SMN transcript levels in leukocytes of SMA patients determined by absolute real-time PCR

Article · August 2009with40 Reads
DOI: 10.1038/ejhg.2009.116 · Source: PubMed
Abstract
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous mutations of the SMN1 gene. Three forms of SMA are recognized (type I-III) on the basis of clinical severity. All patients have at least one or more (usually 2-4) copies of a highly homologous gene (SMN2), which produces insufficient levels of functional SMN protein, because of alternative splicing of exon 7. Recently, evidence has been provided that SMN2 expression can be enhanced by pharmacological treatment. However, no reliable biomarkers are available to test the molecular efficacy of the treatments. At present, the only potential biomarker is the dosage of SMN products in peripheral blood. However, the demonstration that SMN full-length (SMN-fl) transcript levels are reduced in leukocytes of patients compared with controls remains elusive (except for type I). We have developed a novel assay based on absolute real-time PCR, which allows the quantification of SMN1-fl/SMN2-fl transcripts. For the first time, we have shown that SMN-fl levels are reduced in leukocytes of type II-III patients compared with controls. We also found that transcript levels are related to clinical severity as in type III patients SMN2-fl levels are significantly higher compared with type II and directly correlated with functional ability in type II patients and with age of onset in type III patients. Moreover, in haploidentical siblings with discordant phenotype, the less severely affected individuals showed significantly higher transcript levels. Our study shows that SMN2-fl dosage in leukocytes can be considered a reliable biomarker and can provide the rationale for SMN dosage in clinical trials.
4 Figures
ARTICLE
SMN transcript levels in leukocytes of SMA patients
determined by absolute real-time PCR
Francesco Danilo Tiziano*,1, Anna Maria Pinto1, Stefania Fiori1, Rosa Lomastro1, Sonia Messina2,3,
Claudio Bruno4, Antonella Pini5, Marika Pane2, Adele D’Amico6, Alessandro Ghezzo5, Enrico Bertini6,
Eugenio Mercuri2, Giovanni Neri1and Christina Brahe1
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous mutations of the
SMN1 gene. Three forms of SMA are recognized (type I–III) on the basis of clinical severity. All patients have at least one or
more (usually 2–4) copies of a highly homologous gene (SMN2), which produces insufficient levels of functional SMN protein,
because of alternative splicing of exon 7. Recently, evidence has been provided that SMN2 expression can be enhanced by
pharmacological treatment. However, no reliable biomarkers are available to test the molecular efficacy of the treatments. At
present, the only potential biomarker is the dosage of SMN products in peripheral blood. However, the demonstration that SMN
full-length (SMN-fl) transcript levels are reduced in leukocytes of patients compared with controls remains elusive (except for
type I). We have developed a novel assay based on absolute real-time PCR, which allows the quantification of SMN1-fl/SMN2-fl
transcripts. For the first time, we have shown that SMN-fl levels are reduced in leukocytes of type II–III patients compared with
controls. We also found that transcript levels are related to clinical severity as in type III patients SMN2-fl levels are significantly
higher compared with type II and directly correlated with functional ability in type II patients and with age of onset in type III
patients. Moreover, in haploidentical siblings with discordant phenotype, the less severely affected individuals showed
significantly higher transcript levels. Our study shows that SMN2-fl dosage in leukocytes can be considered a reliable biomarker
and can provide the rationale for SMN dosage in clinical trials.
European Journal of Human Genetics advance online publication, 15 July 2009; doi:10.1038/ejhg.2009.116
Keywords: spinal muscular atrophy; real-time PCR; biomarker; SMN; transcripts
INTRODUCTION
Proximal spinal muscular atrophies (SMA) are a group of clinically
variable motor neuron disorders characterized by the degeneration of
the anterior horn cells of the spinal cord. On the basis of age of onset
and severity of the clinical course, childhood-onset SMA can be
classified into three forms (type I–III). SMA III patients can be further
divided into type IIIa and IIIb on the basis of whether the onset is
below or over the age of 3 years, respectively.1SMAI-III are autosomal
recessive, and are caused by loss of function of the survival motor
neuron (SMN1) gene.2SMN1 and a nearly identical copy, SMN2,are
located in a duplicated inverted region at 5q13. Both genes encode the
SMN protein but, because of alternative splicing, the majority of
SMN2 transcripts lack exon 7 (SMN-delta7), and are unable to
produce a sufficient amount of protein to prevent the onset of the
disease. The SMN protein is expressed in most tissues and is localized
in the cytoplasm and in the nucleus. It has been shown that the level of
SMN protein is markedly reduced in SMA patients, both in spinal cord
and in cell cultures and inversely correlate with phenotypic severity.3–5
Patients can carry a variable copy number of the SMN2 gene, higher
copy numbers being generally associated with milder phenotypes.6–8
At present, no cure for SMA is available. Recently, evidence has been
provided that SMN2 gene expression can be enhanced by pharmaco-
logical treatment in vivo and/or in vitro, using different compounds.9–
17 The clinical efficacy of some of these compounds has been tested
also in clinical trials.18–21
The advances in SMA clinical research highlight the need of reliable
biomarkers to monitor the efficacy at the molecular level of treatments
during trials, the dosage of SMN transcripts or protein in peripheral
blood samples being the only one potentially available. However,
possible variations of SMN transcripts/protein levels as evaluated in
leukocytes may not reflect the real effect of pharmacological treatment
in target tissues, such as spinal cord and, eventually, skeletal muscle.
