The Melt Assay SALSA MC002 SMA Newborn Screen is an in vitro diagnostic (IVD) or research use only (RUO) semi-quantitative manual assay for the detection of homozygous deletions of exon 7 in the SMN1 gene in DNA isolated from dry blood spot (DBS) cards. MC002 SMA Newborn Screen is intended to determine a potential cause for spinal muscular atrophy (SMA) through molecular genetic screening of newborns. The MC002 assay cannot determine absolute SMN1 or SMN2 copy numbers with the exception of 0 copies.

For the full intended purpose, see the instructions for use.

Disease overview

Sources: Hendrickson et al. (2009); NCBI GeneReviews.

Symptoms

• Progressive muscle weakness due to degeneration of lower motor neurons.
• Most common symptoms are difficulty in breathing, swallowing, and walking.
• SMA subtypes are distinguished by age of onset and maximum motor function obtained: type I, OMIM# 253300; type II, OMIM# 253550; type III, OMIM# 253400; and type IV, OMIM# 271150.

For more information on SMA: http://www.ncbi.nlm.nih.gov/books/NBK1352/.

Gene overview

* Note: a different exon numbering (exons 1-9) is used by LRG (http://www.lrg-sequence.org/) and NCBI (NG_008691.1; NG_008728.1).

• 5q13.2 is a complicated, highly variable genomic region, containing a 500 kb inverted duplication.
• The only clinically relevant difference between SMN1 and SMN2 is a single nucleotide difference in exon 7 (c.840C>T) affecting mRNA splicing. As a consequence, 90% of SMN2 pre-mRNAs are alternatively spliced and lack exon 7, while 10% are full-length transcripts producing a protein that is identical to the protein coded by SMN1.

In unaffected individuals (including carriers), a complete absence of the SMN2 gene does not have any clinical consequences. SMA patients have at least one SMN2 copy.

SALSA MC002 SMA Newborn Screen provides information on the presence or absence of SMN1 and SMN2 exon 7 specific sequences in a sample. The relative ratio of the SMN1- and SMN2-specific melt peaks reflects the ratio between SMN1 and SMN2 copies in the DNA sample tested. This means that a sample with two copies of both SMN1 and SMN2 will give the same ratio as a sample with only one copy of both SMN1 and SMN2. No carriers are identified with the MC002 assay as it cannot determine absolute SMN1 or SMN2 copy numbers with the exception of 0 copies.

In the assay a single PCR primer pair is used to amplify a 180 nt fragment of the SMN1 and SMN2 genes that includes exon 7. Formation of at least one of these amplicons is expected in all samples as the complete absence of both the SMN1 and SMN2 genes is incompatible with life. In the MC002 PCR reaction, a larger amount of one primer is supplied compared to the other PCR primer. As a result, one strand is formed in excess (asymmetric PCR) (Figure 1A).

A 5' Cy5 fluorescently labelled oligonucleotide probe is also present in each reaction. This fluorescent probe does not affect the PCR reaction. When the probe oligo is free in solution, probe fluorescence is negligible as there is a specific quencher moiety bound to the 3' end of the probe. When the probe oligo is hybridized to a complementary SMN1 or SMN2 amplicon strand, the Cy5 dye and the quencher molecule are separated, resulting in maximal fluorescence.

After the PCR reaction, the reaction temperature is lowered and the fluorescent probe hybridizes to the SMN1 and SMN2 amplicon strands that were produced in excess, resulting in high fluorescence (Figure 1B). When the reaction mixture is slowly heated, the probe will detach from the amplicon strands at a certain temperature (Figure 1C). This is referred to as the "melting temperature" (Tm) and it is dependent on the sequence of the PCR amplicon.

While slowly heating the reaction mixtures, the fluorescence is measured. The Tm of a probe on a specific sample DNA is identified by a rapid drop in fluorescence during the gradual heating of a sample. These are seen in the so-called melt curve (Figure 1D). A derivative of these melt curves is often used to visualize the Tms as peaks (Figure 1E).

