion and SMN1 conversion to SMN2. In fact, a deletion
of these four peaks, together with normal SMN-D01b and
D07b probes specific for SMN2, was expected in the case
of homozygous SMN1 deletion with the presence of two
SMN2 copies; a deletion of the four peaks, together with a
gain of the SMN-D01b and D07b peaks, was expected in
the case of SMN1 deletion in one allele and SMN1
conversion in the other; the detection of normal peaks of
the four probes, together with a gain of the D01b and D07b
peaks, was expected in the case of homozygous SMN1
conversion to SMN2.
For healthy carriers, the presence of a single SMN1 copy
was expected to produce a deletion of the peaks for D01a
and D07a probes, together with either a deletion of the
D04, D05, D06, and D03 peaks (SMN1 deletion), or their
normal size, together with a gain of D01b and D07b peaks
(SMN1 conversion to SMN2).
To improve genotyping of the rearranged SMA critical
region, the peaks of probes specific for NAIP-NAIP genes
(BIRC1-D01 and BIRC1-D02) were evaluated. Because
BIRC-D02 probe is specific for NAIP while BIRC-D01
probe recognizes both NAIP and NAIP genes, the absence
of the D02 peak in the presence of a deleted D01 peak was
interpreted as an extension of a homozygous SMN1
deletion into NAIP in the presence of two NAIP copies.
Each result obtained by MLPA analysis was confirmed
by three independent experiments. Real-time and MLPA
experiments were blindly carried out in two independent
laboratories.
Results
Normal controls
All healthy controls showed normal-sizes peaks with a
mean ratio of 1.00 (range 0.801.24, SD=0.107), confirm-
ing the range of normal size assumed on the basis of the
Coffalyzer program. This range indicates the presence of
two copies for each gene, except for probes SMN-D03,
D04, D05, D06, and BIRC-D01 whose normal range
indicates the presence of four gene copies as described in
the Materials and methods. The mean ratio, range, and
standard deviation in normal controls for the probes
specific for the SMA critical region and for control probes
are reported in Table 1.
Affected patients
All the 19 affected patients disclosed the absence of the
SMN-D01a and D07a peaks with a ratio equal to 0,
indicating the loss of both SMN1 copies.
All the other probes specific for the SMA critical region
showed mean ratios and ranges different from those
evidenced in the group of healthy controls. In fact, the
SMN-D01b and D07b probes, specific for SMN2 gene,
showed a mean ratio of 1.36 (range 0.821.93, SD=0.484)
and 1.40 (range 0.971.86, SD=0.407), respectively. Using
the values observed in the control group as a reference,
peaks with ratio >0.80 and <1.24 were interpreted with the
presence of two SMN2 copies, while peaks showing a ratio
>1.24 were interpreted with the presence of a gain in the
SMN2 copy number. In particular, a ratio >1.24 and <1.60
was interpreted with the presence of three SMN2 copies
while a ratio >1.60 was interpreted with the presence of
four SMN2 copies according to the recommendations of the
manufacturer of MLPA (MRC-Holland). In all cases, the
SMN2 copy number derived by the analysis of the SMN-
D01b probe was the same as showed by the SMN-D07b
probe.
Probes SMN-D03, D04, D05, and D06 showed a mean
ratio of 0.74 (range 0.480.99, SD=0.224), 0.80 (range
0.431.15, SD=0.325), 0.82 (range 0.521.13, SD=0.284),
and 0.84 (range 0.461.17, SD=0.330), respectively. Using
again the values observed in the control group as a
reference, a ratio >0.80 and <1.24 was interpreted with the
presence of four normal SMN1+SMN2 copy numbers,
while a ratio <0.80 was interpreted with the presence of a
reduced SMN1+SMN2 copy number. Because all patients
of this group had loss of both SMN1 copies, the SMN1
+SMN2 copy number actually indicates the SMN2 copy
number only. Thus, by comparison with the SMN2 copy
number established by the analysis of probes SMN-D01b
and D07b as a reference, it was possible to evidence that a
ratio >0.60 and <0.80 corresponds to three copies for
probes SMN-D03, D04, D05, and D06, a ratio >0.42 and
<0.60 to two copies, and a ratio >0 and <0.42 to one copy.