So far, some assays have been developed and validated for SMN2
transcript14,22–24 or protein25–26 quantification. However, to date it
has not been shown whether SMN full-length (SMN-fl) transcript
or protein levels in leukocytes differ significantly among controls,
carriers, and patients. In particular, a reduction of SMN-fl levels has
been shown only for type I patients.14,22 The reported SMN mRNA
assays are mainly based on relative semiquantitative PCR in which
transcript levels are determined by normalizing with respect to house-
keeping gene transcript levels, used as endogenous controls.22–24 How-
ever, it has been shown that the expression levels of these genes vary
widely in the general population and/or can be putatively affected by
pharmacological treatments or metabolic status, thus reducing the
Received 22 September 2008; revised 19 May 2009; accepted 5 June 2009
1Institute of Medical Genetics, Catholic University, Rome, Italy; 2Institute of Neurology, Catholic University, Rome, Italy; 3Institute of Neurology, University of Messina, Messina,
Italy; 4Neuromuscular Disease Operative Unit, ‘G. Gaslini’ Institute, Genova, Italy; 5Ospedale Maggiore, Bologna, Italy; 6Molecular Medicine Unit, Bambino Gesu
`Hospital, Rome,
Italy
*Correspondence: Dr FD Tiziano, Institute of Medical Genetics, Catholic University, Largo Francesco Vito, 1, 00168 Roma – Italy.
Tel: +390 6355 00877; Fax: +390 6305 0031; E-mail: fdtiziano@rm.unicatt.it
European Journal of Human Genetics (2009), 1–7
&
2009 Macmillan Publishers Limited All rights reserved 1018-4813/09 $32.00
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sensitivity of the earlier published assays.14 Brichta et al14 have devel-
oped a real-time PCR assay based on the measurement of SMN levels
relative to the amount of RNA. We have developed an alternative
molecular test based on absolute real-time PCR that allows the
quantification of the number of SMN1-fl and SMN2-fl mRNA mole-
culespernanogramoftotalRNA(mol/ng)andissuitableformeasuring
SMN transcripts, thus avoiding possible biases because of the variations
in endogenous control transcript levels. In our assay, GAPDH transcripts
quantification has been included as positive control for PCR amplifica-
tion and to rule out that possible differences between patients and
controls could be related to PCR efficiency or RNA quality (see
Supplementary information). We have used this novel assay to inves-
tigate whether SMN-fl transcript levels are reduced in patients compared
with controls, and to assess whether transcript levels correlate with
phenotypic severity in patients, which are the prerequisites for using
SMN dosage as a biomarker for future clinical trials in SMA patients.
SUBJECTS AND METHODS
Subjects
Blood samples were obtained from 51 SMA patients (2 type I, 16 type II, and 33
type III), 23 carriers, and 28 controls. The characteristics of age and sex ratio of
the three groups are summarized in Table 1. All patients had homozygous
absence of SMN1 exon 7. Type III SMA patients have been subgrouped
according to the classification proposed by Zerres1in type IIIa and IIIb. For
type II patients between 2.5 and 12 years of age, functional ability was evaluated
using the Hammersmith functional motor scale.27
Among patients, six sib pairs were analyzed: five pairs were phenotypically
discordant (type IIIa sister/II brother; type IIIa/II sisters; asymptomatic/IIIb
sisters; type IIIb brother/IIIa sister; oligosymptomatic/type IIIb brothers), and
one further pair of sibs who were phenotypically similar (type IIIb brother and
sister). Carriers were either parents of patients or were selected among relatives
of patients or other relatives who tested positive for carrier status. For patients
and carriers, both total RNA and genomic DNA were extracted. Controls were
healthy individuals, seen at the Genetics Clinic of the Catholic University
Hospital for karyotype analysis. Blood samples from controls were rendered
anonymous and used for RNA extraction only. Individuals taking drugs known
to modify SMN expression were excluded from our cohorts. For repetitive
samplings, blood samples were drawn approximately at the same hour during
the morning, to rule out possible biases because of circadian variations in SMN/
GAPDH transcript levels or in feeding. Finally, four fibroblast cell cultures were
analyzed in this study, one from a control and three from patients (one for each
type of SMA).
DNA extraction, SMN2 gene copy number assessment, RNA extraction, and
RT-PCR. The DNA, extracted by the standard salting-out procedures, was
quantified by absorbance at 2 60 and 280 nm (GeneQuant Pro, Pharmacia
Biotech, Arlington Heights, IL, USA). SMN2 gene copy number, as well as
carrier status, was determined as reported earlier.12 For RNA extraction from
peripheral blood, PAXgene blood RNA tubes (BD Biosciences, San Jose, CA,
USA) and kit (Qiagen) were used. In the case of fibroblast cultures, total RNA
was extracted by RNeasy mini kit (Qiagen, Duesseldorf, Germany). For all RNA
samples, concentration was established by absorbance determination and quality
was assessed by agarose gel electrophoresis. A total of 2 mg of total RNA were
used for RT-PCR using a High Capacity cDNA Archive Kit (Applied Biosystems,
Carlsbad, CA, USA) in a final react ion volume of 25 ml, using random primers
for reverse transcription.
External standard constructs design. Three plasmids were constructed for
SMN1-fl, SMN2-fl, and GAPDH, respectively, by amplifying a control cDNA.
SMN1 and SMN2 were amplified by using the primer pair: SMN_exst-F:
5¢-GCTTTGGGAAGTATGTTAATTTCA-3¢and SMN_exst-R: 5¢-CTATGCCA
GCATTTCTCCTTAATT-3¢, located in exon 6 and exon 7/8 junction, respec-
tively. For GAPDH, primer pair GAPDH_exst-F: 5¢-CTCTGCTG ATGCCCCC
ATG T TC G T- 3 ¢and GAPDH_exst-R: 5¢-CAAAGTTGTCATGGATGACCTTGG-
3¢, located in exon 5 and exon 6, respectively, was used. For SMN1/SMN2 and
GAPDH genes, PCR products of 129 and 133bp, respectively, were obtained.
