Aims/Hypothesis: Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous monogenic form of diabetes characterized by an autosomal dominant inheritance, an early clinical onset, and a primary defect in β-cell function. The aims of the present study were to examine the prevalence and nature of mutations in the three common MODY genes, HNF4A, GCK, and TCF1, in Danish patients with a clinical diagnosis of MODY and to describe metabolic differences in probands with and without mutations in HNF4A, GCK, and TCF1. Methods: Seventy-eight unrelated subjects of Danish Caucasian origin (29 men, 49 women) and their 351 family members were examined. The promotor and coding regions including intron-exon boundaries of HNF4A, GCK, and TCF1 were examined by denaturing HPLC and/or direct sequencing. Results: We identified 29 different mutations in 38 MODY families. Fifteen of the mutations were novel. The variants segregated with diabetes within the families, and none of the variants were found in 100 nor...

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Half of Clinically Defined Maturity-Onset Diabetes of the Young Patients in Denmark Do Not Have Mutations in HNF4A, GCK, and TCF1

Introduction Molecular diagnosis of monogenic diabetes mellitus is important for individualized patient care. Next-generation sequencing (NGS) enables a simultaneous analysis of multiple genes in a single test. Objectives We aimed to assess the feasibility of using NGS for detecting mutations in a set of known monogenic diabetes gene mutations in a cohort of Polish patients with maturity-onset diabetes of the young (MODY) with earlier negative Sanger sequencing results for HNF1A-MODY or GCK-MODY. Patients and methods We selected a panel of 28 chromosomal genes in which mutations have been reported to cause monogenic diabetes. The MiSeq platform was used for NGS. An exon-capture assay was designed to include coding regions and splice sites. A total of 54 patients with existing negative Sanger sequencing screening results for HNF1A or GCK gene mutations were selected for the study. Results NGS results were generated for all 54 patients and 9 positive controls with previously identified HNF1A or GCK gene mutation. All selected positive controls were confirmed by NGS. Among 28 genes, mutations were detected in 16. The type of the analyzed genetic changes was described in the NGS study as high (n = 3) or moderate (n = 76). Among the detected mutations, there were 4 known GCK gene mutations that had been previously missed in Sanger sequencing. So far, Sanger sequencing allowed us to confirm 21 gene mutations detected by NGS, and segregation with diabetes in 14 pedigrees. Conclusions Our pilot study using NGS for monogenic diabetes screening in the MODY cohort confirmed that it improves the detection of diabetes-related sequence differences. The screening with NGS should also include diabetic patients for whom Sanger-based screening for particular subtypes of MODY provided negative results.

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Genetic testing for monogenic diabetes using targeted next-generation sequencing in patients with maturity-onset diabetes of the young.

Background Maturity-onset diabetes of the young (MODY) is a form of monogenic diabetes with autosomal dominant inheritance. To date, mutations in 11 genes have been frequently associated with this phenotype. In Brazil, few cohorts have been screened for MODY, all using a candidate gene approach, with a high prevalence of undiagnosed cases (MODY-X). Methods We conducted a next-generation sequencing target panel (tNGS) study to investigate, for the first time, a Brazilian cohort of MODY patients with a negative prior genetic analysis. One hundred and two patients were selected, of which 26 had an initial clinical suspicion of MODY-GCK and 76 were non-GCK MODY. Results After excluding all benign and likely benign variants and variants of uncertain significance, we were able to assign a genetic cause for 12.7% (13/102) of the probands. Three rare MODY subtypes were identified (PDX1/NEUROD1/ABCC8), and eight variants had not been previously described/mapped in genomic databases. Important clinical findings were evidenced in some cases after genetic diagnosis, such as MODY-PDX1/HNF1B. Conclusion A multiloci genetic approach allowed the identification of rare MODY subtypes, reducing the large percentage of MODY-X in Brazilian cases and contributing to a better clinical, therapeutic, and prognostic characterization of these rare phenotypes.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/mgg3.962

Targeted sequencing identifies novel variants in common and rare MODY genes

Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which ∼65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies.

Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113–1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) –mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC50 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively [p<0.03]). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively [p<0.001]). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%.

In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening.

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Identification and functional characterisation of novel glucokinase mutations causing maturity-onset diabetes of the young in Slovakia.

A new clinical screening strategy and prevalence estimation for glucokinase variant-induced diabetes in an adult Chinese population

- to: Lindner T, Cockburn BN, Bell GI (1999) Molecular genetics of MODY in Germany. Diabetologia 42: 121–123

BACKGROUND Molecular biological techniques of mutation detection become more and more a focus of interest in clinical medicine because the responsible genes are now known for an increasing number of diseases and the detection is essential to enable reliable predictions and to design individual therapies. Therefore the SSCP (Single Strand Conformation Polymorphism) analysis is one of the most commonly applied methods in detection of point mutations. SSCP ANALYSIS The use of SSCP for detection of the pathogenic mutations of MODY (Maturity Onset Diabetes in the Young) is described as an example. Handling, efficiency and costs are critically analyzed. In a blind test, this method was only partly suitable for reliable analysis. Particularly diseases without a distinct hotspot and with a widespread distribution pattern of mutations throughout the coding sequence require sequencing as the gold standard.

SSCP (single strand conformation polymorphism) analysis for detection of point mutations. A technique and its limits exemplified by dominantly inherited forms of diabetes (MODY)

Focke Ziemssen

Papers

- to: Lindner T, Cockburn BN, Bell GI (1999) Molecular genetics of MODY in Germany. Diabetologia 42: 121–123

SSCP (single strand conformation polymorphism) analysis for detection of point mutations. A technique and its limits exemplified by dominantly inherited forms of diabetes (MODY)