Molecular mechanisms underlying congenital hyperinsulinemia of infancy and its relevance to management – A review

Case 1

A 3.5 kg female baby born of third-degree consanguineous marriage had repeated episodes of seizures since day 1 of life. Blood glucose (BG) was documented to be <45 mg/dL at these episodes. She received a continuous infusion of dextrose-containing fluids but continued to have hypoglycemic episodes with seizures. The glucose infusion rate was increased to 12 mg/kg/min. A “critical” sample taken at the time of hypoglycemia revealed pH 7.3, bicarbonate 23 mEq/mL, blood ketones tested by ketostix 0.5 mmol/L, and no reducing substances in the urine. Administration of glucagon 0.1 mg IV caused a brisk rise of BG by 45 mg/dL. Insulin was detected at 14.4 µIU/mL, and C-peptide was 1 ng/mL. A diagnosis of hyperinsulinism was made, and diazoxide was started at a dose of 10 mg/kg/day and gradually increased to 20 mg/kg/day. However, the baby still needed a high glucose infusion rate to maintain normoglycemia. Considering diazoxide-unresponsiveness, an injection of octreotide was started as an infusion on which the BG improved, and the baby shifted to subcutaneous injections. Molecular genetic analysis by Sanger sequencing revealed a homozygous deletion of exon 7 in the ABCC8 gene. The parents were heterozygous for this variation. The parents were explained the inheritance of the condition and counseled for the chances of its reoccurrence in future conceptions. Unfortunately, the parents could not continue with a further hospital stay, and later, we learned that the baby died soon after.

Case 2

A 3.6 kg male baby, born of non-consanguineous marriage, had repeated episodes of hypoglycemia since the 1^st day of life. He presented with seizures and BG was 35 mg/dL at that time. He required high rates of infusion of glucose at more than 10 mg/kg/min to maintain normoglycemia. A critical sample showed BG 40 mg/dL, insulin 2.78 µIU/mL, cortisol 17.10 ng/mL, ketones 0.2 mmol/L, no acidosis, and brisk response to IV glucagon. With a presumptive diagnosis of hyperinsulinemia, diazoxide was initiated, but hypoglycemia did not resolve even at 20 mg/kg/day. Intravenous infusion of octreotide was started and increased to 18 µg/kg/day to maintain normoglycemia. Genetic analysis revealed a heterozygous mutation in ABCC8 with uniparental disomy. ABCC8 splicing variant c.4415–13G>A was detected on one allele, and the second ABCC8 variant was not found. The mutation was inherited from the father. A focal lesion was very likely, and a fluorine 18L-3,4 dihydroxyphenylalanine positron emission tomography (¹⁸F-DOPA PET) scan was performed. and it revealed a focal lesion at the junction of the body and tail of the pancreas [Figure 1]. A focal pancreatectomy was performed, and the hypoglycemia resolved completely.

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Figure 1:

Fluorine 18L-3,4 dihydroxyphenylalanine positron emission tomography scan images of pancreas showing increased radiotracer uptake (shown by arrows) at junction of body and tail of pancreas suggesting focal lesion. (Case and image credit: Dr Rajni Sharma, Additional Professor, AIIMS, New Delhi).

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What are the major forms of neonatal hypoglycemia?

The three major forms of neonatal hypoglycemia are as follows:

1. Transitional hypoglycemia – This affects all neonates in the first few days of life. The low glucose threshold for insulin secretion that occurs in fetal life persists into postnatal life, resulting in transitional hypoglycemia.

2. Perinatal stress-induced hypoglycemia in high-risk neonates – The perinatal stress causes an exaggeration of the low glucose threshold for insulin release, resulting in hyperinsulinemia

3. Hyperinsulinemia due to genetic defects – This refers to increased insulin release due to mutations that affect the insulin release pathway, the majority acting through inactivation of the ATP-sensitive potassium (K_ATP) channels.

It is important to recognize that hyperinsulinemia is common to all these major causes of hypoglycemia.^[1] At the same time, other significant causes of hypoglycemia, such as fatty acid oxidation defects, hypopituitarism, and glycogen storage disorder, must be ruled out.

