CRISPR/Cas9 ADCY7 Knockout Stimulates the Insulin Secretion Pathway Leading to Excessive Insulin Secretion

• Main Authors: Yazeid Alhaidan, Henrik Thybo Christesen, Elena Lundberg, Mohammed A. Al Balwi, Klaus Brusgaard
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2021-06-01
• Series: Frontiers in Endocrinology
• Subjects: genetics; metabolomics; congenital hyperinsulinism; hyperinsulinemic hypoglycemia; pediatrics; adenylyl cyclase
• Online Access: https://www.frontiersin.org/articles/10.3389/fendo.2021.657873/full

AimDespite the enormous efforts to understand Congenital hyperinsulinism (CHI), up to 50% of the patients are genetically unexplained. We aimed to functionally characterize a novel candidate gene in CHI.PatientA 4-month-old boy presented severe hyperinsulinemic hypoglycemia. A routine CHI genetic panel was negative.MethodsA trio-based whole-exome sequencing (WES) was performed. Gene knockout in the RIN-m cell line was established by CRISPR/Cas9. Gene expression was performed using real-time PCR.ResultsHyperinsulinemic hypoglycemia with diffuse beta-cell involvement was demonstrated in the patient, who was diazoxide-responsive. By WES, compound heterozygous variants were identified in the adenylyl cyclase 7, ADCY7 gene p.(Asp439Glu) and p.(Gly1045Arg). ADCY7 is calcium-sensitive, expressed in beta-cells and converts ATP to cAMP. The variants located in the cytoplasmic domains C1 and C2 in a highly conserved and functional amino acid region. RIN-m(-/-Adcy7) cells showed a significant increase in insulin secretion reaching 54% at low, and 49% at high glucose concentrations, compared to wild-type. In genetic expression analysis Adcy7 loss of function led to a 34.1-fold to 362.8-fold increase in mRNA levels of the insulin regulator genes Ins1 and Ins2 (p ≤ 0.0002), as well as increased glucose uptake and sensing indicated by higher mRNA levels of Scl2a2 and Gck via upregulation of Pdx1, and Foxa2 leading to the activation of the glucose stimulated-insulin secretion (GSIS) pathway.ConclusionThis study identified a novel candidate gene, ADCY7, to cause CHI via activation of the GSIS pathway.