Reasoning
PS3 is met at moderate strength. Three independent peer-reviewed functional studies directly evaluated KCNJ11 p.Arg201His (c.602G>A; R201H) using biologically relevant KATP-channel systems. Lin et al. (PMID:16731837) used mammalian COSm6, HEK293, and N2a cells with wild-type controls, electrophysiology, Western blotting, and surface-expression assays, finding reduced ATP sensitivity and approximately 80% reduced surface expression for R201H. Männikkö et al. (PMID:17065345) used inducible stable INS-1 beta-cell expression with wild-type controls and patch-clamp, membrane-potential, and insulin-secretion assays; R201H increased the ATP IC50 from approximately 20 micromol/l to approximately 2 mmol/l, prevented metabolically induced channel closure and electrical activation, and abolished methyl-succinate-stimulated insulin secretion. Kassem et al. (PMID:15718250) independently tested R201H in Xenopus oocytes expressing Kir6.2/SUR1 and found markedly reduced ATP sensitivity and increased channel current. These findings are concordant, mechanistically linked to KCNJ11/Kir6.2 control of beta-cell electrical activity and insulin secretion, and include appropriate wild-type or comparator controls. Moderate rather than strong strength is assigned because the available studies are research assays and do not demonstrate validation, reproducibility, and robustness in a clinical diagnostic laboratory setting as emphasized by the governing definition.
Rule
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.
Evidence considered
- Lin et al. 2006, Diabetes, PMID:16731837: R201H was tested with wild-type controls in COSm6, HEK293, and N2a cells; electrophysiology and expression assays showed reduced ATP sensitivity and approximately 80% reduced KATP-channel surface expression.
- Männikkö et al. 2006, PMID:17065345: inducible stable INS-1 beta-cell expression of R201H versus wild type showed ATP IC50 increasing from approximately 20 micromol/l to approximately 2 mmol/l, failure of methyl succinate to close KATP channels or stimulate electrical activity and insulin secretion, and rescue of secretion by tolbutamide.
- Kassem et al. 2005, PMID:15718250: Xenopus oocytes expressing Kir6.2/SUR1 showed markedly reduced ATP sensitivity for R201H, with increased current at 1 mmol/l MgATP compared with wild type.
- The studies are biologically relevant because KCNJ11/Kir6.2 forms the pore of beta-cell KATP channels, whose ATP-dependent closure regulates membrane depolarization and insulin release.
Still missing
- No evidence was identified that these assays were validated and shown to be reproducible and robust in a clinical diagnostic laboratory setting.
Sources