I would not automatically assume that any variant has any impact on the gene at all.

However, transcript variants within an intron have many possibilities for regulating genes. Intronic variants might affect alternative splicing of the mRNA, you can see if sequences involved in splicing might be affected (splice donor or acceptor sites, secondary structure, etc.). Intron variants could also be relevant as enhancers that could act on the gene they are found in or could possibly enhance the expression of many genes. For that you can look at predicted protein binding sites and see if there is any differential binding for a specific protein.

This should all be done with caution. If you do not have any reason to think this is regulatory, and you just found it (associated with disease or something) then it is important NOT to assume this SNP is functional.

Remember that a SNP can be associated in some populations because it is correlated with another SNP that actually has an impact on the regulation of the gene.

You can suggest possible mechanisms for this SNP to function and then suggest that future experiments should be done using sequencing to potentially identify other variations that may correlate with this SNP and have a clearer function.

HTH-Good Luck!

I would not automatically assume that any variant has any impact on the gene at all.

However, transcript variants within an intron have many possibilities for regulating genes. Intronic variants might affect alternative splicing of the mRNA, you can see if sequences involved in splicing might be affected (splice donor or acceptor sites, secondary structure, etc.). Intron variants could also be relevant as enhancers that could act on the gene they are found in or could possibly enhance the expression of many genes. For that you can look at predicted protein binding sites and see if there is any differential binding for a specific protein.

This should all be done with caution. If you do not have any reason to think this is regulatory, and you just found it (associated with disease or something) then it is important NOT to assume this SNP is functional.

Remember that a SNP can be associated in some populations because it is correlated with another SNP that actually has an impact on the regulation of the gene.

You can suggest possible mechanisms for this SNP to function and then suggest that future experiments should be done using sequencing to potentially identify other variations that may correlate with this SNP and have a clearer function.

HTH-Good Luck!

This variant can alter splicing of the gene and this ‘splice-variant gene expression’ can change the level of gene expression. This may result in several diseases and has the possibility of halting the regulatory process (eg: Cell Cycle Arrest)

The introns of a gene contains downstream regulatory elements known as DREs and the intron variants either loose these elements or accquire a new type of elements which ultimatelyaffects the gene regulation and signaling including post transcriptional modifications, and in this way the overall expression level as well as pattern of expression to a tissue type get affected

a) Polymorphisms with in intronic region might trigger an abnormal splicing spectrum provided there are located within the splice acceptor/ donor site. There are many free web tools to identify the same. I have previously tried Net2 gene server, it is an easy to use web tool to identify the splice site.

b) It might possibly have a potential role in disease trigger, as few studies have previously implicated intronic variant segregating with familial diseases.

c) Another important role of introns that is currently under investigation is the transcription of the introns to small regulatory RNAs, such as microRNAs, that can regulate gene expression. Probably this polymorphism could have a role in the same as well.

Above mentioned are my modest experiences regarding the same, I suggest you explore you own intuition. Good luck!!!

I would agree with the first answer. Although there are software prediction tools for analysing the effects of variants in introns on splicing, these are not completely reliable. If you have access to RNA then the next experiment is to look at the cDNA around your variant. RT-PCR a couple of exons on either side and if your variant is affecting splicing you should see some aberrant bands in the mutant vs control. However, this is not 100% reliable either. Another approach is to identify a SNP in the transcribed sequence for which your patient is heterozygous and look of allelic expression imbalance. If intronic SNP is affecting splicing it will bias the levels of the SNP from 50:50 which can be detected in sequencing traces or quantitative PCR.

Best of luck.

Thank you Steve. I think working with cDNA will be my next step. Let's say my hypothesis is that the intronic SNP affects levels of gene expression. ie protein in less abundant. Could I test that with quantitative PCR? As you can tell Im quite new into this and I would like to thank each and everyone of you for taking the time to help out!

Maybe as a simple place to start you can do total protein by western and total mRNA by qPCR and do an analysis stratified by genotype. Maybe you will see a dose dependent response where homozygote AA have more mRNA/protein, heterozygotes AB have a middle amount, and homozygote BB have less mRNA/protein.

It is difficult to assay your variant directly because it is in an intron, and I think what Steve is talking about is a good approach. If you can find another sequence variant in the exon region of your gene (probably nearby the intron variant) that can be used as a substitute for your variant (its genotype can be used to determine the genotype of the intronic variant). This will allow you to do some of the allele-specific tests like sequencing the qPCR products to find the amount of transcript produced from each allele in a heterozygote or testing allele-specific transcripts independently by Tm analysis etc. There are a number of approaches to assess "allele-specific expression" that you will be able to find in the literature.

Good luck :)

All of the answers above make good points. Another thing to consider is that an intronic SNP may not change the level of gene expression, but results in the formation of an alternative protein isoform. This may happen when the SNP affects binding of spliceosomal factors to a nearby splice site, and leads to use of alternative sites elsewhere. This eventually results in inclusion of additional sequences in the mRNA, or exclusion of some of the original sequences, most often through skipping of an entire exon. If the change in sequence length is divisible by 3, the mRNA will still be in-frame, and may code a protein with an altered (possibly negative dominant) function. In some cases a (truncated) protein may also be produced even if the reading frame is changed, giving rise to a premature stop codon, although usually such mRNAs are cleared by nonsense-mediated decay.

I agree with Janne J Turunen. The splicing of the intron may play a dominant role in protein level

both splicing and binding are imp factors but in case of splicing more effect can be analyzed at Protein level while binding with transcriptional regulatory factor is imp, so to study impact of intronic region variation is significant in some diseases so  qRT PCR for gene expression and protein level is preferable  

Hi,

I found a heterozygous mutation very close (5 nucleotide) to the splicing site but in the intron. Do you think that might influence any gene expression/ protein expression and do you think that it could be a disease associated?

Thanks,

Mrinal

To Mrinal K,

Could you please be more precise about your variation. you said at position 5, but where (+5) or( _5) because it differs.

Recent discoveries showed introns are the host of enhancers, each associated with the expression of several genes including the host, and mutations in enhancers can cause diseases. For example, see HEDD: Human Enhancer Disease Database.

I agree with Mr. Janne J Turunen.