Abstract
Proximal spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations of the SMN1 gene. Based on severity, three forms of SMA are recognized (types I–III). All patients usually have 2–4 copies of a highly homologous gene (SMN2), which produces insufficient levels of functional survival motor neuron (SMN) protein due to the alternative splicing of exon 7. The availability of potential candidates to the treatment of SMA has raised a number of issues, including the availability of biomarkers. This study was aimed at evaluating whether the quantification of SMN2 products in peripheral blood is a suitable biomarker for SMA. Forty-five adult type III patients were evaluated by Manual Muscle Testing, North Star Ambulatory Assessment scale, 6-min walk test, myometry, forced vital capacity, and dual X-ray absorptiometry. Molecular assessments included SMN2 copy number, levels of full-length SMN2 (SMN2-fl) transcripts and those lacking exon 7 and SMN protein. Clinical outcome measures strongly correlated to each other. Lean body mass correlated inversely with years from diagnosis and with several aspects of motor performance. SMN2 copy number and SMN protein levels were not associated with motor performance or transcript levels. SMN2-fl levels correlated with motor performance in ambulant patients. Our results indicate that SMN2-fl levels correlate with motor performance only in patients preserving higher levels of motor function, whereas motor performance was strongly influenced by disease duration and lean body mass. If not taken into account, the confounding effect of disease duration may impair the identification of potential SMA biomarkers.
Similar content being viewed by others
Introduction
Proximal spinal muscular atrophies (SMAs) are a group of clinically variable motor neuron disorders characterized by degeneration of spinal cord anterior horn cells. SMAs are generally classified into types I to III according to age at onset and highest motor milestone achieved.1, 2 SMA type III is the most clinically variable form, with symptoms onset before (type IIIa) or after (type IIIb) age 3 years,3 normal achievement of motor milestones, variable severity of scoliosis, tendon retractions and joint contractures, and eventual loss of walking ability.
Type I–III SMAs are autosomal recessive conditions caused by loss of function of the survival motor neuron (SMN1) gene.4 Irrespective of phenotypic severity, both copies of the SMN1 gene are absent in about 95% of cases, whereas 2–3% of patients are compound heterozygotes typically with one allele deleted and subtle mutations in the other.5 Complete loss of the SMN protein is embryonically lethal,6 but SMA patients obtain reduced amounts of the protein from a nearly identical gene copy, SMN2, present (with SMN1) in a duplicated and inverted region of 5q13.4 Because of alternative splicing, most SMN2 transcripts lack exon 7 (SMN-del7) so that insufficient amounts of functional protein are produced. In fact, SMN protein levels are reduced in spinal cord and cell cultures from SMA patients, and correlate inversely with phenotypic severity.7, 8, 9 SMN2 copy number can also vary, and patients with high copy number often have a milder phenotype.10, 11, 12
At present, there is no effective treatment for SMA. Some therapeutic approaches aim to increase the amount of SMN protein produced by SMN2 through promoter activation, reduction of exon 7 alternative splicing, or both.13, 14, 15, 16, 17, 18, 19, 20, 21, 22 Some of these approaches are being investigated in ongoing or planned clinical trials, and great efforts have been done to identify the most appropriate clinical outcome measures for patients affected from various severities.23 In this view, it would be very useful to have reliable biomarkers of disease severity and response to treatment.23
In the present study, we investigated associations between clinical phenotype and molecular characteristics in adult patients with type III SMA, with the aim of evaluating available molecular biomarkers for possible use as surrogate endpoints in clinical trials on SMA. Clinical phenotype was assessed by tests of muscle strength and function. Molecular evaluation comprised determination of SMN2 copy number, SMN transcript levels (full length and del7), and SMN protein levels.
Materials and methods
Patients and clinical evaluation
A total of 45 patients (29 male, 16 female, Table 1), mean age 36.8 years (range 18–56 years) with diagnosis confirmed by molecular analysis were recruited to an ongoing double-blind placebo-controlled multicenter trial to assess the safety of salbutamol (EudraCT No. 2007-001088-32). All patients enrolled in the double-blind trial were included in the present study. At clinical evaluation, 26 were ambulant and 19 were wheelchair bound (mean age at loss of walking, 20 years). Based on age of onset, 15 were type IIIa and 30 were type IIIb. No patients reported onset of symptoms over 18 years of age. Written informed consent was obtained from all patients, and the study was approved by the Ethics Committee of each participating Centre.
