Focal congenital hyperinsulinism managed by medical treatment: a diagnostic algorithm based on molecular genetic screening

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cen.12400 This article is protected by copyright. All rights reserved.

Received Date : 18-Sep-2013 Returned for Revision: 24-Oct-2013 Finally Revised Date : 22-Dec-2013 Accepted Date : 31-Dec-2013 Article type : D FOCAL CONGENITAL HYPERINSULINISM MANAGED BY MEDICAL

TREATMENT: A DIAGNOSTIC ALGORITHM BASED ON MOLECULAR

GENETIC SCREENING

Short title: Diazoxide-responsive focal hyperinsulinism

1Arianna Maiorana, 2Fabrizio Barbetti, 1Arianna Boiani, 3Vittoria Rufini, 4 Milena

Pizzoferro, 5Paola Francalanci, 6Flavio Faletra 7Colin G. Nichols, 8Chiara Grimaldi,

8Jean de Ville de Goyet, 9Jacques Rahier, 10Jean-Claude Henquin and 1Carlo Dionisi-

Vici

1Metabolic Unit , Department of Pediatrics, 2Laboratory of Mendelian Diabetes, 4Department

of Nuclear Medicine , 5Department of Pathology, 8Liver Surgery and Transplantation Unit,

IRCCS Bambino Gesù Children’s Hospital, Piazza S. Onofrio 4, 00165, Rome, Italy;

2Department of Experimental Medicine and Surgery, University of Tor Vergata, Section D,

Room 118, Viale Oxford 81, 00133, Rome, Italy; 3Department of Nuclear Medicine,

Catholic University of the Sacred Heart, Via Eugenio Tansi 67, 00135, Rome, Italy; 6Institute

for Maternal and Child Health, IRCCS Burlo Garofalo, Via dell'Istria 65, 34137, Trieste,

Italy; 7Department of Cell Biology and Physiology, Washington University School of

Medicine, St. Louis, Missouri, USA; 9Department of Pathology and 10Unit of Endocrinology

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This article is protected by copyright. All rights reserved.

and Metabolism, Faculty of Medicine, University of Louvain UCL 55.30, Avenue Hippocrate

55, B-1200, Brussels, Belgium.

Corresponding author: Arianna Maiorana, Metabolic Unit, Bambino Gesù Children's

Hospital, Piazza S.Onofrio 4, 00165, Rome, Italy; fax +390668592791: e-mail address:

[email protected]

Key Words: Congenital Hyperinsulinism; Hypoglycemia; Diazoxide; KATP-channel

Declaration of interest and financial disclosure: Nothing to declare

Abstract

Objective: Congenital hyperinsulinism (CHI) requires rapid diagnosis and treatment to avoid

irreversible neurological sequelae due to hypoglycemia. Etiological diagnosis is instrumental

in directing the appropriate therapy. Current diagnostic algorithms provide a complete set of

diagnostic tools including 1) biochemical assays, 2) genetic facility and 3) state-of-the-art

imaging. They consider the response to a therapeutic diazoxide-trial an early, crucial step

before proceeding (or not) to specific genetic testing and eventually imaging, aimed at

distinguishing diffuse versus focal CHI. However, interpretation of the diazoxide-test is not

trivial and can vary between research groups, which may lead to inappropriate decisions.

Objective of this report is proposing a new algorithm in which early genetic screening, rather

than diazoxide trial, dictates subsequent clinical decisions.

Patients, Methods and Results. Two CHI patients weaned from parenteral glucose infusion

and glucagon after starting diazoxide. No hypoglycemia was registered during a 72-h

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continuous glucose monitoring (CGMS) or hypoglycemic episodes were present for no

longer than 3% of 72-h. Normoglycemia was obtained by low-medium dose diazoxide

combined with frequent carbohydrate feeds for several years. We identified monoallelic,

paternally inherited mutations in KATP-channel genes, and 18F-DOPA PET-CT revealed a

focal lesion that was surgically resected, resulting in complete remission of hypoglycemia.

Conclusions: Although rare, some patients with focal lesions may be responsive to

diazoxide. As a consequence, we propose an algorithm that is not based on a “formal”

diazoxide response but on genetic testing, in which patients carrying paternally inherited

ABCC8 or KCNJ11 mutations should always be subjected to 18F-DOPA PET-CT.

