Beschreibung:
<jats:title>Abstract</jats:title>
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<jats:title>Background</jats:title>
<jats:p>Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by insufficient levels of the survival motor neuron (SMN) protein. Almost 95% of SMA cases are caused by the homozygous deletion involving exon 7 of the SMN1 gene. Its incidence is estimated in 1:6000–11 800 live births and it is the most common genetic cause of death in infancy. Symptoms of SMA include several cognitive disabilities leading to death, due to loss of motor functions, rapid denervation, and progressive weakening. In addition, another very common syndrome among newborns is Severe Combined Immunodeficiency (SCID). SCID is a genetic disease that may compromise the Lymphocytes T, B, and/or Natural Killers cells functions causing repeated and persistent infections. Low levels of the recombination excision circles of T and B cells (TREC and KREC, respectively) are indicative of SCID or other lymphopenias. Due to the early onset and rapid progression of SMA and SCID, accurate diagnosis of these syndromes is of utmost importance to perform therapeutic intervention before the onset of symptoms. Therefore, this study aims to describe the analytical and clinical validation of a kit for the implementation of a multiplex SCID/SMA real-time diagnoses routine in dried blood spots (DBS).</jats:p>
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<jats:title>Methods</jats:title>
<jats:p>Commercial quantified positive control and samples were used for the analytical and clinical validation, respectively. The Clinical validation included 67 samples with the SMN1 gene and 7 samples with gene deletion. For the SCID evaluation, 60 samples with normal levels of TREC and KREC and 11 samples with reduced levels were included. DNA isolation was performed with the DNA Extract All Reagents Kit (Thermo Fisher, Massachusetts, EUA) from a 3.2mm disc of newborn DBS. The qPCR assay was performed by the TaqMan SCID/SMA Plus Assay (Thermo Fisher, Massachusetts, EUA) to target TREC, KREC, and SMN1 according to the manufacturer's instructions. The analysis parameters included: (i) Determination of assay efficiency; (ii) Analytical sensitivity (Limit of detection); (iii) Intra-assay and inter-assay precision; and (iv) Clinical validation. All reactions included the endogenous control RNase P.</jats:p>
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<jats:title>Results</jats:title>
<jats:p>The assay demonstrated reaction efficiency &gt;98% and the limit of detection was 30 copies per reaction for TREC, KREC, and SMN1 targets with a 95% confidence interval. The regression equations demonstrated good amplification conditions with a coefficient of determination (r2) = 0.99. The precision experiments demonstrated optimal repeatability and reproducibility. The results obtained in the clinical validation showed 100% agreement with the expected results for the SMN1 target. To the TREC and KREC targets, the assay demonstrated 95% and 100% agreement for normal and reduced levels, respectively.</jats:p>
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<jats:title>Conclusion</jats:title>
<jats:p>An accurate and rapid diagnosis is essential to guide the clinical management of SCID and SMA in newborns. Here, we evaluated and described the TaqMan SCID/SMA Plus Assay performance, a molecular test for detecting SCID and SMA by Quantitative Real-time PCR (qPCR). This assay was highly efficient due offer of a multiplex detection method, allowing the rapid release of results. The use of this tool can help clinicians in rapid newborn screening, providing agility in clinical conduct.</jats:p>
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