Patients were comprehensively evaluated at baseline. Only selected variables are reported here as potential outcome measures. Muscle strength was assessed by manual muscle testing of 18 muscle groups (elbow flexors and extensors, finger flexors and extensors, thigh flexors and extensors, leg flexors and extensors, foot dorsiflexors) and graded from 0 to 5 according to the Medical Research Council (MRC) scale.24 The force of maximum voluntary isometric contraction (Newtons, N) was assessed in elbow flexor, handgrip, three-point pinch, knee flexor, and knee extensor25 for 30 of the 45 patients, using a hand-held myometer (CIT Technics, Groningen, The Netherlands).
In ambulant patients, motor function was assessed by the North Star Ambulatory Assessment (NSAA) scale.26 Ambulant patients also performed the 6-Min walk test (6MWT) recently shown to be reliable for assessing type III SMA patients.27, 28
Forced vital capacity (FVC, % of predicted) was measured in all patients using a standard spirometer in the sitting position. Lean body mass (grams) was assessed by dual X-ray absorptiometry (DXA)29, 30 and normalized to height (expressed in cm); this evaluation was performed in 20 patients only, in those neuromuscular Centres where the tool was available. Furthermore, DXA was feasible only for patients who did not have severe contractures preventing the access to the examination bed of the instrument.
Molecular assessments
Blood samples were collected into EDTA tubes for DNA extraction, sodium citrate tubes for protein extraction, and PAX blood RNA tubes (BD Biosciences, San Jose, CA, USA) for RNA. The samples were analysed at the Institute of Medical Genetics of Catholic University in Roma.
Genomic DNA was extracted by standard salting out, and conventional RFLP-PCR used to verify SMA diagnosis.31 For patients testing negative for SMN1 mutation by RFLP-PCR, SMN1 copy number was determined (same method as SMN2 copy number); for patients with a single SMN1 copy, sequence analysis of exons 1–7 and exon–intron boundaries was performed (sequence of primers and PCR conditions are available on request).
SMN1 and SMN2 copy number was determined by relative real-time PCR as reported elsewhere.14 SMN2 copy number was determined in all patients.
The presence of the p.G287R (c.G859C)32 variant in SMN2 was determined in all patients by RFLP-PCR. Briefly, 50 ng of genomic DNA were amplified with R1114 as forward primer and G287RDdeR: 5′-ATTTAAGGAATGTGAGCACCTTA-3′ as reverse primer. The latter contains a mismatch (bold) that introduces a restriction site for DdeI in the variant allele. Amplification conditions were: 30 cycles of 94 °C for 1 min, 55 °C for 1 min, 72° C for 1 min. The PCR products were digested with 3U of restriction enzyme DdeI overnight at 37 °C. Next day, the digestion products were separated by electrophoresis on 4% agarose gels. If the G287R variant was present, two bands (208 and 185 bp) were obtained.
RNA was extracted by PAX blood RNA extraction kit (Qiagen, Dusseldorf, Germany), according to the manufacturer’s instructions. SMN2 full length (SMN2-fl), lacking exon 7 (SMN-del7) and total (SMN-fl plus SMN-del7, SMN-tot) transcript levels were assessed by absolute real-time PCR (Tiziano et al33 and Angelozzi et al, in preparation). In patients with the G287R variant, full-length transcripts were determined by an alternative set of Taqman MGB probe and primers.33 GAPDH transcript levels were determined as quality control for RT-PCR and real-time PCR.
For SMN protein analysis, time between blood collection and preparation of samples ranged from a few hours to 2 days. Samples from 43 patients were analyzable. PBMCs were separated through Lympholyte M medium (Macherey-Nagel, Duren, Germany). The pellet was washed in PBS and frozen in fetal calf serum containing 10% DMSO. After thawing, PBMCs were counted by NucleoCounter (Chemometec, Allerod, Denmark) and resuspended in lysis buffer at 2 × 106 cells/ml (instead of 108 cells/ml, as in ELISA protocol, Enzo Life Science, Farmingdale, NY, USA); 2 × 105 cells were loaded onto each ELISA plate. The ELISA kits were kindly provided by the SMA Foundation. SMN protein concentrations were expressed as pg of protein/106 cells.