Introduction

Congenital hyperinsulinism (CHI), characterized by uncontrolled or excessive insulin

secretion for the prevailing glucose levels, is the most frequent cause of severe and persistent

hypoglycemia in the neonatal period and early infancy. Patients present with recurrent

episodes of profound hypoglycemia requiring rapid and intensive treatment with dextrose

infusions and intravenous glucagon to prevent neurological sequelae1-4. Mutations in 9 genes

(ABCC8, KCNJ11, GCK, GLUD1, HADH, HNF1A, HNF4Α, SLC16A1, UCP2) have so far

been associated with CHI3-6. Loss-of-function mutations in ABCC8 and KCNJ11, which

respectively encode the SUR1 and Kir6.2 subunits of the pancreatic ATP-sensitive potassium

(KATP) channel, account for more than 50% of CHI cases and are associated with two

histological aspects of the endocrine pancreas: a diffuse form, which is inherited as either an

autosomal recessive or dominant trait and affects all beta cells, and a focal form, which

results from the combination of a paternally inherited germinal mutation and a somatic loss of

heterozygosity of the maternal allele in a restricted group of beta cells7.

[18F]dihydroxyphenylalanine positron emission tomography (18F-DOPA PET) imaging is

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useful to differentiate between the two forms, and to precisely localize focal lesions in the

pancreas8 or in ectopic sites9. Chronic medical treatment to prevent recurrence of

hypoglycemia may include drugs, such as diazoxide (a KATP channel opener) or octreotide (a

long-lasting somatostatin analogue), sometimes in association with frequent carbohydrate-

enriched feeding1-3. Diffuse CHI is first approached with conservative treatment, but when

medical measures are ineffective, near-total pancreatectomy is required, and is associated

with increased risk of developing diabetes. Focal CHI has so far been considered

unresponsive to diazoxide1-3,10,11, and the outcome of a therapeutic diazoxide trial, whose

modalities differ slightly between research groups, represents the critical decision step of

current diagnostic algorithms, that avoids 18F-DOPA PET-CT in those patients whose

hypoglycaemia can be prevented by diazoxide1-3,11. Here, we report two patients with focal

CHI due to paternally inherited mutations in KATP genes, in whom low-medium dose

diazoxide succeeded - as detected by repeated continuous glucose monitoring evaluations

(CGMS) - in maintaining normoglycemia before 18F-DOPA PET-CT localization and

elective resection of the focal lesion. Therefore, we suggest a new algorithm in which genetic

screening, instead of the response to diazoxide, dictates the subsequent decision tree.

Materials and Methods

Ethics

The study was approved by the Ethical Committees of Bambino Gesù Children's Hospital of

Rome, Italy, the Catholic University of Sacred Heart of Rome, Italy, and the University of

Louvain Faculty of Medicine, Brussels, Belgium.

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Continuous glucose monitoring system

Blood glucose control was assessed by pre-meal glucometer measurements, and by 72-h

continuous glucose monitoring (CGMS) (Medtronic; data analysis by MiniMed software)

during the first hospital admission, then at the beginning of treatment, monthly during the

following 3 months, and every 3-6 months afterwards. In general, maintenance of pre- and

post-meal glucose levels between 50 and 150 mg/dl (2.8-8.2 mmol/l), with an average of 70

mg/dl (3.9 mmol/l), was considered as good glycemic control. For this study, we defined a

subject as responsive to diazoxide if his/her glucose was below 50 mg/dl (2.8 mmol/l) for less

than 3% of the time during a 72-h CGMS session (i.e. equal or less than 130 minutes in 3

days) and less than 30 minutes consecutively per single episode. Because CGMS sensitivity

depends on the type of glucometer used for sampling, our Centre compared the accuracy of

blood glucose determination by different glucometers versus plasma glucose (automatically

corrected for the hematocrit) and identified the more sensitive and accurate device that was

selected for the use in our ward. Performing more than 900 GCMS in the last 5 years, we

acquired a fairly good level of performance and of interpretation of data, which makes us

confident about the efficacy and safety of CGMS.

Genetic analysis

Germline mutations were searched in patients and their parents on DNA extracted from

peripheral leukocytes by DNeasy Tissue Isolation kit (Qiagen, Valencia, CA).

ABCC8,KCNJ11,GCK, HNF4A genes were amplified by PCR and directly sequenced.

Genetic analysis of KCNJ11 gene has been performed as described in ref. 16 and analysis of

ABCC8 gene as described in ref. 20.

