Unclassified Variants Flashcards Preview

Genetics of Learning Disorders > Unclassified Variants > Flashcards

Flashcards in Unclassified Variants Deck (26):
1

What has caused variant classification to become even more prominent in Genetics labs?

- Recent technology has increased our capacity to sequence large numbers of genes.
- Our ability to interpret sequence changes lags far behind.
- Hence many of the variants detected by sequencing are reported as variants of unknown clinical significance.
- Nevertheless there are established procedures for investigating the clinical significance of variants and this is carried out by appropriately qualified and experienced staff.

2

How do we go about correctly naming sequence variants?

- Descriptions of sequence variants should use HGVS nomenclature.
- HGVS recommendations for the description of sequence variants are designed to be stable, meaningful, memorable and unequivocal.
- However, every now and then small modifications are made to remove small inconsistencies and/or to clarify confusing conventions and to add any previously uncovered sequence alterations.
- Variants should therefore be described with respect to a HGVS nomenclature and with respect to a reference DNA sequence specified by an accession number and a version.
- RefSeq sequences are derived from GenBank and provide non-redundant curated data representing our current knowledge of known genes. Some records include additional sequence information that was never submitted to an archival database but is available in the literature. Some sequence records are provided through collaboration; the underlying primary sequence data is available in GenBank, but may not be available in any one GenBank record. RefSeq sequences are not submitted primary sequences. RefSeq records are owned by NCBI and therefore can be updated as needed to maintain current annotation or to incorporate additional sequence.

3

What reference sequence is it best to use in nomenclature?

It is best to use the RefSeq as it has been curated and updated by NCBI.

4

What different types of pathogenic mutations exist?

1). Deletions:
- The entire gene
- Part of the gene
- Single / multiple exons

2). Disrupt gene structure
- Translocations
- Inversions
- Insertions

3). Prevent promoter working
- Methylation

4). Prevent correct splicing
- Inactivation of donor splice sites
- Inactivation of acceptor splice sites
- Activating a cryptic splice site

5). Introduction of a frameshift in translation
- Small insertions/deletions - also produce premature stop codons
- Convert a codon into a STOP codon
- Replace/delete an essential amino acid

5

What kind of effects can a missense variant lead to?

Missense changes:
- Amino acid changes of unknown effect
- The effect depends on the chemical difference between old/normal and new amino acid
- Location of the change - is it in the active site?
- Location - is it in the non-functional part but important in maintaining the protein structure/stability. May lead to the degradation of protein or incorrect localisation of the protein which may make the protein less or more flexible which may lead to difference in specificity.
- May give a loss of function or a gain of function.

6

What kind of effects can an intronic variant lead to?

Intronic Variants:
- May or may not affect the splice site.

7

What kind of effects can a synonymous variant lead to?

Synonymous:
- Variant does not change the amino acid
- May create a cryptic splice site
- May disrupt exon splice enhancer/silencer sequences.

8

What kinds of variants might you need to classify?

Missense changes:
- Amino acid changes of unknown effect
- The effect depends on the chemical difference between old/normal and new amino acid
- Location of the change - is it in the active site?
- Location - is it in the non-functional part but important in maintaining the protein structure/stability. May lead to the degradation of protein or incorrect localisation of the protein which may make the protein less or more flexible which may lead to difference in specificity.
- May give a loss of function or a gain of function.

Intronic Variants:
- May or may not affect the splice site.

Synonymous:
- Variant does not change the amino acid
- May create a cryptic splice site
- May disrupt exon splice enhancer/silencer sequences.

9

What do we need to do in order to be able to classify variants of unknown clinical significance?

- We need to accumulate information/evidence to make a decision on the likelihood of a variant being pathogenic or not.
- No one piece of evidence should be used alone.
- CMGS Best Practice Guidelines 2007 help us to interpret these unclassified variants - "interpretation and Reporting of Unclassified Variants (UV's) in Clinical Molecular Genetics".

10

What different lines of evidence can we examine in order to be able to classify variants of unknown clinical significance?

