Intended purpose
The SALSA MLPA Probemix P021 SMA is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplication in the SMN1, SMN2 and exon 5 of the NAIP genes in genomic DNA isolated from human peripheral whole blood specimens, prenatal samples, from either (un)cultured amniotic fluid obtained in week 16 of pregnancy or later, free from blood contamination (un)cultured chorionic villi, free from maternal contamination fetal blood or Dried Blood Spot (DBS) cards. P021 SMA is intended to establish or confirm a potential cause for and clinical diagnosis of Spinal Muscular Atrophy (SMA), carrier testing and for molecular genetic testing of at-risk family members. Secondly, the assay can be used for SMN2 (and NAIP) copy number determination in (pre-symptomatic) SMA patients as an aid in prognosis and for treatment eligibility.

In the majority of SMA patients (>95%), the disease is caused by a homozygous loss of the SMN1 gene, usually detected by the absence of exon 7 specific markers. In a small number of SMA cases, the causative defect concerns a loss of other exon(s) in SMN1. Both defects can be detected by MLPA probemix P021 SMA. Copy number variations (CNVs) detected with P021 SMA should be confirmed with a different technique. In particular, CNVs detected by the SMN1 exon 7 probe always require confirmation by another method. Point mutations, which cause SMA in a small number of cases, will not be detected by MLPA. It is therefore recommended to use this assay in combination with sequence analysis.

Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, parental evaluation, clinical genetic evaluation, and counselling, as appropriate. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.

This device is not intended to be used for standalone diagnostic purposes, pre-implantation screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.

¹ Please note that this probemix is for in vitro diagnostic (IVD) use in the countries specified at the end of the product description. In all other countries, the product is for research use only (RUO).
² To be used in combination with a SALSA MLPA Reagent Kit, SALSA Reference Selection DNA SD082, Coffalyser.Net analysis software.

Clinical background
Spinal Muscular Atrophy (SMA) is a group of autosomal recessive neuromuscular disorders characterized by degeneration of the anterior horn cells of the spinal cord, leading to symmetrical muscle weakness and atrophy. SMA is a heterogeneous disease, its phenotype ranging from early onset with a life expectancy of less than 2 years to a late onset with very mild symptoms. SMA disease severity is strongly correlated with SMN2 copy number. The estimated incidence of SMA is 1:6,000-1:10,000.

Two highly similar genes play a pivotal role in SMA: SMN1 and SMN2. The only clinically relevant difference between the two genes is a single nucleotide difference in exon 7. SMN2 is translated much less efficiently in a functional SMN protein; therefore, it is the SMN1 gene which is the determinant factor in SMA. Someone lacking functional copies of SMN1 is almost always a SMA patient. In most populations, 95-98% of SMA patients show complete absence of at least exon 7 of the SMN1 gene (this percentage is lower in SMA patients from African descent (Labrum et al. 2007)). Most of the remaining patients have a single copy of the SMN1 gene which is inactive due to a point mutation or a deletion of exons 1-6. Please note that rare cases have been described of healthy individuals with homozygous loss of SMN1 exon 7 and only 2 or 3 SMN2 copies (e.g. Helmken et al. 2003).

SMA carriers are symptom-free. The great majority of SMA carriers can be identified by the presence of a single SMN1 exon 7 copy. The presence of more than two SMN1 copies is a relatively frequent phenomenon in healthy individuals, especially in individuals of African descent. For more details, see Interpretation of Results. About 3-8% of SMA carriers (27% of African Americans) have two SMN1 copies on one chromosome and zero copies on the other (2+0) (Alias et al. 2014, Hendrickson et al. 2009). MLPA cannot distinguish '1+1' from '2+0' (silent carriers) arrangements. Both situations are simply detected as having two SMN1 copies leading to false negative results. A thorough molecular analysis should be performed on samples from parents and blood relatives of SMA patients when initial results indicate two SMN1 copies. Luo et al. (2014) reported that a haplotype block specific for SMN1 duplications is present in a large percentage of Ashkenazi Jews and in other ethnic groups. Identifying this haplotype, e.g. with the use of the SALSA MLPA probemix P460 SMA, will help to identify silent carriers.

Most healthy individuals have 0 - 4 copies of SMN2. Provided that at least one functional SMN1 copy is present, complete absence of the centromeric SMN2 gene seems to have no clinical consequences.

Most patients have 1 - 4 copies of SMN2. Establishing the SMN2 copy number is of importance for SMA patients: the more SMN2 copies present, the less severe the disease usually is (Feldkötter et al. 2002). Accurate SMN2 copy number quantification can be important for determining a patient’s eligibility for treatment.

Another factor that influences disease severity is the presence of the c.859G>C polymorphism in SMN2 (Prior et al. 2009). Please note that the SMN2 copy number and the presence of the c.859G>C variant do not completely explain the differences in disease severity between SMA patients. The c.859G>C polymorphism cannot be detected by P021-B1 SMA.

