MRC-Holland b.v/SALSA MLPA Probemix P060 SMA Carrier/SALSA MLPA Probemix P060 SMA Carrier – 50 rxn/P060-050R

Intended use: The SALSA^® MLPA^® probemix P060 SMA is an in vitro diagnostic (IVD)¹or research use only (RUO) assay for the detection of copy number changes of exons 7 and 8 of SMN1 and SMN2 for carrier testing, patient diagnosis and for the confirmation of a potential cause and clinical diagnosis of spinal muscular atrophy (SMA). 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.This assay is for use with human DNA extracted from:

1. Peripheral blood
2. Prenatal samples, from either
a. (un)cultured amniotic fluid obtained in week 16 of the pregnancy or later, free from blood contamination, b. (un)cultured chorionic villi, free from maternal contamination, c. foetal blood
3. Dried Blood Spot (DBS) cards, permitted the DNA has been extracted using the method and type of DBS cards described in Appendix I below.

This assay is not intended to be used as a standalone assay for clinical decisions. The results of this test must be interpreted by a clinical molecular geneticist or equivalent.¹ Please note that this probemix is for In Vitro Diagnostic use (IVD) in the countries specified at the end of this product description. In all other countries, the product is for Research Use Only (RUO).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 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. 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 will help distinguish silent carriers and can be done with the P460 SMA MLPA Probemix.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 seems to have no clinical consequences. More information on spinal muscular atrophy can be found in http://www.ncbi.nlm.nih.gov/books/NBK1352/.P060-B2 probemix content: This SALSA^® MLPA^® probemix P060 SMA contains 21 MLPA probes with amplification products between 154 and 342 nt (Table 2) including 2 probes each for SMN1 and SMN2 (Table 2) and 17 reference probes that detect sequences outside this region. The identity of the genes detected by the reference probes is available online (www.mlpa.com). - The SMN1 Exon 7 probe 14919-L17081 (183 nt) is the most important probe as it can be used to determine SMN1 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 probe and the SMN1 exon 8 probe 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. Figures depicting variation as can be detected by P060 SMA Carrier can be found on page 10 (figure 1-4).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 (see table below). More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mlpa.com. SALSA Reference Selection DNA SD082: The Reference Selection DNA SD082 provided with this probemix can be used to find suitable reference DNA samples from your own sample collection for further use in MLPA experiments. It is strongly advised to use DNA sample and reference DNA samples extracted with the same method and derived from the same source of tissue. For certain applications, the selection of suitable reference DNA samples is complicated. Inclusion of one reaction with 5 μl SD082 Reference Selection DNA facilitates the identification of suitable reference DNA samples. We recommend the use of this SD082 Reference Selection DNA (or Coriell sample HG01701) only for initial experiments on DNA samples from healthy individuals with the intention to identify suitable reference DNA samples. We do not recommend it for use in all experiments. For further instructions, consult the Reference Selection DNA SD082 product description provided. More information regarding the selection and use of reference samples can be found in the MLPA General Protocol.