271
Finally, the BIRC-D01 and D02 probes, specific for
NAIP and NAIP genes, showed a mean ratio of 0.70
(range 0.470.99, SD=0.198) and 0.38 (range 00.91,
SD=0.389), respectively. Results showed by the BIRC-
D01 probe were interpreted using the same value ranges
observed for the SMN-D03, D04, D05, and D06 probes;
while in the case of the BIRC-D02 probe, cases with a ratio
equal to 0 were interpreted with the absence of any NAIP
copy, cases with ratio >0 and <0.80 with presence of a
single NAIP copy, and cases with ratio >0.80 with the
presence of two NAIP copies.
Control probes showed in the group of SMA patients a
mean ratio of 1.01 (range 0.751.25, SD=0.124), very
similar to the one detected in the group of healthy controls.
The mean ratios, ranges, and standard deviations showed
by all probes in the group of SMA-affected patients are
reported in Table 2.
Based on the described calculation above on the SMN2,
NAIP, and NAIP gene copies, MLPA analysis allowed us
to characterize the SMN1 alteration evidenced in all the
affected patients. In fact, three patients (patients 2, 4, and 5)
showed two SMN2 copies, absence of NAIP, and two
NAIP copies. This pattern corresponds to a homozygous
SMN1 deletion. Two cases (patients 1 and 15) had two
SMN2 copies and one copy each of the NAIP and NAIP
genes. This pattern was taken as an evidence for deletion in
one allele (involving SMN1, SMN2, NAIP, and NAIP) and
conversion in the other allele. Six patients (patients 3, 6, 7,
10, 12, and 18) had three SMN2 copies, one copy of NAIP,
and two copies of NAIP, a pattern consistent with deletion
in one allele (involving SMN1 and NAIP, but not SMN2 and
NAIP) and conversion in the other one. Seven patients
(patients 8, 9, 11, 13, 14, 16, and 19) had four SMN2 copies
and two copies each of NAIP and NAIP, in agreement
with a homozygous SMN1 conversion to SMN2. Finally,
patient 17 disclosed four SMN2 copies, no NAIP copy, and
three NAIP copies, a pattern consistent with a complex
rearrangement of the SMA region, likely a homozygous
SMN1 deletion with four SMN2 copies, including two
inherited from one parent together with two NAIP copies,
and the chromosome inherited from the other parent
carrying a duplication of a single SMN2 copy together with
a single NAIP copy. However, because the extent of
deletions or gene conversions is not always the same and
the orientation of NAIP and SMN copies in the repeated
segment is not always identical, another mechanism
leading to the above described patterns cannot be ruled out.
The SMN1, SMN2, NAIP, and NAIP copy number in
the SMA patients are summarized in Table 3.
Table 2 Mean ratio, range, and standard deviation showed by each probe specific for the SMA critical region and by control probes in the
group of SMA-affected patients after MLPA analysis
Probe Gene Mean ratio Range Standard deviation Gene copy number
SMND01a SMN1 0 0 0 0
SMN-D07a SMN1 0 0 0 0
SMN-D01b SMN2 1.36 0.821.93 0.484 24
SMN-D07b SMN2 1.40 0.971.86 0.407 24
SMN-D03 SMN1+SMN2 0.74 0.480.99 0.224 24
SMN-D04 SMN1+SMN2 0.80 0.431.15 0.325 24
SMN-D05 SMN1+SMN2 0.82 0.521.13 0.284 24
SMN-D06 SMN1+SMN2 0.84 0.461.17 0.330 24
