Diazoxide-responsive hyperinsulinemic hypoglycemia caused by HNF4A gene mutations.

Abstract

The phenotype associated with heterozygous HNF4A gene mutations has recently been extended to include diazoxide responsive neonatal hypoglycemia in addition to maturity-onset diabetes of the young (MODY). To date, mutation screening has been limited to patients with a family history consistent with MODY. In this study, we investigated the prevalence of HNF4A mutations in a large cohort of patients with diazoxide responsive hyperinsulinemic hypoglycemia (HH).
We sequenced the ABCC8, KCNJ11, GCK, GLUD1, and/or HNF4A genes in 220 patients with HH responsive to diazoxide. The order of genetic testing was dependent upon the clinical phenotype.
A genetic diagnosis was possible for 59/220 (27%) patients. K(ATP) channel mutations were most common (15%) followed by GLUD1 mutations causing hyperinsulinism with hyperammonemia (5.9%), and HNF4A mutations (5%). Seven of the 11 probands with a heterozygous HNF4A mutation did not have a parent affected with diabetes, and four de novo mutations were confirmed. These patients were diagnosed with HI within the first week of life (median age 1 day), and they had increased birth weight (median +2.4 SDS). The duration of diazoxide treatment ranged from 3 months to ongoing at 8 years.
In this large series, HNF4A mutations are the third most common cause of diazoxide responsive HH. We recommend that HNF4A sequencing is considered in all patients with diazoxide responsive HH diagnosed in the first week of life irrespective of a family history of diabetes, once K(ATP) channel mutations have been excluded.

Full text

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CLINICAL STUDY
Diazoxide-responsive hyperinsulinemic hypoglycemia caused by
HNF4A gene mutations
S E Flanagan1, R R Kapoor2,3, G Mali1, D Cody4, N Murphy5, B Schwahn6, T Siahanidou7, I Banerjee8, T Akcay9,
O Rubio-Cabezas1,10, J P H Shield11, K Hussain2,3and S Ellard1
1Peninsula Medical School, Institute of Biomedical and Clinical Science, University of Exeter, Barrack Road, Exeter EX2 5DW, UK,2London Centre for
Paediatric Endocrinology and Metabolism, Great Ormond Street Hospital for Children NHS Trust, London WC1N 3JH, UK,3Institute of Child Health,
University College London WC1N 1EH, UK,4Department of Endocrinology, Our Lady’s Childrens Hospital, Dublin, 12 Ireland,5Children’s University
Hospital, Dublin, 1 Ireland,6Department of Metabolic Medicine, Royal Hospital for Sick Children, NHS Greater Glasgow and Clyde, Glasgow, UK,
7Department of Pediatrics, Aghia Sophia Children’s Hospital, University of Athens, Athens, 115 Greece,8Department of Endocrinology, Royal Manchester
Children’s Hospital, Central Manchester and Manchester Children’s University Hospitals NHS Trust, Manchester, M13 9WL, UK,9Department of
Endocrinology, Bakirkoy Maternity and Child Hospital, Istanbul, 34142 Turkey,10Department of Endocrinology, Hospital Infantil Universitario Nin ˜o
Jesus, Madrid 28009, Spain and11Department of Child Health, Bristol Royal Hospital for Children, Bristol BS2 8BJ, UK
(Correspondence should be addressed to S Ellard; Email: sian.ellard@rdeft.nhs.uk)
Abstract
Objective: The phenotype associated with heterozygous HNF4A gene mutations has recently been
extended to include diazoxide responsive neonatal hypoglycemia in addition to maturity-onset diabetes
of the young (MODY). To date, mutation screening has been limited to patients with a family history
consistent with MODY. In this study, we investigated the prevalence of HNF4A mutations in a large
cohort of patients with diazoxide responsive hyperinsulinemic hypoglycemia (HH).
