Intended purpose
The SALSA MLPA Probemix P460 SMA (Silent) Carrier is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of 1. copy number changes of exons 7 and 8 of the SMN1 gene for carrier testing, patient diagnosis and for the confirmation of a potential cause and clinical diagnosis of spinal muscular atrophy (SMA), and 2. the presence of the g.27134T>G and g.27706-27707delAT polymorphisms in SMN1 that are a risk factor for silent carriership of SMA in genomic DNA isolated from human peripheral whole blood specimens. P460 SMA (Silent) carrier is also intended for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P460 SMA (Silent) Carrier should be confirmed with a different technique. A small amount of defects in the SMN1 gene are point mutations, none of which will 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, prenatal or pre-implantation testing, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.

¹Please note that this probemix is for in vitro diagnostic (IVD) use in the countries specified at the end of this product description. In all other countries, the product is for research use only (RUO).
²To be used in combination with a SALSA MLPA Reagent Kit, Coffalyser.Net analysis software and SALSA Reference Selection and Binning DNA SD084.

Clinical background
The SALSA MLPA Probemix P460 SMA (Silent) Carrier is an assay for the detection of deletions or duplications in the SMN1 gene, which are associated with Spinal Muscular Atrophy (SMA). This probemix can also be used to detect the presence of two SMN1 polymorphisms, g.27134T>G and g.27706-27707delAT, for haplotype identification.

Spinal Muscular Atrophy (SMA) is a neuromuscular disorder characterised by degeneration of the anterior horn cells of the spinal cord, leading to symmetrical muscle weakness and atrophy. SMA is the second most common lethal autosomal recessive disorder in Caucasians, after cystic fibrosis. SMA is usually divided into three clinical groups. Patients with type I SMA (OMIM# 253300) show onset at birth or before six months, and usually die of respiratory insufficiency within two years. Type I SMA patients are never able to sit or walk. Patients with type II SMA (OMIM# 253550) show onset after six months. They can sit but are never able to walk unaided, and their life expectancy is significantly reduced. Type III SMA (OMIM# 253400) patients show the first symptoms after 18 months and are able to stand and walk, but often become wheelchair-bound during youth or adulthood.

There are two (highly-similar) genes playing a pivotal role in SMA: SMN1 and SMN2. The telomeric SMN1 and the centromeric SMN2 genes are located in a complicated inverted repeat area spanning ~500 kb on chromosome 5q13.2. This area displays high instability, leading to frequent deletions and gene conversions. Most individuals have two copies each of SMN1 and SMN2, both consisting of nine exons (exons 1, 2a, 2b, and 3-8). The SMN1 and SMN2 genes can only be distinguished by two single nucleotide differences: one in exon 7 and one in exon 8. The exon 8 difference has no effect on the transcript, however, the exon 7 difference disrupts splicing in SMN2 most of the time leading to loss of functionality. Only 10-15% of the SMN2 transcripts are functional. This SALSA MLPA Probemix P460 SMA (Silent) Carrier detects the copy number of exons 7 and 8 of the SMN1 gene.

Absence of any functional SMN1 copy results in insufficient amounts of full length transcripts. More than 95% of SMA patients show homozygous deletion of at least exon 7 of the telomeric SMN1 gene. Individuals with only one functional SMN1 copy are carriers of the disease. The great majority of SMA carriers can be identified by the presence of only a single SMN1 exon 7 copy. The one copy frequency in the US is estimated to be 1:37 for Caucasians, 1:46 for Ashkenazi Jews, 1:56 for Asians, 1:91 for African-Americans and 1:125 for Hispanics (Hendrickson et al. 2009).

Although the great majority of SMA carriers can be detected by copy number analysis of the SMN1 exon 7 sequence, some carriers remain undetected. These include carriers with (1) a defective SMN1 allele due to a point mutation in the SMN1 gene or a copy number change of exons 1-6 or 8, and (2) individuals that have two SMN1 copies on one allele and none on the other allele, the so-called “Silent Carriers” (2+0 genotype). The P460 probemix increases the detection rate of the latter group.

