Diazoxide-Induced Neutropenia and Long-Term Follow-up in a Patient with Hyperinsulinemia-Hyperammonemia due to GLUD1 Mutation

Abstract

Hyperinsulinism/hyperammonemia (HI/HA) syndrome is caused by activating mutations in GLUD1 gene, and causes fasting as well as protein sensitive symptomatic hypoglycemia, in addition to persistently elevated plasma ammonia levels. First-line treatment is diazoxide, and most patients respond well to this agent, however side effects may be observed. The most frequent side effect of diazoxide is fluid retention and hypertrichosis, while hyperuricemia and hematologic side effects are observed less often. Herein, we report a case who had a heterozygous mutation of GLUD1 gene and who developed diazoxide related neutropenia 8 years after the start of treatment. On follow-up, leucopenia and mild neutropenia persisted and the treatment was changed to somatostatin analogues. However, she developed persistent severe symptomatic hypoglycemia and required diazoxide retreatment. A lower dose of diazoxide (6 mg/kg/day) successfully controlled hypoglycemia and cell counts increased even though they were not normalized. Neutropenia in current case presented after a long period of time of diazoxide use and this period is the longest defined in the literature. Long-term endocrine and hematologic follow-up of this patient up to 18 years old will also be presented.

Introduction

Hyperinsulinism/hyperammonemia (HI/HA) syndrome is a condition which develops as a result of activating mutations in GLUD1 gene encoding glutamate dehydrogenase enzyme. It leads to fasting as well as protein sensitive symptomatic hypoglycemia, in addition to persistently elevated plasma ammonia levels. This condition accounts for 5% of all persistent hyperinsulinemic hypoglycemia of infancy (PHHI). Hypoglycemia is milder than that seen in congenital hyperinsulinism (CHI) due to KATP channel mutations. First-line treatment is diazoxide, and most patients respond well to this agent, however side effects may be observed with higher doses. The most frequent side effect of diazoxide is fluid retention and hypertrichosis, while hyperuricemia and hematologic side effects are observed less often. Herein we report a case with HI/HA syndrome due to a heterozygous mutation of GLUD1 who developed diazoxide related neutropenia 8 years later. Long-term endocrine and hematologic follow-up of this patient up to 18 years old will also be presented.

Case presentation

The patient was born at term with a birth weight of 3050 gr after an uneventful pregnancy. She was the second child of healthy parents with no history of consanguinity. At the age of six months she developed afebrile seizures which persisted under phenobarbital treatment. She was referred to our clinic at the age of 20 months. Upon physical examination, body weight, height, and head circumference were 10.8kg (25–50p) and 89cm (>97p), 47.2cm (25p) respectively. She had no other neurological findings. Biochemical tests revealed a fasting blood glucose of 41 mg/dL and simultaneous insulin level of 3.19 μIU/mL and ammonia level of 220 μg/dL. A diagnosis of HI/HA was considered (Table 1). Diazoxide was commenced and achieved normoglycemia with a dose of 8 mg/kg/day. Her electroencephalography (EEG) was within normal limits thus phenobarbital was discontinued. Patient was lost to follow-up for 8 years and she was readmitted at the age of 9 years. She was being followed at another medical center during this period and continued to use diazoxide at the same dose. There were no side effects observed due to diazoxide during her follow-up at the other center with a normal white blood cell count (WBC) and normal electrolytes levels. There was no further history of seizures, and she was a low intermediate grade at school. On admission recent blood glucose was between 40-70 mg/dL thus the patient was hospitalized and the dose of diazoxide was gradually increased up to 12 mg/kg/day. During her follow-up in inpatient clinics and at her 1 month visit her complete blood count and serum electrolytes levels were normal. Sequence analysis of GLUD1 gene demonstrated a previously reported heterozygous missense mutation at exon 12 (c.1342T>C;p.Ser448Pro(p.S448P)) (1).

Table 1.

Laboratory findings at the time of diagnosis of hypoglycemia

	Patient’s results	Normal range
Serum glucose (mg/dL)	41	70-100
Insulin (μIU/mL)	3.19	1.9-23
C-peptide (ng/mL)	1.5	0.9-7.1
ACTH (pg/mL)	12.5	0-46
Cortisol (μg/dL)	6.28	6.7-22.6
Growth hormone (ng/mL)	8.01	>10
Lactic acid (mg/dL)	14	4.5-19.8
Pyruvate (mg/dL)	0.3	0.3-0.7
Ammonia (μg/dL)	220	20-120
Urine ketone	Negative	Negative
Blood gas analysis pH HCO3 (mmol/L)	7.41 24.9	7.35-7.45 22-26
Low dose ACTH stimulation test Peak cortisol (μg/dL)	28.6	>19.6

