Popmax allele frequency >0.0038 (0.38%) BA1

Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder with full penetrance expected at an early age.
•Two or more variant positive individuals with a negative IVCT/CHCT test BS2

Popmax allele frequency >0.0008 (0.08%) BS1

The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls
•1 MH case point. Probands with a personal or family historyb of an MH event are awarded 0.5 points, probands with a personal or family history of a positive (MHS) IVCT/CHCT are awarded an additional 0.5 points. Popmax in gnomAD ≤0.00006. For variants with popmax MAF in gnomAD >0.00006, an odds ratio of ≥2.08 when comparing MH case points to allele count in gnomAD can qualify. Popmax in gnomAD must be <0.0038 PS4-Supporting

The prevalence of the variant in affected individuals is significantly increased compared with the prevalence in controls
•2-6 MH case points. Probands with a personal or family historyb of an MH event are awarded 0.5 points, probands with a personal or family history of a positive (MHS) IVCT/CHCT are awarded an additional 0.5 points. Popmax in gnomAD ≤0.00006 * For variants with popmax MAF in gnomAD >0.00006, an odds ratio of ≥4.33 when comparing MH case points to allele count in gnomAD can qualify. Popmax in gnomAD must be <0.0038 PS4-Moderate

The prevalence of the variant in affected individuals significantly increased compared with the prevalence in controls
•≥7 MH case points. Probands with a personal or family history of an MH event areawarded 0.5 points, probands with a personal or family historyb of a positive (MHS)IVCT/CHCT are awarded an additional 0.5 points. Popmax in gnomAD ≤0.00006. * For variants with popmax MAF gnomAD >0.00006, an odds ratio of ≥18.7 when comparing MH case points to allele count in gnomAD can qualify. Popmax in gnomAD must be <0.0038 PS4

A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site AND the nucleotide is not highly conserved BP7

Computational evidence suggest no impact on gene or gene product, REVEL score of <0.5 BP4

Multiple lines of computational evidence support a deleterious effect on the gene or gene product
•Use REVEL score of >0.85 PP3-Moderate

Missense change at an amino acid residue where a different missense varaint previously determined to be pathogenic
•Previously established pathogenic variant must reach a classification of pathogenicity without PM5 * Grantham score for alternate pathogenic variant must be less than for variant being assessed PM5

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change
•Previously established pathogenic variant must reach a classification of pathogenicwithout PS1 PS1

Well-established functional studies show no damaging effect on protein function
•No significant increased sensitivity to RYR1 agonist in an approved in vitro assay, Ca2+ release measured, n≥3 * One or two independent ex vivo studies, NO significant release of Ca2+ in response to agonist * Knock-in mouse showing no MH reaction in response to RYR1 agonist AND no increased sensitivity to RYR1 agonists in ex vivo tissue/cells BS3-Supporting

Located in a mutational hot spot and/or critical and well established functional domain
•Residues 4,631-4,991 (C-terminal region) PM1-Supporting

Well-established functional studies supportive of a damaging effect on protein function
•Two independent ex vivo studies all showing release of Ca2+ in response to RYR1 agonist PS3-Supporting

Well-established functional studies supportive of a damaging effect on protein function
•Increased sensitivity to RYR1 agonist in HEK293 in vitro assay, Ca2+ release significantly increased compared to WT, controls to include known pathogenic and benign variants, n≥3. * Three or more independent ex vivo studies all showing release of Ca2+ in response to RYR1 agonist * Knock-in mouse showing MH reaction in response to RYR1 agonist OR increased sensitivity to RYR1 agonists in ex vivo tissue/cells (but not both, which would be PS3_strong) PS3-Moderate

Located in a mutational hot spot and/or critical and well established functional domain
•Residues 1-552 (N-terminal region) and 2,101-2,458 (central region) PM1

Well-established functional studies supportive of a damaging effect on protein function
•Knock-in mouse showing MH reaction in response to RYR1 agonist AND increasedsensitivity to RYR1 agonists in ex vivo tissue/cells PS3

Co-segregation with disease in 3-4 reported meioses PP1

•Co-segregation with disease in 5-6 reported meioses PP1-Moderate

Co-segregation with disease in ≥7 reported meioses PP1-Strong

Each proven de novo case, 2 points, each assumed de novo case, 1 point, a total of 1 point PM6-Supporting

Each proven de novo case, 2 points, each assumed de novo case, 1 point, a total of 1 point PS2-Supporting

Each proven de novo case, 2 points, each assumed de novo case, 1 point, a total of 2-3 points PS2-Moderate

Each proven de novo case, 2 points, each assumed de novo case, 1 point, a total of 2-3 points PM6

Each proven de novo case, 2 points, each assumed de novo case, 1 point, a total of 4-7 points PM6-Strong

Each proven de novo case, 2 points, each assumed de novo case, 1 point, a total of 4-7 points PS2

Each proven de novo case, 2 points, each assumed de novo case, 1 point, ≥8 points PM6-Very Strong

Each proven de novo case, 2 points, each assumed de novo case, 1 point, ≥8 points PS2-Very Strong

Observed in cis with a pathogenic variant in any inheritance pattern BP2