Just wanted to know if any one one had come across multiple missense mutations in a single exon for a single sample.
Just got back my Sanger Sequencing results and one of the reads has like 3 missense mutations in a single read (both directions). These do not look like some artifact and have been very clearly called. So, just wanted to know what are the chances that something like this might happen.
This can happen when you find polymorphisms. Are you sure that all the variants that you found are real pathogenetic mutations?
Does your gene has a pseudogene? Chromatid exchange between gene and pseudogene can form fusion genes or chimeric genes
If the missense mutations found are heterozygous, well, the sample could be a compound heterozygote for any pair of mutations. A third mutation in any of the above alleles with a milder consequence is not very rare. This is often found in HBB gene sequence tested for beta thalassemia. As commented above, common polymorphisms may be a reason too. Do look up pseudogenes as well.
Dear All,
Thank you for your replies.
Yes my gene does have a pseudo gene. How can I know if the sequence I have obtained is from a chimeric gene?
One of the mutations is pathogenic and well reported in literature.
Chimeric genes are a result of an unequal crossing over event between the gene and its pseudogene. So the first half is gene and second half is derived from the pseudogene for instance. Sometimes these chimeric genes are functional and sometimes they are not functional because of a splcing problem or a premature termination codon present in the pseudogene part.
We use an MLPA technique, which meaures the copy number of genes, and part of genes. You should make MLPA probes specific for genes and pseudogens (SNP specific). In case of chemiric genes you will find copy number other then2 copies, so part of the gene is present ones and other parts are found to have three copies.
Dear Martin,
Thank you for your reply. I cannot set up an MLPA for this reaction.
So, there is no way of actually knowing the seqeunce is from the chimeric gene.
Would the pseudogene also get amplified? I had used Exon Primer (available through UCSC) to get these primer sequences. Is there any chance that the software accounts for the presence of the pseudogene while designing primers?
No, you have to design your primers manualy.
So you have to find out which SNP's are specific for your gene and not for the pseudogene.
You have to set up an allele specific PCR with one or both primers designed in such a way that the most 3' base of the primer binds only to the gene specific SNP. Unfortunately the UCSC data base but also all other databases, are contaminated with NGS data that don't take into acount that pseudogenes exist, so dbSNP is a big mess, even sequencing errors are in there, due to low coverage (2-4 x coverage) data.
You were telling about a termination codon, most probably this is derived from the pseudogene.
So you could start to develope a specific primer on the ORF sequence, and use cDNA as a template. You will have to do this with 10 or more diferent samples to know which SNP's are gene-specific and which are not.
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