What is the incidence of CHI, and how is it classified?

The incidence of CHI is estimated at 1:50,000 live births, though it is much higher in populations with consanguineous marriages.^[2] It can be classified as:

1. Channelopathies – These involve defects in ion channels located on the beta(β)-cell membrane, commonly affecting the K_ATP channels. Mutations affecting the voltage-gated calcium channels (VGCAs) also present with hyperinsulinemia

2. Metabolopathies – These involve defects in the metabolic pathways leading to insulin release. These include activating mutations (e.g., for glutamate dehydrogenase [GDH]) or inactivating mutations (as for hydroxyacyl coenzyme A dehydrogenase) of enzymes that affect insulin release.

What is the molecular mechanism of insulin release?

The K_ATP channel is composed of four subunits of sulfonylurea receptor 1 (SUR1) and four subunits of Kir6.2 (inward rectifier potassium channel).^[3] The Kir6.2 forms the pore of the channel, while SUR1 subunits act as regulatory subunits. The genes coding for the K_ATP channel include ABCC8, which codes for SUR1, and KCNJ11, which codes for Kir6.2, both localized to chromosome 11p15.1.^[4,5]

The secretion of insulin from the β-cells is directly affected by the BG concentrations. With elevated BG concentrations, the glucose uptake by β-cells increases, and the rate of glucose oxidation increases, resulting in an increased ATP/ADP ratio. This inhibits the SUR1 subunits, causing the closure of K_ATP channels. The cell membrane gets depolarized, and VGCA opens, causing an influx of Ca²⁺ that causes exocytosis of insulin-laden storage granules [Figure 2]. On the other hand, when glucose concentrations are low, the K_ATP channels open, allowing K⁺ to diffuse freely, the membrane potential remains hyperpolarized, and no insulin release occurs.^[6]

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Figure 2:

Diagrammatic representation of molecular mechanisms causing CHI in a pancreatic beta-cell. (The red oval represents the mitochondria). CHI: Congenital hyperinsulinemia of infancy, αKG: Alpha-ketoglutarate, ATP: Adenosine triphosphate, GCK: Glucokinase, GDH: Glutamate dehydrogenase, GLU: Glucose, HADH: Hydroxyacyl-coenzyme A dehydrogenase, HNF4α: Hepatocyte Nuclear Factor 4-alpha, INS: Insulin, KATP channel: ATP-sensitive potassium channel, MCT1: Monocarboxylate transporter 1, UCP2: Uncoupling protein 2.

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Why is there a risk of neurologic injury in hyperinsulinemia?

Insulin increases the intracellular uptake of glucose, inhibits gluconeogenesis and glycolysis, and inhibits lipolysis and ketone body production. With hypoglycemia and the absence of alternative energy sources (ketones), the brain is at high risk of hypoglycemic brain injury. However, there is no correlation between the level of serum insulin and the severity of hypoglycemia.^[7]

What are the clinical features of hyperinsulinemic hypoglycemia?

These neonates are born macrosomic (as a result of the growth-promoting effect of insulin), and symptoms start early in life, within a few days of life, or sometimes later in infancy. The symptoms include lethargy, poor feeding, apnea, hypothermia, seizures, and altered sensorium. They may have hepatomegaly and cardiomegaly as a result of fetal hyperinsulinemia and storage of glycogen.^[2,8] The requirement of high glucose infusion rates beyond 8 mg/kg/min is almost synonymous with CHI.

What is the pathologic classification of CHI?

CHI could be classified into three types on the basis of β-cell involvement:

1. Diffuse – The β-cells that hypersecrete insulin is present throughout the pancreas. This accounts for 60–70% of all cases.

