RescueModel Mus Musculus

GeneAlteration knock-in strain was generated by homologous recombination of the targeting vector with 129Sv/J embryonic stem cells genome. A 450-bp DNA segment encompassing 5′ UTR and exon 1 of the CASQ2 was used to screen RPCI-21 PAC mouse genomic library. Positive clones were sequenced to define the structure of an 8.5-Kb region encompassing promoter, exon 1, and part of intron 1 of mouse CASQ2. This region was cloned in 3 parts and assembled into the targeting vector pFlrt (supplemental Figure I). The linearized targeting vector was electroporated into 129Sv/J embryonic stem cells. The clone selected with G418 and gancyclovir was injected into C57BL/6NCrL blastocyts and transferred to pseudopregnant CD-1 females. Genotype was determined by sequencing of DNA extracted from tail biopsy specimens (DNasy Tissue Kit, Qiagen).

RescueVariant murine cardiac CASQ2 , WT?

RescuePhenotype HP:0004756

RescueMethod The AAV recombinant virus (AAV9-CASQ2) and the empty virus (AAV9-GFP) were produced by the AAV Vector Core of the Telethon Institute of Genetics and Medicine (Napoli, Italy) according to a previously published protocol.18 The viral titer was 2.6×1012 genome copies per milliliter for AAV9-CASQ2 and 4.3×1012 genome copies per milliliter for AAV9-GFP. The infection was performed by intraperitoneal injection in pups on postnatal day 3 (100 μL), which recovered until euthanasia was performed at 6, 9, or 12 months. Three-month-old mice were anesthetized with Avertin (0.025 mg/kg), and viral particles (200 μL) were injected into the tail vein and studied 2 months later. ECG recordings in mice of either sex were performed with implantable loop recorders (Data Sciences International).

RescueEvidence (Fig.5&8) Epinephrine/Isoprenol administration had lead to diminished incidence of VT in CASQ infected mice vs R33Q mice.

ModelVariant NM_001232.3(CASQ2):c.97C>T (p.Arg33Ter), ClinVar: 41043

NonPatientCell Sarcoplasmic reticulum of cardiac muscle cell, FMA:62390

GeneAlteration The D307H mutant was generated using Quick Change (Life Sciences). Substitution of the CAC codon with GAC resulted in a BamH1 restriction site that was used along with complete sequencing of the DNA inserts to verify sequence.

FunctionalEvidence (fig. 3) Diminished sensitivity to Ca2+ leading to more intercellular Ca2+

FAVariant NM_001232.3(CASQ2):c.919G>C (p.Asp307His), 17610

CaseAddInfo Fig 4&5 show evidence for the conformation change responsible for the sensitivity difference.

gnomAD 0.00003249 link

Case# Postma 3:1, male, 10yo, white

CasePresentingHPOs HP:0004758, HP:0000980, HP:0005184, HP:0000961, HP:0002789

CasePreviousTesting no

CaseMethod1 PCR

CaseGenotypingMethod amplified/screen of CASQ2, SCN5A, HRC, PLN, RYR2, KCNQ1, KCNH2, and KCNK1 genes

Variant NM_001232.3:c.532+1G>A

CAID CA341763774 link

VariantEvidence not in this article

Case# Postma 2:2, female, 6yo, white

CasePresentingHPOs HP:0004758

CasePreviousTesting no

CaseMethod1 PCR

CaseGenotypingMethod amplified/screen of CASQ2, SCN5A, HRC, PLN, RYR2, KCNQ1, KCNH2, and KCNK1 genes

Variant NM_001232.3(CASQ2):c.62delA (p.Glu21Glyfs)

ClinVar 41042 link

VariantEvidence not in this article

Case# Postma 3:3, female, 11yo, white

CasePresentingHPOs HP:0004758, HP:0001688

CasePreviousTesting no

CaseMethod1 PCR

CaseGenotypingMethod amplified/screen of CASQ2, SCN5A, HRC, PLN, RYR2, KCNQ1, KCNH2, and KCNK1 genes

Variant NM_001232.3(CASQ2):c.97C>T (p.Arg33Ter)

ClinVar 41043 link

gnomAD 0.000008968 link

VariantEvidence not in this article

NonPatientCell myocardium, UBERON:0002349

GeneAlteration isolated adult rat ventricular myocytes infected with adenoviruses for expression of either the wild-type (WT) or mutant forms of CASQ2. Cytosolic and intra-SR [Ca2+]

FunctionalEvidence Causes leakage of Ca2+ transients from SR (fig. 5)

FAVariants NM_001232.3(CASQ2):c.97C>T (p.Arg33Ter), 41043 link

Lahat Family IV

Family#PresentingHPOs HP:0004758

CountryOrigin Israel

Ethnicity Arab

Race White

Family#Method1 whole genome

Family#Method2 PCR

Affected 3

Unaffected 4

Segregations 3, consanguineous (fig 1.)