Statistical analysis
Means, medians, and SD for continuous variables and proportions for categorical variables were calculated. Associations of SMN2-fl, SMN-del7, SMN-tot transcript levels, and SMN protein levels, with clinical characteristics were assessed by linear regression models. A multivariate model was used to take account of the influence of other covariates. Because of small sample size and non-normal distribution of SMN transcript levels,33 the non-parametric Kruskal–Wallis ‘ANOVA’ by ranks (KW) and Mann–Whitney U-test (MW) were used to compare transcript levels between groups (ambulant vs non-ambulant; type IIIa vs type IIIb). Correlations between clinical characteristics were evaluated by Pearson’s r (R) test. The t-test for paired samples was used to compare the performance of groups at different time-points of the 6MWT. Statgraphics (Centurion XV.II, Statpoint Technologies, Warrenton, VA, USA) and SPSS 18.0 (SPSS, Inc., Chicago, IL, USA) were used to carry out the analyses. Differences associated with P<0.05 or, after Bonferroni correction for multiple testing, with P<0.016 were considered significant.
Results
Genotypic characterization of patients at the SMN locus
In 43 of the 45 patients, SMN1 exon 7 was absent. The remaining two patients were compound heterozygotes, missing one copy of SMN1, and with the W102X mutation34 in one case, and the S262I mutation35 in the other (Supplementary Figure 1a).
SMN2 gene copy number was determined in all patients. There were five SMN2 copies in 2 patients, 4 in 29 patients, three in 13 patients and a single copy in the patient with the S262I mutation. Among type IIIa patients, 7 out of 15 (46.7%) had three SMN2 copies, the others had 4 genes. Of the 30 type III b subjects, 21 had 4 SMN2 genes (70%).
The G287R variant (Supplementary Figure 1b) of SMN2 was found in 4/45 (8.9%) patients, all type IIIb with three SMN2 copies. One of these patients was homozygous for the G287R variant, being present in both parents.
Correlations between clinical characteristics
Selected baseline clinical features and molecular characteristics of the patients are shown in Table 1. Total MRC score correlated with handgrip (R=0.78, P<0.00001, n=30, data not shown), elbow flexion (R=0.68, P<0.00001, n=30, data not shown), knee extension (R=0.59, P=0.0006, n=30, data not shown), and knee flexion (R=0.74, P<0.00001, n=30, Figure 1a). Total MRC score correlated weakly with predicted forced vital capacity (R=0.28, P=0.06, n=45, Figure 1b). In ambulant patients, total MRC score correlated strongly with NSAA (R=0.77, P<0.00001, n=26, Figure 1c) and 6MWT (R=0.67, P=0.0002, n=26, Figure 1d). Distance covered during the sixth minute (mean 60.37±20.36 m) was significantly less (P=0.001, n=26) than in the first minute (mean 67.39±18.96 m).
There was no correlation between muscle strength (total MRC scale score) or motor function (NSAA score, ambulant patients only) and patient age (MRC: R=−0.065, P=0.67; NSAA: R=−0.26, P=0.18). However, total MRC and NSAA scores (in ambulant patients) did correlate inversely with disease duration (MRC: R=−0.57, P<0.00001, n=45; NSAA: R=−0.48, P=0.01, n=26, Figures 2a and b). Forced vital capacity also correlated inversely with disease duration (R=−0.31, P=0.038, n=45; data not shown).
Correlations between motor performance and lean body mass
In all patients tested, lean body mass correlated with total MRC score (R=0.66, P=0.0015, n=20, Figure 2c). In ambulant patients, correlations of lean body mass with other aspects of motor performance were strong (MRC: R=0.82, P=0.0005, n=11; NSAA: R=0.71, P=0.006, n=11; 6MWT: R=0.69; P=0.009, n=11; Figure 2d and data not shown). Lean body mass also correlated inversely with disease duration (R=−0.50, P=0.025, n=20) and the correlation remained after correcting for height (R=−0.52, P=0.019, n=20, data not shown).
Associations between clinical and molecular data
By linear regression modeling, SMN2 copy number was unrelated to any clinical variable (in all cases P≥0.27, data not shown), or to SMN2 transcript or SMN protein levels (in all cases P≥0.08, data not shown). Similarly, neither SMN2 transcript nor protein levels were influenced by age, years from symptoms onset, or lean body mass (in all cases P≥0.12, data not shown). Transcript and protein levels did not differ between SMA types IIIa and IIIb (MW and KW, P>0.20, data not shown).
