Search

Aims/hypothesis: Monogenic diabetes is caused by rare mutations in genes usually implicated in beta cell biology. Common variants of monogenic diabetes genes (MDG) may jointly influence the risk of young-onset type 2 diabetes (YOD, diagnosed before the age of 40 years) and cardiovascular and kidney events. Methods: Using whole-exome sequencing data, we constructed a weighted polygenic risk score (wPRS) consisting of 135 common variants (minor allele frequency >0.01) of 34 MDG based on r2>0.2 for linkage disequilibrium in a discovery case–control cohort of 453 adults with YOD (median [IQR] age 39.7 [34.9–46.9] years) and 405 without YOD (median [IQR] age 56.7 [50.3–61.0] years), followed by validation in an independent cross-sectional cohort with array-based genotyping for YOD and a prospective cohort of individuals with type 2 diabetes for cardiovascular and kidney events. Results: In the discovery cohort, the OR of the 135 common variants for YOD ranged from 1.00 to 2.61. In the validation cohort (920 YOD and 4910 non-YOD), top-10%-wPRS was associated with an OR of 1.42 (95% CI 1.03, 1.95, p=0.033) for YOD compared with bottom-10%-wPRS. In 2313 individuals with type 2 diabetes (median [IQR]: age 53.4 [45.4–61.7] years; disease duration 4.0 [1.0–9.0] years) observed for a median (IQR) of 17.5 (14.4–21.8) years, standardised wPRS was associated with increased HR for incident cardiovascular events (1.16 [95% CI 1.06, 1.27], p=0.001), kidney events (1.09 [95% CI 1.02, 1.16], p=0.013) and cardiovascular–kidney events (1.10 [95% CI 1.03, 1.16], p=0.003). Using the 'bottom-20%-wPRS plus baseline disease duration <5 years' group as referent, the 'top-20%-wPRS plus baseline disease duration 5 to <10 years' group had unadjusted and adjusted HR of 1.60 (95% CI 1.17, 2.19, p=0.003) and 1.62 (95% CI 1.16, 2.26, p=0.005), respectively, for cardiovascular–kidney events compared with 1.38 (95% CI 0.97, 1.98, p=0.075) and 1.06 (95% CI 0.72, 1.57, p=0.752) in the 'bottom-20%-wPRS plus baseline disease duration ≥10 years' group. Conclusions/interpretation: Common variants of MDG increased risk for YOD and cardiovascular–kidney events. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Correctly diagnosing MODY is important, as individuals with this diagnosis can discontinue insulin injections; however, many people are misdiagnosed. We aimed to develop a robust approach for determining the pathogenicity of variants of uncertain significance in hepatocyte nuclear factor-1 alpha (HNF1A)-MODY and to obtain an accurate estimate of the prevalence of HNF1A-MODY in paediatric cases of diabetes. Methods: We extended our previous screening of the Norwegian Childhood Diabetes Registry by 830 additional samples and comprehensively genotyped HNF1A variants in autoantibody-negative participants using next-generation sequencing. Carriers of pathogenic variants were treated by local healthcare providers, and participants with novel likely pathogenic variants and variants of uncertain significance were enrolled in an investigator-initiated, non-randomised, open-label pilot study (ClinicalTrials.gov registration no. NCT04239586). To identify variants associated with HNF1A-MODY, we functionally characterised their pathogenicity and assessed the carriers' phenotype and treatment response to sulfonylurea. Results: In total, 615 autoantibody-negative participants among 4712 cases of paediatric diabetes underwent genetic sequencing, revealing 19 with HNF1A variants. We identified nine carriers with novel variants classified as variants of uncertain significance or likely to be pathogenic, while the remaining ten participants carried five pathogenic variants previously reported. Of the nine carriers with novel variants, six responded favourably to sulfonylurea. Functional investigations revealed their variants to be dysfunctional and demonstrated a correlation with the resulting phenotype, providing evidence for reclassifying these variants as pathogenic. Conclusions/interpretation: Based on this robust classification, we estimate that the prevalence of HNF1A-MODY is 0.3% in paediatric diabetes. Clinical phenotyping is challenging and functional investigations provide a strong complementary line of evidence. We demonstrate here that combining clinical phenotyping with functional protein studies provides a powerful tool to obtain a precise diagnosis of HNF1A-MODY. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: In pregnancies where the mother has glucokinase-MODY (GCK-MODY), fetal growth is determined by fetal genotype. When the fetus inherits a maternal pathogenic GCK variant, normal fetal growth is anticipated, and insulin treatment of maternal hyperglycaemia is not recommended. At present, fetal genotype is estimated from measurement of fetal abdominal circumference on ultrasound. Non-invasive prenatal testing of fetal GCK genotype (NIPT-GCK) using cell-free DNA in maternal blood has recently been developed. We aimed to compare the diagnostic accuracy of NIPT-GCK with that of ultrasound, and determine the feasibility of using NIPT-GCK to guide pregnancy management. Methods: We studied an international cohort of pregnant women with hyperglycaemia due to GCK-MODY. We compared the diagnostic accuracy of NIPT-GCK with that of measurement of fetal abdominal circumference at 28 weeks' gestation (n=38) using a directly genotyped offspring sample as the reference standard. In a feasibility study, we assessed the time to result given to clinicians in 43 consecutive pregnancies affected by GCK-MODY between July 2019 and September 2021. Results: In terms of diagnostic accuracy, NIPT-GCK was more sensitive and specific than ultrasound in predicting fetal genotype (sensitivity 100% and specificity 96% for NIPT-GCK vs sensitivity 53% and specificity 61% for fetal abdominal circumference 75th percentile). In terms of feasibility, a valid NIPT-GCK fetal genotype (≥95% probability) was reported in all 38 pregnancies with an amenable variant and repeated samples when needed. The median time to report was 5 weeks (IQR 3–8 weeks). For the 25 samples received before 20 weeks' gestation, results were reported at a median gestational age of 20 weeks (IQR 18–24), with 23/25 (92%) reported before 28 weeks. Conclusions/interpretation: Non-invasive prenatal testing of fetal genotype in GCK-MODY pregnancies is highly accurate and is capable of providing a result before the last trimester for most patients. This means that non-invasive prenatal testing of fetal genotype is the optimal approach to management of GCK-MODY pregnancies. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Monogenic forms of diabetes (MODY, neonatal diabetes mellitus and syndromic forms) are rare, and affected individuals may be misclassified and treated suboptimally. The prevalence of type 1 diabetes is high in Finnish children but systematic screening for monogenic diabetes has not been conducted. We assessed the prevalence and clinical manifestations of monogenic diabetes in children initially registered with type 1 diabetes in the Finnish Pediatric Diabetes Register (FPDR) but who had no type 1 diabetes-related autoantibodies (AABs) or had only low-titre islet cell autoantibodies (ICAs) at diagnosis. Methods: The FPDR, covering approximately 90% of newly diagnosed diabetic individuals aged ≤15 years in Finland starting from 2002, includes data on diabetes-associated HLA genotypes and AAB data (ICA, and autoantibodies against insulin, GAD, islet antigen 2 and zinc transporter 8) at diagnosis. A next generation sequencing gene panel including 42 genes was used to identify monogenic diabetes. We interpreted the variants in HNF1A by using the gene-specific standardised criteria and reported pathogenic and likely pathogenic findings only. For other genes, we also reported variants of unknown significance if an individual's phenotype suggested monogenic diabetes. Results: Out of 6482 participants, we sequenced DNA for 152 (2.3%) testing negative for all AABs and 49 (0.8%) positive only for low-titre ICAs (ICAlow). A monogenic form of diabetes was revealed in 19 (12.5%) of the AAB-negative patients (14 [9.2%] had pathogenic or likely pathogenic variants) and two (4.1%) of the ICAlow group. None had ketoacidosis at diagnosis or carried HLA genotypes conferring high risk for type 1 diabetes. The affected genes were GCK, HNF1A, HNF4A, HNF1B, INS, KCNJ11, RFX6, LMNA and WFS1. A switch from insulin to oral medication was successful in four of five patients with variants in HNF1A, HNF4A or KCNJ11. Conclusions/interpretation: More than 10% of AAB-negative children with newly diagnosed diabetes had a genetic finding associated with monogenic diabetes. Because the genetic diagnosis can lead to major changes in treatment, we recommend referring all AAB-negative paediatric patients with diabetes for genetic testing. Low-titre ICAs in the absence of other AABs does not always indicate a diagnosis of type 1 diabetes. [ABSTRACT FROM AUTHOR]

The historical subclassification of diabetes into predominantly types 1 and 2 is well appreciated to inadequately capture the heterogeneity seen in patient presentations, disease course, response to therapy and disease complications. This review summarises proposed data-driven approaches to further refine diabetes subtypes using clinical phenotypes and/or genetic information. We highlight the benefits as well as the limitations of these subclassification schemas, including practical barriers to their implementation that would need to be overcome before incorporation into clinical practice. [ABSTRACT FROM AUTHOR]

