Next- Generation Sequencing Is An Effective Method For Diagnosing Patients With Different Forms Of Monogenic Diabetes

Open AccessPublished:November 23, 2021DOI:https://doi.org/10.1016/j.diabres.2021.109154

      Abstract

      Aim

      Monogenic diabetes (MD) represents 5-7% of antibody-negative diabetes cases and is a heterogeneous group of disorders.

      Methods

      We used targeted next-generation sequencing (NGS) on Illumina NextSeq 550 platform involving the SureSelect assay to perform genetic and clinical characteristics of a study group of 684 individuals, including 542 patients referred from 12 Polish Diabetes Centers with suspected MD diagnosed between December 2016 and December 2019 and their 142 family members (FM).

      Results

      In 198 probands (36.5%) and 66 FM (46.5%) heterozygous causative variants were confirmed in 11 different MD-related genes, including 31 novel mutations, with the highest number in the GCK gene (206/264), 22/264 in the HNF1A gene and 8/264 in the KCNJ11 gene. Of the 183 probands with MODY1-5 diabetes, 48.6% of them were diagnosed at the pre-diabetes stage and most of them (68.7%) were on diet only at the time of genetic diagnosis, while 31.3% were additionally treated with oral hypoglycaemic drugs and/or insulin.

      Conclusions

      In summary, the results obtained confirm the efficacy of targeted NGS method in the molecular diagnosis of patients with suspected MD and broaden the spectrum of new causal variants, while updating our knowledge of the clinical features of patients defined as having MD.

      Keywords

      1. Introduction

      Monogenic diabetes (MD) is a heterogeneous group of disorders caused by mutations in a single gene, most of which regulate pancreatic β cells function. MD include: neonatal diabetes mellitus (NDM), maturity-onset diabetes in the young (MODY) and syndromic forms of diabetes such as Wolfram, Alström and Bardet-Biedl (WABB) syndromes [
      • Hattersley A.T.
      • Greeley S.A.W.
      • Polak M.
      • Rubio-Cabezas O.
      • Njølstad P.R.
      • Mlynarski W.
      • et al.
      The diagnosis and management of monogenic diabetes in children and adolescents.
      ].
      NDM occurs in the first 6-12 months of a child's life, whereas MODY diabetes manifests itself in adolescents and adults, but both are inherited autosomal dominantly [
      • Hattersley A.T.
      • Greeley S.A.W.
      • Polak M.
      • Rubio-Cabezas O.
      • Njølstad P.R.
      • Mlynarski W.
      • et al.
      The diagnosis and management of monogenic diabetes in children and adolescents.
      ,
      • McDonald T.J.
      • Ellard S.
      Maturity onset diabetes of the young: identification and diagnosis.
      ,
      • Letourneau L.R.
      • Greeley S.A.W.
      Congenital Diabetes: Comprehensive Genetic Testing Allows for Improved Diagnosis and Treatment of Diabetes and Other Associated Features.
      ]. In NDM, the most commonly affected genes are KCNJ11 and ABCC8, encoding the two subunits of the ATP-sensitive potassium channel of the β cells [
      • De Franco E.
      • Flanagan S.E.
      • Houghton J.AL.
      • Allen H.L.
      • Mackay D.JG.
      • Temple I.K.
      • et al.
      The effect of early, comprehensive genomic testing on clinical care in neonatal diabetes: an international cohort study.
      ]. MODY diabetes, as the most common type of MD, can be caused by mutations in at least 14 genes. However, pathogenic variants in three genes (GCK, HNF1A and HNF4A) are responsible for the majority of cases among patients with MD [
      • Hattersley A.T.
      • Greeley S.A.W.
      • Polak M.
      • Rubio-Cabezas O.
      • Njølstad P.R.
      • Mlynarski W.
      • et al.
      The diagnosis and management of monogenic diabetes in children and adolescents.
      ]. Clinical characteristics of patients with MODY diabetes typically include: onset before 25 years of age, absence of autoantibodies characteristic for autoimmune forms of diabetes, normal BMI and a family history of diabetes, whereas GCK-MODY can be identified even in early childhood [
      • Hattersley A.T.
      • Greeley S.A.W.
      • Polak M.
      • Rubio-Cabezas O.
      • Njølstad P.R.
      • Mlynarski W.
      • et al.
      The diagnosis and management of monogenic diabetes in children and adolescents.
      ,
      • McDonald T.J.
      • Ellard S.
      Maturity onset diabetes of the young: identification and diagnosis.
      ,
      • Brahm A.J.
      • Wang G.
      • Wang J.
      • McIntyre A.D.
      • Cao H.
      • Ban M.R.
      • et al.
      Genetic Confirmation Rate in Clinically Suspected Maturity-Onset Diabetes of the Young.
      ]. On the other hand, WABB syndromes are extremely rare, autosomal recessively inherited disorders of many organs requiring multidisciplinary medical care [
      • Farmer A.
      • Aymé S.
      • de Heredia M.L.
      • Maffei P.
      • McCafferty S.
      • Młynarski W.
      • et al.
      EURO-WABB: an EU rare diseases registry for Wolfram syndrome, Alström syndrome and Bardet-Biedl syndrome.
      ].
      It seems evident that the prevalence of MD depends on the population studied. However, only a few years ago, the prevalence of MD was estimated to be only 1% to 2% of all diabetes cases [
      • Irgens H.U.
      • Molnes J.
      • Johansson B.B.
      • Ringdal M.
      • Skrivarhaug T.
      • Undlien D.E.
      • et al.
      Prevalence of monogenic diabetes in the population-based Norwegian Childhood Diabetes Registry.
      ,
      • Shepherd M.
      • Shields B.
      • Hammersley S.
      • Hudson M.
      • McDonald T.J.
      • Colclough K.
      • et al.
      UNITED Team. Systematic Population Screening, Using Biomarkers and Genetic Testing, Identifies 2.5% of the U.K. Pediatric Diabetes Population With Monogenic Diabetes.
      ]. Consequently, many patients were not diagnosed with MD or were misdiagnosed with type 1 or type 2 diabetes despite overlapping clinical features [
      • Shields B.M.
      • Hicks S.
      • Shepherd M.H.
      • Colclough K.
      • Hattersley A.T.
      • Ellard S.
      Maturity-onset diabetes of the young (MODY): how many cases are we missing?.
      ]. The growing awareness of this clinical problem and the availability of genetic testing have therefore led to an increased frequency of MD diagnoses in recent years. The development of DNA sequencing methods and the widespread use of next-generation sequencing (NGS) have enabled remarkable progress in the molecular diagnosis of MD. It is now estimated that monogenic forms of diabetes account for up to 5-7% of antibody-negative diabetes cases [
      • Johansson B.B.
      • Irgens H.U.
      • Molnes J.
      • Sztromwasser P.
      • Aukrust I.
      • Juliusson P.B.
      • et al.
      Targeted next-generation sequencing reveals MODY in up to 6.5% of antibody-negative diabetes cases listed in the Norwegian Childhood Diabetes Registry.
      ,

