AUTHOR=Alhaidan Yazeid , Christesen Henrik Thybo , Lundberg Elena , Balwi Mohammed A. Al , Brusgaard Klaus 

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

JOURNAL=Frontiers in Endocrinology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2021.657873

DOI=10.3389/fendo.2021.657873

ISSN=1664-2392

ABSTRACT=<sec><title>Aim</title><p>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.</p></sec><sec><title>Patient</title><p>A 4-month-old boy presented severe hyperinsulinemic hypoglycemia. A routine CHI genetic panel was negative.</p></sec><sec><title>Methods</title><p>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.</p></sec><sec><title>Results</title><p>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, <italic>ADCY7</italic> gene p.(Asp439Glu) and p.(Gly1045Arg). <italic>ADCY7</italic> 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<sup>(-/-</sup><italic><sup>Adcy7</sup></italic><sup>)</sup> 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 <italic>Adcy7</italic> loss of function led to a 34.1-fold to 362.8-fold increase in mRNA levels of the insulin regulator genes <italic>Ins1</italic> and <italic>Ins2</italic> (<italic>p ≤</italic> 0.0002), as well as increased glucose uptake and sensing indicated by higher mRNA levels of <italic>Scl2a2</italic> and <italic>Gck via</italic> upregulation of <italic>Pdx1</italic>, and <italic>Foxa2</italic> leading to the activation of the glucose stimulated-insulin secretion (GSIS) pathway.</p></sec><sec><title>Conclusion</title><p>This study identified a novel candidate gene, <italic>ADCY7</italic>, to cause CHI <italic>via</italic> activation of the GSIS pathway.</p></sec>