N1C Variant Eligibility Calculator

Information Page

This calculator complements the N1C VARIANT guidelines for eligibility assessment of pathogenic variants to antisense oligonucleotide treatments. Please use the guidelines alongside this calculator. The calculator will direct you towards the relevant sections to read within the guidelines to help you with the assessment. For an understanding of how the assessments work, please also take a look at our training videos. By using this calculator, you agree to our license and the conditions indicated in our disclaimer. The full code of the calculator is available on GitHub.

If you use this calculator, please cite our publication: Cheerie et al., 2025

If you have any questions about the calculator or suggestions for improvement, please reach out to Marlen Lauffer (marlen@n1collaborative.org).

Answer log

In case you want to log your answers and have a written confirmation of your assessment and the classification, please input your variant for assessment here and click submit. This will open a new tab and enable a system that records your answers. You can then start the assessment. Logging your answers is optional. The assessments will not be recorded on N1C side and are solely for you.

Disclaimer

This document is the work product of the N=1 Collaborative (the "N1C"). The N1C is not providing legal or regulatory advice for N=1 trials. This document should not be construed as legal or regulatory advice for any particular purpose. These guidelines focus on the evaluation of eligibility of a genetic variant for ASO treatment only. To assess a patient case for ASO amenability, the disease, phenotype, and patient have to also be considered, which is beyond the scope of these guidelines. The information is based on the best available knowledge and practice at the time of publication. Users of these documents should exercise their own judgment and discretion in applying these practices to their specific situations.

Step 0 - Variant check

Is the variant description correct and conform with the HGVS nomenclature?

For guidance on how to check for HGVS conform nomenclature, please read “Step 0” of the N1C VARIANT guidelines. You can check the variant description using VariantValidator or Mutalyzer.

Is the variant the disease-causing variant?

We recommend only assessing likely pathogenic and pathogenic variants for eligibility towards ASO treatments as discussed in our guidelines. An exception are VUSes that are identified in trans with a (likely) pathogenic variant in a recessive disorder. For more information on pathogenicity assessment of variants, we recommend following the current ACMG-AMP guidelines.

Step 0 - Variant check

Eligibility: Unable to assess

Please check with the referring clinician or patient (family) to receive the correct variant description or information on the disease-causing variant.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 0 - Variant check

Is the variant of a type applicable to be evaluated with these guidelines?

Variants applicable to these guidelines are:

single nucleotide variants
small indels
one or multi-exon deletions or duplications
full gene deletions or duplications)

Currently excluded are:

variants in non-coding genes
deletions/duplications spanning multiple genes (i.e., contiguous gene syndromes)
imprinting defects/uniparental disomy
structural rearrangements (e.g., translocations)
aneuploidies
Mitochondrially inherited disorders (mtDNA variants)

Please also see “Step 0” of the N1C VARIANT guidelines for further explanations.

Step 0 - Variant check

Eligibility: Unable to assess

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 0 - Variant check - CNVs

Is the variant a duplication or deletion of a whole gene (CNV)?

Step 0 - Variant check - CNVs - Whole gene duplication

Consider variant for knockdown approach. Please review “Section B” in the N1C VARIANT guidelines. Is the gene associated with haploinsufficiency?

For guidance on how to assess for haploinsufficiency and dosage sensitivity, please read “Step 2” of the N1C VARIANT guidelines or “Section B”. For a quick evaluation, we recommend using the Dosage Sensitivity curations from ClinGen.

Step 0 - Variant check - CNV - Whole gene duplication

Is there already an ASO/RNAi/siRNA approach developed for this gene?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.

Step 0 - Variant check - CNV - Whole gene duplication

Eligibility: Eligible for a knockdown approach

For further information on the classification, please see the classification table for knockdown.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 0 - CNV - Whole gene duplication

Eligibility: Likely eligible for a knockdown approach

For further information on the classification, please see the classification table for knockdown.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 0 - Variant check - CNV - Whole gene duplication

Consider an allele-selective approach. Is an ASO/RNAi/siRNA approach already developed for this gene?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.