Subsequently, PCR products were cloned by using TA cloning kit (Qiagen).
Plasmid DNA was extracted by the QIAprep Spin Miniprep Kit (Qiagen) and
Table 1 Characteristics of each group included in this study (number of individuals, age range, sex ratio, and transcript levels)
Controls Carriers SMA patients
Type I Type II Type III Total
n (M/F) 28 (14/14) 23 (9/14) 2 (1/1) 16 (8/8) 33 (16/17) 51 (25/26)
Age range (years) 19–45 18–73 0.5–1.5 3–33 2–68 0.5–68
SMN1-fl
Mean±SD 78.27±50.61 72.28±36.72 —
Median 65.13 68.88
Min–max 28.25–217.25 18.05–178.00
SMN2-fl
Mean±SD 41.65±25.62 58.73±41.63 37.13±4.77 52.50±24.78 73.67±29.68 67.33±29.36
Median 37.75 47.50 37.13 47.50 72.50 61.25
Min–max 9.95–100.75 13.08–209.50 33.75–40.50 26.75–102.75 28.50–123.00 26.75–123.00
SMN-fl
Mean±SD 119.92±72.98 127.44±67.28 —
Median 107.63 120.88
Min–max 39.45–318.00 43.55–323.75
GAPDH
Mean±SD 5714±2929 5121±1566 5412±760 4879±1820 5392±1469 5227±1552
Median 4725 5100 5412 4500 5600 5225
Min–max 1092–13025 2975–8975 4875–5950 2500–8925 2310–7925 2310–8925
SMN-fl, SMN1-fl, SMN2-fl, and GAPDH indicate transcript levels, measured as no. of molecules per nanogram of total RNA.
SMN full-length mRNA in SMA patients
FD Tiziano et al
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European Journal of Human Genetics
quantified both by absorbance and by agarose gel electrophoresis with scaling
serial dilution of lambda DNA. The presence of possible sequence variations in
plasmids, randomly introduced by Taq polymerase, was ruled out by sequence
analysis of the clones, performed using the ABI-Prism 3130 instrument and
BigDye terminator v3.1 Cycle Sequencing kit (Applied Biosystems). On the
basis of plasmid length (3980 and 3984bp for SMN1/SMN2 and GAPDH,
respectively), the molecular weight and the number of plasmid molecules per
nanogram of DNA (around 2.29108/ng for the three plasmids) were deter-
mined. Single use serial dilutions of the three external standards, ranging from
104to 107molecules, were aliquoted and kept frozen at 801C.
Primers and MGB-probes. Primer Express v1.5 software (Applied Biosys-
tems) was used to design optimized minor groove binder (MGB) probes and
primers for real-time RT-PCR. SMN1 and SMN2-fl transcripts were amplified
by using the same primer pair (SMN_abs-F: 5¢-TACATGAGTGGCTATCA
TACTGGCTA-3¢and SMN_abs-R: 5¢-AATGTGAGCACCTTCCTTCTTTTT-3¢,
located in exons 6 and 7, respectively), obtaining 72 bp PCR products. Full-
length transcripts of the two genes were specifically distinguished by two
different Taqman MGB probes, labeled with different fluorochromes, on the
basis of the C-T transition located in exon 7 (SMN1_abs: 5¢-NED-
TATGGGTTTCAGACAAA-NFQ-3¢and SMN2_abs: 5¢-VIC-ATATGGGTTT
TAGACAAAA-NFQ-3¢). For GAPDH, an amplicon of 73 bp was obtained by
using the primer pair GAPDH_abs-F: 5¢-GGGTGTGAACCATGAGAAGTAT
GA-3¢and GAPDH_abs-R: 5¢-CTAAGCAGTTGGTGGTGCAGG-3¢.MGB
probe sequence was: 5¢-FAM-CAAGATCATCAGCAATGC-NFQ-3¢.
Real-time PCR assay and construction of standard curves. The PCR
reactions were performed in a final volume of 20ml, containing
2Taqman Universal Mastermix (Applied Biosystems), 40 ng of cDNA (or
appropriate dilutions of external standards), appropriate concentrations of
SMN primers, SMN1 and SMN2 probes, or of GAPDH primers and probe.
Each sample was amplified in quadruplicate and each experiment repeated at
least twice. All reactions were performed using 7900HT Fast Real-Time PCR
System (Applied Biosystems). Optimal primer and probe concentrations were
as follows: SMN_abs_F and SMN_abs_R: 900nM;GAPDH_abs_F
and GAPDH_abs_R: 30 nM; SMN1_abs, SMN2_abs, GAPDH_abs: 200nM.
Serial dilutions of external standards, ranging from 104to 107copies were
used to construct the standard curves. We did not use plasmid concentrations
lower than 104copies because of the poor stability of such dilutions. The
number of SMN1-fl, SMN2-fl, and GAPDH mRNA molecules was extrapolated
automatically by the Sequence Detection System v2.2.2 software (Applied
Biosystems).
Statistical analysis. Statistical analysis was performed by using
Statgraphics-Centurion XV.II (Statpoint Technologies, Warrenton, VA, USA)
software. The experimental variability and the reproducibility of the real-time
PCR assay were assessed by determining the mean and standard deviation (SD)
of coefficient of variation (CV) of repeated experiments. For each sample, the CV
wasdeterminedastheratiobetweenthe SD and mean transcript levels of
repeated amplifications. The distribution of SMN1-fl, SMN2-fl, total SMN-fl
(SMN1-fl plus SMN2-fl), and GAPDH transcripts was analyzed by using
Kolmogorov–Smirnov, Shapiro–Wilks’ W, and Lilliefors tests for normality.
