General information
The SALSA MLPA
Probemix P497 Opsin is a
research use only (RUO) assay for the detection of deletions or duplications in the
OPN1LW and
OPN1MW genes and their locus control region (LCR). This probemix can also be used to detect the wild type sequence of the p.Cys203Arg (c.607T>C) mutation in
OPN1LW and
OPN1MW.
Copy number changes in the X-chromosomal genes
OPN1LW and
OPN1MW result in a number of cone photoreceptor disorders, including red-green colour blindness, deuteranopia, protanopia, X-linked cone dystrophy, blue cone monochromatism, and Bornholm eye disease. Red-green colour blindness is the most common form and affects males much more often than females. Among populations with Northern European ancestry, red-green colour blindness occurs in about 1 in 12 males and 1 in 200 females. Affected individuals have trouble distinguishing between shades of red, yellow, and green. The majority of affected individuals are thought to have a stable condition, however, some develop evidence of progression with increasing visual acuity loss, macular atrophy, retinal pigmentation, and electrophysiological evidence of S cone and rod involvement (Gardner et al. 2014; Gardner 2016).
The close proximity (~23 kb) and extensive sequence identity (96-98%) of the
OPN1LW and
OPN1MW genes promotes meiotic mispairing of the genes and non-homologous recombination, and less commonly gene conversion events. This might lead to the formation of a hybrid pigment gene that contains part of both
OPN1LW and
OPN1MW. Also, some unusual combinations of individually benign nucleotide variations in exon 3 that can arise from recombination between
OPN1LW and
OPN1MW are associated with vision disorders. These pathogenic SNP interchange haplotypes include variants abbreviated as LIAVA, LVAVA and MIAVA for the amino acids specified by positions p.153, p.171, p.174, p.178, and p.180 where L is leucine, V is valine, I is isoleucine, A is alanine and M is methionine. These variants have shown to result in aberrant splicing and exon 3 skipping (Gardner et al. 2014; Greenwald et al. 2017; Verelli et al. 2004). Because of the frequent exchange, X chromosomes can have up to five
OPN1MW copies, whereas individuals rarely have more than one
OPN1LW copy per X chromosome.
Transcription of the
OPN1LW and
OPN1MW genes is regulated by a nearby region of DNA, known as the LCR (Figure 1). Only the two opsin pigment genes nearest the LCR, generally the
OPN1LW gene and the first copy of the
OPN1MW gene, are active in the retina and contribute to colour vision. Figure 2 gives an overview of the copy number changes and variants in
OPN1LW and
OPN1MW related to disease.
The
OPN1LW gene (6 exons) spans ~14.8 kb of genomic DNA and the
OPN1MW gene (6 exons) spans ~14.3 kb of genomic DNA, both are located on chromosome Xq28.
More information is available at
https://ghr.nlm.nih.gov/condition/color-vision-deficiency.
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 P497-A1 Opsin contains 32 MLPA probes with amplification products between 130 and 364 nucleotides (nt). This includes six probes specific for the
OPN1LW gene, six probes specific for the
OPN1MW gene, two probes for the LCR and ten probes that detect both genes, including one probe that detects the wild type sequence of the p.Cys203Arg (c.607T>C) mutation (see Figure 3 in Interpretation of results). In addition, eight reference probes are included that detect locations on the X-chromosome. 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 Reference Selection DNA SD098
The selection of suitable reference DNA samples that can be used with P497 Opsin is complicated. To facilitate the selection of suitable reference DNA samples from your own sample collection, a Reference Selection DNA sample (catalogue number SD098) is provided with this probemix from MRC Holland. Reference Selection DNA SD098 is a male DNA sample with one copy of
OPN1LW and one copy of
OPN1MW. We recommend the use of SD098 for initial experiments on DNA samples from healthy individuals with the intention to select suitable reference samples. For further details, consult the Reference Selection DNA SD098 product description provided.
This product is for research use only (RUO).
Sample DNA
Sample DNA developed for this product: