In vitro recovery of ATP-sensitive potassium channels in β-cells from patients with congenital hyperinsulinism of infancy.

Abstract

Congenital hyperinsulinism in infancy (CHI) is characterized by unregulated insulin secretion from pancreatic β-cells; severe forms are associated with defects in ABCC8 and KCNJ11 genes encoding sulfonylurea receptor 1 (SUR1) and Kir6.2 subunits, which form ATP-sensitive K(+) (K(ATP)) channels in β-cells. Diazoxide therapy often fails in the treatment of CHI and may be a result of reduced cell surface expression of K(ATP) channels. We hypothesized that conditions known to facilitate trafficking of cystic fibrosis transmembrane regulator (CFTR) and other proteins in recombinant expression systems might increase surface expression of K(ATP) channels in native CHI β-cells.
Tissue was isolated during pancreatectomy from eight patients with CHI and from adult cadaver organ donors. Patients were screened for mutations in ABCC8 and KCNJ11. Isolated β-cells were maintained at 37°C or 25°C and in the presence of 1) phorbol myristic acid, forskolin and 3-isobutyl-1-methylxanthine, 2) BPDZ 154, or 3) 4-phenylbutyrate. Surface expression of functional channels was assessed by patch-clamp electrophysiology.
Mutations in ABCC8 were detected for all patients tested (n = 7/8) and included three novel mutations. In five of eight patients, no changes in K(ATP) channel activity were observed under different cell culture conditions. However, in three patients, in vitro recovery of functional K(ATP) channels occurred. Here, we report the first cases of recovery of defective K(ATP) channels in human β-cells using modified cell culture conditions.
Our study establishes the principle that chemical modification of K(ATP) channel subunit trafficking could be of benefit for the future treatment of CHI.

Full text

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In Vitro Recovery of ATP-Sensitive Potassium Channels in
b-Cells From Patients With Congenital Hyperinsulinism
of Infancy
Philippa D. Powell,1Christine Bellanné-Chantelot,2Sarah E. Flanagan,3Sian Ellard,3
Raoul Rooman,4Khalid Hussain,5Mars Skae,6Peter Clayton,6Pascale de Lonlay,7
Mark J. Dunne,1and Karen E. Cosgrove1
OBJECTIVE—Congenital hyperinsulinism in infancy (CHI) is
characterized by unregulated insulin secretion from pancreatic
b-cells; severe forms are associated with defects in ABCC8 and
KCNJ11 genes encoding sulfonylurea receptor 1 (SUR1) and
Kir6.2 subunits, which form ATP-sensitive K+(KATP) channels
in b-cells. Diazoxide therapy often fails in the treatment of CHI
and may be a result of reduced cell surface expression of KATP
channels. We hypothesized that conditions known to facilitate
trafficking of cystic fibrosis transmembrane regulator (CFTR)
and other proteins in recombinant expression systems might in-
crease surface expression of KATPchannels in native CHI b-cells.
RESEARCH DESIGN AND METHODS—Tissue was isolated
during pancreatectomy from eight patients with CHI and from
adult cadaver organ donors. Patients were screened for muta-
tions in ABCC8 and KCNJ11. Isolated b-cells were maintained at
37°C or 25°C and in the presence of 1) phorbol myristic acid,
forskolin and 3-isobutyl-1-methylxanthine, 2) BPDZ 154, or 3) 4-
phenylbutyrate. Surface expression of functional channels was
assessed by patch-clamp electrophysiology.
RESULTS—Mutations in ABCC8 were detected for all patients
tested (n = 7/8) and included three novel mutations. In five of
eight patients, no changes in KATPchannel activity were ob-
served under different cell culture conditions. However, in
three patients, in vitro recovery of functional KATPchannels
occurred. Here, we report the first cases of recovery of defec-
tive KATPchannels in human b-cells using modified cell culture
conditions.
CONCLUSIONS—Our study establishes the principle that
chemical modification of KATPchannel subunit trafficking could
be of benefit for the future treatment of CHI. Diabetes 60:1223–
1228, 2011
C
pancreas (focal CHI) as a result of somatic loss of mater-
nal alleles and expression of paternal mutations or may be
diffuse and inherited with Mendelian genetics (1). The
most severe forms of CHI are caused by loss-of-function
mutations in the genes encoding the subunits of the ATP-
sensitive K+(KATP) channel: ABCC8 (encoding sulfonyl-
urea receptor 1 [SUR1]) and KCNJ11 (encoding Kir6.2);
both genes are located on chromosome 11p15 (1,2). In
b-cells, these channels are complexes consisting of four
SUR1 and four Kir6.2 subunits, which assemble in the
endoplasmic reticulum (ER) and are glycosylated and
modified as they pass through the cis-, medial-, and trans-
golgi network before being expressed at the cell surface.
