HNF1A Mutations Research Articles

Abstract 5132: Assessing tumor mutational burden and profiling variants from FFPE samples using a PCR-based next-generation sequencing assay

Abstract Introduction: High Tumor Mutational Burden (TMB) has shown association with benefit from immune checkpoint blockade therapies. While TMB as computed from whole exome sequencing (WES) is still a gold standard, the high input material (tumor and germline DNA) requirement and complex bioinformatics refrains exploring this biomarker in individual labs. Herein, we develop a PCR-based targeted panel for computing TMB and detecting important variants from FFPE research samples. Methods: A targeted panel was designed that covered 1.7 Mb of genomic region from 409 key cancer genes. First an UDG treatment is applied to repair samples with FFPE fixation error. Utilizing Ion AmpliSeq chemistry, the workflow requires very little (only 20 ng) input DNA. The assay enables a 3-day turn-around time from sample to the final report. The workflow enables < 60 minutes of hands-on time for automated library preparation and templating on a batch of 4 samples. Sequencing is performed on high throughput semiconductor sequencing platform to achieve sufficient depth (~1200x coverage) and accuracy. The analysis pipeline accompanies variant caller parameters optimized for high accuracy in variant detection. The workflow is tumor sample only, therefore, TMB (nonsynonymous somatic mutations/Mb) is calculated by removing germline variants using population databases. Eight NSCLC FFPE tumors were analyzed. Samples were also tested without UDG repair. Count of somatic C:G>T:A mutations below 15% allelic frequency was called deamination. Results: In-silico analyses using 466 lung adenocarcinoma, 375 skin cutaneous melanoma, and 274 colon adenocarcinoma samples from TCGA MC3 dataset displayed high correlation between WES TMB and predicted panel TMB (r2 = 0.90 on lung, r2 = 0.96 on melanoma, and r2 = 0.98 on colon). Deamination reduced by average 85.67% (SD 16.87) on FFPE samples after applying UDG repair. Mean TMB of first six samples was 6.97 (SD 2.00), and TMB of last two samples was 43.58 and 87.14. Among two high TMB samples, first had gain-of-function and loss-of-function mutations in BRAF and NF1 genes and had 59.4% C:G>A:T somatic mutations relating to molecular smoking signature. On the second high TMB sample, the assay detected gain-of-function and loss-of-function mutations in FGFR3, NOTCH1, KRAS, HNF1A, and CREBBP genes and estimated 64.7% C:G>T:A somatic mutations at CpG sites consistent with deamination of 5-methylcytosine. Conclusions: We have developed a workflow on the Ion Torrent sequencing platform with Ion AmpliSeq chemistry to estimate TMB from FFPE research samples. This solution will advance research in immuno-oncology. Citation Format: Ruchi Chaudhary, Charles Scafe, Dinesh Cyanam, Vinay Mittal, Warren Tom, Janice Au-Young, Seth Sadis, Fiona Hyland. Assessing tumor mutational burden and profiling variants from FFPE samples using a PCR-based next-generation sequencing assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5132.

Maturity onset diabetes of the young (MODY) in Chinese children: genes and clinical phenotypes.

