Table_1_CRISPR/Cas9 ADCY7 Knockout Stimulates the Insulin Secretion Pathway Leading to Excessive Insulin Secretion.xlsx

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.

尽管在理解先天性高胰岛素血症（CHI）方面投入了巨大的努力，但高达50%的患者其基因遗传原因尚不明确。本研究旨在对CHI中一种新型候选基因进行功能表征。患者A为4个月大的男婴，表现为严重的胰岛素性低血糖症。常规的CHI基因检测面板结果为阴性。研究方法包括基于家系的三代全外显子测序（WES），以及通过CRISPR/Cas9技术在RIN-m细胞系中建立基因敲除。基因表达分析采用实时PCR技术。研究结果证实，患者在弥漫性β细胞受累的情况下表现出胰岛素性低血糖症，对地塞米松有反应。全外显子测序发现，腺苷酸环化酶7（ADCY7）基因存在复合杂合突变，分别为p.(Asp439Glu)和p.(Gly1045Arg)。ADCY7是一种钙敏感蛋白，在β细胞中表达，并能将ATP转化为cAMP。这些突变位于高度保守且功能性的胞浆域C1和C2区域。与野生型相比，RIN-m(-/-Adcy7)细胞在低糖和高糖浓度下胰岛素分泌显著增加，分别达到54%和49%。在遗传表达分析中，Adcy7功能丧失导致胰岛素调节基因Ins1和Ins2的mRNA水平增加34.1至362.8倍（p ≤ 0.0002），以及通过Pdx1的上调和Foxa2的激活，导致Scl2a2和Gck的mRNA水平升高，从而指示葡萄糖摄取和感知增加，最终激活了葡萄糖刺激胰岛素分泌（GSIS）通路。结论：本研究鉴定出一种新型候选基因ADCY7，通过激活GSIS通路导致CHI。