Hypercalcemia and co-occurring TBX1 mutation in Glycogen Storage Disease Type Ib: case report

Abstract

Glycogen Storage Disease Type Ib (GSD-Ib) is a rare autosomal recessive metabolic disorder caused by mutations in SLC37A4, leading to a deficiency in glucose-6-phosphate translocase. This disorder is characterized by impaired glycogenolysis and gluconeogenesis, resulting in clinical and metabolic manifestations. We report a three-month-old Moroccan female patient presenting with doll-like facies, hepatomegaly, dysmorphic features, and developmental delays. Laboratory analysis revealed hypoglycemia, elevated triglyceride levels, hypercalcemia, and neutropenia. Genetic testing confirmed a homozygous pathogenic variant in SLC37A4 and a heterozygous variant of uncertain significance in TBX1. Initial management included a lactose-free and galactose-free diet, multivitamin supplementation, and granulocyte colony-stimulating factor (G-CSF) therapy to address neutropenia. A novel aspect of this case involves hypercalcemia as an unusual finding in GSD-Ib and the co-occurrence of a variant in the TBX1 gene, which is not typically associated with the disease but may contribute to the patient’s clinical presentation. These findings add a new dimension to our understanding of GSD-Ib and suggest potential avenues for future research to elucidate these genetic interactions and their impact on clinical outcomes.

Introduction

Glycogen Storage Disease Type I (GSD-I) is a rare autosomal recessive metabolic disorder characterized by impaired glycogenolysis and gluconeogenesis. GSD-I includes two significant subtypes: GSD-Ia (OMIM #232200), caused by a deficiency in glucose-6-phosphatase (G6Pase), and GSD-Ib (OMIM #232220), resulting from a deficiency in glucose-6-phosphate translocase (G6PT) [1]. The incidence rate of GSD I is approximately 1 in 100,000 live births, with GSD-Ib accounting for about 20% of these cases [2].

The GSD-Ib is caused by mutations in the SLC37A4 gene, which encodes the G6PT, a component of the glucose-6-phosphatase system and which consists of three types of proteins: an enzyme G6Pase, a G6PT, and the catalytic subunit 3 of glucose-6-phosphatase (G6PC3). This protein complex, associated with the endoplasmic reticulum, is responsible for the dephosphorylation of glucose-6-phosphate to glucose, a key step in glucose catabolism, thereby facilitating the utilization of glucose stored in the liver as glycogen. This phosphatase is also involved in the dephosphorylation of another sugar, 1,5-anhydroglucitol, which is present in small amounts in the diet. Indeed, it has been shown that G6PT and G6PC3 collaborate to degrade 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), which would otherwise accumulate in the neutrophils of these patients, causing toxicity due to its potent inhibition of the glucose-phosphorylating enzyme hexokinase. When G6PT (in patients with GSD-Ib) is deficient, 1,5-AG6P accumulates in the neutrophils of these patients. The cytosolic accumulation of 1,5-AG6P inhibits glycolysis, which is the sole energy source for mature neutrophils. This mechanism explains the dysfunction and apoptosis of neutrophils in GSD-Ib [3]. So, the mutation leads to defective G6PT, which not only affects the metabolic pathways within neutrophils, causing chronic neutropenia and increased susceptibility to infections, but also contributes to systemic inflammation and gut-related immune dysfunctions, thereby potentially leading to IBD. The presence of IBD in GSD-Ib is thus considered a downstream consequence of these interconnected metabolic and immunological disruptions rather than a separate, isolated result of a single mutation.

This deficiency leads to glycogen accumulation and other metabolic intermediates in the liver, kidneys, and intestines, resulting in a broad spectrum of clinical manifestations. Key symptoms include a ‘Doll-like’ facies, hypoglycemia, Hyperuricemia, Hyperlipidemia, hepatomegaly, short stature, neutropenia, and neutrophil dysfunction, contributing to increased susceptibility to bacterial infections and IBD [1].