Background To investigate the clinical and molecular characteristics of Chinese children with maturity onset diabetes of the young (MODY). Methods A total of 42 Chinese patients suspected MODY referred to our unit from 2014 to 2018 were enrolled. Mutational analysis of monogenic diabetes mellitus genes was performed by next-generation sequencing and confirmed by Sanger sequencing. Results There were 28 males (66.7%) and 14 females (33.3%) with a mean age of 9.49 ± 3.46 years (range, 1.4-15.3 years) and a mean birth weight of 3.38 ± 0.49 kg (range, 2.55-4.90 kg). Among these patients, 15 patients had polyuria, polydipsia or weight loss. Two patients (4.8%) were obese and six (14.3%) were overweight. Moreover, 13 patients (30.9%) had a family history of diabetes. Thirty variants were identified in 28 patients. Twenty-six variants in 25 patients were pathogenic or likely pathogenic genes (59.5%, 25/42), including 15 patients (60.0%, 15/25) with GCK mutation, four (16.0%, 4/25) with PAX4 mutation, three (12.0%, 3/25) with HNF4A mutation, one (4.0%, 1/25) with INS mutation, one (4.0%, 1/25) with NEUROD1 mutation and one (4.0%, 1/25) with HNF1A mutation. Nine mutations (36.0%, 9/25) were novel. There was no difference between mutation-suspected patients and MODY-confirmed patients except for a 2-h glucose increment in an oral glucose tolerance test (OGTT), while the GCK-MODY had lower glycated hemoglobin (HbA1c) and a significantly smaller 2-h glucose increment in an OGTT compared with transcription factor MODYs. The GCK-MODY was identified by incidental hyperglycemia without glycosuria. GCK-MODY without drug management and hepatocyte nuclear factor-1 alpha (HNF4A) or HNF1A-MODY with sulfonylurea therapy obtained good glucose controlling. Conclusions Mutation of the GCK gene is the most common in MODY patients in China followed by PAX4. The screening criteria can improve the cost-effectiveness of disease diagnosis and treatment. A precise molecular diagnosis would lead to optimal treatment of the patients.

Using Clinical Indices to Distinguish MODY2 (GCK Mutation) and MODY3 (HNF1A Mutation) from Type 1 Diabetes in a Young Chinese Population.

IntroductionAccurate diagnosis of maturity-onset diabetes of the young (MODY) is required in order to select appropriate treatment options and to assess prognosis. The aim of this study was to explore potential clinical indicators that could be used to differentiate MODY2, MODY3, and type 1 diabetes (T1D) in young subjects.MethodsTwelve patients with MODY3 and 29 patients with MODY2 were characterized and compared to 26 patients with T1D. These three groups were matched for age and gender. Clinical profiles of the 67 patients were collected. Receiver operating characteristic (ROC) curves were used to identify the optimal cutoff values of clinical indicators.ResultsCompared to patients with T1D, subjects with MODY3 had higher fasting C-peptide levels (1.34 ± 1.51 vs. 0.29 ± 0.22 ng/mL; P < 0.001) and lower high-sensitivity C-reactive protein (hsCRP) levels (0.18 ± 0.15 vs. 1.22 ± 1.49 mg/L, P = 0.004); patients with MODY2 had lower hsCRP (0.37 ± 0.39 vs. 1.22 ± 1.49 mg/L; P = 0.003), total cholesterol (4.12 ± 0.68 vs. 4.61 ± 0.81 mmol/L, P = 0.034), and low-density lipoprotein cholesterol (LDL-C) (2.24 ± 0.68 vs. 2.67 ± 0.79 ng/L, P = 0.002) levels and higher fasting C-peptide levels (0.96 ± 0.42 vs. 0.29 ± 0.22 ng/mL, P = 0.002). The ROC-derived hsCRP values for discriminating MODY2 from T1D, MODY3 from T1D, and MODY3 from MODY2 were 0.675, 0.833, and 0.763, respectively. The ROC-derived fasting C-peptide levels for discriminating MODY2 from T1D and MODY3 from T1D were 0.951 and 0.975, respectively. The ROC-derived total cholesterol and LDL-C values for discriminating MODY2 from T1D were 0.670 and 0.662, respectively; the ROC-derived triglyceride value for discriminating MODY3 from MODY2 was 0.756. Additionally, a combination of indicators permitted better discrimination of MODY subtypes than any single parameter.ConclusionOur findings suggest that fasting C-peptide, hsCRP, and lipid levels permit good discrimination among MODY2, MODY3, and T1D. These clinical indicators could be used as markers of MODY2 and MODY3 in young patients with diabetes.Electronic Supplementary MaterialThe online version of this article (10.1007/s13300-019-0647-x) contains supplementary material, which is available to authorized users.

HNF4A Haploinsufficiency in MODY1 Abrogates Liver and Pancreas Differentiation from Patient-Derived Induced Pluripotent Stem Cells

SummaryMaturity-onset diabetes of the young 1 (MODY1) is a monogenic diabetes condition caused by heterozygous HNF4A mutations. We investigate how HNF4A haploinsufficiency from a MODY1/HNF4A mutation influences the development of foregut-derived liver and pancreatic cells through differentiation of human induced pluripotent stem cells from a MODY1 family down the foregut lineage. In MODY1-derived hepatopancreatic progenitors, which expressed reduced HNF4A levels and mislocalized HNF4A, foregut genes were downregulated, whereas hindgut-specifying HOX genes were upregulated. MODY1-derived hepatocyte-like cells were found to exhibit altered morphology. Hepatic and β cell gene signatures were also perturbed in MODY1-derived hepatocyte-like and β-like cells, respectively. As mutant HNF4A (p.Ile271fs) did not undergo complete nonsense-mediated decay or exert dominant negativity, HNF4A-mediated loss of function is likely due to impaired transcriptional activation of target genes. Our results suggest that in MODY1, liver and pancreas development is perturbed early on, contributing to altered hepatic proteins and β cell defects in patients.

Exceptional diazoxide sensitivity in hyperinsulinaemic hypoglycaemia due to a novel HNF4A mutation.

SummaryDiazoxide is the first-line treatment for patients with hyperinsulinaemic hypoglycaemia (HH). Approximately 50% of patients with HH are diazoxide resistant. However, marked diazoxide sensitivity resulting in severe hyperglycaemia is extremely uncommon and not reported previously in the context of HH due to HNF4A mutation. We report a novel observation of exceptional diazoxide sensitivity in a patient with HH due to HNF4A mutation. A female infant presented with severe persistent neonatal hypoglycaemia and was diagnosed with HH. Standard doses of diazoxide (5 mg/kg/day) resulted in marked hyperglycaemia (maximum blood glucose 21.6 mmol/L) necessitating discontinuation of diazoxide. Lower dose of diazoxide (1.5 mg/kg/day) successfully controlled HH in the proband, which was subsequently confirmed to be due to a novel HNF4A mutation. At 3 years of age, the patient maintains age appropriate fasting tolerance on low dose diazoxide (1.8 mg/kg/day) and has normal development. Diagnosis in proband’s mother and maternal aunt, both of whom carried HNF4A mutation and had been diagnosed with presumed type 1 and type 2 diabetes mellitus, respectively, was revised to maturity-onset diabetes of young (MODY). Proband’s 5-year-old maternal cousin, also carrier of HNF4A mutation, had transient neonatal hypoglycaemia. To conclude, patients with HH due to HNF4A mutation may require lower diazoxide than other group of patients with HH. Educating the families about the risk of marked hyperglycaemia with diazoxide is essential. The clinical phenotype of HNF4A mutation can be extremely variable.Learning points:Awareness of risk of severe hyperglycaemia with diazoxide is important and patients/families should be accordingly educated.Some patients with HH due to HNF4A mutations may require lower than standard doses of diazoxide.The clinical phenotype of HNF4A mutation can be extremely variable.

SUN-272 Persistent Hyperinsulinemic Hypoglycemia Due to Mutation in Hepatocyte Nuclear Factor 4A

Background: Congenital hyperinsulinism (HI) is the most common cause of hypoglycemia in children. Delay in diagnosis and or inadequate management can lead to brain damage. Persistent hyperinsulinemic hypoglycemia of infancy is a genetic disorder with both familial and sporadic form. Aim: To present the case of an infant with persistent hyperinsulinenic hypoglycemia due to HNF4A gene mutation, inherited by his mother who was perceived to have Diabetes Mellitus Type 2. Clinical presentation: A premature boy born at 35 weeks gestation by caesarian section due to mother’s hypertension, proteinuria and peripheral edema with birth weight of 2565gr and 44.5cm length. He was the first child of family and mother is referred to suffer from diabetes mellitus type 2 since 17 years of age treated with oral antidiabetic drugs. During pregnancy, optimal glycemic control was achieved after initiation of insulin therapy. After birth, at first 2 hours of life, blood glucose was 23mg/dl. Asymptomatic hypoglycemia persisted. During hypoglycemia arterial pH, lactic acid and cortisol were within reference range and insulin as ell c-peptide levels were elevated. Synachten test was normal. 30 minutes after glucagon administration, glucose level was increased by 48mg/dl and after 60 minutes glucose level was increased by 34 mg/dl. Five hours after feeding, glucose was 47mg/dl and diazoxide treatment was started (7.5 mg/kg/day). He responded well to diazoxide and daily dosage was decreased to 1mg/kg/day. Genetic testing was performed. Heterozygous mutation in c.998G>C, pR333P of HNF4A gene was identified to infant and his mother. The infant is now 8 months old and he continuous on diazoxide therapy growing well. The identification of the mutation clarified that the mother suffers from maturity- onset diabetes of the young (MODY) and not Diabetes Mellitus type 2. Conclusion: Early detection of HI is very important in order to prevent permanent sequelae. Genetic mutation testing should be performed in order to define best treatment approach. Family history of diabetes can elucidate that the affected gene is HNF4A or HNF1a. HI due to HNF4A mutations may be transient or persistent into childhood and is usually responsive to diazoxide therapy. MODY can develop during the second or third decade of life that is usually responsive to sulfonylurias. Reference: Stanley CA Perspective on the genetics and diagnosis of congenital hyperinsulinism disorders JCEM 2016; 101: 815-26

Functional characterization of p.Pro409His variant in HNF1A, a hypomorphic mutation involved in pancreatic β-cell dysfunction.

HNF1A is a gene coding for the transcription factor HNF1-α, mutated in some forms of MODY and type 2 diabetes mellitus characterized by a strong genetic component. The penetrance of HNF1A variants differs considerably; thus, to assess the genetic risk of diabetes in carrier subjects of a HNF1A mutant allele, a functional characterization of mutant forms is of paramount importance. The HNF1A gene was sequenced in two patients with partly discordant diabetic phenotype, carrying the p.Pro409His variant. To evaluate the pathogenicity of the variant, we measured the transactivation power of the corresponding P408H HNF1-α mutant mouse form on HNF1-α target promoters. We found a lower but detectable activity of transactivation of the mutant form compared with the wild-type form and we excluded mechanisms of protein degradation or nuclear mislocalization. The HNF1A mutation p.Pro409His can be considered a mild variant that confers a moderate risk of type 2 diabetes mellitus in heterozygous carriers.

A decision algorithm to identify patients with high probability of monogenic diabetes due to HNF1A mutations

PurposeTo investigate the utility of biomarkers of maturity-onset diabetes of the young (MODY), high-sensitivity C-reactive protein (hsCRP), and 1,5-anhydroglucitol (1,5-AG) in conjunction with other clinical and laboratory features to improve diagnostic accuracy and provide a diagnostic algorithm for HNF1A MODY.MethodsWe examined 77 patients with HNF1A MODY, 88 with GCK MODY mutations, 99 with type 1 diabetes, and 92 with type 2 diabetes. In addition to 1,5-AG and hsCRP, we considered body mass index (BMI), fasting glucose, and fasting serum C-peptide as potential biomarkers. Logistic regression and receiver operating characteristic curves were used in marker evaluation.ResultsConcentration of hsCRP was lowest in HNF1A MODY (0.51 mg/l) and highest in type 2 diabetes (1.33 mg/l). The level of 1,5-AG was lowest in type 1 diabetes and HNF1A MODY, 3.8 and 4.7 μg/ml, respectively, and highest (11.2 μg/ml) in GCK MODY. In the diagnostic algorithm, we first excluded patients with type 1 diabetes based on low C-peptide (C-statistic 0.98) before using high BMI and C-peptide to identify type 2 diabetes patients (C-statistic 0.92). Finally, 1,5-AG and hsCRP in conjunction yielded a C-statistic of 0.86 in discriminating HNF1A from GCK MODY. We correctly classified 92.9% of patients with type 1 diabetes, 84.8% with type 2 diabetes, 64.9% HNF1A MODY, and 52.3% GCK MODY patients.ConclusionsPlasma 1,5-AG and serum hsCRP do not discriminate sufficiently HNF1A MODY from common diabetes types, but could be potentially useful in prioritizing Sanger sequencing of HNF1A gene.

Genetic characterization of suspected MODY patients in Tunisia by targeted next-generation sequencing.

Maturity Onset Diabetes of the Young (MODY) is a monogenic form of diabetes with autosomal dominant inheritance pattern. The diagnosis of MODY and its subtypes is based on genetic testing. Our aim was investigating MODY by means of next-generation sequencing in the Tunisian population. We performed a targeted sequencing of 27 genes known to cause monogenic diabetes in 11 phenotypically suspected Tunisian patients. We retained genetic variants passing filters of frequency in public databases as well as their probable effects on protein structures and functions evaluated by bioinformatics prediction tools. Five heterozygous variants were found in four patients. They include two mutations in HNF1A and GCK that are the causative genes of the two most prevalent MODY subtypes described in the literature. Other possible mutations, including novel frameshift and splice-site variants were identified in ABCC8 gene. Our study is the first to investigate the clinical application of targeted next-generation sequencing for the diagnosis of MODY in Africa. The combination of this approach with a filtering/prioritization strategy made a step towards the identification of MODY mutations in the Tunisian population.

Body mass index and C-peptide are important for the promptly differential diagnosis of maturity-onset diabetes from familial type 2 diabetes in outpatient clinic.

Type 2 diabetic patients are becoming younger and having a tendency to family aggregation, they are easily suspected as maturity-onset diabetes of young (MODY) in the outpatient clinic and send to genetic testing. 9 diabetic families were compared in our outpatient clinic who met the primary diagnosis criteria of MODY. Detailed clinical features and laboratory data including gene sequence were collected and analyzed. The patients met the primary clinical diagnostic criteria of MODY for genetic testing at the first look. However, members of families A1 to A3 had normal Body mass index (BMI) and a lower C-peptide level which indicated impaired pancreatic islet function. In contrast, the members with diabetes of families B1 to B6 had normal or increased C-peptide level which indicated insulin resistance and were overweight with BMI. Genetic testing showed that the mutations in HNF1A, INS, KCNJ11 and so on in families A were consistent with the diagnosis of MODY. No pathogenic mutation was found in the members of families B which were diagnosed with familial T2D. Before the clinical laboratory testing and the further gene test, BMI and the concentration of C-peptide are important for the promptly differential diagnosis of MODY from familial type 2 diabetes and medication instruction in the outpatient clinic which could help to alleviate the burden of genetic testing for them.

Frequency and Characteristics of MODY 1 (HNF4A Mutation) and MODY 5 (HNF1B Mutation): Analysis From the DPV Database.

To characterize the initial presentation and clinical course of patients with hepatocyte nuclear factor (HNF) 4A‒ and HNF1B‒MODY in a multinational registry. Within the Diabetes Patienten Verlaufsdokumentation (DPV) registry, 44 patients with HNF4A- and 35 patients with HNF1B-MODY were characterized and compared with patients <20 years old with type 1 diabetes (T1D)/type 2 diabetes (T2D). Clinical and laboratory parameters, therapy, metabolic control, and extrapancreatic symptoms in patients with HNF1B-MODY. Patients with both MODY types were significantly older than patients with T1D at diagnosis (HNF4A, 13.8 years, and HNF1B, 13.5 years, vs T1D, 8.8 years; P < 0.0001). Mean C-peptide at diagnosis was higher for HNF4A-MODY than for T1D (1.8 vs 0.9 ng/mL; P < 0.01); 36.4% of patients with HNF4A-MODY and 65.7% of patients with HNF1B-MODY were treated with insulin, whereas 20.5% and 8.6% received oral antidiabetics only (P < 0.05 and P < 0.01 vs T2D). At the most recent visit, glycated hemoglobin levels were lower in HNF4A- and HNF1B-MODY (mean, 6.5% and 6.1%) than in T1D (7.9%; P < 0.0001). In 40% of patients with HNF1B-MODY, extrapancreatic symptoms were reported. Several clinical predictors previously described to differentiate between MODY and T1D or T2D were revalidated by logistic regression analyses in this cohort. The DPV registry enabled us to precisely characterize phenotype and treatment in these two rare MODY types. Although phenotype of HNF4A- and HNF1B-MODY showed distinct differences from those of T1D and T2D, 38% of patients were initially misclassified as having T1D or T2D.

