Intended purpose
The SALSA MLPA Probemix P060 SMA Carrier is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplication in exons 7 and 8 of the SMN1 gene 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. P060 SMA Carrier 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. This probemix can also be used for the detection of copy number changes of exons 7 and 8 of the SMN2 gene, as an interpretation aid for SMN1 copy number determination.

Copy number variations (CNVs) detected with P060 SMA Carrier 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 and Coffalyser.Net analysis software.

Clinical background
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. The estimated incidence of SMA is 1:6,000-1:10,000: the second most common lethal autosomal recessive disorder in Caucasians, after cystic fibrosis (Ben-Shachar et al. 2011, Smith et al. 2007). SMA is usually divided into four clinical groups based on age of onset and maximum function obtained (type I, OMIM# 253300; type II, OMIM# 253550; type III, OMIM# 253400; and type IV, OMIM# 271150).

Two (highly-similar) genes play a pivotal role in SMA: SMN1 and SMN2. Most individuals have two copies of each gene. The SMA region on 5q13.2, containing the telomeric SMN1 and the centromeric SMN2, is a complicated inverted repeat area displaying high instability, leading to frequent deletions and gene conversions. SMN1 and SMN2 can only be distinguished by two single nucleotide differences: one in exon 7 and one in exon 8. The single nucleotide difference in exon 7 of SMN2 affects mRNA splicing resulting in an altered SMN protein with a limited half-life and function.

A total of 95-98% of SMA patients (this percentage is lower in SMA patients from African descent) show homozygous deletion of at least exon 7 of the telomeric SMN1 gene (Labrum et al. 2007). The remaining 3-5% present compound heterozygosity with a point mutation on one chromosome and a deletion/gene conversion on the other. Such a point mutation will not be detected by this P060 SMA Carrier MLPA assay and should be identified by sequencing. In a small number of patients, the SMN1 defect is a copy number change of SMN1 exons 1-6 which can be detected with the P021 SMA MLPA probemix (Arkblad et al. 2006).

The great majority of SMA carriers can be identified by the presence of 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. Approximately 3-8% of SMA carriers (27% of African Americans) have one functional and one defective SMN1 copy, or have two SMN1 copies on one chromosome and 0 copies on the other (2+0) (Alias et al. 2014, Ben-Shachar et al. 2011, Hendrickson et al. 2009, Miskovic et al. 2011, Smith et al. 2007). Dosage analysis cannot determine the difference between '1+1' and '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 in parents of SMA patients who have 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.

The SMN2 copy number is very variable with only 60-70% of individuals having two copies. Provided that at least one functional SMN1 copy is present, complete absence of the centromeric SMN2 gene has no known clinical consequences. More information on spinal muscular atrophy can be found in http://www.ncbi.nlm.nih.gov/books/NBK1352/.

Probemix content
This SALSA MLPA Probemix P060 SMA Carrier contains 21 MLPA probes with amplification products between 154 and 342 nt including two probes each for SMN1 and SMN2 and 17 reference probes that detect sequences outside this region. The identity of the genes detected by the reference probes is 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 important 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 14881-L17082 (218 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 chimeric 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, which aids in the detection of gene-conversion events. The SMN2 copy number has no influence on SMA carrier status.

- The SMN2 Exon 8 probe 14878-L17084 (301 nt) identifies the SMN2 exon 8 copy number, which aids in the detection of gene-conversion events. The SMN2 copy number has no influence on SMA carrier status.

The summary of these findings and what they mean for carrier/patient status can be found in Table B on page 8 and 9 of the Product Description. Figures depicting variation as can be detected by P060 SMA Carrier can be found on page 12 (Figures 1-4) of the Product Description.

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 P060 SMA Carrier 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 or can be ordered separately. 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. 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. 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.