Step 0 - CNV - Whole gene duplication

Eligibility: Eligible for a knockdown approach

For further information on the classification, please see the classification table for knockdown.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 0 - CNV - Whole gene duplication

Eligibility: Unlikely eligible for a knockdown approach

For further information on the classification, please see the classification table for knockdown.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 0 - CNV - Whole gene deletion

Consider variant for upregulation from WT allele. Please review “Section C” in the N1C VARIANT guidelines. For upregulation from the WT allele, we recommend a manual assessment of the different strategies. These cannot be assessed using this calculator. Please note that upregulation from the WT allele for whole gene deletions only applies if there is still a functional wildtype copy. In case there is already an approach available for this variant, then this variant is considered eligible.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 1 - Inheritance pattern

What is the inheritance pattern of this variant?

For guidance on how to determine the inheritance pattern, please see “Step 1” of the N1C VARIANT guidelines.

Optional: Please provide any evidence, e.g. link, publication, website, or written explanation, for your choice of answer. This evidence will be shown in the answer log alongside with all your other answers.

Step 2 - Pathomechanism - Autosomal dominant

What is the pathomechanism of the variant under assessment?

For guidance on how to determine the pathomechanism of a given variant, please read “Step 2” of the N1C VARIANT guidelines.

Step 2 - Pathomechanism - Autosomal dominant - Loss of function

What is the variant type?

Step 3 - Splice assessment - Autosomal dominant - Loss of function - Intronic variant

Is there functional evidence of a splicing effect?

For guidance on how to determine whether a variant affects splicing, please see “Step 3” of the N1C VARIANT guidelines. There should be functional evidence of a splicing effect based on e.g., RNAseq, cDNA data. Splice predictions are not considered sufficient evidence.

Step 3 - Splice assessment - Intronic variant

The variant can be assessed for splice correction ASO approaches. Please tick all boxes that apply.

To identify which boxes should be ticked, please carefully read “Step 3” of the N1C VARIANT guidelines.

Canonical splicing is destroyed Canonical splicing is weakened Canonical splicing is not weakened Variant within 5 bp of canonical splice site Intronic variant >100 bp upstream of acceptor splice site Intronic variant >50 bp downstream of donor splice site Intronic variant within -6 to -100 bp or +6 to +50 bp of the canonical splice sites

Step 3 - Splice assessment

Has there already been an ASO developed for this variant?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.

Step 3 - Splice assessment

Has there already been an ASO developed for this variant?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.

Step 3 - Splice assessment

Eligibility: Not eligible for splice correction

For further information, please see the classification table for splice correction.

Under rare circumstances, the variant could be considered for exon skipping. Please see guidelines “Step 3” for more detailed information. Variant could also be considered for upregulation from the WT allele. Please pick the next guideline:

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment

Eligibility: Eligible for splice correction

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment

Eligibility: Likely eligible for splice correction

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment

Eligibility: Eligible for splice correction

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment

Eligibility: Unlikely eligible for splice correction

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Intronic variant

Eligibility: Unable to assess

The variant cannot be assessed for splice correction. If this variant is predicted to have an effect on splicing, please obtain functional evidence for a splicing effect.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Intronic variant

Eligibility: Unable to assess.

The variant cannot be assessed for splice correction. Please obtain evidence for or against a splicing effect.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section C - Considerations for upregulation from the WT-allele

Please review “Section C” in the N1C VARIANT guidelines. For upregulation from the WT allele, we recommend a manual assessment of the different strategies. These cannot be assessed using this calculator.

In case there is an upregulation from the WT allele already available for the disorder with sufficient functional evidence, the variant is considered eligible.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Synonymous variant

Is there functional evidence of a splicing effect?

Step 3 - Splice assessment - Synonymous variant

The variant can be assessed for splice correction ASO approaches. Please tick all boxes that apply.

To identify which boxes should be ticked, please carefully read “Step 3” of the N1C VARIANT guidelines.

Canonical splicing is destroyed Canonical splicing is weakened Variant within 5 bp of canonical splice site Variant within 6 to 15 bp of canonical splice site None

Step 3 - Splice assessment - Synonymous variant

Eligibility: Unable to assess

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Synonymous variant

Eligibility: Unable to assess

The variant cannot be assessed for splice correction. Please obtain evidence for or against a splicing effect.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Gain-of-function - Missense variants or in-frame indels

Is there functional evidence of a splicing effect?