Maturity Onset Diabetes of the Young due to Glucokinase, HNF1-A, HNF1-B, and HNF4-A Mutations in a Cohort of Turkish Children Diagnosed as Type 1 Diabetes Mellitus

Background/Aims: Maturity onset diabetes of the young (MODY) is a rare condition often misdiagnosed as type 1 diabetes (T1D). The purposes of this study were: to identify any patients followed in a large Turkish cohort as T1D, with an atypical natural history, who may in fact have MODY, and to define the criteria which would indicate patients with likely MODY as early as possible after presentation to allow prompt genetic testing. Methods: Urinary C-peptide/creatinine ratio (UCPCR) was studied in 152 patients having a diagnosis of T1D for at least 3 years. Those with a UCPCR ≥0.2 nmol/mmol were selected for genetic analysis of the Glucokinase (GCK), Hepatocyte nuclear factor 1a (HNF1A), Hepatocyte nuclear factor 4a (HNF4A), and Hepatocyte nuclear factor 1b (HNF1B) genes. This UCPCR cut-off was used because of the reported high sensitivity and specificity. Cases were also evaluated using a MODY probability calculator. Results: Twenty-three patients from 152 participants (15.1%) had a UCPCR indicating persistent insulin reserve. The mean age ± SD of the patients was 13.6 ± 3.6 years (range 8.30–21.6). Of these 23, two (8.7%) were found to have a mutation, one with HNF4A and one with HNF1B mutation. No mutations were detected in the GCK or HNF1A genes. Conclusion: In Turkish children with a diagnosis of T1D but who have persistent insulin reserve 3 years after diagnosis, up to 9% may have a genetic mutation indicating a diagnosis of MODY.

Quality of life assessment in patients with HNF1A-MODY and GCK-MODY

AimThe impact of maturity onset diabetes of the young (MODY) on quality of life (QoL) has never been examined. We assessed disease impact on QoL among patients with HNF1A-MODY and GCK mutation carrier status.MethodsThe study included 80 patients with HNF1A-MODY and 89 GCK gene mutation carriers. We also examined 128 type 1 diabetes (T1DM) patients for comparison. Diabetes-specific QoL was assessed using the Audit of Diabetes Dependent Quality of Life questionnaire.ResultsHNF1A-MODY and GCK-MODY groups had similar mean age (41.7 vs. 38.0 years, respectively) and BMI (24.1 vs. 24.3 kg/m2), whereas T1DM patients were on average younger (34.2 years) with similar BMI (25.0 kg/m2). Less than a third of GCK mutation carriers were on pharmacotherapy (n = 20, 31%), while the majority of HNF1A mutation carriers used oral drugs or insulin (n = 66, 82.5%). While current QoL was similar across the three groups (p = 0.66), two other major indices—the impact of diabetes on QoL and the average weighted impact (AWI)—differed among them (p < 0.001 for both comparisons). The impact of diabetes on patient QoL and AWI observed in both MODY groups was smaller than in T1DM. Etiological diagnosis of diabetes and a diagnosis of retinopathy were the only independent factors influencing the impact of diabetes on QoL and AWI in regression analysis. In HNF1A-MODY, all three major indices of QoL were more heavily influenced for patients on insulin in comparison to other treatment sub-groups.ConclusionMODY has a smaller negative impact on QoL compared to T1DM. Mode of treatment further stratifies QoL decline for HNF1A-MODY subjects.

Screening of HNF1A and HNF4A mutation and clinical phenotype analysis in a large cohort of Chinese patients with maturity-onset diabetes of the young

