Detection of Survival Motor Neuron Protein in Buccal Cells Through Electrochemiluminescence-Based Assay

Steinkellner, Hannes; Etzler, Julia; Gmeiner, Bernhard; Laccone, Franco

doi:10.1089/adt.2015.635

Cited by 7 publications

(5 citation statements)

References 28 publications

(34 reference statements)

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“…In humans, similar findings have been reported in a smaller number of tissues that include brain [43], muscle [43], whole blood [32, 36], PBMCs [12, 15, 18, 22, 29, 33, 34], fibroblasts [20, 26, 29] and buccal cells [36, 37]. SMN protein levels have also been investigated in body fluids, most notably in cerebrospinal fluid as an exploratory biomarker in a phase 1 study of intrathecal administration of antisense oligonucleotides [44], but also in urine, plasma and saliva [33, 36, 37]. However, the extent to which tissues differ in SMN mRNA and protein concentrations in humans is still largely unknown [45].…”

Section: Introductionsupporting

confidence: 73%

“…qPCR [22, 31, 32] and ELISA [12, 15, 18, 22, 33–35] have shown their applicability in larger studies with patients participating in randomized controlled trials with SMN inducing therapies such as valproic acid and salbutamol [11, 15, 19]. Recently, electrochemiluminescence-based immunoassay (ECLIA or ECL) was introduced for measurements of SMN levels in small amounts of whole blood [32, 36, 37]. …”

Section: Introductionmentioning

confidence: 99%

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A Comparative Study of SMN Protein and mRNA in Blood and Fibroblasts in Patients with Spinal Muscular Atrophy and Healthy Controls

et al. 2016

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BackgroundClinical trials to test safety and efficacy of drugs for patients with spinal muscular atrophy (SMA) are currently underway. Biomarkers that document treatment-induced effects are needed because disease progression in childhood forms of SMA is slow and clinical outcome measures may lack sensitivity to detect meaningful changes in motor function in the period of 1–2 years of follow-up during randomized clinical trials.ObjectiveTo determine and compare SMN protein and mRNA levels in two cell types (i.e. PBMCs and skin-derived fibroblasts) from patients with SMA types 1–4 and healthy controls in relation to clinical characteristics and SMN2 copy numbers.Materials and methodsWe determined SMN1, SMN2-full length (SMN2-FL), SMN2-delta7 (SMN2-Δ7), GAPDH and 18S mRNA levels and SMN protein levels in blood and fibroblasts from a total of 150 patients with SMA and 293 healthy controls using qPCR and ELISA. We analyzed the association with clinical characteristics including disease severity and duration, and SMN2 copy number.ResultsSMN protein levels in PBMCs and fibroblasts were higher in controls than in patients with SMA (p<0.01). Stratification for SMA type did not show differences in SMN protein (p>0.1) or mRNA levels (p>0.05) in either cell type. SMN2 copy number was associated with SMN protein levels in fibroblasts (p = 0.01), but not in PBMCs (p = 0.06). Protein levels in PBMCs declined with age in patients (p<0.01) and controls (p<0.01)(power 1-beta = 0.7). Ratios of SMN2-Δ7/SMN2-FL showed a broad range, primarily explained by the variation in SMN2-Δ7 levels, even in patients with a comparable SMN2 copy number. Levels of SMN2 mRNA did not correlate with SMN2 copy number, SMA type or age in blood (p = 0.7) or fibroblasts (p = 0.09). Paired analysis between blood and fibroblasts did not show a correlation between the two different tissues with respect to the SMN protein or mRNA levels.ConclusionsSMN protein levels differ considerably between tissues and activity is age dependent in patients and controls. SMN protein levels in fibroblasts correlate with SMN2 copy number and have potential as a biomarker for disease severity.

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Section: Introductionsupporting

confidence: 73%

Section: Introductionmentioning

confidence: 99%

A Comparative Study of SMN Protein and mRNA in Blood and Fibroblasts in Patients with Spinal Muscular Atrophy and Healthy Controls

et al. 2016

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“…A similar assay (ECLIA) based on MSD technology was recently published. The assay, sensitivity of which is 10-fold less compared to the SMN-ECL, was used for the measurement of SMN protein in buccal cells obtained from four healthy controls, one SMA carrier, and one SMA patient [ 20 ]. The SMA patient sample had approximately twice as much SMN protein as was measured in the control samples.…”