Possible alternative distributions of SMN transcripts were also evaluated by
using goodness-of-fit tests.
To compare transcript levels in the three populations, both parametric (t-test
for independent variables and one-way ANOVA) and non-parametric tests
(Kruskal–Wallis ANOVA by ranks (KW) and Mann–Whitney U-test (MW))
were used. Possible correlations between SMN1-fl and SMN2-fl transcript
levels, between SMN2 gene copy number and SMN2-fl transcript levels, as
well as between Hammersmith’s functional motor scale score and SMN2-fl
transcript levels were analyzed by a linear regression model. Contingency tables
and two-tailed Fisher’s F-test were used to calculate the relative risk (RR) and
95% confidence interval (CI) for correlations of age of onset and SMN2-fl
transcript levels. To evaluate possible differences in SMN2-fl levels in siblings,
the hypothesis test was used: pairs were divided on the basis of phenotype and
the relatively less severe sibs were compared with more severe ones. For all tests,
significance cutoff was fixed at P-values r0.05.
RESULTS
Validation, specificity, and reproducibility of the assay are described
in Supplementary information. The exact number of SMN2 genes,
SMN-fl, SMN1-fl, SMN2-fl, and GAPDH transcript levels of single
individuals are indicated in Supplementary Table 1.
SMN-fl transcripts do not show a normal distribution
The normality tests reported in the Subjects and methods section
indicated that in carriers and controls, SMN1-fl, SMN2-fl, and SMN-fl
levels do not show a normal distribution (Po0.05, Supplementary
Figure 1a-b and data not shown). Similar results were obtained for
SMN2-fl levels in patients (Po0.04, Supplementary Figure 1c and
Table 1). Therefore, we considered median, quartiles, minimum and
maximum as more appropriate to describe SMN levels than
mean±SD.
SMN2-fl levels are more stable over time than SMN1-
To evaluate physiological fluctuations of SMN1-fl, SMN2-fl, and
GAPDH transcripts, we performed 2–4 blood samplings in seven
controls during a period of 1 month (at days 0, 1, 14, and 30) and two
blood draws in six patients (at day 0 and 30). The results are
summarized in Figure 1. Although a certain degree of fluctuation
was observed, SMN2-fl and GAPDH transcript levels seemed to be
more stable over time than SMN1-fl. SMN2-fl levels seemed to be less
variable in SMA patients than in controls. To confirm this observation,
we have evaluated the mean CV (0.19±0.11 and 0.14±0.06 for
SMN2-fl and GAPDH, respectively), which was more similar to that
expected for experimental variation of the assay (see Assay develop-
ment and validation section in online Supplementary information). In
contrast, SMN1-fl transcripts showed wide day-to-day variations
(mean CV: 0.35±0.17). We also evaluated the total SMN-fl level
variations and observed that transcript level fluctuations reflect that
observed for SMN1-fl levels (data not shown). As SMN1-fl and
SMN2-fl transcripts are amplified by the same primer pair, the
different mean CV of the two amplicons cannot be ascribed to the
PCR artifact.
Patients have lower SMN levels compared with controls and
carriers
To assess whether SMN-fl transcripts are reduced in SMA subjects, we
compared SMN2-fl levels in patients (n¼51) with SMN-fl in controls
(n¼28, Table 1 and Figure 2). The difference between the two groups
was statistically significant (MW: P¼4.3105,KW:P¼4.2105);
also, when excluding the two type I patients, the P-values remained
highly significant (MW¼KW, P¼8105). We subsequently subdi-
vided the patients according to their SMA type. Although the number
of samples from type I patients (n¼2) was insufficient for statistical
analysis, the difference in transcript levels between patients and
controls was statistically significant both for SMA type II (n¼16,
MW: 2.4105,KW:P¼2.22105) and type III (n¼33, MW and
KW: P¼0.0042). No significant differences in SMN2-fl levels were
observed when dividing patients by sex (MW and KW: P¼0.16) or age
(oor Z14 years, MW: P¼0.07, KW: P¼0.06).
SMN2-fl levels are not related to SMN2 gene copy number
To evaluate whether SMN2-fl levels correlate with SMN2 gene copy
number, we have determined the gene copies in 35 of 51 patients. The
two SMA type I patients had two copies, 25 SMA type II and III
patients had three copies, and eight type III patients had four copies.
By using a linear correlation model, indicating SMN2 copy number
as an independent variable, no evidence for a correlation between
SMN full-length mRNA in SMA patients
FD Tiziano et al
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European Journal of Human Genetics
SMN2-fl levels and gene copy number was found (one-way ANOVA
P¼0.52). Moreover, when comparing patients with three and four
SMN2 genes (Figure 3), no statistical difference was found
(MW¼KW: P¼0.35), although individuals with four copies had
slightly higher median SMN2-fl transcript levels (3 SMN2:
56.25 mol/ng, range: 26.75–121.5; 4 SMN2: 63.75 mol/ng, range:
41.25–112.25).
SMN2-fl transcript levels are related to phenotypic severity
To assess whether SMN2-fl levels are related to clinical severity,
different parameters were evaluated: type of SMA, age at onset, and
the Hammersmith motor scale score (in type II patients with age
range 2.5–12 years). Type II patients showed median SMN2-fl tran-
script levels lower than type III, and the difference between the two
groups was highly significant (MW¼KW: P¼0.0034, Table 1 and
Figure 2). Subsequently, SMN2-fl transcript levels were related to the
age of onset of type II and III patients. Although in type II patients no
correlation was found between the two variables (data not shown), in
the case of SMA type III (n¼23, 1.5–19 years) age of onset was
inversely related to SMN2-fl levels. Although it was not possible to
identify a linear regression model between the two variables, we found
that SMN2-fl levels Z58 mol/ng are related to a threefold lower risk of
disease onset before the age of 3 (RR: 0.31, CI: 0.12–0.76, Fisher’s exact
test P¼0.02, Figure 4a). A total of 6 out of 12 type IIIb and 2 out of 10
type IIIa patients had four SMN2 copies. The other patients had three
SMN2 genes.
