Test Name
Spinal Muscular Atrophy Carrier Screening and Diagnostic Assay (SMAGEN)
CPT Code
81329
Methodology
Multiplex Fluorescent Polymerase Chain Reaction (PCR)
Turnaround Time
4–7 days
Specimen Requirements
Type:
Peripheral blood
Volume:
4 mL
Minimum Volume:
0.5 mL
Tube/Container:
Lavender BD Hemogard™ K2EDTA Tubes
Transport Temperature:
Ambient (transported within 24 hours)
Due to difficulties associated with newborn and infant draws, smaller volumes may be submitted (note: a minimum of 0.5 mL is required).
Stability
Ambient:
48 hours
Refrigerated:
7 days (at 2-8 °C)
Frozen:
Unacceptable
Additional Information
Background Information
Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder, with an incidence of approximately 1 in 10,000 births. The condition has variable severity and age of onset and has been categorized into clinical types 0-IV (OMIM# 253300, 253550, 253400, 271150).
- SMA I accounts for 60% of all SMA and has an onset of symptoms in infancy.
- SMA 0 is rare and at the most severe end of the spectrum, correlating with prenatal onset of muscular weakness and neonatal respiratory failure.
- SMA IV, while also rare, has the mildest presentation and correlates with adult-onset of muscle symptoms.
Treatment was previously limited to supportive care but is now available to prevent or slow the progression of SMA.
In all types, the genetic cause maps to an inverted duplication on chromosome 5q13.2. The complexity of the 5q13 region increases the likelihood of errors during DNA replication, resulting in a relatively high risk of deletions, gene conversions, and new mutations. SMN1 and SMN2 are two highly homologous genes located in this region, sharing more than 99% nucleotide identity. One difference between these genes affects protein coding, with alternative splicing of exon 7 in SMN2 that results in decreased production of the full length, functional SMN protein compared to SMN1. Both SMN1 and SMN2 produce the survival motor neuron (SMN) protein, with SMN2 producing just a small percentage of this protein, due to this alternate splicing. Therefore, with SMN1 accounting for the majority of SMN production, mutations in or deletions of SMN1 are the cause of the SMA phenotype.
The majority (95%) of patients with SMA have homozygous deletions of the SMN1 gene (noted as 0+0 genotype). Of the remaining 5%, most are heterozygous for a deletion of SMN1 on one chromosome and a small pathogenic, or disease-causing, variant in the SMN1 copy on the other chromosome (1+0 genotype). Pathogenic sequence variants are not detected by this test. There is no correlation between SMN1 copy number or presence of pathogenic variants and disease severity. Disease severity is inversely correlated with
the SMN2 gene copy number. In addition, the presence of the disease modifier variant c.859G>C is associated with reduced disease severity due to improved SMN2 splicing.
SMN1 copy number varies among healthy non-carrier individuals (1+1 or 2+1 genotypes). The SMA carrier rate is about 1 in 50 and varies with ethnicity (see table below). SMA carriers are also healthy. Most SMA carriers have a single copy of SMN1 on one chromosome, while the other copy is deleted (1+0 genotype). At least 4% of the population has two SMN1 copies on one chromosome (2+0 genotype), although the 2+0 genotype is more frequent in some ethnic groups, such as up to 27.5% in African-Americans. There are also reports of some carrier individuals having three copies of SMN1 on the same chromosome (3+0 genotype). Thus, the presence of three or more copies of SMN1 reduces, but does not entirely eliminate, the residual carrier risk.
Carrier risk estimates for individuals with 2 copies of SMN1 (2+0 genotype) may be further refined using the presence or absence of two common benign variants associated with SMN1 duplication, c.*3+80T>G in intron 7 and c.*211_*212del in exon 8. The presence of either variant indicates an increased risk of being a silent carrier (2+0 genotype) in some ethnicities. The presence or absence of these variants does not adjust the silent carrier risk for individuals with three copies of SMN1 (3+0 genotype). Neither SMN2 copy number nor presence or absence of the disease modifier variant c.859G>C impact carrier status.
Table 1: Residual SMA Carrier Risk after Negative Carrier Screen and Negative Family History
Residual risk for unlisted ethnicities is unknown.