Although studies of b-cells from patients with CHI have
proved the link between ABCC8 and KCNJ11 gene defects
and loss-of-function of KATPchannels (3,4), recombinant
techniques have been used to further understand the
mechanisms of this loss. Disease-causing mutations engi-
neered in rodent SUR1 and Kir6.2 have been expressed in
mammalian and nonmammalian expression systems (e.g.,
COSm6 cell line, Xenopus oocytes) and found to cause
incorrect assembly of the channel complex, impaired
trafficking from the ER, or loss of nucleotide regulation (4–
8). These experiments also demonstrated the importance
of specific amino acid motifs present on both SUR1 and
Kir6.2 for anterograde and retrograde trafficking of KATP
channels (reviewed in [1,9]). Similar approaches have been
used to demonstrate that some CHI-related defects can be
overcome by altering the cell culture environment
(6,10,11). However, to date no studies have examined
methods to recover defective KATP channels in native
tissue, which could be of relevance in the future treat-
ment of CHI. We now report for the first time rescue of
KATPchannels in patient b-cells using chemical media-
tors, kinase activators, and reduced temperature.
ongenital hyperinsulinism in infancy (CHI) is
characterized by severe hypoglycemia, which
manifests in the neonatal period. The disease
may be limited to a localized region of the
RESEARCH DESIGN AND METHODS
Tissue was isolated (with permission) from cadaver human organ donors and
from eight patients with CHI who required subtotal pancreatectomy for in-
tractable hypoglycemia. Table 1 summarizes patient details. Islets of Langer-
hans were isolated as previously described (3,12). Total RNA was extracted
from islets and cells using TRIzol reagents (Invitrogen, Paisley, U.K.) and
subjected to RT-PCR using primers designed and tested in-house. All PCR
reactions consisted of an initial denaturation step of 94°C for 5 min followed
by 35 cycles of 94°C for 1 min, Ta°C for 1 min, and 72°C for 1 min followed by
From the1Faculty of Life Sciences, University of Manchester, Manchester,
U.K.; the2Department of Genetics, Groupe Hospitalier Pitié-Salpétrière,
Université Pierre et Marie Curie, Paris, France; the3Institute of Biomedical
and Clinical Science, Peninsula Medical School, University of Exeter, Exe-
ter, U.K.; the4Department of Pediatrics, Antwerp University Hospital, Uni-
versiteitsplein 1, Wilrijk, Antwerp, Belgium; the5Institute of Child Health,
Great Ormond Street Hospital, London, U.K.; the6Department of Endocri-
nology, Royal Manchester Children’s Hospital, Manchester, U.K.; and the
7Hôpital Necker Enfants Malades, Reference Centre of Metabolic Diseases,
Faculté de Médecine, Paris, France.
Corresponding author: Karen E. Cosgrove, karen.e.cosgrove@manchester.
ac.uk.
Received 12 October 2010 and accepted 28 January 2011.
DOI: 10.2337/db10-1443
? 2011 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
diabetes.diabetesjournals.orgDIABETES, VOL. 60, APRIL 2011 1223
BRIEF REPORT

Page 2

a final elongation stage of 72°C for 10 min. For detection of mRNA encoding
KCNJ11 (Genbank Accession number NM_000525), primer sequences were as
follows: (F) ACA AGA ACA TCC GGG AGC, (R) ACA CGT AGC ATG AAG
CAG AGG with Ta 60°C. For detection of three different regions of ABCC8
(Genbank Accession number AF087138), primer sequences were as follows:
(F) AGA CTG CCC ACA AGA AGC (bases 748–765), (R) AGA AGA AAA ACC
ACA TGA (bases 1335–1317) with Ta 58°C; (F) GAC CCA CAA GCT ACA GTA
CC (bases 2693–2712), (R) CAC TCC ACA GTG ACA GAC G (bases 3295–3276)
with Ta 58°C; (F) TCT CGA ATA CAC AGA CTC C (bases 3713–3731), (R) ACA
GTG TGC TAT CTG AGC (bases 4386–4368) with Ta 60°C. PCR products were
resolved on 1.2% agarose gel prepared with Tris-borate EDTA buffer (Fisher
Scientific, Loughborough, U.K.) containing 1.2 mg/mL ethidium bromide
(Promega, Madison, WI). Bands were visualized and photographed under ul-
traviolet light.
