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

Front Endocrinol (Lausanne). 2021 Jun 11:12:657873. doi: 10.3389/fendo.2021.657873. eCollection 2021.

Abstract

Aim: Despite 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.

Patient: A 4-month-old boy presented severe hyperinsulinemic hypoglycemia. A routine CHI genetic panel was negative.

Methods: A 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.

Results: Hyperinsulinemic 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.

Conclusion: This study identified a novel candidate gene, ADCY7, to cause CHI via activation of the GSIS pathway.

Keywords: adenylyl cyclase; congenital hyperinsulinism; genetics; hyperinsulinemic hypoglycemia; metabolomics; pediatrics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenylyl Cyclases / deficiency
  • Adenylyl Cyclases / genetics*
  • Amino Acid Sequence
  • Animals
  • CRISPR-Cas Systems
  • Cell Line
  • Child, Preschool
  • Congenital Hyperinsulinism / enzymology*
  • Congenital Hyperinsulinism / genetics
  • Congenital Hyperinsulinism / metabolism*
  • Gene Knockout Techniques
  • Glucose / metabolism
  • Hepatocyte Nuclear Factor 3-beta / genetics
  • Hepatocyte Nuclear Factor 3-beta / metabolism
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Humans
  • Insulin / genetics
  • Insulin / metabolism*
  • Insulin Secretion
  • Insulin-Secreting Cells / metabolism*
  • Male
  • Rats
  • Sequence Alignment
  • Trans-Activators / genetics
  • Trans-Activators / metabolism

Substances

  • Foxa2 protein, rat
  • Homeodomain Proteins
  • Insulin
  • Trans-Activators
  • pancreatic and duodenal homeobox 1 protein
  • Hepatocyte Nuclear Factor 3-beta
  • Adenylyl Cyclases
  • adenylyl cyclase 7
  • Glucose