Step 3 - Splice assessment - Gain of function - Missense variants or in-frame indels

The variant can be assessed for splice correction ASO approaches. Please tick all boxes that apply.

To identify which boxes should be ticked, please carefully read Step 3 of the N1C VARIANT guidelines. For missense variants and in-frame indels, please pay special attention to the effect of these variants on protein function independent of the splice-altering effect.

Canonical splicing is destroyed Canonical splicing is weakened Variant within 5 bp of canonical splice site Variant within 6 to 15 bp of canonical splice site Effect of amino acid change on protein function is not known (independent of the splice-altering effect) Change in amino acid sequence causes GoF or DN effect Change in amino acid sequence causes complete loss of function Change in amino acid sequence causes partial loss of function/reduced protein function Change on protein/amino acid level known to be benign

Step 3 - Gain-of-function/dominant-negative variants - Splice assessment

Eligibility: Not eligible for splice correction

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - GoF/DN variants.

Has there already been an ASO developed for this variant?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.

Step 3 - Splice assessment - GoF/DN variants.

Has there already been an ASO developed for this variant?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.

Step 3 - Splice assessment - GoF/DN variants.

Eligibility: Eligible for splice correction

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - GoF/DN variants.

Eligibility: Likely eligible for splice correction

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - GoF/DN variants.

Eligibility: Eligible for splice correction

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - GoF/DN variants.

Eligibility: Unlikely eligible for splice correction

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Missense variants or in-frame indels

Eligibility: Not eligible for a splice correction.

Under certain circumstances, the variant can be assessed for an exon skipping approach.

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Missense variants or in-frame indels

Eligibility: Unable to assess

The variant cannot be assessed for splice correction until further evidence is available. Under certain circumstances, variant could be assessed for exon skipping.

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Missense variants or in-frame indels

Eligibility: Not eligible for splice correction

The variant cannot be assessed for splice correction. The variant can be considered for exon skipping or a upregulation from the WT allele.

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Missense variants or in-frame indels

Eligibility: Unable to assess

The variant cannot be assessed for splice correction. Please obtain evidence for or against a splicing effect.

Under certain circumstances, the variant can be considered for exon skipping or upregulation from the WT allele.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section A - Exon skipping assessment

For exon skipping assessment, please review “Section A” of the N1C VARIANT guidelines. Do any of the following criteria apply to the exon?

Each of these criteria is a "not eligible" criterion.

Exon is first coding exon
Exon is last coding exon
Exon is out of frame
Skipping the exon will generate a STOP codon
Gene contains only one coding exon

Section A - Exon skipping assessment

Eligibility: Not eligible for exon skipping

Please check for exceptions described in “Section A” of the N1C VARIANT guidelines.

For further information on the classification, please see the classification table for exon skipping.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section A - Exon skipping assessment

Eligibility: Not eligible for exon skipping

Please note exceptions listed in “Section A” of the N1C VARIANT guidelines.

For further information on the classification, please see the classification table for exon skipping.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section A - Exon skipping assessment

Special considerations apply for repeat domains.

Please see “Section A” of the N1C VARIANT guidelines. Most suitable are in-frame exons that contain a single, full repeat of the tandem repeats and no additional domains. If an ASO has already been developed for this exon, we consider it eligible for exon skipping.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section A - Exon skipping assessment

Is there already an exon skipping therapy available?

Please check for the exon in general and not only the variant in question. A suggestion of a possible search term in Pubmed is: ABCA4 AND ((ASO) OR (AON) OR (antisense oligonucleotide)) AND (Exon 17). Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.

Section A - Exon skipping assessment

Is there already an ASO exon skipping therapy available?

