Molecular Genetics (TRANSFERRED) Flashcards
Reference: Genetics residents in-training examination - March 2020 (Spring 2020)
You are seeing a 6 year old girl who is homozygous for a pathogenic mutation in a recessive gene, which explains her symptoms.
The couple wants to know their reproductive risk. You test the parents and only the father is carrier of this mutation. Name two possible explanations; for each explanation, propose a diagnostic test and state the recurrence risk for the couple if this is the actual cause.
- Deletion of the maternal allele: test for deletions, RR 25%.
- isoUPD pat: UPD trio or SNP-array the patient, RR<1%
- The mother is low level somatic/gonadal cell mosaic for the mutation: NGS 500x in order to detect it/digital qPCR, RR <1-25%
- De novo mutation on the maternal allele, test for deletions is negative, R/O lab error, (+/- ID test (microsatelittes) to prove that she is the biological mother), RR<1%
- Lab error : test on another blood sample, or in another lab. Troubleshooting.
- Egg donor/fertility clinic error.
Reference: Genetics residents in-training examination - March 2020 (Spring 2020)
Synonymous variants are usually expected to be tolerated. Describe one exception to this.
Synonymous variants are tolerated except when they affect splicing. This usually occurs when the synonymous change occurs near the exon/intron boundary i.e. near or in the canonical splice site.
Reference: Genetics residents in-training examination - March 2020 (Spring 2020)
Name two mechanisms by which a deep-intronic variant can be pathogenic.
- The mutation creates a new splice donor site, or a new splice acceptor site leading to pseudo-exon inclusion
- The mutation creates a splicing enhancer or interferes with a silencer leading to pseudo-exon inclusion
- The mutation interferes with transcription regulatory motifs
- The mutation inactivates non-coding RNA genes
- A genomic rearrangement can insert an exon from another gene within an intron leading to inclusion of this exon in the mRNA
(ref: Hum Genet (2017) 136:1093–1111)
Reference: Genetics residents in-training examination - March 2020 (Spring 2020)
Describe the variant illustrated on the following figure.
See Genetics residents in-training examination
March 2020 - page 14.
What type of variant is this?
Do you expect this variant to be pathogenic?
Name three different online resources that may help you correctly assign a pathogenicity prediction to this variant.
- Substitution
- Nonsense
- Yes
- ClinVar, Locus-specific database (LSDB), gnomAD/ExAC, MutationTaster and other in silico prediction tools, as well as any other answer that is deemed adequate
Reference: Genetics residents in-training examination – Fall 2020
A. How would you describe the variant c.2606C>G, p.Ser869* details? What type of mutation is it?
B. How would you describe the variant c.179A>G, p.Asn60Ser details? What type of mutation is it?
C. You just received a laboratory report indicating your patient has a variant of uncertain clinical significance (VUS). How would you summarize the meaning of a VUS to your patient?
A. a C to G substitution at nucleotide 2606, amino acid 869 changed from a serine to a stop codon (nonsense change). Frameshift mutation.
B. an A to G substitution at nucleotide 179, amino acid 60 changed from a asparagine to a serine (nucleotide substitution). Missense mutation.
C. A variant for which there is insufficient information either towards pathogenic or benign to confidently determine the variant’s significance, or a variant for which conflicting evidence (both apparent pathogenic and benign) exists.
