Intended use: SALSA® MLPA® Probemix P055 is an in vitro diagnostic (IVD)1 or research use only (RUO) assay for the identification of the cause of phenylalanine hydroxylase deficiency in patients diagnosed for phenylketonuria (PKU). The probemix detects deletions/duplications of one or more exons of the PAH gene. When a deletion or duplication has been identified in patient DNA, this product can also be used for carrier screening of family members.
This assay is optimised for use with peripheral blood derived genomic DNA and DNA from cells collected with buccal swabs. Deletions or duplications obtained with the P055 PAH probemix must be verified by another technique. In particular, copy number changes detected by only a single probe always require validation by another method. This SALSA® MLPA® probemix is not intended to be used for initial diagnosis of PKU or as a standalone assay for clinical decisions. Most defects in the PAH gene are point mutations, the majority of which will not be detected by MLPA. It is therefore recommended to use this SALSA® MLPA® probemix in combination with sequence analysis. The results of this test are intended to be interpreted by a clinical molecular geneticist or equivalent.
1Please 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: Phenylketonuria (PKU), as well as the less severe forms of the condition (sometimes called variant PKU and non-PKU hyperphenylalaninemia), is an inborn error of metabolism characterised by hyperphenylalaninemia resulting from a deficiency of phenylalanine hydroxylase (98% of cases) or impaired synthesis or recycling of tetrahydrobiopterin (2% of cases) (Scriver and Kaufman 2001). Untreated PKU can lead to microcephaly, epilepsy, severe mental retardation and behavioural problems. More information on PKU is available on http://www.ncbi.nlm.nih.gov/books/NBK1504/.
The majority of PKU cases are due to defects in the PAH gene. Among these defects are deletions and duplications of complete exons, which are usually missed by standard sequence analysis. The MLPA technique can detect most of these deletions and duplications and therefore complements sequence analysis of the PAH gene. The expected number of PAH chromosomal rearrangements that can be detected with this MLPA probemix is between 1 and 5% of all PKU cases in most populations (see below for publications on probemix P055 PAH).
Probemix content: This SALSA® MLPA® probemix P055 PAH contains 38 MLPA probes with amplification products between 128 and 427 nt including 22 probes for the PAH gene region, one probe upstream and downstream of PAH (product description Table 2), and 14 reference probes that detect sequences outside this region. The identity of the genes detected by the reference probes is available online (www.mlpa.com).
Several partially conserved and possibly regulatory elements are located just upstream of PAH exon 1. These DNaseI hypersensitive sites have been described by Bristeau et al. (2001). Two probes are located in these upstream sequences (418 nt HSS3 and 154 nt HSS2).
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 product description). 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.