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18F-DOPA PET-CT scan

Anesthetized patients (fasted for at least 6h, and receiving i.v. glucose) underwent a PET

scan of the abdomen using a hybrid machine (Gemini GXL, Philips Medical Systems) 45 min

after i.v. administration of 18F-DOPA (4 MBq/Kg). A contrast-enhanced CT was also

sequentially performed to accurately localize the focal lesion on the basis of the vascular

map. To back up the visual identification of the focus, a standardized uptake value (SUV)

was obtained. A SUV ratio >1.2 was considered indicative of a focal lesion12.

Histology

Pancreatic specimens were obtained intra-operatively and prepared as previously described13.

Immunohistochemistry for CDKN1C peptide (p57) was subsequently performed on formalin-

fixed, paraffin-embedded tissue14.

In vitro functional studies of pancreas

On the basis of intra-operative examinations, the pathologist sampled small fragments of

healthy pancreas and focal lesion for functional studies. The two fragments, immersed in

culture medium at 4°C, were transported from Rome to Brussels within 10 hours. They were

then treated as recently described in detail, to measure the dynamics of insulin secretion and

estimate the insulin content of the tissue15.

Results

Patient 1

Clinical history

The clinical history of patient 1 has been reported16. Briefly, she was born with normal

weight at 38 weeks of gestation from unrelated parents. At the age of 2 months, she displayed

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cyanosis and tremor. The diagnosis of CHI was made on the basis of hypoketotic (<0.05

µmol/l) fasting hypoglycaemia (40 mg/dl, 2.2 mmol/l), relative hyperinsulinemia (31 µU/ml),

low free fatty acids (220 µmol/l), and good response to glucagon. At presentation, she needed

intravenous glucose infusion, glucagon infusion and enteral feeding to maintain

normoglycemia. Treatment with diazoxide (15 mg/kg/day, in three daily doses) and low-dose

chlorothiazide (2 mg/day, to avoid fluid retention) was started, allowing immediate weaning

from glucagon and parenteral glucose infusions. The patient was discharged on a diazoxide

dose of 7.5 mg/kg/day and a low amount of milk in enteral nutrition (12 ml/h) to reassure the

family. After about two months, nocturnal enteral feeding was stopped and free diet was

started. During that period, glucometer measurements were performed before each meal,

during night and before the first meal of the next day (i.e. after nocturnal fasting), for more

than five consecutive days, by parents at home, while the patient was on diazoxide, and by

nurses during subsequent hospitalization. Glycaemic values were consistently >3 mmol/L. In

addition, more refined assessment of glycemic control was obtained by 72-h CGMS (about

200 blood glucose determinations per day) during hospitalization; after discharge, the

procedure was repeated as stated in Methods. Four months after hypoglycaemia onset,

CGMS showed no hypoglycemic episodes (diazoxide dose 7 mg/kg/day), even between

nocturnal meals (Fig.1). This result was confirmed at 72-h CGMS sessions performed 12 and

24 months after discharge, while the patient was on diazoxide. Thus, the patient was

responsive at almost half the recommended initial diazoxide dose3,17,18, i.e. 10 mg/kg/day for

an infant. Subsequently, only single asymptomatic episodes of borderline hypoglycemia

(between 40-50 mg/dl, 2.2-2.8 mmol/l ) were recorded in each 3-days session of CGMS

performed 12, 8 and 3 months before surgery and which prevented us from withdrawing

diazoxide, but which progressively tapered off to 4.0 mg/kg/day (Fig. 1 suppl).

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Mutational analysis

A heterozygous KCNJ11/V290M mutation was found in the proband and in her unaffected

father16. DNA analysis of the mother was negative. No mutations in ABCC8, GCK and

HNF4Α genes were detected.

18F-DOPA PET-CT scan, surgery and histology

18F-DOPA PET-CT, performed at the age of 4 years and 11months, documented a focal

lesion in the pancreatic body, which was surgically removed 2 months later16. Histology on

intraoperative frozen pieces and postoperative fixed specimens was typical of a focal lesion.

CDKN1C (p57) was negative within the lesion, but positive in islets outside the lesion. This

pattern14,19 is consistent with loss of maternal 11p15.5 as the cause of a focal islet cell

adenomatous hyperplasia7,14. After operation the patient no longer required medical therapy

as attested by CGMS16.

