VUS: How Far Can We Interpret It? [Part 2]

Take a look at the previous post, ‘[Part 1] Quantitative Interpretation of ACMG/AMP Variant Classification’

— Practical Applications and Clinical Use After the Point System

The ACMG/AMP guidelines define VUS (Variant of Uncertain Significance) as a single category, and there is currently no official framework for subdividing it.

However, in real-world interpretation, treating all VUS as a single homogeneous group has clear limitations.
Even within VUS, some variants are closer to pathogenic, while others are closer to benign.

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A “natural extension” enabled by the point system

When applying the point-based framework proposed by Tavtigian et al. (2020), VUS remains a single category, but in practice it spans a broad spectrum.

As a result, recent practice has introduced an internal sub-tiering approach:

• VUS (High) — closer to pathogenic
• VUS (Mid) — neutral
• VUS (Low) — closer to benign

This categorization is not formally defined in guidelines, but emerges naturally when applying a point-based interpretation framework.

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Changes in the 3billion report: introduction of VUS sub-tiering

To improve clarity in interpretation, 3billion has introduced a system that reports VUS with sub-tiering (Low / Mid / High).

Key points are:

• The final classification remains VUS
• Sub-tiering is an additional interpretation layer
• It serves as a supportive indicator for clinical decision-making

In other words, this is not a change in classification, but a way of more precisely indicating where a variant lies within the VUS spectrum.

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Limitations observed in real-world studies

Recent studies applying point-based variant classification systems to hereditary cancer genes have shown that:

• Some VUS are reclassified as Likely Pathogenic or Likely Benign
• The overall proportion of VUS decreases
• Sub-tiering within VUS becomes feasible

However, these studies also demonstrate that:

VUS subcategorization is useful for describing directionality,
but has limited power in predicting future reclassification.

Other reanalysis studies have similarly shown that variant reclassification is driven primarily by the accumulation of new evidence—such as clinical reports, functional studies, or additional case data—rather than by the initial interpretation status. In fact, most VUS remain unchanged over time.

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Then why is this still important?

The value of this approach lies not in predicting reclassification, but in supporting clinical decision-making.

It is particularly useful for:

• Prioritizing variants for segregation studies
• Selecting candidates for additional testing
• Identifying variants for reanalysis
• Guiding report interpretation

In short, even within the same VUS category, it helps determine which variants should be followed up first.

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Practical clinical examples

1) VUS (High): potential for upgrade with additional evidence

Consider a variant reported as VUS (High).

If:

• The phenotype is consistent, and
• Segregation analysis reveals de novo occurrence (PM6) or co-segregation (PP1),

→ It may be realistically reclassified as Likely Pathogenic.

In such cases:

• Parental testing is recommended
• Family studies should be actively considered
• The variant is prioritized for follow-up

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2) VUS (Mid): a single additional piece of evidence is often insufficient

For VUS (Mid), the situation differs.

Even if PM6 (de novo) is identified: → Reclassification often remains difficult

This is because:

• The evidence is not decisive
• Additional functional studies or accumulated case data are needed

In these cases:

• Immediate family testing may not be the priority
• Data accumulation and periodic reanalysis are more appropriate

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3) VUS (Low): lower clinical priority

For VUS (Low):

• Some benign evidence is already present
• Additional pathogenic evidence is unlikely to significantly change interpretation

In practice:

• Lower priority for additional testing
• Limited clinical impact
• Typically managed through long-term reanalysis

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Important considerations

When applying this approach, several principles should be kept in mind:

First, subcategorization is not an official classification. Variants must still be reported as VUS.

Second, the criteria are not standardized. Interpretation may vary between institutions.

Third, overinterpretation must be avoided. Phrases such as “almost pathogenic” can lead to clinical misunderstanding.

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Summary

VUS subcategorization is not yet defined in formal guidelines, but has emerged as a practical approach alongside the adoption of point-based interpretation.

Current evidence suggests that this approach is not a strong predictor of reclassification, but rather a tool that enhances clinical decision-making.

Therefore, VUS sub-tiering should not be viewed as a replacement for variant classification, but as a supportive layer that helps better understand VUS and prioritize meaningful follow-up actions.

Reference
1. ACMG 2025 Annual Meeting abstract (O51)
(conference abstract, non-peer-reviewed full article)

2. Eldomery MK, Maciaszek JL, Cain T, Loyola VP, Mothi SS, Wheeler DA, Tang L, Wang L, Klco JM, Blackburn PR. Evaluation of Bayesian point-based system on the variant classification of hereditary cancer predisposition genes. Genet Med. 2024 Dec;26(12):101276. doi: 10.1016/j.gim.2024.101276. Epub 2024 Sep 18. PMID: 39306722.

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