Overview #
The PS2 and PM6 criteria from the ACMG/AMP (2015) guidelines assess de novo variant occurrence, providing crucial evidence for pathogenicity when a variant arises spontaneously rather than being inherited.
These criteria are particularly important for dominant disorders where new mutations are a common cause of disease.
SeqSMART implements both criteria using its integrated family analysis tools, SMART algorithm, and AI-assisted clinical correlation, ensuring accurate and reproducible evaluation of de novo events.
1. What Are PS2 and PM6? #
| Criterion | Definition | Evidence Direction |
| PS2 – Pathogenic Strong 2 | “De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.” | Provides strong evidence of pathogenicity when a variant is confirmed as de novo in the affected individual. |
| PM6 – Pathogenic Moderate 6 | “Assumed de novo, but without confirmation of paternity and maternity.” | Provides moderate evidence of pathogenicity when the variant is likely de novo but parental data are unavailable. |
Both criteria assess whether the observed variant could reasonably explain the disease phenotype, considering the inheritance pattern and available parental data.
2. SeqSMART Approach to PS2 #
PS2 applies when the variant is confirmed de novo, meaning it is absent in both parents and present in the affected child.
SeqSMART performs this analysis automatically when complete trio (child + both parents) genomic data are available.
Step 1: Input Data #
To enable PS2 evaluation, users should upload VCF files for:
- The affected child (proband)
- The father
- The mother
Step 2: De Novo Detection via the SMART Algorithm #
SeqSMART’s SMART algorithm compares the child’s variants against parental genotypes to detect any variants present only in the proband.
Key parameters analyzed:
- Allelic balance and read depth to rule out sequencing artifacts
- Variant quality metrics
- Population frequency (to exclude common variants)
Only high-confidence de novo variants pass to the next validation step.
Step 3: Genome-Wide Haplotype Confirmation #
To ensure biological accuracy, SeqSMART performs a genome-wide haplotype comparison to confirm paternity and maternity.
This step verifies that the variant’s absence in parents is genuine and not due to misidentified parental data or sample contamination.
Step 4: PS2 Assignment #
If:
- The variant is present only in the child,
- Absent in both parents, and
- Paternity/maternity is confirmed,
SeqSMART assigns PS2 (Strong) to the variant, supporting a pathogenic classification based on verified de novo occurrence.
3. SeqSMART Approach to PM6 #
PM6 applies when a variant is assumed to be de novo, but parental genetic data are unavailable.
Step 1: Clinical Context Evaluation #
If a variant occurs in a dominant disease gene and the proband’s phenotype matches the expected presentation:
- SeqSMART evaluates available clinical, phenotypic, and inheritance information.
- When the case strongly suggests a new (de novo) mutation with no family history, PM6 may be applied automatically at a moderate strength level.
Step 2: Manual Assignment #
When SeqSMART cannot automatically confirm or infer a de novo event due to missing parental data:
- Users can manually assign PM6 in the variant interface.
- Supporting notes or literature references can be attached to document clinical reasoning.
4. Parental Data Availability #
The ability to apply PS2 or PM6 depends directly on parental genotype availability:
| Parental Data Available | Applicable Criterion | Assessment Type |
| Both parents (VCFs) available and confirmed | PS2 | Automated (Strong) |
| Only proband data available | PM6 | Assumed (Moderate) |
| No reliable family data | Neither | Not assessed |
Even when parental data are absent, SeqSMART provides flexibility — users can manually assign, downgrade, or remove these criteria based on their own clinical assessment.
5. Transparency and User Control #
- All PS2/PM6 assignments are logged in the variant audit trail, including evidence source, version, and reasoning.
- Users may override system-generated decisions at any time.
- SeqSMART displays a detailed explanation of how each de novo call was made, including supporting evidence (read depth, phasing data, and quality metrics).
6. Example #
Confirmed De Novo (PS2):
A child presents with a dominant developmental disorder. Trio sequencing confirms the variant is absent in both parents → PS2 (Strong) is applied.
Assumed De Novo (PM6):
A single affected individual has a variant in a dominant gene and no family history of the disease, but parental data are unavailable → PM6 (Moderate) is applied.
SeqSMART automates and enhances the evaluation of de novo inheritance using advanced trio analysis and haplotype validation.
By combining genomic data verification with clinical context analysis, SeqSMART ensures that PS2 and PM6 are applied with scientific rigor, traceability, and user flexibility.
SeqSMART Inheritance Principle:
Trust the data. Verify the origin.