Groups of dispersed islet cells were cultured at either 37°C or 25°C in
a humidified atmosphere of 5% CO2/air mixture for a minimum of 16 h with or
without addition of 10 mmol/L BPDZ 154 (B. Pirotte, Universite de Liege, Bel-
gium), 100 mmol/L diazoxide (Sigma, Poole,U.K.), or 2.5 mmol/L 4-phenylbutyrate
(4-PB) (Triple Crown America, Perkasie, PA). Alternatively, cells were in-
cubated for 1 h before experimentation at 37°C with 100 mmol/L 3-isobutyl-1-
methylxanthine (IBMX), 2 mmol/L forskolin, and 10 nmol/L phorbol myristic
acid (PMA; Sigma, Poole, U.K.). All data were obtained using the cell-attached
or inside-out recording configurations of the patch-clamp technique as pre-
viously described (3,12). In all experiments, the integrity of the recording
configuration was assessed by inducing activation of the high conductance
Ca2+- and voltage-gated K+channel by perfusing the inside face of the cell
membrane with a Ca2+-containing solution (3,12). To select for KATPchannel
currents, the pipette contained a standard NaCl-rich bathing solution con-
taining (in mol/L) 140 NaCl, 4.7 KCl, 2.5 CaCl2, 1.13 MgCl2, 10 HEPES, 2.5
glucose (pH 7.4 with NaOH), and the bath solution contained (in mol/L) 140
KCl, 10 NaCl, 1.13 MgCl2, 1 EGTA, 2.5 glucose, and 10 HEPES (pH 7.2 with
KOH) for all recordings. Reagents were added at the concentrations indicated
in the text. Diazoxide and BPDZ 154 were added to working solutions from
a stock solution dissolved in DMSO (Sigma) giving a final concentration of less
than 0.1% vol/vol DMSO. Statistical comparisons were made using Student
t tests or ANOVA with values of P , 0.05 taken to be significant. All data are
expressed as mean values 6 SEM.
RESULTS
In this study, insulin-secreting cells were investigated from
eight patients with CHI who underwent pancreatectomy
following failure to respond to medical treatment. Per-
mission for genotype analysis was obtained for seven
patients (#2–#8) and all were found to carry defects in
ABCC8 (Table 1; Fig. 2A). Genotyping was declined for
patient #1; therefore RNA was isolated from dispersed
islets and RT-PCR analysis for three regions of ABCC8,
and KCNJ11 carried out. Primers directed to discrete
regions of the N-terminus of ABCC8 and for KCNJ11
amplified appropriate products, but failed to do so when
primers were directed toward the center or COOH-terminal
regions of ABCC8 mRNA.
In isolated inside-out patches of membrane from control
human b-cells, KATPchannels were spontaneously open and
activated in the presence of ATP by ADP (500 mmol/L) and
diazoxide (200 mmol/L) (Fig. 1A). In all eight CHI cases, we
found that SUR1 defects were linked to a functional loss of
KATPchannels in b-cells since the average peak current
varied between patients from zero to approximately 10%
(n = 109 recordings) of the control values obtained from
human adult b-cells (n = 189). These data are similar to
our previously reported findings (3,4,12). Figure 1A (center
panel) shows typical recordings of b-cells from patient #1
when the cells were maintained under standard cell cul-
ture conditions (i.e., at 37°C without additional supple-
mentation of the tissue culture medium). In contrast with
control recordings, no operational or normally regulated
channels were recorded in either intact cells (n = 8/8) or
isolated inside-out patches (n = 8/8; Fig. 1A). However,
when b-cells from the patient were maintained at 25°C for
a minimum of 16 h, KATPchannels were restored (n = 7/7)
and were found to be responsive to ADP and diazoxide
(Fig. 1A and B). This modification did not affect the ac-
tivity of KATPchannels in control human b-cells (Fig. 1C).
Following incubation at 25°C with 2.5 mmol/L 4-PB, no
further improvements were observed for patient #1 b-cells.
In contrast, we did see an increase in KATPchannel activity
in ABCC8 (Arg998X/Ser1449dup) b-cells (patient #3)
maintained at 37°C with 4-PB. This procedure led to the
appearance of KATPchannel currents, which were increased
by 4.4 ± 2-fold in 3/4 cells compared with control values, but
was not associated with the restoration of nucleotide-
dependent activation of channels (n = 3). Due to limited
tissue availability, we did not test effects of incubation at
25°C in patient #3 b-cells.
A similar pattern involving recovery of functional KATP
channel activity was found in ABCC8 (c.1467+5G.A)
b-cells (patient #2), first when exposed to a combination
of IBMX (100 mmol/L), PMA (10 nmol/L), and forskolin
(2 mmol/L) for 1 h at 37°C immediately before the experi-
ment (n = 4/4; Fig. 1D) and second when cells were main-
tained at 37°C for 24–48 h with BPDZ 154 (10 mmol/L)
added to the cell culture medium (n = 6/6; Fig. 1D). BPDZ
154-induced recovery of channel activity was also seen in
ABCC8 (Arg998X/Ser1449dup) b-cells (patient #3; n = 4
recordings, 4.3 6 1-fold increase in activity), but this was
not associated with 0.5 mmol/L ATP-induced inhibition of
channels (n = 4).
DISCUSSION
In healthy b-cells, glucose-induced closure of KATPchan-
nels is a key regulator of stimulus-secretion coupling. The
TABLE 1
CHI patient tissue details
Patient (#)Age at surgeryHistology Gene defectGenotypeReference
1
2
3
4
5
6
7
8
10 weeks
12 weeks
12 weeks
12 weeks
7 weeks
3.5 years
12 months
4 weeks
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Focal
Presumed Homozygous, ABCC8
Homozygous, ABCC8
Compound Heterozygous, ABCC8
Homozygous, ABCC8
Homozygous, ABCC8
Compound Heterozygous, ABCC8
Compound Heterozygous, ABCC8
Paternal uniparental isodisomy, ABCC8
Unknown
c.1467+5G.A
p.Arg998X/p.Ser1449dup
c.3992–9G.A
c.3992–9G.A
p.Gly70Glu/p.Arg1419Gly
p.Lys242fs/p.Arg1437X
p.Arg598X
—
Novel mutation
25
3
3
3
Novel mutations
3
Seven patients were found to have diffuse CHI, and one patient was defined as focal CHI. Consent for genotyping was obtained from seven
patients, and all were found to carry defects in the ABCC8 gene. Islet mRNA was isolated from patient #1, and RT-PCR suggested that CHI was
caused by defects in ABCC8 expression and not KCNJ11. Where CHI causing mutations have been previously described, see references
indicated.
KATPCHANNEL RESCUE IN CHI
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FIG. 1. In vitro recovery of KATPchannels in CHI b-cells. A: Data from control (adult) human b-cells to illustrate the modulation of ATP-inhibited
channels (open probability [Po] = 0.03 6 0.02, n = 11 cells), by ADP (0.5 mol/L, Po = 0.1 6 0.03, n = 9) and diazoxide (0.2 mol/L, Po = 0.3 6 0.05,
n = 5) and from CHI b-cells maintained at either 37°C (standard conditions) or at 25°C. Note the marked increase in channel activity in CHI b-cells
maintained at low temperature and how ADP and diazoxide induce an increase in the activity of channels in the presence of ATP (0.5 mol/L). B: The
effects of cells maintained at 25°C on channel open probability. In CHI b-cells, no KATPchannels were recorded under standard conditions (open
probability = 0, ■) but were readily observed in cells maintained at the lower temperature (◇). C: Maintaining control b-cells at 25°C had no
effect on the average magnitude of KATPchannels in isolated patches (n = 7/7). D: The recovery of KATPchannels in ABCC8 (c.1467+5G>A) b-cells
following short-term exposure to IBMX (0.1 mmol/L), forskolin (Fsk; 2 mmol/L), and PMA (10 nmol/L) for 1 h or long-term exposure to BPDZ 154
(10 mmol/L). Representative single-channel current data are shown alongside amplitude histogram profiles following Gaussian fitting of the data
(smoothed lines). Note how in control b-cells >99% of the events occur at 0 pA (indicated by the dotted line) consistent with the absence of
functional channels, but that following treatment open events are apparent, which are sensitive to ATP (0.5 mmol/L).
