Apply PVS1_Moderate for truncating variants between codons 582 and 620, for which nonsense-mediated decay is not predicted. This region includes the subunits assembly domain (SAD) between residues 589-620, which mediates tetramerization, so that truncating variants that remove this region are reportedly unable to assemble and are disease-causing (PMID: 10654932).

https://docs.google.com/presentation/d/1-Dz9jmvebv1z1QoSBdON3vbbNaH-V2-v/edit#slide=id.p1

• PS1 is met at the PS1_Moderate level if the comparison variant reaches a likely pathogenic classification using these rule specifications, without the use of the PS1 code.
• This code can also be met at the PS1_Moderate level when the corresponding variant in the paralogous KCNQ2 gene meets criteria to be classified as Pathogenic by the KCNQ Channel Brain Disorders Variant Curation Expert Panel specifications. Curators should access paralogue data and regions of alignment and non-alignment between KCNQ1 and KCNQ2 at the site below: https://www.cardiodb.org/paralogue_annotation/gene.php?name=KCNQ1.
• The corresponding variant in the paralogous gene must substitute the same amino acid as the variant being considered, not a different amino acid.
• An example is that the NM_000218.3(KCNQ1):c.430A>G (p.Thr144Ala) has a paralogous variant in NM_172107.4(KCNQ2):c.340A>G (p.Thr114Ala). If the latter variant in KCNQ2 is classified Pathogenic in the future by the KCNQ Channel Brain Disorders Variant Curation Expert Panel specifications, the paralogous p.Thr144Ala variant in KCNQ1 will meet PS1_Moderate.
• The paralogous variant must have reached Pathogenic classification without the use of the PS1 code.
• While this paralogue-based strategy has been approved for KCNQ2 only, this criterion may eventually apply to the KCNQ3, KCNQ4, and KCNQ5 genes too, following future specifications and variant classifications by the KCNQ Channel Brain Disorders Variant Curation Expert Panel.

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history. Note: Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity.

• For PS2, both maternity and paternity must be confirmed, with no family history of disease (no evidence of QT-prolongation in parents or family history of sudden, unexplained death under the age of 40 years). Confirmation of paternity only is insufficient. Egg donation, surrogate motherhood, errors in embryo transfer, etc. can contribute to non-maternity. Cases and parents genotyped by trio whole exome sequencing are considered to have confirmed maternity and paternity.
• The de novo variant in question must be coding or flanking.
• The PS2 strength level depends on the clinical phenotype specificity and number of probands as defined in Tables 1 and 2.
• When using Table 1 to find the number of points per proband, if the proband has a phenotype sufficient to diagnose LQTS (prolonged QTc interval >480ms), the row used should usually be "phenotype consistent with gene but not highly specific". During the pilot phase, curators should also note the genotyping method and whether comprehensive testing of all/other LQTS genes has been performed.
• When using Table 1 to find the number of points per proband, if the proband meets PP4, so that the phenotype is sufficient to diagnose _KCNQ1_-specific LQTS (LQT 1), the number of points corresponding to "phenotype highly specific for gene" should be used instead. Note: This would require QTc prolongation above 480ms AND either swimming-associated events OR treadmill stress test result (PMID: 21699858) OR T-wave morphology characteristic of LQT1 (PMID: 7586261, 29141844).

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product. Note: Functional studies that have been validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting are considered the most well-established.

Please refer to the linked table below for strength level specifications:

https://docs.google.com/presentation/d/1YA_82T8orad_nAy_fjapiFFz3ZSITVwZ/edit#slide=id.p1

Caveats:

1. Conflicting evidence from different papers - no points
2. Same types of evidence (e.g RNA and Protein Metabolism) coming from the same paper - count 1
3. No more than 2 pieces of evidence can be counted from the same paper
4. Same finding in 2 papers from the same group – count 1
5. Electrophysiology result can only meet PS3 if the variant is co-expressed with KCNE1 and the current magnitude is outside the normal range defined by the paper and is statistically significantly different from the normal control.
6. Please count Experimental / Structural / Functional Simulation (PMID: 29021305) only when the results align with a electrophysiology experiment.

