Identification of a novel Scn3b mutation in a Chinese Brugada syndrome pedigree: implications for Nav1.5 electrophysiological properties and intracellular distribution of Nav1.5 and Navβ3

Background The Scn3b gene encodes for Navβ3, a pivotal regulatory subunit of the fast sodium channel in cardiomyocytes. However, its mutation status in the Chinese population suffering from Brugada Syndrome (BrS) has not been characterized, and the contributory pathophysiological mechanisms to disease pathology remain undefined. Methods and Results A Scn3b (c.260C>T, p.P87l) mutation was identified in a patient with BrS of Chinese descent. Functional analyses demonstrated that sodium channel activation for the wild type, mutant samples, and co-expression of both commenced at −55 mv and peaked at −25 mv. The mutant group exhibited a notable reduction, approximately 60%, in peak sodium channel activation current (INa) at −25 mv. The parameters for half-maximal activation voltages (V1/2) and slope factors (k) showed no significant differences when comparing wild type, mutant, and combined expression groups (P = 0.98 and P = 0.65, respectively). Additionally, no significant disparities were evident in terms of the steady-state sodium channel inactivation parameters V1/2 and k (with P-values of 0.85 and 0.25, respectively), nor were there significant differences in the activation time constant τ (P = 0.59) and late sodium current density (P = 0.23) across the wild-type, mutant, and co-expressed groups. Confocal imaging and Western blot analysis demonstrated decreased plasma membrane localization of SCN3B and SCN5A in the P87l group. Computational simulations of cardiac action potentials suggested that SCN3B P87l can alter the morphology of the action potentials within the endocardium and epicardium while reducing the peak of depolarization. Conclusions The pathogenic impact of the Scn3b P87l mutation predominantly originates from a reduction in peak INa activation current coupled with decreased cell surface expression of Nav1.5 and Navβ3. These alterations may influence cardiac action potential configurations and contribute to the risk of ventricular arrhythmias in individuals with BrS.

M-CAP: Mendelian Clinically Applicable Pathogenicity Score, Bejerano Lab, Stanford University Mendelian Clinically Applicable Pathogenicity (M-CAP) Score M-CAP is the first pathogenicity classifier for rare missense variants in the human genome that is tuned to the high sensitivity required in the clinic (see Table ).By combining previous pathogenicity scores (including SIFT, Polyphen-2 and CADD) with novel features and a powerful model, we attain the best classifier at all thresholds, reducing a typical exome/genome rare (<1%) missense variant (VUS) list from 300 to 120, while never mistaking 95% of known pathogenic variants as benign.Further details can be found here (http://rdcu.be/lPtd).M-CAP source code M-CAP public git repository can be found here (https://bitbucket.org/bejerano/mcap_public).
M-CAP train and test data can be found here (downloads/dat/).Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.See manuscript for detailed guidance.

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Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence.Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc.)

Moderate evidence of pathogenicity
Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion.BP4 can be used only once in any evaluation of a variant.