2. Focal – There is nodular hyperplasia of islet-like cell clusters, including dutculoinsular complexes and giant nuclei at a localized area of the pancreas, with surrounding pancreatic tissue being normal. This accounts for 30–40% of cases

3. Atypical – This could be described as a combination of the above two patterns.

The identification of the histological type may be crucial, as the focal type responds well to focal pancreatectomy with preservation of the exocrine pancreas and the uninvolved endocrine part as well. The gold standard test is the ¹⁸F-DOPA PET scan. It has a sensitivity of 89% and a specificity of 98% in differentiating the diffuse and focal forms.^[2]

What is the genotype-phenotype correlation seen in K_ATP CHI?

CHI can be categorized into familial and non-familial forms. Monogenic forms of familial CHI are primarily attributed to genetic defects in the pathways regulating insulin secretion from pancreatic β-cells. Many genes have been known to play a significant role in familial monogenic CHI, including ABCC8, KCNJ11, GLUD1, glucokinase (GCK), hydroxyacylcoenzyme A dehydrogenase (HADH), uncoupling protein 2 (UCP2), INSR, HNF1A, HNF4A, SLC16A1, and PGM1. ADK, ALG3, AKG6, CACNAIC, CACNA 1D, CDKN1C, CREBBP, DIS3L2, EIF253, EP300, FAH, Forkhead box protein (FOXA2), GPC3, HK1, HRAS, KCNJ11, KCNQ1, KMT2D, KDM6A, and MPI.^[9,10]

Recessive inactivating mutations in ABCC8 and KCNJ11 are the most common causes of CHI and result in reduced activity or complete loss of function of the K_ATP channels. Persistent unregulated insulin release despite low BG causes severe hypoglycemia. More than 150 homozygous, compound heterozygous, and heterozygous inactivating mutations in ABCC8 and more than 24 in KCNJ11 have been reported.^[11] These cause defects in K_ATP channel synthesis, turnover, or transport from the endoplasmic reticulum and Golgi apparatus to the plasma membrane or alteration in open-state frequency. They typically cause diffuse CHI, need high glucose infusion rates, and are unresponsive to diazoxide. Dominant inactivating mutations usually present a milder phenotype and respond to diazoxide, though they may sometimes be more severe and medically unresponsive.^[6]

The focal form involves two independent events. The first is the inheritance of a paternal heterozygous mutation in ABCC8 or KCNJ11, followed by the second event, which is the bodily loss of the maternal 11p allele (11p15.1–11p15.5), with consequent loss of cell repressor genes, forming the focal lesion. This paternal uniparental disomy unmasks the paternally inherited K_ATP channel mutation, which leads to altered expression of a number of imprinted genes, including the maternally expressed tumor suppressor genes H19 and CDKN1C and the paternally expressed growth factor insulin-like growth factor 2.^[12,13] These result in focal proliferation of ß-cells, that lack the K_ATP channel, leading to focal adenomatous hyperplasia.^[14] The focal involvement is always sporadic in origin.^[6,12]

Atypical CHI is characterized by morphological mosaicism with hyperfunctional islets restricted to certain pancreatic areas, making it difficult to classify as a diffuse or localized type.^[15,16] While one of the genetic pathways for chromosome 11p15.1 with an ABCC8 gene mutation has been shown to be explained by mosaic interstitial paternal uniparental isodisomy,^[15] the genetic basis of the majority of individuals with atypical CHI is still unknown.^[16]

What are the other molecular mechanisms causing CHI?

Hepatocyte nuclear factor 4alpha (HNF4α)

This belongs to the hormone receptor superfamily, and inactivating mutations in the encoding gene may cause a reduction in the expression of K_ATP channel subunit Kir6.2 or a reduction in the levels of PPARα.^[21,22] The latter effect causes a decrease in β-oxidation of fatty acids and accumulation of malonyl coenzyme A in the cytosol, which inhibits carnitinepalmitoyltransferase 1. This increases the concentration of long-chain acyl-CoA levels, signaling insulin release.^[23] The phenotypic presentation is quite variable, ranging from transient neonatal CHI to persistent hypoglycemia, macrosomia, and often family history of maturity-onset diabetes in young. The histological involvement is diffuse and responds to diazoxide.

