The elective controlled fasting study in our patient

The elective controlled fasting study in our patient provides further metabolic profiling in patients with GCGR defect. Despite her lean body mass with low weight, at the age of 7 years old she demonstrated a good tolerance for fasting with hypoglycemia <50 mg/dL occurring at 21.5 h fasting (Fig. 3). During fasting, ketone production, lactic Tasquinimod level, insulin, and counter-regulatory hormones levels, such as growth hormone and cortisol, were all appropriate. Not surprisingly, she had no response to synthetic exogenous glucagon injection. The normal lactate at the time of hypoglycemia suggested unimpaired gluconeogenesis. We reviewed the spectrum of GCGR mutations in adult cases as well as our case of GCGR defect and demonstrated it in Fig.5 [2,4,7,14]. Notably, all adult patients, except one, carry mutations that lead to a complete loss of GCGR function. The nonsense, frameshift or splice acceptor site mutations (purple and green arrows, Fig.5) introduce premature stop codons and produce no protein or truncated protein. Two missense mutations (yellow arrows, Fig.5), c.256C > T (p.P86S) and c.187G > A (p.D63N), affect GCGR protein intracellular trafficking and abnormal localization to the endoplasmic reticulum rather than the cell membrane. So all these adult GCGR mutations cause complete loss of GCGR function. The patient reported by Sipos et al. carries two homozygous (blue arrows, Fig. 5) variants in GCGR, c.674G > A (p.R225H) and c.1102G > A (p.V368 M) [14]. The significance of either variant is uncertain. First, the glucagon level in this patient is unknown and the diagnosis of Mahvash disease is uncertain. Secondly, these two missense variants are predicted to be damaging by in silico analysis. No functional studies were conducted. Lastly, individuals homozygous for either variant are seen in gnomAD. There are 41 GCGR c.674G > A (p.R225H) alleles in gnomAD with 2 homozygotes (minor allele frequency 0.175 in South Asians) and 10 alleles of c.1102G > A (p.V368 M) in gnomAD with 1 homozygote (highest minor allele frequency 0.026). Based on the above information, the clinical significance of both variants is unknown underscoring the importance of functional studies as we have done. The novel variant identified in our patient leads to a single amino acid deletion that does not affect GCGR production nor cell membrane localization (see Fig.4). The conformational change in TMD5 predicted by computer modeling may result in intracellular altered G protein binding and signaling. Although we demonstrated the loss of function mediated by attenuated cAMP signaling but this does not rule out other cAMP-independent intracellular pathways are still able to be activated through this mutant GCGR, especially exposed to supraphysiological glucagon level. Notably, our patient also had hypercholesterolemia. Her lipid profile of high LDL-C, normal HDL-C and triglyceride is consistent with type IIa hypercholesterolemia. Her exome analysis did not reveal pathogenic variants in any of the known genes associated with hypercholesterolemia, including LDLR [MIM: 606945], APOB [MIM: 107730], and PCSK9 [MIM: 607786]. Both Gcgr and liver specific siRNA-mediated Gcgr inhibition in mice showed elevations of cholesterol, especially LDL-C. Similar LDL-C elevations were observed in patients with type 2 diabetes who were treated with a GCGR antagonist [[15], [16], [17]]. The elevated LDL-C in our patient is likely secondary to the GCGR defect, but there may also be an aspect of familial hyperlipidemia as shown in Table 2.
Conclusion
Financial disclosure
Conflict of interest This manuscript has not been published. It will not be submitted elsewhere while under consideration and, should it be published in Molecular Genetics and Metabolism, it will not be published elsewhere – either in similar form or verbatim – without permission of the editors.
Acknowledgements
Functional studies were supported by grants the Youth Innovation Promotion Association and Natural Science Foundation of Shanghai, China (18ZR1447800 to L.H.Z.), National Institutes of Health, United State (DK071662 to H.E.X.). It was also partially supported by the Fudan-SIMM Joint Research Fund, China (FU-SIMM-20174003) and the Support of SA-SIBS Scholarship Program.