The motor ability of type II patients was evaluated by the
Hammersmith scale score.27 The cutoff of 12 years was chosen to
avoid bias because of the presence of complications, such as severe
scoliosis and contractures, which are more frequent after this age.
Only 10 out of 16 type II patients were below the age of 12 years. We
compared SMN2-fl levels and motor function by using a linear
regression model, indicating transcript levels as an independent
variable, and found a significant correlation between the two variables
(b¼0.64, P¼0.04, Figure 4b), indicating a moderately strong asso-
ciation between the Hammersmith score and transcript levels. The
Figure 1 (a) Day-to-day variations of SMN-fl, SMN1-fl, and SMN2-fl transcript levels in seven controls (ctrl 1–7) and of SMN2-fl 2 type III patients (pt 1–6).
SMN2-fl level fluctuations were similar to that expected for experimental variability, whereas in controls SMN1-fl levels and, consequently, SMN-fl levels
varied up to threefold. (b)GAPDH transcript level fluctuations were in the range of experimental variability.
Figure 2 SMN2-fl levels in patients vs SMN-fl in controls. SMN2-fl trans-
cript levels in patients are significantly reduced compared with SMN-fl levels
in controls. Also median SMN2-fl levels in patients are significantly
lower compared with controls when considering type II and III patients
separately. Type II patients showed significantly lower SMN2-fl transcript
levels compared with type III subjects.
Figure 3 SMN2-fl transcript levels are not related to SMN2 gene copy
number. Although patients with four SMN2 copies showed higher median
SMN2-fl levels compared with individuals with three SMN2, this difference
was not statistically significant.
SMN full-length mRNA in SMA patients
FD Tiziano et al
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European Journal of Human Genetics
relationship between the two variables can be described by using the
following expression:
SMN2 f1 ¼18:6455+1:61056Hammersmith score
Subsequently, we subgrouped patients on the basis of functional scores
r20 (n¼5) or 420 (n¼5), and observed that the more severely
affected patients had median lower SMN2-fl levels (29.25 mol/ng,
range: 23.60–33.50), compared with the other group (median:
57.00 mol/ng, range: 45.5–102.75); the difference was statistically
significant (MW: P¼0.01, KW: P¼0.009, Figure 4c).
Finally, to assess whether differences in SMN2-fl levels exist between
haploidentical SMA siblings, we evaluated transcript levels in six sib
pairs, five of which showed marked phenotypic differences, whereas
one further sib pair was similar in disease severity. We found that in
discordant couples, the less severely affected sib showed significantly
higher SMN2-fl levels compared with the more severely affected one
(P¼0.002). The phenotypically similar sib pair showed only slightly
different SMN2-fl levels (Figure 4d).
DISCUSSION
The recent move in SMA research from basic to clinical has raised the
necessity to develop reliable and reproducible clinical tools, and to
identify biomarkers useful for monitoring the response of SMA
patients to pharmacological treatments. Although validated clinical
tools have been developed, at present the quantification of SMN2 gene
products in blood leukocytes, either at protein or transcript levels, is
the only potential biomarker available. However, except for type I
SMA,14,22 no clear differences in SMN levels between patients and
controls have been shown, thus questioning the reliability of transcript
analysis as a biomarker for SMA and its usefulness in monitoring the
molecular effects of pharmacological treatment. We have developed
and validated a new real-time PCR assay, based on the use of absolute
standard curves, which allows the quantification of SMN-fl transcripts
Figure 4 Correlation between SMN2-fl levels and clinical severity (a) Transcript levels are related to the age of onset of type III patients: patients with
SMN2-fl levels o58mol/ng of total RNA showed a threefold higher risk of disease onset below the age of 3 years (type IIIa). (b) In type II patients (ranging:
2.5–12 years), a linear correlation was found between SMN2-fl levels and Hammersmith functional scale scores. Dots indicate SMN2-fl levels and the
corresponding functional score for individual patients; the black line is the graphic representation of the equation describing the linear regression model; the
dark gray lines indicate 95% confidence interval and the outer light gray lines are 95% prediction limits for new observations. (c) Patients with a functional
score r20 showed significantly lower SMN2-fl levels. (d) In haploidentical sib pairs with discordant SMA phenotype, the less severely affected sib (white
columns) showed significantly higher transcript levels compared with the more severely affected one (black columns), whereas phenotypically concordant sibs
(gray columns) had similar SMN2-fl levels. However, SMN2nn-fl transcript quantification is not predictive of phenotypic severity in individual cases, as less
severely affected patients may have higher transcript levels. Error bars indicate SD of repeated experiments
SMN full-length mRNA in SMA patients
FD Tiziano et al
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European Journal of Human Genetics
as the number of mRNA molecules per nanogram of total RNA,
independently from the use of endogenous controls. We have
exploited techniques that are widely used to determine the plasmatic
load of some RNA viruses, such as HIV28 or HCV,29 or of prion
protein.30
One interesting finding of our study is the difference in day-to-day
variability of SMN1-fl and SMN2-fl levels. Although we have observed
moderate variations in SMN2-fl levels, which were similar to that
expected for experimental variability and comparable with that of
GAPDH transcripts, SMN1-fl levels varied up to threefold from one
day to the other. Owing to SMN1-fl variability, the total amount of
SMN-fl transcripts also varied markedly (Figure 1a and b and data not
shown; see also Supplementary information). The relevance of this
observation is related to the strategy to test the in vivo effect of
different compounds on SMN expression by administering a given
drug to parents of SMA patients, who often offer spontaneously their
own collaboration.13,14 In a study on the molecular effect of valproic
acid, 7 of 10 carriers had increased levels of SMN-fl transcripts after
treatment, whereas only in one-third of the patients a molecular effect
was shown.14 The discrepancy between these observations may be
explained by spontaneous fluctuations in SMN1-fl transcript levels, as
the assay used by these authors does not allow the discrimination of
SMN1 from SMN2 transcripts. Thus, the results of molecular studies
of the in vivo efficacy of a given compound on SMN expression in
carriers or controls should be interpreted cautiously, and only data
relative to SMN2 transcripts should be taken into account.