Electrophysiology and Experimental / Structural / Functional Simulation assays approved by the VCEP for PS3 are:

(1) Manual patch-clamp (e.g. PMIDs: 21380488, 30571187, 11162126, 19959132, 30591322, 17053194)

(2) Automated patch-clamp (e.g. PMID: 30571187)

(3) Microelectrode array analysis of hIPSC-cardiomyocytes (e.g. PMID: 35765105) 

(4) Experimental / Structural / Functional Simulation (e.g. PMIDs: 29021305, 35442947, 32096762)

RNA or Protein Metabolism assays approved by the VCEP for PS3 are:

(5) Cell Surface Localization by Flow Cytometry (e.g. PMID: 29532034)  

(6) Mislocalization by Immunofluorescence of KCNQ1 (e.g. PMIDs: 21380488, 19114714, 11162126, 17053194) or KCNH2 (e.g. PMIDs: 19959132, 30591322)

(7) Total Cell Expression by Flow Cytometry (e.g. PMID: 29532034)

(8) Western Blotting (e.g. PMIDs: 21380488, 19114714)

(9) RNA Metabolism showing partial / incomplete disruption of splicing (e.g. PMIDs: 17292394, 28264985)

• Functional assays are described at the following link: https://docs.google.com/spreadsheets/d/12iWEYVxD5-wMutqqW10u-7RKupCoElij/edit#gid=1933102446

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls

• In order to be evaluated for this criterion, the variant must not meet BS1.
• PS4_moderate is Met by 3-5 probands.
• This code is not considered mutually exclusive with PM3. For example, variants associated with both dominant and recessive cases can meet both PS4 and PM3.
• If probands are reported in at least 2 papers, and the author lists from the two papers contain any of the same authors names, definitely the patients are the same, so count only one case for PS4.
• If the two lists of authors are totally distinct / different, usually you can count both cases for PS4.
• Patients reported as affected must have QTc measurement greater than or equal to 460ms in order to be counted. Diagnosis of long QT syndrome by itself is not sufficient for inclusion.

Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation 

• The variant must be rare (meeting PM2_Supporting) in order to be considered for PM1.
• Met by a rare variant within the pore helix (amino acids 300 to 320). This region is known to be a critical region of KCNQ1 and has been confirmed to show an absence of likely benign or benign variants listed in gnomAD.

For recessive disorders, detected in trans with a pathogenic variant  Note: This requires testing of parents (or offspring) to determine phase

• Can be used for variants seen in patients with Jervell and Lange-Nielsen syndrome, if the phenotype includes both long QT interval and congenital deafness.
• This code is not considered mutually exclusive with PS4. For example, variants associated with both recessive and dominant cases can meet both PM3 and PS4.
• Use the ClinGen SVI recommendations to determine evidence weight for this rule code.
• Both variants must be classified using these rule specifications.

Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants

• This code is mutually exclusive with PVS1 (PMID: 30192042) and PP3 (in order not to double-count in silico predictor data) but can be used together with PM1.
• In-frame insertions or deletions of any size will meet PM4 at the moderate (default) level, due to the greater importance of the location of the variant rather than the size.

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

• Caveat: Residue must be highly conserved across all 5 human KCNQ paralogues in order to be considered for PM5. To confirm conservation, curators should access paralogue data and regions of alignment and non-alignment between KCNQ1 and KCNQ2, KCNQ3, KCNQ4, and KCNQ5  at the site below: https://www.cardiodb.org/paralogue_annotation/gene.php?name=KCNQ1.
• Poor conservation (ineligibility for PM5) is defined whenever 1 or more KCNQ’s show a different amino acid at the position. 
• Comparison variants must have a Pathogenic or Likely Pathogenic classification reached using these VCEP-specified rules without using PM5.
• Variants at any codon where a likely benign / benign variant has been classified by the group are not eligible for PM5.
• SpliceAI must be used to examine both variants for similar predicted effect or lack of effect on splicing (SpliceAI Δ score <0.2).
• Not used at the PM5_Strong level.

Assumed de novo, but without confirmation of paternity and maternity.

• For PM6, maternity and paternity are not confirmed but assumed, with no family history of disease (no evidence of QT-prolongation in parents or family history of sudden, unexplained death under the age of 40 years).
• The de novo variant in question must be coding or flanking.
• The PM6 strength level depends on the clinical phenotype specificity and number of probands as defined in Tables 1 and 2.
• When using Table 1 to find the number of points per proband, if the proband has a phenotype sufficient to diagnose LQTS (prolonged QTc interval >480ms), the row used should usually be "phenotype consistent with gene but not highly specific". During the pilot phase, curators should also note the genotyping method and whether comprehensive testing of all/other LQTS genes has been performed.
• When using Table 1 to find the number of points per proband, if the proband meets PP4, so that the phenotype is sufficient to diagnose _KCNQ1_-specific LQTS (LQT 1), the number of points corresponding to "phenotype highly specific for gene" should be used instead. Note: This would require QTc prolongation above 480ms AND either swimming-associated events OR treadmill stress test result (PMID: 21699858) OR T-wave morphology characteristic of LQT1 (PMID: 7586261, 29141844).

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease  

• Each affected family member must have Schwartz score (PMID: 36017572, Table 10) >3 or QTc greater than or equal to 480ms or syncope in order to be included / counted.
• The proband + 5 affected family members with the variant meets PP1_Moderate.
• The numbers above have been informed by PMID: 27236918.
• If any family member is affected but does not harbor the variant, use BS4 (non-segregation) instead.