Diagnosing GSD-Ib typically involves genetic testing to identify mutations in the SLC37A4 gene. Differentiating between GSD Ia and Ib can be challenging due to overlapping clinical features, but genetic analysis provides a definitive diagnosis. Current treatments focus on managing hypoglycemia and neutropenia, with granulocyte colony-stimulating factor (G-CSF) being a mainstay. Recent advancements include using sodium-glucose co-transporter 2 (SGLT2) inhibitors, such as empagliflozin, which have shown promise in improving neutrophil function and reducing infection rates [4,5,6].

While diagnosing our patient with GSD-Ib, we identified not only the known pathogenic variant in the SLC37A4 gene responsible for the syndrome but also a variant of uncertain significance (VUS) in the TBX1 gene. The TBX1 gene, located on chromosome 22q11.21, encodes a member of the T-box family of transcription factors involved in developing various tissues and organs. Mutations in TBX1 are typically associated with DiGeorge syndrome and other congenital anomalies. Although TBX1 is not traditionally associated with GSD-Ib, its role in the development of various tissues, including those involved in metabolic regulation, suggests potential indirect effects of the disease. The co-occurrence of variants in SLC37A4 and TBX1 in a single patient presents a unique opportunity to explore the possible interaction between these genetic alterations and their possible combined impact on the clinical presentation of GSD-Ib. These findings contribute to a deeper understanding of GSD-Ib, highlighting potential areas for further investigation that could inform future therapeutic approaches.

In this case report, we present a unique and intriguing case of a three-month-old infant girl with a rare combination of clinical manifestations. Her presentation, including hepatomegaly, dysmorphic features, developmental delay, severe neutropenia, and hypoglycemia, led to the confirmation of GSD-Ib through molecular diagnosis. We aim to highlight the novelty of her case and discuss the co-occurrence of two variants on SLC37A4 and TBX1, which adds a new dimension to our understanding of GSD-Ib.

Clinical presentation

The patient is a three-month-old Moroccan female referred to our pediatric unit for further management due to a suspected Inborn Error of Immunity (IEI). She was born to a first-degree consanguineous marriage at full term via cesarean section following an uneventful pregnancy. She is the youngest of three siblings. Notably, there is a history of a brother who died at four days old due to a congenital heart disease and a family history of diabetes on both maternal and paternal sides. The patient had been hospitalized twice before in a clinic, the first one at the age of one month for bacteremia and the second at three months for sepsis. Unfortunately, we couldn’t access the documentation concerning these two hospitalizations (Fig. 1).

Upon physical examination, the patient presented with a doll-like face, round cheeks, a domed forehead, an enlarged anterior fontanelle, a flattened nasal bridge, and growth retardation (weight and height below − 2 SD) (Fig. 2). There was a noted delay in psychomotor development, with the patient not reaching the expected milestones for her age. The pleuropulmonary examination revealed polypnea and supraclavicular retractions, although no rales were present. The patient was conscious, weighing 4.5 Kg, with a temperature of 37 °C, heart rate of 120 beats per minute, respiratory rate of 40 breaths per minute, and oxygen saturation of 98% on room air. Systemic examination showed good muscle tone, a slightly bulging fontanelle, and no sensory-motor deficits on neurological assessment. Pleuropulmonary examination confirmed polypnea with supraclavicular retractions and no rales. The abdominal examination revealed a distended abdomen with hepatomegaly (9 cm from the costal margin) and splenomegaly (2 cm below the costal margin). No lymphadenopathy was noted in the lymph node examination. Cardiovascular examination revealed well-perceived heart sounds (B1 and B2) with no murmurs. Ear, Nose, and Throat (ENT) examination showed a clean throat, no otitis, and the presence of oral thrush. The mucocutaneous examination noted diaper rash and desquamative erythroderma on the face. Glucose measurement revealed hypoglycemia at 0.3 mmol/L with a urine dipstick showing one ketone cross.

