I sequenced the first exon of the MC1R gene of 15 labradors (genomic DNA) to look for the loss-of-function mutation (C.916C>T) and as expected, it was where it should have been (916th base pair). As i compared the SNPs with the phenotype of the dogs i found a second mutation that also correlated 100% with the phenotype of the dogs. The mutation is located 45 base pairs before the start codon of the first exon. As far as i'm concerned the region that lies right before the first exon should not be part of the coding DNA. If so, why could this mutation have been evolutionary conserved? Or am i simply mistaken in that the mutation before the first exon could also be part of the gene for MC1R (meaning there would be an intron before the first exon which i'm not sure if that's possible)?
$\begingroup$ Is that in a regulatory region of the gene or within a splicing region? $\endgroup$– AlexDeLargeJun 21, 2016 at 18:06
$\begingroup$ I only sequenced the first exon of the gene $\endgroup$– LukeceptionJun 21, 2016 at 18:08
$\begingroup$ That is contradictory. How can you sequence protein-coding parts only and find a mutation in a non-coding part? $\endgroup$– AlexDeLargeJun 21, 2016 at 18:12
$\begingroup$ I sequenced a little more than just the first exon of course because the results towards the ends of the sequencing product are usually not so clear $\endgroup$– LukeceptionJun 21, 2016 at 18:15
3$\begingroup$ @AlexDeLarge exon need not be protein coding. UTRs are part of exons. $\endgroup$– WYSIWYGJun 21, 2016 at 18:18
If you have 2 different mutations (irrespectively of where they sit in regards to the final gene-product), and both correlate perfectly with a given phenotype, (and you have dismissed any other potential cause), you can not formally say, if the first mutation, or the second, or both mutations are required for your phenotype. (Though you can make educated guesses or rely on external information...)
Loss-of-function mutations do not have to be in the protein-coding region (but simply in one part of the genome, which is required for the expression of a gene towards a phenotype).
Polymorphisms can also be neutral - especially in artificially bred / selected animals, such as the wolf-like animals we now call labradors. You could easily envision a scenario where some neutral mutations was present in an individual, which would have a desirable / selectable mutation. If that neutral mutation is within a few 100bp of the selected mutation, it unlikely will have been uncoupled from the selected trait / mutation.
The region in the mRNA upstream of the start site is 5' UTR; 5' untranslated region. Sometimes, UTRs have regulatory functions. Or, the UTR mutation might just be along for the ride, and have no effect at all. You generally can't look at a non coding SNP and tell if it's functional with no other information unless it messes with one of the first few bases of a known splice site.
EDIT: I still don't understand. Is the mutation in the first exon, before the start codon, or is it upstream of the first exon? There is no splice site upstream of the first exon.
Just to clarify is the mutation is 45bp upstream of the protein start site, and outside the first exon. (the first exon needed not contain the protein start site. There are mRNA where the protein start site is in the second exon)
1 - Your mRNA does not start where you think it does. Mammals proteins have multiple isoforms. And perhaps your protein of interest has functional isoform with an alternative start site further upstream.
2- Labradors are inbred line. Most dog breeds are inbred. Perhaps the mutation outside the exon is just there for the ride.
3- The mutation has damaged the promoter. Your gene is fine. But since the promoter has been mutation, gene expression is now below levels needed to main normal phenotype.
4- The start of the first exon has been miss annotated. The mutation is in the exon, specifically the 5UTR. And this mutation has effected ribosome binding in some manner