The MC002 probe forms a perfect amplicon-probe hybrid with amplicons that contain the SMN1 exon 7 wildtype sequence resulting in a Tm of approximately 63°C. When bound to an SMN2 amplicon, the probe-amplicon hybrid has a 1 nt mismatch, resulting in a Tm that is approximately 7°C lower (~56°C).

Figure 1. SALSA Melt assay technical explanation (click image to enlarge). (A) The exon 7 sequence of SMN1 and SMN2 is amplified using a single PCR primer pair, with one primer in excess (asymmetric PCR). (B) A fluorescent probe binds to one strand of the SMN1 and SMN2 amplicons. The 5' fluorescent group is now separated from its 3' quencher at the other end of the probe oligo, resulting in strong fluorescence emittance. (C) In the upper part of the image, the fluorescent probe binds to the SMN1 amplicon that has no sequence difference with the probe. In the lower part of the image, the probe binds to the SMN2 amplicon that has a single nucleotide difference with the probe. When the reaction is slowly heated, the probe-amplicon binding becomes unstable. The probe will dissociate from the SMN2 amplicons at a lower temperature than from the SMN1 amplicons, which have no mismatch. When the probe dissociates from its target sequence, fluorescence decreases, as the fluorescent group is quenched by the 3' quencher. (D) A plot showing the fluorescence vs. temperature of a sample with both SMN1 and SMN2 amplicons. As the temperature increases, more probe molecules dissociate from the amplicons and the fluorescence decreases. This probe-amplicon dissociation occurs at a lower temperature for the probe-SMN2 amplicon hybrid that contains a mismatch. (E) The first derivative, -d(RFU)/dT of the curve shown in °C. The graph shows a peak at each temperature with a rapid drop in fluorescence. These temperatures are the Tm for the probe-SMN2 amplicon hybrid with a 1 nt mismatch and the probe-SMN1 amplicon hybrid with a perfect match.

When copies of both SMN1 and SMN2 are present, two separate Tms will be generated for the probe, visualised as two separate peaks (Figure 2A). If only copies of SMN1, or only copies of SMN2, are present in a sample, a single Tm/peak will be generated. Please note that an extra, Q-fragment specific peak with a clearly lower Tm (49°C) may be visible in reactions that contain a low amount of sample DNA.

Melt curve profiles obtained are compared to the melt curves obtained on the SALSA SD074 Threshold DNA known to have a single SMN1 copy and five SMN2 copies (Figure 2B). Samples where the SMN1 peak is absent, or the SMN1:SMN2 peak ratio is lower* as compared to the SALSA SD074 Threshold DNA, must be further tested by an independent technique, such as SALSA MLPA Probemix P021 SMA. SALSA SD075 gives an example of what a SMA patient profile would look like (Figure 2C). The number of samples requiring further testing in newborn screening programs is expected to be below 0.2%.

* A very low SMN1-specific melt peak might be due to contamination of a sample with DNA from another sample. For this reason, not only samples where the SMN1 peak is absent, but also samples with a very low SMN1 peak should be further tested.

Figure 2. Typical MC002 results (click image to enlarge). Expected melt peak profiles for standard samples. (A) Melt peak profile of an unaffected individual with specific SMN1 (63°C) and SMN2 (56°C) peaks showing an SMN1:SMN2 ratio of 1:1. (B) SALSA SD074 Threshold DNA melt peak profile with specific SMN1 (63°C) and SMN2 (56°C) peaks showing a high SMN2:SMN1 ratio. (C) SALSA SD075 Positive DNA melt peak profile showing an SMN2 (56°C) peak and absence of the SMN1 peak with known SMN1:SMN2 ratio of 0:2; indicative of an SMA patient.

The MC002 probemix contains a small amount of a Q-fragment. This is an oligonucleotide that is amplified with the same PCR primer pair as the SMN1 and SMN2 amplicons and contains a sequence that is similar to the sequence detected by the Cy5-labelled probe. Binding of the MC002 probe to the Q-fragment amplicon results in a probe-amplicon hybrid with a Tm of 49°C, approximately 7°C lower than the SMN2 melt peak.

