UV Analysis

Question 1

Describe the 4 main classes of genetic variation that can arise

Answer 1

1. SNPs which can have no effect on the protein product,
2. Structural variants that affect more than one base pair and can involve insertions or deletions of genomic sequences
3. Copy Number Variation involves chromosomal deletions or duplications in which a gain or loss of a stretch of DNA or an entire chromosome occurs
4. Microdeletions or duplications can cause Developmental Delay and Intellectual Disability

Question 2

What is a rare disease, how many have been described and give an example

Answer 2

It affects less than 1 in 2000 individuals, ~7,000 have been described and an example is Familial hypercholesterolemia

Question 3

What are monogenic disorders

Answer 3

They arise due to a highly penetrant variant in a single gene, examples include Huntington’s disease

Question 4

What is Penetrance

Answer 4

Probability that individual with a variant will express a phenotypic difference

Question 5

What is Expressivity

Answer 5

The magnitude of the phenotypic difference

Question 6

What are autosomal dominant variants and give an example of a disorder

Answer 6

One mutated copy of a gene is required to cause disease i.e. heterozygous carriers are effected. Disease is usually highly penetrant. Familial Hypercholesterolemia

Question 7

What are autosomal recessive variants and give an example of a disorder

Answer 7

Variants in both copies of a gene lead to disease i.e., homozygous for a variant, or compound heterozygous for two different variants within the same gene. Cystic Fibrosis

Question 8

What are X-linked dominant variants and give an example of a disorder

Answer 8

A single copy of the mutated gene within the X chromosome causes disease; both males and females are affected. Less common than recessive. Fragile X syndrome

Question 9

What are X-linked recessive variants and give an example of a disorder

Answer 9

Associated with mutations in genes on the X chromosome. Males will be affected; female can compensate with normal gene on their second X chromosome. Duchenne Muscular Dystrophy

Question 10

What are Y-linked recessive variants and give an example of a disorder

Answer 10

Associated with mutations in genes on the Y chromosome. Only males are affected and dominance of gene is irrelevant. Swyer syndrome

Question 11

What are mitochondrial variants and give an example of a disorder

Answer 11

Variants in the mitochondrial DNA consisting of 37 genes believed to be exclusively inherited maternally. Leber’s hereditary optic neuropathy

Question 12

What is a de novo variant

Answer 12

They arise in a family member for the first time; unique form of rare genetic variation, extremely low incidence.

Question 13

Explain Mosaicism

Answer 13

Offspring are born to phenotypically normal parents
Germline; de novo variant in germ cell during early embryonic development of the parent.
Somatic; variants occurring during postzygotic development; early vs late.

Question 14

What is a missense variant

Answer 14

A change in the nucleotide sequence results in an alterative amino acid which can lead to structural or functional changes in the translated protein

Question 15

What is a synonymous variant

Answer 15

A change in the nucleotide sequence gives rise to the same amino acid, but this can disrupt splicing, transcription, co-translational folding and mRNA stability

Question 16

What is a nonsense variant

Answer 16

A change in the nt sequence results in a premature stop codon which can lead to a truncated and potentially non-functional protein

Question 17

What are insertions and deletions

Answer 17

They involve an Insertion or deletion of a number of nts. If indivisible by 3, can lead to changes in the reading frame of the gene -> original aa sequence of protein is lost.

Question 18

What is an inversion

Answer 18

Two breaks occurring in a chromosome; region between rotates 180 degrees and re-joins with the two end fragments. The original sequence is replaced by a reverse complement. Does not change overall amount of genetic material, however breakpoints occurring in essential genes can be lethal.

Question 19

What is a duplication

Answer 19

A copy of one or more nts are inserted directly 3’ of the original sequence copy. May affect phenotype by altering gene dosage. i.e. extra gene leading to excess protein.

Question 20

What is a conversion

Answer 20

Deletion-insertion where the original sequence is replaced by nts of a sequence copied from another genomic location. Pseudogene-mediated conversions can introduce pathogenic mutations into functional genes.

Question 21

What are Deletion-insertions (delins/indel)

Answer 21

One or more nts replaced by one or more other nts which is not a substitution, inversion or conversion. This is a common mechanism of kinase activation in cancer.

Question 22

What are the professional guidelines for variant nomenclature

Answer 22

The HGVS guidelines are the standard nomenclature in molecular diagnostics accepted world-wide. This allows for the standardisation of variant description to prevent inconsistencies and miscommunications. The standards are designed to ensure stability, meaningful, memorable and unequivocal and should be used when reporting clinically, in the literature or in databases

Question 23

Who wrote the professional guidelines for somatic variant interpretation and why are they important?

Answer 23

The guidelines have been outlined by the Association of Molecular Pathology (AMP) and these guidelines are devised to standardise variant interpretation. The classifications are treatment driven, therefore the aim is to determine eligibility for therapy or predict therapy response.

