The IGM N-of-One Diagnostic Interpretation for Genetic Disorders.

1. The IGM N-of-One Diagnostic
Interpretation for Genetic Disorders
Prepared by: Slavé Petrovski and David B. Goldstein
Institute for Genomic Medicine (IGM), Columbia University
sp3347@cumc.columbia.edu

2. The IGM trio framework is divided into Tier 1 and Tier 2:
Tier 1: takes the philosophy that it's unlikely that we will find
individiuals among the general population that share the same
pathogenic genotype(s) as a proband with a severe undiagnosed
genetic disorder.
Tier 2: accepts that above can infrequently happen due to various
biological and artifactual reasons and helps identify such events.
Tier 2 is similarly more permissive to coverage and quality metrics.
The IGM internal controls of convenience for diagnostic
sequencing are selected on the basis of NOT being ascertained for
neurodev., neuropsych., immunological, dysmorphic, early onset,
or severe Mendelian disorders.
The IGM places a strong emphasis on support from
population genetic data and does develop bioinformatics
signatures to aid interpretation of genetic variation. This is
where our philosophy can slightly differ to other Centers.

3. N-of-One Clinical Sequencing for Genetic Disorders
Trio exome sequencing
Identify qualifying genotypes
Genetic diagnosis Genetic candidate (GUS)
Tier 1: Conservative (↑specificity)
• Absent among parents & controls
• Non-synonymous CCDS only
• Highest QC (coverage, etc.).
Tier 2: Liberal (↑sensitivity)
•Low Genotype frequency in controls
•Include IEB & non-CCDS transcripts
•Reduce Coverage and QC thresholds
Generally only done for:
Previously reported variants or LoF
variants in known HI genes.
Research N-of-One Candidate
• Follow Literature
• Gene Matchmaking
• Functional Work?
1) Visually Inspect
2) Sanger Validate
Bioinformatics Signature GUS

4. Making A Genetic Diagnosis
• Based on known gene-disease associations in human
1. Known human disease gene (OMIM)
2. Disease phenotype overlaps with the patient’s clinical features
3. Genetic model is consistent with known genotype-phenotype
relationship
4. Qualifying mutation has been reported or is of the same class as
those that have been reported (e.g., loss-of-function mutation in a
haploinsufficient gene)
5. Has to be agreement between genetics team and treating clinician(s)
6. Independent Sanger validation (CLIA)
• Richards S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the
American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in Medicine (2015)
• MacArthur DG, et al. Guidelines for investigating causality of sequence variants in human disease. Nature (2014)

5. Bioinformatics Signatures
Rank FLAG DNMs HEMIZ HOMOZ CMPDHET
High KnownVar Yes Yes Yes Yes
High Indel KnownVar Yes Yes Yes Yes
High LoF_inLoFClinVar/Gene Yes Yes Yes Yes
High HotZone CCDS Essential Yes Yes Yes Double
High Non-Syn in MGI Seizure Ortholog Yes Yes Yes Yes
High Exceptional recessive LoF genotype - - Yes Double
Moderate 2bp +/- KnownVar Yes Yes Yes Yes
Moderate HotZone CCDS Yes Yes Yes Double
Moderate LoF_inLoFintol [FDR<0.01] Yes Yes Yes Yes
Moderate Non-Syn in Intolerant, Essential, Constrain gene Yes Yes Yes Double
Moderate Exceptional recessive LoF genotype; 1 LoF allele - - Yes Yes
Low Non-Syn with Global Intolerance score <50%tile Yes Yes Yes Double
Low Variant in OMIM disease-associated gene Yes Yes Yes Yes
Low Recessive Non-Syn genotype in a Carrier Gene - Yes Yes Double

6. Additional Screens (Periodically assessed)
1. Non-trio interpretation.
2. Regenerate qualifying genotypes and bioinformatics signatures
based on most recent literature and knowledge.
3. Relink to most recent instances of pathogenic variant
databases.
4. Relink to most recent OMIM gene-disease associations.
5. Run a Parental Mosaic inheritance screen.
6. Generate an incomplete penetrance screen (permitted to be
present in unaffected parent).