Lines of evidence:
- Mutation database
- Presence/absence in SNP database
- Testing matched controls
- Literature search
- Co-occurrence in trans with a known pathogenic mutation
- Co-segregation with the disease in a family
- Loss of heterozygosity in tumour (loss of wt allele)
- Occurrence of a new variant with sporadic incidence of the disease
- Species conservation
- In silico prediction software programs

11

Describe how we can use mutation databases when investigating unknown sequence changes.

- We can refer to a number of databases. These include locus specific databases.
- The most commonly used general database is DMuDB (managed by NGRL in Manchester).
- DMuDB is a database containing variants and known mutations for many genes.
- DMuDB is an excellent source. Can investigate if change has been found before by another diagnostic lab.
- DMuDB is also linked to dbSNP which is an SNP database which may have frequencies in normal populations. Also has known mutations.
- Can also look at variants detected NHLBI Exome Sequencing Project.

12

Describe how we can use the testing of matched controls when investigating unknown sequence changes.

- Testing matched controls is particularly useful for ethnic populations in which little information is known.
- This approach involves screening a number of individuals.
- This is usually a tremendous and costly effort. To have a 95% chance of observing a variant with an allele frequency of 1 in 100 at least once we would have to screen 298 chromosomes. Still may not identify rare variants.

13

Describe how we can use literature searches when investigating unknown sequence changes.

- Use search engines and be aware of any legacy nomenclature.
- May show functional studies. Requires a knowledge of all possible functions of the protein, requires a suitable assay, usually performed only in research labs, just because the variant in question leads to a reduction or abolition of function does not necessarily mean that it would lead to the phenotype in question.
- Need to read any publications critically. Many of these will be from researchers. Need to decide if there is sufficient evidence for clinical use.

14

Describe how we can use co-segregation analysis when investigating unknown sequence changes.

Co-segregation analysis can be undertaken to see if the variant segregates with the disease in the family.

1). Co-segregation analysis with disease in the family may be tricky for a number of reasons:
- Limited by partial penetrance
- The disorder may be late onset
- Phenocopies - individuals displaying similar symptoms
- Requires samples from enough family members
- Is the variant linked to the unidentified pathogenic varian on the same allele? Need functional assay on the observed sequence variant

2). Co-occurrence in trans with a known pathogenic mutation is also something that needs to be investigated:
- Has this variant ever occurred in cis or trans with a pathogenic mutation
- Need to determine if on opposite chromosome (in trans) by testing parents or other relatives.
- Principle - 2 mutations may be embryonic lethal
- Principle - 2 mutations homozygous state or in compound heterozygote state may give distinct phenotype
- Depends on penetrance of heterozygote genotypes which is impossible to determine

3). Occurrence of a new variant concurrent with the disease:
- Is this a sporadic variant or has it been inherited from the parents
- Requires parental samples
- Issues surrounding imprinting and partial penetrance

15

Why does co-occurrence in trans with a known pathogenic mutation is also something that needs to be investigated when classifying variants?

Co-occurrence in trans with a known pathogenic mutation is also something that needs to be investigated:
- Has this variant ever occurred in cis or trans with a pathogenic mutation
- Need to determine if on opposite chromosome (in trans) by testing parents or other relatives.
- Principle - 2 mutations may be embryonic lethal
- Principle - 2 mutations homozygous state or in compound heterozygote state may give distinct phenotype
- Depends on penetrance of heterozygote genotypes which is impossible to determine

16

Describe how we can use species conservation when investigating unknown sequence changes.

Species conservation:
- Principle: the more conserved an amino acid is then the less likely an amino acid change will be tolerated
- Requires reference sequence alignments. Requires careful design and may need to vary with gene type.
- Recommended to include at least 5 mammalian homologues over 300 million years.
- Need to cover an appropriate amount of evolutionary time.

17

Describe how personal and family history can be used to investigate unknown sequence changes for some disorders.