Subjects and methods: We sequenced the ABCC8, KCNJ11, GCK, GLUD1, and/or HNF4A genes in 220
patients with HH responsive to diazoxide. The order of genetic testing was dependent upon the clinical
phenotype.
Results: A genetic diagnosis was possible for 59/220 (27%) patients. KATPchannel mutations were
most common (15%) followed by GLUD1 mutations causing hyperinsulinism with hyperammonemia
(5.9%), and HNF4A mutations (5%). Seven of the 11 probands with a heterozygous HNF4A mutation
did not have a parent affected with diabetes, and four de novomutations were confirmed. These patients
were diagnosed with HI within the first week of life (median age 1 day), and they had increased birth
weight (median C2.4 SDS). The duration of diazoxide treatment ranged from 3 months to ongoing
at 8 years.
Conclusions: In this large series, HNF4A mutations are the third most common cause of diazoxide
responsive HH. We recommend that HNF4A sequencing is considered in all patients with diazoxide
responsive HH diagnosed in the first week of life irrespective of a family history of diabetes, once KATP
channel mutations have been excluded.
European Journal of Endocrinology 162 987–992
Introduction
Hyperinsulinemic hypoglycemia (HH) is characterized
by unregulated secretion of insulin, despite hypoglycemia.
HH most commonly presents in the neonatal period
with the clinical presentation ranging from mild,
moderate to severe medically unresponsive hypoglyce-
mia(1).Diazoxideisthefirstlineoftreatmentforpatients
with HH. In the pancreatic b-cell, glucose metabolism is
coupled to insulin secretion by the adenosine tripho-
sphate-sensitive potassium channels (ATP-sensitive
KATP channels). Glucose metabolism results in an
increase in the concentration of intracellular ATP,
which binds to the Kir6.2 subunits to effect channel
closure. This results in membrane depolarization and
ultimatelyinsulinsecretion.DiazoxidebindstotheSUR1
regulatory subunits and acts by keeping the KATP
channels open, thereby preventing insulin secretion.
Loss-of-function mutations in the ABCC8 and
KCNJ11 genes encoding the SUR1 and Kir6.2 subunits
of the KATPchannel lead to either a reduction in the
number of channels within the b-cell membrane or a
decrease in channel activity (2–5), and hence diazoxide
treatment is often ineffective. Some of these patients
may be managed with octreotide, but many will require
a partial or near total pancreatectomy. A minority of
cases have mutations with residual function that
correlate with response to diazoxide (6).
Diazoxide responsive HH can also result from
activating mutations in the GLUD1 gene which encodes
European Journal of Endocrinology (2010) 162 987–992ISSN 0804-4643
q 2010 European Society of EndocrinologyDOI: 10.1530/EJE-09-0861
Online version via www.eje-online.org
This is an Open Access article distributed under the terms of the European Journal of Endocrinology’s Re-use Licence which permits unrestricted
non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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the intra-mitochondrial enzyme, glutamate dehydro-
genase. The majority of patients with GLUD1 mutations
have hyperammonemia (HA) (7, 8). Rarer causes of
diazoxide responsive HH include mutations in the GCK
or HADH genes (9, 10). Activating GCK mutations show
a variable phenotype but patients may be diagnosed
outside the neonatal period, and some are responsive
to diazoxide (11). HADH mutations have been reported
in five patients, and these mutations typically cause
HH with associated defects in fatty acid oxidation
(10, 12, 13).
We have recently shown that loss-of-function
mutations in the HNF4A gene can also cause HH
(14). The clinical severity ranges from mild transient
hypoglycemia that does not require pharmacological
treatment to persistent HH treated with diazoxide for up
to 3 years (15, 16). Heterozygous HNF4A mutations
result in increased birth weight (median increase
790 g), macrosomia in 56%, and a form of maturity-
onset diabetes of the young (HNF4A MODY) that shows
sensitivity to low-dose sulfonylureas (14, 16). All
previous studies have described patients with hypogly-
cemia in families with known HNF4A mutations
recruited because of their history of diabetes (14, 15),
or selected for testing due to neonatal HH in a proband,
where the family history was consistent with HNF4A
MODY (16). The prevalence of HNF4A mutations in
patients referred for genetic testing due to a diagnosis of
HH has not been investigated. We now report the
genetic and clinical characteristics in a large cohort
(nZ220) of patients with diazoxide responsive HH.