The most relevant finding of our study is that SMN2-fl transcripts
are significantly reduced in type II and III patients. As indicated in
Figure 2 and the Table 1, both considering all patients as a group or
divided on the basis of the type of SMA, SMN2-fl transcripts are
significantly reduced in the majority of SMA patients, compared with
SMN-fl in controls. Moreover, type III patients have significantly
higher SMN2-fl transcript levels than type II. To our knowledge, this is
the first demonstration of a statistically significant reduction of SMN
levels in blood leukocytes of type II–III patients. The partial overlap of
SMN-fl levels between patients and controls is not unexpected: blood
leukocytes are not target cells in SMA, and it is conceivable that in
target tissues, the cutoff between patients and controls could be
sharper. It would be important to study the key cell types involved
in SMA pathophysiology, such as motor neurons and/or muscle cells
and to relate SMN2-fl levels in these cells with those found in blood.
To gain further insights into a possible correlation between trans-
cript levels and clinical severity, we investigated whether SMN2-fl
levels are related to the age of onset of type II and III patients, and to
the Hammersmith motor scale score (for type II patients below the
age of 12 years). We did not observe any correlation between age of
onset of type II patients and SMN2-fl transcript levels; however, in
these patients, the first symptoms of the disease are often misrecog-
nized by parents and may be interpreted as a slight delay in ambula-
tion achievement. In the case of type III patients, we observed that
higher transcript levels are related to more advanced age at onset
(Figure 4a). In particular, we found that patients with SMN2-fl levels
Z58 mol/ng have a threefold lower risk of disease onset below the age
of 3 years. The onset of the disease below or over this age (type IIIa
and b) was earlier indicated as an important prognostic factor for
walking ability maintenance:1in an earlier study, Wirth et al8reported
that the 60% of type IIIb and 35% of type IIIa patients have four
SMN2 copies (50 and 20%, respectively, in our cohort), confirming
the correlation between phenotypic severity and SMN2 gene number.
A correlation between clinical severity and SMN2-fl transcript levels is
further supported by the finding that type II children with higher
scores of the Hammersmith functional motor scale show significantly
higher SMN2-fl transcript levels compared with those with lower
scores (Figure 4b-c). In addition, in haploidentical SMA siblings with
discordant phenotype, the less severely affected sib showed signifi-
cantly higher SMN2-fl levels compared with the more severely affected
one, whereas in a phenotypically similar pair, both sibs had similar
SMN2-fl levels (Figure 4d).
We and others have earlier found a correlation between SMN2 copy
number and phenotypic severity;6,7,8 however, SMN2 gene number
alone is not sufficient to explain the phenotypic variability of SMA, as
patients with the same gene number have different phenotypes, and
haploidentical sibs can be markedly discordant for disease severity. To
our knowledge, this is the first in vivo molecular study on SMA
discordant siblings; only one in vitro study has been published
earlier,31 showing that the more severely affected sibs had lower
SMN levels in lymphoblastoid cell lines but not in fibroblasts. We
compared the SMN2-fl transcript levels and SMN2 gene number and
did not find a significant correlation (Figure 3). Similar results were
also found in the study by Simard et al,23 whereas Sumner et al22 and
Vez a i n et al24 found that SMN2 transcript levels are related to the
number of SMN2 genes. The discrepancy among different studies can
be at least partially accounted for by the use of relative quantification
and of different endogenous internal standards that vary widely
among different individuals. Our data suggest that the regulation of
SMN2-fl transcript production (at transcriptional or splicing levels)
has greater influence on phenotypic modulation than SMN2 copy
number per se, which is further supported by the finding that in
discordant sib pairs with identical gene copy number, the less severely
affected sib higher transcript levels. However, although SMN2-fl
transcript quantification appears to be more tightly related to pheno-
typic variability than SMN2 copy number assessment, it should not be
used as a prognostic marker for individual patients, because of the
overlapping between the different phenotypic groups. It would be of
interest to have further data on a possible correlation between SMN
transcript and protein levels in leukocytes by means of an ELISA assay,
which is currently available for cell cultures only.26
In conclusion, our data indicate that SMN2-fl quantification in
blood leukocytes by absolute real-time PCR can be considered suitable
as a biomarker in SMA clinical trials as SMN2-fl transcripts are both
reduced in patients compared with SMN-fl in controls and their level
may at least partially reflect SMN levels in target tissues, being related
to disease severity. The present assay, compared with that published
earlier, offers the opportunity to standardize and optimize SMN2-fl
transcript quantification in different laboratories, in the view of
upcoming international multicentric clinical trials. Further studies
are still necessary to assess whether possible SMN2-fl transcript
increments may correlate with clinical improvements of SMA patients
included in clinical trials, and/or if SMN2-fl dosage can predict clinical
response to pharmacological treatment.
