Germline and somatic mutations in histologically atypical congenital hyperinsulinism

Annette Larsen^1,2,3Evgenia Globa⁴Ditte Caroline Andersen^2,5Catarina De Castro Blanco Limbert⁶Åsa Löfgren Mattsson⁷Anne Lerberg Nielsen⁸Michael Bau Mortensen⁹Eva Kildall Hejbøl¹⁰Klaus Brusgaard^11,12,13,2Sönke Detlefsen¹⁰Henrik Thybo Christesen^1,14,2*

• ¹Hans Christian Andersen Children's Hospital, Odense University Hospital, Odense, Denmark
• ²Syddansk Universitet Klinisk Institut, Odense, Denmark
• ³Department of Clinical Genetics, Odense Universitetshospital, Odense, Denmark
• ⁴Ukrainian Scientific and Practical Center of Endocrine Surgery, Transplantation of Endocrine Organs and Tissues of the Ministry of Health of Ukraine, Department of Pediatric Endocrinology, Kyiv, Ukraine
• ⁵DCA-group, Syddansk Universitet, Odense, Denmark
• ⁶CHLC, Unit for Pediatric Endocrinology and Diabetes, Hospital Dona Estefania, Lisbon, Portugal
• ⁷Department of Pediatrics, Helsingborgs lasarett, Helsingborg, Sweden
• ⁸Department of Nuclear Medicine, Odense Universitetshospital, Odense, Denmark
• ⁹Odense Universitetshospital Kirurgisk Afdeling A, Odense, Denmark
• ¹⁰Department of Pathology, Odense Universitetshospital, Odense, Denmark
• ¹¹Odense pancreas Center (OPAC), Odense Universitetshospital, Odense, Denmark
• ¹²Odense Universitetshospital, Department of Clinical Genetics, Odense, Denmark
• ¹³Odense Universitetshospital Steno Diabetes Center Odense, Odense, Denmark
• ¹⁴Odense Universitetshospital, Odense Pancreas Center, Odense, Denmark

Background: In histologically atypical congenital hyperinsulinism (CHI), the correlations between clinical, histological and genetic features are largely unknown. Laser-capture microdissection may be used to identify low-grade mosaic DNA variants in the islets of Langerhans. Aim: To investigate genotype-histotype-phenotype correlations in atypical CHI. Methods: In our single-center cohort of hyperinsulinemic hypoglycemia (HH) patients, 77 underwent pancreatic surgery. In those with histologically atypical CHI, genetic analyses included sequencing of frequent CHI genes from blood and bulk pancreatic tissue and tests for Beckwith-Wiedemann Syndrome (BWS) where appropriate. If negative, a 140-gene targeted panel including the non-coding region of HK1 was performed in blood, pancreatic bulk tissue and islets isolated by laser-capture microdissection. Histological, immunohistochemical and morphometric analyses were performed on pancreatic tissue. Results: The 77 HH patients were classified histologically as KATP-channel focal CHI (n=48), KATP-channel diffuse CHI (n=14), insulinoma (n=6), non-insulinoma HH in teenagers (n=1), BWS (n=1), unclassified (n=2). Histologically atypical CHI patients (n=5/70; 7.1%) had a median (range) birth weight of 2965 (2650-3385) grams and a clinical disease onset at 93 (1-259) days. 18F-DOPA PET/CT showed diffuse tracer uptake. In three patients, genetic analysis showed HK1 intron 2 variants, of which one was present in germline (de novo heterozygous) while the other two had somatic low-grade mosaic alterations in bulk pancreatic tissue (n=1) or exclusively in islets after isolation by laser-capture microdissection (n=1). Patient 4 showed a CACNA1D frameshift mutation suggesting Cav1.3 channel gain-of-function properties. No relevant genetic changes were found in Patient 5. In all five atypical CHI specimens, pancreatic histology showed slight changes with areas having pronounced occurrence of large islets of Langerhans, while small islets and endocrine cell clusters were evenly distributed. Giant cell nuclei were observed, but at much lower frequencies compared to KATP-channel diffuse CHI. Conclusion: Histologically atypical CHI was seen in 7.1% of surgery-treated CHI patients and characterized by discrete changes with enlarged islets of Langerhans and a low frequency of giant nuclei in endocrine cells. Genetics showed heterozygous or low-grade mosaic HK1 intron 2 DNA variants in three patients. Low-grade mosaic pancreatic genetic changes may only be detectable after islet isolation by laser-capture microdissection.