Development of an exoglycosidase plate-based assay for detecting α1-3,4 fucosylation biomarker in individuals with HNF1A-MODY.

Maturity-onset diabetes of the young due to hepatocyte nuclear factor-1 alpha variants (HNF1A-MODY) causes monogenic diabetes. Individuals carrying damaging variants in HNF1A show decreased levels of α1-3,4 fucosylation, as demonstrated on antennary fucosylation of blood plasma N-glycans. The excellent diagnostic performance of this glycan biomarker in blood plasma N-glycans of individuals with HNF1A-MODY has been demonstrated using liquid chromatography methods. Here, we have developed a high-throughput exoglycosidase plate-based assay to measure α1-3,4 fucosylation levels in blood plasma samples. The assay has been optimized and its validity tested using 1000 clinical samples from a cohort of individuals with young-adult onset diabetes including cases with HNF1A-MODY. The α1-3,4 fucosylation levels in blood plasma showed a good differentiating power in identifying cases with damaging HNF1A variants, as demonstrated by receiver operating characteristic curve analysis with the AUC values of 0.87 and 0.95. This study supports future development of a simple diagnostic test to measure this glycan biomarker for application in a clinical setting.

Interlaboratory evaluation of plasma N-glycan antennary fucosylation as a clinical biomarker for HNF1A-MODY using liquid chromatography methods.

Antennary fucosylation alterations in plasma glycoproteins have been previously proposed and tested as a biomarker for differentiation of maturity onset diabetes of the young (MODY) patients carrying a functional mutation in the HNF1A gene. Here, we developed a novel LC-based workflow to analyze blood plasma N-glycan fucosylation in 320 diabetes cases with clinical features matching those at risk of HNF1A-MODY. Fucosylation levels measured in two independent research centers by using similar LC-based methods were correlated to evaluate the interlaboratory performance of the biomarker. The interlaboratory study showed good correlation between fucosylation levels measured for the 320 cases in the two centers with the correlation coefficient (r) of up to 0.88 for a single trait A3FG3S2. The improved chromatographic separation allowed the identification of six single glycan traits and a derived antennary fucosylation trait that were able to differentiate individuals carrying pathogenic mutations from benign or no HNF1A mutation cases, as determined by the area under the curve (AUC) of up to 0.94. The excellent (r = 0.88) interlaboratory performance of the glycan biomarker for HNF1A-MODY further supports the development of a clinically relevant diagnostic test measuring antennary fucosylation levels.

Altered cortisol metabolism in individuals with HNF1A-MODY.

OBJECTIVE AND CONTEXT: Maturity onset diabetes of the young due to variants in HNF1A (HNF1A-MODY) is the most common form of monogenic diabetes. Individuals with HNF1A-MODY usually have a lean phenotype which contrasts with type 2 diabetes (T2DM). Data from hepatocytes derived from Hnf1a knock-out mice demonstrated dysregulation of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which regulates glucocorticoid availability and action in target tissues, together with 11ß-HSD2 and steroid A-ring reductases, 5α- and 5ß-reductase. We proposed that altered glucocorticoid metabolism might underpin some of the phenotypic differences between patients with HNF1A-MODY and those with T2DM. DESIGN: A retrospective matched cohort study. PATIENTS AND MEASUREMENTS: 24-hours urine steroid metabolome profiling was carried out by gas chromatography-mass spectrometry in 35 subjects with HNF1A-MODY, 35 individuals with T2DM and 35 healthy controls matched for age, sex and BMI. The steroid metabolites were expressed as µg/L in all groups and measured in mid-morning urine in diabetic subjects and 24-hour urine collection in healthy controls. Hence, only ratios were compared not the individual steroids. Established ratios of glucocorticoid metabolites were used to estimate 11ß-HSD1/2 and 5α- and 5ß-reductase activities. RESULTS: While 11ß-HSD1 activity was similar in all groups, 11ß-HSD2 activity was significantly lower in subjects with HNF1A-MODY and T2DM than in healthy controls. The ratio of 5ß- to 5α-metabolites of cortisol was higher in subjects with HNF1A-MODY than in T2DM and healthy controls, probably due to increased activity of the 5ß-reductase (AKR1D1) in HNF1A-MODY. CONCLUSIONS: This is the first report of steroid metabolites in HNF1A-MODY. We have identified distinct differences in steroid metabolism pathways in subjects with HNF1A-MODY that have the potential to alter steroid hormone availability. Further studies are required to explore whether these changes link to phenotype.

Rathke's Cleft Cyst Abscess with a Very Unusual Course.

Infected Rathke's cleft cysts (RCC) are extremely rare with only a few published cases. We report the case of a 31-year-old man who presented with headaches, visual disturbance, and hypopituitarism secondary to an infected RCC with extension of abscesses along the optic tract. Magnetic resonance imaging showed ring enhancing cystic lesions within an expanded sella with suprasellar and intraparenchymal extension. The radiological appearance suggested a high-grade optic glioma, but an endoscopic transsphenoidal biopsy revealed frank pus in the pituitary fossa, which subsequently grew Staphylococcus aureus . Pathological examination of the cyst wall showed an inflamed RCC. Following a prolonged course of intravenous antibiotics, the infection resolved and vision improved. RCC abscesses are rare and the intracranial extension of the infection in our case makes it unique.

Fucosylated AGP glycopeptides as biomarkers of HNF1A-Maturity onset diabetes of the young.

AIMS: We previously demonstrated that antennary fucosylated N-glycans on plasma proteins are regulated by HNF1A and can identify cases of Maturity-Onset Diabetes of the Young caused by HNF1A variants (HNF1A-MODY). Based on literature data, we further postulated that N-glycans with best diagnostic value mostly originate from alpha-1-acid glycoprotein (AGP). In this study we analyzed fucosylation of AGP in subjects with HNF1A-MODY and other types of diabetes aiming to evaluate its diagnostic potential. METHODS: A recently developed LC-MS method for AGP N-glycopeptide analysis was utilized in two independent cohorts: a) 466 subjects with different diabetes subtypes to test the fucosylation differences, b) 98 selected individuals to test the discriminative potential for pathogenic HNF1A variants. RESULTS: Our results showed significant reduction in AGP fucosylation associated to HNF1A-MODY when compared to other diabetes subtypes. Additionally, ROC curve analysis confirmed significant discriminatory potential of individual fucosylated AGP glycopeptides, where the best performing glycopeptide had an AUC of 0.94 (95% CI 0.90-0.99). CONCLUSIONS: A glycopeptide based diagnostic tool would be beneficial for patient stratification by providing information about the functionality of HNF1A. It could assist the interpretation of DNA sequencing results and be a useful addition to the differential diagnostic process.

Effect of COVID-19 on the clinical course of diabetic ketoacidosis (DKA) in people with type 1 and type 2 diabetes.

OBJECTIVE: COVID-19 in people with diabetes is associated with a disproportionately worse prognosis. DKA is an acute complication of diabetes with a mortality rate of approximately 0.67%. Little is known about the natural history of DKA in the presence of COVID-19. This study aimed to explore the effects of COVID-19 on presentation, clinical course and outcome in patients presenting with DKA. DESIGN: Retrospective cohort study. METHODS: All patients treated for DKA between 1 March 2020 and 30 May 2020 were included. Patients were categorised as COVID-positive or COVID-negative based on the swab test. A pre-COVID group was established using data from 01 March 2019 to 30 May 2019 as external control. Data regarding demographics, diabetes type, pH, bicarbonate, lactate, glucose, DKA duration, complications and outcome were collected. RESULTS: A total of 88 DKA episodes were included in this study. There was no significant difference in the severity or duration of DKA between the three groups. COVID-positive T1DM were more hyperglycaemic on admission compared to COVID-negative and pre-COVID patients. There was an over representation of T2DM in COVID-positive patients with DKA than in pre-COVID or COVID-negative groups. CONCLUSION: COVID-19 appears to influence the natural history of DKA differently in T1DM and T2DM. Patients with T1DM and COVID-19 presented with more hyperglycaemia (60 mmol/L (35.9-60.0) vs 31.4 mmol/L (28.0-39.1) vs 24 mmol/L (20.2-33.75), respectively). Patients with T2DM were unusually presenting in DKA when infected with COVID-19 with greater ICU need and higher mortality rates. A collaborative, multi-centre study is needed to provide more definitive results.

Homozygous Hypomorphic HNF1A Alleles Are a Novel Cause of Young-Onset Diabetes and Result in Sulfonylurea-Sensitive Diabetes.