Question 24

Briefly outline the framework for somatic UV classification

Answer 24

The framework for determining the disease-causing likelihood of a somatic variant is based on weighted evidence criteria related to somatic, germline and population databases, in silico bioinformatics tools, professional guidelines, prognostic/diagnostic/therapeutic significance, clinical trials, approved therapies.

Question 25

What are the tiers that a somatic driver variant can fall under

Answer 25

Driver variants are classified into a 4-tiered system: i. Tier I, driver variants of strong clinical significance ii. Tier II, driver variants of potential clinical significance iii. Tier III, driver variants of unknown significance iv. Tier IV, passenger variants of known insignificance

Question 26

Explain the difference between a driver and passenger variant

Answer 26

Driver mutations confer growth advantage on the cells carrying them and have been positively selected during the evolution of the cancer. They reside in the subset of genes known as ‘cancer genes’. Passenger variants that do not directly drive cancer initiation and progression

Question 27

Are diver variants always clinically actionable?

Answer 27

Driver variants are not always actionable and can have no direct impact upon clinical care i.e. not clinically actionable.

Question 28

Describe some of the criteria for a driver variant classification

Answer 28

The variant is present in a germline database as pathogenic or likely pathogenic associated or not associated with an inherited cancer The variant is present in a somatic database associated with patient tumour type. The variant is absent or at less than 1% AF in population databases

Question 29

Describe some of the criteria for passenger variant classification

Answer 29

The variant is functionally benign i.e., synonymous variant Variant is present in > 1% of the healthy population in a population database i.e., gnomAD Not listed in somatic variant database or seen at low numbers

Question 30

Describe some of the actionability assessment criteria for a Tier IA or Tier IB driver variant

Answer 30

A) There is an approved therapy available associated with the variant for the tumour type A) The significance of this variant has been listed in professional guidelines B) The variant has proven diagnostic or prognostic significance in the tumour type B) The variant predicts response or resistance to therapies with specific tumour type with strong evidence

Question 31

Describe some criteria for Tier II variants

Answer 31

There is an approved therapy with this variant for a different tumour type Inclusion in a clinical trial (Phase 2/3)

Question 32

Who wrote the professional guidelines for germline variant interpretation and why are they important?

Answer 32

The American College of Medical Genetic (ACMG) Standards and guidelines for the interpretation of sequence variants which has been interpreted for us in the UK by the Association for Clinical Genomic Science (ACGS) Best Practice Guidelines for Variant Classification in Rare Disease. The purpose of germline variant analysis is diagnostic driven; aim of the classification is to identify the disease-causing variant.

Question 33

Describe the framework for determining the disease-causing likelihood of a germline variant

Answer 33

Based on weighted evidence criteria related to patient phenotype, segregation data, population data, reports in clinical databases, in silico bioinformatics tools, functional studies and disease penetrance.

Question 34

What are the tiers a germline variant can fall under

Answer 34

1. Benign, 2. Likely Benign, 3. Uncertain Significance, 4. Likely Pathogenic, 5. Pathogenic.

Question 35

Give an example of very strong or strong evidence that indicates a pathogenic variant

Answer 35

The prevalence in affected individuals is significantly higher than controls Well-established functional studies show a deleterious effect

Question 36

Give an example of very strong or strong evidence that indicates a benignvariant

Answer 36

The MAD is too high for the disorder or the observation in controls is inconsistent with disease penetrance Well-established functional studies show no deleterious effect Non-segregation with disease

Question 37

What is the purpose of a Clinical Genetics Service

Answer 37

To assist with making an accurate diagnosis of a genetic condition by arranging and interpreting genetic tests and referring families to other professionals, if appropriate Aims to provide individuals affected or at risk of a genetic condition with accurate information, counselling support and genetic testing.

Question 38

What is the purpose of the Genetics Laboratory

Answer 38

To delivers a wide range of genetic and genomic analysis services for both rare disease and cancer, to the UK and internationally. The aim is to receive samples, perform the appropriate genomic tests, report results and dispatch DNA samples for specialist testing.

Question 39

Can you briefly outline the care pathway for patients with a genetic disorder

Answer 39

1. The patient has a consultation and is referred to clinical genetics 2. Consent for testing is obtained by a genetics counsellor who sends the request for testing is sent with the sample 3. The sample is receipted by the genetics laboratory and is put on the appropriate assay and resulting data undergoes bioinformatics processing 4. Variant interpretation is carried out by the scientists and an MDT meeting determines the outcome of the variant 5. Variants are reported if benign, likely benign, likely pathogenic or pathogenic 6. A clinical review is carried out based on the results. Clinician can give a diagnosis, offer treatment, familial genetic testing, further genetics counselling or if test is inconclusive further investigations may be undertaken.

