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Genetic testing Report (VCGS)
mRNA transcript
NM_000257.2(MYH7):c.1208G>A
Protein
(NP_000248.2(MYH7):p.Arg403Gln)
Chromosome
(NC_000014.8:g.23898487C>T) (chr14:g.23898487C>T)
mRNA transcript
(NM_000257.2:c.1208G>A)
Protein
(NP_000248.2:p.Arg403Gln) (MYH7 Arg403Gln) MYBPC3 R542Q
Ensembl Mutalyzer
Seattle Seq Annotation
Conversion between variant annotation levels
Mutalyzer: https://mutalyzer.nl/position-converter
Seattle Seq Annotation: http://snp.gs.washington.edu/SeattleSeqAnnotation138/
Ensembl: http://www.ensembl.org/index.html
Mutalyzer conversion between mRNA and DNA
Step 1: select genome
Step 2: enter variant (DNA or mRNA)
Step 3: convert
Variant annotation
Annotation class Characteristic Source Feature name
Inheritance pattern Number of meiosis, obligate carriers, de
novo Family pedigree N/A
Mutation consequence
Missense, premature termination, gross deletion, splice site
Seattle Seq Annotation functionGVS
Mutation location alter initiation codon,
last nucleotide of exon, functional domain
Seattle Seq Annotation
proteinPosition, distance to splice
Conservation Nucleotide
conservation, protein conservation
Seattle Seq Annotation
scorePhastCons, consScoreGERP
Prediction of pathogenicity
In silico model prediction
Seattle Seq Annotation, Provean
polyPhen, scoreCADD,
granthamScore, SIFT, Provean
Frequency in general population
Frequency in general population
Seattle Seq Annotation, ExAc,
dbSNP genomesExAc, rsID
Occurrence in additional cases
Reported in another patient with same
disease
Clinvar, pubmed, in-house patients, google N/A
Inheritance pattern
2 x genotype + phenotype + 1 x genotype + phenotype – Pathogenic/likely pathogenic variant may be useful for cascade geneIc tesIng There is scope for addiIonal clinical tesIng in this family
Seattle Seq Annotation tool
Step 1: select addiIonal opIons
Step 2: enter variant informaIon
Step 3: submit
Seattle Seq Annotation tool results
Annotation Result Annotation class chromosome 14 Descriptive
position 23898487 Descriptive referenceBase C Descriptive sampleAlleles C/T Descriptive
accession NM_000257.2 Descriptive cDNAPosition 1624 Descriptive
geneList MYH7 Descriptive functionGVS missense Mutation consequence aminoAcids ARG,GLN Mutation consequence
proteinPosition 403/1936 Mutation location distanceToSplice 50 Mutation location scorePhastCons 0.998 Conservation consScoreGERP 4.04 Conservation
scoreCADD 23.6 Prediction of pathogenicity polyPhen 1 Prediction of pathogenicity
granthamScore 43 Prediction of pathogenicity inDBSNPOrNot dbSNP_133 Frequency in general population
rsID rs121913624 Frequency in general population genomesESP C=13006 Frequency in general population genomesExAC unknown Frequency in general population
Provean/SIFT Annotation tool
Step 1: enter variant informaIon Comma separated, DNA
Step 2: submit
Provean/SIFT Annotation results
Variation ROW_NO. 1 INPUT 14,23898487,C,T
Protein sequence change
PROTEIN_ID ENSP00000347507
LENGTH 1935 STRAND -1
CODON_CHANGE CCT C[G/A]G GTG POS 403
RESIDUE_REF R RESIDUE_ALT Q
TYPE Single AA Change
provean prediction
SCORE -3.47 PREDICTION (cutoff=-2.5) Deleterious
#SEQ 542 #CLUSTER 30
SIFT prediction
SCORE 0 PREDICTION (cutoff=0.05) Damaging
MEDIAN_INFO 3.68 #SEQ 389
Annotation dbSNP_ID rs121913624
Exome Aggregate Consortium Database (ExAc)
Step 1: enter gene
Step 2: scroll down search for variant
Step 3: if present, click to view populaIon frequency
ExAc results
Check the ethnic specific allele frequency
Clinvar
Step 1: enter variant
NC_000014.8:g.23898487C>T NM_000257.2:c.1208G>A NP_000248.2:p.Arg403Gln MYH7 Arg403Gln
Clinvar result Step 1: SupporIng observaIons
Step 2: Read summary descripIons
Other sources: Pubmed Google Scholar Gene specific mutaIon databases (e.g. LVOD)
Variant Classification Classification Category Criteria Exceptions Check
Pathogenic
A 1 needed