In vitro insulin secretion

In healthy pancreas, insulin secretion rate was low in 1mM glucose, and 15mM glucose

induced a large biphasic secretory response that was abolished by subsequent addition of

diazoxide and eventually restored by tolbutamide (Fig.2). In tissue from the focal lesion, the

rate of insulin secretion was very high in 1mM glucose and paradoxically decreased slightly

on stimulation with 15 mM glucose, whereas 100 μM diazoxide and 100 μM tolbutamide had

no effect. The estimated insulin content of the focal lesion was ~50-fold higher than that of

normal pancreas (246.0 vs 5.0 ng/mg).

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Patient 2

Clinical history

Patient 2 was a female, born with normal weight at 41 weeks of gestation, from unrelated

parents. At the age of 3 months she first manifested generalized seizures. On the basis of

hypoketotic (<0.05 µmol/l) hypoglycemia (32 mg/dl,1.75 mmol/l), associated with

suppressed free fatty acids (108 µmol/l), detectable insulin (3.8 µU/ml) and appropriate

glycemic response to glucagon injection, a diagnosis of CHI was made. Intravenous infusion

providing 15 mg/kg/min of glucose was initially necessary to maintain euglycemia.

Thereafter, diazoxide (12 mg/kg/day divided in three doses) was started, allowing a gradual

withdrawal of parenteral treatment. The patient also received low-dose chlorothiazide (2

mg/day) to prevent fluid retention. During the first seven months after diagnosis the child

manifested feeding difficulties, gastroesophageal reflux, with frequent vomiting, which

complicated management of disease because meals were not completed and administration of

therapy was unsafe. This situation prompted the use of drip feeding with a carbohydrate rich

diet to minimize the risk of hypoglycaemia.

Follow up during long-term diazoxide treatment and on a free diet was performed by

CGMS, with the same scheme as in patient 1. As shown by a representative CGMS profile

obtained after 1.8 years of treatment (Fig.3), average glycemia was 90 mg/dl (5 mmol/l),

with a single recorded episode just below the threshold of 50 mg/dl (48 mg/dl, 2.6 mmol/l)

of 10 minutes duration (corresponding to <0.25% of 3-days CGMS registration) while

receiving diazoxide at a dose of 10 mg/kg/day. Over 2.5 years of follow-up, blood glucose

remained unchanged as detected at CGMS, and diazoxide was tapered to 6.6 mg/kg/day right

before surgery, whilst the child was on a free diet.

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Genetic analysis

DNA sequencing identified a frameshift mutation c.1580_1581dupGG, p.K528Gfs*4 in the

ABCC8 gene20 which produces a premature stop codon with generation of a truncated SUR1

protein. The same mutation was found in the asymptomatic father whereas the mother

showed a wild type ABCC8 sequence. No mutations were found in KCNJ11, GCK and

HNF4A genes.

18F-DOPA PET-CT scan, surgery and histology

18F-DOPA PET-CT performed at the age of 2 years and 3 months showed a large focal lesion

of the pancreatic head (Fig.4A). Removal by elective surgery 4 months later led to rapid

recovery of the patient to euglycemia, as ascertained by CGMS performed at 1.5 months after

surgery, while she was on normal diet and free of diazoxide. The resected lesion appeared as

a polyp at the surface of the pancreas (Fig.4B), and intra-operative examination of frozen

sections showed a typical picture of focal CHI13 (Fig.4C). The lesion, poorly delimited,

seemed to result from the confluence of apparently normally structured islets separated by a

few exocrine acini maintaining an organoid pattern. Nuclei appeared polymorphous with size

variations and hyperchromatism. Outside the lesion, islets were small, containing beta-cells

with a small nucleus and scanty cytoplasm, both characteristic of non-stimulated beta cells.

Post-operative immunohistochemistry on fixed tissue showed that CDKN1C peptide labelled

outside but not within the lesion.

In vitro insulin secretion

In vitro insulin secretion by healthy pancreatic tissue adjacent to the focal lesion was

stimulated ~2-fold when the glucose concentration was increased from 1 to 15mM (Fig.2).

Addition of diazoxide (to open KATP channels) inhibited the effect of glucose, and subsequent

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addition of tolbutamide (to close KATP channels) antagonized this inhibition in a reversible

manner. Again, characteristic of tissue lacking KATP channels, insulin secretion was not

stimulated by high glucose or affected by any of the two drugs (Fig.2), and the estimated

insulin content of the focal lesion was ~100-fold higher than that of healthy pancreas (426.0

vs 4.3 ng/mg).