Question 40

What are the differences between somatic and germline variants

Answer 40

Somatic variants are acquired and can occur within all cells but not in germ cells; are not inherited by subsequent generations Constitutional variants occur within all cells, including the germ cells and are inherited by subsequent generations

Question 41

Give examples of germline variant interpretation databases you may refer to while performing UV analysis

Answer 41

HGMDpro - core database comprising known/published variants for genetically inherited disease. Variants are categorised according to type i.e. missense or according to the disease/phenotype. ClinVar - hosted by NCBI, annotates variants with clinical significance based on previous report of variants found in patient samples, assertions made regarding their clinical significance, information about the submitter, and other supporting data. DECIPHER - Database of genomic variation and phenotype in humans using Ensembl resources is an interactive web-based database which incorporates a suite of tools designed to aid the interpretation of genomic variants. In addition to copy number variation, the DECIPHER also displays open-access patient sequence variants and DDD research sequence variants. Protein plots are also available that display missense constraint, gnomAD frequency, domains and ClinVar.

Question 42

Give examples of somatic variant interpretation databases you may refer to while performing UV analysis

Answer 42

COSMIC - The cBio Cancer Genomics Portal is an open-access resource for interactive exploration of multidimensional cancer genomics data sets. My Cancer Genome - My Cancer Genome is a cancer knowledge resource which contains up to date information on mutations associated with therapeutic implications. Cancer Genome Interpreter - Designed to support identification of actionable variants, CGI collates knowledge from various resources. Includes evidence levels for variants.

Question 43

What are examples of copy number variation interpretation databases used while analysing an array

Answer 43

HGMDpro - core database comprising known/published variants for genetically inherited disease. Variants are categorised according to type i.e. missense or according to the disease/phenotype. ClinVar - hosted by NCBI, annotates variants with clinical significance based on previous report of variants found in patient samples, assertions made regarding their clinical significance, information about the submitter, and other supporting data. DECIPHER - Database of genomic variation and phenotype in humans using Ensembl resources is an interactive web-based database which incorporates a suite of tools designed to aid the interpretation of genomic variants. In addition to copy number variation, the DECIPHER also displays open-access patient sequence variants and DDD research sequence variants. Protein plots are also available that display missense constraint, gnomAD frequency, domains and ClinVar. OMIM - Online Mendelian Inheritence in Man (OMIM) is a database of human genes and genetic phenotypes. The full-text, referenced overviews in OMIM contain information on all known mendelian disorders and over 15,000 genes. OMIM focuses on the relationship between phenotype and genotype. ClinGen - Clinical Genome Resource (ClinGen) consortium is curating genes and regions of the genome to assess whether there is evidence to support that these genes/regions are dosage sensitive and should be targeted on a cytogenomic array.

Question 44

What ethical issues are associated with genetic testing

Answer 44

Incidental findings - the potential for the recognition and reporting of incidental/secondary findings unrelated to indication for genomic testing that have medical implications for patient care. ACMG guidelines for incidental findings advise reporting of some IFs; medical benefit for the patients and families. Consent - must be given voluntarily by an adult with capacity, who has sufficient and appropriate information to make a decision. Confidentiality - Duty of confidentiality is balanced by a duty of disclosure under certain circumstances. Disclosing results to family members in the case of familial disease - there is an obligation to respect an individual’s confidence vs obligation to communicate potentially clinically relevant information to other family members. Misattributed paternity - Nondisclosure often guaranteed in consent forms, implications for family planning and screening.

Question 45

Which sources of evidence can be used to classify a variant

Answer 45

``` o In silico tools such as SIFT, CADD o Databases of “normal” such as gnomAD o Gene/disorder specific databases o ClinVar o Published literature ```

Question 46

What is a phylogenetic analysis

Answer 46

Estimates the evolutionary relationships among organisms using a sequence of a common gene or protein to assess the evolutionary relationship of species. Multiple Sequence Alignment (MSA) is generally the alignment of three or more biological sequences (protein or nucleic acid) of similar length. From the output, homology can be inferred and the evolutionary relationships between the sequences studied. Highly conserved sequences indicate importance and any changes could potentially be deleterious to function. MSA tools include MUSCLE (MUltiple Sequence Comparison by Log- Expectation), T-Coffee and Clustal Omega.

Question 47

What is protein function prediction and give an example of a tool

Answer 47

It involves the use of computational tools to predict the biological roles of proteins and can be based on homology, sequence motfis, structure, genomic-context, network based methods or computational solvent mapping BLAST, FASTA

Question 48

What examples of genome sequence databases are available

Answer 48

RefSeq - NCBI database of annotated sets of reference sequences for genomic, transcriptomic and protein sequences Ensemble - genome browser and database of reference sequences - genomic, transcript and protein

Question 49

Explain your tole to a non expert