Integration of genomic and other data has begun to stratify type 2 diabetes in prognostically meaningful ways, but this has yet to impact on mainstream diabetes practice. The subgroup of diabetes caused by single gene defects thus provides the best example to date of the vision of 'precision diabetes'. Monogenic diabetes may be divided into primary pancreatic beta cell failure, and primary insulin resistance. In both groups, clear examples of genotype-selective responses to therapy have been advanced. The benign trajectory of diabetes due to pathogenic GCK mutations, and the sulfonylurea-hyperresponsiveness conferred by activating KCNJ11 or ABCC8 mutations, or loss-of-function HNF1A or HNF4A mutations, often decisively guide clinical management. In monogenic insulin-resistant diabetes, subcutaneous leptin therapy is beneficial in some severe lipodystrophy. Increasing evidence also supports use of 'obesity therapies' in lipodystrophic people even without obesity. In beta cell diabetes the main challenge is now implementation of the precision diabetes vision at scale. In monogenic insulin-resistant diabetes genotype-specific benefits are proven in far fewer patients to date, although further genotype-targeted therapies are being evaluated. The conceptual paradigm established by the insulin-resistant subgroup with 'adipose failure' may have a wider influence on precision therapy for common type 2 diabetes, however. For all forms of monogenic diabetes, population-wide genome sequencing is currently forcing reappraisal of the importance assigned to pathogenic mutations when gene sequencing is uncoupled from prior suspicion of monogenic diabetes. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Monogenic diabetes is caused by rare mutations in genes usually implicated in beta cell biology. Common variants of monogenic diabetes genes (MDG) may jointly influence the risk of young-onset type 2 diabetes (YOD, diagnosed before the age of 40 years) and cardiovascular and kidney events., Methods: Using whole-exome sequencing data, we constructed a weighted polygenic risk score (wPRS) consisting of 135 common variants (minor allele frequency >0.01) of 34 MDG based on r 2 >0.2 for linkage disequilibrium in a discovery case-control cohort of 453 adults with YOD (median [IQR] age 39.7 [34.9-46.9] years) and 405 without YOD (median [IQR] age 56.7 [50.3-61.0] years), followed by validation in an independent cross-sectional cohort with array-based genotyping for YOD and a prospective cohort of individuals with type 2 diabetes for cardiovascular and kidney events., Results: In the discovery cohort, the OR of the 135 common variants for YOD ranged from 1.00 to 2.61. In the validation cohort (920 YOD and 4910 non-YOD), top-10%-wPRS was associated with an OR of 1.42 (95% CI 1.03, 1.95, p=0.033) for YOD compared with bottom-10%-wPRS. In 2313 individuals with type 2 diabetes (median [IQR]: age 53.4 [45.4-61.7] years; disease duration 4.0 [1.0-9.0] years) observed for a median (IQR) of 17.5 (14.4-21.8) years, standardised wPRS was associated with increased HR for incident cardiovascular events (1.16 [95% CI 1.06, 1.27], p=0.001), kidney events (1.09 [95% CI 1.02, 1.16], p=0.013) and cardiovascular-kidney events (1.10 [95% CI 1.03, 1.16], p=0.003). Using the 'bottom-20%-wPRS plus baseline disease duration <5 years' group as referent, the 'top-20%-wPRS plus baseline disease duration 5 to <10 years' group had unadjusted and adjusted HR of 1.60 (95% CI 1.17, 2.19, p=0.003) and 1.62 (95% CI 1.16, 2.26, p=0.005), respectively, for cardiovascular-kidney events compared with 1.38 (95% CI 0.97, 1.98, p=0.075) and 1.06 (95% CI 0.72, 1.57, p=0.752) in the 'bottom-20%-wPRS plus baseline disease duration ≥10 years' group., Conclusions/interpretation: Common variants of MDG increased risk for YOD and cardiovascular-kidney events., Competing Interests: Acknowledgements: We thank all the involved research and clinical staff in the Hospital Authority and Department of Medicine and Therapeutics, the Chinese University of Hong Kong, for their professionalism and dedication. Special thanks are extended to all patients, their relatives and volunteers for participating in these studies. We are grateful to the support of the Hong Kong Hospital Authority. Some data in this manuscript were presented in the American Diabetes Association 84th Scientific Sessions in the form of a published abstract and a poster in 2024. Data availability: Due to local law and regulation, no data can be shared with external parties. Summary statistics may be shared upon reasonable request to the corresponding author. Funding: The study is funded by the Hong Kong Genome Institute, Hong Kong Government Health and Medical Research Fund Commissioned Research (CFS-CUHK2) and Research Grants Council Impact Research Fund. Author’s relationships and activities: JCNC has received research grants (through institutions) and/or honoraria for consultancy and/or giving lectures from Applied Therapeutics, AstraZeneca, Bayer, Boehringer Ingelheim, Eli Lilly, Hua Medicine, Powder Pharmaceuticals, Merck Serono, MSD, Pfizer, Sanofi, Viatris and Zuellig Pharma. AOYL has served as an advisory committee member for AstraZeneca, Boehringer Ingelheim, Sanofi and Amgen, and has received research grants and travel grants from AstraZeneca, Boehringer Ingelheim, MSD, Novartis, Novo Nordisk, Sanofi and Amgen. RCWM has received research grants for clinical trials from AstraZeneca, Bayer, MSD, Novo Nordisk, Sanofi, Roche and Tricida and honoraria for consultancy or lectures from AstraZeneca and Boehringer Ingelheim. APSK has received research grants and/or speaker honoraria from Abbott, AstraZeneca, Bayer, Boehringer Ingelheim, Eli Lilly, Merck Serono, Nestle, Novo Nordisk and Sanofi. JCNC, WYS and RCWM hold patents for using biogenetic markers to predict risk of diabetes and its complications. JCNC, CKPL, RCWM and WYS are co-founders of GemVCare, a biotech start-up supported by the Technology Start-up Support Scheme for Universities of the Hong Kong Government Innovation and Technology Commission. RCWM is an advisory board member of the editorial board of Diabetologia. The authors declare that there are no other relationships or activities that might bias, or be perceived to bias, their work. Contribution statement: JCNC and AOYL conceptualised the research question. CKO and BF performed the data analysis. CKO, BF, JCNC, AOYL, STFT, CHTT, RW, ESHL, MS, CKPL, GY, JPYH, EYKC, APSK, RO, WYS and RCWM contributed to the interpretation of the data. CKO wrote the first draft. All authors critically reviewed the manuscript and approved the final version. JCNC is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis., (© 2024. The Author(s).)

Aims/hypothesis: Current clinical guidelines for childhood-onset monogenic diabetes outside infancy are mainly focused on identifying and testing for dominantly inherited, predominantly MODY genes. There are no systematic studies of the recessively inherited causes of monogenic diabetes that are likely to be more common in populations with high rates of consanguinity. We aimed to determine the contribution of recessive causes of monogenic diabetes in paediatric diabetes clinics and to identify clinical criteria by which to select individuals for recessive monogenic diabetes testing. Methods: We conducted a cross-sectional study of 1093 children from seven paediatric diabetes clinics across Turkey (a population with high rates of consanguinity). We undertook genetic testing of 50 known dominant and recessive causes of monogenic diabetes for 236 children at low risk of type 1 diabetes. As a comparison, we used monogenic diabetes cases from UK paediatric diabetes clinics (a population with low rates of consanguinity). Results: Thirty-four children in the Turkish cohort had monogenic diabetes, equating to a minimal prevalence of 3.1%, similar to that in the UK cohort (p = 0.40). Forty-one per cent (14/34) had autosomal recessive causes in contrast to 1.6% (2/122) in the UK monogenic diabetes cohort (p < 0.0001). All conventional criteria for identifying monogenic diabetes (parental diabetes, not requiring insulin treatment, HbA1c ≤ 58 mmol/mol [≤7.5%] and a composite clinical probability of MODY >10%) assisted the identification of the dominant (all p ≤ 0.0003) but not recessive cases (all p ≥ 0.2) in Turkey. The presence of certain non-autoimmune extra-pancreatic features greatly assisted the identification of recessive (p < 0.0001, OR 66.9) but not dominant cases. Conclusions/interpretation: Recessively inherited mutations are a common cause of monogenic diabetes in populations with high rates of consanguinity. Present MODY-focused genetic testing strategies do not identify affected individuals. To detect all cases of monogenic paediatric diabetes, it is crucial that recessive genes are included in genetic panels and that children are selected for testing if they have certain non-autoimmune extra-pancreatic features in addition to current criteria. [ABSTRACT FROM AUTHOR]

