04 and 05 Interpreting Genomic Data

What is a Variant Call File (VCF), and what information does it contain?

A Variant Call File (VCF) indicates variations in the genome between individuals. It lists variants, often specifying changes like "on chromosome 1, position 10177, there should be an 'A' but an 'AC' is present". This file is used to list variants related to a disease, provided the information is not too extensive for a computer to handle.

What is the primary goal of filtering mutation lists in genomic data analysis?

The primary goal of filtering mutation lists is to identify which specific variant is the causative one for a disease. This process relies on prior knowledge and involves asking several key questions about the variants.

How is "functionality" considered when filtering variants to find a causative one?

Regarding functionality, a key question is whether the variant causes a change in function. Non-functional variants, such as non-coding and synonymous variants, are often filtered out early in the process, leaving primarily exons and coding regions for further analysis.

How is "frequency" used as a criterion for filtering variants, and what role do databases play?

When considering frequency, one asks if the variant's frequency in the population is comparable to the disease prevalence. Databases are utilized to determine population frequencies, and it's noted that frequencies can differ across various populations. If a variant causes a rare disease, it is expected to be a rare variant in the population.

How do "zygosity" and "mode of inheritance" help in identifying a causative variant?

The zygosity of the variant (e.g., homozygous or heterozygous) is checked to see if it aligns with the known mode of inheritance for the condition, such as autosomal recessive, autosomal dominant, or X-linked.

What role do "patients vs. controls" and "family occurrence" play in identifying a causative variant?

A causative variant should be found only in patients having the condition and not in controls. Additionally, it's considered whether the variant is found in only one family or in multiple, unrelated families.

Explain how "segregation with disease" is used to identify a causative variant.

To identify a causative variant, it must segregate (track) with the disease within the family. This means all affected individuals within the family should possess the same genotype for the variant, while all unaffected individuals should have a different genotype.

What additional factors, "biological fit" and "incomplete penetrance," are considered when identifying causative variants?

It's important to consider if the variant fits with the known biology of the disease. Incomplete penetrance is also a factor, occurring when some patients exhibit the condition and symptoms while others with the same variant do not.

Describe the typical variant classification system.

Variants are commonly classified using a 5-tier system: Pathogenic, Likely pathogenic, Variant of uncertain significance, Likely benign, and Benign. It's important to note that not all coding variants, loss-of-function variants, or rare variants necessarily cause disease.

Outline the first three steps for finding a pathogenic variant in a mitochondrial disease case using genomic sequencing.

1. Genome Focus: Filter the data to focus specifically on the mitochondrial genome. 2. Quality Control: Remove data that is of low quality or has low coverage. 3. Variant Presence: Keep only variants present in affected individuals and absent in unaffected individuals.

What are the final steps in filtering for a pathogenic variant in a mitochondrial disease case after initial QC and presence checks?

4. Remove Synonymous Variants. 5. Remove Frequent Variants. 6. Database Check for known disease associations.

List the methods used to confirm a potential pathogenic variant after identification.

• Case-control and population studies - Protein work (expression studies, functional assays) - Co-segregation studies (preferably in unrelated families) - iPSC and organoid studies - Model organism studies

What are "Incidental Findings" (IFs) in genomics?

Incidental Findings (IFs) are "other variants" discovered during genomic analysis that are not directly related to the primary reason for sequencing but may still possess clinical significance.

What are the key considerations for returning Incidental Findings (IFs) to individuals?

• Burden of knowledge - Potential for life-altering decisions - Impact on family - Effect on insurability and employability

Under what conditions should Incidental Findings (IFs) be returned to research subjects?

IFs should be returned if: - They are scientifically valid and confirmed - They have health implications - A clear course of action is available

What is "Variant Reclassification"?

Variant reclassification is the process of updating a variant’s classification (e.g., from pathogenic to benign or vice versa) based on new data, meaning classifications are not permanent.