✘ BP5
Variant found in a case with an alternate molecular basis for disease ✘ BP6 Reputable source recently reports variant as benign but the evidence is not available to the laboratory to perform an independent evaluation ✘ BP7 A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of anew splice site AND the nucleotide is not highly conserved p r e d i c t i o n o f f u n c t i o n a l e f f e c t s o f h u m a n Default threshold is -2 .5 , that is: -Variants with a score equal to or below -2 .5 are considered " deleterious, " -Variants with a score above -2 .5 are considered " neutral ."( 2 ) How to use a more stringent threshold .3 8 : 4 0 EDT, Saturday, May 22 , 20 2 1 * The results are kept for 48 hours.
Attribution-NonCommercial 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0nonsense,frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease Caveats: • Beware of genes where LOF is not a known disease mechanism (e.g.GFAP, MYH7) • Use caution interpreting LOF variants at the extreme 3' end of a gene • Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact • Use caution in the presence of multiple transcripts Strong evidence of pathogenicity ✘ PS1 Same amino acid change as a previously established pathogenic variant regardless of nucleotide change Example: Val->Leu caused by either G>C or G>T in the same codon.Caveat: Beware of changes that impact splicing rather than at the amino acid/protein level ✘ PS2 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 ✘ PS3 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 ✘ PS4 The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls.
silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of anew splice site AND the nucleotide is not highly conserved CTNNA3 (G793E): Rules for Combining Criteria to Classify Sequence Variants PS1-PS4) AND 1-2 Moderate (PM1-PM6) OR ✘ 3 1 Strong (PS1-PS4) AND ≥2 Supporting (PP1-PP5) OR nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease Caveats:• Beware of genes where LOF is not a known disease mechanism (e.g.GFAP, MYH7)• Use caution interpreting LOF variants at the extreme 3' end of a gene• Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact• Use caution in the presence of multiple transcripts caused by either G>C or G>T in the same codon.Caveat:Beware of changes that impact splicing rather than at the amino acid/protein level ✘ PS2 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 ✘ PS3 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 ✘ PS4 The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls.Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.See manuscript for detailed guidance.Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence.Moderate evidence of pathogenicity ✘ PM1 Located in a mutational hot spot and/or critical and well-established functional domain (e.g.active site of an enzyme) without benign variation ✔ PM2 Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or ExAC Caveat: Population data for indels may be poorly called by next generation sequencing ✘ PM3 For recessive disorders, detected in trans with a pathogenic variant.Note: This requires testing of parents (or offspring) to determine phase ✘ PM4 Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants ✘ PM5 Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before Example: Arg156His is pathogenic; now you observe Arg156CysCaveat: Beware of changes that impact splicing rather than at the amino acid/protein level ✘ PM6 Assumed de novo, but without confirmation of paternity and maternity Supporting evidence of pathogenicity ✘ PP1 Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease Note: May be used as stronger evidence with increasing segregation data ✘ PP2 Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease ✘ PP3 Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion.PP3 can be used only once in any evaluation of a variant.✘ PP4 Patient's phenotype or family history is highly specific for a disease with a single genetic etiology ✘ PP5 Reputable source recently reports variant as pathogenic but the evidence is not available to the laboratory to perform an independent evaluation DPP6 (A20T): Criteria for Classifying Benign Variants Stand-Alone evidence of benign impact ✘ BA1 Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes, or ExAC Strong evidence of benign impact ✘ BS1 Allele frequency is greater than expected for disorder ✘ BS2 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 ✘ BS3 Well-established in vitro or in vivo functional studies shows no damaging effect on protein function or splicing ✔ BS4 Lack of segregation in affected members of a family Caveat: The presence of phenocopies for common phenotypes (i.e.cancer, epilepsy) can mimic lack of segregation among affected individuals.Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation.Supporting evidence of benign impact ✘ BP1 Missense variant in a gene for which primarily truncating variants are known to cause disease ✘ BP2 Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder; or observed in cis with a pathogenic variant in any inheritance pattern ✘ BP3 In-frame deletions/insertions in a repetitive region without a known function ✔ BP4 Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc.) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion.BP4 can be used only once in any evaluation of a variant.✘ BP5 Variant found in a case with an alternate molecular basis for disease ✘ BP6 Reputable source recently reports variant as benign but the evidence is not available to the laboratory to perform an independent evaluation ✘ BP7 A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of anew splice site AND the nucleotide is not highly conserved DPP6 (A20T): Rules for Combining Criteria to Classify Sequence Variants nonsense, frameshift, canonical +/−1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease Caveats:• Beware of genes where LOF is not a known disease mechanism (e.g.GFAP, MYH7)• Use caution interpreting LOF variants at the extreme 3' end of a gene• Use caution with splice variants that are predicted to lead to exon skipping but leave the remainder of the protein intact• Use caution in the presence of multiple transcripts caused by either G>C or G>T in the same codon.Caveat:Beware of changes that impact splicing rather than at the amino acid/protein level ✘ PS2 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 ✘ PS3 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 ✘ PS4 The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls.Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.See manuscript for detailed guidance.Note 2: In instances of very rare variants where case-control studies may not reach statistical significance, the prior observation of the variant in multiple unrelated patients with the same phenotype, and its absence in controls, may be used as moderate level of evidence.Moderate evidence of pathogenicity ✘ PM1 Located in a mutational hot spot and/or critical and well-established functional domain (e.g.active site of an enzyme) without benign variation ✘ PM2 Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or ExAC Caveat: Population data for indels may be poorly called by next generation sequencing ✘ PM3 For recessive disorders, detected in trans with a pathogenic variant.Note: This requires testing of parents (or offspring) to determine phase ✘ PM4 Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants ✘ PM5 Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before Example: Arg156His is pathogenic; now you observe Arg156CysCaveat: Beware of changes that impact splicing rather than at the amino acid/protein level ✘ PM6 Assumed de novo, but without confirmation of paternity and maternity Supporting evidence of pathogenicity ✘ PP1 Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease Note: May be used as stronger evidence with increasing segregation data ✘ PP2 Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease ✘ PP3 Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion.PP3 can be used only once in any evaluation of a variant.✘ PP4 Patient's phenotype or family history is highly specific for a disease with a single genetic etiology ✘ PP5 Reputable source recently reports variant as pathogenic but the evidence is not available to the laboratory to perform an independent evaluation healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder with full penetrance expected at an early age ✘ BS3 Well-established in vitro or in vivo functional studies shows no damaging effect on protein function or splicing ✔ BS4 Lack of segregation in affected members of a family Caveat: The presence of phenocopies for common phenotypes (i.e.cancer, epilepsy) can mimic lack of segregation among affected individuals.Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation.with a pathogenic variant for a fully penetrant dominant gene/disorder; or observed in cis with a pathogenic variant in any inheritance pattern ✘ BP3 In-frame deletions/insertions in a repetitive region without a known function ✔ BP4