Ethnicity
Carrier Rate
2 Copies SMN1
exon 7
3 Copies SMN1
exon 7
2 Copies SMN1
No variant detected
2 Copies SMN1
At least one variant detected
African American/Black
1:71
1:132
1:6997
1:375
1:39
Ashkenazi Jewish
1:56
1:514
1:5899
1:580
SMA carrier
Asian
1:50
1:719
1:5185
1:779
1:57
Asian Indian
1:50
1:428
1:5252
Not reported
Not reported
Caucasian/European
1:45
1:604
1:4719
1:814
1:12
Hispanic
1:83
1:641
1:7574
1:906
1:99
Iranian
1:16
1:96
1:1604
Not reported
Not reported
Israeli Jewish
1:38
1:450
1:4004
Not reported
Not reported
Spanish
1:40
1:781
Not reported
1:888
SMA carrier
Carrier Screening
Both the American College of Medical Genetics and Genomics and the American College of Obstetricians and Gynecologists recommend that SMA carrier screening be offered to all women/couples who are planning a pregnancy or are currently pregnant.
In individuals with a family history of SMA, it is best to obtain genetic test reports from family members before testing, to confirm the diagnosis and type of mutation. In a pan-ethnic U.S. population studied by Sugarman et al., the carrier detection rate through SMN1 dosage analysis is estimated at an average of 91%, though it ranges from 70.5% to 94.8% with ethnicity. The addition of the c.*3+80T>G and c.*211_*212del variants increases the detection of silent carriers (2+0 genotype).
Ethnicity-specific carrier and detection rates compiled from multiple studies are provided in the Table below.
Table 1: Residual SMA Carrier Risk after Negative Carrier Screen and Negative Family History
Residual risk for unlisted ethnicities is unknown.
Ethnicity
Carrier Rate
2 Copies SMN1
exon 7
3 Copies SMN1
exon 7
2 Copies SMN1
No variant detected
2 Copies SMN1
At least one variant detected
African American/Black
1:71
1:132
1:6997
1:375
1:39
Ashkenazi Jewish
1:56
1:514
1:5899
1:580
SMA carrier
Asian
1:50
1:719
1:5185
1:779
1:57
Asian Indian
1:50
1:428
1:5252
Not reported
Not reported
Caucasian/European
1:45
1:604
1:4719
1:814
1:12
Hispanic
1:83
1:641
1:7574
1:906
1:99
Iranian
1:16
1:96
1:1604
Not reported
Not reported
Israeli Jewish
1:38
1:450
1:4004
Not reported
Not reported
Spanish
1:40
1:781
Not reported
1:888
SMA carrier
Methodology
Multiplex Fluorescent Polymerase Chain Reaction (PCR) followed by Capillary Electrophoresis is used to detect SMN1 (NM_000344.3) and SMN2 (NM_017411.3) based on fragment size.
This test detects copy number of SMN1 exon 7, which is homozygously deleted in 95% of SMA patients, and copy number of SMN2 exon 7, which influences the severity of disease in affected patients. The test also detects three variants; c.*3+80T>G and c.*211_*212del associated with SMN1 gene duplication and c.859G>C associated with reduced disease severity due to improved SMN2 splicing. The c.*3+80T>G and c.*211_*212del variants are reported only for individuals with 2 copies of SMN1 as the presence of either variant indicates an increased risk of being a silent carrier (2+0 genotype). The disease modifier variant c.859G>C is reported only when zero copies of SMN1 are noted.
Variants interrogated using assembly GRCh38 hg38 (legacy name):
- SMN1 NM_000344.3; rs143838139, c.*3+80T>G, g.70952074T>G (g.27134T>G)
- SMN1 NM_000344.3; rs200800214, c.*211_*212del, g.70952646_70952647del
- SMN2 NM_017411.3; rs121909192, c.859G>C, p.Gly287Arg, g.70076545G>C
Interpretation
Among patients with a clinical presentation suggestive of SMA, detection of zero SMN1 copies confirms the diagnosis. In symptomatic patients with one SMN1 copy, SMN1 gene sequencing should be considered to identify the small percentage of patients with heterozygous sequence variants or small deletions. Symptomatic patients with two SMN1 copies are unlikely to have SMA, though very rare cases of homozygous sequence variants have been reported.