      Delvecchio M, Mozzillo E, Salzano G, Iafusco D, Frontino G, Patera PI, Rabbone I, Cherubini V, Grasso V, Tinto N, Giglio S, Contreas G, Di Paola R, Salina A, Cauvin V, Tumini S, d'Annunzio G, Iughetti L, Mantovani V, Maltoni G, Toni S, Marigliano M, Barbetti F. Monogenic Diabetes Accounts for 6.3% of Cases Referred to 15 Italian Pediatric Diabetes Centers During 2007 to 2012. J Clin Endocrinol Metab. 2017;102:1826-1834. 10.1210/jc.2016-2490.

      ,

      Urrutia I, Martínez R, Rica I, Martínez de LaPiscina I, García-Castaño A, Aguayo A, Calvo B, Castaño L; Spanish Pediatric Diabetes Collaborative Group. Negative autoimmunity in a Spanish pediatric cohort suspected of type 1 diabetes, could it be monogenic diabetes? PLoS One. 2019;14:e0220634. 10.1371/journal.pone.0220634. eCollection 2019.

      ]. It should be emphasized that correct enrollment of patients and molecular diagnosis of MD is crucial for the initiation of appropriate treatment, prognosis of patients and identification of diabetes among relatives of the patient [
      • Shields B.M.
      • Hicks S.
      • Shepherd M.H.
      • Colclough K.
      • Hattersley A.T.
      • Ellard S.
      Maturity-onset diabetes of the young (MODY): how many cases are we missing?.
      ]. To date, epidemiological studies in the Polish population have been conducted on the pediatric population and mainly by Sanger sequencing, allowing MODY2 (GCK-MODY) diabetes to be identified as the most common in this group of patients [
      • Fendler W.
      • Borowiec M.
      • Baranowska-Jazwiecka A.
      • Szadkowska A.
      • Skala-Zamorowska E.
      • Deja G.
      • et al.
      Prevalence of monogenic diabetes amongst Polish children after a nationwide genetic screening campaign.
      ,
      • Małachowska B.
      • Borowiec M.
      • Antosik K.
      • Michalak A.
      • Baranowska-Jaźwiecka A.
      • Deja G.
      • et al.
      Monogenic diabetes prevalence among Polish children-Summary of 11 years-long nationwide genetic screening program.
      ].
      The aim of this study was to evaluate the genetic and clinical characteristics of Polish patients with suspected monogenic diabetes using the NGS method.

      1.1 Material and methods

      1.1.1 Patients

      The study group consisted of 684 subjects (F/M; 48.2%/51.8%), including 542 patients (median age: 14.0 years; IQR 10.0-18.0) consecutively referred to the Genetic Outpatient Clinic of the Centre for Rare Diseases in Lodz, Poland from 12 Polish Diabetes Centers (mainly paediatric) with suspected monogenic diabetes diagnosed between December 2016 and December 2019 and their 142 first-degree relatives (median age: 39.0 years; IQR 34.0-43.0) – family members (FM).
      Patients were included in the study based on the presence of hyperglycaemia (H) or diabetes mellitus (DM) recognized according to the WHO definition, positive family history of diabetes, absence of autoantibodies, preserved insulin secretion based on fasting C-peptide or insulin values and absence of additional organ-specific symptoms. Hyperglycaemia or pre-diabetes was defined as impaired fasting blood glucose and/or impaired glucose tolerance. HbA1c levels were reported as a percentage, both at the time of clinical diagnosis of H/DM and as an average value for the year prior to genetic diagnosis of MD. Patients with NDM, syndromic forms of monogenic diabetes (Wolfram syndrome and WFS-like syndrome) and with rare MODY variants found in the genes analyzed were subsequently excluded from the presented clinical report [
      • Płoszaj T.
      • Antosik K.
      • Jakiel P.
      • Zmysłowska A.
      • Borowiec M.
      Screening for extremely rare pathogenic variants of monogenic diabetes using targeted panel sequencing.
      ].
      The study protocol was approved by the Bioethics Committee of the Medical University of Lodz, Poland (RNN/148/13/KE). Patients and/or their parents gave written informed consent for participation in the study.

      1.1.2 Molecular analysis

      The DNA was isolated from peripheral blood cells (drawn into EDTA-coated vials) using automated Maxwell system (Promega, Madison, USA). The identification of pathogenic variants was performed by using targeted NGS method on Illumina NextSeq 550 platform involving the SureSelect assay (Agilent, SantaClara, USA). A custom panel was designed using SureDesign platform (www.earray.chem.agilent.com/suredesign/) and included 35 genes that have been associated with monogenic diabetes: ABCC8, AIRE, APPL1, BLK, CEL, EIF2AK3, FOXP3, GATA4, GATA6, GCK, GLIS3, GLUD1, HADH, HNF1A, HNF1B, HNF4A, INS, ISL1, KCNJ11, GCKR, KLF11, MAFA, MAFB, MNX1, NEUROD1, NEUROG3, NKX2-2, NKX6-1, PAX4, PAX6, PDX1, PTF1A, RFX6, WFS1, CISD2. Sequencing included coding regions, splice site and intron regions up to 150 bp deep. UTR and promoter regions up to 500 bp and downstream to 500 bp were also sequenced.
      Alignments, variant calling and annotation were performed using SureCall software version 4.2 (Agilent, SantaClara, USA). Pathogenicity of the detected genetic variants was evaluated according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) [
      • Richards S.
      • Aziz N.
      • Bale S.
      • Bick D.
      • Das S.
      • Gastier-Foster J.
      • et al.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      ]. The identified variants of uncertain significance (VUS) were mostly novel variants for which only population prevalence data and predictive in-silico analyses were available. Thus, some of VUS changed their classification to pathogenic or probably pathogenic variants when they occurred de novo or segregated with disease in affected family members. The absence of the variant in the ClinVar, dbSNP, Gnomad and HGMD databases and in available publications was used as a criterion for considering the variant as a “novel mutation”. All pathogenic/likely pathogenic variants detected by the NGS method were then confirmed by Sanger sequencing with Applied Biosystems 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) or by MLPA method for large mutations and by co-segregation with phenotype and family mutation linkage analysis. Dedicated primers have been designed separately to verify each case. The obtained chromatograms were computer analyzed using Sequencher 5.0 software. The presence of the same variant in FM of the patients was also checked using the Sanger sequencing method.

      1.1.3 Statistical analysis

      The normality of linear values distribution was assessed with Shapiro-Wilk test. For continuous variables presentation medians (Me) and interquartile ranges (IQR 25%-75%) were used. The U Mann-Whitney test was used for non-parametric linear data comparisons. Nominal values were presented as a percentage (%) for the study group. Pearson’s chi-square test was used to compare nominal values. The cut-off point for statistical significance was p level lower than 0.05. Statistical analysis was performed using the STATISTICA 13.1 programme (Statsoft, Tulsa, OK, USA).

      1.2 Results

      Of the 684 patients - 542 probands and 142 FM - included in the study group, 198 probands (36.5%) and 66 FM (46.5%) were diagnosed with different forms of monogenic diabetes (Figure 1).
      Figure thumbnail gr1
      Figure 1Details of molecular diagnosis in probands with suspected monogenic diabetes and their family members undergoing genetic testing.F-female; M-male; MODY- maturity-onset diabetes in the young; NDM – neonatal diabetes mellitus; MD – monogenic diabetes.
      Heterozygous causative variants in patients were found in 11 different genes associated with MD, with the highest number in the GCK gene (206/264 of positive subjects), 22/264 in the HNF1A gene and 8/264 in the KCNJ11 gene. The detailed number of heterozygous variants reported among positive patients is shown in Table 1. In 198 positive probands, 31 pathogenic and likely pathogenic variants (15.6%) were assessed as novel mutations not previously described in available databases (Table 2).
      Table 1Number of causative variants found in positive patients in monogenic diabetes-related genes.
      Genen%
      ABCC820.76
      APPL120.76
      GCK20678.03
      HNF1A228.33
      HNF4A72.65
      HNF1B72.65
      KCNJ1183.03
      MAFA10.38
      PDX151.89
      RFX610.38
      WFS131.14
      Table 2Summary of all novel mutations detected in the genes.
      GeneNucleotide changeProtein changePathogenicity according to the ACMG classification
      GCKNM_033507.3:c.1021A>CNP_277042.1:p.Ser341Argpathogenic
      NM_033507.1:c.518delANP_277042.1:p.Lys173ArgfsTer32pathogenic
      NM_033507.1:c.664G>TNP_277042.1:p.Glu222Terpathogenic
      NM_033507.1:c.716T>GNP_277042.1:p.Met239Arglikely pathogenic
      NM_033507.1:c.1366G>TNP_277042.1:p.Val456Leulikely pathogenic
      NM_033507.1:c.163A>GNP_277042.1:p.Ser55Glylikely pathogenic
      NM_033507.1:c.236A>TNP_277042.1:p.Asp79Vallikely pathogenic
      NM_033507.1:c.782T>ANP_277042.1:p.Phe261Tyrpathogenic
      NM_033507.1:c.1334G>ANP_277042.1:p.Gly445Asppathogenic
      NM_033507.3:c.751C>GNP_277042.1:p.Arg251Glypathogenic
      NM_033507.3:c.989C>ANP_277042.1:p.Ala330Asplikely pathogenic
      NM_033507.3:c.163delANP_277042.1:p.Ser55ValfsTer2pathogenic
      NM_033507.1:c.143dupTNP_277042.1:p.Glu49GlyfsTer4pathogenic
      NM_033507.1:c.233T>ANP_277042.1:p.Leu78Glnlikely pathogenic
      NM_033507.1: c.867-2A>Csplice_donor_variantpathogenic
      NM_033507.1:c.610_614delGTGAANP_277042.1:p.Val204Terpathogenic
      NM_033507.1:c.1290_1296delGCTGACGNP_277042.1:p.Arg430SerfsTer183pathogenic
      NM_033507.1:c.622G>ANP_277042.1:p.Val208Metlikely pathogenic
      NM_033507.1:c.1149C>GNP_277042.1:p.Cys383Trppathogenic
      NM_033507.1:c.800T>CNP_277042.1:p.Leu267Prolikely pathogenic
      NM_033507.1:c.1334G>ANP_277042.1:p.Gly445Asplikely pathogenic
      NM_033507.1:c.770A>GNP_277042.1:p.Glu257Glypathogenic
      NM_033507.1:c.125T>GNP_277042.1:p.Met42Argpathogenic
      HNF1ANM_000545.5:c.1187_1206delTCAACCAGCAGCCCCAGAACNP_000536.5:p.Leu396ProfsTer16pathogenic
      NM_000545.8:c.615G>TNP_000536.5:p.Lys205Asnlikely pathogenic
      NM_000545.5:c.900delCNP_000536.5:p.Ala301LeufsTer41pathogenic
      NM_000545.5:c.824A>TNP_000536.5:p.Glu275Vallikely pathogenic
      HNF4ANM_000457.4:c.24_25delCGNP_000448.3:p.Asp9HisfsTer20pathogenic
      NM_000457.4:c.603G>TNP_000448.3:p.Trp201Cyslikely pathogenic
      NM_000457.4:c.984T>ANP_000448.3:p.Tyr328Terpathogenic
      KCNJ11NM_000525.3:c.987C>GNP_000516.3:p.Asp329Glulikely pathogenic
      PAX6NM_001258462.1:c.829G>ANP_001245391.1:p.Ala277Thrlikely pathogenic
      ACMG - American College of Medical Genetics and Genomics
      The most common types of monogenic diabetes diagnosed among patients were MODY2 (GCK-MODY) (78.03%) and MODY3 (HNF1A-MODY) diabetes (8.33%). These were followed by: NDM diabetes with mutations in KCNJ11 gene (3.03%), MODY 5 (HNF1B-MODY) (2.65%), MODY 1 (HNF4A-MODY) (2.65%), other rare MODY diabetes forms (2.65%; with variants in ABCC8, APPL1, MAFA, RFX6, WFS1 genes), MODY 4 (PDX1-MODY) diabetes (1.89%) and MD syndromes (0.76%; Wolfram syndrome and WFS-like syndrome) (Figure 2).
      Figure thumbnail gr2
      Figure 2Frequency of different forms of monogenic diabetes among positive patients.
      Next, a clinical assessment of patients with selected common forms of MODY diabetes: MODY1-5 was performed. The median age of clinical diagnosis of hyperglycaemia or diabetes mellitus was 11.0 years in the probands (IQR 8.0-15.0) and was 2 years lower than the age of genetic diagnosis of monogenic diabetes (Me=13.0 years; IQR 10.0-17.0), whereas in their FM the median age of clinical diagnosis was 25.0 years (IQR 16.0-36.5; p<0.0001) but genetic diagnosis occurred at 38.0 years of age (IQR 26.0-43.0; p<0.0001). Moreover, probands with MODY1-5 diabetes had a lower age at genetic diagnosis compared to negative patients (Me=14.0 years; IQR 10.0-18.0; p=0.042). At the time of genetic diagnosis, HbA1c levels were significantly higher in probands with MODY1-5 diabetes (Me=6.3%; IQR 6.0-6.6) than in patients without monogenic diabetes (Me=6.1%; IQR 5.5-7.1; p=0.035), whereas at the time of diagnosis of glucose tolerance disorders there was only a tendency towards lower HbA1c values (p=0.064). Patients without genetic confirmation of MD more often had DKA at the time of clinical diagnosis of diabetes (p=0.003) (Table 3).
      Table 3Comparison of clinical features of probands diagnosed with MODY1-5 (n=183) and negative patients (n=344).
      ParameterProbands withMODY1-5 diabetesMe (IQR 25%-75%) or %Negative probandsMe (IQR 25%-75%) or %p value
      Age at clinical diagnosis of H/DM (years)11.0 (8.0-15.0)11.0 (8.0-16.0)0.526
      Age at genetic testing (years)13.0 (10.0-17.0)14.0 (10.0-18.0)0.042
      HbA1c level at clinical diagnosis of H/DM (%/mmol/mol)6.4 (6.0-6.7)46 (42-50)6.8 (5.7-10.1)51 (39-87)0.064
      HbA1c level at genetic testing (%/mmol/mol)6.3 (6.0-6.6)45 (42-49)6.1 (5.5-7.1)43 (37-54)0.035
      Ketoacidosis at clinical diagnosis * (yes/no)1.1%/98.9%13.3%/86.7%0.003
      Type of clinical diagnosis:
      Symptoms and random glycaemia>200 mg/dL10.7%62.8%<0.0001
      Fasting glycaemia>126 mg/dL1.4%1.4%
      OGTT87.9%35.8%
      Clinical diagnosis:
      Hyperglycaemia48.6%39.9%0.213
      Diabetes mellitus51.4%60.1%
      Treatment at genetic testing:
      Insulin16.7%38.1%<0.0001
      Oral hypoglycaemic agents14.6%8.1%
      Diet only68.7%53.8%
      H-hyperglycaemia; DM-diabetes mellitus; OGTT-oral glucose tolerance test. *DM patients only. Cases with data missing excluded from analysis.
      The most common way to diagnose diabetes mellitus or pre-diabetes stage in MODY1-5 probands was the oral glucose tolerance test (OGTT) (87.9%) and, interestingly, only 51.4% of them were then confirmed to have diabetes, whereas in 62.8% of the negative patients the diagnosis of diabetes was associated with the presence of symptoms and a random glucose level above 200 mg/dL (p<0.0001) (Table 3).
      In the group of patients with genetically confirmed MODY1-5 diabetes, the majority of probands (68.7%) were exclusively on a nutrition therapy at the time of genetic diagnosis, while 31.3% of them were additionally treated with oral hypoglycaemic agents and/or insulin. However, in the subgroup of their family members, differences in the frequency of treatment were not so evident (respectively: 52.0% and 48.0%; p=0.033). Furthermore, treatment differences were also observed between MODY1-5 probands and patients without MD (p<0.0001) (Table 3).
      Clinically comparing probands diagnosed with the two most frequent types of MD, MODY2 and MODY3 diabetes, an older age at both clinical (p=0.005) and genetic (p<0.0001) diagnosis was observed in patients with MODY3 diabetes. Furthermore, the clinical diagnosis was more often hyperglycaemia in MODY2 patients, whereas it was already diabetes in MODY3 probands, consistent with treatment at genetic testing, with a predominance of nutrition therapy alone in MODY2 patients and oral hypoglycaemic drugs in MODY3 probands (both p<0.0001) (Suppl. Table 1).
      After a proper genetic diagnosis, each MD patient and family members were given appropriate recommendations for further management, including nutrition and pharmacological therapy, if necessary, and genetic counselling.

      1.3 Discussion

      For the first time in Poland, monogenic diabetes was searched exclusively by NGS method in both pediatric and young adult patients from 12 diabetes centers, selected mainly on the basis of positive family history, preserved insulin secretion and absence of autoantibodies characteristic for autoimmune diabetes. In addition, we also carried out a genetic analysis in family members of positive MD patients. This showed a high frequency of monogenic diabetes of more than 36% in the selected group of patients and more than 46% in the first-degree relatives of probands. These findings are consistent with observations made in the USA and Canada, indicating a 35-40% contribution of various forms of monogenic diabetes among all diabetes cases in patients [
      • Brahm A.J.
      • Wang G.
      • Wang J.
      • McIntyre A.D.
      • Cao H.
      • Ban M.R.
      • et al.
      Genetic Confirmation Rate in Clinically Suspected Maturity-Onset Diabetes of the Young.
      ,
      • Bennett J.T.
      • Vasta V.
      • Zhang M.
      • Narayanan J.
      • Gerrits P.
      • Hahn S.H.
      Molecular genetic testing of patients with monogenic diabetes and hyperinsulinism.
      ]. A study conducted in Europe confirmed monogenic diabetes in about 16% of patients, and among patients of Euro-Caucasian origin even in more than 23% of patients [
      • Donath X.
      • Saint-Martin C.
      • Dubois-Laforgue D.
      • Rajasingham R.
      • Mifsud F.
      • Ciangura C.
      • et al.
      Next-generation sequencing identifies monogenic diabetes in 16% of patients with late adolescence/adult-onset diabetes selected on a clinical basis: a cross-sectional analysis.
      ]. Interestingly, some recent NGS studies in selected pediatric and/or young adult patients have reported more than 40% of cases of monogenic diabetes [
      • Stankute I.
      • Verkauskiene R.
      • Blouin J.-L.
      • Klee P.
      • Dobrovolskiene R.
      • Danyte E.
      • et al.
      Systematic Genetic Study of Youth With Diabetes in a Single Country Reveals the Prevalence of Diabetes Subtypes, Novel Candidate Genes, and Response to Precision Therapy.
      ,