The study aimed to screen the HNF1A and HNF4A mutation in a large Chinese cohort of high clinical suspicion of maturity-onset diabetes of the young (MODY) patients and characterize the clinical features of those patients. The performance of hsCRP as a biomarker to differentiate MODY3 from early onset T2DM was also evaluated. A total of 74 patients with a strong clinical suspicion of MODY from 59 families and 33 newly diagnosed early-onset T2DM were included. HNF1A and HNF4A mutations were analyzed by Sanger sequencing. ROC curves were used to identify the optimal cutoff of hsCRP. One novel (c.864_865insG) and six recurrent HNF1A mutations (R203H, R263H, P379T, L422P, P519L and c.873delC) in 17 patients from 8 families (13.6%), as well as one novel HNF4A (R331H) mutation were identified. Nonspecific clinical presentations were observed in MODYX compared to MODY3 patients. MODY3 subjects exhibited with younger, lower BMI, TG, fasting and postprandial C-peptide, higher HDL than T2DM. Particularly, we confirmed serum hsCRP was lower in MODY3 than T2DM. ROC curve showed a good discrimination with an AUC of 0.852 and identified a cutoff hsCRP of 0.79 (75% sensitivity and 83% specificity). Good glycemic control was observed in all identified patients after switching to glimepiride therapy. The prevalence of HNF1A mutation was relatively lower in Mainland China and HNF4A mutation was rare. Serum hsCRP concentrations performed well in discriminating MODY3 from T2DM. Molecular diagnosis of MODY3/1 did transform management in clinical practice and facilitated the glycemic control.

PS02.046: UNRAVELING TUMOR HETEROGENEITY OF ESOPHAGEAL ADENOCARCINOMA (EAC) THROUGH HIGH-THROUGHPUT OF SORTED TUMOR CELL POPULATIONS

Abstract Background In EAC clinical, histological, immune-histochemical and genetic patterns were documented, which support the existence of biologically different sub populations. We studied genetic intra/inter tumor heterogeneity of EAC with a new technology based on sorting of specific cell populations. Methods Formalin embedded material obtained from 16 EAC surgical specimens, classified according to the presence/absence of intestinal metaplasia in esophagus (BIM) and stomach (GIM) in BIM + GIM- (Barret's like), BIM-GIM- (cardio-pyloric like), BIM—GIM + (gastric like) types, was processed using a high-throughput cell sorting technology. Stromal and tumor cell populations were sorted based on antibodies against vimentin/pan-cytokeratin and on DNA content. Targeted resequencing on DNA extracted from the sorted cells was performed for 63 cancer-related genes (OncoSeek panel). Results In 11/16 (68.75%) of cases a mutation in TP53 was detected, and in 2 we observed CDKN2A (TP53 regulator) mutations. In Barret's like and Gastric like sub types only mutations of TP53 or TP53regulator genes were present. In pyloric like type TP53 and/or other different mutations of whom 2 in HNF1A were detected (Figure 1). Conclusion Selective sorting led to new patterns of EAC tumor mutational status and heterogeneity, among others somatic mutations in HNF1A not previously found. Parallel analysis of unsorted samples failed to detect these specific mutations. The cardio-pyloric like sub type contains mutational patterns different than those of Barret's and gastric like types. Further research on additional cases is necessary to confirm these findings. Disclosure All authors have declared no conflicts of interest.

A Novel De Novo Missense Mutation in HNF4A Resulting in Sulfonylurea-Responsive Maturity-onset Diabetes of the Young

A Novel De Novo Missense Mutation in HNF4A Resulting in Sulfonylurea-Responsive Maturity-onset Diabetes of the Young

Hnf4a deletion in the mouse kidney phenocopies Fanconi renotubular syndrome.

Different nephron tubule segments perform distinct physiological functions, collectively acting as a blood filtration unit. Dysfunction of the proximal tubule segment can lead to Fanconi renotubular syndrome (FRTS), with major symptoms such as excess excretion of water, glucose, and phosphate in the urine. It has been shown that a mutation in HNF4A is associated with FRTS in humans and that Hnf4a is expressed specifically in proximal tubules in adult rat nephrons. However, little is known about the role of Hnf4a in nephrogenesis. Here, we found that Hnf4a is expressed in both presumptive and differentiated proximal tubules in the developing mouse kidney. We show that Hnf4a is required for the formation of differentiated proximal tubules but is dispensable for the formation of presumptive proximal tubules. Furthermore, we show that loss of Hnf4a decreased the expression of proximal tubule-specific genes. Adult Hnf4a mutant mice presented with FRTS-like symptoms, including polyuria, polydipsia, glycosuria, and phosphaturia. Analysis of the adult Hnf4a mutant kidney also showed proximal tubule dysgenesis and nephrocalcinosis. Our results demonstrate the critical role of Hnf4a in proximal tubule development and provide mechanistic insight into the etiology of FRTS.