Section A - Exon skipping assessment

Eligibility: Eligible for exon skipping

For further information on the classification, please see the classification table for exon skipping.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section A - Exon skipping assessment

Eligibility: Unlikely eligible for exon skipping

For further information on the classification, please see the classification table for exon skipping.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section A - Exon skipping assessment

Eligibility: Likely eligible for exon skipping

For further information on the classification, please see the classification table for exon skipping.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - whole exon duplication and deletion

Eligibility: Not eligible for splice correction

If the deleted exon is in-frame, the variant is also not applicable to an exon skipping treatment. The variant can be considered for upregulation from the WT allele in AD and possibly XLD disorders. If the variant is an out-of-frame exon deletion, check if any of the two adjacent exons could be skipped to get the transcript back into frame, then an exon skipping approach could be considered. If the deleted exon is the first or last coding exon, exon skipping is not a viable option (rare exceptions apply). Exon skipping might have to be allele-selective; please review “Section C” of the N1C VARIANT guidelines.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section A - Exon skipping assessment for whole exon deletion variants

For exon skipping assessment, please review “Section A” of the N1C VARIANT guidelines. Please keep in mind that the aim is to skip an adjacent exon to get the transcript back into frame. Do any of the following criteria apply to the exon?

Each of these criteria is a "not eligible" criterion.

Exon is first coding exon
Exon is last coding exon
Skipping the exons will generate a STOP codon
Gene contains only the two coding exons (deleted one and the one to be skipped)

Section A - Exon skipping assessment for whole exon deletion variants

Eligibility: Not eligible for exon skipping

Please check for exceptions as described in “Section A” of the N1C VARIANT guidelines.

For further information on the classification, please see the classification table for exon skipping.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Whole exon duplication - Loss of function

Eligibility: Not eligible for splice correction

If an exon duplication leads to a loss of function in a (autosomal) dominant disorder, an upregulation from the WT allele can be considered. Please review “Section C” in the N1C VARIANT guidelines. For upregulation from the WT allele, we recommend a manual assessment of the different strategies. These cannot be assessed using this calculator.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Autosomal dominant - Gain of function

Gain of function variants can be analyzed using the assessment for splice correction or for knockdown. In rare circumstances, an exon skipping approach can be employed. Review “Section A” of the N1C guidelines to identify in which cases GoF variants can be targeted with an exon skipping approach. Note that exon skipping of out-of-frame exons can also be applied for GoF variants. Please pick your assessment:

Step 3 - Splice assessment - Gain of function and dominant negative variants

Is there functional evidence of a splicing effect?

Step 3 - Splice assessment - Gain of function and dominant negative variants

What is the variant type?

Step 3 - Splice assessment - Gain of function and dominant negative variants - Nonsense and frameshift variants

Nonsense and frameshift variants leading to splice aberrations and causing a GoF or DN effect can be corrected with a splice correction ASO. By correcting splicing, the variants will cause a loss of function effect on protein level due to the amino acid changes. In such cases, it is also less relevant whether canonical splicing is affected, as a reduction of the toxic GoF effect is the goal, however, attention to haploinsufficiency should be paid. Is the disorder associated with haploinsufficiency?

Note that it is also possible to target these variants with a knockdown approach or exon skipping.

Step 3 - splice assessment - Gain of function and dominant negative variants - Nonsense and frameshift variants

Is there already an ASO approach developed for this variant?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.

Step 3 - splice assessment - Gain of function and dominant negative variants - Nonsense and frameshift variants

Is there already an ASO approach developed for this variant?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.

Step 3 - Splice assessment - Gain of function and dominant negative variants - Nonsense and frameshift variants

Eligibility: Eligible for splice correction.

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Gain of function and dominant negative variants - Nonsense and frameshift variants

Eligibility: Unlikely eligible for splice correction.

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Gain of function and dominant negative variants - Nonsense and frameshift variants

Eligibility: Likely eligible for splice correction

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Gain of function and dominant negative variants - Whole exon deletions and duplications

For whole exon deletions and duplications that cause a splicing effect and lead to a toxic GoF of a protein, multiple approaches are possible. Splicing can be corrected if the correction leads to either a LoF of that transcript or a benign effect. Exon skipping can be used to correct the reading frame or to cause downregulation of the transcript of a knockdown approach using siRNA/gapmer ASOs can be used. Please also check whether the gene is associated with haploinsufficiency. Please choose the guideline you want to follow:

Step 3 - Splice assessment - Gain of function and dominant negative variants

Eligibility: Unable to assess

The variant can be considered for a knockdown approach or in certain circumstances for exon skipping. Please choose the preferred assessment:

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Synonymous variant

The variant can be assessed for splice correction ASO approaches. Please tick all boxes that apply.

