General information
The SALSA MLPA
Probemix P285 LRP5 is a
research use only (RUO) assay for the detection of deletions or duplications in the
LRP5,
DKK1,
NDP and
FZD4 genes, which are associated with osteoporosis-pseudoglioma syndrome (OPPG / OPPS) and exudative vitreoretinopathy (EVR). This probemix can also be used to detect the presence of the
LRP5 G171V point mutation.
OPPG or OPPS and EVR are caused by defects in the same genes:
LRP5,
NDP and
FZD4 genes, amongst others. Gong et al. (2001) demonstrated in situ low density lipoprotein receptor (LRP5
) expression by osteoblasts, which showed that LRP5 can transduce Wnt signalling in vitro via the canonical pathway. They further showed that a mutant secreted form of LRP5 could reduce bone thickness in mouse calvarial explant cultures. These data indicated that Wnt-mediated signalling via LRP5 affects bone accrual during growth and is important for the establishment of peak bone mass.
LRP5 deletions result in decreased bone mass. Gain of function
LRP5 mutations resulting in increased bone density have also been described. A probe for one gain of function mutation, G171V, is included in this probemix. Dickkopf-1 (DKK1) is a soluble inhibitor of Wnt/beta-catenin signalling required for embryonic head development. DKK1 regulates Wnt signalling by binding to the Wnt co-receptor LRP5. Mice heterozygous for
DKK1 deletion display an increased bone formation and a high bone mass phenotype. Defects in
NDP have been reported in Norrie disease and in X-linked exudative vitreoretinopathy. Finally, heterozygous mutations in the frizzled-4 gene (
FZD4), which encodes 7-transmembrane domain proteins that are receptors for Wnt signaling proteins, are also reported to cause familial exudative vitreoretinopathy-1 (EVR1).
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 P285-C3 LRP5 contains 46 MLPA probes with amplification products between 128 and 481 nucleotides (nt). This includes 24 probes for
LRP5 gene, four probes for
DKK1 gene, three probes for
FZD4 gene, and three probes for
NDP gene. Furthermore, this probemix also contains one probe specific for the G171V mutation located in exon 3 of the
LRP5 gene, which will only generate a signal when the mutation is present. In addition, 11 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 mutation-specific probe (202 nt probe 09270-SP0044-L09500 G171V mutation). 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.
This product is for research use only (RUO).
Sample DNA
Sample DNA developed for this product: