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.
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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- Lefebvre S, Bürglen L, Reboullet S, et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell. 1995; 80(1):155-165.
- Mahajan R. Onasemnogene Abeparvovec for Spinal Muscular Atrophy: The Costlier Drug Ever. Int J Appl Basic Med Res. 2019; 9(3):127-128.
- Pearn J. Incidence, prevalence, and gene frequency studies of chronic childhood spinal muscular atrophy. J Med Genet. 1978; 15(6):409-13.
- Prior TW. Carrier screening for spinal muscular atrophy. Genet Med 2008;10:840-2.
- Prior T, Finanger E: Spinal Muscular Atrophy. Gene Reviews. Update Dec 22, 2016.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.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.
Sara Cook, M.D., Ph.D.
Resident, Anatomic and Clinical Pathology
Daniel Anderson, D.O.
Senior Associate Consultant, Neurology