To identify which boxes should be ticked, please carefully read “Step 3” of the N1C VARIANT guidelines.

Canonical splicing is destroyed Canonical splicing is weakened Variant within 5 bp of canonical splice site Variant within 6 to 15 bp of canonical splice site None

Step 3 - Splice assessment - GoF/DN - Intronic variant

The variant can be assessed for splice correction ASO approaches. Please tick all boxes that apply.

To identify which boxes should be ticked, please carefully read “Step 3” of the N1C VARIANT guidelines.

Step 3 - Splice assessment - Gain of function and dominant negative variants

Eligibility: Unable to assess

The variant can be considered for a knockdown approach or in certain circumstances for exon skipping. Please choose the preferred assessment:

Section B - Knockdown assessment - Gain of function and dominant negative variants

Please tick all that apply

Please see “Section B” in the N1C VARIANT guidelines for further information.

Loss of one allele tolerated Complete loss of function (e.g., both alleles) tolerated Gene associated with haploinsufficiency Hypomorphic alleles are a cause of disease Intolerant to reduction (very strict control of gene dosage) None

Section B - Knockdown assessment

Eligibility: Not eligible for a knockdown approach

For further information on the classification, please see the classification table for knockdown.

The exon the variant is located in could be considered for exon skipping should it fulfill all criteria.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section B - Knockdown assessment

Eligibility: Unable to assess

More information needs to be obtained to make an assessment. Should there already be a knockdown approach available for this variant or any other variant in this gene with sufficient functional evidence, then the variant is eligible.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section B - Knockdown assessment

Is there already an ASO/RNAi/siRNA approach developed for this gene?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.

Section B - Knockdown assessment

Eligibility: Eligible for a knockdown approach.

For further information on the classification, please see the classification table for knockdown.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section B - Knockdown assessment

Eligibility: Likely eligible for a knockdown approach

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section B - Knockdown assessment

Is there already an ASO/RNAi/siRNA approach developed for this gene?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.

Section B - Knockdown assessment

Eligibility: Eligible for a knockdown approach

For further information on the classification, please see the classification table for knockdown.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section B - Knockdown assessment

Eligibility: Unlikely eligible for a knockdown approach

Please consider an allele-selective knockdown.

For further information on the classification, please see the classification table for knockdown.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 2 - Pathomechanism - Autosomal dominant - Dominant negative variant

Dominant negative variants can be analyzed using the assessment for splice correction or downregulation. Please choose your preferred assessment:

Section B - Knockdown assessment - Dominant negative variant

Please tick all that apply

Please see “Section B” in the N1C VARIANT guidelines for further information.

Section B - Knockdown assessment - Autosomal/X-linked dominant - Dominant negative variant

Eligibility: Not eligible for a knockdown approach.

For further information on the classification, please see the classification table for knockdown.

The exon the variant is located in could be considered for exon skipping should it fulfill all criteria. Please be cautious as dominant negative effects can also appear with skipped exons.

Section B - Knockdown assessment - Autosomal/X-linked dominant - Dominant negative variant

Eligibility: Unable to assess

More information needs to be obtained to make an assessment. Should there already be a knockdown approach available for this variant or any other variant in this gene, the variant is eligible, otherwise the eligibility is “unable to assess”.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section B - Knockdown assessment - Autosomal/X-linked dominant - Dominant negative variant

Has there already been a knockdown approach developed?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on ASO/siRNA availability.

Section B - Knockdown assessment - Autosomal/X-linked dominant - Dominant negative variant

Eligibility: Eligible for a knockdown approach

For further information on the classification, please see the classification table for knockdown.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section B - Knockdown assessment - Autosomal/X-linked dominant - Dominant negative variant

Eligibility: Likely eligible for a knockdown approach

Please consider an allele-selective approach for this dominant negative variant.>

For further information on the classification, please see the classification table for knockdown.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section B - Knockdown assessment - Autosomal/X-linked dominant - Dominant negative variant

Has there already been a knockdown approach developed?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on ASO/siRNA availability.>

Section B - Knockdown assessment - Autosomal/X-linked dominant - Dominant negative variant

Eligibility: Eligible for a knockdown approach

For further information on the classification, please see the classification table for knockdown.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section B - Knockdown assessment - Autosomal/X-linked dominant - Dominant negative variant

Eligibility: Unlikely eligible for a knockdown approach

Please consider an allele-selective approach for this dominant negative variant.>

For further information on the classification, please see the classification table for knockdown.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 2 - Pathomechanism

Eligibility: Unable to assess

Please obtain further evidence to determine pathomechanism.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 2 - Pathomechanism - Autosomal recessive

What is the pathomechanism of the variant under assessment?