The precision medicine approach of tailoring treatment to the individual characteristics of each patient or subgroup has been a great success in monogenic diabetes subtypes, MODY and neonatal diabetes. This review examines what has led to the success of a precision medicine approach in monogenic diabetes (precision diabetes) and outlines possible implications for type 2 diabetes. For monogenic diabetes, the molecular genetics can define discrete aetiological subtypes that have profound implications on diabetes treatment and can predict future development of associated clinical features, allowing early preventative or supportive treatment. In contrast, type 2 diabetes has overlapping polygenic susceptibility and underlying aetiologies, making it difficult to define discrete clinical subtypes with a dramatic implication for treatment. The implementation of precision medicine in neonatal diabetes was simple and rapid as it was based on single clinical criteria (diagnosed <6 months of age). In contrast, in MODY it was more complex and slow because of the lack of single criteria to identify patients, but it was greatly assisted by the development of a diagnostic probability calculator and associated smartphone app. Experience in monogenic diabetes suggests that successful adoption of a precision diabetes approach in type 2 diabetes will require simple, quick, easily accessible stratification that is based on a combination of routine clinical data, rather than relying on newer technologies. Analysing existing clinical data from routine clinical practice and trials may provide early success for precision medicine in type 2 diabetes. [ABSTRACT FROM AUTHOR]

MODY is an early-onset monogenic form of diabetes. Correctly identifying MODY is of considerable importance as diagnosing the specific genetic subtype can inform the optimal treatment, with many patients being able to discontinue unnecessary insulin treatment. Diagnostic molecular genetic testing to confirm MODY is expensive, so screening strategies are required to identify the most appropriate patients for testing. In this issue of Diabetologia, Johansson and colleagues (DOI ) describe a nationwide systematic screening approach to identify individuals with MODY in the paediatric age range. They focused testing on patients negative for both GAD and islet antigen 2 (IA-2) islet autoantibodies, thereby ruling out those with markers of type 1 diabetes, the most common form of diabetes in this age group. This commentary discusses the advantages and limitations of the approach, and the caution required when interpreting variants of uncertain pathogenicity identified from testing whole populations rather than targeting only patients with a strong MODY phenotype. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: MODY can be wrongly diagnosed as type 1 diabetes in children. We aimed to find the prevalence of MODY in a nationwide population-based registry of childhood diabetes. Methods: Using next-generation sequencing, we screened the HNF1A, HNF4A, HNF1B, GCK and INS genes in all 469 children (12.1%) negative for both GAD and IA-2 autoantibodies and 469 antibody-positive matched controls selected from the Norwegian Childhood Diabetes Registry (3882 children). Variants were classified using clinical diagnostic criteria for pathogenicity ranging from class 1 (neutral) to class 5 (pathogenic). Results: We identified 58 rare exonic and splice variants in cases and controls. Among antibody-negative patients, 6.5% had genetic variants of classes 3-5 (vs 2.4% in controls; p = 0.002). For the stricter classification (classes 4 and 5), the corresponding number was 4.1% (vs 0.2% in controls; p = 1.6 × 10). HNF1A showed the strongest enrichment of class 3-5 variants, with 3.9% among antibody-negative patients (vs 0.4% in controls; p = 0.0002). Antibody-negative carriers of variants in class 3 had a similar phenotype to those carrying variants in classes 4 and 5. Conclusions/interpretation: This is the first study screening for MODY in all antibody-negative children in a nationwide population-based registry. Our results suggest that the prevalence of MODY in antibody-negative childhood diabetes may reach 6.5%. One-third of these MODY cases had not been recognised by clinicians. Since a precise diagnosis is important for treatment and genetic counselling, molecular screening of all antibody-negative children should be considered in routine diagnostics. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: We aimed to identify microRNAs (miRNAs) under transcriptional control of the HNF1β transcription factor, and investigate whether its effect manifests in serum. Methods: The Polish cohort ( N = 60) consisted of 11 patients with HNF1B-MODY, 17 with HNF1A-MODY, 13 with GCK-MODY, an HbA-matched type 1 diabetic group ( n = 9) and ten healthy controls. Replication was performed in 61 clinically-matched British patients mirroring the groups in the Polish cohort. The Polish cohort underwent miRNA serum level profiling with quantitative real-time PCR (qPCR) arrays to identify differentially expressed miRNAs. Validation was performed using qPCR. To determine whether serum content reflects alterations at a cellular level, we quantified miRNA levels in a human hepatocyte cell line (HepG2) with small interfering RNA knockdowns of HNF1α or HNF1β. Results: Significant differences (adjusted p < 0.05) were noted for 11 miRNAs. Five of them differed between HNF1A-MODY and HNF1B-MODY, and, amongst those, four (miR-24, miR-27b, miR-223 and miR-199a) showed HNF1B-MODY-specific expression levels in the replication group. In all four cases the miRNA expression level was lower in HNF1B-MODY than in all other tested groups. Areas under the receiver operating characteristic curves ranged from 0.79 to 0.86, with sensitivity and specificity reaching 91.7% (miR-24) and 82.1% (miR-199a), respectively. The cellular expression pattern of miRNA was consistent with serum levels, as all were significantly higher in HNF1α- than in HNF1β-deficient HepG2 cells. Conclusions/interpretation: We have shown that expression of specific miRNAs depends on HNF1β function. The impact of HNF1β deficiency was evidenced at serum level, making HNF1β-dependent miRNAs potentially applicable in the diagnosis of HNF1B-MODY. [ABSTRACT FROM AUTHOR]

Diabetes is not a single homogeneous disease but composed of many diseases with hyperglycaemia as a common feature. Four factors have, historically, been used to identify this diversity: the age at onset; the severity of the disease, i.e. degree of loss of beta cell function; the degree of insulin resistance and the presence of diabetes-associated autoantibodies. Our broad understanding of the distinction between the two major types, type 1 diabetes mellitus and type 2 diabetes mellitus, are based on these factors, but it has become apparent that they do not precisely capture the different disease forms. Indeed, both major types of diabetes have common features, encapsulated by adult-onset autoimmune diabetes and maturity-onset diabetes of the young. As a result, there has been a repositioning of our understanding of diabetes. In this review, drawing on recent literature, we discuss the evidence that autoimmune type 1 diabetes has a broad clinical phenotype with diverse therapeutic options, while the term non-autoimmune type 2 diabetes obscures the optimal management strategy because it encompasses substantial heterogeneity. Underlying these developments is a general progression towards precision medicine with the need for precise patient characterisation, currently based on clinical phenotypes but in future augmented by laboratory-based tests. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: MODY is mainly characterised by an early onset of diabetes and a positive family history of diabetes with an autosomal dominant mode of inheritance. However, de novo mutations have been reported anecdotally. The aim of this study was to systematically revisit a large collection of MODY patients to determine the minimum prevalence of de novo mutations in the most prevalent MODY genes (i.e. GCK, HNF1A, HNF4A). Methods: Analysis of 922 patients from two national MODY centres (Slovakia and the Czech Republic) identified 150 probands (16%) who came from pedigrees that did not fulfil the criterion of two generations with diabetes but did fulfil the remaining criteria. The GCK, HNF1A and HNF4A genes were analysed by direct sequencing. Results: Mutations in GCK, HNF1A or HNF4A genes were detected in 58 of 150 individuals. Parents of 28 probands were unavailable for further analysis, and in 19 probands the mutation was inherited from an asymptomatic parent. In 11 probands the mutations arose de novo. Conclusions/interpretation: In our cohort of MODY patients from two national centres the de novo mutations in GCK, HNF1A and HNF4A were present in 7.3% of the 150 families without a history of diabetes and 1.2% of all of the referrals for MODY testing. This is the largest collection of de novo MODY mutations to date, and our findings indicate a much higher frequency of de novo mutations than previously assumed. Therefore, genetic testing of MODY could be considered for carefully selected individuals without a family history of diabetes. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Current genetic tests for diagnosing monogenic diabetes rely on selection of the appropriate gene for analysis according to the patient's phenotype. Next-generation sequencing enables the simultaneous analysis of multiple genes in a single test. Our aim was to develop a targeted next-generation sequencing assay to detect mutations in all known MODY and neonatal diabetes genes. Methods: We selected 29 genes in which mutations have been reported to cause neonatal diabetes, MODY, maternally inherited diabetes and deafness (MIDD) or familial partial lipodystrophy (FPLD). An exon-capture assay was designed to include coding regions and splice sites. A total of 114 patient samples were tested-32 with known mutations and 82 previously tested for MODY ( n = 33) or neonatal diabetes ( n = 49) but in whom a mutation had not been found. Sequence data were analysed for the presence of base substitutions, small insertions or deletions (indels) and exonic deletions or duplications. Results: In the 32 positive controls we detected all previously identified variants (34 mutations and 36 polymorphisms), including 55 base substitutions, ten small insertions or deletions and five partial/whole gene deletions/duplications. Previously unidentified mutations were found in five patients with MODY (15%) and nine with neonatal diabetes (18%). Most of these patients (12/14) had mutations in genes that had not previously been tested. Conclusions/interpretation: Our novel targeted next-generation sequencing assay provides a highly sensitive method for simultaneous analysis of all monogenic diabetes genes. This single test can detect mutations previously identified by Sanger sequencing or multiplex ligation-dependent probe amplification dosage analysis. The increased number of genes tested led to a higher mutation detection rate. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Monogenic diabetes (MD) might be misdiagnosed as type 1 diabetes. The prevalence of MD among children with apparent type 1 diabetes has not been established. Our aim was to estimate the prevalence of common forms of MD in childhood diabetes. Methods: We investigated 2,756 children aged 0-14 years with newly diagnosed diabetes who had been recruited to the nationwide population-based Norwegian Childhood Diabetes Registry (NCDR), from July 2002 to March 2012. Completeness of ascertainment was 91%. Children diagnosed with diabetes who were under12 months of age were screened for mutations in KCNJ11, ABCC8 and INS. Children without GAD and protein tyrosine phosphatase-like protein antibodies were screened in two ways. Those who had a parent with diabetes were screened for mutations in HNF1A, HNF4A, INS and MT-TL1. Children with HbA <7.5% (<58 mmol/mol) and no insulin requirement were screened for mutations in GCK. Finally, we searched the Norwegian MODY Registry for children with genetically verified MD. Results: We identified 15 children harbouring a mutation in HNF1A, nine with one in GCK, four with one in KCNJ11, one child with a mutation in INS and none with a mutation in MT-TL1. The minimum prevalence of MD in the NCDR was therefore 1.1%. By searching the Norwegian MODY Registry, we found 24 children with glucokinase-MODY, 15 of whom were not present in the NCDR. We estimated the minimum prevalence of MD among Norwegian children to be 3.1/100,000. Conclusions/interpretation: This is the first prevalence study of the common forms of MD in a nationwide, population-based registry of childhood diabetes. We found that 1.1% of patients in the Norwegian Childhood Diabetes Registry had MD. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: MODY is believed to be caused by at least 13 different genes. Five rare mutations at the BLK locus, including only one non-synonymous p.A71T variant, were reported to segregate with diabetes in three MODY families. The p.A71T mutation was shown to abolish the enhancing effect of BLK on insulin content and secretion from pancreatic beta cell lines. Here, we reassessed the contribution of BLK to MODY and tested the effect of BLK-p.A71T on type 2 diabetes risk and variations in related traits. Methods: BLK was sequenced in 64 unelucidated MODY samples. The BLK-p.A71T variant was genotyped in a French type 2 diabetes case-control study including 4,901 cases and 4,280 controls, and in the DESIR (Data from an Epidemiological Study on the Insulin Resistance Syndrome) and SUVIMAX (Supplementation en Vitamines et Mineraux Antioxydants) population-based cohorts ( n = 6,905). The variant effects were assessed by logistic and linear regression models. Results: No rare non-synonymous BLK mutations were found in the MODY patients. The BLK p.A71T mutation was present in 52 normoglycaemic individuals, making it very unlikely that this loss-of-function mutation causes highly penetrant MODY. We found a nominal association between this variant and increased type 2 diabetes risk, with an enrichment of the mutation in the obese diabetic patients, although no significant association with BMI was identified. Conclusions/interpretation: No mutation in BLK was found in our MODY cohort. From our findings, the BLK-p.A71T mutation may weakly influence type 2 diabetes risk in the context of obesity; however, this will require further validation. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: The aim of this study was to study dynamic changes in the prevalence of different types of diabetes in paediatric populations in Poland, with a specific focus on monogenic diabetes (MD). Methods: Using epidemiologic data (PolPeDiab Collaboration) and nationwide genetic test results (TEAM Programme), we compared the prevalence of type 1, type 2 and cystic fibrosis-related diabetes (CFRD) and MD. Genetically confirmed MD included MODY, neonatal diabetes and Wolfram and Alström syndromes. The study covered all children aged 0-18 years treated for diabetes between 2005 and 2011 in three regions, inhabited by 23.7% (1,989,988) of Polish children, with a low prevalence of childhood obesity (<5%). Results: The prevalence of type 1 diabetes showed a continuous increase, from 96 to 138/100,000 children. The prevalence of type 2 diabetes and CFRD also increased, from 0.3 to 1.01/100,000 children and from 0.1 to 0.95/100,000 children, respectively. The prevalence of MD was stable at between 4.2 and 4.6/100,000 children, accounting for 3.1-4.2% of children with diabetes, with glucokinase ( GCK)-MODY being the most frequent type, amounting to 83% of patients with MD. The percentage of positive test results decreased with the number of referrals, suggesting that children with the highest probability of MD were referred initially, followed by those with a less clear-cut phenotype. The prevalence of neonatal diabetes equalled 1 in 300,000 children. Conclusions/interpretation: The prevalence of MD in a paediatric population with a low prevalence of obesity remains stable and is nearly fivefold higher than that of type 2 diabetes and CFRD, justifying a need for increased access to genetic diagnostic procedures in diabetic children. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Diagnosing MODY is difficult. To date, selection for molecular genetic testing for MODY has used discrete cut-offs of limited clinical characteristics with varying sensitivity and specificity. We aimed to use multiple, weighted, clinical criteria to determine an individual's probability of having MODY, as a crucial tool for rational genetic testing. Methods: We developed prediction models using logistic regression on data from 1,191 patients with MODY ( n = 594), type 1 diabetes ( n = 278) and type 2 diabetes ( n = 319). Model performance was assessed by receiver operating characteristic (ROC) curves, cross-validation and validation in a further 350 patients. Results: The models defined an overall probability of MODY using a weighted combination of the most discriminative characteristics. For MODY, compared with type 1 diabetes, these were: lower HbA, parent with diabetes, female sex and older age at diagnosis. MODY was discriminated from type 2 diabetes by: lower BMI, younger age at diagnosis, female sex, lower HbA, parent with diabetes, and not being treated with oral hypoglycaemic agents or insulin. Both models showed excellent discrimination (c-statistic = 0.95 and 0.98, respectively), low rates of cross-validated misclassification (9.2% and 5.3%), and good performance on the external test dataset (c-statistic = 0.95 and 0.94). Using the optimal cut-offs, the probability models improved the sensitivity (91% vs 72%) and specificity (94% vs 91%) for identifying MODY compared with standard criteria of diagnosis <25 years and an affected parent. The models are now available online at . Conclusions/interpretation: We have developed clinical prediction models that calculate an individual's probability of having MODY. This allows an improved and more rational approach to determine who should have molecular genetic testing. [ABSTRACT FROM AUTHOR]

To test, or not to test, that is often the question in diabetes genetics. This is why the paper of Shields et al in the current issue of Diabetologia is so warmly welcomed. MODY is the most common form of monogenic diabetes. Nevertheless, the optimal way of identifying MODY families still poses a challenge both for researchers and clinicians. Hattersley's group in Exeter, UK, have developed an easy-to-use MODY prediction model that can help to identify cases appropriate for genetic testing. By answering eight simple questions on the internet (), the doctor receives a positive predictive value in return: the probability that the patient has MODY. Thus, the classical binary (yes/no) assessment provided by clinical diagnostic criteria has been substituted by a more rational, quantitative estimate. The model appears to discriminate well between MODY and type 1 and type 2 diabetes when diabetes is diagnosed before the age of 35 years. However, the performance of the MODY probability calculator should now be validated in other settings than where it was developed-and, as always, there is room for some improvements and modifications. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: The ABCC8 gene encodes the sulfonylurea receptor 1 (SUR1) subunit of the pancreatic beta cell ATP-sensitive potassium (K) channel. Inactivating mutations cause congenital hyperinsulinism (CHI) and activating mutations cause transient neonatal diabetes (TNDM) or permanent neonatal diabetes (PNDM) that can usually be treated with sulfonylureas. Sulfonylurea sensitivity is also a feature of HNF1A and HNF4A MODY, but patients referred for genetic testing with clinical features of these types of diabetes do not always have mutations in the HNF1A/4A genes. Our aim was to establish whether mutations in the ABCC8 gene cause MODY that is responsive to sulfonylurea therapy. Methods: We sequenced the ABCC8 gene in 85 patients with a BMI <30 kg/m, no family history of neonatal diabetes and who were deemed sensitive to sulfonylureas by the referring clinician or were sulfonylurea-treated. All had tested negative for mutations in the HNF1A and HNF4A genes. Results: ABCC8 mutations were found in seven of the 85 (8%) probands. Four patients were heterozygous for previously reported mutations and four novel mutations, E100K, G214R, Q485R and N1245D, were identified. Only four probands fulfilled MODY criteria, with two diagnosed after 25 years and one patient, who had no family history of diabetes, as a result of a proven de novo mutation. Conclusions/interpretation: ABCC8 mutations can cause MODY in patients whose clinical features are similar to those with HNF1A/4A MODY. Therefore, sequencing of ABCC8 in addition to the known MODY genes should be considered if such features are present, to facilitate optimal clinical management of these patients. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: An accurate molecular diagnosis of diabetes subtype confers clinical benefits; however, many individuals with monogenic diabetes remain undiagnosed. Biomarkers could help to prioritise patients for genetic investigation. We recently demonstrated that high-sensitivity C-reactive protein (hsCRP) levels are lower in UK patients with hepatocyte nuclear factor 1 alpha (HNF1A)-MODY than in other diabetes subtypes. In this large multi-centre study we aimed to assess the clinical validity of hsCRP as a diagnostic biomarker, examine the genotype-phenotype relationship and compare different hsCRP assays. Methods: High-sensitivity CRP levels were analysed in individuals with HNF1A-MODY ( n = 457), glucokinase (GCK)-MODY ( n = 404), hepatocyte nuclear factor 4 alpha (HNF4A)-MODY ( n = 54) and type 2 diabetes ( n = 582) from seven European centres. Three common assays for hsCRP analysis were evaluated. We excluded 121 participants (8.1%) with hsCRP values >10 mg/l. The discriminative power of hsCRP with respect to diabetes aetiology was assessed by receiver operating characteristic curve-derived C-statistic. Results: In all centres and irrespective of the assay method, meta-analysis confirmed significantly lower hsCRP levels in those with HNF1A-MODY than in those with other aetiologies ( z score −21.8, p < 5 × 10). HNF1A-MODY cases with missense mutations had lower hsCRP levels than those with truncating mutations (0.03 vs 0.08 mg/l, p < 5 × 10). High-sensitivity CRP values between assays were strongly correlated ( r ≥ 0.91, p ≤ 1 × 10). Across the seven centres, the C-statistic for distinguishing HNF1A-MODY from young adult-onset type 2 diabetes ranged from 0.79 to 0.97, indicating high discriminative accuracy. Conclusions/interpretation: In the largest study to date, we have established that hsCRP is a clinically valid biomarker for HNF1A-MODY in European populations. Given the modest costs and wide availability, hsCRP could translate rapidly into clinical practice, considerably improving diagnosis rates in monogenic diabetes. [ABSTRACT FROM AUTHOR]

