Query regarding variant calling and reporting

Question

[luffy]

Hello,

I have a few queries, if anyone would kindly classify it would be really helpful.

1. when a heterozygous single variant for an autosomal recessive disorder classified as likely pathogenic and could not find second significant heterozygous variant, but this variant matches the phenotype of the patient, in this case should this variant be reported as a causative mutation or as actionable mutation? and what would be the next step? validate the variant with sanger or any new strategy to find the second significant heterozygous variant?

2. What can be done when no significant variant was detected using clinical exome sequencing? How do we decide the next strategy to find the causative mutation?

3. I have a few SNVs and CNVs found using clinical exome sequencing, i want validate these variants for publication what are the methods/strategies can i use to validate these variants? for example for SNV sanger and for CNV MLPA, what are the other methods?

Any help would be much appreciated

Thank you

[desouzareis.r]

Hi,

1. Any pathogenic, likely pathogenic or even unknown variants that matches the phenotype and has passed on quality control should be reported. Maybe the ACMG guidelines for the interpretation of sequence variants could help.
2. I think WGS would be the next strategy as you can investigate the presence of SVs/CNVs, ROH, expansions. However is more expensive.
3. Sanger and MLPA would be the most used strategies for SNVs and CNVs respectively. However, it's also possible to validate SNVs with Nextera technology using NGS and validate CNV using Microarray/CGH-Array or Fish depending on the CNV size and characteristics.

I am not expert in variant interpretation, however I hope I helped.

[luffy]

Hello @desouzareis.r, Thank you for your response,

1. Yes, i have been following ACMG/AMP guidelines, however i was bit confused since i was not able to find the second significant heterozygous variant which pass all the filters such as population frequency etc, anyway my question is whether to do something to find the second heterozygous variant or just go about validating the variant?

2. Since i am doing this as a part of research project WGS is bit expensive since i also have few more samples, what is your thoughts on doing WES instead of WGS. For ROH should i also sequence parents samples as well correct? so like WES trio analysis?

Thank you for your time

[desouzareis.r]

Hi,

1. I had a little help to answer this question and yes, you should find the other variant at any cost... it should be somewhere. However you should be absolutely sure that that is the phenotype. Maybe the second variant could be with a higher population frequency or with low coverage, so would be a good idea to change the filters you are using. Another strategy would be a digenic approuch.
2. The trio analysis is a good option that could help, you could try to find Structural Variants others than CNVs although is harder using WES. For ROH you doesn't need to sequence parents samples, but I have no experience with ROH analysis.