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 manuscript: Cheerie et al., 2024
If you have any questions about the calculator or suggestions for improvement, please reach out to Marlen Lauffer (marlen@n1collaborative.org).
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.
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
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.
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
Please check with the referring clinician or patient (family) to receive the correct variant description or information on the disease-causing variant.
Step 0 - Variant check
Variants applicable to these guidelines are:
Currently excluded are:
Please also see “Step 0” of the N1C VARIANT guidelines for further explanations.
Step 0 - Variant check
Step 0 - Variant check - CNVs
Step 0 - Variant check - CNVs - Whole gene duplication
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
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
For further information on the classification, please see the classification table for knockdown.
Step 0 - CNV - Whole gene duplication
For further information on the classification, please see the classification table for knockdown.
Step 0 - Variant check - CNV - Whole gene duplication
Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.
Step 0 - CNV - Whole gene duplication
For further information on the classification, please see the classification table for knockdown.
Step 0 - CNV - Whole gene duplication
For further information on the classification, please see the classification table for knockdown.
Step 0 - CNV - Whole gene deletion
Step 1 - Inheritance pattern
For guidance on how to determine the inheritance pattern, please see “Step 1” of the N1C VARIANT guidelines.
Step 2 - Pathomechanism - Autosomal dominant
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
Step 3 - Splice assessment - Autosomal dominant - Loss of function - Intronic variant
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
To identify which boxes should be ticked, please carefully read “Step 3” of the N1C VARIANT guidelines.
Step 3 - Splice assessment
Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.
Step 3 - Splice assessment
Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.
Step 3 - Splice assessment
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:
Step 3 - Splice assessment
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment - Intronic variant
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.
Step 3 - Splice assessment - Intronic variant
The variant cannot be assessed for splice correction. Please obtain evidence for or against a splicing effect.
Section C - Considerations for upregulation from the WT-allele
In case there is an upregulation from the WT allele already available for the disorder with sufficient functional evidence, the variant is considered eligible.
Step 3 - Splice assessment - Synonymous variant
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 - Synonymous variant
To identify which boxes should be ticked, please carefully read “Step 3” of the N1C VARIANT guidelines.
Step 3 - Splice assessment - Synonymous variant
Step 3 - Splice assessment - Synonymous variant
The variant cannot be assessed for splice correction. Please obtain evidence for or against a splicing effect.
Step 3 - Splice assessment - Gain-of-function - Missense variants or in-frame indels
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 - Gain of function - Missense variants or in-frame indels
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 - Gain-of-function/dominant-negative variants - Splice assessment
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 knockdown. Please pick the next guideline:
Step 3 - Splice assessment - GoF/DN variants.
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.
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.
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment - GoF/DN variants.
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment - GoF/DN variants.
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment - GoF/DN variants.
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 knockdown. Please pick the next guideline:
Step 3 - Splice assessment - Missense variants or in-frame indels
Under certain circumstances, the variant can be assessed for an exon skipping approach.
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment - Missense variants or in-frame indels
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.
Step 3 - Splice assessment - Missense variants or in-frame indels
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.
Step 3 - Splice assessment - Missense variants or in-frame indels
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.
Section A - Exon skipping assessment
Each of these criteria is a "not eligible" criterion.
Section A - Exon skipping assessment
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.
Section A - Exon skipping assessment
Section A - Exon skipping assessment
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.
Section A - Exon skipping assessment
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.
Section A - Exon skipping assessment
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
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
For further information on the classification, please see the classification table for exon skipping.
Section A - Exon skipping assessment
For further information on the classification, please see the classification table for exon skipping.
Section A - Exon skipping assessment
For further information on the classification, please see the classification table for exon skipping.
Step 3 - Splice assessment - whole exon duplication and deletion
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.
Section A - Exon skipping assessment for whole exon deletion variants
Each of these criteria is a "not eligible" criterion.
Section A - Exon skipping assessment for whole exon deletion variants
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.
Section A - Exon skipping assessment for whole exon deletion variants
Please also be aware of exceptions mentioned in “Section A” of the N1C VARIANT guidelines that are not all covered by this calculator.
Step 3 - Splice assessment - Whole exon duplication - Loss of function
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.
Step 3 - Splice assessment - Autosomal dominant - Gain of function
Step 3 - Splice assessment - Gain of function and dominant negative variants
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 - Gain of function and dominant negative variants
Step 3 - Splice assessment - Gain of function and dominant negative variants - Nonsense and frameshift variants
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.
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
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
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
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment - Gain of function and dominant negative variants - Nonsense and frameshift variants
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment - Gain of function and dominant negative variants - Nonsense and frameshift variants
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment - Gain of function and dominant negative variants - Whole exon deletions and duplications
Step 3 - Splice assessment - Gain of function and dominant negative variants
The variant can be considered for a knockdown approach or in certain circumstances for exon skipping. Please choose the preferred assessment:
Step 3 - Splice assessment - Synonymous variant
To identify which boxes should be ticked, please carefully read “Step 3” of the N1C VARIANT guidelines.