Fig. 1

The patient’s family tree. Circle: female; Square: male; Black filled square: deceased brother; Red half-filled square: relatives suffering from diabetes

Fig. 2

The patient presenting a doll-like face and domed forehead

Investigations and diagnosis

Blood chemistry showed hypoglycemia, hypercalcemia, hypertriglyceridemia, normal cholesterol levels, hepatic cytolysis with AST at 128 U/L, ALT at 59 U/L, normal albumin levels at 48 g/L, and average coagulation profile. There was rpre-prandial hyperlactatemia at 67 mg/dL and postprandial hyperlactatemia at 83 mg/dL, normal uric acid at 38 mg/L, and normal renal function with urea at 0.13 g/L and low creatinine level at 3.7 mg/L. The urinary analysis showed a positive albumin/creatinine ratio at 30 mg/mmol, and urinary electrolyte balance indicated calcinuria > 240 mg/L. Blood count revealed anemia, neutropenia, a C-reactive protein (CRP) of 8.7 mg/l, HbA1c at 4.2%, and vitamin D deficiency at 22 ng/mL. Ferritin levels were normal at 83.3 ng/mL (Table 1). The immunological investigations (HIV testing and assessment of immunoglobulin and lymphocyte subtypes) were all within normal ranges. An indicative renal involvement was pointed with 24-hour proteinuria of 29 mg/l and an albumin/creatinine ratio of 30 mg/ml. The abdominal ultrasound showed hepatomegaly with diffuse hepatic steatosis. Fluorescence in situ hybridization (FISH) did not show any microdeletion.

Since her third and last hospitalization in our pediatric infectious unit, the patient underwent a series of assessments and follow-ups at the Hepato-Gastro-Enterology pediatric department. A platelet function using the platelet function aggregation test (PFA) was performed and found to be normal. Additionally, fecal calprotectin was tested due to a history of chronic diarrhea, and the result was negative. An echocardiogram was conducted and showed normal findings. Vitamin levels were measured, revealing average values. On her last check-up, she presented with a skin abscess on the anterior surface of the thigh.

Table 1 Patient’s biological findings at admission

Genetic result

Our patient’s genetic basis of GSD-Ib was confirmed through comprehensive genetic testing. The analysis identified a homozygous missense variant in the SLC37A4 gene; NM_001164277.1:c.446G > A (p.Gly149Glu). This variant causes a missense change involving the alteration of a conserved nucleotide, substituting glycine for glutamine at position 149 of the G6PT protein encoded by the SLC37A4 gene. The variant allele was identified at a frequency of 0.00000892 in 1,457,168 control chromosomes in the gnomAD database, with no observed homozygous cases. The variant is reported in ClinVar as Pathogenic.

We also identified a heterozygous missense mutation in the TBX1 gene, specifically NM_080647.1:c.517G > A (p.Ala173Thr). This variant results in the substitution of alanine to threonine at position 173 of the TBX1 protein. The variant allele was found at a frequency of 0.00000992 in 1,612,698 control chromosomes in the GnomAD database, with no homozygous occurrence. The in-silico tool predicts a pathogenic outcome for this variant (Table 2). The variant has been reported in ClinVar as VUS.

Table 2 Results of predictive tools for TBX1 variant

Management and outcome

The initial management of the patient involved the immediate placement on a lactose-free and galactose-free diet, supplemented with multivitamins and vitamin D. She was administered desmopressin spray (an analog of the antidiuretic hormone vasopressin) alongside tranexamic acid to manage a potential bleeding disorder. Desmopressin is commonly used to increase plasma levels of von Willebrand factor and factor VIII, which helps improve clotting function and reduce bleeding episodes. On the other hand, Tranexamic acid is an antifibrinolytic agent that inhibits the breakdown of blood clots, thereby enhancing clot stability. There is a hemorrhagic tendency in GSD-Ib, the clinical manifestations of which are relatively minor, marked by a significant frequency of epistaxis, especially in females [7]. No spontaneous hemorrhagic complications were observed in the patient. However, this hemorrhagic tendency can complicate minor surgical procedures. At baseline, the usual coagulation parameters were not altered. A coagulation evaluation should be performed if invasive dental care, a biopsy, or a surgical procedure is needed. She also received antibiotic prophylaxis (cotrimoxazole) for neutropenia, along with an angiotensin-converting enzyme inhibitor for renal impairment at a dose of 0.2 mg/kg/day. However, because of the persistence of neutropenia (0.55 × 10³/µl) on her last CBC, the patient will be placed under G-CSF therapy.