In the entire group, neither total nor delta7 SMN2 transcript levels correlated significantly with any clinical characteristic, although SMN2-fl levels correlated weakly with total MRC score (R=0.29; P=0.052, n=45, data not shown), as well as with lower limb MRC score (R=0.29; P=0.049, n=45, data not shown). In ambulant patients only, SMN2-fl levels correlated with total MRC score (R=0.46, P=0.02, n=26, Figure 3a), and with lower limb MRC score (R=0.49, P=0.01, n=26, Figure 3b), and weakly with 6MWT (R=0.37), although this correlation was not significant (P=0.07, n=26, Figure 3c). SMN protein levels did not correlate with motor performance (P≥0.31, data not shown) or with SMN2-fl levels (R=0.23, P=0.18, n=43, Figure 4a); however protein levels did correlate with the SMN2-fl/SMN2-delta7 ratio (R=0.40, P=0.016, n=43, Figure 4b).
Discussion
Several potential therapeutic approaches to SMA are undergoing development or have been tested in recent years.36 Reliable clinical outcome measures and biomarkers are essential to effectively evaluate these approaches. Different motor function measures have been used and validated in SMA patients,23, 26 but some of them are too long to administer, include tasks unbearable for adult patients, or may be used only for patients with moderate phenotypes due to floor or ceiling effects. Moreover, some complications related to the disease, such as scoliosis, retractions, and weight gain, can further impair motor function. Thus, the identification of reliable biomarkers as surrogate endpoints of disease progression and response to treatment has become a matter of urgency.23 The aim of our study was to evaluate the applicability of SMN transcript and protein levels, as surrogate outcome measures in adult type III SMA patients, by relating clinical and molecular data. The clinical variables chosen have been previously validated or used in other SMA studies.23 The molecular techniques we used (absolute real time for transcript analysis, ELISA for protein quantification) are currently considered the most suitable tools for SMN quantification, as they do not make use of normalization against endogenous controls and are therefore unaffected by possible variations in the expression levels of housekeeping genes.
We found that clinical measures correlated strongly with each other, as expected. Similarly, Glanzman et al37 recently found that modified Hammersmith scale score correlated strongly with myometry-measured muscle strength. The motor performance was significantly affected by disease duration but not by age at evaluation, suggesting that in the design of clinical trials this variable could be useful to enroll more homogenous cohorts of patients, rather than age.
Montes et al28 recently evaluated the 6MWT in type III SMA patients spanning a wide age range and found that they showed progressive motor fatigue. We observed similar fatigability in the present series, so it may be also worth investigating whether increased resistance to motor fatigue can be used as a marker of treatment efficacy.
In the present study, we found no correlation between motor performance and SMN2 copy number. However, similar to our own previous data11 and those of others,10, 12, 37 a higher number of SMN2 genes was found in type IIIb patients, but was not predictive of the clinical phenotype in individual subjects. In the patient bearing the S262I mutation in exon 6, we found a single SMN2 copy, suggesting that this point mutation determines only a mild reduction in SMN protein function. This hypothesis is supported by the previous report of the same mutation in another patient,35 affected by SMA type III as well; however, in that patient, SMN2 gene copy number was not assessed. Also, the frequency of the G287R variant in our cohort was much higher than previously reported (about 9% vs 1%38). This variant has a positive effect in the inclusion of exon 7 in SMN2 transcripts.32, 38 All our patients bearing the G287R variant were type IIIb subjects, thus raising the prevalence of the mutation in this group of patients up to 16% (4/25 individuals). Because of the positive effect of this variant in exon 7 inclusion in SMN2 transcripts, this finding is not unexpected. It is noteworthy that these four subjects had three SMN2 copies, supporting the positive modulating effect of the G287R variant on SMA severity.