Calcium voltage-gated channel subunit alpha-1D (CACNA1D)

The CACNA1D gene encodes the VGCAs that are expressed in pancreatic β-cells, and the influx of calcium causes exocytosis of insulin. An activating mutation causes the channels to remain open at a lower membrane potential, leading to dysregulated insulin secretion. There are associated heart defects and severe hypotonia.^[26]

Monocarboxylate transporter 1

Encoded by the SLC16A1 gene mapped on chromosome 1p13.2-p12, it transports monocarboxylates (pyruvate and lactate) into the cells. Dominant activating mutations cause increased pyruvate uptake, increased ATP production, and, thus, increased insulin release.^[28] The condition is characterized by hypoglycemic episodes, which usually occur within 30–45 min after exercise, in response to the accumulation of pyruvate and lactate. Hence, carbohydrate intake before and during exercise is important to prevent episodes of hypoglycemia. The defect is upstream of the K_ATP pathway, and diazoxide usually works.

What are the metabolic conditions leading to CHI?

Tyrosinemia type I and CHI

CHI may be part of the symptomatology of tyrosinemia type 1, though the precise mechanism is not known. This could be related to the accumulation of toxic metabolites due to a deficiency of the enzyme fumaryl acetoacetate hydrolase. Severe liver and kidney disease are the main features, and hypoglycemia persists despite dietary treatment.^[30] Diazoxide does improve hypoglycemia.

What are the syndromes associated with CHI?

While CHI mostly presents as an isolated entity, it may be associated with certain syndromes, most commonly seen with Beckwith–Wiedemann syndrome. Typical features such as prenatal and postnatal onset macrosomia, macroglossia, organomegaly, and hemihypertrophy may be present. Early neonatal hypoglycemia is present in 50% of these cases with variable diazoxide responsiveness.^[31]

Kabuki syndrome is characterized by various dysmorphic features, such as elongated palpebral fissures with eversion of the lateral lower eyelid. Thick finger pads may be associated with CHI in <7% of cases that usually respond to diazoxide. Other syndromes with hyperinsulinemia include Turner syndrome, Sotos syndrome, and others listed in Table 1. The exact mechanism causing hypoglycemia is not well understood. They generally respond to diazoxide and resolve with time.^[19]

What is postprandial hyperinsulinemia (PPHI)?

PPHI typically occurs a few hours after meals in children who have undergone Nissen fundoplication/gastric bypass. These children have an exaggerated secretion of glucagon-like peptide 1 (GLP1), which acts as an insulin secretagogue, causing exaggerated insulin surge and hypoglycemia.^[32] PPHI has also been reported in children with insulin autoimmune syndrome. Children newly exposed to exogenous insulin develop insulin-binding autoantibodies.

What is insulinoma?

Insulinoma, or insulin-secreting tumor of the pancreas, is a rare cause of hyperinsulinism in children or adolescents.^[33] The intracellular mammalian target of the rapamycin (mTOR) pathway is involved in increased insulin secretion. Its possibility should be considered in children older than 2 years with acquired hyperinsulinemia and CT or MRI should be done. Careful family history is important as it could be part of multiple endocrine neoplasia. Surgical removal of the tumor is indicated.

What is the diagnostic approach to CHI?

Further evaluation

Once the diagnosis of CHI is reached, diazoxide is initiated as it is a K_ATP channel opener. The dose is 5–15 mg/kg/day in three divided doses. If doses of 15 mg/kg/day or more for 5 days do not resolve the hypoglycemia, it is considered diazoxide-unresponsive. Other medical therapies are initiated, and genetic testing is ordered to look for mutations typical of focal CHI, in which case radionuclide imaging with 18F-DOPA PET/CT is performed [Figure 3]. It is based on the principle of selective uptake and conversion into dopamine by the DOPA decarboxylase enzyme, which is present in β-cells. There is sustained retention of the radiotracer by hyperplastic clonally expanded β-cells as compared to the rest of the pancreas.

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Figure 3:

Diagnostic and management plan for congenital hyperinsulinemia of infancy.

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What is the approach to genetic testing in CHI?