      Glotov OS, Serebryakova EA, Turkunova ME, Efimova OA, Glotov AS, Barbitoff YA, Nasykhova YA, Predeus AV, Polev DE, Fedyakov MA, Polyakova IV, Ivashchenko TE, Shved NY, Shabanova ES, Tiselko AV, Romanova OV, Sarana AM, Pendina AA, Scherbak SG, Musina EV, Petrovskaia-Kaminskaia AV, Lonishin LR, Ditkovskaya LV, Zhelenina LA, Tyrtova LV, Berseneva OS, Skitchenko RK, Suspitsin EN, Bashnina EB, Baranov VS. Whole–exome sequencing in Russian children with non–type 1 diabetes mellitus reveals a wide spectrum of genetic variants in MODY–related and unrelated genes. Mol Med Rep. 2019;20:4905-4914. 10.3892/mmr.2019.10751.

      ].
      In all the above studies the most common forms of diagnosed MD remained MODY2 and MODY3 diabetes, which is consistent with the results presented here [
      • Brahm A.J.
      • Wang G.
      • Wang J.
      • McIntyre A.D.
      • Cao H.
      • Ban M.R.
      • et al.
      Genetic Confirmation Rate in Clinically Suspected Maturity-Onset Diabetes of the Young.
      ,
      • Bennett J.T.
      • Vasta V.
      • Zhang M.
      • Narayanan J.
      • Gerrits P.
      • Hahn S.H.
      Molecular genetic testing of patients with monogenic diabetes and hyperinsulinism.
      ,
      • Donath X.
      • Saint-Martin C.
      • Dubois-Laforgue D.
      • Rajasingham R.
      • Mifsud F.
      • Ciangura C.
      • et al.
      Next-generation sequencing identifies monogenic diabetes in 16% of patients with late adolescence/adult-onset diabetes selected on a clinical basis: a cross-sectional analysis.
      ,
      • Stankute I.
      • Verkauskiene R.
      • Blouin J.-L.
      • Klee P.
      • Dobrovolskiene R.
      • Danyte E.
      • et al.
      Systematic Genetic Study of Youth With Diabetes in a Single Country Reveals the Prevalence of Diabetes Subtypes, Novel Candidate Genes, and Response to Precision Therapy.
      ,

      Glotov OS, Serebryakova EA, Turkunova ME, Efimova OA, Glotov AS, Barbitoff YA, Nasykhova YA, Predeus AV, Polev DE, Fedyakov MA, Polyakova IV, Ivashchenko TE, Shved NY, Shabanova ES, Tiselko AV, Romanova OV, Sarana AM, Pendina AA, Scherbak SG, Musina EV, Petrovskaia-Kaminskaia AV, Lonishin LR, Ditkovskaya LV, Zhelenina LA, Tyrtova LV, Berseneva OS, Skitchenko RK, Suspitsin EN, Bashnina EB, Baranov VS. Whole–exome sequencing in Russian children with non–type 1 diabetes mellitus reveals a wide spectrum of genetic variants in MODY–related and unrelated genes. Mol Med Rep. 2019;20:4905-4914. 10.3892/mmr.2019.10751.

      ]. Only the relatively high proportion of mutations found in the KCNJ11 gene (3%) may seem surprising, indicating that NDM is the third most common MD observed in our study. However, similar findings were made by Donath et al. in their study, in which the found mutation rate both in the ABCC8 and KCNJ11 genes was over 4% [
      • Donath X.
      • Saint-Martin C.
      • Dubois-Laforgue D.
      • Rajasingham R.
      • Mifsud F.
      • Ciangura C.
      • et al.
      Next-generation sequencing identifies monogenic diabetes in 16% of patients with late adolescence/adult-onset diabetes selected on a clinical basis: a cross-sectional analysis.
      ].
      Furthermore, in the presented study we found 31 novel mutations, mainly in the GCK gene, but also in genes: HNF1A, HNF4A and KCNJ11, which significantly expands the spectrum of causative variants in MD patients [
      • Bansal V.
      • Gassenhuber J.
      • Phillips T.
      • Oliveira G.
      • Harbaugh R.
      • Villarasa N.
      • et al.
      Spectrum of mutations in monogenic diabetes genes identified from high-throughput DNA sequencing of 6888 individuals.
      ,
      • Sanyoura M.
      • Letourneau L.
      • Knight Johnson A.E.
      • del Gaudio D.
      • Greeley S.A.W.
      • Philipson L.H.
      • et al.
      GCK-MODY in the US Monogenic Diabetes Registry: Description of 27 unpublished variants.
      ].
      The clinical characteristics of Polish patients with confirmed MODY1-5 diabetes showed that at the time of clinical diagnosis of hyperglycaemia or diabetes, they were on average 11 years old and had already received a confirmed genetic diagnosis of MD after only 2 years, while their first-degree relatives with diabetes diagnosed on average at 25 years of age waited as long as 13 years for a correct genetic diagnosis of MD. The average age of onset of diabetes in probands corresponds to the observations of other authors and ranges between 9 and 14 years [

      Delvecchio M, Mozzillo E, Salzano G, Iafusco D, Frontino G, Patera PI, Rabbone I, Cherubini V, Grasso V, Tinto N, Giglio S, Contreas G, Di Paola R, Salina A, Cauvin V, Tumini S, d'Annunzio G, Iughetti L, Mantovani V, Maltoni G, Toni S, Marigliano M, Barbetti F. Monogenic Diabetes Accounts for 6.3% of Cases Referred to 15 Italian Pediatric Diabetes Centers During 2007 to 2012. J Clin Endocrinol Metab. 2017;102:1826-1834. 10.1210/jc.2016-2490.

      ,
      • Stankute I.
      • Verkauskiene R.
      • Blouin J.-L.
      • Klee P.
      • Dobrovolskiene R.
      • Danyte E.
      • et al.
      Systematic Genetic Study of Youth With Diabetes in a Single Country Reveals the Prevalence of Diabetes Subtypes, Novel Candidate Genes, and Response to Precision Therapy.
      ,
      • Chambers C.
      • Fouts A.
      • Dong F.
      • Colclough K.
      • Wang Z.
      • Batish S.D.
      • et al.
      Characteristics of maturity onset diabetes of the young in a large diabetes center.
      ]. Our study also observed both a later age of clinical diagnosis of carbohydrate tolerance disorders and referral for genetic testing in probands with MODY3 diabetes compared with those with MODY2, which in both subgroups of patients represented an older age than in studies by other authors [
      • Gandica R.G.
      • Chung W.K.
      • Deng L.
      • Goland R.
      • Gallagher M.P.
      Identifying monogenic diabetes in a pediatric cohort with presumed type 1 diabetes.
      ]. In addition, hyperglycaemia was the more common clinical diagnosis in our MODY2 patients, whereas it was already diabetes in MODY3 probands, which corresponded to the treatment used, with a predominance of diet alone in MODY2 patients and oral hypoglycaemic drugs in MODY3 probands.
      The mean HbA1c value at the time of diabetes diagnosis in Polish patients with MODY diabetes was 6.4%, which is similar to the results of studies in patients of Euro-Caucasian origin (6.6%) [
      • Donath X.
      • Saint-Martin C.
      • Dubois-Laforgue D.
      • Rajasingham R.
      • Mifsud F.
      • Ciangura C.
      • et al.
      Next-generation sequencing identifies monogenic diabetes in 16% of patients with late adolescence/adult-onset diabetes selected on a clinical basis: a cross-sectional analysis.
      ] and significantly lower than the observations made by the authors of the USA study (7.9%) [
      • Chambers C.
      • Fouts A.
      • Dong F.
      • Colclough K.
      • Wang Z.
      • Batish S.D.
      • et al.
      Characteristics of maturity onset diabetes of the young in a large diabetes center.
      ]. The tendency towards lower HbA1c values observed in these patients compared to patients without confirmed MD corresponds with the results of other authors [
      • Chambers C.
      • Fouts A.
      • Dong F.
      • Colclough K.
      • Wang Z.
      • Batish S.D.
      • et al.
      Characteristics of maturity onset diabetes of the young in a large diabetes center.
      ], who reported lower HbA1c values at the onset of diabetes and persistently lower HbA1c levels in the subsequent course of the disease. However, our study showed higher HbA1c levels at the time of genetic diagnosis in patients with MODY1-5 diabetes compared to negative patients, which may suggest the need for more intensive treatment in patients with suspected monogenic diabetes. Moreover, the significantly less frequent presence of DKA and symptoms of diabetes at clinical diagnosis in patients with MODY1-5 diabetes compared with negative patients noticed both in our study and by other authors [
      • Chambers C.
      • Fouts A.
      • Dong F.
      • Colclough K.
      • Wang Z.
      • Batish S.D.
      • et al.
      Characteristics of maturity onset diabetes of the young in a large diabetes center.
      ] may support the suspicion of monogenic diabetes.
      An interesting observation made in our study is the presence of diabetes at the time of clinical diagnosis in just over half of the MODY patients studied (about 51%), and made mainly on the basis of the OGTT test. This seems to be supported both by the study by Delvecchio et al, in which only 27% of patients with MODY had diabetes at the time of clinical diagnosis [

      Delvecchio M, Mozzillo E, Salzano G, Iafusco D, Frontino G, Patera PI, Rabbone I, Cherubini V, Grasso V, Tinto N, Giglio S, Contreas G, Di Paola R, Salina A, Cauvin V, Tumini S, d'Annunzio G, Iughetti L, Mantovani V, Maltoni G, Toni S, Marigliano M, Barbetti F. Monogenic Diabetes Accounts for 6.3% of Cases Referred to 15 Italian Pediatric Diabetes Centers During 2007 to 2012. J Clin Endocrinol Metab. 2017;102:1826-1834. 10.1210/jc.2016-2490.