ACKNOWLEDGEMENTS
This study has been granted by ASAMSI, Famiglie SMA, and FSMA USA. We
are grateful to the patients and their families for participating in the study.
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Supplementary information accompanies the paper on European Journal of Human Genetics website (http://www.nature.com/ejhg)
SMN full-length mRNA in SMA patients
FD Tiziano et al
7
European Journal of Human Genetics
    • There is a strong inverse correlation between SMN protein levels and disease severity in SMA fibroblasts and lymphoblastoid cells as well (Coovert et al., 1997;Lefebvre et al., 1997;Kolb et al., 2006). Changes in SMN mRNA and protein levels observed in SMA patient-derived PBMCs mirror those observed in SMA cell lines (Sumner et al., 2006;Simard et al., 2007;Vezain et al., 2007;Tiziano et al., 2010;Crawford et al., 2012). SMN protein is present within the nuclei in discreet foci known as gems (Liu and Dreyfuss, 1996).
    [Show abstract] [Hide abstract] ABSTRACT: Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an early-onset, autosomal recessive neurodegenerative disease characterized by the loss of spinal α-motor neurons. This loss of α-motor neurons is associated with muscle weakness and atrophy. SMA can be classified into five clinical grades based on age of onset and severity of the disease. Regardless of clinical grade, proximal SMA results from the loss or mutation of SMN1 (survival motor neuron 1) on chromosome 5q13. In humans a large tandem chromosomal duplication has lead to a second copy of the SMN gene locus known as SMN2. SMN2 is distinguishable from SMN1 by a single nucleotide difference that disrupts an exonic splice enhancer in exon 7. As a result, most of SMN2 mRNAs lack exon 7 (SMNΔ7) and produce a protein that is both unstable and less than fully functional. Although only 10-20% of the SMN2 gene product is fully functional, increased genomic copies of SMN2 inversely correlates with disease severity among individuals with SMA. Because SMN2 copy number influences disease severity in SMA, there is prognostic value in accurate measurement of SMN2 copy number from patients being evaluated for SMA. This prognostic value is especially important given that SMN2 copy number is now being used as an inclusion criterion for SMA clinical trials. In addition to SMA, copy number variations (CNVs) in the SMN genes can affect the clinical severity of other neurological disorders including amyotrophic lateral sclerosis (ALS) and progressive muscular atrophy (PMA). This review will discuss how SMN1 and SMN2 CNVs are detected and why accurate measurement of SMN1 and SMN2 copy numbers is relevant for SMA and other neurodegenerative diseases.
    Full-text · Article · Mar 2016
    • Both assays have been compared to existing methods for SMN2 mRNA and protein analysis and the results show that both methods are valid to measure SMN mRNA and protein in human blood samples. The results of the study are in line with previously published data ([10], [14])) showing that the expression of SMN mRNA and protein in blood greatly overlap between different disease types and thus cannot be used as a marker for clinical severity. Analysis of SMN1 and SMN2 mRNAs and analysis of the corresponding SMN copy number in healthy subjects resulted in new and interesting findings.
    [Show abstract] [Hide abstract] ABSTRACT: Spinal muscular atrophy is caused by a functional deletion of SMN1 on Chromosome 5, which leads to a progressive loss of motor function in affected patients. SMA patients have at least one copy of a similar gene, SMN2, which produces functional SMN protein, although in reduced quantities. The severity of SMA is variable, partially due to differences in SMN2 copy numbers. Here, we report the results of a biomarker study characterizing SMA patients of varying disease severity. SMN copy number, mRNA and Protein levels in whole blood of patients were measured and compared against a cohort of healthy controls. The results show differential regulation of expression of SMN2 in peripheral blood between patients and healthy subjects.
    Full-text · Article · Oct 2015
    • Total cDNA were diluted 1:4 (representing ∼10 ng of cDNA per quantification). Published TAQMAN probes were used for the SMN transcript assays run on the ABI 7500 Real-Time PCR System (Applied Biosystems) [29]. Quantification of GAPDH transcripts was done to monitor the quality of the RNA/cDNA preparations and amplification reactions.
    [Show abstract] [Hide abstract] ABSTRACT: Background: Clinical trials of therapies for spinal muscular atrophy (SMA) that are designed to increase the expression the SMN protein ideally include careful assessment of relevant SMN biomarkers. Objective: In the SMA VALIANT trial, a recent double-blind placebo-controlled crossover study of valproic acid (VPA) in ambulatory adult subjects with SMA, we investigated relevant pharmacodynamic biomarkers in blood samples from SMA subjects by direct longitudinal measurement of histone acetylation and SMN mRNA and protein levels in the presence and absence of VPA treatment. Methods: Thirty-three subjects were randomized to either VPA or placebo for the first 6 months followed by crossover to the opposite arm for an additional 6 months. Outcome measures were compared between the two treatments (VPA and placebo) using a standard crossover analysis. Results: A significant increase in histone H4 acetylation was observed with VPA treatment (p = 0.005). There was insufficient evidence to suggest a treatment effect with either full length or truncated SMN mRNA transcript levels or SMN protein levels. Conclusions: These measures were consistent with the observed lack of change in the primary clinical outcome measure in the VALIANT trial. These results also highlight the added benefit of molecular and pharmacodynamic biomarker measurements in the interpretation of clinical trial outcomes.
    Full-text · Article · May 2015
    • The quantification of the Chit1 and AMCase mRNA levels and the comparison of those levels with the levels of well-known reference genes are important steps in gaining insight into the in vivo regulation of mammalian chitinases. Recently, real-time RT-PCR has been used to quantify mRNA levels in many gene expression studies[17][18][19][20]because this method is sufficiently sensitive to detect mRNA from even a single cell. Real-time PCR commonly involves the normalization of the expression levels of the gene of interest with those of the housekeeping genes that are thought to be consistently expressed in all of the samples.