Quality of Life Assessment in Patients with HNF1A- and GCK-MODY Compared with T1DM

Maturity onset diabetes of the young (MODY) constitutes a few percent of this disease, with HNF1A- and GCK-MODY being its most common forms. We examined the impact on QoL (Quality of Life) of monogenic diabetes among patients with HNF1A-MODY or GCK mutation carrier status and compared it to type 1 diabetic (T1DM) individuals. Methods: The study included 80 patients with HNF1A-MODY and 89 GCK gene mutation carriers (45 patients with diabetes and 44 prediabetic). We also examined 128 T1DM patients as a control group. Diabetes-specific QoL was assessed using the Audit of Diabetes Dependent Quality of Life questionnaire. Results: Both HNF1A-MODY and GCK-MODY groups had similar mean age (41.7 vs. 38.0 years, respectively) and BMI (24.1 vs. 24.3 kg/m2), whereas T1DM patients were younger (34.2 years) with similar BMI (25.0 kg/m2). Less than a third of GCK mutation carriers were on pharmacotherapy (31%), while the majority of HNF1A mutation carriers used oral drugs or insulin (82.5%) (p&amp;lt;0.0001). While QoL were similar in three groups (p=0.66), both the impact of diabetes on the patient’s QoL and the average weighted impact (AWI) differed between three groups (p&amp;lt;0.001; p&amp;lt;0.001, respectively). For MODY patients, both indices were larger in HNF1A-MODY patients than in GCK-MODY (-0.96 ± 1.vs. -0.63 ± 0.88, p&amp;gt;0.and -1.52 ± 1.44 vs. -0.95±1.06, p=0.01, respectively). However, the largest impact of diabetes on the patient’s QoL and AWI were observed in the T1DM patients (-1.6 ± 1.05; -1.99 ± 1.39, respectively). The highest impact of diabetes (&amp;gt;-1.5) was observed in all groups on ^‘freedom to eat‘, ‘feelings about future‘, and ‘freedom to drink‘. In the HNF1A-MODY group, all three major indices of QoL were more affected for patients on insulin as compared to the other individuals. Conclusions: Diagnosis of the most frequent subtypes of MODY has a negative impact on QoL for affected individuals, although, it is smaller than in T1DM. Mode of treatment seems to influence QoL for HNF1A-MODY subjects. Disclosure M. Szopa: None.

Loss-of-function mutations in Zn-finger DNA-binding domain of HNF4A cause aberrant transcriptional regulation in liver cancer.

Hepatocyte nuclear factors (HNF) are transcription factors that crucially regulate cell-specific gene expression in many tissues, including the liver. Of these factors, HNF4A acts both as a master regulator of liver organogenesis and a tumor suppressor in the liver. In our whole genome sequencing analysis, we found seven somatic mutations (three Zn-finger mutations, three deletion mutants, and one intron mutation) of HNF4A in liver cancers. Interestingly, three out of seven mutations were clustered in its Zn-finger DNA-binding domain; G79 and F83 are positioned in the DNA recognition helix and the sidechain of M125 is sticking into the core of domain. These mutations are likely to affect DNA interaction from a structural point of view. We then generated these mutants and performed in-vitro promoter assays as well as DNA binding assays. These three mutations reduced HNF4 transcriptional activity at promoter sites of HNF4A-target genes. Expectedly, this decrease in transcriptional activity was associated with a change in DNA binding. RNA-Seq analysis observed a strong correlation between HNF4A expression and expression of its target genes, ApoB and HNF1A, in liver cancers. Since knockdown of HNF4A caused a reduction in ApoB and HNF1A expression, possibly loss of HNF4 reduces the expression of these genes and subsequently tumor growth is triggered. Therefore, we propose that HNF4A mutations G79C, F83C, and M125I are functional mutations found in liver cancers and that loss of HNF4A function, through its mutation, leads to a reduction in HNF1A and ApoB gene expression with a concomitant increased risk of liver tumorigenesis.

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

IntroductionMaturity 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 methodsWe 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.ResultsOur 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.ConclusionsThe 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.