Detection of Spinal Muscular Atrophy Patients Using Dried Saliva Spots.

Yogik Onky Silvana Wijaya,Toshio Saito,Haruo Shintaku,Masakazu Shinohara,Kentaro Okamoto,Hisahide Nishio,Yasuhiro Takeshima,Hiroyuki Awano,Emma Tabe Eko Niba

doi:10.3390/genes12101621

Abstract

Spinal muscular atrophy (SMA) is a lower motor neuron disease, once considered incurable. The main symptoms are muscle weakness and muscular atrophy. More than 90% of cases of SMA are caused by homozygous deletion of survival motor neuron 1 (SMN1). Emerging treatments, such as splicing modulation of SMN2 and SMN gene replacement therapy, have improved the prognoses and motor functions of patients. However, confirmed diagnosis by SMN1 testing is often delayed, suggesting the presence of diagnosis-delayed or undiagnosed cases. To enable patients to access the right treatments, a screening system for SMA is essential. Even so, the current newborn screening system using dried blood spots is still invasive and cumbersome. Here, we developed a completely non-invasive screening system using dried saliva spots (DSS) as an alternative DNA source to detect SMN1 deletion. In this study, 60 DSS (40 SMA patients and 20 controls) were tested. The combination of modified competitive oligonucleotide priming-polymerase chain reaction and melting peak analysis clearly distinguished DSS samples with and without SMN1. In conclusion, these results suggest that our system with DSS is applicable to SMA patient detection in the real world.

Highlights

Spinal muscular atrophy (SMA) is one of the most devastating neuromuscular disorders, characterized by motor neuron degeneration [1]
The patients had been diagnosed as having survival motor neuron 1 (SMN1)-deleted SMA based on an SMN1 deletion test using the PCR restriction fragment length polymorphism (PCR-RFLP) method [6]
Nested PCR products of these samples were electrophoresed on an agarose gel (Figure 2A)

Summary

Introduction

Spinal muscular atrophy (SMA) is one of the most devastating neuromuscular disorders, characterized by motor neuron degeneration [1]. The majority of SMA cases (~95%) involve homozygous deletion of survival motor neuron 1 (SMN1), while some others (~5%) carry a deleterious SMN1 mutation, SMN1 is considered the disease-causing gene [1,2]. The homologous gene, SMN2, serves as an SMA-modifying gene because a high SMN2 copy number is generally associated with a milder phenotype [4,5]. SMN1 and SMN2 are identical except for five nucleotides located in intron 6, exon 7, intron 7, and exon 8 [1]. Molecular diagnosis of SMA requires the detection of SMN1specific nucleotides, especially the one located in exon 7. Several methods have been developed to achieve this, including single-stranded conformation polymorphism (SSCP) analysis [1], restriction enzyme digestion analysis [6], modified competitive oligonucleotide priming-polymerase chain reaction (mCOP-PCR) [7,8], multiplex ligation-dependent probe amplification (MLPA) [9], digital droplet PCR (ddPCR) [10] and next-generation sequencing (NGS) [11]

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Discussion

Conclusion