September 2021 – Molecular Pathology

Siblings, a 15-year-old girl and a 16-year-old boy, present with progressive muscle weakness. Their history is notable for delayed motor milestones and proximal muscle weakness, progressing to loss of ambulation. Spinal muscular atrophy (SMA) deletion duplication analysis reveals both siblings to have one copy of the SMN1 gene and two copies of the SMN2 gene. Results of the SMN1 full gene analysis are included in the table below.

Figure 1: SMN1 full gene analysis 

Can a definitive molecular diagnosis of SMA be made, and what other molecular tests may be needed?

  • A diagnosis of SMA can be made. The c.5C>G variant in the SMN1 or SMN2 gene is pathogenic.
  • A diagnosis of SMA can be made. The patients only have one copy of SMN1.
  • A diagnosis of SMA cannot be made. A definitive molecular diagnosis would require full gene analysis that distinguishes SMN1 exon 1 from SMN2 exon 1.
  • A diagnosis of SMA cannot be made. Further testing with Neuromuscular Genetic Panels by Next Generation Sequencing is needed.

The correct answer is ...

A molecular diagnosis of SMA cannot be made.  A definitive molecular diagnosis would require full gene analysis that distinguishes SMN1 exon 1 from SMN2 exon 1.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease which occurs in one out of every 10,000 live births, making it the leading genetic cause of infant mortality. SMA is characterized by degeneration of the alpha motor neurons of the spinal cord leading to progressive muscle weakness, and in cases of Type I SMA, progression to respiratory failure and death before age 2 if supportive therapies are not initiated.  

However, SMA is a clinically heterogeneous disease, with Type I being the most severe of the four types. Patients with Type II SMA present with marked delay in gross motor development by 6-18 months of life, never become ambulatory, and suffer from progressive weakness, scoliosis, and restrictive lung disease. Life expectancy of SMA Type II patients ranges from 2 years of age to 40 years of age. Patients with Type III SMA have variable age of onset and clinical course. They often require wheelchair assistance by adolescence; however, their life expectancy does not significantly differ from the general population. The mildest type of SMA, Type IV, generally presents in the second or third decade of life, with mild proximal limb girdle weakness that progresses slowly.

All four types of SMA are caused by a mutation or deletion in the survival motor neuron 1 (SMN1) gene. Humans also have the survival motor neuron 2 (SMN2) gene, which differs from SMN1 gene by five nucleotides.  All of these nucleotides are in non-coding regions, except for the cysteine to thymine variation in exon 7. Ultimately, this variation is translationally silent and the amino acid sequence of SMN2 is identical to SMN1; however, the variation affects the alternative splicing such that 90% of transcripts are missing exon 7 and result in an unstable protein that is rapidly degraded. Accordingly, there is an inverse relationship between SMN2 copy number and disease severity. In general, type I SMA patients have two copies of SMN2, type II SMA patients have three copies of SMN2, and type III SMA patients have three or four copies of SMN2.

Molecular analysis of SMA patients has become increasingly important, as there are now three new therapies that have recently been FDA-approved for the treatment of SMA. The SMN1 gene exon 7 is homozygously absent in approximately 95% of affected patients. The great majority of the remainder of SMA cases is heterozygous for the exon 7 deletion and a small more subtle mutation in the other allele; compound heterozygotes. SMA cases resulting from homozygous point mutations are exceedingly rare. Therefore, the first-line molecular analysis for the diagnosis of SMA consists of detecting the homozygous loss of exon 7 of the SMN1 gene. This can be done using techniques such as MLPA, qPCR, or ddPCR, which typically detect the presence of exon 7 of the SMN1 gene, thereby quantifying the number of copies of the SMN1 gene. If the result from these techniques is inconclusive or negative, it is recommended to perform full gene analysis of the SMN1 gene. This analysis typically consists of long-range-PCR of SMN1 exons 2-8, followed by bidirectional Sanger sequence analysis. SMN1 exon 1 is also PCR-amplified and bidirectionally Sanger-sequenced; however, the primers used to amplify SMN1 exon 1 will also amplify SMN2 exon 1. Therefore, if a pathogenic variant is detected in exon 1, a definitive molecular diagnosis cannot be made. An individual with one copy of SMN1 and a variant in exon 1 of SMN2 gene does not have SMA, whereas an individual with one copy of SMN1 with a pathologic variant in exon 1 of SMN1 has SMA. 

In this case, both siblings tested positive for an exon 1 variant. Due to the limitations of the test, it cannot be determined if this variant is in SMN1 or SMN2. However, both siblings have the same variant and both have clinical histories suggestive of SMA. Therefore, a clinical diagnosis can be made. Importantly, with this diagnosis, both patients are eligible for disease modifying therapy. Of note, there are reports of efficient long-range PCR methods that are able to amplify exons 1-8 of SMN1. This method would be able to distinguish pathogenic variants in exon 1 of SMN 1 from variants in exon 1 of SMN2.


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Sara Cook, M.D., Ph.D.

Resident, Anatomic and Clinical Pathology
Mayo Clinic

Daniel Anderson, D.O.

Senior Associate Consultant, Neurology
Mayo Clinic

MCL Education

This post was developed by our Education and Technical Publications Team.