September 2020 — Molecular

A 17 year old boy presented with symptoms consistent with motor neuron disease. He has no known family history of neuromuscular disease. Molecular analysis from an outside institution reported 2 copies of SMN1, 2 copies of SMN2, and a homozygous point mutation. However it could not be determined whether this mutation was in the SMN1 or SMN2 gene. Importantly, microarray analysis revealed consanguinity. SMN1 full gene analysis revealed a homozygous pathogenic mutation in exon 6 (c.683T>A).

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What is the correct diagnosis and what other molecular test may have been applicable?

  • Further Molecular Testing Needed, Spinal Muscular Atrophy Diagnostic Assay by Deletion/Duplication Analysis
  • Further Molecular Testing Needed, Neuromuscular Genetic Panels by Next Generation Sequencing
  • Spinal Muscular Atrophy, Familial Mutation Targeted Testing
  • Spinal Muscular Atrophy, SMN2 Full Gene Analysis

The correct answer is...

The correct answer is Spinal Muscular Atrophy, Familial Mutation Targeted Testing.

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 the age of two 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 two 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 two new therapies that have recently been FDA-approved for the treatment of SMA. In December of 2016, Spinraza was FDA-approved to treat all types of SMA. Spinraza is an antisense oligonucleotide that binds downstream of exon 7 promoting the inclusion of exon 7, thereby increasing the amount of full-length SMN2 mRNA and, subsequently, increasing the level of full-length SMN protein. In May 2019, the FDA approved the use of Zolgensma for the treatment of SMA patients under the age of two. Zolgensma is gene replacement therapy using the AAV9 viral vector to replace the SMN1 gene. Molecular diagnosis of SMA is imperative to be able to initiate therapy in a timely manner as to maximize the protection of motor function in these patients.

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, typically via long-range-PCR of SMN1 exons 2-8, followed by bidirectional Sanger sequence analysis. Lastly, if the results from SMN1 full gene analysis is negative, a neuromuscular genetic panel analysis, typically via next-generation sequencing (NGS) and/or Sanger sequencing, could be considered.

In cases in which one or more variants have been identified in the patient or a family member, familial targeted testing (FMTT) can be considered. In this case, the patient was reported to have a known pathogenic variant. Furthermore, it was reported that consanguinity was determined via microarray analysis. While FMTT was not needed to make the SMA diagnosis for this patient, it could have been utilized to make the diagnosis in the patient by targeting this variant as well as determining that each parent was heterozygous for the same variant.

SMN2 full gene analysis is not needed to make the diagnosis of SMA. However, of note, there are known sequence variants of SMN2 that have been shown to be modulators of SMA disease severity. The variant c.859G>C in SMN2 exon 7 creates a new splice enhancer-binding site which in turn increases the exon 7 inclusion. This variant is correlated with a milder SMA phenotype. The A-44G variant in the SMN2 gene is another positive disease modifier. This variant was shown to decrease RNA-binding protein HuR and increase exon 7 inclusion. This variant is also associated with a milder clinical phenotype. Recently, a large study of 217 SMA patients in which the SMN2 gene was fully sequenced, it was determined that the variants A-44G, A-549G, and C-1897T in intron 6 of SMN2 were significantly associated with a clinical presentation that was discordant SMN2 copy number. Therefore, SMN2 full gene analysis may be insightful in SMA patients that have a clinical presentation that is discordant with their SMN2 copy number.

1. Burghes, A., Beattie, C. Spinal muscular atrophy: why do low levels of survival motor neuron protein make motor neurons sick? Nat Rev Neurosci 2009; 10, 597–609.
2. Bürglen L, Lefebvre S, Clermont O, et al. Structure and organization of the human survival motor neurone (SMN) gene. Genomics. 1996;32(3):479-482.
3. Clermont O, Burlet P, Benit P, et al: Molecular analysis of SMA patients without homozygous SMN1 deletions using a new strategy for identification of SMN1 subtle mutations. Hum Mutat 2004; 24:417-427.
4. Faravelli I, Nizzardo M, Comi GP, Corti S. Spinal muscular atrophy--recent therapeutic advances for an old challenge. Nat Rev Neurol. 2015; 11(6):351-9.
5. Feldkötter M, Schwarzer V, Wirth R, Wienker TF, Wirth B. Quantitative analyses of SMN1 and SMN2 based on real-time lightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. Am J Hum Genet. 2002; 70(2):358-368.
6. Kubo Y, Nishio H, Saito K: A new method for SMN1 and hybrid SMN gene analysis in spinal muscular atrophy using long-range PCR followed by sequencing. J Hum Genet 2015; 60: 233-239.
7. Lefebvre S, Bürglen L, Reboullet S, et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell. 1995; 80(1):155-165.
8. Mahajan R. Onasemnogene Abeparvovec for Spinal Muscular Atrophy: The Costlier Drug Ever. Int J Appl Basic Med Res. 2019; 9(3):127-128.
9. Pearn J. Incidence, prevalence, and gene frequency studies of chronic childhood spinal muscular atrophy. J Med Genet. 1978; 15(6):409-13.
10. Prior TW. Carrier screening for spinal muscular atrophy. Genet Med 2008;10:840-2.
11. Prior T, Finanger E: Spinal Muscular Atrophy. Gene Reviews. Update Dec 22, 2016.
12. Prior TW, Krainer AR, Hua Y, et al. A positive modifier of spinal muscular atrophy in the SMN2 gene. Am J Hum Genet. 2009; 85(3):408-413.
13. Ruhno C, McGovern VL, Avenarius MR, et al. Complete sequencing of the SMN2 gene in SMA patients detects SMN gene deletion junctions and variants in SMN2 that modify the SMA phenotype. Hum Genet. 2019; 138(3):241-256.
14. Shorrock, H.K., Gillingwater, T.H. & Groen, E.J.N. Overview of Current Drugs and Molecules in Development for Spinal Muscular Atrophy Therapy. Drugs 2018; 78, 293–305.
15. Sugarman EA, Nagan N, Zhu H, Akmaev VR, Zhou Z, Rohlfs EM, Flynn K, Hendrickson BC, Scholl T, Sirko-Osadsa DA, Allitto BA. Pan-ethnic carrier screening and prenatal diagnosis for spinal muscular atrophy: clinical laboratory analysis of >72,400 specimens. Eur J Hum Genet. 2012; 20(1):27-32.
16. Vidal-Folch N, Gavrilov D, Raymond K, Rinaldo P, Tortorelli S, Matern D, Oglesbee D. Multiplex Droplet Digital PCR Method Applicable to Newborn Screening, Carrier Status, and Assessment of Spinal Muscular Atrophy. Clin Chem. 2018; 64(12):1753-1761.
17. Wirth B: An update of the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA). Hum Mutat 2000; 15:228-237.
18. Wu X, Wang SH, Sun J, Krainer AR, Hua Y, Prior TW. A-44G transition in SMN2 intron 6 protects patients with spinal muscular atrophy. Hum Mol Genet. 2017; 26(14):2768-2780.

Photo of Sara Cook, M.D., Ph.D. Sara Cook, M.D., Ph.D.
Resident, Anatomic and Clinical Pathology
Mayo Clinic
Photo of Ross Rowsey, Ph.D. Ross Rowsey, Ph.D.
Senior Associate Consultant, Laboratory Genetics and Genomics
Mayo Clinic
Assistant Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

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