Intended use: The SALSA MLPA Probemixes P051 and P052 Parkinson are an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplication in SNCA, PARK2, UCHL1, PINK1, PARK7, ATP13A2, LRRK2, GCH1 genes, and the presence of two point mutations, A30P in the SNCA gene and G2019S in the LRRK2 gene, in genomic DNA isolated from human peripheral whole blood specimens. P051 and P052 Parkinson are intended to confirm a potential cause for and clinical diagnosis of Parkinson’s disease and for molecular genetic testing of at-risk family members. Additionally, deletions or duplication in GCH1 gene, covered by P052 Parkinson mix 2, can be used to confirm a potential cause for and clinical diagnosis of GTP cyclohydrolase 1-deficient dopa-responsive dystonia and for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P051 and P052 Parkinson should be confirmed with a different technique. In particular, CNVs detected by only a single probe always require confirmation by another method. Most defects in the SNCA, PARK2, UCHL1, PINK1, PARK7, ATP13A2, LRRK2, GCH1 genes are point mutations, the majority of which will not be detected by MLPA. It is therefore recommended to use this assay in combination with sequence analysis. Not all exons of ATP13A2 and LRRK2 genes are covered.

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, 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 or prenatal 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 Binning DNA SD067.

Clinical background: Parkinson’s disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons of the midbrain. Resting tremor, bradykinesia, rigidity, and postural instability are the main clinical manifestations of the disease. Parkinson’s disease occurs in approximately 13 per 100,000 people. The age of onset in patients with Parkinson’s disease varies over a wide range and can be defined as either early onset (≤50 years) or late onset (>50 years). The majority of Parkinson’s disease cases are sporadic and a family history is reported in approximately 10-20% of patients.

Mutations in multiple genes are associated with autosomal dominant (SNCA, LRRK2, GCH1, UCHL1) or autosomal recessive (PARK2 (also known as PRKN), PINK1, PARK7, ATP13A2) Parkinson’s disease. Mutations in these genes range from point mutations to larger exonic rearrangements including deletions and duplications. The presence of multiple copies of SNCA is known to be associated with Parkinson’s disease and the severity of symptoms increases with the number of copies of the gene (Keyser et al. 2010, Matsumoto et al. 2010). The LRRK2 G2019S mutation (p.Gly2019Ser, c.6055G>A) is the most common Parkinson-associated mutation known today and has been reported in 41% of sporadic and 37% of familial Parkinson patients from the North African Arab population and in 18.3% of Ashkenazi Jewish Parkinson patients (Lesage et al. 2006, Ozelius et al. 2006), while the mutation has been found in only 0.58% Parkinson patients of European and Asian origin (Ross et al. 2011).
Mutations in PARK2 and PINK1 are the most common causes of early onset Parkinson’s disease (EOPD), however the frequencies vary widely across studies. It has been reported that up to 50% of familial and 18% of sporadic EOPD cases had pathogenic PARK2 mutations, whereas more recent studies have reported a pathogenic mutation frequency as low as 1.6%. Frequency estimates for PINK1 mutations tend to fall within a similarly broad range as for PARK2, whereas PARK7 mutations are generally very rare, being estimated in a UK-based study in 0.4% (Kilarski et al. 2012). Parkinson-related mutations in ATP13A2, GCH1 and UCHL1 are very rare.

GTP cyclohydrolase 1-deficient dopa-responsive dystonia (GTPCH1-deficient DRD), also known as autosomal dominant Segawa syndrome (OMIM #128230) is characterised by a childhood-onset dystonia, postural and motor disturbances showing marked diurnal fluctuation, and late development of parkinsonism (Segawa et al. 1976). All individuals with this disorder, are treated with relatively low doses of levodopa and show complete or near-complete reversal of symptoms. The disorder is caused by mutations in the GCH1 gene encoding GTP cyclohydrolase 1.

More information is available on https://www.ncbi.nlm.nih.gov/books/NBK1223/; https://www.ncbi.nlm.nih.gov/books/NBK1478/ and https://www.ncbi.nlm.nih.gov/books/NBK1508/.

Probemix content: The SALSA MLPA Probemixes P051-D2/P052-D2 Parkinson both contain 50 MLPA probes with amplification products between 130 and 500 nucleotides (nt). The P051-D2 probemix includes 38 probes for the PARK7, ATP13A2, PINK1, SNCA and PARK2 genes. The P051-D2 probemix also contains two probes specific for the SNCA A30P and LRRK2 G2019S mutations which will only generate a signal when the mutation is present. The P052-D2 probemix includes 36 probes for the ATP13A2, UCHL1, PARK2, LRRK2 and GCH1 genes. The P052-D2 probemix also contains one probe specific for the LRRK2 G2019S mutation which will only generate a signal when the mutation is present. PARK2 maps close to FRA6E, one of the most active fragile sites in the human genome. Therefore, the P052-D2 probemix also contains two probes that are located at fragile sites detecting the CAV1 and CAV2 genes, which are not related to Parkinson’s disease. The content of the P051 and P052 probemixes per gene can be found in Table 2 a-i of the product description.
In addition, ten and eleven reference probes are included in the P051-D2 and P052-D2 Parkinson probemixes respectively, that detect autosomal chromosomal locations. Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mlpa.com).

Each of these probemixes contain 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.mlpa.com.

SALSA Binning DNA SD067: The SD067 Binning DNA provided with this probemix can be used for binning of SNCA A30P (c.88G>C = p.A30P) and LRRK2 G2019S (c.6055G>A = p.G2019S) mutation-specific probes (SNCA probe 02166-L27543, LRRK2 probe 04575-L27549 (P051-D2) and LRRK2 probe 04574-L27601 (P052-D2)). SD067 Binning DNA is a mixture of genomic DNA from healthy individuals and synthetic DNA that contains the target sequence detected by the above mentioned probe. Inclusion of one reaction with 5 μl SD067 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, 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). Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of mutation signal(s). It is strongly advised that all samples tested are extracted with the same method and derived from the same source of tissue. For further details, please consult the SD067 Binning DNA product description.

Sample DNA
Sample DNA developed for this product:

• SD067 Binning DNA- included with this probemix.