For guidance on how to determine the pathomechanism of a given variant, please read “Step 2” of the N1C VARIANT guidelines.

Step 2 - Pathomechanism - Autosomal recessive - Loss of function

What is the variant type?

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant

Is there functional evidence of a splicing effect?

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant

The variant can be assessed for splice correction ASO approaches. Please tick all boxes that apply.

To identify which boxes should be ticked, please carefully read “Step 3” of the N1C VARIANT guidelines.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant

Has there already been an ASO developed for this variant?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant

Has there already been an ASO developed for this variant?

Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant

Eligibility: Eligible for splice correction

For further information, please see the classification table for splice correction.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant

Eligibility: Likely eligible for splice correction

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant

Eligibility: Unlikely eligible for splice correction

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant

Eligibility: Not eligible for splice correction

For further information, please see the classification table for splice correction.

Under rare circumstances, the variant could be considered for exon skipping. Please see guidelines “Step 3” for more detailed information. Please review “Section A” for information on exon skipping.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant

Eligibility: Unable to assess

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant

Eligibility: Unable to assess

The variant cannot be assessed for splice correction. Please obtain evidence for or against a splicing effect.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Synonymous variant

Is there functional evidence of a splicing effect?

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Synonymous variant

The variant can be assessed for splice correction ASO approaches. Please tick all boxes that apply.

To identify which boxes should be ticked, please carefully read “Step 3” of the N1C VARIANT guidelines.

Canonical splicing is destroyed Canonical splicing is weakened Variant within 5 bp of canonical splice site Variant within 6 to 15 bp of canonical splice site None

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Synonymous variant

Eligibility: Not eligible for splice correction

For further information, please see the classification table for splice correction.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Synonymous variant

Eligibility: Unable to assess

The variant cannot be assessed for splice correction. Please obtain evidence for or against a splicing effect.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Missense variants and in-frame indels

Is there functional evidence of a splicing effect?

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Missense variants and in-frame indels

The variant can be assessed for splice correction ASO approaches. Please tick all boxes that apply.

To identify which boxes should be ticked, please carefully read "Step 3" of the N1C VARIANT guidelines. For missense variants and in-frame indels, please pay special attention to the effect of these variants on protein function independent of the splice-altering effect.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Missense variants and in-frame indels

Eligibility: Unable to assess

Variant cannot be assessed for splice correction until further evidence is available. Under certain circumstances, the variant could be assessed for exon skipping.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Missense variants and in-frame indels

Eligibility: Not eligible for splice correction

For further information, please see the classification table for splice correction.

Variant is not eligible for a splice correction approach but under certain circumstances can be assessed for exon skipping approaches.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Missense variants and in-frame indels

Eligibility: Not eligible for splice correction

For further information, please see the classification table for splice correction.

The variant is not eligible for splice correction. The variant can be considered for exon skipping.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Missense variants and in-frame indels

Eligibility: Unable to assess

The variant cannot be assessed for splice correction. Under certain circumstances, the variant can be considered for exon skipping or upregulation from the WT allele. Please review “Section A” (exon skipping) or "Section C" (upregulation from the WT allele) of the N1C VARIANT guidelines for further information.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 3 - Splice assessment - Autosomal recessive - Loss of function - Exon deletion

Eligibility: Not eligible for splice correction.

If the deleted exon is in-frame, the variant is also not applicable to an exon skipping treatment. If the variant is an out-of-frame exon deletion, please check if any of the two adjacent exons could be skipped to get the transcript back into frame, then an exon skipping approach could be considered. If the deleted exon is the first or last coding exon, exon skipping is not a viable option (rare exceptions apply). Please review “Section A” of the N1C VARIANT guidelines.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Section A - Exon skipping assessment - Autosomal recessive - Loss of function - Exon deletion

For exon skipping assessment, please review “Section A” of the N1C VARIANT guidelines.

