DOI: 10.5061/dryad.95x69p908
title: Supplemental data from: Determinants of hyperinsulinism severity in children with Beckwith-Wiedemann Syndrome

description: Context: Congenital hyperinsulinism (HI) is a serious clinical feature of
 Beckwith-Wiedemann syndrome (BWS) causing severe hypoglycemia. The
 relationship between BWS genotypes and HI severity is not well understood.
 Objective: Investigate the relationship between molecular determinants of
 patients with BWS and HI with measures of HI severity. Design:
 Retrospective cohort study including 85 children from 2009-2024. Setting:
 All patients evaluated at single, tertiary care center. Patients: BWS
 genotype frequency included 41 children with pUPD11, 24 with IC2 LOM,
 eight with 11p15 chromosomal anomalies, six with GWpUPD, four with IC1
 GOM, and two with CDKN1C. Interventions: Retrospectively reviewed
 interventions included maximum glucose infusion rate (max GIR), diazoxide
 responsiveness, and surgery. Main Outcome Measures: Primary outcome was
 association between BWS genotypes and measures of HI severity. Secondary
 outcomes included the relationship between pUPD11 length and presence of
 K-ATP variants with diazoxide responsiveness and surgical need. Results:
 Significant differences presented among genotypes in max GIR (p = 0.004),
 enteral dextrose requirements (p = 0.029), and pancreatectomy (p = 0.012).
 Most patients with IC2 LOM, IC1 GOM or CDKN1C were diazoxide responsive
 and did not require surgery. Patients with pUPD11 were more likely to be
 diazoxide unresponsive and require surgery, especially if pUPD11 length
 extended into the K-ATP gene region and if a pathogenic variant in the
 ABCC8 or KCNJ11 was present. Conclusion: Patients with pUPD11 experience
 more severe HI, while patients with IC2 LOM, IC1 GOM, and CDKN1C exhibit
 milder disease. Based on our findings, we designed a genetic testing
 algorithm to guide clinical management.
description: <em>Patient Cohort</em> All
 patients included in this study received care at the Children’s Hospital
 of Philadelphia Congenital Hyperinsulinism Center. A retrospective chart
 review was conducted on medical records of patients with molecularly
 confirmed BWS and a diagnosis of hyperinsulinism between 2009 and 2024.
 Among all patients with BWS cared for at the Children’s Hospital of
 Philadelphia between 2009 to 2024, only those with a molecularly confirmed
 diagnosis were eligible for inclusion and patients with only a clinical
 diagnosis of BWS were excluded. From this molecularly confirmed BWS
 cohort, patients were included if they also had a diagnosis of
 hyperinsulinism. The diagnosis of HI was established when hypoglycemia
 persisted beyond the first week of life and was supported by additional
 criteria such as increased glucose requirement, inappropriately low plasma
 concentrations of beta-hydroxybutyrate and free fatty acids, and a
 glycemic response to glucagon. Molecular testing via single nucleotide
 polymorphism (SNP) array and chromosome 11p15 methylation analysis was
 performed on various tissue types including blood, skin, pancreas, and/or
 liver. Each patient’s molecular subtype was classified as IC2 LOM, pUPD11,
 IC1 GOM, <em>CDKN1C</em>, GWpUPD, or an 11p15 chromosomal
 anomaly (T<strong>able 1</strong>). Patients with 11p15
 chromosomal anomalies had either a deletion, duplication, partial trisomy,
 or translocation affecting the 11p15 region. Methylation levels at IC1 and
 IC2 were assessed using allele-specific methylated multiplex quantitative
 real-time PCR, with the proportion of cells with exhibiting altered
 methylation calculated as previously described. Methylation testing
 results on blood were reviewed, and the percentage of mosaicism was
 calculated using established methodology^ ^(<strong>Supplementary
 Table 1</strong>). Molecular testing for hyperinsulinism focused on
 Sanger sequencing of <em>ABCC8</em>,
 <em>KCNJ11</em>, and <em>GCK</em> which accounted
 for all pathogenic variants identified in this cohort, despite expansion
 of additional genes to hyperinsulinism panels as the study period
 progressed. BWS clinical scores were calculated for each patient according
 to the BWS international consensus guidelines. BWS cardinal and suggestive
 features are also listed in <strong>Supplementary Table
 2.</strong> Continuous enteral dextrose support via gastrostomy tube
 to maintain euglycemia was administered alone or in combination with other
 therapies to allow for at-home management in patients who could not be
 weaned from glucose support in the hospital, and the total number of
 patients was captured for analysis (<strong>Table 2</strong>).
