Coding and noncoding variants of disease-causing genes of maturity-onset diabetes of the young: phenotype modulators and regulators of gene expression

Dijabetes adultnog tipa kod mladih (MODY) je nasledni, autozomno dominantni oblik dijabetesa, klinički i genetički veoma heterogen, koji nastaje kao posledica primarne disfunkcije β-ćelija pankreasa. MODY nastaje usled genetičkih varijanti u jednom od brojnih gena asociranih sa MODY dijabetesom, te je metodom sekvenciranja nove generacije i metodom istovremenog umnožavanja vezanih proba, prvi put u Srbij, analizirano 13 gena uzročnika MODY dijabetesa kod 29 klinički suspektnih pedijatrijskih pacijenata. Kombinovanjem ove dve metode detektovano je 20 različitih varijanti kod 75,9% pacijenata u 4 različita gena. Varijante u dva gena, GCK i HNF1B, detekotvane redom, kod 50% i 22,7% pacijenata, predstavljale su glavne uzročnike MODY dijabetesa u ovoj grupi pacijenata. U ovoj studiji identifikovana je i jedna nova prethodno neprijavljena varijanta u GCK genu, c.596T>C; p.Val199Ala, za koju su in silico predikcije nedvosmisleno pokazale da je patogena. Kako bi se detektovale varijante u promotorskom regionu, koje takođe dovode do MODY dijabetesa, analizirani su promotorski regioni četiri najčešća gena uzročnika MODY dijabetesa. Varijantni set u promotrskom regionu GCK gena (−282C>T; −194A>G; 402C>G) i varijanta c.-154-160insTGGGGGT u promotoru HNF1A gena, odabrane su kako bi se u funkcionalnim esejima u ćelijskoj kulturi ispitao njihov uticaj na ekspresiju datih gena. Analizirane su i interakcije ovih potencijalnih regulatornih elemenata sa transkripcionim faktorima u esejima usporene elektroforetske pokretljivosti. Rezultati funkcionalne analize odabarnih promotorskih varijanti ukazali su da varijante u promotoru, osim potencijalno patogenog efekta, mogu da imaju i ulogu modifikatora fenotipa, čime su dopunjena postojeća znanja o varijantama u promotorima MODY gena.Maturity-onset diabetes of the young (MODY) is an inherited, autosomal dominant form of diabetes, clinically and genetically very heterogeneous, resulting from primary β-cell dysfunction. Since MODY diabetes is caused by single gene variants in one of the many MODY-related genes, next generation sequencing and multiplex ligation-dependent probe amplification analysis were used to analyze 13 MODY-relate genes in 29 clinically suspected pediatric patients, for the first time in Serbia. Combining these two methods, 20 different variants, found in 4 genes, were identified in 75.9% patients. Variants in the GCK and HNF1B gene, detected in 50% and 22.7% patients, respectively, were the main cause of MODY diabetes in our patients. Also, a novel variant in the GCK gene: c.596T>C, p.Val199Ala was identified and predicted to be pathogenic by in silico algorithms. Due to the fact that promoter variants can also lead to MODY diabetes, promoter regions of the four most common MODY genes were screened. Two variants, the variant set in the promoter region of the GCK gene (−282C>T; −194A>G; 402C>G), and the variant c.-154-160insTGGGGGT in the promoter of the HNF1A gene, were selected to analyze their effect on gene expression in functional cell culture studies. Also, electrophoretic mobility shift assay was carried out to investigate the interaction of potential transcription factors with the promoter region surrounding the variant, as well as whether the variants affect the binding of those transcription factors. The results of the functional analysis of the selected promoter variants indicated that variants in the promoter, in addition to potentially pathogenic effect, may also play the role of a phenotype modifier, thus supplementing the existing knowledge about variants in the promoters of MODY genes

Present and future of next-generation sequencing application for rare diseases

Svaka bolest čija je učestalost manja od 1 u 2000 ljudi definiše se kao retka bolest. Iz tog razloga, broj retkih bolesti je veliki. Do sada je opisano preko 6000 različitih retkih bolesti. Preko 80% retkih bolesti ima genetičku osnovu i to je razlog zašto su znanja iz molekularne biologije od neprocenjivog značaja za istraživanje molekularne osnove retkih bolesti, postavljanje tačne dijagnoze i razvoj inovativnih terapeutika. Cilj ovog rada je da objasni važnost otkrivanja molekularno-genetičke osnove retkih bolesti i da prikaže desetogodišnje iskustvo primene sekvenciranja nove generacije u Srbiji (2014.-2023.) u tu svrhu. U prethodnom periodu za istraživanje retkih bolesti korišćeni su sekvenciranje kliničkog egzoma, sekvenciranje kompletnog egzoma i sekvenciranje kompletnog genoma. Takođe, date su i perspektive za budućnost gde će genomika biti kompletirana tehnologijom sekvenciranja dugačkih fragmenata i komplementirana upotrebom transkriptomike, proteomike, metabolomike i drugih „omika“.Any disease found in less than 1 person out of 2000 people is defined as a rare disease. For this reason, the number of rare diseases is high. Over 6,000 different rare diseases have been described so far. More than 80% of rare diseases have a genetic basis, and this is the reason why knowledge of molecular biology is invaluable for research into the molecular basis of rare diseases, establishing an accurate diagnosis and developing innovative therapeutics. The aim of this paper is to explain the importance of discovering the molecular genetic basis of rare diseases and to present the ten-year experience of applying new generation sequencing in Serbia (2014-2023) for this purpose. During this period, clinical exome sequencing, complete exome sequencing and complete genome sequencing were used for research of rare diseases. In the future, it is expected that genomics, which until now was based mainly on the technology of shortread fragments, will be broaden with the long-reads sequencing technology, and complemented by the use of transcriptomics, proteomics, metabolomics and other omics.Rad je rezultat naučne aktivnosti Centra za genetičku dijagnostiku retkih bolesti IMGGI

The molecular basis of monogenic diabetes

Monogenski dijabetes je heterogen oblik dijabetesa u čiji nastanak je uključen veliki broj gena. Promene u genima uzročnicima monogenskog dijabetesa narušavaju funkciju β-ćelije pankreasa dovodeći do smanjene ili oštećene sekrecije insulina i, posledično, hiperglikemije kod pacijenata. Produkte ovih gena uglavnom čine transkripcioni faktori, zatim membranski kanali i proteini sa specifičnom funkcijom u ćeliji. Mnogi transkripcioni faktori monogenskog dijabetesa imaju plejotropno dejstvo, te se kod nosioca promena u ovim genima mogu uočiti ne samo poremećaji u funkcionisanju β-ćelija, već i defekti u drugim organima ili multisistemski poremećaji. Dva glavna oblika monogenskog dijabetesa su neonatalni dijabetes, koji se javlja u prvih 6 meseci života, i mnogo češći, MODY dijabetes koji nastaje u mlađem odraslom dobu. Najčešći geni uzročnici neonatalnog dijabetesa su geni KCNJ11, ABCC8 i INS kao i lokus 6q24, dok se u slučaju MODY dijabetesa izdvajaju dva dominantna gena HNF1A i GCK. Iako je monogenski dijabetes veoma redak, njegovo prepoznavanje među rasprostranjenijim oblicima dijabetesa je od izuzetnog značaja, s obzirom na to da su terapija, klinička prezentacija, subklasifikacija i prognoza toka bolesti specifični prema genu uzročniku. Savremene tehnologije sekvenciranja (NGS) su pronašle svoje mesto u dijagnozi monogenskog dijabetesa, budući da je ova metoda nezamenjiva kada je u pitanju analiza velikog broja gena i heterogen fenotip koji se sreće kod ovog tipa dijabetesa. Rastuća saznanja o genima uzročnicima monogenskog dijabetesa su izdvojila ovaj oblik dijabetesa kao dobrog kandidata za implementaciju relativno novog koncepta, precizne medicine dijabetesa, čiji bi krajnji cilj bio postizanje boljeg kvaliteta života pacijenata.Monogenic diabetes is a heterogeneous form of diabetes resulting from defects in a single gene. Defects in monogenic diabetes-related genes disrupt the β-cells function, leading to reduced or impaired insulin secretion and, consequently, hyperglycemia in patients. Products of these genes are mainly transcription factors, followed by membrane channels and proteins with a specific cell function. Most transcription factors involved in monogenic diabetes have a pleiotropic effect expanding the β-cells disfunction with defects in other organs or multisystem disorders. Two main forms of monogenic diabetes are neonatal diabetes, appearing in the first 6 months of life, and the more common, MODY diabetes, that occurs in young adulthood. The most common genes involved in neonatal diabetes are KCNJ11, ABCC8 and INS, followed by the 6q24 lokus, while in the case of MODY diabetes the two dominant genes HNF1A and GCK stand out. Although monogenic diabetes is very rare, its recognition among the more common forms of diabetes is of great importance, since therapy, clinical subclassification and presentation, as well as disease prognosis are gene-specific. Modern sequencing technologies (NGS) have found their place in the diagnosis of monogenic diabetes, as this method is irreplaceable when it comes to the analysis of a large number of genes and the heterogeneous phenotype encountered in these patients. Growing knowledge about monogenic diabetes-related genes has singled out this form of diabetes as a good candidate for the implementation of a relatively new concept, the precision diabetes medicine, whose ultimate goal would be achieving a better quality of life for the patients

The importance of combined NGS and MLPA genetic tests for differential diagnosis of maturity onset diabetes of the young

Introduction: Maturity onset diabetes of the young (MODY) is a rare form of monogenic diabetes. Being clinically and genetically heterogeneous, it is often misdiagnosed as type 1 or type 2 diabetes, leading to inappropriate therapy. MODY is caused by a single gene mutation. Thirteen genes, defining 13 subtypes, have been identified to cause MODY. A correct diagnosis is important for the right therapy, prognosis, and genetic counselling. Material and methods: Twenty-nine unrelated paediatric patients clinically suspected of having MODY diabetes were analysed using TruSight One panel for next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) assay. Results: In this study we identified variants in MODY genes in 22 out of 29 patients (75.9%). Using two genetic tests, NGS and MLPA, we detected both single nucleotide variants and large deletions in patients. Most of the patients harboured a variant in the GCK gene (11/22), followed by HNF1B (5/22). The rest of the variants were found in the NEUROD1 and HNF1A genes. We identified one novel variant in the GCK gene: c.596T gt C, p.Val199Ala. The applied genetic tests excluded the suspected diagnosis of MODY in two patients and revealed variants in other genes possibly associated with the patient's clinical phenotype. Conclusions: In our group of MODY patients most variants were found in the GCK gene, followed by variants in HNF1B, NEUROD1, and HNF1A genes. The combined NGS and MLPA-based genetic tests presented a comprehensive approach for analysing patients with suspected MODY diabetes and provided a successful differential diagnosis of MODY subtypes

The importance of combined NGS and MLPA genetic tests for differential diagnosis of maturity onset diabetes of the young

Introduction: Maturity onset diabetes of the young (MODY) is a rare form of monogenic diabetes. Being clinically and genetically heterogeneous, it is often misdiagnosed as type 1 or type 2 diabetes, leading to inappropriate therapy. MODY is caused by a single gene mutation. Thirteen genes, defining 13 subtypes, have been identified to cause MODY. A correct diagnosis is important for the right therapy, prognosis, and genetic counselling.Material and methods: Twenty-nine unrelated paediatric patients clinically suspected of having MODY diabetes were analysed using TruSight One panel for next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) assay.Results: In this study we identified variants in MODY genes in 22 out of 29 patients (75.9%). Using two genetic tests, NGS and MLPA, we detected both single nucleotide variants and large deletions in patients. Most of the patients harboured a variant in the GCK gene (11/22), followed by HNF1B (5/22). The rest of the variants were found in the NEUROD1 and HNF1A genes. We identified one novel variant in the GCK gene: c.596T>C, p.Val199Ala. The applied genetic tests excluded the suspected diagnosis of MODY in two patients and revealed variants in other genes possibly associated with the patient’s clinical phenotype.Conclusions: In our group of MODY patients most variants were found in the GCK gene, followed by variants in HNF1B, NEUROD1, and HNF1A genes. The combined NGS and MLPA-based genetic tests presented a comprehensive approach for analysing patients with suspected MODY diabetes and provided a successful differential diagnosis of MODY subtypes

Molecular genetic strategy for diagnosis of congenital adrenal hyperplasia in Serbia