Subjects and methods
We studied 220 patients with diazoxide responsive HH
who did not require pancreatectomy. Diazoxide respon-
siveness was defined as the ability to come off i.v. glucose
and maintain normoglycemia. Patients with evidence of
perinatal asphyxia were excluded from the cohort. The
cohort included referrals via the UK Genetic Testing
Network (http://www.ukgtn.nhs.uk) and international
cases (nZ111). Clinical data were provided via a
standard request form (www.diabetesgenes.org),
clinical letter of referral, or by case note review. The
age at diagnosis ranged from birth to 15 years (median
1 week), and 61% of the cohort were male (see Table 1
for clinical characteristics of the cohort). Macrosomia
was defined as a birth weight of R1.3 SDS (equivalent
to the 90th centile). The study was conducted in
accordance with the Declaration of Helsinki (2000).
Genetic analysis
Genomic DNAwas extracted from peripheral leukocytes
using standard procedures, and the coding exons
and intron/exon boundaries of the ABCC8, KCNJ11,
GCK, GLUD1, and HNF4A genes were amplified by
PCR (primers available on request). HNF4A analysis
included the coding exons 1d–10 and the P2 pancreatic
promoter. PCR products were sequenced using standard
methods on an ABI 3730 (Applied Biosystems,
Warrington, UK), and were compared to the published
sequence NM_000457.3 (exons 2–10) and AY680697
(exon 1d only) (17) using Mutation Surveyor v3.2
(SoftGenetics, State College, PA, USA). The order of
genetic testing depended on the clinical phenotype with
sequencing of the GLUD1 gene performed in all the
patients with HA. ABCC8, KCNJ11, GCK, and HNF4A
mutations were excluded in all the patients whose
genetic diagnosis was not known. No patients in our
cohort were reported to have defects in fatty acid
oxidation (increased levels of 3-hydroxyglutaric acid or
3-hydroxybutyryl-carnitine), and therefore genetic
analysis of the HADH gene was not indicated.
When an HNF4A mutation was identified, parents
were tested (if available) to establish the mode of
inheritance, and microsatellite analysis (PowerPlex 16
System, Promega) was undertaken to confirm de novo
mutations. Novel non-synonymous variants were tested
in ethnically matched control chromosomes.
Clinical studies
Clinical characteristics were obtained from patients’
hospital records with assistance from their physician.
HH was defined as a blood glucose level !3 mmol/l
Table 1 Clinical characteristics for the 220 patients with diazoxide responsive hyperinsulinemic hypoglycemia.
Total
cohort
HNF4A mutation
positive
KATPchannel
positive
GLUD1 mutation
positive
GCK mutation
positive
Unknown
etiology
Number of
patients
Age at
diagnosis
22011 33 132161
1 week
(1 day–24
weeks)
C0.17
(K1.1G1.28)
1 day
(1–2 days)
4 days
(1 day–4
weeks)
C1.27
(K0.03G2.94)
24 weeks
(5 days–30
weeks)
K0.29
(K1.08G1.13)
9 years
(3–15 years)
1 week
(1 day–26
weeks)
K0.19
(K1.2G0.97)
Birth weight
(SDS)
C2.4
(C1.4G3.8)
C0.74
(K0.88G2.4)
Data are provided for the total cohort and for probands grouped by their genetic etiology. Unless otherwise indicated, the data are represented by the median
(interquartile range). SDS for birth weights were calculated by comparing with the data from Child Growth Foundation LMS (19).
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with detectable serum insulin and/or c-peptide.