Method Authors' Recommended Pathogenicity threshold Misclassified known variants pathogenic SIFT
M-CAP only scores rare missense variants: hg19, ENSEMBL 75 missense, ExAC v0.3 ( public/legacy/exacv1_downloads/release0.3)where no super population has minor allele frequency above 1%.If a missense variant has no M-CAP score, the M-CAP prediction should be assumed to be likely benign.M-CAP v1.0 scores 10/24/2016 (downloads/dat/mcap_v1_1.txt.gz)please do not use M-CAP v1.3 raw scores 10/30/2018 (downloads/dat/mcap_v1_3.txt.gz)M-CAP v1.4 raw and normalized scores 3/25/2019 (downloads/dat/mcap_v1_4.txt.gz)(v1.3 raw score of 0.025 converted to sensitivity score 0.95, to facilitate interpretation) ) can mimic lack of segregation among affected individuals.Also, families may have more than one pathogenic variant contributing to an autosomal dominant disorder, further confounding an apparent lack of segregation.As many in silico algorithms use the same or very similar input for their predictions, each algorithm cannot be counted as an independent criterion.BP4 can be used only once in any evaluation of a variant.
domain (e.g.active site of an enzyme) without benign variation ✔ PM2 Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or ExAC Caveat: Population data for indels may be poorly called by next generation ✔ PP3 Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.) Caveat: As many in silico algorithms use the same or very similar input for their predictions, each algorithm should not be counted as an independent criterion.PP3 can be used only once in any evaluation of a variant.✘ PP4 Patient's phenotype or family history is highly specific for a disease with a single genetic etiology ✘ PP5 Reputable source recently reports variant as pathogenic but the evidence is not available to the laboratory to perform an independent evaluation CTNNA3(G793E): Criteria for Classifying Benign Variants ✘ BS3 Well-established in vitro or in vivo functional studies shows no damaging effect on protein function or splicing ✔ BS4 Lack of segregation in affected members of a family Caveat: The presence of phenocopies for common phenotypes (i.e.cancer, epilepsy