Regular blood gas analysis was conducted to prevent metabolic acidosis, and continuous monitoring of liver function and glucose levels was maintained. The patient was scheduled for monthly follow-up visits to monitor growth, nutritional status, and metabolic control. Family counseling on dietary regimens and infection prevention strategies was provided.

Discussion

GSD-Ib is a rare autosomal recessive metabolic disorder characterized by the impaired ability to break down glycogen into glucose, an impairment due to a deficiency in the glucose-6-phosphatase system, specifically in the G6PT encoded by the SLC37A4 gene. This disruption in glucose-6-phosphate transport impedes average glucose production from glycogenolysis and gluconeogenesis, contributing to hypoglycemia and glycogen accumulation in various tissues, particularly the liver and kidneys. Additionally, variants in this gene are associated with neutropenia and increased susceptibility to infections, as G6PT is also involved in the proper functioning of neutrophils [8]. Besides the classic symptoms, our case report revealed two notable findings: hypercalcemia and genetic variants in both the SLC37A4 and TBX1 genes, which add a new dimension to our understanding of GSD-Ib.

The clinical presentation of GSD-Ib can vary widely. Still, most patients exhibit Metabolic manifestations, including a doll-like face with excess adipose tissue in the cheeks, hypoglycemia, hyperlipidemia, lactic acidemia, hepatomegaly, and nephromegaly. Moreover, these patients exhibit neutropenia and impaired neutrophil function, leading to recurrent bacterial infections, IBD, and aphthous stomatitis. In patients with GSD-Ib, the diagnosis of IBD often relies on a thorough clinical evaluation, as demonstrated in this case. Despite normal fecal calprotectin levels, which may occur due to intermittent or variable manifestations of intestinal inflammation in GSD-Ib, the patient’s symptoms, including chronic diarrhea, recurrent abdominal pain, and signs of systemic inflammation, were pivotal in supporting an IBD diagnosis. This highlights the complexity of diagnosing IBD in the context of metabolic disorders, where conventional biomarkers may not always align with clinical symptoms. In pediatric patients, where invasive procedures like endoscopy may pose challenges, a comprehensive clinical assessment becomes essential, integrating laboratory data and a careful understanding of the underlying metabolic pathology to guide diagnosis and management. The elevated prevalence of consanguineous marriages in Morocco suggests a high incidence of autosomal recessive disorders. Alongside descending from a consanguineous marriage, our patient showed all typical signs of GSD-Ib. However, we observed dysmorphic features, development delay, and elevated calcium levels in three samples. Hypercalcemia is not commonly associated with GSD-Ib, making this an unusual finding in our patient. In this context, the mechanisms leading to hypercalcemia are poorly understood and warrant further investigation. Potential explanations could include secondary hyperparathyroidism, which is often a response to chronic kidney disease – a known complication of GSD-Ib due to glycogen accumulation in the kidneys [9]. Another plausible cause could be the dysregulation of calcium homeostasis linked to altered vitamin D metabolism or impaired renal function, which are consequences of metabolic derangements seen in GSD-Ib patients [10]. In a recent Moroccan case report [11], they found hypercalcemia in a 2-month-old girl with GSD-I. They highlighted an important finding in a cross-sectional study involving 23 Turkish pediatric subjects with GSD-I, which identified hypercalcemia in 78.3% of the cases, primarily during episodes of acute metabolic decompensation [12]. They linked the condition to the possible association with prolonged lactic acidosis or pseudo-hypercalcemia due to hyperlipidemia, as laboratory tests can occasionally indicate falsely elevated calcium levels in hyperlipidemic serum samples. This phenomenon is observed in GSDI patients with inadequate metabolic control [13]. It is also worth considering that hypercalcemia in our patient might result from a yet unidentified pathogenic mechanism directly linked to the metabolic dysfunctions in GSD-Ib. This could involve aberrant signaling pathways or regulatory mechanisms not previously associated with GSD-Ib. Further studies are necessary to elucidate these potential pathways and to determine whether hypercalcemia could serve as a novel clinical marker for a subset of GSD-Ib patients.

