Spinal Muscular Atrophy (SMA) via MLPA of SMN1 and SMN2

Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by muscle weakness (hypotonia, areflexia/hyporeflexia, tongue fasciculations, history of motor difficulties) that is symmetric, and progressive (Prior and Finanger. 2016. PubMed ID: 20301526). This weakness can affect the ability to crawl, walk, sit up, and control head movements, and additional clinical features include growth failure, restrictive lung disease, scoliosis, joint contractures, and sleep difficulties (Feng et al. 2017. PubMed ID: 28125085). Onset ranges from before birth to young adulthood, with later onset correlating to a less severe phenotype (Prior and Finanger. 2016. PubMed ID: 20301526). SMA affects approximately 1 in 6,000-10,000 births depending on ethnicity (Prior et al. 2011. PubMed ID: 21673580). There are five different classifications of SMA; however, a clear delineation into the subtypes is not always possible and the phenotypes represent more of a spectrum. SMA type 0 presents with severe weakness and hypotonia at birth, and early respiratory failure is common. There may be decreased fetal movement and joint contractures as well. SMA type 1 exhibits onset before 6 months of age. These individuals can only sit with support, and they can have normal or minimal facial weakness, difficulties sucking and swallowing, and mild joint contractures. SMA type 2 has an onset between 6-18 months. These individuals can sit independently after they are placed and can have postural finger tremors. SMA type 3 has onset after 18 months of age. These individuals can walk and move independently. SMA type 4 has onset in adulthood. Patients present with a milder form of muscle weakness (Prior and Finanger. 2016. PubMed ID: 20301526).

SMA is inherited in an autosomal recessive manner. However, approximately 2% of SMA patients have de novo pathogenic variants (Prior and Finanger. 2016. PubMed ID: 20301526). About 95-98% of individuals with SMA have a homozygous deletion of exon 7 of the SMN1 gene, which encodes the full length survival motor neuron protein (Prior and Finanger. 2016. PubMed ID: 20301526). The remaining 2% to 5% percent of individuals with SMA are compound heterozygotes for the SMN1 exon 7 deletion and an intragenic missense, nonsense or frameshift variant in SMN1 (Ogino and Wilson. 2004. PubMed ID: 14711346).

SMN1 is located in a complex region of chromosome 5 at band q13.2 that includes the homologous SMN2 gene. SMN1 and SMN2 are arranged in an inverted duplication, with SMN1 being telomeric to SMN2. These two genes differ by only 5 nucleotides, which includes a single variant (c.840C>T) in the coding sequence of SMN2 that affects splicing of SMN2. Consequently, SMN2 can only produce a fraction of the amount of full length transcripts compared to SMN1. In patients with a homozygous deletion of SMN1, the number of copies of SMN2 can modulate the clinical severity of the disorder. This observation has led to the development of new therapies for SMA designed to increase expression from SMN2. Consequently, assessment of the copy number of both SMN1 and SMN2 is clinically relevant in affected individuals (Talbot and Tizzano. 2017. PubMed ID: 28644430).

SMN1 deletions will be detected in over 99% of individuals with Spinal Muscular Atrophy in either the homozygous state (95% to 98% of affected individuals) or in the compound heterozygous state with another pathogenic variant (2% to 5% of affected individuals) (Rudnik-Schoneborn et al. 2012. PubMed ID: 22510849).

Multiplex Ligation-Dependent Probe Amplification (MLPA) enables the detection of deletion and duplications of single and multiple exons within a given gene (Eijk-Van Os and Schouten 2011. PMID: 20938835). It is a semi-quantitative technique to determine relative copy number using a multiplex PCR-based reaction. Only hybridized and ligated adjacent probe oligonucleotides of approximately 60 nucleotides in length are amplified using PCR, and thus are specific for the sequence of interest. A stuffer sequence attached to the probe ensures a particular length for deciphering the probe target. For additional information please see www.mlpa.com. This test involves copy number analysis of both the SMN1 and SMN2 genes.

This test is validated for blood, saliva, cell culture, and the DNA Genotek OCD-100 kit.