The SALSA MLPA Probemix P055 PAH is an in vitro diagnostic (IVD)1
or research use only (RUO) semi-quantitative assay2
for the detection of deletions or duplications in the PAH
gene in genomic DNA isolated from human peripheral whole blood specimens and DNA from cells collected with buccal swabs. P055 PAH is intended to confirm a potential cause for and clinical diagnosis of phenylalanine hydroxylase deficiency in patients diagnosed with phenylketonuria (PKU) and for molecular genetic testing of at-risk family members.
Copy number variations (CNVs) detected with P055 PAH 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 PAH
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 initial diagnosis of PKU, standalone diagnostic purposes, pre-implantation or prenatal testing, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.
Please 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).
To be used in combination with a SALSA MLPA Reagent Kit and Coffalyser.Net analysis software.
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).
This SALSA MLPA probemix P055-D1 PAH contains 38 MLPA probes with amplification products between 128 and 427 nucleotides (nt). This includes 22 probes for the PAH
gene region, one probe upstream and downstream of PAH
(Table 2), and 14 reference probes 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)
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 below). 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