    [Show abstract] [Hide abstract] ABSTRACT: Chitinases hydrolyze the β-1-4 glycosidic bonds of chitin, a major structural component of fungi, crustaceans and insects. Although mammals do not produce chitin or its synthase, they express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). These mammalian chitinases have attracted considerable attention due to their increased expression in individuals with a number of pathological conditions, including Gaucher disease, Alzheimer's disease and asthma. However, the contribution of these enzymes to the pathophysiology of these diseases remains to be determined. The quantification of the Chit1 and AMCase mRNA levels and the comparison of those levels with the levels of well-known reference genes can generate useful and biomedically relevant information. In the beginning, we established a quantitative real-time PCR system that uses standard DNA produced by ligating the cDNA fragments of the target genes. This system enabled us to quantify and compare the expression levels of the chitinases and the reference genes on the same scale. We found that AMCase mRNA is synthesized at extraordinarily high levels in the mouse stomach. The level of this mRNA in the mouse stomach was 7- to 10-fold higher than the levels of the housekeeping genes and was comparable to that the level of the mRNA for pepsinogen C (progastricsin), a major component of the gastric mucosa. Thus, AMCase mRNA is a major transcript in mouse stomach, suggesting that AMCase functions as a digestive enzyme that breaks down polymeric chitin and as part of the host defense against chitin-containing pathogens in the gastric contents. Our methodology is applicable to the quantification of mRNAs for multiple genes across multiple specimens using the same scale.
    Full-text · Article · Nov 2012
    • However, all the patients with subtle mutations significantly reduced fl-SMN transcript levels compared with normal controls. These results were similar to those seen in patients with a homozygous deletion of SMN1 [26]. We have attempted to assess the correlation between the fl-SMN levels and the clinical severity, while the difference of fl-SMN transcript levels in these patients were not significant.
    [Show abstract] [Hide abstract] ABSTRACT: Background Proximal spinal muscular atrophy (SMA) is a common neuromuscular disorder resulting in death during childhood. Around 81 ~ 95% of SMA cases are a result of homozygous deletions of survival motor neuron gene 1 (SMN1) gene or gene conversions from SMN1 to SMN2. Less than 5% of cases showed rare subtle mutations in SMN1. Our aim was to identify subtle mutations in Chinese SMA patients carrying a single SMN1 copy. Methods We examined 14 patients from 13 unrelated families. Multiplex ligation-dependent probe amplification analysis was carried out to determine the copy numbers of SMN1 and SMN2. Reverse transcription polymerase chain reaction (RT-PCR) and clone sequencing were used to detect subtle mutations in SMN1. SMN transcript levels were determined using quantitative RT-PCR. Results Six subtle mutations (p.Ser8LysfsX23, p.Glu134Lys, p.Leu228X, p.Ser230Leu, p.Tyr277Cys, and p.Arg288Met) were identified in 12 patients. The p.Tyr277Cys mutation has not been reported previously. The p.Ser8LysfsX23, p.Leu228X, and p.Tyr277Cys mutations have only been reported in Chinese SMA patients and the first two mutations seem to be the common ones. Levels of full length SMN1 (fl-SMN1) transcripts were very low in patients carrying p.Ser8LysfsX23, p.Leu228X or p.Arg288Met compared with healthy carriers. In patients carrying p.Glu134Lys or p.Ser230Leu, levels of fl-SMN1 transcripts were reduced but not significant. The SMN1 transcript almost skipped exon 7 entirely in patients with the p.Arg288Met mutation. Conclusions Our study reveals a distinct spectrum of subtle mutations in SMN1 of Chinese SMA patients from that of other ethnicities. The p.Arg288Met missense mutation possibly influences the correct splicing of exon 7 in SMN1. Mutation analysis of the SMN1 gene in Chinese patients may contribute to the identification of potential ethnic differences and enrich the SMN1 subtle mutation database.
    Full-text · Article · Sep 2012
    • Attempts have been made to add more reference genes in the estimation to increase its reliability; still, the practice does not improve the power of interpretation, nor has it the theoretical basis to do so. Besides fold changes, gene expressions can also be evaluated for a known quantity of cDNA [11] or RNA [12,13] as previously tried. In comparison to RNA, cDNAs are more stable during dilution procedure as observed in environmental samples [14].
    [Show abstract] [Hide abstract] ABSTRACT: Temporal and tissue-specific patterns of gene expression play important roles in functionality of a biological system. Real-time quantitative polymerase chain reaction (qPCR) technique has been widely applied to single gene expressions, but its potential has not been fully released as most results have been obtained as fold changes relative to control conditions. Absolute quantification of transcripts as an alternative method has yet to gain popularity because of unresolved issues. We propose a solution here with a novel procedure, which may accurately quantify the total cDNA conventionally prepared from a biological sample at the resolution of ~70 pg/μl, and reliably estimate the absolute numbers of transcripts in a picogram of cDNA. In comparison to the relative quantification, cDNA-based absolute (CBA) qPCR method is found to be more sensitive to gene expression variations caused by factors such as developmental and environmental variations. If the number of target transcript copies is further normalized by reference transcripts, cell-level variation pattern of the target gene expression may also be detectable during a developmental process, as observed here in cases across species (Ipomoea purpurea, Nicotiana benthamiana) and tissues (petals and leaves). By allowing direct comparisons of results across experiments, the new procedure opens a window to make inferences of gene expression patterns across a broad spectrum of living systems and tissues. Such comparisons are urgently needed for biological interpretations of gene expression variations in diverse cells.
    Full-text · Article · Mar 2012
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