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common endocrine diseases, yet genetic diagnosis is among the most complicated of all monogenic disorders. It has an overall incidence of 1: 10000-1: 20000, it is inherited in autosomal recessive pattern and caused by mutations affecting CYP21A2 gene. Based on the phenotypic expression, this disease is categorized into severe, classical form revealed at birth and mild, non-classical form. Although diagnosis could be established based on biochemical tests and distinctive clinical features, molecular genetic testing is crucial for diagnosis confirmation, detection of carriers and asymptomatic patients, disease prognosis, as well as for providing proper genetic counselling and prenatal diagnosis. Based on CYP21A2 mutational spectrum and frequencies in Serbia, in this paper we propose an optimal molecular genetic diagnostic algorithm for CAH and discuss genetic mechanisms underlying the disease. The complete diagnostic procedure combines multiplex minisequencing technique (SNaPshot PCR) as a method for rapid detection of common point mutations, direct sequencing of whole CYP21A2 gene and PCR with sequence specific primers (PCR-SSP) for large gene rearrangements detection (CYP21A1P/CYP21A2 chimeras). While SNaPshot PCR assay analyses ten common mutations (c. 290-13A/C gt G, p.P30L, p.R356W, p.G110fs, p.V281L, p.Q318X, p.L307fs, p.I172N, Cluster p.[I236N;V237E;M239K] and p.P453S) which account for over 80% of all CYP21A2 mutations in Serbian population, direct sequencing of CYP21A2 gene is needed to identify potential rare or novel mutations present in Serbian population with frequency of 1.8%. Additionally, large gene rearrangements which are present with frequency of 16.7% make PCR-SSP analysis an unavoidable part of molecular characterization of CAH in Serbia. Described molecular genetic strategy is intended to facilitate correct diagnosis assessment in CAH affected individuals and their families in Serbia but it will also contribute to molecular genetic testing of CAH patients across Europe

The importance of genomic profiling for differential diagnosis of pediatric lung disease patients with suspected ciliopathies

Uvod/Cilj Izmenjena funkcija aksonemalne strukture dovodi do ciliopatija (motornih i senzornih), koje su do sada povezane sa brojnim pedijatrijskim poremećajima, uključujući i respiratorne. Primarna cilijarna diskinezija (PCD) najčešća je ciliopatija, koja nastaje kao posledica poremećaja u motornim cilijama. Promenjena struktura i/ ili funkcija motornih cilija dovodi do neonatalnog respiratornog distresa, hroničnog vlažnog kašlja, simptoma nazalne sekrecije, bronhoektazija, hronične upale sinusa i uha, a 50% bolesnika ima i situs inversus. Ovi simptomi su prilično uobičajeni kod male dece i u drugim stanjima; stoga je uspostavljanje precizne dijagnoze otežano. Cilj ovog istraživanja je ukazivanje na značaj genomskog profilisanja bolesnika i dizajniranje strategije za genetičku analizu podataka kod bolesnika suspektnih na ciliopatije sa kliničkom slikom sličnom drugim bolestima pluća. Metode Sproveli smo bioinformatičku analizu podataka dobijenih metodom sekvenciranja nove generacije 21 bolesnika sa potvrđenom ili suspektnom dijagnozom PCD-a. Analizirano je 93 gena: 29 PCD gena, 45 gena asociranih sa pojedinačnim simptomima plućnih bolesti i 19 gena asociranih sa senzornim ciliopatijama. Rezultati Dizajnirani algoritam za genetičku analizu NAM je omogućio da potvrdimo kliničku i uspostavimo genetičku dijagnozu kod 17/21 (80,95%) bolesnika, među kojima je 11/21 (52,38%) PCD bolesnika. Kod 3/21 (14,28%) bolesnika detektovane su monoalelske varijante u PCD genima, kod 6/21 (28,57%) bolesnika detektovane su varijante u genima relevantnim za druga plućna oboljenja, dok je kod 1/21 (4,76%) bolesnika genetička osnovna bolesti ostala nerazjašnjena. Zaključak Dizajniranje strategije za lakše i brže uspostavljanje konačne dijagnoze ciliopatija je obavezno i uključuje i kliničku i genetičku potvrdu bolesti.Introduction/Objective Dysfunction of the axonemal structure leads to ciliopathies. Sensory and mo-tile ciliopathies have been associated with numerous pediatric diseases, including respiratory diseases. Primary ciliary dyskinesia (PCD) is ciliopathy linked to the dysfunction of motile cilia. Motile ciliary dys-function in childhood leads to chronic rhinosinusitis, persistent cough, neonatal respiratory distress, bronchiectasis, and situs inversus (SI) have 50% of patients. These symptoms are common among pediatric lung diseases, which additionally makes it difficult to establish the accurate diagnosis. The aim of the study was to point out the significance of genomic profiling for patients with suspected ciliopathies and to design a strategy for genomic analysis relevant for differential diagnosis of lung disease patients with suspected ciliopathies. Methods We conducted a bioinformatic analysis of data generated by New Generation Sequencing (NGS) approach of 21 patients with final or suspected diagnosis of PCD. It was analyzed 93 genes: 29 PCD genes, 45 genes related to individual symptoms of lung diseases, and 19 genes related to sensory ciliopathies. Results the algorithm we have designed, enabled us to establish the clinical and genetic diagnosis for 17/21 (80.95%) patients, among which 11/21 (52.38%) were PCD patients. In 3/21 (14.28%) patients we detected monoallelic variants in PCD disease-causing genes. In 6/21 (28.57%) patients, variants in genes for other pulmonary diseases were detected, and for one patient, genetic background of disease remained unclear. Conclusion an improved strategy for easier and faster establishment of final diagnosis of ciliopathies is mandatory and includes both, clinical and genetic confirmation of disease