Routine screening at 3-month visit revealed a WBC count of 2800/μL and absolute neutrophil count (ANC) of 1000/μL. On follow-up leucopenia and mild neutropenia persisted (WBC:2600–3800/μL, ANC:1000–1300/μL). She had no clinical signs suggesting infection or any other cause for neutropenia. Viral serology as well as blood marrow aspiration biopsy were all within normal limits (Table 2). Thus, leucopenia and neutropenia were considered as side effects of diazoxide. Diazoxide was stopped and somatostatin analogue octreotide (15-30 μg/kg/day) therapy commenced. Nevertheless, she developed persistent severe symptomatic hypoglycemia (blood glucose being between 30-50 mg/dL) during this time although the dose of octreotide was increased up to a maximum dose of 30 μg/kg/day. Thus, her treatment was reverted back to diazoxide. A lower dose (6 mg/kg/day) of diazoxide successfully achieved normoglycemia and cell counts increased even though they were not normalized (WBC:3300-4300/μL,ANC:1200-1490/μL). She did not develop any infection during this time period.

Table 2.

Laboratory findings for the initial hematologic assessment and follow-up

	Initial	During follow-up
Hemoglobin(g/dL)	14	13.8-15.0
Hematocrit (%)	42	41-46
WBC(x109/L)	2.8	3.3-4.3
ANC(x109/L)	1.0	1.2-1.5
ALC(x109/L)	1.6	1.6-1.8
Platelet(x109/L)	219	196-310
Viral infections (EBV, CMV, Hepatitis A,B,C,Herpes type 1&2 and HIV serology, Parvovirus PCR	Negative	Negative
ANA	Negative	Negative
Compleman 3(C3) (mg/dL)	Normal	Normal
Compleman 4(C4) (mg/dL)	Normal	Normal
Vit B12 level (pg/mL)	Normal	Normal
Serum Ig A level(mg/dL) Serum IgG level(mg/dL) Serum IgM level(mg/dL)	Normal Normal Normal	Normal Normal Normal
Lymphocyte subsets	NA	Normal
Bone marrow aspiration	Normocellularity without myeloid arrest	NA

NA:non available ANC: Absolute neutrophil count ALC: Absolute lymphocyte count.

During follow-up, there was a period when the patient quit using diazoxide herself and it was seen that WBC and ANC counts returned to normal at that time (WBC:7500/μL, ANC:4600/μL). However, symptomatic hypoglycemia reoccurred and diazoxide was readministered at the dose of 5 mg/kg/day which successfully controlled hypoglycemia. After re-starting the diazoxide a mild decrease in the cell counts was observed. The patient has been followed-up at our hospital for nine years since the start of her first neutropenia episode, and her hematological work-up during follow-up is presented in Table 2. On diazoxide treatment, she did not experience any severe hypoglycemia and her HbA1c levels were within normal ranges during follow-up (Table 3). To evaluate long term neurological sequels of hypoglycemia in this patient we performed Wechsler Intelligence Scale for Children-Revised (WISC-R test). Full scale, verbal scale and performance scale IQ of patient revealed a score of 100 which was consistent with average intelligence.

Table 3.

Laboratory findings for the endocrine follow-up

	Patient’s results	Normal ranges
Glucose (fasting) (mg/dL)	71-85	70-100
Glucose (posprandial) (mg/dL)	71-87	<140
Insulin (fasting) (μIU/mL)	8.5-21.42	1.9-23
Insulin (postprandial) (μIU/mL)	42.02-120.51	1.9-23
HbA1c (%)	4.4-4.8	4-6.5
Ammonia (μg/dL)	67.9-114.76	20-120

Discussion

We report a case with HI/HA syndrome due to a heterozygous mutation of GLUD1 who developed diazoxide associated neutropenia after 8 years of diazoxide use, the longest duration reported so far. In previous reports, diazoxide-induced neutropenia has been shown to develop within days or at most months of diazoxide use (2,3). In the present case we observed neutropenia following an increase in dosage, after a long time of treatment.

Heterozygous mutation in GLUD1 gene is the second most common cause of CHI (1). Hypoglycemia due to GLUD1 mutations has a milder clinic compared to CHI due to KATP channel mutations. Current case had a normal birth weight and there was no hypoglycemia at birth. She was brought to hospital at age of six months after suffering seizures. Hypoglycemia and hyperammonemia were detected while evaluating etiology of seizures, and she was diagnosed at early infancy similar to the cases in the literature (4).

First-line treatment in HI/HA syndrome is diazoxide, a KATP channel agonist, owing to its good response rate, and the current patient also responded well to diazoxide (4-6). The most common side effects of diazoxide are hypertrichosis and fluid retention. Fluid retention secondary to diazoxide may be very severe and life-threatening (7-10). On the other hand, hematological side effects of diazoxide use are rarer, and have mostly been reported as specific case reports since first hematological abnormalities (mild neutropenia and eosinophilia) due to diazoxide have been reported in 1967 (11,12). Neutropenia and thrombocytopenia are among more prominent hematological side effects, while anemia has been reported to a lesser degree (13).