Phenotypic data are presented as median (interquartile
range),and comparative
Mann–Whitney U test.
statisticsusedthe
Results
Genetic results
The genetic etiology was determined in 59/220 (27%)
probands (Table 1). Thirty-three patients had a
mutation in one of the KATPchannel genes (5 KCNJ11
and 28 ABCC8; 4 with biallelic mutations). Thirteen
probands were heterozygous for a GLUD1 mutation
(patients previously reported (8)), and activating GCK
mutations were identified in two cases.
HNF4A mutations
A total of 11 different heterozygous HNF4A mutations
wereidentifiedin11probands(Fig.1andSupplementary
Table 1, see section on supplementary data given at
the end of this article). Two of these patients, with
IVS2-21AOG(c.264-21AOG)
(c.987_1003del) mutations have been reported pre-
viously (16). One mutation, Y16X (c.48COG), has
previously been identified in another patient with
hyperinsulinism (HI) (16), while the remaining eight
mutations are novel: R76W (c.226COT), R80W
(c.238COT), C106S (c.317GOC), M116I (c.317GOA),
andL330fsdel
L263P
L331_L332dup (c.992_997dupTGCTGC), and Q362X
(c.1084COT). L331_L332dup is likely to be pathogenic
since a single leucine duplication mutation (p.Leu332d-
up) has been identified in three unrelated MODY
probands (Sian Ellard, unpublished data and (18)).
Analysis of seven orthologous sequences demonstrated
that the five novel missense mutations occurred at
residues that are conserved through evolution, and
these mutations were not present in 300 ethnically
matched (Caucasian) control chromosomes.
(c.789TOC),Y319fs (c.953dupA),
HNF4A variants of uncertain significance
Four novelheterozygousHNF4Avariantswereidentified
in a further four probands: c.621C4AOG (IVS5nt
C4AOG), V94M (c.280GOA), S371R (c.1113COA),
and H378del (c.1133_1135delACC) (Supplementary
Table 1). In one patient, the c.621C4AOG variant was
inherited from their unaffected father (current age 33
years), in silico splicing prediction software suggested no
effect on splicing (http://www.fruitfly.org), and it was
present in 2/76 ethnically matched (Bangladeshi) control
chromosomes. Family member testing for the three re-
maining patients demonstrated inheritance of the variant
fromanunaffectedgrandparent(aged71,50,and52years
respectively) and parent. Normal blood glucose levels
and HbAlc were confirmed in two of these grandparents
(S371R and H378del; V94M not tested). The penetran-
ce of HNF4A mutations causing diabetes is estimated at
74% by the age of 50 years (Sarah Flanagan, Sian Ellard,
Y16X
L263Pc.264-21A>G*Y319fsL330fsdel17ins9* L331_L332dup
R76WR80WC106S M1161Q362X
N/N
N/N
M/N M/NM/N
M/N
M/N
M/N
N/N
M/N
N/N
N/N
N/N
N/NN/N N/NN/N
N/N
N/N
M/N
M/N
M/N
M/N
M/N
M/N
M/N
M/N
M/N
M/N
M/N
M/N
M/N
N/N
M/N
N/N
Not
tested
D
3.7kg (36 weeks)
>1 year
3.8kg (33 weeks)
4 years
Diabetes diagnosed at 12 yrs
3.8kg (40 weeks)
3 months
4.1kg (38 weeks)
>8 years
4.1kg (36 weeks)
>4 years
5.9kg (40 weeks)
>2 years
3.9kg (38 weeks)
18 months
3.5kg (35 weeks)
>3 years
3.6kg (39 weeks)
>10 months
4.6kg (40 weeks)
>10 months
4.1kg (40 weeks)
6 months
Not
tested
Not
tested
Not
tested
Not
tested
Figure 1 Partial pedigrees showing inheritance of HNF4A mutations in the 11 families. Circles represent females, and squares indicate
males. A circle with the letter D denotes an ovum donor. Probands are indicated by an arrow. Diagonal hatching denotes patients with
hyperinsulinism, vertical hatching represents gestational diabetes, and filled symbols show diabetic individuals. The genotype is given
below each symbol: M/N denotes a heterozygous HNF4A mutation, and N/N denotes a normal genotype. For each proband, birth weight
(gestation in weeks) and duration of diazoxide treatment are provided, Oindicates the minimum duration when treatment is ongoing. The
HNF4A mutation identified in each family is shown above each pedigree. Previously reported pedigrees are denoted by an asterisk* (16).