Discussion

We describe two patients with focal CHI, both of whom achieved good metabolic control

with low-medium dose diazoxide therapy, according to our criteria of diazoxide

responsiveness. Such medical treatment is currently deemed adequate in mild, diffuse forms

of CHI caused by dominant mutations in ABCC8 and KCNJ11 genes, which lead to partial

inactivation of KATP channels, or by mutations in other genes1. In contrast, patients with focal

CHI frequently do not respond to even a maximal diazoxide dosage (as shown in Fig.5),

which most of the leading research groups on CHI have set at 15 mg/kg/day for neonates and

10 mg/kg/day for infants1-4. Clinical criteria to assess diazoxide responsiveness may vary, but

a plasma glucose constantly equal to or above 60-70 mg/dl (3.3-3.8 mmol/l) without support

of carbohydrate-enriched diet appears to be the gold standard for most groups; in addition,

some researchers advocate the use of fasting for a variable duration at the end of a 5-days

diazoxide trial to definitely classify patients as responders1,3,4,11. Conversely, non-responders

are defined as those with at least two confirmed blood glucose values below 3 mmol (i.e. 54

mg/dl) in a 24h period, after 5 days of diazoxide4,10. Algorithms proposed by these groups

prescribe that diazoxide-responders should not be subjected to 18F-DOPA imaging2,3,11. These

algorithms do not prescribe the frequency at which blood glucose determinations should be

performed during the diazoxide-test and we believe that rare (say 1-2 per day) glucose values

below 60 mg/dl (3.3 mmol/l), or even short, asymptomatic, severely hypoglycemic (< 50

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mg/dl, < 2.8 mmol/l) episodes could have been missed when glucose determinations are

performed by standard frequency sampling. For this reason we have implemented the use of

CGMS in patients with CHI in our metabolic ward in 2003. CGMS, with over 200

determinations/day, allows detection of low glucose values of quite short duration,

independent of meals or period of the day (i.e. sleep hours, fed/fasting state). As a result,

CGMS allows a more detailed determination of daily glucose profiles and better assessment

of the outcome of diazoxide therapy. Relying on “frequent sampling”, instead of on CGMS to

monitor glucose, might easily miss asymptomatic and/or short hypoglycemic episodes when

evaluating CHI patients at follow-up, but in our two patients, numerous 72-h CGMS sessions

either certified no hypoglycemic episodes (defined as glucose < 50 mg/dl, < 2.8 mmol/l) or

borderline low glucose values (50-54 mg/dl, 3-5 mmol/l) that represented less than 3% of the

total glucose determinations, with single episodes no longer than 30 minutes. On these bases,

we considered these two focal CHI patients to be diazoxide-responsive. Their therapeutic

outcome was fully divergent from other patients with focal lesion we observed, who failed to

respond at all to maximal doses of diazoxide (Fig.5).

These observations raise questions regarding definition of clinical responsiveness to

diazoxide. Our managing approach to the 86 CHI patients that we have followed during the

last 20 years was inspired by Glaser et al.21,22, who advocated medical treatment for all CHI

patients whenever possible. To this end, we have aggressively pursued diazoxide therapy, and

have considered all patients who weaned from parenteral glucose infusion and/or i.v.

glucagon, who stop presenting sustained hypoglycaemia after five days of diazoxide at initial

maximal dose combined, if needed, with nutritional measures, to be diazoxide-sensitive.

Responsiveness to diazoxide was then confirmed by CGMS. With this approach, only 21% of

our patients with instrumental and genetic diagnosis of diffuse CHI underwent near-total

pancreatectomy (Maiorana A. and Dionisi-Vici C. unpublished observations), in keeping with

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the 28% reported by Glaser et al.22. We also consider that, owing to its side-effects, diazoxide

should be tailored to the minimal effective dose. Hence, in the second patient, the initial

treatment combined a lower dose of diazoxide and an enriched carbohydrate diet, eliciting a

satisfactory clinical outcome. The efficacy of the treatment was then assessed by CGMS,

rendering fasting tolerance tests unnecessary. It also avoided repeated blood sampling to

measure free fatty acids and ketone bodies23, both of which may be of questionable utility

during diazoxide therapy and are of discomfort for patients, especially in infancy and

childhood. These considerations are important since, according to current algorithms, the

clinical determination of diazoxide-responsiveness is instrumental for subsequent execution

of 18F-DOPA PET-CT2,3,11. Only recently, the group from Great Ormond Street Hospital for

Children in London, UK, has slightly changed the decision tree, advocating 18F-DOPA PET-

CT for diazoxide-responsive patients, but only when paternally inherited heterozygous

mutations of KATP are detectable24.