More information is available at: https://www.ncbi.nlm.nih.gov/books/NBK1352/.

Probemix content
The SALSA MLPA Probemix P021-B1 SMA contains 32 MLPA probes with amplification products between 175 and 445 nt. This includes four probes specific for sequences in exon 7 or 8 of either SMN1 or SMN2, 17 probes detecting sequences that are present in both SMN1 and SMN2 (at least one probe per exon; one additional probe for exon 1, 2b, and 3; seven probes in total for the intron 6-exon 7-intron 7-exon 8 region of both SMN1 and SMN2) and 1 probe for the NAIP gene.

In addition, ten reference probes are included that detect autosomal chromosomal locations. Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.com).

• The SMN1-specific exon 7 probe 21488-L30891 (274 nt) and the SMN2-specific exon 7 probe 21489-L30892 (281 nt) are the most important probes in this mix. These two probes distinguish SMN1 from SMN2 by having their ligation site at the critical single nucleotide difference between these genes in exon 7, which is a site that affects RNA splicing. These probes can be used to quantify SMN1 (important in determining carrier status) and SMN2 (important for disease prognosis) respectively.

• The SMN1-specific exon 8 probe 21490-L29983 (295 nt) and the SMN2-specific exon 8 probe 21491-L29984 (301 nt) distinguish SMN1 and SMN2 at the exon 8 G-to-A transition. In approximately 5-10% of cases, the copy number detected by these exon 8 probes does not correspond to that found by the aforementioned exon 7 probes, due to gene conversion. In such cases, only the exon 7 probes should be used to quantify SMN1 and SMN2 copy number.

• There are seven SMN exon 7 & 8 probes that detect the combined copy number of SMN1 and SMN2 genes in the intron 6, exon 7, intron 7 and exon 8 region. In normal individuals carrying two copies of SMN1 and two copies of SMN2, these probes detect four gene copies in total. In case of a homozygous SMN1 deletion, these probes can be used to more accurately determine the SMN2 copy number.

• The ten SMN exon 1-6 probes are useful to identify patient samples with a gain or loss of these exons as well as rare carriers who have two SMN1 exon 7 sequences but one of the SMN1 genes is non-functional due to a deletion of exons 1-6 (Arkblad et al. 2006; Thauvin-Robinet C et al. 2012). As these probes detect both SMN1 and SMN2, an exon 1-6 deletion detected by these probes should only be considered pathogenic if this is suggested by the individual’s clinical context. Please note that many healthy individuals have an extra copy of exons 1-6 (SMN1/2Δ7-8) without known clinical significance.

This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mrcholland.com.

SALSA Reference Selection DNA SD082
The selection of suitable reference DNA samples that can be used with P021 SMA is crucial. To facilitate the selection of suitable reference DNA samples from your own sample collection, a reference selection DNA sample (catalogue number SD082) is provided with this probemix from MRC Holland and can also be ordered separately. SALSA Reference Selection DNA SD082 should only be used for initial experiments on DNA samples from healthy individuals with the intention to identify suitable reference samples from your own collection. When the SD082 reactions are set as reference samples in the data analysis of an experiment with possible suitable reference samples from your own collection, suitable reference DNA samples will be those samples from healthy individuals that have a final ratio between 0.80 and 1.20 for all probes included in the Probemix. Suitable reference DNA samples selected as described can subsequently be used as reference DNA samples in experiments with patient samples. SD082 should not be used as a reference sample in subsequent experiments. For further details, consult the SALSA Reference Selection DNA SD082 product description, available online: www.mrcholland.com.

Sample DNA
Sample DNA developed for this product:

• SD082 Reference Selection DNA - included with this probemix.

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.

			Currently Viewing
		MC002	P021	P060	P460
Properties	CE-marked	yes	yes	yes	yes
	Technique	Melt Assay	MLPA	MLPA	MLPA
Used for	Neonatal Screening	●	○	○
	Patient Detection		●	○	○
	Carrier Detection		○	●	●
	Silent Carrier # Detection				●
Coverage	SMN1 exon 7	✓ Δ	✓	✓	✓
	SMN1 exon 8		✓	✓	✓
	SMN2 exon 7	✓ Δ	✓	✓	✓
	SMN2 exon 8		✓	✓
	SMN1+2 exon 1-8		✓
	Silent Carrier SNP Probes				✓
	Patient Detection Confirmation		P021; P060; P460	MC002 *	MC002	MC002
Product page		MC002	P021	P060	P460

● Primary test.
○ Secondary test.
^# Increased detection of silent carriers: carriers with 2 SMN1 copies on one allele + 0 on the other.
^Δ MC002: no absolute copy numbers aside from 0 determined.
^* MC002 cannot detect exon 1-6 deletions.