HNF4A gene mutations
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& Andrew Hattersley, unpublished data), and these
variantswerenotidentifiedinethnicallymatchedcontrol
chromosomes (300 Caucasian control chromosomes
tested for V94M; 130 Turkish control chromosomes
tested for S371R and H378del). We conclude that these
four novel variants are unlikely to be pathogenic.
Inheritance of HNF4A mutations
In 4/11 families, the mutation was inherited from a
diabeticparent(for pedigreesseeFig.1).Onepatienthad
inherited a Y319fs mutation from her unaffected father
(current age 39 years), but her paternal aunt who had
gestational diabetes at 30 years was also found to carry
the mutation. The proband with the L331_L332dup
mutation had inherited it from her unaffected mother,
but the maternal grandparents were not available for
testing. Four mutations, R76W, C106S, M116I, and
Q362X, were proven by microsatellite analysis to have
arisen de novo. In the remaining family, with a Y16X
mutation, the mode of inheritance could not be
establishedasthechildwasconceivedbyovumdonation.
Clinical characteristics of HNF4A mutation
carriers
Age at diagnosis was provided for 10/11 probands with
a HNF4A mutation, and all ten patients were diagnosed
within the first week of life (median age 1 day, range
1–7 days; Table 1). The majorityof patients (9/11) were
macrosomic as defined by a corrected birth weight of
R1.3 S.D.s from the mean. The median birth weight for
the 11 cases was C2.4 SDS (see Table 1 and Fig. 1). The
duration of diazoxide treatment for all 11 patients
ranged from 3 months to ongoing at 8 years, with seven
patients having persistent HI as defined by a require-
ment for diazoxide at the age of 1 year. Two of the
remaining probands are currently under 12 months of
age, and are still requiring diazoxide. One proband
subsequently developed diabetes at the age of 12 years
(Fig. 1). Two unaffected parents were found to be
heterozygous mutation carriers, but in the absence of a
formal OGTT, impaired glucose tolerance cannot be
excluded. None of the ten heterozygous relatives
reported a history of neonatal hypoglycemia.
Clinical characteristics by genetic etiology
The clinical characteristics of the probands were
compared according to genetic etiology (Table 1).
Patients with a HNF4A mutation presented earlier,
and were born heavier than patients with a GLUD1
mutation (1 day versus 24 weeks, PZ0.0006 and
C2.4 SDS versus K0.29 SDS, PZ0.0003 respectively).
No differences in the age at diagnosis or birth weight
were observed between patients with an HNF4A or KATP
channel mutations (1 day versus 4 days, PZ0.084 and
C2.4 SDS versus C1.27 SDS, PZ0.052).
Discussion
We identified a genetic etiology in 27% of patients with
diazoxide responsive HH. KATPchannel mutations were
most common, accounting for 15% of cases. HNF4A
mutations have only been reported previously in five
probands with diazoxide responsive neonatal hypogly-
cemia (14, 16), but we found HNF4A mutations in a
further nine cases, making this the third most common
genetic etiology within the cohort and the second most
common cause of isolated diazoxide responsive HH.
A further four novel heterozygous HNF4Avariants (one
intronic and three non-synonymous amino acid
substitutions) were identified, but are thought unlikely
to be pathogenic mutations.