The G6PT protein encoded by the SLC37A4 gene is integral to glucose homeostasis. It plays a crucial role in the endoplasmic reticulum, where it transports glucose-6-phosphate from the cytoplasm into the lumen of the endoplasmic reticulum. This process is essential for the final steps of gluconeogenesis and glycogenolysis, pathways vital for maintaining blood glucose levels. In our patient, we identified a missense variant in the SLC37A4 gene. This variant leads to a glycine-to-serine substitution at position 149 (p.Gly149Glu) in the G6PT protein. Glycine at this position is highly conserved and crucial for the proper function of the G6PT protein. Functional studies on this variant have already been demonstrated [14]. Since GSD-Ib is a relatively rare condition, only a few cases have been reported in Morocco. As far as we know, only 3 cases with a molecular diagnosis have been reported [15], all showing the same mutation (c.1042_1043delCT), making our patient the fourth Moroccan case of GSD-Ib with a genetic diagnosis. However, a TBX1 gene variant is particularly intriguing, as it is not typically associated with GSD-Ib. The TBX1 gene is a member of the T-box family of transcription factors, which play critical roles in developing the pharyngeal arches and other structures during embryogenesis. Mutations in TBX1 are more commonly associated with DiGeorge syndrome, which involves a spectrum of congenital anomalies, including cardiac defects, palatal abnormalities, and thymic hypoplasia [16]. Recent research has explored its potential involvement in adipose tissue function, energy homeostasis, and glucose metabolism. Indeed, Markan et al. demonstrated that TBX1 expression in adipose tissue is necessary for maintaining glucose homeostasis and proper insulin signaling in subcutaneous adipose tissue. At the same time, loss of TBX1 in adipose tissue impairs glucose homeostasis, indicating its crucial role in metabolic regulation [17]. The variant found in our patient is classified as VUS and computational scores unanimously predict a pathogenic effect. The Combined Annotation Dependent Depletion (CADD) score, which has a high sensitivity to predict molecular pathogenicity of variants, was 30. It is a widely utilized tool for assessing the pathogenicity of genetic variants. It integrates a range of annotations, including evolutionary conservation metrics and functional impact predictions, to provide a comprehensive assessment of a variant’s likelihood of being deleterious. A high CADD score indicates that a variant is more likely to have a detrimental effect on gene function or to contribute to disease. Due to its sensitivity and broad applicability, the CADD score is an invaluable resource for prioritizing variants in both clinical and research settings [18]. Since its higher values indicate a more severe impact, we had to consider this variant. We believe that the co-occurrence of the TBX1 variant in a GSD-Ib patient may suggest a possible interaction or shared pathway that could influence the clinical presentation of the disease. TBX1 could potentially impact the regulation of genes involved in metabolic pathways or the development of tissues affected by glycogen accumulation. The TBX1 variant might also contribute to an overlapping phenotype that includes GSD-Ib and DiGeorge syndrome features. This overlap might manifest as atypical clinical symptoms, such as the observed dysmorphic features, developmental delay, and family history of a deceased sibling with congenital heart disease.

Conclusion

In conclusion, despite the valuable information obtained, there are some limitations, including the absence of familial genetic testing and the functional exploration for the TBX1 variant. However, our case report expands the clinical spectrum of GSD-Ib by identifying hypercalcemia and mutations in the SLC37A4 and TBX1 genes. These findings underscore the complexity of genetic interactions in metabolic disorders and emphasize the importance of a holistic approach to diagnosis and management. As we unravel the genetic underpinnings of GSD-Ib, it is crucial to integrate genetic, biochemical, and clinical data to improve patient outcomes and develop targeted therapies.