We also found a weak correlation between motor performance and SMN2-fl transcript levels, when considering all patients, which was much stronger in ambulant patients. It is likely that in non-ambulant patients, the presence of long-term complications of the condition further worsened motor performance. An alternative hypothesis is that SMN2-fl levels in blood do not reflect those found in target tissues of the disease, such as spinal cord and/or skeletal muscle. However, in our opinion, this hypothesis is less likely, as the correlation of transcript levels with motor function is stronger in the less severely affected patients. As no other transcript assessed (SMN2-del7, total SMN2 transcripts, or SMN2-fl/ del7 ratio) correlated with any baseline clinical characteristic, even in ambulant patients, SMN2-fl appears to have the strongest relation to phenotype. Very recently, some of us (FDT and LR) have collaborated to the BforSMA study.39, 40 Also in that large cohort of young patients spanning the whole phenotypic spectrum of the disease, SMN2-fl levels were significantly higher in the less severely affected subjects, although they were not predictive of the motor performance of single individuals. We found similar results also in our previous study.33 The main difference with the cohort included in the present study is related to the long duration of the disease of our patients and to the associated complications, which may impair the clinical evaluation. Moreover, to our knowledge, longitudinal data regarding SMN level variations with age are not available.
We also found that SMN protein levels were unrelated to baseline clinical characteristics and SMN mRNA levels, except for a weak correlation with the SMN2-fl/SMN-del7 ratio, whose biological significance remains unclear. Lack of correlation between SMN protein levels and motor performance was also found in the study of Crawford et al39 and remains unexplained. It is possible that SMN protein levels do not reflect those found in target tissues of the disease, or that the ELISA method we used requires optimization. It is noteworthy that stabilization buffers are not available for protein samples. On the other hand, for RNA extraction, these buffers allow to preserve samples for relatively long time and provide a ‘snapshot’ of gene expression at the time of sampling. As time between blood sampling and protein extraction (in the context of a multicenter clinical trial) varied considerably, levels of SMN protein could be affected by different variables, such as cell death, sample preservation, higher extractability of SMN protein over time, or post-translational modifications. Indeed, putative loss or increase in SMN protein levels hypothetically related to the factors above cannot be ruled out.
Our finding of strong correlations between several aspects of motor performance and lean body mass is potentially important and suggests that lean body mass, as measured by DXA, might be worth further investigation as an outcome measure in clinical trials on potential therapeutic agents in SMA. On the other side, DXA is not easily feasible in patients with severe contractures, the longitudinal variation of lean body mass in relation to age and disease course is at present unavailable, and the time required to observe a lean body mass increase in response to a potentially effective intervention is also unknown.
To conclude, the results of our study suggest that, in adults with type III SMA, SMN2 copy number, SMN2-del7 transcripts, and SMN protein levels in blood cells are not suitable as markers of phenotypic severity and hence as indicators of response to treatment. SMN2-fl transcript levels appear potentially more useful, as they correlate satisfactorily with motor performance in ambulant patients. Importantly we found that lean body mass shows promise as marker of disease severity and possibly also response to treatment. These findings require verification in larger series patients, of wider range of disease severity and age range (including children). Finally, our data suggest that if not taken into account, the confounding effect of disease duration may impair the identification of potential SMA biomarkers.
References
Pearn J : Classification of spinal muscular atrophies. Lancet 1980; 1: 919–922.
Munsat TL, Davies KE : International SMA consortium meeting (26–28 June 1992, Bonn, Germany). Neuromuscul Disord 1992; 2: 423–428.
Zerres K, Rudnik-Schöneborn S, Forrest E et al: A collaborative study on the natural history of childhood and juvenile onset proximal spinal muscular atrophy (type II and III SMA): 569 patients. J Neurol Sci 1997; 146: 67–72.
Lefebvre S, Bürglen L, Reboullet S et al: Identification and characterization of a spinal muscular atrophy-determining gene. Cell 1995; 80: 155–165.
Wirth B : An update of the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA). Hum Mut 2000; 15: 228–237.
Frugier T, Tiziano FD, Cifuentes-Diaz C et al: Nuclear targeting defect of SMN lacking the C-terminus in a mouse model of spinal muscular atrophy. Hum Mol Genet 2000; 9: 849–858.
Lefebvre S, Burlet P, Liu Q et al: Correlation between severity and SMN protein level in spinal muscular atrophy. Nat Genet 1997; 16: 265–269.
Coovert DD, Le TT, McAndrew PE et al: The survival motor neuron protein in spinal muscular atrophy. Hum Mol Genet 1997; 6: 1205–1214.