Use of this test to predict the likelihood of disease in offspring must also take into consideration that 2% of SMN1 disease-causing variants occur de novo rather than being inherited. Due to the complex inheritance of SMA by SMN1 copy number, de novo variant, and/or pathogenic variant, SMA carrier testing will never provide 100% reassurance that carrier status is eliminated or zero.
SMA carrier testing of both reproductive partners will provide the best estimate of reproductive risk and is most useful for individuals with an increased residual carrier risk following this test. Genetic counseling may be appropriate based on clinical or family history.
References
1. Alías L, Bernal S, Calucho M, Martínez E, March F, Gallano P, Fuentes-Prior P, Abuli A, Serra-Juhe C, Tizzano EF. Utility of two SMN1 variants to improve spinal muscular atrophy carrier diagnosis and genetic counselling. Eur J Hum Genet. 2018 Oct;26(10):1554–57.
2. Bürglen L, Lefebvre S, Clermont O, Burlet P, Viollet L, Cruaud C, Munnich A, Melki J. Structure and organization of the human survival motor neurone (SMN2) gene. Genomics. 1996 Mar 15;32(3):479–82.
3. Carrier screening for genetic conditions. Committee Opinion No. 691. American College of Obstetricians and Gynecologists. Obstet Gynecol. 2017;129:e41–55.
4. Chen X, Sanchis-Juan A, French CE, Connell AJ, Delon I, Kingsbury Z, Chawla A, Halpern AL, Taft RJ; NIHR BioResource, et al. Spinal muscular atrophy diagnosis and carrier screening from genome sequencing data. Genet Med. 2020 May;22(5):945–53.
5. Feng Y, Ge X, Meng L, Scull J, Li J, Tian X, Zhang T, Jin W, Cheng H, Wang X, et al. The next generation of population-based spinal muscular atrophy carrier screening: comprehensive pan-ethnic SMN1 copy-number and sequence variant analysis by massively parallel sequencing. Genet Med. 2017 Aug;19(8):936–44.
6. Lefebvre S, Bürglen L, Reboullet S, Clermont O, Burlet P, Viollet L, Benichou B, Cruaud C, Millasseau P, Zeviani M, et al. Identification and characterization of a spinal muscular atrophy-d termining gene. Cell. 1995 Jan 13;80(1):155–65.
7. Luo M, Liu L, Peter I, Zhu J, Scott SA, Zhao G, Eversley C, Kornreich R, Desnick RJ, Edelmann L. An Ashkenazi Jewish SMN1 haplotype specific to duplication alleles improves panethnic carrier screening for spinal muscular atrophy. Genet Med. 2014 Feb;16(2):149–56.
8. MacDonald WK, Hamilton D, Kuhle S. SMA carrier testing: a meta-analysis of differences in test performance by ethnic group. Prenat Diagn. 2014 Dec;34(12):1219–26.
9. Mailman MD, Heinz JW, Papp AC, Snyder PJ, Sedra MS, Wirth B, Burghes AHM, Prior TW. Molecular analysis of spinal muscular atrophy and modification of the phenotype by SMN2. Genet Med. 2002 Jan;4(1):20–26.
10. Monani UR, Lorson CL, Parsons DW, Prior TW, Androphy EJ, Burghes AH, McPherson JD. A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. Hum Mol Genet. 1999 Jul;8(7):1177–83.
11. Prior TW, Nagan N, Sugarman EA, Batish SD, Braastad C. Technical standards and guidelines for spinal muscular atrophy testing. Genet Med. 2011 Jul;13(7):686–94.
12. 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 20:27–32.
13. 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(3):228–37.
14. Wirth B, Schmidt T, Hahnen E, Rudnik-Schöneborn S, Krawczak M, Müller-Myhsok B, Schönling J, Zerres K. De novo rearrangements found in 2% of index patients with spinal muscular atrophy: mutational mechanisms, parental origin, mutation rate, and implications for genetic counseling. Am J Hum Genet. 1997 Nov;61(5):1102–11.
15. For more information about SMA, please consult GeneReviews.org.