is this a potential mosaicism?

Question

[lait]

Hi,

I have found a denovo mutation in the child:

• coverage: 356
• Ref allele: G
• Alt allele: A
• ref allele: 48% (109+, 63-)
• alt allele: 52% (118+, 66-)

Father:

• coverage: 254
• Ref allele: G
• Alt allele: -
• ref allele: 100% (151+, 103-)
• alt allele: 0%

Mother:

• coverage: 211
• Ref allele: G
• Alt allele: A
• ref allele: 97% (125+, 79-)
• alt allele: 3% (5+, 2-)

this is a link showing some screenshots for father/mother/child in IGV:

questions:

1- Do you think this could be a hint for a low-parental mosaicism in the mother that appeared as denovo in the child?

2- Could you please give me your thoughts on: how to investigate further in this case to make sure if this is/is not real mosaicism?

any hints are much appreciated!

EDIT:

this image shows the 7 reads of the mom carrying the mutation.

[russhh]

So the child is (G, A), the father is (G, G) and the mother is (A, A), but the mother has some low-frequency reference reads at the same position. It doesn't look like a de novo mutation to me.

[lait]

Sorry, there was a mistake in the AF of the mother, I just corrected it. the mother is being reported as 0/0 by varscan2, because of the low AF of 'A' that is why this mutation is considered denovo in the child

So: child: 0/1, mother: 0/0, father: 0/0

[russhh]

Cool - sorry about the pedantry. Yeah that's pretty interesting - are there any other technical issues that might explain it though: Did you have to PCR the DNA? were the samples sequenced in separate batches?

[lait]

I am not sure what do you mean by PCR the DNA, I can ask our lab staff though if you give me more details. What I can say is: we always sequence our samples (WGS, illumina 2500) twice in the same run, on 2 different lanes, and then we collect the samples across different lanes. We do this to get a higher coverage.

[finswimmer]

It might be a low-parental mosaicism.

I would try to confirm this with sanger sequencing. But with this low frequency it is not sure if it's possible. So if you see this mosaic with sanger than it is confirmed, but if you don't see it you cannot exclude it.

fin swimmer

[h.mon]

Just out of curiosity / thinking out loud:

Did you check if the Alt reads from the mother are PCR / optical duplicates? Did you check dbSNP for this variant? I am under the impression there is some strand bias, am I wrong?

[lait]

Thanks h.mon, please think out even louder, those are the sort of things that I need to know: how to investigate more, manually.

• Please check this image , it shows the 7 reads of the mother having this mutation.

• No duplicates as far as shown, or do consider the 5th and 6th reads as duplicates with opposite strand orientation ?

• All 7 reads have a RMSMappingQuality (MQ) = 60.

[h.mon]

Are your reads single- or paired-end?

[lait]

they are paired-end reads

Answer 1

[Matt Shirley]

For the mother's mosaic allele to be passed to the child you she would need to have the mosaicism present in her germline cells. What tissue are you sequencing? If it's a buccal sample or skin punch then it's unlikely that the same developmental progenitor cell that gave rise to the sample you sequenced also gave rise to the mother's egg cells. I'd try to validate the mother's alternate allele using taqman probes, ddPCR, or an amplicon sequencing assay to be sure it's biological, and even then it's a hard sell to determine if the mother actually passed that allele to the child.

[lait]

thanks for ur answer Matt. the sequencing is done for blood samples for the child, mother and father.

[Matt Shirley]

It's a little more interesting to see this in blood tissue, since there's definitely more mosaicism in blood. If you have access to assays that sequence cell-free DNA, it might be interesting to see if there's an enrichment of the allele in the cfDNA, since this may be shed from some other maternal tissue (not present in blood cells themselves).

[finswimmer]

For the mother's mosaic allele to be passed to the child you she would need to have the mosaicism present in her germline cells.

That's why my first intuitive answer was: "No" But than I've found several sources that say it is possible that germline mutation could be present in other somatic cells.

As h.mon says one should first quality check those reads that have the variant.

fin swimmer

Answer 2

[swbarnes2]

I think there are other simpler, more likely explanations than mosaicism. Do you have enough other genetic data to be confident the reported father is really the father?

[lait]

Yes, we have enough genetic data for this trio and we made sure the parents are the real biological mother & father for the child