The first pediatric case of glucagon receptor defect due to biallelic mutations in GCGR is identified by newborn screening of elevated arginine

Abstract

Glucagon receptor (GCGR) defect (Mahvash disease) is an autosomal recessive hereditary pancreatic neuroendocrine tumor (PNET) syndrome that has only been reported in adults with pancreatic α cell hyperplasia and PNETs. We describe a 7-year-old girl with persistent hyperaminoacidemia, notable for elevations of glutamine (normal ammonia), alanine (normal lactate), dibasic amino acids (arginine, lysine and ornithine), threonine and serine. She initially was brought to medical attention by an elevated arginine on newborn screening (NBS) and treated for presumed arginase deficiency with a low protein diet, essential amino acids formula and an ammonia scavenger drug. This treatment normalized plasma amino acids. She had intermittent emesis and anorexia, but was intellectually normal. Arginase enzyme assay and ARG1 sequencing and deletion/duplication analysis were normal. Treatments were stopped, but similar pattern of hyperaminoacidemia recurred. She also had hypercholesterolemia type IIa, with only elevated LDL cholesterol, despite an extremely lean body habitus. Exome sequencing was initially non-diagnostic. Through a literature search, we recognized the pattern of hyperaminoacidemia was strikingly similar to that reported in the Gcgr ^-/- knockout mice. Subsequently the patient was found to have an extremely elevated plasma glucagon and a novel, homozygous c.958_960del (p.Phe320del) variant in GCGR. Functional studies confirmed the pathogenicity of this variant. This case expands the clinical phenotype of GCGR defect in children and emphasizes the clinical utility of plasma amino acids in screening, diagnosis and monitoring glucagon signaling interruption. Early identification of a GCGR defect may provide an opportunity for potential beneficial treatment for an adult onset tumor predisposition disease.

Keywords: GCGR mutation; Glucagon receptor; Hyperaminoacidemia; Mahvash disease; Newborn screening; Pancreatic neuroendocrine tumor (PNET); Pancreatic α cell hyperplasia (ACH).

Fig. 1

The hepatic α cell axis feedback loop underlies the pathogenesis of glucagon receptor defect in Gcgr^−/− mice. Interrupted glucagon signaling secondary to glucagon receptor defect in hepatocytes leads to decreased hepatic amino acids uptake and catabolism, and increased plasma amino acids. The hyperaminoacidemia, especially glutamine (*), activates pancreatic islet α cell proliferation partially through mTOR dependent mechanisms. This activation results in pancreatic α cell hyperplasia (ACH) with or without pancreatic neuroendocrine tumors (PNETs), and increases glucagon production (hyperglucagonemia). mTOR: mechanistic target of rapamycin. Adapted from [5,6].

Fig. 2

Height, weight and BMI growth curves. After discontinuation of treatment (Rx–low protein diet supplemented with Cyclinex-2 and ammonia scavenger), marked by big blue arrow, there has been almost no weight increase, accompanied by decreased growth velocity and BMI. Small red arrow indicates BMI 11. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Controlled fasting data. A. The patient demonstrated 21.5 h fasting tolerance until glucose <50 mg/dL.; glucose level was unresponsive to glucagon stimulation test; B. Appropriate ketone production, lactate and hormone levels in response to fasting glucose level (at 21.5 h.).

Fig. 4

Functional studies of GCGR p.Phe320del. A. Schematic representation of constructs. Fused: fusion between glucagon peptide hormone (GCG, red curve) to full length glucagon receptor (GCGR), serves as a self-activated GCGR construction. FL: full length glucagon receptor, ECD: extracellular domain, TMD: transmembrane domain, N: N terminus, C: c-terminus. B. Activation of GCGR measured by cAMP signals, which are relative luciferase (RLU) activities of the different constructs in the absence (0 nM) or presence (500 nM) of exogenous glucagon peptide hormone (GCG). Note the virtual absence of cAMP signal of the mutant GCGR p.Phe320del. C. GCG dose response curves of WT and p.Phe320del mutant FL human GCGR, and WT and p.Phe320del mutant fused human GCGR constructs, the EC50 value for GCG is 2.041 nM(log [2.041] = 0.3), for both FL and fused p.Phe320del, there is very low activation up to 1x10⁵nM (log [1 × 10⁵] = 5) GCG. n = 3, error bars = S.D. D. Localization of sfGFP-GCGR wild type (WT) and GCGR mutant p.Phe320del in HEK293 suspension cells. WT and p.Phe320del GCGR are both expressed on the cell surface. E. Expression levels of GCGR WT and GCGR p.Phe320del protein from lysate and precipitate, determined by immunoblotting using anti-FLAG antibody. GCGR p.Phe320del protein was only detected in precipitate with a considerable degradation, which may suggest its instability. F. Computer modeling of wild type (F320, left) and p.Phe320 deletion (right) GCGR and the transmembrane domain 5 (TMD5) regions are shown. Residues with conformational change were labeled and shown as sticks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Human GCGR mutation spectrum. All solid arrow indicates homozygous variant in each reported case. The two linked dot arrows indicate compound heterozygous variants in the same patient. Yellow arrow: the variant affects GCGR protein intracellular trafficking and abnormal GCGR localization to endoplasmic reticulum rather than the cell membrane; Green arrow: the variant is either nonsense or frameshift mutation predicted to produce no protein or truncated protein; Purple arrow: the splice acceptor site variant leads to altered splicing and introduces a premature stop codon; Blue arrow: The two homozygous variants were identified in the same patient. Both variants are predicted to be damaging but no functional studies were done. Homozygotes for either allele are found in gnomAD (see below); Red arrow: variant identified in our case that does not affect GCGR protein production nor membrane localization. The nomenclature is based on reference sequence NM_000160.4. ^a. nucleotide change (c. nomenclature) was not provided in published report and, therefore, has been inferred from population data; ^b. 1 allele in gnomAD; ^c. 4 alleles in gnomAD; ^d. DNA change cannot be inferred from published paper; ^e. 41 alleles in gnomAD with 2 homozygotes (minor allele frequency 0.175 in South Asians); ^f. 10 alleles in gnomAD with 1 homozygote (highest minor allele frequency 0.026).