HNF4A mutations were associated with an early age
of diagnosis (median 1 day) and increased birth weight
(median birth weight C2.4 SDS with macrosomia in
9/11) which is likely to result from increased insulin
secretion in utero. Seven of the eleven probands (64%)
did not have a diabetic parent, and in four cases, a
de novo mutation was confirmed. Therefore, the absence
of a family history of diabetes should not preclude
sequencing of the HNF4A gene in patients presenting
with diazoxide responsive HH.
Neonatal hypoglycemia has been reported in only a
minority of patients (11%) with HNF4A mutations
who were ascertained by their family history of MODY
(14, 15), and none of the ten heterozygous relatives in
our study were known to have had neonatal hypogly-
cemia. The reason(s) for the incomplete penetrance of
symptomatic hypoglycemia are not known, although it
appears to be a general feature rather than mutation
specific. It is also possible that some patients had
unrecognized hypoglycemia in the neonatal period. The
hyperinsulinemic HNF4A phenotype ranges from
increased birth weight (macrosomia in w50% mutation
carriers) to neonatal hypoglycemia managed by i.v.
glucose only for 1–9 days (14, 15), or neonatal
hypoglycemia requiring diazoxide therapy for between
3 months and 8 years (14, 16). It is therefore likely that
other environmental and genetic factors are influencing
the severity of the hyperinsulinemic phenotype associ-
ated with HNF4A mutations. The mechanism under-
lying the biphasic phenotype of neonatal hypoglycemia
with later diabetes is not known. It has been speculated
to result from differences in HNF-4a-dependent
temporal gene expression, or early hypersecretion of
insulin resulting in later b-cell exhaustion (14).
Patients with an HNF4A mutation were diagnosed
with HH within the first week of life (data available for
10/11 patients). The overlap in age at diagnosis and
birth weight between patients with HNF4A and KATP
channel mutations means that it is not possible to
distinguish between these two etiologies on an individ-
ual patient basis. Although diagnosis in the first week of
life and a family history of young-onset diabetes suggest
a HNF4A mutation, given the higher prevalence of KATP
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channel mutations and the high rate of diabetes
phenocopies in the population, we recommend sequen-
cing KCNJ11 and ABCC8 first, followed by HNF4A.
The frequency of HNF4A mutations approached that
ofGLUD1mutations(5vs5.9%).PatientswithaGLUD1
mutation were diagnosed later (median 24 weeks), were
of normal birth weight, and most (12/13) had HA.
However, the recent description of a patient with a
GLUD1 mutation, extreme protein sensitivity but
normal serum ammonium suggests that this prevalence
could be an underestimate (8). A genetic diagnosis was
onlypossiblefor27%ofpatients inthisstudy,suggesting
that there are more gene(s) harboring causative
mutations which remain to be identified in patients
with HH.
Inconclusion,wehaveshownthatHNF4Amutations
are a relatively common cause of diazoxide responsive
HH diagnosed in the first week of life. A genetic
diagnosis is important for these patients as it predicts
the likelihood of later sulfonylurea-sensitive diabetes
and a high risk of having macrosomic babies. We
therefore propose that HNF4A should be sequenced in
all the patients without a KATPchannel mutation who
present with diazoxide–responsive HH in the first week
of life irrespective of a family history of diabetes.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/
10.1530/EJE-09-0861.
Declaration of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of the research reported.
Funding
S E Flanagan is the Sir Graham Wilkins, Peninsula Medical School
Research Fellow, and O Rubio-Cabezas is supported by an ‘ Ayuda
para contratos post-Formacio ´n Sanitaria Especializada’ from the
‘Instituto de Salud Carlos III’ (FIS CM06/00013), Spain. S Ellard is a
member of the core staff within the NIHR funded Peninsula Clinical
Research Facility. This study was funded by the Wellcome Trust
(081188/A/06/Z).
Acknowledgements
The authors would like to thank Andrew Parrish, Annet Damhuis,
and Kevin Colclough for their technical assistance.
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