PublishedLOD 8.24 combined

eLOD 1.70

ProbandLabel Lahat 2:2, female

Variant NM_001232.3(CASQ2):c.919G>C (p.Asp307His)

ClinVar 17610

gnomAD 0.00003249 link

FamilyPMIDs 11704930, links disease to a variant in CASQ2

Lahat Family I

Family#PresentingHPOs HP:0004758

CountryOrigin Israel

Ethnicity Arab

Race White

Family#Method1 whole genome

Family#Method2 PCR

Affected 3

Unaffected 5

Segregations 3, consanguineous (fig 1.)

PublishedLOD 8.24 combined

eLOD 1.82

ProbandLabel Lahat 2:6, female

Variant NM_001232.3(CASQ2):c.919G>C (p.Asp307His)

ClinVar 17610

gnomAD 0.00003249 link

FamilyPMIDs 11704930, links disease to a variant in CASQ2

11704930 connects these cases to CASQ2

Variant NM_001232.3(CASQ2):c.919G>C (p.Asp307His)

ClinVar 17610

gnomAD 0.00003249 link

Case# Barletta 3:1, female, 17yo

CasePresentingHPOs HP:0004758

CasePreviousTesting no

CaseMethod1 PCR

CaseGenotypingMethod amplified 11 exons of CASQ2

Variant NM_001232.3(CASQ2):c.500T>A (p.Leu167His), NM_001232.3(CASQ2):c.339_354del16 (p.Ser113Argfs)

ClinVar 17612, 17611

VariantEvidence (fig5.) L167H variant had reduced Ca2+ sparks in infected myoctes comparable to levels in G112+5X and both significantly less than WT.

CaseAddInfo Authors admit that the functional evaluation of L167H is not as convincing as with G112+5X, the latter showing complete functional impairment and the former preserving major biochemical and biophysical properties of CASQ2.

Case# Barletta 3:2, male, 6yo

CasePresentingHPOs HP:0004758

CasePreviousTesting no

CaseMethod1 PCR

CaseGenotypingMethod amplified 11 exons of CASQ2

Variant NM_001232.3(CASQ2):c.339_354del16 (p.Ser113Argfs)

ClinVar 17611

VariantEvidence G112+5X variant decrease in Ca2+ transients and decrease in SR ability to store Ca2+ (fig.4&5)

NonPatientCell myocardium of ventricle, UBERON:0001083

GeneAlteration The D307H mutation was introduced into the coding sequence of canine CASQ2 by site-directed mutagenesis. The canine CASQ2D307H coding sequence was inserted into an adenoviral vector to generate Ad-CASQ2D307H for subsequent gene transfer into adult rat ventricular myocytes.

FunctionalEvidence (fig. 2) More intracellular Ca2+ due to reduced ability of SR to store Ca2+

Add Fig. 4 Increased transients through Ca2+ sparks

FAVariants NM_001232.3(CASQ2):c.919G>C (p.Asp307His), 17610 link

NonHumanModel Mus Musculus

GeneAlteration The knock-in strain was generated by homologous recombination of the targeting vector with 129Sv/J embryonic stem cells genome. A 450-bp DNA segment encompassing 5′ UTR and exon 1 of the CASQ2 was used to screen RPCI-21 PAC mouse genomic library. Positive clones were sequenced to define the structure of an 8.5-Kb region encompassing promoter, exon 1, and part of intron 1 of mouse CASQ2. This region was cloned in 3 parts and assembled into the targeting vector pFlrt (supplemental Figure I). The linearized targeting vector was electroporated into 129Sv/J embryonic stem cells. The clone selected with G418 and gancyclovir was injected into C57BL/6NCrL blastocyts and transferred to pseudopregnant CD-1 females. Genotype was determined by sequencing of DNA extracted from tail biopsy specimens (DNasy Tissue Kit, Qiagen).

ModelPhenotype HP:0004758

HumanPhenotype HP:0004758

ModelEvidence Fig. 1 ECG during stimuli shows VT in only the mutant mice

ModelVariant NM_001232.3(CASQ2):c.97C>T (p.Arg33Ter), 41043 link