Please keep in mind that the aim is to skip an adjacent exon to get the transcript back into the reading frame. Do any of the following criteria apply to the exon?

Each of these is a “not eligible” criterion. Please check for exceptions as described in “Section A” of the N1C VARIANT guidelines.

Exon is first coding exon
Exon is last coding exon
Exon is out of frame
Skipping both exons will generate a STOP codon
Gene contains only the two coding exon (deleted one and the one to be skipped).

Section A - Exon skipping assessment - Autosomal recessive - Loss of function - Exon deletion

Eligibility: Not eligible for exon skipping

Please check for exceptions as described in “Section A” of the N1C VARIANT guidelines.

For further information on the classification, please see the classification table for exon skipping.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 2 - Autosomal recessive - Loss of function - Exon duplication

Eligibility: Not eligible for ASO treatment

This variant cannot be corrected with an ASO approach.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 2 - Autosomal recessive - Gain of function variants

Gain of function variants can be analyzed using the assessment for splice correction or for knockdown.

In rare circumstances, also an exon skipping approach can be employed. We recommend reviewing “Section A” of the N1C guidelines to identify in which GoF variants can be targeted with an exon skipping approach. Please choose your preferred assessment:

Step 2 - Autosomal recessive - Unknown pathomechanism

Eligibility: Unable to assess

Please obtain further evidence to determine pathomechanism.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

What is the pathomechanism of the variant under assessment?

For guidance on how to determine the pathomechanism of a given variant, please read “Step 2” of the N1C VARIANT guidelines.

Step 2 - Pathomechanism - X-linked dominant - Loss of function

What is the variant type?

Step 2 - Pathomechanism - X-linked dominant - Loss of function

Eligibility: Not eligible for splice correction and exon skipping

If an exon duplication leads to a loss of function in an XLD disorder, upregulation from the WT allele can be considered. Please review “Section C” in the N1C VARIANT guidelines. For upregulation from the WT allele, we recommend a manual assessment of the different strategies. These cannot be assessed using this calculator. Please also note that for XLD disorders further considerations apply due to X-inactivation. Upregulation from the WT allele is definitely not possible in hemizygotes.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 2 - Pathomechanism - X-linked dominant - Gain of function

Gain of function variants can be analyzed using the assessment for splice correction or for knockdown. In rare circumstances, also an exon skipping approach can be employed. We recommend to review “Section A” of the N1C guidelines to identify in which cases GoF variants can be targeted with an exon skipping approach. Please choose your preferred assessment:

Step 2 - Pathomechanism - X-linked dominant - Dominant negative

Dominant negative variants can be analyzed using the assessment for splice correction or for downregulation. Please choose your preferred assessment:

Step 2 - Pathomechanism - X-linked dominant - Unknown pathomechanism

Eligibility: Unable to assess

Please obtain further evidence to determine pathomechanism.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 2 - Pathomechanism - X-linked recessive

What is the pathomechanism of the variant under assessment?

For guidance on how to determine the pathomechanism of a given variant, please read “Step 2” of the N1C VARIANT guidelines.

Step 2 - Pathomechanism - X-linked recessive - Loss of function

What is the variant type?

Step 2 - Pathomechanism - X-linked recessive - Gain of function

Gain of function variants can be analyzed using the assessment for splice correction or for knockdown. In rare circumstances, also an exon skipping approach can be employed. We recommend to review “Section A” of the N1C guidelines to identify in which cases GoF variants can be targeted with an exon skipping approach. Please choose your preferred assessment:

Step 2 - Pathomechanism - X-linked recessive - Unknown pathomechanism

Eligibility: Unable to assess.

Please obtain further evidence to determine pathomechanism.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 1 - Inheritance pattern - Mitochondrial

Eligibility: Unable to assess

Mitochondrially inherited disorders cannot be analyzed using these guidelines.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

Step 1 - Inheritance pattern - Unknown

Eligibility: Unable to assess

Please check with referring clinician or patient (family) on more information regarding the inheritance pattern.

Optional: Please provide any final thoughts, comments, and evidence for the assessment.