ims/hypothesis: Maturity-onset diabetes of the young is frequently misdiagnosed as type 1 or type 2 diabetes. A correct diagnosis of MODY is important for determining treatment, but can only be confirmed by molecular genetic testing. We aimed to compare the regional distribution of confirmed MODY cases in the UK and to estimate the minimum prevalence. Methods: UK referrals for genetic testing in 2,072 probands and 1,280 relatives between 1996 and 2009 were examined by region, country and test result. Referral rate and prevalence were calculated using UK Census 2001 figures. Results: MODY was confirmed in 1,177 (35%) patients, with HNF1A (52%) and GCK mutations (32%) being most frequent in probands confirmed with MODY. There was considerable regional variation in proband referral rates (from <20 per million in Wales and Northern Ireland to >50 per million for South West England and Scotland) and patients diagnosed with MODY (5.3 per million in Northern Ireland, 48.9 per million in South West England). Referral rates and confirmed cases were highly correlated ( r = 0.96, p < 0.0001). The minimum prevalence of MODY was estimated to be 108 cases per million. Conclusions/interpretation: Assuming this minimal prevalence throughout the UK then >80% of MODY is not diagnosed by molecular testing. The marked regional variation in the prevalence of confirmed MODY directly results from differences in referral rates. This could reflect variation in awareness of MODY or unequal access to genetic testing. Increased referral for diagnostic testing is required if the majority of MODY patients are to have the genetic diagnosis necessary for optimal treatment. [ABSTRACT FROM AUTHOR]

Little of the genetic basis for type 2 diabetes has been explained, despite numerous genetic linkage studies and the discovery of multiple genes in genome-wide association (GWA) studies. To begin to resolve the genetic component of this disease, we searched for sites at which genetic results had been corroborated in different studies, in the expectation that replication among studies should direct us to the genomic locations of causative genes with more confidence than the results of individual studies. We have mapped the physical location of results from 83 linkage reports (for type 2 diabetes and diabetes precursor quantitative traits [QTs, e.g. plasma insulin levels]) and recent large GWA reports (for type 2 diabetes) onto the same human genome sequence to identify replicated results in diabetes genetic ‘hot spots’. Genetic linkage has been found at least ten times at 18 different locations, and at least five times in 56 locations. All replication clusters contained study populations from more than one ethnic background and most contained results for both diabetes and QTs. There is no close relationship between the GWA results and linkage clusters, and the nine best replication clusters have no nearby GWA result. Many of the genes for type 2 diabetes remain unidentified. This analysis identifies the broad location of yet to be identified genes on 6q, 1q, 18p, 2q, 20q, 17pq, 8p, 19q and 9q. The discrepancy between the linkage and GWA studies may be explained by the presence of multiple, uncommon, mildly deleterious polymorphisms scattered throughout the regulatory and coding regions of genes for type 2 diabetes. [ABSTRACT FROM AUTHOR]

Mutations in the HNF1A (previously known as TCF1) gene encoding hepatocyte nuclear factor-1α (HNF1A) lead to the development of maturity-onset diabetes of the young, type 3 (HNF1A-MODY), characterised by impaired insulin secretion and a reduction in beta cell mass. HNF1A plays an important role in pancreatic beta cell differentiation and survival. The mammalian target of rapamycin (mTOR) is a central growth factor- and nutrient-activated protein kinase controlling cell metabolism, growth and survival. We investigated the role of mTOR inactivation in the decline in beta cell mass in a cellular model of HNF1A-MODY. Previously we showed that suppression of HNF1A function via expression of a dominant-negative mutant (DN-HNF1A) decreases insulin gene transcription in insulinoma (INS-1) cells. We investigated the signalling of two distinct mTOR protein complexes, mTORC1 and mTORC2, in response to DN-HNF1A induction. We observed delayed inactivation of mTORC2 48 h after DN-HNF1A induction, evidenced by a reduction in serine 473 phosphorylation of thymoma viral proto-oncogene 1 (AKT1). We also observed an early inactivation of mTORC1 24 h after DN-HNF1A induction, which was detected by decreases in threonine 389 phosphorylation of p70 ribosomal protein S6 kinase (S6K1) and serine 65 phosphorylation of translational inhibitor eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). Flow cytometry and gene expression analysis demonstrated a pre-apoptotic decrease in INS-1 cell size in response to DN-HNF1A induction, and an increase in the level of the mTORC1-regulated cell-cycle inhibitor, cyclin-dependent kinase inhibitor 1B p27. Our data suggest that mTOR kinase and signalling through mTORC1 are highly sensitive to suppression of HNF1A function, and may contribute to disturbance of cell-size regulation and cell-cycle progression in HNF1A-MODY. [ABSTRACT FROM AUTHOR]

Mutations in the GCK and HNF1A genes are the most common cause of the monogenic forms of diabetes known as ‘maturity-onset diabetes of the young’. GCK encodes the glucokinase enzyme, which acts as the pancreatic glucose sensor, and mutations result in stable, mild fasting hyperglycaemia. A progressive insulin secretory defect is seen in patients with mutations in the HNF1A and HNF4A genes encoding the transcription factors hepatocyte nuclear factor-1 alpha and -4 alpha. A molecular genetic diagnosis often changes management, since patients with GCK mutations rarely require pharmacological treatment and HNF1A/4A mutation carriers are sensitive to sulfonylureas. These monogenic forms of diabetes are often misdiagnosed as type 1 or 2 diabetes. Best practice guidelines for genetic testing were developed to guide testing and reporting of results. A workshop was held to discuss clinical criteria for testing and the interpretation of molecular genetic test results. The participants included 22 clinicians and scientists from 13 countries. Draft best practice guidelines were formulated and edited using an online tool (). An agreed set of clinical criteria were defined for the testing of babies, children and adults for GCK, HNF1A and HNF4A mutations. Reporting scenarios were discussed and consensus statements produced. Best practice guidelines have been established for monogenic forms of diabetes caused by mutations in the GCK, HNF1A and HNF4A genes. The guidelines include both diagnostic and predictive genetic tests and interpretation of the results. [ABSTRACT FROM AUTHOR]

Heterozygous mutations of glucokinase ( GCK) and hepatocyte nuclear factor-1 alpha ( HNF1A; also known as hepatic transcription factor 1 [ TCF1]) genes are the most common cause of MODY. Genomic deletions of the HNF1B (also known as TCF2) gene have recently been shown to account for one third of mutations causing renal cysts and diabetes syndrome. We investigated the prevalence of partial and whole gene deletions in UK patients meeting clinical criteria for GCK or HNF-1α/-4α MODY and in whom no mutation had been identified by sequence analysis. A multiplex ligation-dependent probe amplification (MLPA) assay was developed using synthetic oligonucleotide probes for 30 exons of the GCK, HNF1A and HNF4A genes. Partial or whole gene deletions were identified in 1/29 (3.5%) probands using the GCK MLPA assay and 4/60 (6.7%) of probands using the HNF1A/-4A MLPA assay. Four different deletions were detected: GCK exon 2, HNF1A exon 1, HNF1A exons 2 to 10 and HNF1A exons 1 to 10. An additional Danish pedigree with evidence of linkage to HNF1A had a deletion of exons 2 to 10. Testing other family members confirmed co-segregation of the deletion mutations with diabetes in the pedigrees. Large deletions encompassing whole exons can cause GCK or HNF-1α MODY and will not be detected by sequencing. Gene dosage assays, such as MLPA, are a useful adjunct to sequence analysis when a diagnosis of MODY is strongly suspected. [ABSTRACT FROM AUTHOR]