As the Q-fragment is present in very low quantities, the Q-fragment specific melt peak will be absent, or very low, in reactions with sufficient sample DNA as the Q-fragment will be outcompeted by SMN1 and SMN2 copies (Figure 3A). When an insufficient amount of sample DNA is present (below 0.25 ng), the Q-fragment specific melt peak will be the highest melt peak present (Figure 3B). When this occurs, reliable conclusions cannot be made. In no DNA reactions, the Q-fragment specific melt peak is the only peak expected (Figure 3C).

Figure 3. Example MC002 melt peak patterns (click image to enlarge). (A) A sample that contains 1 copy of both the SMN1 and SMN2 genes; the amount of sample DNA is rather low but sufficient, resulting in a visible, but low, Q-fragment specific peak. (B) A sample that has insufficient sample DNA, resulting in the Q-fragment melt peak being the highest of the three peaks. (C) A no DNA reaction where the Q-fragment melt peak is the only peak present.

SALSA MC002 SMA Newborn Screen is CE-marked under the IVDR for in vitro diagnostic (IVD) use in Europe. This assay has also been registered for IVD use in Israel and Thailand.

This assay is for research use only (RUO) in all other territories.

Each MC002 kit contains probemix, polymerase and control DNA. Reagents do not have to be purchased separately.

In case more extensive MC002 validation is required, DNA samples available from the Coriell Institute can be used. The following Coriell cell line-derived DNA samples have been tested at MRC Holland and verified for the listed SMN1 and SMN2 exon 7 copy numbers (using MLPA). Please note that the quality of cell lines can change.

Comparison of SMA products available from MRC Holland

MRC Holland offers four different assays for SMA that fit the complete range of genetic testing needs. The table below indicates which product can best be used for which purpose.

● Primary test.
○ Secondary test.
^# Increased detection of silent carriers: individuals with 2 SMN1 copies on one allele + 0 on the other.
^Δ MC002: no absolute copy numbers aside from 0 determined.
^† P060 cannot be used to determine SMN2 copy numbers for diagnostic purposes. The SMN2 probes in P060 can only be used as an interpretation aid for SMN1 copy number determination. SMN2 copy number determination for diagnostic purposes can only be done with P021.

Key related products

• P021 SMA: Patient testing for spinal muscular atrophy. Quantification of exons 7 and 8 of SMN1 and SMN2. Quantification of the combined SMN1+SMN2 copy number of each other exon.
• P060 SMA Carrier: Carrier testing for spinal muscular atrophy. Quantification of exons 7 and 8 of SMN1.
• P460 SMA (Silent) Carrier: Carrier testing for spinal muscular atrophy. Quantification of exons 7 and 8 of SMN1 with increased chance of detection of silent SMA carriers by inclusion of probes for a specific SNP haplotype.

Other related products

• P058 IGHMBP2: Autosomal recessive distal spinal muscular atrophy 1 (DSMA1). Gene included IGHMBP2.

MC002 SMA Newborn Screen

• MC002 flyer
• MC002 demosheet

Newborn Screening for Spinal Muscular Atrophy (SMA)

• Information page about newborn screening for SMA

General resources

• Melt Assays technique overview

Learning

• Help centre with other learning resources

Selected publications using MC002 SMA Newborn Screen

• Brkušanin M et al. (2024). Our Journey from Individual Efforts to Nationwide Support: Implementing Newborn Screening for Spinal Muscular Atrophy in Serbia. Int J Neonatal Screen. 10:57.
• Gos M et al. (2024). 129P Newborn screening for spinal muscular atrophy in Poland – a summary of 3-year experience. Neuromuscul Disord. 43:104441.27.
• Strunk A et al. (2019). Validation of a Fast, Robust, Inexpensive, Two-Tiered Neonatal Screening Test algorithm on Dried Blood Spots for Spinal Muscular Atrophy. Int J Neonatal Screen. 5:21.

References

• Hendrickson BC et al. (2009). Differences in SMN1 allele frequencies among ethnic groups within North America. J Med Genet. 46:641-4.