The Role of Autophagy and Apoptosis in Affected Skin and Lungs in Patients with Systemic Sclerosis

Systemic sclerosis (SSc) is a complex autoimmune inflammatory disorder with multiple organ involvement. Skin changes present the hallmark of SSc and coincide with poor prognosis. Interstitial lung diseases (ILD) are the most widely reported complications in SSc patients and the primary cause of death. It has been proposed that the processes of autophagy and apoptosis could play a significant role in the pathogenesis and clinical course of different autoimmune diseases, and accordingly in SSc. In this manuscript, we review the current knowledge of autophagy and apoptosis processes in the skin and lungs of patients with SSc. Profiling of markers involved in these processes in skin cells can be useful to recognize the stage of fibrosis and can be used in the clinical stratification of patients. Furthermore, the knowledge of the molecular mechanisms underlying these processes enables the repurposing of already known drugs and the development of new biological therapeutics that aim to reverse fibrosis by promoting apoptosis and regulate autophagy in personalized treatment approach. In SSc-ILD patients, the molecular signature of the lung tissues of each patient could be a distinctive criterion in order to establish the correct lung pattern, which directly impacts the course and prognosis of the disease. In this case, resolving the role of tissue-specific markers, which could be detected in the circulation using sensitive molecular methods, would be an important step toward development of non-invasive diagnostic procedures that enable early and precise diagnosis and preventing the high mortality of this rare disease

Rare metabolic diseases in the genomics era

Introduction: All inborn metabolic diseases are rare, having a prevalence less than 1:2000. Vast majority of them are monogenic and finding pathogenic genetic variantsis needed to setthe correct diagnosis, enable adequate treatment and provide genetic counseling to members of affected family. Thisstudy is an overview of genomic studies of rare metabolic diseases in Serbia. Methods: Since 2005, more than 300 patients suspected to have a rare metabolic or neurometabolic disease have been analyzed using sanger sequencing, clinical-exome sequencing, whole-exome sequencing or whole-genome sequencing in order to find disease-causing or disease-modifying variants. Novel variants were characterized using in silico modelling or in in vitro eukaryotic assays (standard or CRISPR/Cas9 developed). Results: Disease-causing variants were found in more than 60 different genes associated with a metabolic or neurometabolic disease. The most frequent disease was phenylketonuria (109 patients), followed by glycogen storage disease Ib (30 patients), while majority of diseases is seen only in a single patient. More than 40 new genetic variants were comprehensively characterized in silico or in vitro. For the first time, candidate modifiers (SHANK gene family) were identified in a group of phenylketonuria patients with an unusual phenotype. Conclusion: In the genomics era, next-generation sequencing significantly shortens time to diagnosis and allowsstudying genetic modifiers of monogenic diseases and genotype-phenotype correlation. Furthermore, characterization of novel genetic targets boosts development of precision medicin

Improving the diagnostics of rare lung disorders using a uniquely designed pipeline for analysis of ngs data

Rare lung diseases (RLDs) are a group of diseases that individually affect one in 2,000 people, with an estimate that about 80% of RLDs have a genetic origin. Despite the variations among RLDs in clinical characteristics and manifestations, most of these diseases similarly damage the lungs, making diagnosis difficult. The utility of NGS technology in RLDs for diagnostic purposes allows a better understanding of the genetic background, however, the identification and classification of disease-causing variants are challenging. Further, numerous VUS (variants of uncertain significance) that cannot be precisely defined and classified are produced. The main goal of this study was to create a unique guideline that will enable the standardization of the assessment of novel genetic variants in RLDs causative genes. The designed pipeline consists of three main steps: (1) sequencing, detection, and identification of genes/variants, (2) classification of variants, and (3) characterization of variants using in silico structural and functional analysis. The pipeline validation was performed through the analysis of variants detected in a disease-causing and candidate genes of one of the RLDSs, and detected VUS variants have gained diagnostic significance. The application of this pipeline resulted in the identification and classification of novel variants, through analysis at the transcriptional, translational, and posttranslational levels, and led to accurate diagnosis.Book of abstracts: International Conference of Biochemists and Molecular Biologists in Bosnia and Herzegovina - ABMBBIH May, 202