BIRC1-D01 NAIP+NAIP 0.70 0.470.99 0.198 24
BIRC1-D02 NAIP 0.38 00.91 0.389 02
Control probes 1.01 0.751.25 0.124
Table 1 Mean ratio, range, and standard deviation showed by each probe specific for the SMA critical region and by control probes in the
group of normal controls after MLPA analysis
Probe Gene Gene copy number Mean ratio Range Standard deviation
SMN-D01a SMN1 2 0.96 0.841.06 0.089
SMN-D07a SMN1 2 1.02 0.891.13 0.085
SMN-D01b SMN2 2 0.98 0.831.09 0.090
SMN-D07b SMN2 2 1.01 0.941.10 0.057
SMN-D03 SMN1+SMN2 4 1.00 0.921.09 0.062
SMN-D04 SMN1+SMN2 4 1.06 0.941.19 0.116
SMN-D05 SMN1+SMN2 4 1.02 0.861.17 0.131
SMN-D06 SMN1+SMN2 4 1.01 0.841.15 0.136
BIRC1-D01 NAIP+NAIP 4 1.02 0.901.15 0.099
BIRC1-D02 NAIP 2 0.99 0.881.16 0.108
Control Probes 1.00 0.801.24 0.112
272
Subjects at risk to be carriers
MLPA analysis of SMN-D01a and D07a probes in the 57
relatives of SMA patients showed a mean ratio of 0.87
(range 0.481.58, SD=0.402) and 0.84 (range 0.391.46,
SD=0.375). Using the range values observed in healthy
control as a reference, we assumed that subjects showing a
ratio >0.80 and <1.24 had two SMN1 copies, those
showing a ratio >1.24 had three SMN1 copies, and those
showing a ratio >0 and <0.80 had one SMN1 copy. On this
basis, 33 subjects (58%) had a single SMN1 functional
copy, supporting the status of healthy carrier (patients 21
53). In 23 subjects (40%), MLPA analysis disclosed either
two (18 cases) or three (5 cases) SMN1 copies, excluding
their carrier status (patients 5476). Finally, in patient 20,
the 18-year-old sister of a SMA IIIb patient aged 17 years
at diagnosis (patient 13), MLPA analysis disclosed the
absence of both SMN1 copies, thus showing that the girl
who was still asymptomatic at time of analysis, was in fact,
affected.
In the 35 individuals analyzed also by real-time PCR, the
two techniques provided overlapping results in the
estimation of the SMN1 and SMN2 genes copy number.
MLPA histograms, illustrating different patterns of homo-
zygous wild type, homozygous SMN1 loss, and heterozy-
gous healthy carrier, are shown in Fig. 1.
Discussion
SMA is one of the most common lethal Mendelian
disorders of children with a high frequency of healthy
carriers in the general population. The identification of
these subjects is critical for genetic counseling. The PCR-
RLFP technique currently used for diagnosing the homo-
zygous loss of SMN1 is unable to identify the heterozygous
carriers. Therefore, several protocols based on the quan-
titative analysis of SMN1 copy number were developed [1,
5, 11, 14, 17, 20]. In the present study, we investigated the
ability of the MLPA technique to genotype SMA patients
and healthy carriers. MLPA analysis confirmed the loss of
homozygous SMN1 previously detected by PCR-RFLP in
the 19 affected patients and in the presymptomatic sister of
a SMA IIIb patient. SMA IIIb can manifest a late onset,
despite the loss of homozygous SMN1 [4]. In fact, the
affected brother of this girl registered the onset of SMA at
age of 17. Both sibs had four SMN2 copies and two NAIP
and NAIP copies, proving that these genes are modulating
the disease phenotype.