Step 3 - Splice assessment - GoF/DN - Intronic variant
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
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 see “Section B” in the N1C VARIANT guidelines for further information.
Section B - Knockdown assessment
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.
Section B - Knockdown assessment
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.
Section B - Knockdown assessment
Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.
Section B - Knockdown assessment
For further information on the classification, please see the classification table for knockdown.
Section B - Knockdown assessment
Section B - Knockdown assessment
Please follow the recommendations and criteria outlined in the N1C VARIANT guidelines for sufficient evidence on oligonucleotide availability.
Section B - Knockdown assessment
For further information on the classification, please see the classification table for knockdown.
Section B - Knockdown assessment
Please consider an allele-selective knockdown.
For further information on the classification, please see the classification table for knockdown.
Step 2 - Pathomechanism - Autosomal dominant - Dominant negative variant
Section B - Knockdown assessment - Dominant negative variant
Please see “Section B” in the N1C VARIANT guidelines for further information.
Section B - Knockdown assessment - Autosomal/X-linked dominant - Dominant negative variant
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
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”.
Section B - Knockdown assessment - Autosomal/X-linked dominant - Dominant negative variant
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
For further information on the classification, please see the classification table for knockdown.
Section B - Knockdown assessment - Autosomal/X-linked dominant - Dominant negative variant
Please consider an allele-selective approach for this dominant negative variant.>
For further information on the classification, please see the classification table for knockdown.
Section B - Knockdown assessment - Autosomal/X-linked dominant - Dominant negative variant
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
For further information on the classification, please see the classification table for knockdown.
Section B - Knockdown assessment - Autosomal/X-linked dominant - Dominant negative variant
Please consider an allele-selective approach for this dominant negative variant.>
For further information on the classification, please see the classification table for knockdown.
Step 2 - Pathomechanism
Please obtain further evidence to determine pathomechanism.
Step 2 - Pathomechanism - Autosomal recessive
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
Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant
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 - Autosomal recessive - Loss of function - Intronic variant
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
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
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
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant
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.
Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant
Step 3 - Splice assessment - Autosomal recessive - Loss of function - Intronic variant
The variant cannot be assessed for splice correction. Please obtain evidence for or against a splicing effect.
Step 3 - Splice assessment - Autosomal recessive - Loss of function - Synonymous variant
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 - Autosomal recessive - Loss of function - Synonymous variant
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 - Synonymous variant
For further information, please see the classification table for splice correction.
Step 3 - Splice assessment - Autosomal recessive - Loss of function - Synonymous variant
The variant cannot be assessed for splice correction. Please obtain evidence for or against a splicing effect.
Step 3 - Splice assessment - Autosomal recessive - Loss of function - Missense variants and in-frame indels
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 - Autosomal recessive - Loss of function - Missense variants and in-frame indels
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
Variant cannot be assessed for splice correction until further evidence is available. Under certain circumstances, the variant could be assessed for exon skipping.
Step 3 - Splice assessment - Autosomal recessive - Loss of function - Missense variants and in-frame indels
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.
Step 3 - Splice assessment - Autosomal recessive - Loss of function - Missense variants and in-frame indels
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.
Step 3 - Splice assessment - Autosomal recessive - Loss of function - Missense variants and in-frame indels
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.
Step 3 - Splice assessment - Autosomal recessive - Loss of function - Exon deletion
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.
Section A - Exon skipping assessment - Autosomal recessive - Loss of function - Exon deletion
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.
Section A - Exon skipping assessment - Autosomal recessive - Loss of function - Exon deletion
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.
Section A - Exon skipping assessment - Autosomal recessive - Loss of function - Exon deletion
Step 2 - Autosomal recessive - Loss of function - Exon duplication
This variant cannot be corrected with an ASO approach.
Step 2 - Autosomal recessive - Gain of function variants
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
Please obtain further evidence to determine pathomechanism.
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
Step 2 - Pathomechanism - X-linked dominant - Loss of function
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.
Step 2 - Pathomechanism - X-linked dominant - Gain of function
Step 2 - Pathomechanism - X-linked dominant - Dominant negative
Step 2 - Pathomechanism - X-linked dominant - Unknown pathomechanism
Please obtain further evidence to determine pathomechanism.
Step 2 - Pathomechanism - X-linked recessive
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
Step 2 - Pathomechanism - X-linked recessive - Gain of function
Step 2 - Pathomechanism - X-linked recessive - Unknown pathomechanism
Please obtain further evidence to determine pathomechanism.
Step 1 - Inheritance pattern - Mitochondrial
Mitochondrially inherited disorders cannot be analyzed using these guidelines.
Step 1 - Inheritance pattern - Unknown
Please check with referring clinician or patient (family) on more information regarding the inheritance pattern.