I found a new variant (a substitution) in exon 20 of BRCA1 gene. This is not reported in HGVS, BIC or any other mutation database. The variant was found after sequencing with both the forward and reverse primer in case of only one patient. How can I proved that it was not a polymorphism but a mutation? Plz suggest.
Hi Abhijit,
A 'polymorphism' is normally defined as a variant found in >1% of the population, (the exact percentage used by different people can vary). However, it usually means a fairly common benign variant.
The best resources to check to see if your variant is common, rare, or novel are
http://exac.broadinstitute.org/
http://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/
A 'mutation' is normally defined as a variant that has a pathogenic effect. These are normally very rare or novel, but can also be common. There are many computational tools which can predict the possible pathogenicity of a nonsynonymous variant, and a good way of running several of them together would be via Annovar
http://wannovar.usc.edu/
The following paper is a good guide to whether a variant should be described as benign or pathogenic, based on available evidence:
http://www.ncbi.nlm.nih.gov/pubmed/25741868
Hi Abhijit,
A 'polymorphism' is normally defined as a variant found in >1% of the population, (the exact percentage used by different people can vary). However, it usually means a fairly common benign variant.
The best resources to check to see if your variant is common, rare, or novel are
http://exac.broadinstitute.org/
http://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/
A 'mutation' is normally defined as a variant that has a pathogenic effect. These are normally very rare or novel, but can also be common. There are many computational tools which can predict the possible pathogenicity of a nonsynonymous variant, and a good way of running several of them together would be via Annovar
http://wannovar.usc.edu/
The following paper is a good guide to whether a variant should be described as benign or pathogenic, based on available evidence:
http://www.ncbi.nlm.nih.gov/pubmed/25741868
I agree with Simon that ExAC and 1000G are the most comprehensive accessible human genetic variation databases to see whether a variant is polymorphic (MAF>0.01).
However, given that you have resequenced a patient (presumably with breast cancer) and the gene is a BRCA gene, I suggest you identify the codon and the respective aminoacid change (nonsense, missense or sense) and then see whether it is present in the BRCA mutation database which is especially curated for this purpose.
http://arup.utah.edu/database/BRCA/Variants/BRCA1
There are currently five substitutions (missense or nosense) reported for Exon 20 of BRCA1.
Hope it helps.
This review on predicting the impact of non-synonymous mutations may help: http://www.ncbi.nlm.nih.gov/pubmed/25234433
Evidence for the pathogenicity of the variant can be obtained by performing functional studies. Since BRCA1 is involved in DNA repair and its disruption results in hypersensitivity to DNA damaging agents, such as irradiation and DNA alkylating agents you could use any of these two options:
1) Generate full length BRCA1 with the variant, introduce the mutant BRCA1 construct in BRCA1 deficient cell line, assess sensitivity of the cell line to DNA damaging agents. With proper controls, the sensitivity/resistance of the cell line expressing mutant BRCA1 variant should indicate whether variant is pathogenic/benign, respectively.
2) If a cell line is avilable from the organism in which you found the variant, you could CRISPR to substitute the variant for wild type sequence and assess sensitivity of cell line to DNA damaging agents as for option 1.
Najim
Ihssane, Sift and Polyphen are old tools, and whilst they're still the most widely used, are greatly outperformed by newer methods such as DANN, CADD, and FATHMM. This was my finding from benchmarking 20 of them, but agrees with other independent tests (sorry, don't have a reference handy).
My apologies Ihssane if I appeared overly critical of your comment. It's just a personal frustration of mine that I still regularly see new publications which *only* cite SIFT and PolyPhen results!
Thank you all.. All the suggestions are really helpful for me and enrich my knowledge.
Polymorphism is natural variations found in sequence where as mutation is variation in sequence due to epigenetic or gene deletion, addition.....
Mutation results due to DNA sequence changes specifically that happen once an allele is transfered from one generation to another and initiate alterations in the allele status from normal to abnormal. In contrast, gene polymorphism is defined as a variation that occurs in allele in a DNA sequence.
Gene polymorphisms are caused by duplications, deletions, and a mutation of triplication of high quantity of DNA base pairs sequences. In addition, Polymorphisms may occur due to changes inside introns or changes in regions that one or multiple DNA bases that are between genes. If the changes occur in a gene’ coding sequence, then different phenotypes may appear as a result of protein variation that is caused by sequence changes. These changes are located exactly in genes’ coding sequence
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