      ], and by Donath et al, in which only 10% of patients had symptoms of diabetes [
      • Donath X.
      • Saint-Martin C.
      • Dubois-Laforgue D.
      • Rajasingham R.
      • Mifsud F.
      • Ciangura C.
      • et al.
      Next-generation sequencing identifies monogenic diabetes in 16% of patients with late adolescence/adult-onset diabetes selected on a clinical basis: a cross-sectional analysis.
      ]. In our study, less than 11% of patients with MODY were diagnosed with clinical diabetes on the basis of coexisting symptoms and incidental hyperglycaemia above 200 mg/dL, whereas in the study performed by Chambers et al the initial blood glucose in patients with MODY diabetes averaged 201 mg/dL [
      • Chambers C.
      • Fouts A.
      • Dong F.
      • Colclough K.
      • Wang Z.
      • Batish S.D.
      • et al.
      Characteristics of maturity onset diabetes of the young in a large diabetes center.
      ]. These findings suggest that the genetic diagnosis of MODY diabetes can often be made at the stage of hyperglycaemia and therefore precede the clinical diagnosis of diabetes, further emphasizing the importance of early genetic testing.
      The young age of clinical diagnosis and the significant contribution of hyperglycaemia as an early phase of impaired glucose tolerance found in our MODY1-5 patients was related to the treatment given. The majority of probands (nearly 69%) required only a low GI diet, whereas in their adult first-degree relatives almost half of them (48%) were already treated with oral hypoglycaemic agents and/or insulin. These results are consistent with observations made by other authors, in which 48-50% of children and young adults with MODY diabetes were treated with oral agents or insulin therapy [
      • Shepherd M.
      • Shields B.
      • Hammersley S.
      • Hudson M.
      • McDonald T.J.
      • Colclough K.
      • et al.
      UNITED Team. Systematic Population Screening, Using Biomarkers and Genetic Testing, Identifies 2.5% of the U.K. Pediatric Diabetes Population With Monogenic Diabetes.
      ,
      • Chambers C.
      • Fouts A.
      • Dong F.
      • Colclough K.
      • Wang Z.
      • Batish S.D.
      • et al.
      Characteristics of maturity onset diabetes of the young in a large diabetes center.
      ]. Similar findings were observed in paediatric MODY-positive patients, who were also more likely to be prescribed no pharmacologic treatment early after diagnosis compared to patients without monogenic diabetes [
      • Chambers C.
      • Fouts A.
      • Dong F.
      • Colclough K.
      • Wang Z.
      • Batish S.D.
      • et al.
      Characteristics of maturity onset diabetes of the young in a large diabetes center.
      ].
      Our study has several limitations. Not all patients with suspected monogenic diabetes underwent gene deletion/duplication analysis using MLPA method. Because it is not population-based, it did not allow us to calculate the prevalence of monogenic diabetes in Poland. Due to the lack of both fasting and stimulated C-peptide values in all patients (most only fasting C-peptide or insulin values), we could only qualitatively comment on the presence or absence of insulin secretion disorders. Similarly, we did not have BMI values, insulin doses or individual oral agents in all patients, which could have enriched the clinical characteristics of the patients presented.
      Concluding, the results obtained seem to confirm the effectiveness of the NGS method for selected patients qualified for the study with suspected monogenic diabetes and extend the spectrum of new pathogenic variants. They also enlarge our knowledge of the clinical features of patients with various forms of monogenic diabetes.

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgments

      We express our gratitude to patients and their families participating in the study for their contribution. We also thank the medical centers referring patients for testing at our facility, and physicians helping in collecting clinical data, in particular Prof. P. Fichna, Dr. A. Brandt, Dr M. Okońska, Dr M. Stelmach, Dr B. Florys, Dr E. Skała-Zamorowska, Dr H. Kamińska, Dr B. Wysocka-Łukasik, Dr B. Banecka, Dr J. Jabłońska, Dr D. Charemska, Dr A. Hogendorf, Dr A. Horodnicka-Józwa, Dr K. Dżygało, Dr B. Salmonowicz, Dr J. Chrzanowska, Dr A. Zubkiewicz-Kucharska and Dr W. Stankiewicz.
      Author Contributions
      A.Z. collected data and wrote the draft of the manuscript. P.J., K.G. and T.P. performed genetic analyses. A.M. performed statistical analyses. I.B-S., G.D., B.G-O., P.J-Ch., B.K., I.K., W.M., M.M., J.N., A.N., K.R-K., E.S-Z., B.S., A.Sz., A.Sz., M.W. collected clinical data. M.B. performed genetic analyses and contributed to writing the manuscript.
      Human Studies Statement
      All procedures followed were in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.
      Data accessibility Statement
      The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.
      Funding Statement
      This study is supported by National Science Centre grants No 2018/29/B/NZ5/00330 and 2015/19/B/NZ5/02243 and Medical University of Lodz grants No: 502-03/2-159-02/502-24-306 and: 502-03/2-159-02/502-24-307.

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