General information
The SALSA MLPA
Probemix P305 AGXT is a
research use only (RUO) assay for the detection of deletions or duplications in the
AGXT and
GRHPR genes, which are associated with primary hyperoxaluria type 1 and 2. This probemix can also be used to detect the presence of the
AGXT c.731T>C (p.I244T), c.33_34insC (c.33dupC; p.Lys12GlnfsX156) and c.508G>A (p.G170R) point mutations.
Primary hyperoxaluria type 1 and 2 are each caused by a deficiency of an enzyme that catalyses glyoxylate conversion (alanine-glyoxylate aminotransferase encoded by
AGXT and glyoxylate reductase/hydroxypyruvate reductase encoded by
GRHPR, respectively). This deficiency results in the production of oxalate, which accumulates in the form of calcium oxalate crystals. Persistent hyperoxaluria causes nephrolithiasis and nephrocalcinosis, leading to renal failure, followed by tissue oxalosis with life-threatening complications. There are three types of primary hyperoxaluria, of which type 1 is the most severe, followed by type 2. Both type 1 and type 2 are inherited in an autosomal recessive manner (Howles and Thakker. 2020).
The
AGXT gene (11 exons) spans ~11.7 kb of genomic DNA and is located on 2q37.3, 241.5 Mb from the p-telomere (only 1.5 Mb from the q-telomere). The
GRHPR gene (9 exons) spans ~14.3 kb of genomic DNA and is located on 9p13.2, 37.4 Mb from the p-telomere.
More information is available at
https://www.ncbi.nlm.nih.gov/books/NBK1283/ and
https://www.ncbi.nlm.nih.gov/books/NBK2692/.
This SALSA MLPA probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited license for research purposes.
Probemix content
The SALSA MLPA Probemix P305-B3 AGXT contains 31 MLPA probes with amplification products between 132 and 382 nucleotides (nt). This includes 12 probes for the
AGXT gene (one probe for each exon and two probes for exon 1) and eight probes for the
GRHPR gene (one for each exon except exon 5). Furthermore, this probemix also contains three probes specific for the
AGXT c.731T>C (in exon 7), c.33_34insC (in exon 1) and c.508G>A (in exon 4) point mutations which will only generate a signal when the mutation is present. In addition, eight reference probes are included 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).
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 Binning DNA SD030
The SD030 Binning DNA provided with this probemix can be used for binning of all probes including the
AGXT c.731T>C, c.33_34insC and c.508G>A mutation-specific probes (
AGXT probe 09743-L32674, probe 21829-L32675 and probe 09740-L10150, respectively). SD030 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequence detected by the above mentioned probe. Inclusion of one reaction with 5 μl SD030 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 SD030 Binning DNA product description, available online:
www.mrcholland.com.
Sample DNA
Sample DNA developed for this product:
The product description of this Sample DNA can be found on this page under
Online available downloads >
Other product documentation.