Reference: Genetics residents in-training examination – Fall 2020
Give one example of a genetic disorder caused by the mechanism listed:
a. Gene Copy Number Variations
b. Epigenetic Modifications
c. Repeat Expansions
d. Recurrent Single Nucleotide Changes
e. Non-recurrent Single Nucleotide Changes
List two testing technologies used in molecular testing for each of the chosen disorders
Give one example of a genetic disorder caused by the mechanisms listed:
a. Spinal muscular atrophy, hereditary breast cancer HER2, DiGeorge syndrome, various cancers and MYC (ie. neuroblastoma + MYCN), any disease with loss of function as the mechanism (ie. Duchenne and Becker muscular dystrophy)
b. Beckwith-Wiedemann syndrome, Prader-Willi syndrome, Angelman syndrome, Silver-Russel syndrome
c. Fragile X syndrome, myotonic dystrophy type 1 and 2, oculopharyngeal muscular dystrophy, amyotrophic lateral sclerosis/frontotemporal dementia, some inherited ataxias
d. Hereditary hemochromatosis, thrombophilia, achondroplasia, cystic fibrosis
e. Any genetically heterogeneous disorder; hearing loss, hypertrophic cardiomyopathy, dilated cardiomyopathy, epilepsy, lysosomal storage disease, neuromuscular disorders, RASopathies, skeletal dysplasias, autism
List two testing technologies used in molecular testing for each of the chosen disorders
a. Gene Copy Number Variations: FISH, microarray, MLPA, qPCR, next generation sequencing
b. Epigenetic Modifications: methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), bisulfite sequencing
c. Repeat Expansions: traditional PCR, triplet repeat-primed PCR, Methylation-specific PCR, Southern blot
d. Recurrent Single Nucleotide Changes: restriction fragment length polymorphism (RFLP), allele specific oligonucleotide, Sanger sequencing
e. Non-recurrent Single Nucleotide Changes: Sanger sequencing, next generation sequencing
Reference: Genetics residents in-training examination – Fall 2020
You identify one pathogenic variant in an autosomal recessive disease gene that fits with the broad phenotypic presentation of your patient. What are four possible explanations for this and how would you test for them in either a clinical or research environment?
Missing deletion/duplication and need to do del/dup analysis (MLPA or exon-level microarray)
Splice variant (RT-PCR or RNASeq – research)
Noncoding promotor variant (genome and RNASeq or RT-PCR)
Might not be the gene (exome or genome)
Reference: Genetics residents in-training examination – Fall 2020
A) Give one disease example for each of the following mechanisms of disease:
- Dominant negative
- Loss of function
- Gain of function
B) Briefly describe molecular pathogenesis for each of the three disease mechanisms listed above.
A)
- Dominant negative: CFTR, Achondroplasia (FGFR3), Osteogenesis imperfecta
- Loss of function: Hereditary cancers (BRCA1/2), Lots
- Gain of function: Noonan (PTPN11), Huntington’s
B)
1. Dominant negative mutations arise when the mutant allele interferes with the function of the remaining wild-type allele, causing a >50% loss of function. This can occur when the gene product forms active complexes with itself (and possibly with other proteins)
- Loss-of-function mutations result in reduced or abolished protein function.
- Gain of function mutations confers new or enhanced activity on a protein.
Reference: Genetics residents in-training examination – Fall 2020
You have identified a variant in a known disease gene.
1. Describe steps you will take to assess clinical significance of this variant.
- Give examples of evidence sufficient to make the following conclusions:
a. This variant is likely pathogenic
b. This variant is likely benign
- Follow the ACMG variant interpretation guidelines to score the evidence collected to date to arrive to the final variant classification (PMID 25741868).
Some examples of the relevant evidence:
- Determine if allele frequency of the variant is significantly lower in presumed healthy individuals (gnomAD) comparing to individuals with the disease (ClinVar).
- Determine if in silico prediction programs (e.g. MutationTaster, SIFT, Polyphen etc) predict this variant to be damaging
- Review literature citing this variant and determine strength of genetic, functional and segregation evidence collected to date (if any)
- a. PVS1+PM2
- The variant is predicted to cause a frameshift resulting in a truncated and absent protein
- The variant alters a known disease-causing gene and loss-of-function variants in that gene have been definitively associated with the disease.
- The variant has not been previously identified in presumed healthy individuals (absent from gnomAD)
b. BP4+BP7
- Synonymous variant for which splicing prediction algorithms predict no splicing impact
- Multiple lines of computational evidence predict on impact on gene or gene product (conservation, evolutionary, in silico etc)