P.D. POWELL AND ASSOCIATES
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pivotal role of these channels in this process is central to
our understanding of how loss-of-function mutations in
ABCC8 or KCNJ11 cause CHI (1), whereas gain-of-function
mutations lead to neonatal diabetes (2). Gene defects that
render KATPchannels less sensitive to ATP prevent correct
ATP:ADP sensing and can cause impaired insulin secretion
and diabetes (2). By contrast, decreased KATP channel
density and/or loss of ADP sensitivity is associated with
inappropriate electrical activity and uncontrolled insulin
release leading to CHI (3). Here we report recovery of
KATPchannel activity in b-cells from patients with CHI
following incubation with chemical mediators, kinase
activators, and reduced temperature to increase trafficking
of the channels to the cell membrane. In the field of cystic
fibrosis (CF) it has long been recognized that enhancement
of cell surface trafficking of the defective ATP-binding
cassette (ABC) protein cystic fibrosis transmembrane
regulator (CFTR) may be of therapeutic value (13). Most
CF patients (90%) carry a deletion of phenylalanine at
position 508 (DF508) of ABCC7, which encodes CFTR
(14). This mutation results in misfolding of the protein
leading to ER retention where it is subsequently poly-
ubiquitinated and targeted for degradation (15). Incubation
of cells engineered to express DF508 CFTR at 25°C was
found to result in recovery of CFTR activity by stabilizing
protein folding in the ER (16). We found that low tem-
perature incubation also led to recovery of mutant KATP
channel activity in CHI patient tissue. By analogy with
studies of the processing of mutant CFTR (16), we believe
that low temperature incubation of CHI b-cells stabilized
KATPchannel subunits during folding and assembly in the
ER, and thereby allowed the protein to escape the ER
quality control measures. During the biogenesis of KATP
channels, it has recently been shown that SUR1 can in-
teract with heat shock cognate (HSC)70, heat shock pro-
tein (HSP)90, and HSP40 (17). We speculate that the
success of low temperature incubation arises through
disruption of mutant KATPchannel degradation involving
HSC70, and this results in increased functional expression
of the channel at the cell membrane (Fig. 2B). We explored
this potential mechanism further using 4-PB, which
increases the trafficking of mutant CFTR via down-
regulation of HSC70 expression (18). Similarly, in our
studies 4-PB also led to an increase in KATPchannel ac-
tivity providing further support to the role of HSC70 in
degradation of mutant KATPchannels (17).
In a separate set of experiments, we found that in-
cubation of CHI b-cells with a kinase activating cocktail
also led to KATPchannel recovery. This effect could be due
to signaling pathways associated with activation of protein
kinase C, or the downstream effects of increased cytosolic
cAMP, such as activation of protein kinase A or other
cAMP-dependent signaling pathways including cAMP ki-
nase, A-kinase anchor proteins, cAMP regulatory element–
binding protein (CREB), and cAMP-regulated guanine nu-
cleotide exchange factors (cAMP-GEFs). Modulation of
kinase activity has previously been reported to increase
trafficking of other ion channels and transporters such as
the bile salt export pump (also a member of the ABC pro-
tein family; 19), aquaporin-2 (20), and CFTR (21). It has also
been reported that PKC activation is associated with a re-
duction in surface KATPchannel expression as a result of
a reduction in recycling of endocytic vesicles and the di-
version of KATPchannel-containing vesicles to lysosomal
FIG. 2. KATPchannel structure and trafficking in b-cells. A: A consensus model of the structure of SUR1 (top) with predicted transmembrane
domains (numbered 1–17), extracellular NH2terminus, and intracellular COOH terminus. Shaded rectangles represent the intracellular nucleotide
binding domains. Predicted sites of mutations for patient #3 are indicated (black stars); note that the intronic mutation c.1467+5G>5 does not
have a predicted site on SUR1. ○, known anterograde (forward) trafficking motifs on SUR1; ●, the RKR retention motif, which must be masked by
correct channel assembly to permit forward trafficking. The intron/exon structure of ABCC8 is shown below with shaded rectangles marking the
exons predicted to encode the nucleotide binding domains. B: The trafficking of KATPchannel proteins (black elipses) in b-cells. Gray arrows
indicate suggested mechanisms of compounds and conditions described in this study to increase forward trafficking of KATPchannels. PM, plasma
membrane; Endo, endosome; Lys, lysosome.