Confirmed de novo (in the setting of a new disease in the family)
Confirmed de novo alteration in a novel gene with possible disease implications ☐
Likely de novo alteration (i.e. paternity not confirmed with known disease alteration) ☐
Alterations resulting in premature truncation (e.g. reading frame shift, nonsense)
Truncation in close proximity to 3’ terminus ☐ LOF has not been established as a mechanism of pathogenicity (e.g. MYH7) ☐
Other ACMG defined mutation (e.g. initiation codon or gross deletion)
In frame gross deletion of a single exon not in a known protein functional domain ☐
Strong segregation with disease (LOD ≥ 3 = >10 meiosis) ☐
Functionally validated splice mutation In frame skipping a single exon not in a known protein function domain ☐
B 4 needed
Significant disease association in appropriately sized case-control studies ☐
Reported in another patient satisfying established diagnostic criteria for classic disease without a clear mutation.
Can count as 2 if in multiple cases and absent from controls ☐
Last nucleotide of exon ☐ Good segregation with disease (LOD 1.5-3 =5-9 meiosis) ☐ Deficient protein function in appropriate functional assay(s) ☐
Well characterised mutation at same position Can count as 2 if alteration impacts hotspot with 2 or more pathogenic mutations at same position
☐
Other strong data supporting pathogenic classification ☐
Likely pathogenic
1 needed Alterations at the canonical donor/acceptor sites (+/- 1,2) without additional data in support of pathogenicity ☐
C Rarity in general population databases (minor allele frequency <0.1% or 1/1000)
Dependant on disease penetrance and inheritance (eg. HCM: 1/1000 alleles) ☐
4 needed In silico models in agreement AND/OR completely conserved in appropriate places Polyphen + SIFT for most genes ☐
Moderate segregation with disease (At least 3 informative meiosis) ☐
Other data supporting pathogenic classification e.g alterations in a well-defined functional
domain ☐
3 of B ☐ 2 of B and at least 1 of C ☐ 1 of B and at least 3 of C ☐
Variant Classification
Classification Category Criteria Exceptions Check
VUS Insufficient or conflicting evidence ☐ Gross duplications without strong evidence for pathogenic or benign ☐
Likely benign
D Subpopulation frequency in support of benign classification ☐
1 needed Intact protein function observed in appropriate functional assay(s) ☐ Intronic alteration with no splicing impact by RT-PCR analysis or other
splicing assay ☐
Other strong data supporting benign classification ☐
Co-occurrence with mutation in same gene Genes without a defined, severe, biallelic phenotype ☐
E Co-occurrence with a mutation in another gene that explains phenotype ☐
2 needed In silico models in agreement benign ☐ Does not segregate with disease in family studies (genes with incomplete
penetrance) ☐
No disease associate in small case control study ☐ Other data supporting benign classification ☐
Benign
F General pop frequency is too high (based on disease prevalence and penetrance) ☐
1 needed Does not co-segregate with affected’s ☐ Internal frequency is too high to be a pathogenic mutation based on
disease prevalence and penetrance ☐
Seen in trans with a mutation or in homozygous state in individual without severe disease for that gene
Genes without a defined, severe, biallelic phenotype ☐
No disease association in appropriately sized case control studies ☐
1 of D and at least 2 of E ☐ 2 or more of D ☐ > 3 of E w/o conflicting data ☐ >4 of E without conflicting data ☐
Summary
• Converted between DNA, mRNA and protein nomenclature
• Used on-line websites to annotate variants for functional impact on the protein
• Searched a variant database to determine the allele frequency in the general population
• Searched for reports of the variant in unrelated patients with the same disease
• Used guidelines to record the pathogenicity classification