In a recent meta-analysis in 2015, a total of 664 patients had been evaluated for short and long term side effects of diazoxide (14). Diazoxide related side effects were detected in 325 cases (≈50%). Hypertrichosis (52%), fluid retention/electrolyte imbalances/edema (30%), and vomiting/poor appetite (12%) were the most common side effects. Bone marrow suppression (leucopenia, isolated neutropenia or thrombocytopenia) was seen in 3% (n:11) of cases. In another recent survey from Japan, 384 patients with hyperinsulinemic hypoglycemia who used diazoxide from 165 institutions were monitored and 30.5% (117/384) experienced at least one adverse reaction, 1% (4/384) had neutrophil count decreased whereas 0.8% (3/384) had platelet count decreased (15). In a single-center study in 2019, 165 of 194 (85.1%) patients with hyperinsulinism were treated with diazoxide and pulmonary hypertension was observed in 8 of these cases and neutropenia was seen in other eight (3). In a study evaluating 295 children with hyperinsulinemic hypoglycemia treated with diazoxide, hematological side effects have been reported among the most common diazoxide-related side effects (2). Fourty-six (15.6%) patients were diagnosed with neutropenia and 14 (4.7%) with thrombocytopenia.

Studies on neutropenia due to diazoxide use show that neutropenia usually develops within days after diazoxide initiation (2). Median duration of diazoxide use has been reported as 48 (14-207) days in 137 cases whose treatment need to be discontinued due to all causes (2). In our case, neutropenia was observed after 8 years of diazoxide use, only after increasing the dose up to 12 mg/kg/day. Similarly, studies in the literature show that this side effect occurs at high doses (2,14). In the current case, after cessation of treatment due to neutropenia, neutropenia improved, but diazoxide treatment was readministered as hypoglycemia could not be achieved with other drugs. Hypoglycemia was controlled with low-dose (6 mg/kg/day) of diazoxide when readministered the second time, however, mild neutropenia developed again. This mild neutropenia persisted while continuing to receive low-dose diazoxide. Neutropenia in the current case again improved in the period when the patient discontinued the drug use at her own will, however neutropenia recurred when diazoxide treatment was readministered due to recurrent hypoglycemia. All these findings support the neutropenia seen in this patient is drug-related and dose dependent. At the present time, pathophysiology of diazoxide-associated neutropenia remains unclear, but it has been suggested that the mechanism behind hematological side effects may be a possible immune mediated hypersensitivity reaction (12,16-18). It is interesting that neutropenia developed after a very long time in the current case, and this should also be taken into account when investigating the underlying mechanisms of diazoxide-related neutropenia.

In literature there is a case who had hyperinsulinism due to Munchausen syndrome by proxy and neutropenic fever secondary to diazoxide use (13). No serious infection associated with neutropenia has been reported in any other case. Although there have been case reports indicating that neutropenia improved shortly after discontinuation of diazoxide in previous years, the number of studies saying that neutropenia usually regresses without need of cessation of diazoxide increases recently. U.S. Food and Drug Administration states that in patients with diazoxide-induced neutropenia, there is no increased susceptibility to infections and neutropenia usually regresses without need of cessation of diazoxide (19). Herrera et al. reported that 29 of 42 cases had repeated hemogram evaluations and the mean time to resolution of neutropenia in these cases was 10 (3-40) days (2). Diazoxide was discontinued before resolution in 14 of 29 cases. Diazoxide was continued in the current case, since there was no history of severe infection secondary to neutropenia, and glycemic control failed with alternative treatments. Patient has been followed-up for nine years since the development of neutropenia, and continues to have mild neutropenia. No other side effects of diazoxide or any complications secondary to neutropenia have been observed.

In conclusion, the present case had a diazoxide responsive hypoglycemia and despite neutropenia diazoxide could not be stopped completely but the dose could be reduced, since hyperinsulinemic hypoglycemia is a severe condition that could have severe neurological consequences. The recurrence of hypoglycemias was prevented in this way and WISC-R test had shown that the patient had no severe neurological insult. The present case is important because it provides a long-term follow-up of a case with diazoxide induced neutropenia. This case also suggests that care should be taken in long-term follow-up of patients using diazoxide in terms of hematological side effects, even though side effects may not necessarily be observed in early period, and that patients should be followed-up closely especially after dose changes. Neutropenia caused by diazoxide is milder and benign without serious complications such as infections or neutropenic fever even in the long term follow-up.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

Authors would like to thank the team of the Exeter Genetics Laboratory and the member of the Department of Molecular Genetics, University of Exeter Medical School, UK, especially Prof. Sian Ellard and Dr Sarah Flanagan for genetic analysis.