Case Report: Two Distinct Focal Congenital Hyperinsulinism Lesions Resulting From Separate Genetic Events

Focal hyperinsulinism (HI) comprises nearly 50% of all surgically treated HI cases and is cured if the focal lesion can be completely resected. Pre-operative localization of the lesion is thus critical. Few cases of hyperinsulinism with multiple focal lesions have been reported, and assessment of the molecular mechanisms driving this rare occurrence has been limited. We present two cases of multifocal HI, each resulting from two independent, pancreatic focal lesions. 18Fluoro-dihydroxyphenylalanine positron emission tomography/computed tomography detected both lesions preoperatively in one patient, whereas identification of the second lesion was an incidental finding during surgical exploration in the other. Complete resection of the focal lesions resulted in cure of the HI in both cases. In each patient, genetic testing of the individual focal lesions revealed different regions of loss of heterozygosity for the maternal 11p15 allele, confirming that each lesion arose from independent somatic events in the setting of a paternally inherited germline ABCC8 mutation. These cases highlight the importance of a multidisciplinary and personalized approach to the management of infants with HI.


INTRODUCTION
Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycemia in infants and children (1). Prompt diagnosis and initiation of appropriate treatment are crucial to mitigate risk of permanent hypoglycemic brain damage. Inactivating mutations in the genes encoding the β-cell adenosine triphosphate (ATP)-sensitive potassium (K ATP ) channel, ABCC8 (SUR1) and KCNJ11 (KIR6.2), located on chromosome 11p15.1, are responsible for the most common forms of HI (2). K ATP -HI can be classified into two distinct histological forms: a diffuse form, in which all of the pancreatic β-cells are affected, and a focal form of localized islet hyperplasia and dysfunction.
While focal HI is more likely to present at an older age and with hypoglycemic seizures than diffuse HI, these two histological forms cannot be distinguished by clinical presentation alone (3). Differentiating between focal and diffuse HI is of paramount importance as surgical excision of the focal lesion is curative. In contrast, the severity of diffuse HI is ameliorated, but not cured, by near-total pancreatectomy, which carries additional risks of diabetes and exocrine pancreatic insufficiency (4).
Focal HI occurs via a "two-hit" mechanism, following the model originally described by Knudson (5), requiring paternal transmission of a recessive loss-of-function mutation in ABCC8 or KCNJ11 and a pancreas-limited somatic loss of the maternal 11p15 region compensated by paternal isodisomy (6). Somatic loss of heterozygosity (LOH) leads to imbalanced expression of imprinted genes that regulate cell growth, including IGF2, H19, and CDKN1C, resulting in the histological findings of focal adenomatous hyperplasia (6). A single paternally inherited recessive K ATP mutation has a 94% positive predictive value for focal HI (2). In these cases, preoperative 18 fluoro-dihydroxyphenylalanine positron emission tomography/computed tomography ( 18 F-DOPA PET/CT) is utilized to localize the focal lesion and guide surgical excision (7). Owing to the mechanism of focal lesion development, the occurrence of multiple focal lesions is exceedingly rare. Few cases of HI with multiple focal lesions have been reported to date (8)(9)(10)(11)(12). Of these reports, only one included molecular genetic information confirming that the two lesions, one pancreatic and one ectopic, arose from independent somatic events (8). We describe two cases of HI due to distinct pancreatic lesions which broaden our understanding of the pathogenic mechanisms underlying multifocal HI.

MATERIALS AND METHODS
Clinical information was extracted from the electronic medical records as part of a study approved by our Institutional Review Board. 18 F-DOPA PET/CT was performed as previously described under an Investigational New Drug Application with Food and Drug Administration oversight and Institutional Review Board approval (NCT01916148) (13).

Genetic Analysis
Genetic testing was performed in CLIA-approved laboratories. DNA extracted from peripheral blood from patient 1 was subjected to polymerase chain reaction (PCR) amplification and Sanger sequencing of the ABCC8, KCNJ11, and GCK genes, with deletion/duplication analyses for ABCC8 performed using multiplex ligation-dependent probe amplification. Chromosomal single nucleotide polymorphism (SNP) microarrays were performed on DNA extracted from pancreas tissue from patient 1, and from blood and pancreas tissue from patient 2, using the Illumina CytoSNP850Kv1.1 BeadChip. Whole exome sequencing was performed on DNA extracted from peripheral blood from patient 2 using the xGen Whole Exome Panel kit, and findings were confirmed by Sanger sequencing. In addition, deletion and duplication analysis was performed on peripheral blood DNA from patient 2 by next-generation sequencing for a panel of 23 genes, including ABCC8. DNA extracted from pancreas samples from patient 2 underwent PCR amplification and next generation sequencing of the ABCC8, KCNJ11, GCK, GLUD1, HADH, HNF1A, HNF4A, SLC15A1, and UCP2 genes. Methylation analyses of DNA extracted from blood, skin, and pancreas tissue from patient 2 were performed using sodium bisulfite treatment followed by quantitative methylation sensitive PCR (14). The nucleotides of ABCC8 were numbered according to the sequence reported by Nestorowicz et al. that includes the alternatively spliced exon 17 sequence (NCBI accession no. L78224) (15).

Histological Analysis
Histology studies were performed on pancreatic tissue obtained at the time of surgery from which formalin-fixed, paraffinembedded sections were mounted on positively charged slides. Sections were stained with hematoxylin and eosin and primary mouse monoclonal antibodies directed against chromogranin (Dako, clone DAK-A3, 1:200) and p57 (Thermo Scientific, p57 Kip2 Ab-6, 1:800). Immunohistochemical staining was performed using the Leica Bond Immunohistochemistry Stainer (Leica Biosystems, Inc., Buffalo Grove, IL) following standard protocols and using the Polymer Refine Detection Brown Detection System and DAB Enhancer.

Patient 1
An infant male was born at 38 weeks gestation via spontaneous vaginal delivery complicated by nuchal cord. He had Apgar scores of 4 and 9 at 1 and 5 min of life, respectively, and a birth weight of 3.55 kg (66th percentile, appropriate for gestational age). Shortly after birth, plasma glucose was checked due to jitteriness and was found to be 1.55 mmol/L (28 mg/dL). He was admitted to the neonatal intensive care unit where he had repeated episodes of hypoglycemia. Critical specimens confirmed a diagnosis of HI (Table 1), and treatment was initiated with diazoxide and chlorothiazide, without clinical response even after the diazoxide dose was titrated up to 15 mg/kg/day. Genetic testing of peripheral blood DNA of the patient and his parents revealed a single heterozygous paternally inherited recessive ABCC8 mutation c.3992-9 G>A. 18 F-DOPA PET/CT, performed to localize the suspected focal lesion prior to surgery, demonstrated increased uptake in the inferior pancreatic head extending into the uncinate region (Figures 1A,B). He underwent 20% pancreatectomy in which a focal lesion in the inferior aspect of the pancreatic head measuring 1 cm in its largest dimension was excised. Intraoperatively, an additional, non-contiguous lesion in the anterior aspect of the proximal pancreatic body measuring 0.4 cm was identified by palpation and excised. Histopathology showed that both resected regions demonstrated localized islet cell hyperplasia with occasional nucleomegaly surrounded by normal pancreas, consistent with focal HI (Figure 2). Chromosomal SNP array analyses performed on the excised pancreatic tissue revealed a 47.74 Mb mosaic region of LOH of 11p15.5p11.2 (chr11:198,510-47,940,925, hg19) in the head lesion and a 34.1 Mb mosaic C>A) of the ABCC8 gene was also identified in peripheral blood DNA; parent of origin was not determined. 18 F-DOPA PET/CT revealed two separate foci of increased uptake: an exophytic lesion arising from the posterior aspect of the pancreatic body and a lesion on the medial aspect of the pancreatic head (Figures 1C-F). She subsequently underwent 10% pancreatectomy in which a 0.9 cm lesion in the posterior pancreatic body and an 0.8 cm in the pancreatic head were locally excised with clear surgical margins by frozen section. Final histopathology was consistent with focal islet cell hyperplasia (Figure 3).

DISCUSSION
Identification of patients with focal HI is of critical importance, as these patients can be cured if the lesion is localized and fully resected. Over the past several decades, improvements in the diagnosis of focal HI have been bolstered both by advances in molecular diagnostic testing and the implementation of 18 F-DOPA PET/CT for localization of focal lesions. Focal HI comprises roughly 50% of all surgically treated congenital HI cases (4). In contrast, the presence of multiple focal HI lesions is rare, and few patients with multifocal lesions have been reported to date. Goossens et al. first reported three cases of "multifocal nesidioblastosis" in 1989 (9). Limited histopathological data is provided, and this report predated our understanding of the genetic and molecular mechanisms responsible for congenital HI. Since then, Ni et al. described a patient with a KCNJ11 mutation found to have one focal lesion in the pancreatic head and another in the pancreatic body (11). Our group previously reported two separate cases, one suspected and one confirmed, of focal HI in which there were both pancreatic  and ectopic intestinal focal lesions (10,12). In each of these cases, the presence of multiple focal lesions was ultimately identified on 18 F-DOPA PET/CT imaging, accentuating the utility of this technique in the surgical management of focal HI. However, genetic analysis of the resected tissue was not reported.
To our knowledge, the only report including genetic analyses of tissue from multiple focal lesions is that of Giurgea et al. describing two patients, each with one pancreatic and one ectopic lesion (8). Both patients carried a heterozygous, paternally inherited ABCC8 gene mutation and were cured after complete lesionectomy. Genotyping of affected tissue by fluorescent PCR assay for chromosome 11p markers revealed somatic deletions of the maternal 11p15 region with different deletion break points within the pancreatic and ectopic lesions. No LOH was detected in samples from unaffected pancreatic tissue.
This report provides a detailed description of multiple focal lesions confined to the pancreas, adding to the limited body of literature on multifocal HI. In both patients presented here, analyses of resected pancreatic tissue revealed two distinct focal lesions with different sizes of LOH within 11p15, confirming that the lesions resulted from distinct somatic events in the setting of a paternally inherited, germline recessive ABCC8 mutation. The independence of the somatic events leading to multifocal HI is further supported by the distinct anatomic location of the focal lesions identified in patient 1, which reflects independent spatial origin. In pancreas embryogenesis, the inferior pancreatic head and uncinate process arise from the ventral pancreatic bud, and the remainder of the pancreas is formed from the dorsal pancreatic bud (16). In addition, the relative timing of the somatic event may determine focal lesion size (8,17).
Thus, the somatic event leading to the larger focal lesion in the inferior pancreatic head likely occurred within the ventral pancreatic bud earlier in development, whereas the smaller lesion in the pancreatic body may have resulted from a later somatic event within the dorsal bud. These findings augment those of Giurgea et al. and affirm the "two-hit" pathogenetic mechanism of focal HI.
The c.3992-9 G>A recessive mutation in ABCC8 observed in patient 1 is a well-recognized, founder mutation within the Ashkenazi Jewish population that affects splicing (18). In contrast, the pathogenicity of the ABCC8 changes detected in patient 2 are not well-established. The paternally inherited heterozygous deletion in exon 36 would be predicted to result in a shift in the mRNA reading frame, resulting in an early termination codon. This would affect the nucleotide-binding fold 2 (NBF2) domain of SUR1, mutations in which represent a common molecular mechanism of diazoxide-unresponsive HI (19). The single base change in intron 16 (ABCC8: c.2222+15 C>A) is not predicted to affect splicing [Human Splicing Finder (20)]. However, this variant is rare [allele frequency 0.0007%, gnomAD v3.1 (21)] and has been previously reported (22). Thus, either one of these ABCC8 changes may act as a recessive loss-offunction mutation and represent the first "hit" in this patient.
Based upon the experience at our institution, multiple focal lesions represent ∼1% of all focal HI cases (n = 3/264) and 0.3% of all non-syndromic HI cases (n = 3/937) seen. The risk of focal HI in a fetus carrying a paternally inherited recessive K ATP mutation (i.e., the risk of somatic paternal uniparental disomy within the pancreas) has been estimated to be 0.37% (1:270) (23). This may suggest that the risk of a second LOH event within the pancreas would also be 0.37%. The incidence of multifocal HI within focal HI cases seen at our institution (1.1%) is nearly three-fold higher than this estimate. The reason for this difference is not clear and may be explained by case ascertainment bias. Alternatively, this may indicate that the risk of pancreas-limited paternal uniparental disomy among carriers of recessive K ATP channel mutations is higher than previously estimated.
These cases illuminate the pivotal role of collaboration across the fields of pediatric endocrinology, genetics, radiology, surgery, and pathology in optimizing care for patients with HI. Notably, while 18 F-DOPA PET/CT accurately identified both pancreatic focal lesions in patient 2, this was not the case for patient 1 in whom the second focal lesion was identified intraoperatively owing to the surgical expertise and experience at our center. Through this multidisciplinary approach, both patients were cured.

CONCLUSION
Rarely, patients with the focal form of HI may have more than one focal lesion. This occurs due to separate somatic events resulting in varying lengths of 11p15 LOH unique to each lesion. A multidisciplinary team approach, including specialists with extensive experience caring for patients with HI, plays a critical role in the effective diagnosis and treatment of congenital HI. The importance of this approach to patient care is especially highlighted by rare, atypical, and challenging cases as presented herein.

DATA AVAILABILITY STATEMENT
The relevant original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Children's Hospital of Philadelphia Institutional Review Board. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

AUTHOR CONTRIBUTIONS
ER wrote the first draft of the manuscript. LM, KB, SB, HM, LB, AA, JK, TB, LS, NA, and KL contributed to the areas relevant to their expertise and edited the manuscript. DDDL conceptualized the work and edited the manuscript. All authors were involved in preparation of the manuscript.

FUNDING
This work was supported by National Institutes of Health Grants R01DK098517 (DDDL) and T32 NS091006-6 (ER).