The in vitro secretory behaviour of pancreatic fragments from our two patients was compared

with that of 18 diazoxide-resistant focal cases17. This large series showed that control

pancreas adjacent to the lesion secretes insulin in similar pattern to normal islets25 in spite of

the presence of a mutated allele, a result that fits in with the lack of symptoms in the

heterozygous fathers. In our two cases insulin secretion was also qualitatively normal in

healthy pancreatic tissue but secretion and content were very different in tissue from within

the lesion. In both patients, insulin secretion from the focal lesion was not stimulated (even

slightly decreased) in high glucose, and was unaffected by diazoxide and tolbutamide. These

characteristics have been observed in many previously reported diazoxide-resistant focal

cases with mutations in ABCC8 or KCNJ1117. The insulin content of both focal lesions was

very high, within the range of previously reported cases, whereas the insulin content of the

adjacent healthy pancreas was much lower17.

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The rapidity of blood glucose control at onset of the treatment can be interpreted as

efficient overall inhibition of insulin secretion from the intact pancreas by diazoxide in both

patients since the only other drug present was chlorothiazide, which does not affect insulin

secretion from normal beta-cells or from beta-cells lacking KATP channels26.

The discrepancy between these clinical findings and the lack of inhibition of secretion by

diazoxide in focal tissue in vitro is thus the reverse of situations in which diazoxide was

ineffective in vivo at maximally tolerable doses but able to open mutated KATP channels at

high concentrations in vitro29. In children (0.5 – 5 years)27 taking diazoxide at doses of 4-7

mg/kg, plasma concentrations of the drug remained below 200 µmol/l, of which >85% is

bound to albumin28. As we used diazoxide at 100 µmol/l in the presence of a ~40-fold lower

concentration of albumin than in plasma, the free drug concentration was not plausibly higher

in vivo than in vitro. Notably, the expected phenotype of the mutation, at least in patient 1, is

reduced – but not complete lack of - functional KATP channel activity16. It is therefore

possible that, owing to their complex regulation by intracellular nucleotides and other factors,

mutated channels maintained a weak sensitivity to diazoxide in vivo, which escaped detection

during strong glucose stimulation in vitro. One should also bear in mind that the global

therapeutic response depends on the balance between insulin secretion from diazoxide-

sensitive normal islets and abnormal β-cells within the lesion. Our findings cannot be

considered totally unusual. In a previous cohort of 35 diazoxide-sensitive patients, half were

found to exhibit focal forms of CHI at surgery17. Diazoxide-sensitive CHI displaying a focal

aspect by PET imaging has recently been described30. However, in contrast to our two cases,

no mutation in ABCC8 or KCNJ11 or loss of heterozygosity was found in these patients.

Their β-cell hyperfunction could also be reversed by diazoxide in vitro, because it was caused

by an activating mutation in GCK or undue expression of hexokinase I in a subset of islets

concentrated in a few lobules of their pancreas31. In addition, Kapoor et al. recently described

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partial or complete diazoxide responsiveness in some patients carrying compound

heterozygous, and recessive heterozygous ABCC8 mutations32. Remarkably, focal CHI may

be clinically heterogeneous, with sometimes responsiveness or resistance to diazoxide in

patients with the same mutation in KATP channels33. Alternatively, unidentified genetic

factors may modulate the expression of the phenotype, or off-target actions of diazoxide may

be responsible for the in vivo responsivity.

In an effort to rationalize the genetic diagnostic screening of patients with CHI, most

algorithms rely on the diazoxide test to distinguish between responders and non-responders1-

3,11, and hence whether or not to perform 18F-DOPA PET-CT. Nevertheless, at least two

major research groups (re)introduced KATP genes sequencing at a subsequent step of their

algorithms2,3. All groups, including ours, have found that most patients with CHI carry a

KATP mutation20,32,34 and it is conceivable that most groups agree in principle regarding the

usefulness of molecular genetic studies in all patients with CHI, both for appropriate

therapeutic decisions and genetic counselling in familial cases. For these reasons, we propose

a revised algorithm, based on a reasoned combination of metabolic investigation and genetic

analysis, that does not utilize diazoxide response at the beginning of the decision tree (Fig.6).