Glucokinase (GCK) acts as a glucose sensor in the pancreatic beta cell and regulates insulin secretion. In the gene encoding GCK the heterozygous mutations that result in enzyme inactivation cause MODY2. Functional studies of naturally occurring GCK mutations associated with hyperglycaemia provide further insight into the biochemical basis of glucose sensor regulation. Identification of GCK mutations in selected MODY patients was performed by single-strand conformation polymorphism and direct sequencing. The kinetic parameters and thermal stability of recombinant mutant human GCK were determined, and in pull-down assays the effect of these mutations on the association of GCK with glucokinase (hexokinase 4) regulator (GCKR, also known as glucokinase regulatory protein [GKRP]) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB1, also known as PFK2) was tested. We identified three novel GCK mutations: the insertion of an asparagine residue at position 161 (inserN161) and two missense mutations (M235V and R308W). We also identified a fourth mutation (R397L) reported in a previous work. Functional characterisation of these mutations revealed that insertion of asparagine residue N161 fully inactivates GCK, whereas the M235V and R308W mutations only partially impair enzymatic activity. In contrast, GCK kinetics was almost unaffected by the R397L mutation. Although none of these mutations affected the interaction of GCK with PFKFB1, we found that the R308W mutation caused protein instability and increased the strength of interaction with GCKR. Our results show that different MODY2 mutations impair GCK function through different mechanisms such as enzymatic activity, protein stability and increased interaction with GCKR, helping further elucidate the regulation of GCK activity. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Impaired beta cell function is the hallmark of gestational diabetes mellitus (GDM) and MODY. In addition, women with MODY gene mutations often present with GDM, but it is not known whether common variants in MODY genes contribute to GDM. Subjects and methods: We genotyped five common variants in the glucokinase (GCK, commonly known as MODY2), hepatocyte nuclear factor 1-α (HNF1A, commonly known as MODY3) and 4-α (HNF4A commonly known as MODY1) genes in 1,880 Scandinavian women (648 women with GDM and 1,232 pregnant non-diabetic control women). Results: The A allele of the GCK -30G→A polymorphism was more common in GDM women than in control subjects (odds ratio [OR] 1.28 [95% CI 1.06.1.53], p=0.008, corrected p value, p=0.035). Under a recessive model [AA vs GA+GG], the OR increased further to 2.12 (95% CI 1.21.3.72, p=0.009). The frequency of the L allele of the HNF1A I27L polymorphism was slightly higher in GDM than in controls (1.16 [95% CI 1.001.1.34], p=0.048, corrected p value, p=0.17). However, the OR increased under a dominant model (LL+IL vs II; 1.31 [95% CI 1.08.1.60], p=0.007). The rs2144908, rs2425637 and rs1885088 variants, which are located downstream of the primary beta cell promoter (P2) of HNF4A, were not associated with GDM. Conclusions/interpretation: The -30G→A polymorphism of the beta-cell-specific promoter of GCK and the I27L polymorphism of HNF1A seem to increase the risk of GDM in Scandinavian women. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Heterozygous mutations in the gene of the transcription factor hepatocyte nuclear factor 4α (HNF-4α) are considered a rare cause of MODY with only 14 mutations reported to date. The description of the phenotype is limited to single families. We investigated the genetics and phenotype of HNF-4α mutations in a large European Caucasian collection. Methods: HNF-4α was sequenced in 48 MODY probands, selected for a phenotype of HNF-1α MODY but negative for HNF-1α mutations. Clinical characteristics and biochemistry were compared between 54 HNF-4α mutation carriers and 32 familial controls from ten newly detected or previously described families. Results: Mutations in HNF-4α were found in 14/48 (29%) probands negative for HNF-1α mutations. The mutations found included seven novel mutations: S34X, D206Y, E276D, L332P, 1314F, L332insCTG and IVS5nt+1G>A. 1314F is the first reported de novo HNF-4α mutation. The average age of diagnosis was 22.9 years with frequent clinical evidence of sensitivity to sulphonylureas. Beta cell function, but not insulin sensitivity, was reduced in diabetic mutation carriers compared to control subjects (homeo stasis model assessment of beta cell function 29% p<0.001 vs controls). HNF-4α mutations were associated with lower apolipoprotein A2 (p=0.001), Al (p=0.04) and total HDL-cholesterol (p=0.02) than in control subjects. However, in contrast to some previous reports, levels of triglycerides and apolipoprotein C3 were normal. Conclusions/interpretation: HNF-4α mutations are common when no HNF-1α mutation is found in strictly defined MODY families. The HNF-4α clinical phenotype and beta cell dysfunction are similar to HNF- 1α MODY and are associated with reduced apolipoprotein A2 levels. We suggest that sequencing of HNF-4α should be performed in patients with clinical characteristics of HNF-1α MODY in whom mutations in HNF-1α are not found. [ABSTRACT FROM AUTHOR]

Aims/hypothesis. Mutations in the hepatocyte nuclear factor-1 beta (HNF-1β) gene result in disorders of renal development, typically involving renal cysts and early-onset diabetes (the RCAD syndrome/ MODY5). Sixteen mutations have been reported, including three splicing mutations of the intron 2 splice donor site. Because tissues showing abundant expression (kidney, liver, pancreas, gut, lung and gonads) are not easily accessible for analysis in living subjects, it has previously proven difficult to determine the effect of HNF-1β mutations at the mRNA level. This is the aim of the present study. Methods. We have developed a nested RT-PCR assay that exploits the presence of ectopic HNF-1β transcripts in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines derived from subjects carrying HNF-1β splice site mutations. Results. We report a fourth mutation of the intron 2 splice donor site, IVS2nt+2insT. Sequence analysis of ectopic HNF-1β transcripts showed that both IVS2nt+2insT and IVS2nt+1G>T result in the deletion of exon 2 and are predicted to result in premature termination of the HNF-1β protein. Mutant transcripts were less abundant than the normal transcripts but there was no evidence of nonsense-mediated decay. Conclusions/interpretation. This is the first study to define the pathogenic consequences of mutations within the HNF-1β gene by mRNA analysis. This type of approach is a useful and important tool to define mutational mechanisms and determine pathogenicity. [ABSTRACT FROM AUTHOR]

Aims/hypothesis. After screening 16 Korean families with early onset Type 2 diabetes in search for hepatocyte nuclear factor (HNF)-1α gene mutation, we identified a novel missense mutation (R263L) associated with MODY phenotype. We studied the biological characteristics of the mutation and the potential functional consequences based on the crystallographic structure of HNF-1α in complex with DNA. Methods. DNA from subjects with a familial form of early onset diabetes was isolated and HNF-1α was sequenced. The R263L substitution was generated by PCR-based sited-directed mutagenesis. Functional and biochemical studies were conducted by reporter assay using glucose-transporter type 2 (GLUT2) or insulin promoters and electrophoretic mobility shift assay, respectively. Results. Transfection of wild-type HNF-1α increased the reporter activities of GLUT2 and insulin promoters in NIH3T3 and SK-Hep1 cells, while R263L mutant was defective in transactivation of those promoters. Both wild-type HNF-1α and R263L mutant could not transactivate GLUT2 and insulin promoters in MIN6N8 insulinoma cells. R263L mutant had a defective cooperation with its heterodimeric partner HNF-1β or coactivator p300. R263L mutant protein displayed greatly reduced DNA binding ability, despite its comparable protein stability to the wild-type HNF-1α. Conclusion/interpretation. These results suggest that the mutation of HNF-1α at codon 263 from arginine to leucine leads to the development of MODY3 through decreased insulin production and defective glucose sensing. These findings are in good agreement with the crystal structure in which R263 makes hydrogen bonds with phosphorus atoms of DNA backbone to mediate the stable binding of HNF-1α homeodomain to the promoter. [ABSTRACT FROM AUTHOR]

Aims/hypothesis. The aim of this study was to examine the prevalence and nature of mutations in HNF4α/MODY1, GCK/MODY2 and HNF-1α/MODY3 genes in Czech subjects with clinical diagnosis of MODY. Methods. We studied 61 unrelated index probands of Czech origin (28 males, 33 females) with a clinical diagnosis of MODY and 202 family members. The mean age of probands was 22.7±12.0 years (range, 6–62) and the mean age at the first recognition of hyperglycaemia was 14.7±6.0 years (range, 1–25). The promotor and coding regions inclusive intron exon boundaries of the HNF-4α, GCK and HNF-1α genes were examined by PCR-dHPLC (HNF-1α and GCK) and direct sequencing. Results. We identified 20 different mutations in the HNF-4α, GCK and HNF-1α in 29 families (48% of all families studied), giving a relative prevalence of 5% of MODY1, 31% of MODY2 and 11.5% of MODY3 among the Czech kindred with MODY. Three of 3, 10 of 11 and 1 of 6 of the mutations identified in HNF-4α, GCK and HNF-1α respectively, were new. Conclusion/interpretation. Of the families 48% carried mutations in the MODY1–3 genes and of the identified mutations 70% were new. In 52% of Czech families with clinical characteristics of MODY, no mutations were found in the analysed genes. This finding shows that the majority of MODY mutations in a central European population are local and that other MODY genes could be responsible for autosomal dominant transmission of diabetes mellitus. [ABSTRACT FROM AUTHOR]