OBJECTIVE: Heterozygous loss-of-function mutations in HNF1A cause maturity-onset diabetes of the young (MODY). Affected individuals can be treated with low-dose sulfonylureas. Individuals with homozygous HNF1A mutations causing MODY have not been reported. RESEARCH DESIGN AND METHODS: We phenotyped a kindred with young-onset diabetes and performed molecular genetic testing, a mixed meal tolerance test, a sulfonylurea challenge, and in vitro assays to assess variant protein function. RESULTS: A homozygous HNF1A variant (p.A251T) was identified in three insulin-treated family members diagnosed with diabetes before 20 years of age. Those with the homozygous variant had low hs-CRP levels (0.2-0.8 mg/L), and those tested demonstrated sensitivity to sulfonylurea given at a low dose, completely transitioning off insulin. In silico modeling predicted a variant of unknown significance; however, in vitro studies supported a modest reduction in transactivation potential (79% of that for the wild type; P < 0.05) in the absence of endogenous HNF1A. CONCLUSIONS: Homozygous hypomorphic HNF1A variants are a cause of HNF1A-MODY. We thus expand the allelic spectrum of variants in dominant genes causing diabetes.

Plasma Fucosylated Glycans and C-Reactive Protein as Biomarkers of HNF1A-MODY in Young Adult-Onset Nonautoimmune Diabetes.

OBJECTIVE: Maturity-onset diabetes of the young (MODY) due to variants in HNF1A is the most common type of monogenic diabetes. Frequent misdiagnosis results in missed opportunity to use sulfonylureas as first-line treatment. A nongenetic biomarker could improve selection of subjects for genetic testing and increase diagnosis rates. We previously reported that plasma levels of antennary fucosylated N-glycans and high-sensitivity C-reactive protein (hs-CRP) are reduced in individuals with HNF1A-MODY. In this study, we examined the potential use of N-glycans and hs-CRP in discriminating individuals with damaging HNF1A alleles from those without HNF1A variants in an unselected population of young adults with nonautoimmune diabetes. RESEARCH DESIGN AND METHODS: We analyzed the plasma N-glycan profile, measured hs-CRP, and sequenced HNF1A in 989 individuals with diabetes diagnosed when younger than age 45, persistent endogenous insulin production, and absence of pancreatic autoimmunity. Systematic assessment of rare HNF1A variants was performed. RESULTS: We identified 29 individuals harboring 25 rare HNF1A alleles, of which 3 were novel, and 12 (in 16 probands) were considered pathogenic. Antennary fucosylated N-glycans and hs-CRP were able to differentiate subjects with damaging HNF1A alleles from those without rare HNF1A alleles. Glycan GP30 had a receiver operating characteristic curve area under the curve (AUC) of 0.90 (88% sensitivity, 80% specificity, cutoff 0.70%), whereas hs-CRP had an AUC of 0.83 (88% sensitivity, 69% specificity, cutoff 0.81 mg/L). CONCLUSIONS: Half of rare HNF1A sequence variants do not cause MODY. N-glycan profile and hs-CRP could both be used as tools, alone or as adjuncts to existing pathways, for identifying individuals at high risk of carrying a damaging HNF1A allele.

Maturity onset diabetes of the young due to HNF1A variants in Croatia.

INTRODUCTION: Maturity onset diabetes of the young due to HNF1A mutations (HNF1A-MODY) is the most frequent form of monogenic diabetes in adults. It is often misdiagnosed as type 1 or type 2 diabetes, but establishing genetic diagnosis is important, as treatment differs from the common types of diabetes. HNF1A-MODY has not been investigated in Croatia before due to limited access to genetic testing. In this study we aimed to describe the characteristics of young adults diagnosed with diabetes before the age of 45 years, who have rare HNF1A allele variants, and estimate the prevalence of HNF1A-MODY in Croatia. MATERIALS AND METHODS: We recruited 477 C-peptide positive and beta cell antibody negative subjects through the Croatian Diabetes Registry. HNF1A was sequenced for all participants and systematic assessment of the variants found was performed. The prevalence of HNF1A-MODY was calculated in the study group and results extrapolated to estimate the proportion of diabetic individuals with HNF1A-MODY in Croatia and the population prevalence. RESULTS: Our study identified 13 individuals harbouring rare HNF1A allelic variants. After systematic assessment, 8 were assigned a diagnosis of HNF1A-MODY. Two individuals were able to discontinue insulin treatment following the diagnosis. We estimated that HNF1A-MODY in Croatia has a prevalence of 66 (95% CI 61 - 72) cases per million. CONCLUSIONS: The estimated prevalence of HNF1A-MODY in Croatia is similar to that reported in other European countries. Finding cases lead to important treatment changes for patients. This strongly supports the introduction of diagnostic genetic testing for monogenic diabetes in Croatia.

The management of Cushing's disease - from investigation to treatment.

Cushing's disease (CD) is caused by an adrenocorticotrophin (ACTH) secreting pituitary adenoma and it is the commonest cause of endogenous hypercortisolism. When high suspicion of Cushing's syndrome (CS) exists, recommended screening tests include the overnight dexamethasone suppression test, the low-dose dexamethasone suppression test, or late night salivary cortisol. If the initial test is positive on two occasions, the patient should be referred to a specialist endocrinologist for in-patient assessment, while elevated midnight serum cortisol and a low dose dexamethasone suppression test will confirm endogenous hypercortisolaemia. Plasma ACTH measurement at 9 am follows and, if elevated, MRI scan of the pituitary should be performed. Corticotrophin releasing hormone (CRH) test helps to distinguish pituitary from ectopic ACTH-dependent CS, though bilateral petrosal sinus sampling remains the gold standard. Transsphenoidal surgery is the recognised first-line treatment of CD, and can be repeated if unsuccessful. Second line therapy includes pituitary radiotherapy, bilateral adrenalectomy and medical treatment. Pituitary radiotherapy is very effective but it usually takes several years for its full effect to be seen. Bilateral adrenalectomy is useful in acutely unwell patients, who are unable to tolerate medical therapy. The most effective medical agents inhibit adrenal steroidogenesis and include metyrapone, ketoconazole, mitotane and etomidate. They are used in preparation for surgery, when an operation has been unsuccessful, or when the effects of radiotherapy are being awaited. Cabergoline and pasireotide decrease ACTH production, but are effective in only 30% and 25% of patients, respectively. It is crucial for patients with CD to be managed in specialist endocrine centres, as the expertise of multidisciplinary team members predicts the best outcome.

The investigation of Cushing syndrome: essentials in optimizing appropriate diagnosis and management.

The investigation of Cushing syndrome (CS) should start with careful history taking and clinical examination, and exogenous steroid usage must be excluded. It is essential to confirm hypercortisolism before further investigations are undertaken. The recommended first-line tests include midnight salivary cortisol and/or the 1 mg overnight or low-dose dexamethasone suppression tests. The next step is to differentiate adrenocorticotrophin (ACTH)-dependent from ACTH-independent CS by measuring ACTH. With ACTH-dependence, further investigations should differentiate pituitary-dependent from ectopic ACTH-dependent CS. Many dynamic tests may be considered, but we suggest that bilateral inferior petrosal sinus sampling should be performed in almost all patients with ACTH-dependent CS, except for patients with a pituitary macroadenoma. Imaging should include MR scanning of the pituitary, and CT scanning of the chest and abdomen to look for an ectopic source. Confirmation of the diagnosis of CS and accurate localization of its source are vital to optimize therapy for this complex disorder.

Ipilimumab: a novel immunomodulating therapy causing autoimmune hypophysitis: a case report and review.

Ipilimumab (Yervoy; Medarex and Bristol-Myers Squibb) is a human MAB against cytotoxic T-lymphocyte antigen 4, which enhances co-stimulation of cytotoxic T-lymphocytes, resulting in their proliferation and an anti-tumour response. It is licensed for the treatment of unresectable or metastatic malignant melanoma, while multiple clinical trials using this medication in the treatment of other malignancies are ongoing. As a clinical response to ipilimumab results from immunostimulation, predictably it generates autoimmunity as well, causing immune-related adverse events in the majority of patients. Of those, endocrinopathies are frequently seen, and in particular, autoimmune lymphocytic hypophysitis with anterior panhypopituitarism has been reported a number of times in North America. We present a case of a male referred to our department with manifestations of anterior panhypopituitarism after his third dose of ipilimumab for metastatic malignant melanoma, and we discuss the management of his case in the light of previous reports. We also review the published literature on the presenting symptoms, time to presentation, investigations, imaging, treatment and follow-up of ipilimumab-induced autoimmune lymphocytic hypophysitis.