Overview #
Zygosity and variant configuration play a crucial role in variant interpretation for autosomal recessive disorders.
Two ACMG/AMP criteria — PM3 and BP2 — evaluate whether a variant occurs in trans (on opposite chromosomes) or in cis (on the same chromosome) with another pathogenic variant.
SeqSMART integrates literature-derived evidence, AI-driven text mining, and case-based phasing analysis to identify and validate these relationships with high precision.
1. What Are PM3 and BP2? #
| Criterion | Definition | Evidence Direction |
| PM3 – Pathogenic Moderate 3 | “Detected in trans with a pathogenic variant for a recessive disorder.” | Supports pathogenicity when the variant is found in trans with a known pathogenic variant in an affected individual. The strength increases with the number of independent trans observations. |
| BP2 – Benign Supporting 2 | “Observed in cis with a pathogenic variant.” | Supports benignity when the variant is in cis with a known pathogenic variant in a recessive gene, since two pathogenic alleles on the same chromosome cannot explain the phenotype. |
In practice, determining cis/trans configuration is essential for accurate variant interpretation in recessive diseases, yet such data are often unavailable from standard population databases.
2. SeqSMART’s Approach to PM3 and BP2 #
Because phasing and inheritance data are rarely included in public variant repositories, SeqSMART uses a two-pronged strategy to identify evidence supporting PM3 and BP2:
- Automated literature and database mining via NLP
- Case-based phasing and pedigree analysis using user-supplied data or trio sequencing
2.1 NLP-Based Evidence Extraction from Scientific Resources #
SeqSMART’s Natural Language Processing (NLP) pipeline automatically scans scientific resources and variant databases to extract relevant cis/trans information.
Sources: #
- ClinVar and other curated variant databases
- PubMed (titles, abstracts, and full-text articles)
- Supplementary materials and clinical case reports
Data Extraction Focus: #
- Compound heterozygosity reports
- Parental origin or phasing descriptions (e.g., “in trans” or “in cis”)
- Reported variant combinations and their associated phenotypes
After extraction, the results undergo expert curation to verify:
- The accuracy of zygosity and phasing information
- Relevance to the disease mechanism and inheritance model
- Consistency with observed phenotypes
⚠️ Note: Literature-derived data are often limited to well-studied variants. For many novel or rare variants, PM3 and BP2 rely primarily on case-level evidence.
2.2 Case-Based Evaluation via Pedigree and Trio Analysis #
SeqSMART provides two robust mechanisms for evaluating PM3 and BP2 in user-submitted cases:
- Manual pedigree-based input
- Automated trio-based phasing analysis
A. Manual Entry via Pedigree Tool #
In the interactive pedigree builder, users can manually specify variant configuration based on laboratory results (e.g., Sanger sequencing of parents).
Workflow:
- Assign each family member’s variant genotype (heterozygous, homozygous, or wild type).
- Indicate cis or trans configuration between variants, when known.
- Annotate phenotype using HPO or OMIM references.
SeqSMART then:
- Analyzes whether the variant configuration supports the observed phenotype.
- Applies PM3 when variants are in trans in a recessive gene context.
- Applies BP2 when variants are in cis, indicating reduced pathogenic likelihood.
All entries are stored and visualized within the pedigree structure, providing a clear graphical overview of inheritance patterns.
B. Automated Trio-Based Phasing from VCF Files #
If the case includes VCF files for the child and both parents, SeqSMART performs fully automated phasing and compound heterozygosity detection.
Step-by-step process:
- VCF Upload & Parsing
- Child, father, and mother VCFs are processed simultaneously.
- Variants are grouped per gene.
- Child, father, and mother VCFs are processed simultaneously.
- Compound Heterozygosity Detection
- The system identifies rare variant pairs within the same gene.
- Each variant’s parental origin is traced.
- The system identifies rare variant pairs within the same gene.
- Cis/Trans Configuration Determination
- If one variant is inherited from each parent → variants are in trans → PM3 met (pathogenic support).
- If both variants originate from the same parent → variants are in cis → BP2 met (benign support).
- If one variant is inherited from each parent → variants are in trans → PM3 met (pathogenic support).
- Classification Integration
- SeqSMART automatically applies PM3 or BP2 to the case report.
- Confidence level depends on the quality and completeness of the genotype data.
- SeqSMART automatically applies PM3 or BP2 to the case report.
This automated approach significantly reduces the need for manual phasing, improving efficiency and minimizing human error.
3. Practical Example #
| Scenario | Observation | Applied Criterion |
| Recessive gene; variant A from mother, variant B from father | Variants are in trans, both detected in affected individual | PM3 (Supporting or Moderate depending on cases) |
| Both variants inherited from same parent | Variants are in cis and cannot explain recessive phenotype | BP2 (Supporting) |
| Only one parent available, or phase unclear | Configuration uncertain | Criterion remains Unassessed |
4. Best Practices for Users #
To achieve the most accurate results for PM3 and BP2:
- Provide trio VCFs whenever possible — this enables automatic phasing.
- Confirm phasing results with Sanger sequencing or validated lab methods.
- Use the Pedigree Tool to annotate genotype and phenotype data for all family members.
- Review SeqSMART’s automatically applied evidence to verify biological plausibility.
These practices ensure the highest precision in applying cis/trans evidence to variant classification.
Summary #
PM3 and BP2 provide essential evidence for evaluating variant phase in recessive disorders.
SeqSMART combines AI-driven literature mining, manual pedigree input, and automated trio-based phasing to assess cis/trans configurations accurately and reproducibly.
This hybrid system supports both large-scale automated workflows and detailed expert validation.
SeqSMART Zygosity Principle:
Phase matters — because inheritance tells the truth.