Overview #
The PM4 and BP3 criteria evaluate in-frame insertions or deletions (indels) that alter protein length without disrupting the reading frame.
These variants can either impact critical functional domains (PM4) or occur in repetitive, functionally neutral regions (BP3).
By distinguishing between these contexts, SeqSMART ensures precise classification of variants that change protein structure but not its translation frame.
1. What Are PM4 and BP3? #
| Criterion | Definition | Evidence Direction |
| PM4 – Pathogenic Moderate 4 | “Protein length changes as a result of in-frame deletions/insertions in a non-repeat region or stop-loss variants.” | Supports pathogenicity when an in-frame indel or stop-loss variant alters a critical region of the protein or affects its structural integrity. |
| BP3 – Benign Supporting 3 | “In-frame deletions/insertions in a repetitive region without a known function.” | Supports benignity when the variant lies within a repetitive, low-complexity region that is not functionally important. |
Both criteria apply only to in-frame variants, since frameshift changes typically lead to loss-of-function and are evaluated under PVS1 or related criteria.
2. Step-by-Step SeqSMART Evaluation #
SeqSMART uses a multi-layered algorithm to distinguish functional, neutral, and repetitive sequence contexts for in-frame indels, determining whether PM4 or BP3 applies.
Step 1: Identify In-Frame vs. Frameshift Indels #
SeqSMART’s variant parser first classifies the indel by its effect on the reading frame:
- In-frame insertion or deletion: length is a multiple of three base pairs → eligible for PM4/BP3.
- Frameshift variant: alters the reading frame → handled under PVS1 or other loss-of-function criteria.
PM4 and BP3 are never applied to frameshift variants, as their interpretation follows a separate decision framework.
Step 2: Evaluate Functional Consequences of In-Frame Indels #
SeqSMART examines whether the in-frame indel or stop-loss variant might cause structural or functional disruption.
Possible outcomes include:
- Loss of function (LoF) if the indel removes a critical domain or structural motif.
- Gain of abnormal function, such as an elongated protein due to stop-loss extension.
- Creation of an internal premature stop codon, affecting downstream stability or folding.
If the indel occurs outside repeat regions and impacts critical structure or length, PM4 is applied as Moderate pathogenic evidence.
Step 3: Detect and Analyze Repetitive Regions #
To determine whether a region is repetitive or functionally neutral, SeqSMART employs its custom repetitive region detector, which analyzes both sequence composition and genomic context.
The algorithm scans ±100 bp around the variant to identify:
- Tandem repeats
- Homopolymer tracts (e.g., poly-A, poly-G)
- Low-complexity sequences
Then it compares the results against internal gene models of known non-functional repetitive regions.
Interpretation:
- If the variant occurs within a repetitive or low-complexity region → BP3 is applied (Benign Supporting).
- If the variant occurs outside repeats and affects structured, conserved regions → PM4 is applied (Pathogenic Moderate).
3. Functional Domain Consideration (Future Enhancement) #
In upcoming SeqSMART versions, UniProt-based functional annotations will be fully integrated into PM4/BP3 evaluation.
This will allow the algorithm to automatically check whether the affected region overlaps with:
- Active sites
- Transmembrane or signal domains
- Catalytic or cofactor-binding regions
- Protein–protein interaction motifs
This feature will further refine PM4 specificity and prevent over-interpretation of indels in regions with ambiguous function.
4. Notes for Users #
- Frameshift variants are excluded from PM4/BP3 assessment, as they are classified under PVS1 or related LoF-based rules.
- Functional consequence prediction for in-frame indels is inherently complex; SeqSMART assigns moderate evidence strength by default unless supported by experimental validation.
- Stop-loss variants extending the coding sequence are evaluated under PM4 when the extended region likely disrupts protein stability or degradation.
- Users may override automatic classifications based on laboratory results, structural modeling, or published functional studies.
- SeqSMART logs all decisions transparently, including repeat detection metrics and protein feature overlap.
5. Data Transparency #
Each PM4 or BP3 call in SeqSMART is accompanied by:
- Variant type classification (in-frame or frameshift)
- Repetitive region detection result
- Reference to affected protein coordinates
- Supporting rationale for criterion assignment
This ensures complete traceability and allows expert reviewers to audit or modify system-derived conclusions easily.
Summary #
PM4 and BP3 enable accurate interpretation of in-frame indels by distinguishing between functionally critical and repetitive, nonfunctional protein regions.
SeqSMART’s automated assessment combines structural context, sequence complexity analysis, and functional impact modeling, offering a robust and reproducible framework for evaluating these criteria.
SeqSMART Structural Integrity Principle:
When protein length changes, context defines consequence.