 Diazoxide responsiveness was defined as the ability to maintain plasma
 glucose concentrations &gt;70 mg/dL (3.9 mmol/L), after an adequate
 dose of diazoxide (ranging from 5 – 15 mg/kg/day depending on the
 patient’s age and divided into two doses), for at least 12 hours of
 fasting and/or generate appropriate hyperketonemia (plasma
 beta-hydroxybutyrate &gt;1.8 mmol/L) before development of plasma
 glucose &lt;50-60 mg/dL (2.8-3.3 mmol/L). Chlorothiazide, a diuretic,
 was commonly used concurrently with diazoxide to mitigate side effects
 such as fluid retention. All clinical data was abstracted from medical
 charts in adherence with IRB 19-016549 and IRB 13-010658.
 <em>Statistical Analysis</em>
 Statistical analyses were performed using R v4.4.2. Associations
 between the BWS molecular subtype and continuous variables including
 maximum glucose infusion rate (GIR), percentage requiring enteral
 dextrose, frequency of pancreatectomy, BWS clinical score, and blood
 mosaicism percentage were examined using Kruskal-Wallis or chi-square
 tests. Potential outliers were assessed using Grubbs’ test and the
 interquartile range (Tukey) method. All extreme values were verified
 against source records. Logistic regression analysis
 was conducted to examine factors influencing diazoxide responsiveness and
 the need for pancreatectomy in this patient population. Firth’s logistic
 regression, which applies penalized maximum likelihood estimation to
 mitigate bias in rare disease data and unbalanced sample distributions,
 was used to model likelihood of diazoxide responsiveness and the need for
 pancreatectomy. To refine analysis, the pUPD11 subtype was further
 stratified into three groups (<strong>Figure
 1</strong>): 1.    
 <strong>pUPD11-LOH/KATP</strong>: Patients with the pUPD11
 subtype with extended loss of heterozygosity encompassing the
 <em>ABCC8</em> & <em>KCNJ11</em> gene
 region and with a confirmed pathogenic variant in
 <em>ABCC8</em> or <em>KCNJ11</em>.
 2.     <strong>pUPD11-LOH</strong>: Patients with the
 pUPD11 subtype exhibiting extended LOH encompassing the
 <em>ABCC8</em> & <em>KCNJ11</em> gene
 region, but without a detected pathogenic <em>ABCC8</em> or
 <em>KCNJ11</em> variant. 3.    
 <strong>pUPD11</strong>: Patient with the pUPD11 subtype in
 which the LOH does not extend into the <em>ABCC8</em> or
 <em>KCNJ11</em> gene region and without a pathogenic
 <em>ABCC8</em> or <em>KCNJ11</em>
 variant. The stratification of pUPD11 was made to
 compare the effects of each molecular determinant on specific outcomes of
 hyperinsulinism severity. Patients in the IC2 LOM group were chosen as the
 reference group due to the sizeable cohort and their predominantly
 diazoxide-responsive nature, with some variation. SNP arrays, performed at
 our institution, were analyzed to confirm the length of heterozygosity
 loss in each patient with pUPD11. Pre-specified exclusion criteria
 comprised the exclusion of patients with a diagnosis of 11p15 chromosomal
 anomaly and GWpUPD from this regression analysis due to the increased
 complexity of these subtypes, which made meaningful comparisons
 challenging. For the diazoxide responsiveness analysis, patients who had
 not undergone a diazoxide trial or had their treatment with diazoxide
 discontinued due to side effects, were excluded.
description: # Supplemental data from: Determinants of hyperinsulinism severity in
 children with Beckwith-Wiedemann Syndrome Dataset DOI:
 [10.5061/dryad.95x69p908](https://doi.org/10.5061/dryad.95x69p908) ##
 Description of the data and file structure Supplemental tables and figures
 are included with legends, this includes all data for this publication.
 Supplementary Table 1. Percent Mosaicism Calculations, csv document
 Supplementary Table 2: Cardinal and Suggestive Features of BWS, csv
 document Supplementary Table 3. Clinical Features of BWS-Hyperinsulinism
 Cohort by Molecular Subtype, csv document Supplementary Table 4. HI
 Pathogenic Variants in Novel Cohort, csv document Supplementary Table 5.
 Pancreatectomy Extent and Post-operative Treatments, csv document
 Supplementary Table 6. Sensitivity and Specificity of Firth’s Logistic
 Regression, csv document Supplementary Figure 1. HI Therapeutic
 Considerations by BWS Molecular Subtype, pdf figure Supplementary Figure
 2. Receiver operating characteristic curves of Diazoxide Responsive and
 Pancreatectomy Analysis, pdf figure ## Code/software None ## Access
 information Other publicly accessible locations of the data: * None Data
 was derived from the following sources: * n/a ## Human subjects data We
 received explicit consent from our participants to publish the
 de-identified data in the public domain, and all data when entered into
 the database are de-indentifed and the data tables are generated with
 these deidentifed data that cannot be re-linked to specific patients.