Guidance for connecting a rare phenotype to a genotype in a small family sample

Question

[tac-a-cat]

Hello all, long time reader, first time poster.

I'm looking for guidance on how to connect a rare phenotype to a genotype in my small sample of 5 related bovines (3 parents, 2 offspring). I have WGS of all 5 individuals (12x average coverage).

The pedigree is as follows (explanations under the graph in case of difficulties interpreting the color scheme):

• Cow 1 and Bull 1 have 1 healthy female offspring, Yearling 1.
• Bull 1 has 1 affected offspring (Yearling 2) with another female, Cow 2.
• There is a case of inbreeding between Bull 1 and his offspring Yearling 1, producing another affected individual, Yearling 3.

From this I can conclude that:

• The phenotype is recessive. All bovines have several healthy offspring each prior to these events.

  • The phenotype is not X-linked (despite the affected offspring being both male, the father is healthy.)

  • The phenotype is likely monogenic. The ancestors of Bull 1 and Cow 2 can be traced to the same farm several generations prior. So a polygenic recessive factor would probably not survive all these crossing-over events leading up to the affected offspring. Plus the phenotype appears the same in both of the yearlings.

I am looking for guidance on how to proceed in finding the genetic cause for this mutation. I already attempted basic analyses with PLINK, GATK and similar, but the results are all inconclusive, likely due to the small sample size. I can get more WGS from NCBI and similar, but how do I treat my original samples - would they all be cases, or should the carriers be in a separate group? Could the inbreeding help somehow? I imagine the genetic cause and its surrounding genomic context are super homozygous, but when I ran a ROH analysis, many many regions were like that, due to the inbreeding being such a recent event.

I'm open to suggestions for tools, methods or papers, I'll do the actual code and analysis myself of course, not asking for premade solutions here.

Thanks!

[Ankit]

From the pedigree,

C1: may be a carrier
B1: Carrier
Y1: Carrier
C2: Carrier
Y2: recessive
Y1: recessive

You can extract all genetic variation from VCF where Y2 and Y3 are 1/1 and check segregation with their respective parents. Take only segregating variants (inherited from parents) and see overlap between Y2 and Y3. May be you will see some overlap variants and then evaluate the deleteriousness of these genetic variants. After annotation have a look SIFT, Polyphen scores, Stop gain, loss frameshifts, nonsynonymous etc. You will get some hints from this analysis. If you don't find anything you can call indels from your bams and perform similar analysis.

I hope it helps.

[tac-a-cat]

Thanks for the reply, I will have a look. Some of the variants are marked as 2/2, or 1/3 or 2/4 etc. I imagine those are alternative alleles marked in order from most common to least common? So that means that extraction and filtering should be repeated for 2/2, 3/3, 4/4 etc. sites.?

[Ankit]

This is bit complicated. So,

0: Reference
1: Alternate allele
2: Second alternate allele
3: Third alternate allele
4: Fourth alternate allele

Given that disease is recessive, If both parents are 1/3 then either 1/1 or 3/3 may cause disease.

But if one parent is 1/3 and other parent 1/2 then 1/1, 3/2, 2/3 or 1/2 might cause disease.

So just 2/2 , 3/3 , 4/4 will not be sufficient.

To analyze this, you'll need to focus on segregating variants and identify cases where the offspring has a genotype associated with disease (overlap strategy between affected) based on the combination of alleles inherited from each parent.

Dealing with alternate allele will add another layer of complexity.

May be some segregating software exist for predicting this scenario (I'm not aware).

By manually doing this needs you need to be very careful while elimination. Try