Aim/hypothesis. We analysed Japanese MODY patients for mutations in the HNF-1α gene. Methods. Fifty unrelated Japanese patients with early-onset diabetes (diagnosed at 25 years of age or younger) or with a strong family history of diabetes were screened for mutations in the HNF-1α gene. Functional studies of the mutant HNF-1α were carried out. Results. We identified three new mutations in the HNF-1α gene in the families with a strong family history for diabetes. One mutation (L518P519fsTCC→A) was identified in three unrelated families, while the other two mutations (T521I and V617I) were identified in one family. We also identified the A site of the promoter (+102G-to-C), which was reported previously. We examined the functional properties of the mutant HNF-1α. By increasing the amount of L518P519fsTCC→A-HNF-1α, increasing inhibition of the transcription of human transthyretin (TTR) was observed (up to 61% of the control). Increasing amounts of T521I-HNF-1α or V617I-HNF-1α mutant proteins increased TTR promoter transcription up to 4.3-fold and 2.4-fold, respectively, whereas both increased transcription up to 12.4-fold of the control. Conclusion/interpretation. The L518P519fsTCC→A was identified for the first time and this mutation might be a common cause of Japanese MODY3 in Okinawa area. In addition, both the T521I and V617I mutations were present in two patients in the same family. Since the prevalence of these mutations is relatively high (10%, 5/50), the HNF-1α gene needs to be screened for mutations in patients either with early-onset diabetes or with a strong family history for diabetes. [ABSTRACT FROM AUTHOR]

Aims/hypothesis. Maturity onset diabetes of the young type 3 (MODY3) is a monogenic form of diabetes mellitus caused by mutations in the gene encoding for hepatocyte nuclear factor 1 alpha, HNF1α. In this study we have examined the in vivo and in vitro effects of a mutation (L107I) outside the DNA binding and dimerization domains in the N terminal part of the HNF1α gene. Methods. Beta-cell function of the affected family members was assessed by an oral glucose tolerance test. Functional tests were carried out to explain the role of the mutation in vitro by transcriptional activity assay, Western blotting, DNA-binding assays and subcellular localization experiments. Results. Affected family members showed an 86% decreased insulin response to glucose when compared to age-matched healthy control subjects. In vitro the mutation showed a 79% decrease in transcriptional activity as compared to wild type HNF1α in HeLa cells lacking HNF1α. The transcriptional activity was not suppressed when the mutant was co-expressed with wild type HNF1α suggesting that the decreased activity was not mediated by a dominant negative mechanism. The L107I/HNF1α protein showed normal nuclear targeting but impaired binding to an HNF1α consensus sequence. Conclusion/interpretation. Our results suggest that the L107I substitution represents a MODY3 mutation which impairs beta-cell function by a loss-of-function mechanism. [ABSTRACT FROM AUTHOR]

Aims/hypothesis. Mutations in the HNF-1α gene result in maturity-onset diabetes of the young (MODY); an early-onset, dominantly inherited form of diabetes caused by pancreatic β-cell dysfunction. Splice site mutations represent approximately 10% of reported HNF-1α mutations. No studies to date have investigated the effect of splice site mutations on mRNA processing because the tissues with abundant HNF-1α expression (liver, pancreas, kidney and gut) are not easily accessible for analysis. This study aimed to define the pathogenic mechanism in three novel splice site mutations by analysing illegitimate transcripts. Methods. To assess the consequence of potential HNF-1α splice site mutations we developed a nested reverse transcriptase PCR (RT-PCR) assay for the amplification of illegitimate HNF-1α transcripts in Epstein Barr virus transformed lymphoblastoid cell lines. Results. Sequencing the illegitimate HNF-1α transcripts showed that the splice donor site mutation IVS8nt+1G>A leads to complete skipping of exon 8, the splice acceptor site mutation IVS4nt-2A>G causes skipping of exon 5 with the recruitment of a cryptic splice acceptor site within intron 5 and the cryptic splice acceptor site mutation (IVS7nt-6G>A) resulted in the skipping of exon 7. All three changes are predicted to result in premature termination of the HNF-1α protein, providing further evidence for their role as pathogenic mutations. Conclusion/interpretation. We conclude that the sequencing of illegitimate transcripts from lymphoblastoid cell lines is helpful in the assessment of intronic variation in HNF-1α that could alter splicing. This analysis of the mRNA is required to define mutational mechanisms and confirm pathogenic status. [ABSTRACT FROM AUTHOR]

Aims/hypothesis. Glucokinase plays a key role in glucose homeostasis and the expression of its gene is differentially regulated in pancreatic beta cells and in the liver through distinct promoters. The factors that determine the tissue-specific expression of the glucokinase gene are not known. Putative binding sites for hepatocyte nuclear factor (HNF)-6, the prototype of the ONECUT family of transcription factors, are present in the hepatic promoter of the glucokinase gene and in diabetic hnf6 knockout mice. We hypothesized that HNF-6 controls the activity of the hepatic glucokinase promoter. Methods. We tested the binding of recombinant HNF-6 to DNA sequences from the mouse hepatic glucokinase promoter in vitro and the effect of HNF-6 on promoter activity in transfected cells. We investigated in vivo the role of HNF-6 in mice by examining the effect of inactivating the hnf6 gene on glucokinase gene-specific deoxyribonuclease I hypersensitive sites in liver chromatin and on liver glucokinase mRNA concentration. Results. HNF-6 bound to the hepatic promoter of the glucokinase gene and stimulated its activity. Inactivation of the hnf6 gene did not modify the pattern of deoxyribonuclease I hypersensitive sites but was associated with a decrease of liver glucokinase mRNA to half the control value. Conclusions/interpretation. Although HNF-6 is not required to open chromatin of the hepatic promoter of the glucokinase gene, it stimulates transcription of the glucokinase gene in the liver. This could partly explain the diabetes observed in hnf6 knockout mice. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: We assessed how the role of genes genetic causation in causing maturity-onset diabetes of the young (MODY) alters the response to an oral glucose tolerance test (OGTT). Methods: We studied OGTT in 362 MODY subjects, from seven European centres; 245 had glucokinase gene mutations and 117 had Hepatocyte Nuclear Factor –1 alpha (HNF-1α) gene mutations. Results: BMI and age were similar in the genetically defined groups. Fasting plasma glucose (FPG) was less than 5.5 mmol/l in 2 % glucokinase subjects and 46 % HNF-1α subjects (p < 0.0001). Glucokinase subjects had a higher FPG than HNF-1α subjects ([means ± SD] 6.8 ± 0.8 vs 6.0 ± 1.9 mmol/l, p < 0.0001), a lower 2-h value (8.9 ± 2.3 vs 11.2 ± 5.2 mmol/l, p < 0.0001) and a lower OGTT increment (2-h – fasting) (2.1 ± 2.3 vs 5.2 ± 3.9 mmol/l, p < 0.0001). The relative proportions classified as diabetic depended on whether fasting (38 % vs 22 %, glucokinase vs HNF-1α) or 2-h values (19 % vs 44 %) were used. Fasting and 2-h glucose values were not correlated in the glucokinase subjects (r = –0.047, p = 0.65) but were strongly correlated in HNF-1α subjects (r = 0.8, p < 0.001). Insulin concentrations were higher in the glucokinase subjects throughout the OGTT. Conclusion/interpretation: The genetic cause of the beta-cell defect results in clear differences in both the fasting glucose and the response to an oral glucose load and this can help diagnostic genetic testing in MODY. OGTT results reflect not only the degree of hyperglycaemia but also the underlying cause. [Diabetologia (2002) 45: 427–435] [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Five different types of maturity-onset diabetes of the young (MODY) have been identified until now but mutation screening suggests that more MODY genes exist. Mutations in genes encoding transcription factors essential for normal development and function of pancreatic beta cells has recently become important in studying the genetics of Type II (non-insulin-dependent) diabetes mellitus. Patients with MODY and their families in Iceland were screened for mutations in the transcription factor genes. Methods: Clinical and biochemical information on individuals with MODY was collected and their family trees constructed. Linkage analysis was carried out on chromosomal regions known to harbour genes previously shown to be associated with MODY. Mutations were identified by direct sequencing. Results: Three families were identified. Two of these showed linkage to chromosome 12 and carried mutations in exon 4 of the HNF-1α gene (290fsdelC and R272C). However, the third family showed no linkage to the previously described MODY genes but shared a novel mutation in the NeuroD1 gene on chromosome 2q32. This mutation, a glutamate to lysine substitution at codon 110, resides in the basic domain of the protein. Conclusion/interpretation: Mutations in MODY subjects have been identified in the Icelandic population. In addition this study identified the NeuroD1 gene as the gene responsible for the sixth type of MODY. [Diabetologia (2001) 44: 2098–2103] [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Maturity-onset-diabetes of the young (MODY) is caused by mutations in at least five different genes. Our aim was to determine the prevalence of the most common MODY genes in Italian families with early-onset Type II (non-insulin-dependent) diabetes mellitus. Methods: We screened 28 Italian early-onset Type II diabetic families (diagnosis < 35 years) for mutations in the hepatic nuclear factor-4α, (MODY1), glucokinase (MODY2) and hepatic nuclear factor-1α (MODY3). Both strands of exons, flanking introns and minimal promoter regions of the above-mentioned genes were amplified using polymerase chain reaction and were sequenced directly. Results: We identified four different mutations, three of which are not described, (W113X, G42P43fsCC→A, H514R) and four new polymorphisms (G184G, T513T, IVS3-nt47delG, IVS1- nt53C→G) in the hepatic nuclear factor-1α gene, two new potential mutations (G44S, IVS4nt + 7C→T) and three new polymorphisms (promoter-nt84C→G, IVS9 + nt8C→T, IVS9 + nt49G→A) in the glucokinase gene, and a new polymorphism (IVS1c-nt11T→G) in the hepatic nuclear factor-4α gene. Conclusion/interpretation: Mutations in the hepatic nuclear factor-1α and glucokinase are associated with Type II diabetes in 14 % and 7 % of Italian families, respectively. Our findings provide an impetus for screening Italian MODY and early-non Type II diabetic families for mutations in the above mentioned genes to identify relatives at risk who could benefit from primary prevention care. [Diabetologia (2001) 44: 1326–1329] [ABSTRACT FROM AUTHOR]