Genetic testing is of paramount importance for making molecular diagnosis of CHI and the recent guidelines recommend it for all patients with hyperinsulinemia persisting beyond 3 months age (except those likely to have an acquired cause).^[34] However, it is typically unwarranted for clinical management in diazoxide-responsive patients, especially in resource constrained settings. The yield of genetic testing in CHI patients ranges from 45.3% to 79%, and the detection rate of diazoxide-unresponsive CHI patients is almost 90%.^[34,35] In cases where CHI is associated with a histologically diffused form, next-generation sequencing (NGS) is typically the initial test indicated, followed by Sanger validation.^[36,37]

NGS with copy number variation (CNV) analysis is increasingly being employed for the screening of small copy number variants, providing an accessible and cost-effective option. In instances where a mutation remains undetected in blood samples, and pancreatectomy has been conducted, re-evaluating the known CHI genes to identify variants exclusive to pancreatic DNA warrants consideration.

Methylation studies should be considered for cases exhibiting histologically focal variants or syndromic variants like Beckwith-Wiedemann syndrome. Karyotyping remains the gold standard genetic test for conditions such as CHI associated with monosomy X, trisomy 13, and other chromosomal disorders suspected in certain cases.

Multiplex-ligation-dependent probe amplification (MLPA) has limited utility in the identification of deletions and duplications in individual genes, for example, ABCC8, KCNJ11, GLUD1, GCK, HADH, UCP2, INSR, HNF1A, HNF4A, and SLC16A1. MLPA has enormous significance in detecting mosaicism, particularly in conditions where low-level somatic mosaicism plays a role in disease manifestation or progression.

Microarray-based comparative genomic hybridization (array CGH) is very useful for the identification of large deletions, duplications, and aneuploidies; in contrast to MLPA, array CGH lacks the capability to detect low-level mosaicism, particularly mosaicism below 30% for deletions and duplications, and below 10% for aneuploidies. Nevertheless, this method permits the examination of CNV across the whole genome.

Beyond offering insights into coding and non-coding regions, genome sequencing enables the detection of structural changes in copy number variants, including large deletions, duplications, aneuploidies, and mosaic variants. Nonetheless, the sensitivity of this approach for detecting low-level mosaic variants is diminished compared to targeted NGS, attributed to the lower read depth achieved. Long-read sequencing holds promise for concurrently detecting sequence variation and DNA methylation status, although their clinical utilization remains limited. It has primarily been employed for genes challenging to sequence using conventional methodologies.

Optical genome mapping, as a next-generation cytogenetic technique, holds incredible potential. However, its widespread clinical use requires further validation.

Can genetic testing guide the dietary management?

GLUD1 gene-associated hyperinsulinism-hyperammonemia syndrome may benefit from a protein-restricted diet to manage both hypoglycemia and hyperammonemia. Similarly, mutations in the HADH gene, which cause hyperinsulinism due to short-chain L-3-hydroxyacyl-CoA dehydrogenase deficiency, may respond to a diet high in carbohydrates and low in fat, as well as frequent feeding to prevent hypoglycemia.^[38]

In cases where genetic testing reveals mutations that are associated with diazoxide-unresponsive CHI, such as those in the ABCC8 or KCNJ11 genes, dietary management in conjunction with medical therapy or surgical intervention is of immense importance. Somatic mutations within the GCK gene represent a rare yet significant etiology of diazoxide-unresponsive atypical CHI.^[39]

What is the role of genetic counseling?

It provides families with information about the nature of the disorder, the implications for the affected individual, and the risks for other family members. This is essential for making informed decisions about family planning and management options. If a child has a recessive form of CHI, there is a 25% chance of recurrence in each subsequent pregnancy if both parents are carriers. In cases of dominant mutations, the risk of recurrence can be 50% if one parent is affected. However, if the mutation is de novo, the risk of recurrence is significantly lower but not zero due to the possibility of germline mosaicism.

Paternally inherited missense variations in the ABCC8 or KCNJ11 genes can lead to a severe form of CHI with an early presentation.

What is the management of CHI?