N1C Variant Eligibility Calculator

Information Page

Answer log

Disclaimer

Is the variant description correct and conform with the HGVS nomenclature?

Is the variant the disease-causing variant?

Eligibility: Unable to assess

Is the variant of a type applicable to be evaluated with these guidelines?

Eligibility: Unable to assess

Is the variant a duplication or deletion of a whole gene (CNV)?

Consider variant for knockdown approach. Please review “Section B” in the N1C VARIANT guidelines. Is the gene associated with haploinsufficiency?

Is there already an ASO/RNAi/siRNA approach developed for this gene?

Eligibility: Eligible for a knockdown approach

Eligibility: Likely eligible for a knockdown approach

Consider an allele-selective approach. Is an ASO/RNAi/siRNA approach already developed for this gene?

Eligibility: Eligible for a knockdown approach

Eligibility: Unlikely eligible for a knockdown approach

What is the inheritance pattern of this variant?

What is the pathomechanism of the variant under assessment?

What is the variant type?

Is there functional evidence of a splicing effect?

The variant can be assessed for splice correction ASO approaches. Please tick all boxes that apply.

Has there already been an ASO developed for this variant?

Has there already been an ASO developed for this variant?

Eligibility: Not eligible for splice correction

Eligibility: Eligible for splice correction

Eligibility: Likely eligible for splice correction

Eligibility: Eligible for splice correction

Eligibility: Unlikely eligible for splice correction

Eligibility: Unable to assess

Eligibility: Unable to assess.

Please review “Section C” in the N1C VARIANT guidelines. For upregulation from the WT allele, we recommend a manual assessment of the different strategies. These cannot be assessed using this calculator.

Is there functional evidence of a splicing effect?

The variant can be assessed for splice correction ASO approaches. Please tick all boxes that apply.

Eligibility: Unable to assess

Eligibility: Unable to assess

Is there functional evidence of a splicing effect?

The variant can be assessed for splice correction ASO approaches. Please tick all boxes that apply.

Eligibility: Not eligible for splice correction

Has there already been an ASO developed for this variant?

Has there already been an ASO developed for this variant?

Eligibility: Eligible for splice correction

Eligibility: Likely eligible for splice correction

Eligibility: Eligible for splice correction

Eligibility: Unlikely eligible for splice correction

Eligibility: Not eligible for a splice correction.

Eligibility: Unable to assess

Eligibility: Not eligible for splice correction

Eligibility: Unable to assess

For exon skipping assessment, please review “Section A” of the N1C VARIANT guidelines. Do any of the following criteria apply to the exon?

Eligibility: Not eligible for exon skipping

Please tick all boxes that apply. Please also be aware of exceptions mentioned in “Section A” of the N1C VARIANT guidelines that are not all covered by this calculator.

Eligibility: Not eligible for exon skipping

Special considerations apply for repeat domains.

Is there already an exon skipping therapy available?

Is there already an ASO exon skipping therapy available?

Eligibility: Eligible for exon skipping

Eligibility: Unlikely eligible for exon skipping

Eligibility: Likely eligible for exon skipping

Eligibility: Not eligible for splice correction

For exon skipping assessment, please review “Section A” of the N1C VARIANT guidelines. Please keep in mind that the aim is to skip an adjacent exon to get the transcript back into frame. Do any of the following criteria apply to the exon?

Eligibility: Not eligible for exon skipping

Variant can be assessed for exon skipping. Please tick all boxes that apply.

Eligibility: Not eligible for splice correction

Is there functional evidence of a splicing effect?

What is the variant type?

Is there already an ASO approach developed for this variant?

Is there already an ASO approach developed for this variant?

Eligibility: Eligible for splice correction.

Eligibility: Unlikely eligible for splice correction.

Eligibility: Likely eligible for splice correction

Eligibility: Unable to assess

The variant can be assessed for splice correction ASO approaches. Please tick all boxes that apply.

The variant can be assessed for splice correction ASO approaches. Please tick all boxes that apply.

Eligibility: Unable to assess

Please tick all that apply

Eligibility: Not eligible for a knockdown approach

Eligibility: Unable to assess

Is there already an ASO/RNAi/siRNA approach developed for this gene?

Eligibility: Eligible for a knockdown approach.