Aim/hypothesis. Neurogenin 3 (NEUROG3) is a member of the subfamily of basic-helix-loop-helix (bHLH) transcription factors involved in differentiation of the endocrine pancreas and therefore a possible candidate gene for maturity-onset diabetes of the young (MODY) and Type II (non-insulin-dependent) diabetes mellitus.¶Methods. Using Polymerase-chain-reaction single-stranded-conformation polymorphism, we examined the coding region including the 5'-untranslated and 3'- untranslated regions of the NEUROG3 in a group of 133 diabetic patients comprising 19 MODY patients, 19 patients with dominant Type I diabetes, and 31 early-onset and 64 late-onset Type II diabetic patients.¶Results. We found two missense mutations, Gly167Arg and Ser199Phe, as well as two non-coding variants in the 5' UTR, a c → t nucleotide variant at position –10 upstream of the start codon in one MODY patient and a 2 base pair (CA) deletion polymorphism at position –44/-45. Allele frequencies measured in 377 diabetic patients and in 217 glucose-tolerant control subjects were: Gly167Arg, 0.04 (95 % CI: 0.02–0.06) and 0.04 (0.02–0.06); Ser199Phe, 0.31 (0.26–0.36) and 0.30 (0.24–0.36); –44–45delCA, 0.33 (0.31–0.35) and 0.35 (0.32–0.38), respectively. Both Ser199Phe and the –44–45delCA were in linkage disequilibrium (χ2 > 60) but the Ser199Phe and the –44–45delCA polymorphism were not associated with consistent changes in fasting- or glucose-induced insulin secretion in 249 glucose-tolerant offspring (first-degree relatives) of Type II diabetic parents or in 217 unrelated middle-aged glucose tolerant subjects.¶Conclusion/interpretation. Genetic variability in NEUROG3 is not associated with dominant Type I diabetes, MODY, Type II diabetes or changes in insulin secretion in the Danish Caucasians examined subjects. [Diabetologia (2001) 44: 123–126] [ABSTRACT FROM AUTHOR]

Aims/hypothesis. To evaluate insulin sensitivity and insulin secretion in prediabetic and diabetic subjects with mutations in MODY1 (HNF-4α) and MODY3 (HNF-1α) genes, in subjects with GAD antibodies, latent autoimmune diabetes in adults and in subjects with the common form of Type II (non-insulin-dependent) diabetes mellitus. Methods. Insulin secretion was measured as the incremental 30-min insulin (I30) and insulin glucose ratio (I:G30) during OGTT whereas insulin sensitivity was measured as the insulin sensitivity index during OGTT in 131 carriers of MODY mutations [NGT = 38, IFG/IGT = 21, diabetes mellitus (DM) = 72], in 293 subjects with GADA (NGT = 47, IFG/IGT = 29, DM = 217) and in 2961 subjects with a family history of the common form of Type II diabetes but without MODY mutations or GADA (NGT = 1360, IFG/IGT = 857, DM = 744). A subgroup of the subjects underwent a euglycaemic clamp (n = 210) and intravenous glucose tolerance test (n = 337) for the estimation of insulin sensitivity and first-phase insulin secretion. Results. Non-diabetic subjects with MODY mutations had pronounced impaired insulin secretion (I30, I:G30) compared with the two other groups (p = 0.005). Normal or non-diabetic glucose tolerance was maintained by enhanced insulin sensitivity compared with the other two groups (p < 0.05 and p < 0.005). In contrast to patients with Type II diabetes and with adult latent autoimmune diabetes, MODY patients showed only a modest deterioration in insulin sensitivity at onset of diabetes. The 2-h glucose values inversely correlated with insulin sensitivity in subjects with GADA (r = –0.447, p < 0.001) and subjects from Type II diabetic families (r = –0.426, p < 0.001), whereas no such relation was observed in subjects with MODY mutations (r = 0.151, p = NS). There were no statistically significant differences in insulin secretion or insulin sensitivity between subjects with GADA or subjects with a family history of Type II diabetes, either at the NGT or the IFG/IGT stage. Conclusion/interpretation. Glucose-tolerant carriers of MODY mutations are characterised by a severe impairment in insulin secretion. Enhanced insulin sensitivity is the most likely explanation for the normal glucose tolerance. Whereas subjects with positive GADA or Type II diabetes have impaired insulin sensitivity with increasing glucose concentrations, MODY mutation carriers seem to be protected from the effect of glucose toxicity. [Diabetologia (2000) 43: 1476–1483] [ABSTRACT FROM AUTHOR]

Aims/hypothesis. Mutations in the transcription factor hepatocyte nuclear factor (HNF)-4α are the cause of one form of maturity-onset diabetes of the young, MODY1. The HNF-4γ is structurally related to HNF-4α and is expressed together with HNF-4α in pancreatic islets. We therefore tested the hypothesis that genetic variation in the HNF-4γ gene (HNF4G) is associated with MODY in Japanese subjects. Methods. We screened the protein coding region of HNF4G (exons 3–11) for mutations in 57 unrelated Japanese subjects with MODY by amplifying each exon and adjacent intron region using the polymerase chain reaction (PCR) and specific primers and then directly sequencing the PCR products. The frequency of each variant was compared between patients with MODY and a group of non-diabetic subjects. Results. We found ten sequence variants, two of these were located in exons: exon 6, a silent substitution in codon 144, c.432A/G and exon 7, a G-to-A substitution in codon 190 (c.570G/A) resulting in a conservative Met-to-Ile substitution (M/I190) in the putative ligand-binding region of HNF-4γ protein. The remaining eight variants were located in introns. There was no significant difference in the frequency of these polymorphisms between subjects with MODY and non-diabetic control subjects. Conclusion/interpretation. Genetic variation in the coding region of HNF4G is unlikely to be a major cause of MODY in Japanese people. [Diabetologia (2000) 43: 1064–1069] [ABSTRACT FROM AUTHOR]

Aims/hypothesis. Altered fetal insulin secretion caused by fetal or maternal glucokinase mutations influence birth weight. Here, we attempt to answer two additional questions: firstly, whether this variation in birth weight (from low birth weight to macrosomia) has an effect on adult height or weight. Secondly, whether maternal hyperglycaemia during fetal life has an effect on metabolic phenotypes of the adult offspring. Methods. We studied 447 family members from 37 MODY2 kindred, divided into four groups according to the presence or absence of a glucokinase mutation in the subject (S + or S–, respectively) and his/her mother (M + or M–). Birth weight data were obtained from a questionnaire sent to the mothers. Results. Birth weight was reduced in the presence of a fetal mutation (M–S + ) and increased in the presence of a maternal mutation (M + S–). These effects are additive as similar birth weights were observed in M + S + and M–S– offspring. Adult height, weight or body mass index (weight/height2) were, however, similar in the four groups of subjects. Non-diabetic adult offspring, regardless of the glycaemic status of the mothers (M + S– or M–S–), had similar insulin secretion, insulin sensitivity, blood pressures and lipid profiles. These variables as well as the severity of hyperglycaemia were similar in adult M + S + and M–S + MODY2 subjects. Conclusion/Interpretation. Maternal environment and fetal genotypes could alter growth in utero by changing fetal insulin secretion but these effects do not result in a persistent programming in latter life. [Diabetologia (2000) 43: 1060–1063] [ABSTRACT FROM AUTHOR]