According to this algorithm, we sequenced the ABCC8 and KCNJ11 genes first, and then

sequenced the GCK and HNF4A genes in those negative for KATP gene mutations, in 51

patients that were negative to specific metabolic screening indicative of GLUD1, HADH or

CDGs genes defects. We identified 23 KATP mutations16,20 ( Maiorana A., Dionisi-Vici C.,

Barbetti F., unpublished observations). Among these 23, 6 heterozygous, paternally inherited

mutations were found, including the two described in the present study. In all 6 a focal lesion

was identified by 18F-DOPA PET-CT, and all underwent successful surgery and were

effectively cured16,20 ( Maiorana A., Barbetti F., Dionisi-Vici C., unpublished observations).

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We acknowledge that sometimes patients with a focal form of CHI may experience a

progressive decrease in the severity and an eventual cessation of hypoglycemic episodes after

several years of medical treatment, an improvement attributed to apoptosis of beta-cells

within the lesion3,35. However, we would recommend that surgery, in the hands of an

experienced surgeon, should be the elective indication for focal lesions regardless of medical

responsiveness because it allows complete remission of the disease, reduces the clinical

burden and, by relieving the discomfort of chronic medical therapy (and attendant side-

effects) requiring strict monitoring, improves the quality of life of patients and their families.

In conclusion, some patients with focal lesions can be responsive to diazoxide. CGMS

is a very useful tool in evaluating and monitoring therapy outcome, demonstrating the

existence of “boarder-line” situations, with fluctuations of metabolic control during

diazoxide treatment. Defining “partial” sensitivity to diazoxide is not trivial and may lead to

inappropriate diagnostic and therapeutic decisions, whereas responsiveness to diazoxide does

not exclude focal forms17,30-32. Based on our clinical observations, we propose a new

algorithm that is not based on a “formal” diazoxide trial but on genetic testing, one in which

patients carrying paternal ABCC8 or KCNJ11 mutations should then be subjected to 18F-

DOPA PET-CT, as well as those showing a wild type pattern of the known genes associated

with CHI.

Acknowledgments

We are grateful to M. Nenquin for performing the in vitro experiments.

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Figures

Figure 1. Near normal blood glucose levelsachieved by medical treatment in patient 1. Blood

glucose was monitored using the 72-h continuous glucose monitoring system (CGMS) 1

month after the first discharge. CGMS showed a glycemic range of 56-106 mg/dl (3.1-5.8

mmol/l) and an average glucose value of 75 mg/dl (4.1 mmol/l) with no episode of

hypoglycemia. Diazoxide dosage was 7 mg/kg/day. Open stars indicate simultaneous

glucometer values; filled stars in indicate meal event.

Figure 2. Comparison of the effects of glucose and drugs acting on KATP channels on insulin

secretion by the focal lesion and the adjacent healthy pancreas in patient 1 and patient 2 .

The concentration of glucose was increased from 1 to 15 mM (G1 to G15), and 100 µM

diazoxide (Dz) and 100 µM tolbutamide (Tolb) were added as indicated at the top of the

figure. Forskolin (Fk), a direct activator of adenyl-cyclase, was present throughout at 1 µM.

Open circles: beta cells from the lesion; filled circles: beta cells from the healthy pancreas.

Figure 3. Near-normal glucose levels achieved by medical treatment in patient 2. Blood

glucose was monitored by 72-h CGMS, after 20 months of diazoxide treatment, at a

diazoxide dosage of 10 mg/kg/day. CGMS showed a glycemic range of 48-134 mg/dl (2.6-

7.4 mmol/l) and an average glucose value of 90 mg/dl (5 mmol/l) with one single event of

hypoglycemia of 10 minutes (< 0.25% in 3 days). Open stars indicate simultaneous

glucometer values.

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Figure 4. A. 18F-L-DOPA PET-CT scan (transaxial view) showing the presence of a focal

lesion in the medial-dorsal part of the pancreatic head of patient 2 (arrow). The uptake of 18F-

L-DOPA is increased in the head region (max SUV: 4.1) compared to the body and tail

regions (SUV ratio: 1.28). B. A focal lesion (exophytic polypoid mass) is easily identified at

the surface of the pancreatic head resected from patient 2. C. Intra-operative histology

showed adenomatous hyperplasia of endocrine cells within the mass.