Patrizi AL, Tiziano F, Zappata S et al: SMN protein analysis in fibroblast, amniocyte, and CVS cultures from spinal muscular atrophy patients and its relevance for diagnosis. Eur J Hum Genet 1999; 7: 301–309.
Feldkötter M, Schwarzer V, Wirth R et al: Quantitative analysis of SMN1 and SMN2 based on real-time LightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. Am J Hum Genet 2002; 70: 358–368.
Tiziano FD, Bertini E, Messina S et al: The Hammersmith functional score correlates with the SMN2 copy number: a multicentric study. Neuromuscul Disord 2007; 17: 400–403.
Wirth B, Brichta L, Schrank B et al: Mildly affected patients with spinal muscular atrophy are partially protected by an increased SMN2 copy number. Hum Genet 2006; 119: 422–428.
Brichta L, Hofmann Y, Hahnen E et al: Valproic acid increases the SMN2 protein level: a well-known drug as a potential therapy for spinal muscular atrophy. Hum Mol Genet 2003; 12: 2481–2489.
Andreassi C, Angelozzi C, Tiziano FD et al: Phenylbutyrate increases SMN expression in vitro: relevance for treatment of spinal muscular atrophy. Eur J Hum Genet 2004; 12: 59–65.
Brahe C, Vitali T, Tiziano FD et al: Phenylbutyrate increases SMN gene expression in spinal muscular atrophy patients. Eur J Hum Genet 2005; 13: 256–259.
Jarecki J, Chen X, Bernardino A et al: Diverse small-molecule modulators of SMN expression found by high-throughput compound screening: early leads towards a therapeutic for spinal muscular atrophy. Hum Mol Genet 2005; 14: 2003–2018.
Angelozzi C, Borgo F, Tiziano FD et al: Salbutamol increases SMN mRNA and protein levels in spinal muscular atrophy cells. J Med Genet 2008; 45: 29–31.
Pane M, Staccioli S, Messina S et al: Daily salbutamol in young patients with SMA type II. Neuromuscul Disord 2008; 18: 536–540.
Mercuri E, Bertini E, Messina S et al: Randomized, double-blind, placebo-controlled trial of phenylbutyrate in spinal muscular atrophy. Neurology 2007; 68: 51–55.
Tiziano FD, Lomastro R, Pinto AM et al: Salbutamol increases SMN transcript levels in leukocytes of spinal muscular atrophy patients: relevance for clinical trial design. J Med Genet 2010; 47: 856–858.
Hua Y, Sahashi K, Hung G et al: Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model. Genes Dev 2010; 24: 1634–1644.
Singh NN, Shishimorova M, Cao LC et al: A short antisense oligonucleotide masking a unique intronic motif prevents skipping of a critical exon in spinal muscular atrophy. RNA Biol 2009; 6: 341–350.
Kaufmann P, Muntoni F : Issues in SMA clinical trial design. The International Coordinating Committee (ICC) for SMA Subcommittee on SMA Clinical Trial Design. Neuromuscul Disord 2007; 17: 499–505.
Florence JM, Pandya S, King WM et al: Intrarater reliability of manual muscle test (Medical Research Council scale) grades in Duchenne’s muscular dystrophy. Phys Ther 1992; 72: 115–122.
Merlini L, Mazzone ES, Solari A et al: Reliability of hand-held dynamometry in spinal muscular atrophy. Muscle Nerve 2002; 26: 64–70.
Mercuri E, Mayhew A, Muntoni F et al: Towards harmonisation of outcome measures for DMD and SMA within TREAT-NMD; Report of three expert workshops: TREAT-NMD/ENMC workshop on outcome measures, 12th–13th May 2007, Naarden, The Netherlands; TREAT-NMD workshop on outcome measures in experimental trials for DMD, 30th June–1st July 2007, Naarden, The Netherlands; Conjoint Institute of Myology TREAT-NMD Meeting on physical activity monitoring in neuromuscular disorders, 11th July 2007, Paris, France. Neuromuscul Disord 2008; 18: 894–903.
Takeuchi Y, Katsuno M, Banno H et al: Walking capacity evaluated by the 6-minute walk test in spinal and bulbar muscular atrophy. Muscle Nerve 2008; 38: 964–971.