Personal and Family History:
- To find if the variant under investigation falls into individuals with strong family history. For example for breast cancer does the family history resemble that seen for known carriers of BRCA1 mutation (rather than that seen in an individual without a BRCA1 mutation).
- E.g. If a large number of index cases are mutation screened and many of them provide simple family cancer histories. These family histories can be sorted into most severe to least severe. Placing the variant in either category helps in defining the likelihood of it being pathogenic.
- This requires significant number of families and also relies on the assumption that the individual recollection of the family history is accurate.

18

Describe how tumour pathology and IHC can be used to investigate unknown sequence changes for some disorders.

- If details of tumour phenotype differ between clearly BRCA mutations and non-carriers then it should be possible to use tumour phenotype to help assess unclassified variants (e.g. BRCA1).
- Requires good quality tumour sample.
- However, IHC characteristics usually result from truncated proteins and missense mutations don't truncate proteins.

19

Describe how loss of heterozygosity (LOH) can be used to investigate unknown sequence changes for some disorders.

- Loss of variant allele suggests that the unclassified variant is not pathogenic, whereas the loss of the wt allele particularly or no loss at all is consistent with pathogenicity.
- Requires good quality tumour material (no normal tissue) and good quality tumour DNA.

20

Describe how we can use in silico prediction of pathogenic effect when investigating unknown sequence changes.

- Software to predict if an amino acid substitution is tolerated.
- Align GVGD combines protein sequence alignments of multiple species with biophysical characteristics of amino acids. The biochemical distance of the variant amino acid from the observed amino acid at a particular location in different species is calculated (Grantham deviation). A grade, varying from C0 to C65 is given to estimate the probability that a certain variant is pathogenic.
- High risk (>60)
- Low risk (>60)
- SIFT (Sorting Intolerant from Tolerant) - based on sequence evolutionary conservation of the affected residue and the type of amino acid substitution SIFT scores less than 0.05 are predicted to be deleterious (probably pathogenic) and scores greater than or equal to 0.05 are predicted to be tolerant (benign).
- Polyphen - relies on protein sequence and structure to make predictions on the affect of amino acid substitutions.
- The problem is that different programs can give different results.

21

Describe align GVGD.

Align GVGD combines protein sequence alignments of multiple species with biophysical characteristics of amino acids. The biochemical distance of the variant amino acid from the observed amino acid at a particular location in different species is calculated (Grantham deviation). A grade, varying from C0 to C65 is given to estimate the probability that a certain variant is pathogenic.

22

Describe SIFT.

SIFT (Sorting Intolerant from Tolerant) - based on sequence evolutionary conservation of the affected residue and the type of amino acid substitution SIFT scores less than 0.05 are predicted to be deleterious (probably pathogenic) and scores greater than or equal to 0.05 are predicted to be tolerant (benign).

23

Describe Polyphen.

Polyphen - relies on protein sequence and structure to make predictions on the affect of amino acid substitutions.

24

Describe how we can use in silico splice site prediction of when investigating unknown sequence changes.

- Several web based programs
- Not been validated for use in a diagnostic setting
- Settings can be adjusted by user
- Use of four programs
- All missense and intronic variants should be investigated for disruption/creation of splice sites
- Where 2 programs indicate a splice alteration then RNA studies should be performed in labs that offer this service - or alternatively splicing can be investigated by minigene assays.
- Separate splice prediction programs to look for ESE/ESS sites - sequence motifs more variable - not used routinely
- Alamut uses 4 splice site programs and generates a report. Scientist needs to make the final decision.

25

Describe the classification system generally used in labs.

1 - certainly not pathogenic
2 - unlikely to be pathogenic but cannot be formally proven
3 - Likely to be pathogenic but cannot be formally proven
4 - Certainly pathogenic

26

How should unclassified variants be reported?

- Recommended that labs keep patient specific checklists of all documented evidence (mutation/variant report).
- Reports should summarise the evidence for and against pathogenicity.
- RNA analysis may be recommended.
- Co-segregation/parental studies can be recommended.
- Labs should have a system for recording UVs and submitting them to appropriate mutation databases (likely to becom mandatory CPA requirement).
- Labs should have a mechanism to regularly review UVs - additional data may become available to re-classify.