Neonatal diabetes mellitus (NDM) diagnosed before 6 months of age is a rare monogenic condition occurring in 1:100,000 live births in Europe (1, 2), and 1:30,000 (3-fold higher) in the Middle East (3, 4). The incidence of permanent NDM in the Middle East is even higher (10-fold) (1, 4). NDM accounts for 1:4000 cases of diabetes in children (5, 6). It may be either isolated or associated with multiorgan involvement. Genetic mutations causing NDM result in impaired insulin function due to one of the following mechanisms: abnormal pancreatic development, abnormal β-cell function or β-cell destruction. Mutations in over 25 genes have been reported to cause NDM (7–9), with syndromic NDM accounting for over 10% of the cases (9).

GLIS3 encodes the zinc finger protein GLI-similar protein 3 (GLIS3), a transcription factor expressed in the developing pancreas and involved in the development of the thyroid, eyes, liver and kidneys (10–14). GLIS3 can function as a repressor or activator, playing critical roles in the regulation of various cellular processes (15, 16). In the pancreas, GLIS3 is expressed at various stages of ductal and endocrine cell development, regulating β-cell development and insulin expression (12). Mutations in GLIS3 (OMIM 610192) were described in 2006 as the cause of a rare syndrome characterized by NDM, congenital hypothyroidism (CH) and congenital glaucoma (CG) (17, 18). Other features included intrauterine growth retardation (IUGR), cystic renal disease and hepatic cholestasis or fibrosis. In addition, developmental delay, facial dysmorphism, skeletal abnormalities and exocrine pancreatic dysfunction have been described (4, 17–22). To date, 14 GLIS3 mutations have been reported (http://www.hgmd.cf.ac.uk/ac/gene.php) (4, 17, 20–22).

Herein, we report on two non-identical twins presenting with NDM, CG and CH caused by a novel homozygous GLIS3 mutation. This case report expands our knowledge of the clinical phenotype, management approaches and outcome of infants with GLIS3 mutations, indicating the need for additional research to further our understanding of the roles of GLIS3.

We describe non-identical twins who are homozygous for a novel GLIS3 mutation, presenting with NDM, CH, CG, kidney cysts and developmental delay. The management of their diabetes was challenging due to recurrent hypoglycemic episodes.

Since the first description in 2006 of GLIS3 mutations causing a syndromic form of NDM in three siblings of a consanguineous family from Saudi Arabia (17, 18), an additional 16 patients have been reported carrying 14 different mutations ( Table 2 ) (17, 19–23). All patients had NDM presenting between 1 and 31 days of life.

The molecular etiologies of NDM can be divided into three broad groups according to the defect that they cause in pancreatic function: abnormal β-cell function (KCNJ11, ABCC8, INS, GCK, SLC2A2, SLC19A2), β-cell destruction (dominant INS, EIF2AK3, IER3IPI, FOXP3, WFS1, EIF2B1, LRBA) and abnormal pancreatic development (PDX1, PTF1A, HNF1B, RFX6, GATA4, GATA6, NEUROG3, NEUROD1, NKX2-2, MNX1, RFX6, CNOT1) (6). The GLIS3 gene is involved in the development of pancreatic β-cells and as such, belongs to the third group. In particular, in-vivo studies using Glis3-knockout mice have shown that Glis3 plays a critical role in cell-lineage differentiation, particularly in the development of β-cells, in the regulation of mature β-cell function and survival, and in insulin regulation (15).

In most patients with GLIS3 mutations reported to date, and as was noted in our patients, a normal-size pancreas is observed. However, pancreatic hypoplasia and exocrine pancreatic insufficiency requiring pancreatic enzyme replacement and nutrient supplementation have been reported in some patients (21), but not in our cases. These findings support a possible role of GLIS3 in exocrine pancreatic development, as well as in β-cell development.