Montes J, McDermott MP, Martens WB et al. Muscle Study Group and the Pediatric Neuromuscular Clinical Research Network: Six-Minute Walk Test demonstrates motor fatigue in spinal muscular atrophy. Neurology 2010; 74: 833–838.
Khatri IA, Chaudhry US, Seikaly MG et al: Low bone mineral density in spinal muscular atrophy. J Clin Neuromuscul Dis 2008; 10: 11–17.
Kinali M, Banks LM, Mercuri E et al: Bone mineral density in a paediatric spinal muscular atrophy population. Neuropediatrics 2004; 35: 325–328.
van der Steege G, Grootscholten PM, van der Vlies P et al: PCR-based DNA test to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy. Lancet 1995; 345: 985–986.
Prior TW, Krainer AR, Hua Y et al: A positive modifier of spinal muscular atrophy in the SMN2 gene. Am J Hum Genet 2009; 85: 408–413.
Tiziano FD, Pinto AM, Fiori S et al: SMN transcript levels in leukocytes of SMA patients determined by absolute real time PCR. Eur J Hum Genet 2010; 18: 52–58.
Sossi V, Giuli A, Vitali T et al: Premature termination mutations in exon 3 of the SMN1 gene are associated with exon skipping and a relatively mild SMA phenotype. Eur J Hum Genet 2001; 9: 113–120.
Hahnen E, Schonling J, Raschke H et al: Missense mutations in exon 6 of the survival motor neuron gene in patients with spinal muscular atrophy. Hum Mol Genet 1997; 6: 821–825.
Sproule DM, Kaufmann P : Therapeutic developments in spinal muscular atrophy. Ther Adv Neurol Disord 2010; 3: 173–185.
Glanzman AM, O'Hagen JM, McDermott MP et al. Pediatric Neuromuscular Clinical Research Network for Spinal Muscular Atrophy (PNCR); Muscle Study Group (MSG): Validation of the Expanded Hammersmith Functional Motor Scale in Spinal Muscular Atrophy Type II and III. J Child Neurol 2011; 26: 1499–1507.
Vezain M, Saugier-Veber P, Goina E et al: A rare SMN2 variant in a previously unrecognized composite splicing regulatory element induces exon 7 inclusion and reduces the clinical severity of spinal muscular atrophy. Hum Mutat 2010; 31: E1110–E1125.
Finkel RS, Crawford TO, Swoboda KJ et al. Pilot Study of Biomarkers for Spinal Muscular Atrophy Trial Group: Candidate proteins, metabolites and transcripts in the Biomarkers for Spinal Muscular Atrophy (BforSMA) clinical study. PLoS One 2012; 7: e35462.
Crawford TO, Paushkin SV, Kobayashi DT et al Pilot Study of Biomarkers for Spinal Muscular Atrophy Trial Group: Evaluation of SMN protein, transcript, and copy number in the biomarkers for spinal muscular atrophy (BforSMA) clinical study. PLoS One 2012; 7: e33572.
Acknowledgements
The present study is dedicated to the memory of Christina Brahe. We are grateful to the SMA Foundation for kindly providing SMN ELISA kits, and to Dr Dione Kobayashi, Professor Eugenio Mercuri, and Dr Enrico Bertini for critical comments on the manuscript. We are extremely grateful to the patients and their families. The study was funded by the Agenzia Italiana del Farmaco (AIFA). Carla Angelozzi has been granted by ASAMSI.
Disclaimer
FDT and LM had full access to all study data and take responsibility for the integrity of the data and the accuracy of the analyses.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Supplementary Information accompanies the paper on European Journal of Human Genetics website
Supplementary information
Rights and permissions
About this article
Cite this article
Tiziano, F., Lomastro, R., Di Pietro, L. et al. Clinical and molecular cross-sectional study of a cohort of adult type III spinal muscular atrophy patients: clues from a biomarker study. Eur J Hum Genet 21, 630–636 (2013). https://doi.org/10.1038/ejhg.2012.233
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ejhg.2012.233
Keywords
This article is cited by
-
Transcript, methylation and molecular docking analyses of the effects of HDAC inhibitors, SAHA and Dacinostat, on SMN2 expression in fibroblasts of SMA patients
Journal of Human Genetics (2016)
-
Decay in survival motor neuron and plastin 3 levels during differentiation of iPSC-derived human motor neurons
Scientific Reports (2015)