Section: Discussionmentioning

confidence: 99%

“…To support successful clinical development of a treatment for SMA, it is important to be able to readily and accurately measure SMN protein in an accessible tissue, such as whole blood. Existing methods to measure SMN protein levels include an Elecsys platform-based assay developed by Roche Diagnostics to measure SMN in whole blood [ 19 ], an electrochemiluminescence immunoassay (ECLIA) to measure SMN in buccal cells [ 20 ], an SMN-ELISA developed to measure SMN in peripheral blood mononuclear cells [ 21 ], and western blotting and a homogeneous time-resolved fluorescence (HTRF) assay [ 12 ] to measure SMN protein in tissues homogenates. Here we report the development of a sensitive electrochemiluminescence (ECL)-based immunoassay for measuring SMN protein in as little as 5μL of whole blood.…”

Section: Introductionmentioning

confidence: 99%

SMN Protein Can Be Reliably Measured in Whole Blood with an Electrochemiluminescence (ECL) Immunoassay: Implications for Clinical Trials

et al. 2016

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Spinal muscular atrophy (SMA) is caused by defects in the survival motor neuron 1 (SMN1) gene that encodes survival motor neuron (SMN) protein. The majority of therapeutic approaches currently in clinical development for SMA aim to increase SMN protein expression and there is a need for sensitive methods able to quantify increases in SMN protein levels in accessible tissues. We have developed a sensitive electrochemiluminescence (ECL)-based immunoassay for measuring SMN protein in whole blood with a minimum volume requirement of 5μL. The SMN-ECL immunoassay enables accurate measurement of SMN in whole blood and other tissues. Using the assay, we measured SMN protein in whole blood from SMA patients and healthy controls and found that SMN protein levels were associated with SMN2 copy number and were greater in SMA patients with 4 copies, relative to those with 2 and 3 copies. SMN protein levels did not vary significantly in healthy individuals over a four-week period and were not affected by circadian rhythms. Almost half of the SMN protein was found in platelets. We show that SMN protein levels in C/C-allele mice, which model a mild form of SMA, were high in neonatal stage, decreased in the first few weeks after birth, and then remained stable throughout the adult stage. Importantly, SMN protein levels in the CNS correlated with SMN levels measured in whole blood of the C/C-allele mice. These findings have implications for the measurement of SMN protein induction in whole blood in response to SMN-upregulating therapy.

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“…Steinkellner et al . [ 21 ] and Czech et al . [ 8 ] established the Elecsys ® platform’s ECLIA method using buccal cells or whole blood.…”

Section: Discussionmentioning

confidence: 99%

A new biomarker candidate for spinal muscular atrophy: Identification of a peripheral blood cell population capable of monitoring the level of survival motor neuron protein

et al. 2018

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Spinal muscular atrophy (SMA) is a severe genetic neuromuscular disorder caused by insufficiency of functional survival motor neuron (SMN) protein. Several clinical trials have been conducted with the aim of upregulating the expression of the SMN protein in SMA patients. In order to evaluate the efficiency of these SMN-targeted approaches, it has become necessary to verify SMN protein levels in the cells of SMA patients. Accordingly, we have developed a method allowing the evaluation of the functional SMN protein with < 1.5 mL of peripheral blood using imaging flow cytometry. The expression of SMN protein in CD3+, CD19+, and CD33++ cells obtained from SMA patients, was significantly reduced compared with that in cells from control subjects. In spot analysis of CD33++ cells, the intensities of SMN spots were significantly reduced in SMA subjects, when compared with that in controls. Therefore, SMN spots implied the presence of functional SMN protein in the cell nucleus. To our knowledge, our results are the first to demonstrate the presence of functional SMN protein in freshly isolated peripheral blood cells. We anticipate that SMN spot analysis will become the primary endpoint assay for the evaluation and monitoring of therapeutic intervention, with SMN serving as a reliable biomarker of therapeutic efficacy in SMA patients.

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Detection of Survival Motor Neuron Protein in Buccal Cells Through Electrochemiluminescence-Based Assay

Cited by 7 publications

References 28 publications

A Comparative Study of SMN Protein and mRNA in Blood and Fibroblasts in Patients with Spinal Muscular Atrophy and Healthy Controls

A Comparative Study of SMN Protein and mRNA in Blood and Fibroblasts in Patients with Spinal Muscular Atrophy and Healthy Controls

SMN Protein Can Be Reliably Measured in Whole Blood with an Electrochemiluminescence (ECL) Immunoassay: Implications for Clinical Trials

A new biomarker candidate for spinal muscular atrophy: Identification of a peripheral blood cell population capable of monitoring the level of survival motor neuron protein

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