KATPCHANNEL RESCUE IN CHI
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degradation (22). The overall positive effect of kinase acti-
vation in CHI b-cells suggests that activation of cAMP-
dependent kinases or similar mechanisms may have led
to increased expression or function of KATP channels
(Fig. 2B). Further studies are required to characterize
the precise signaling pathways involved.
Although diazoxide has previously been reported to in-
crease trafficking of KATPchannels in recombinant sys-
tems (6), the effects on trafficking of the related compound
BPDZ 154, a more potent KATPchannel agonist (12), have
not been investigated previously. In this study we found
that BPDZ 154 caused recovery of KATPchannels in CHI
b-cells suggesting that the compound, like diazoxide, may
influence channel trafficking in addition to opening KATP
channels. In recombinant cells several channel modulators
have now been reported to act as “chemical chaperones”
by mediating the recovery of defective channels (6,10),
including diabetes-causing mutations (11). Although the
mechanisms responsible for these actions have not been
fully investigated, the agents are thought to stabilize bio-
genesis of the channel complex rather than the SUR1
subunit and thereby facilitate ER exit of the complex. A
similar action may also explain the long-term effects of
BPDZ 154 on CHI b-cells (Fig. 2B).
Our series of experiments also included five cases of CHI
in which either short- or long-term modification of the cell
culture conditions failed to evoke any increase in the
expression or regulation of KATPchannels in CHI b-cells
(n = 51 experiments). This is not surprising since ABCC8
(or KCNJ11) defects will alter the protein in many dif-
ferent ways and not all will have configurations that en-
able the protein to be potentially liberated from the ER.
Indeed, in recombinant studies several ABCC8 mutations
have been shown to be resistant to chemical chaperones
(5,8,10).
Little is known about the effects of splice site mutations
in ABCC8 on the subsequent expression of SUR1 protein.
However, our results demonstrate that the presence of
splice-site mutations does not necessarily lead to the ab-
solute loss of channel protein since patients #4 and #5
demonstrated recordable (but defective) KATP channel
activity in untreated cells. We have no information on the
effects of the mutations reported in our study on tran-
scription and translation efficiency of the ABCC8 gene and
cannot therefore rule out that the culture conditions we
used somehow modified or stabilized these processes,
leading to apparent KATPchannel recovery in some of our
experiments, including patient #2 b-cells expressing a splice
site mutation.
In summary, we have demonstrated rescue of KATP
channel activity for the first time in pancreatic b-cells
isolated from patients with congenital hyperinsulinism.
Clinically, enhancement of mutant KATPchannel traffick-
ing to the cell membrane of b-cells could be beneficial for
the treatment of CHI, especially if these channels retained
regulatory properties or could be activated by KATPchan-
nel agonists. With the introduction of rapid genetic testing
in CHI, pharmacogenomics is becoming increasingly re-
alistic for tailoring drug treatment to individuals. Therefore
it is possible that once the gene mutations causing CHI are
identified, optimization of trafficking conditions for each
patient may permit successful enhancement of KATP
channel activity in some cases. By analogy with CF where
large scale screening efforts for small molecule “correc-
tors” of CFTR trafficking have been successful (23), this
could eventually have an impact on clinical treatment of
CHI. Indeed, several pharmacological modulators of traf-
ficking pathways are currently undergoing clinical trials
for the treatment of other diseases (24).
ACKNOWLEDGMENTS
This work was supported in part by the NIHR Manchester
Biomedical Research Centre (to K.E.C., P.C., and M.J.D.)
and the Wellcome Trust (081188/A/06/Z to K.H., S.E., and
S.E.F.). K.E.C. is a Research Councils U.K. Academic
Research Fellow. S.E.F. is the Sir Graham Wilkins,
Peninsula Medical School Research Fellow, and S.E. is
a member of the core staff within the NIHR-funded
Peninsula Clinical Research Facility.
No potential conflicts of interest relevant to this article
were reported.
P.D.P., C.B.-C., and S.E.F. researched and provided data.
S.E., R.R., and K.H. contributed to data analysis and
discussion. M.S. researched and provided data. P.C. and
P.d.L. contributed to data analysis and discussion. M.J.D.
contributed to data analysis and discussion and wrote the
manuscript. K.E.C. researched and provided data, contrib-
uted to data analysis and discussion, and wrote the
manuscript.
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