Patients with GLIS3 mutations do not typically show any evidence of autoimmune markers, in contrast to type 1 diabetes mellitus in which the autoimmune destruction of β-cells is usually associated with the presence of pancreatic autoantibodies. Interestingly, anti-GAD antibodies were elevated in case 2. Although the presence of anti-GAD antibodies has been reported in rare cases of patients with non-autoimmune NDM (23), it would be interesting to investigate whether other patients with homozygous GLIS3 mutations are positive for these antibodies. It would also be important to follow up our patient to determine whether she might revert to being GAD negative, as previously reported in some non-diabetic individuals (24). Of note, GLIS3 gene polymorphisms have been associated with risk of type 1 diabetes mellitus in genome-wide association studies (25, 26), indicating that GLIS3 is a susceptibility locus for this disease.

Both twins had intermittent episodes of hypoglycemia that resulted in insulin-pump disconnection. Moreover, in case 1, prolonged unresponsive hypoglycemia, resistant to glucagon therapy, was observed during an acute illness, requiring cessation of insulin therapy for 3 days. Other causes for hypoglycemia and adrenal insufficiency were excluded. Recurrent hypoglycemic episodes have been reported in one additional case with a GLIS3 mutation (21). The mechanism through which these patients develop hypoglycemia is currently unknown, and further studies are needed to investigate its causes. Hypoglycemic episodes in the first days of life in small for gestational age infants are attributed to low liver glycogen storage. However, in our cases, the hypoglycemic events occurred during follow-up when the probands were older and at normal weight for their age. One explanation could be that GLIS3 dysfunction directly affects the regulation of insulin secretion. Indeed, GLIS3 expression persists beyond the embryonic period, promoting β-cell proliferation and β-cell survival, and regulating insulin gene (INS) expression by binding to GLIS3-response element in the INS promoter (16, 27). GLIS3 has been shown to activate pancreatic and duodenal homeobox 1 (PDX1), as well as neurogenic differentiation 1 (NEUROD1) expression and potently controls INS transcription (27). It has been shown that mice lacking Glis3 have decreased numbers of glucagon-positive cells (28); however, the fact that no response to glucagon injections was observed in case 1 does not support low glucagon secretion as the cause for recurrent hypoglycemic episodes in these cases. Genome-wide association studies have revealed that GLIS3 variants are associated with type 2, as well as type 1, diabetes mellitus (26), supporting its involvement in insulin sensitivity, which may explain the requirement of high insulin doses under stress conditions in our patients, as well as in other reports (20).

Our twins were homozygous for a novel nonsense mutation located in exon 9 (p.Q798*), which results in a premature stop codon. It is likely that the mutated transcript undergoes nonsense-mediated decay and little or no protein is produced. Five missense mutations of GLIS3 have been reported to date; another eight mutations were intragenic deletions, and one was a duplication resulting in a frameshift ( Table 2 ). A rapid molecular diagnosis of NDM is recommended as the specific etiology assists in clinical management (insulin vs. sulfonylurea), and may also guide longitudinal monitoring for other associated problems in NDM subtypes with syndromic features, as well as for screening family members (5, 9). Indeed, in the family reported on here, prenatal genetic testing revealed heterozygosity for the same GLIS3 mutation in a later pregnancy.

Management of diabetes in our patients was challenging. The use of multiple insulin injections in infants results in labile blood glucose levels, and the use of NPH insulin commonly results in hypoglycemic episodes (5). Moreover, no insulin analogues are currently approved for use in infants (<1 year). CSII has been shown to provide the most effective management of NDM (5, 29, 30). However, insulin dilution is required to prevent pump obstruction. In contrast, the usefulness of CGM in neonates is arguable, since inaccuracy of CGM in newborns may lead to more interventions with potentially adverse effects on outcomes (31). The combination of CSII and CGM enabled the family to maintain blood glucose in a safe range and to prevent severe hypoglycemic events due to the automatic insulin-delivery shutoff system when the CGM predicts hypoglycemic values.

We report a novel GLIS3 mutation in non-identical twins, further characterizing the genotypic and phenotypic spectra in individuals with this rare genetic condition. Defining the clinical features of patients with rare forms of NDM is fundamental to providing insights into the roles of genes such as GLIS3 in the pathophysiology of diabetes mellitus.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The patients’ parents gave written informed consent for the publication of this case report.

SL, GE-A, MB, CF, MP, SW, and YT-R analyzed and interpreted the clinical and biochemical aspects of the patients’ data. EF analyzed and interpreted the molecular genetic data. SL, EF, SW, and YT-R contributed to writing the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.