Although SMN2 and NAIP genes are not directly
involved in the pathogenesis of SMA, their analysis can
differentiate in cases arising from homozygous SMN1
deletion, those due to deletion in one allele and conversion
of the other, and in cases caused by homozygous conver-
sion [2]. Thus, different from PCR-RLFP method, which is
disclosing only the absence of SMN1, MLPA analysis also
detects the mechanism responsible for SMN1 loss. Evalua-
tion of SMN2 and NAIP copy number is also useful for
establishing genotypephenotype correlation in SMA
patients based on the evidence that the SMN2 and NAIP
copy number relates with severity of the disease [13]. In the
present series, all patients affected either by SMA I or SMA
II showed two or three copies of SMN2 and two or three
copies of NAIP+NAIP. On the other hand, SMA III
patients showed three or four copies of SMN2 and three or
four copies of NAIP+NAIP with the only exception of one
case displaying only two copies of SMN2 and one copy
each of NAIP and NAIP. These results confirm that in the
Table 3 SMN1, SMN2, NAIP, and NAIP copy numbers as evidenced by MLPA analysis in SMA patients
Patient Clinical Diagnosis SMN1 SMN2 NAIP NAIP
1 SMA-I 0 2 1 1
2 SMA-I 0 2 0 2
3 SMA-I 0 3 1 2
4 SMA-I 0 2 0 2
5 SMA-II 0 2 0 2
6 SMA-II 0 3 1 2
7 SMA-IIIa 0 3 1 2
8 SMA-IIIa 0 4 2 2
9 SMA-IIIb 0 4 2 2
10 SMA-IIIb 0 3 1 2
11 SMA-IIIb 0 4 2 2
12 SMA-IIIb 0 3 1 2
13 SMA-IIIb 0 4 2 2
14 SMA-IIIb 0 4 2 2
15 SMA-IIIb 0 2 1 1
16 SMA-IIIb 0 4 2 2
17 SMA-IIIb 0 4 0 3
18 SMA-IIIb 0 3 1 2
19 SMA-IIIb 0 4 2 2
20 Healthy carrier 0 4 2 2
273
Fig. 1 Histograms showing results of Coffalyzer analysis in five
SMA-affected patients (patients 1, 3, 8, and 17), five healthy carriers
(patients 21, 22, 36, 41, and 45), and five normal subjects (patients
54, 57, 64, 73, and 75). Numbers on the left of each histogram
indicates the ratio peak area. The relative SMN1, SMN2, NAIP, and
NAIP copy numbers are reported in Table 3
274
majority of SMA patients the lower the copy numbers of
SMN2, NAIP, and NAIP genes, the higher the severity of
disease. However, because this correlation is not present in
all cases, care should be used when applying this kind of
analysis for genotypephenotype correlation in clinical
practice (e.g., in prenatal diagnosis).
A second aim of this study was the identification of
SMA healthy carriers using MLPA assay. To this purpose,
we analyzed 57 first-degree relatives of SMA-affected
patients. In 33 cases MLPA analysis disclosed a single
SMN1 copy, supporting their carrier status. In 23 cases the
number of SMN1 copies was normal, suggesting a wild-
type phenotype. The last case was the presymptomatic
sister of a SMA IIIb patient, described above, in which
MLPA disclosed a homozygous SMN1 loss. To validate the
MLPA results, the carrier status in 35 of the 57 subjects was
also investigated by real-time PCR using TaqMan tech-
nology. In all cases, both techniques disclosed the same
copy number of SMN1 and SMN2 genes, confirming that
MLPA assay has the same sensitivity and specificity of
other already validated techniques.
Based on these results, we suggest that MLPA analysis
could represent the gold standard technique in the molec-
ular diagnosis of SMA, presenting several advantages
compared to other methods. In fact, MLPA is able to
provide information about SMN1, SMN2, and NAIP genes
in a single reaction, providing an easy, fast, and high
throughput system for analyzing the SMA critical region
both in affected patients and in healthy carriers. The
simultaneous analysis of different sequences within and
outside the SMA critical region provides an accurate
control system which lessens the risk of false positive and
false negative results, the identification of molecular
organization of the SMA critical region being determined
through the analysis of different genes. Moreover, the use
of at least 20 ng of DNA for the MLPA reaction reduces the
risk of DNA contamination, which is prevented in the
common PCR reactions. Similar to other gene dosage
techniques, MLPA is unable to detect SMN1 point
mutations and to disclose the presence of two SMN1
copies in the same allele. However, these conditions can
account for less than 5% of SMA cases.
In conclusion, MLPA analysis proved to be an excellent
tool for the molecular diagnosis and characterization of
different forms of SMA. This approach could become a
routine analysis in the genetic testing of this disease and an
accurate method for the identification of healthy carriers,
which, because of their prevalence, are recommend for
population screening.
Acknowledgments Oronzo Scarciolla and Liborio Stuppia con-
tributed equally to this work.
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