Figure 5. Uncontrolled glucose oscillations in a patient with neonatal onset focal CHI and

paternal inherited ABCC8 mutation with no responsiveness to medical therapy. Blood glucose

was monitored using a120-h CGMS which showed hypoglycemia for >20% of the time at 4

months of age. At that time, after no responsiveness to diazoxide dosage of 15 mg/kg/day, the

patient was on therapy with diazoxide at the dosage of 4 mg/kg/day, octreotide at the dosage

of 19 µg/kg/day in combination with continuous enteral feeding for an amount of 13

mg/kg/min of glucose. Open stars indicate simultaneous glucometer values.

Figure 6. Suggested diagnostic and management algorithm for congenital hyperinsulinism

(CHI). Once the diagnosis has been established, patients should immediately be treated with

diazoxide (DZX) then, in case of unresponsiveness, with octreotide. A metabolic diagnostic

workup must be run in parallel with treatment, to identify specific disorders which include

Congenital Disorders of Glycosylation (CDGs), Hyperisulinism-Hyperammonemia syndrome

(HI-HA) due to gain of function mutation of glutamate dehydrogenase gene (GLUD1), and

3-Hydroxyacyl-CoA dehydrogenase (HAD) deficiency due to mutation of HADH gene.

According to familial history and suspected inheritance mode, ABCC8, KCNJ11, GCK,

HNF1α, HNF4α, HADH, INSR, SLC16A1or UCP2 genes can be sequenced (the last two

genes are sequenced only in a few laboratories). Patients with recessive biallelic mutations in

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ABCC8, KCNJ11, or HADH genes (mt/mt), or with dominant mutations (wt/mt) in ABCC8,

KCNJ1, or GCK, HNF1α, HNF4α, INSR, SLC16A1 and UCP2 genes exhibit a diffuse form

of CHI. Patients with a recessive mutation in ABCC8, KCNJ11 genes in the paternal allele

(wt/mt) exhibit a focal form of CHI and should undergo 18F-DOPA PET-CT and elective

partial pancreatectomy. Patients responsive to DZX with no detectable mutations (wt/wt)

should also undergo 18F DOPA-PET-CT to search for atypical focal forms of CHI potentially

treatable by partial pancreatectomy (i.e. bifocal forms). Wt: wild type; mt: mutant; wt/mt

dominant*: already reported in the literature or family tree strongly suggestive of dominant

negative mutation.

Figure 1 supp. Near-normal glucose levels achieved by medical treatment in patient 1. Blood

glucose was monitored by CGMS for 48h, three months before surgery, at the age of 4 years

and 10 months, at a diazoxide dosage of 4 mg/kg/day. CGMS showed a glycemic range of

44-115 mg/dl (2.4-6.3 mmol/l) and an average glucose value of 75 mg/dl (4.2 mmol/l) with

one single event of hypoglycemia of 5 minutes ( < 0.25% in 2 days). Open stars indicate

simultaneous glucometer values; filled stars in indicate meal event. This session lasted less

than 72h for family needs.

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Established diagnosis of CHIStart medical therapy (DZX ⇒Octreotide)

Mutational analysis

Metabolic investigations

Transferrin IEFabnormal

Hyperammonemia ⇑C4-OH-carnitine⇑3-OH-glutarate

HADHGLUD1

CDGs

HI-HAdiffuse HAD

diffuse

ABCC8/KCNJ11HNF1-4α/GK/HADH/

SLC16A1/UCP2wt/wt

DZX responsive

HNF1-4α/GK/SLC16A1/UCP2

wt/mt

ABCC8/KCNJ11mt/mt

wt/mt dominant*

diffuse

ABCC8/KCNJ11wt/mt paternal

18F DOPA PET/TC

Medical therapy (DZX ⇒ Octreotide)Diet

focalatypicaldiffuse

Partial pancreatectomy

responsive

General follow-up:growth, neurological andnutritional development

Non- responsive

Subtotalpancreatectomy

abnormal normal

HADHmt/mt

Mannosetherapy

MPI-CDG

Focal congenital hyperinsulinism managed by medical treatment: a diagnostic algorithm based on molecular genetic screening

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