In most populations, approximately 6.3-15.5% of the individuals have two SMN1 copies on a single chromosome 5 strand, of which 0.07-0.19% are a silent carrier. In the African-American population this percentage is even as high as 47.2%, of which 0.41% is a silent carrier (Hendrickson et al 2009). Luo et al. (2014) has reported that a specific SMN1 haplotype block is present in a large percentage of Ashkenazi Jews who carry an SMN1 duplication. This haplotype was also identified on SMN1 duplication alleles in other ethnic groups, but in lower percentages. Detection of the SMN1 polymorphisms g.27134T>G and g.27706-27707delAT can aid in identifying this haplotype and thereby silent carriers.

More information on Spinal Muscular Atrophy is available at http://www.ncbi.nlm.nih.gov/books/NBK1352/.

Probemix content
The SALSA MLPA Probemix P460-A1 SMA (Silent) Carrier contains 23 MLPA probes with amplification products between 131 and 331 nucleotides (nt). This includes three probes for the SMN1 and SMN2 genes. Furthermore, this probemix also contains two probes specific for the g.27134T>G and g.27706-27707delAT polymorphisms, both of which will only generate a signal when the respective polymorphism is present. In addition, 18 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 Exon 7 probe 14919-L17081 (183 nt) is the most important probe as it can be used to determine SMN1 copy number, which is essential for deducing SMA diagnosis and carrier status. This probe is specific for SMN1 and will give no significant signal on SMN2. The probe has its ligation site at the C-to-T transition in exon 7, which is the site that affects RNA splicing in SMN2.

- The SMN1 Exon 8 probe S0960-L25957 (154 nt) is able to distinguish between SMN1 and SMN2 at exon 8 (G-to-A transition). The signal of this probe indicates the copy number of SMN1 exon 8. In approximately 95% of the samples, the copy number detected by the SMN1 exon 7 and exon 8 probes is identical. This SMN1 exon 8 probe cannot be used to quantify the number of SMN1 copies, as an exon 8 mutation will still result in a functional protein. Only the SMN1 exon 7 probe should be used to determine the SMN1 copy number. In the majority of the remaining 5% of samples, gene conversion between SMN1 and SMN2 has resulted in a hybrid gene containing the SMN1 exon 7 sequence and the SMN2 exon 8 sequence. Such a hybrid gene results in a functionally identical protein to the SMN1 protein.

- The SMN2 Exon 7 probe 14921-L17083 (282 nt) identifies the SMN2 exon 7 copy number. (Determining SMN2 copy number for the purpose of prognosis, using MLPA Probemix P021 SMA is recommended.)

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 & Binning DNA SD084
The SD084 Reference Selection & Binning DNA provided with this probemix has two distinct functions. First, it can be used for binning of the two polymorphism-specific probes (SMN1 probe S0938-L26163 g.27134T>G polymorphism and SMN1 probe S0961-L25586 g.27706-27707delAT polymorphism) during data analysis, and second for the selection of suitable reference samples before an experiment is started. Suitable reference samples contain two copies each of SMN1 and SMN2, but do not contain the two polymorphisms. SD084 Reference Selection & Binning DNA is a mixture of genomic DNA from a cell line and synthetic DNA that contains the target sequences detected by the above mentioned probes.

For the purpose of binning, inclusion of one reaction with 5 μl SD084 Reference Selection & Binning DNA in initial MLPA experiments is essential as it can be used to aid in data binning of the peak pattern using Coffalyser.Net software. Furthermore, SD084 Reference Selection & Binning DNA should be included in the experiment whenever changes have been applied to the set-up of the capillary electrophoresis device (e.g. when capillaries have been renewed). SD084 Reference Selection & Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of polymorphism signal(s). It is strongly advised that all samples tested are extracted with the same method and derived from the same source of tissue.

The selection of suitable reference DNA samples that can be used with P460 SMA (Silent) Carrier is complicated, due to the high frequency of gene conversions and CNVs in the healthy population. For the purpose of facilitating the selection of suitable reference DNA samples (containing two copies each of SMN1 and SMN2 and not containing the two polymorphisms) from your own sample collection, the SD084 Reference Selection & Binning DNA sample can also be used. SD084 Reference Selection & Binning DNA should only be used for initial experiments on DNA samples from healthy individuals with the intention to identify suitable reference samples. SD084 should not be used as a reference sample in subsequent experiments. For further details, consult the SD084 Reference Selection & Binning DNA product description, available online: www.mrcholland.com.

Sample DNA
Sample DNA developed for this product:

• SD084 Reference Selection & Binning 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.