30 research outputs found
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, kliniÄki i genetiÄki veoma heterogen, koji nastaje kao posledica primarne disfunkcije Ī²-Äelija pankreasa. MODY nastaje usled genetiÄkih 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 uzroÄnika MODY dijabetesa kod 29 kliniÄki suspektnih pedijatrijskih pacijenata. Kombinovanjem ove dve metode detektovano je 20 razliÄitih varijanti kod 75,9% pacijenata u 4 razliÄita gena. Varijante u dva gena, GCK i HNF1B, detekotvane redom, kod 50% i 22,7% pacijenata, predstavljale su glavne uzroÄnike 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 takoÄe dovode do MODY dijabetesa, analizirani su promotorski regioni Äetiri najÄeÅ”Äa gena uzroÄnika MODY dijabetesa. Varijantni set u promotrskom regionu GCK gena (ā282C>T; ā194A>G; 402C>G) i varijanta c.-154-160insTGGGGGT u promotoru HNF1A gena, odabrane su kako bi se u funkcionalnim esejima u Äelijskoj 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, Äime su dopunjena postojeÄa 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 Ī²-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 (ā282C>T; ā194A>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 Äija je uÄestalost manja od 1 u 2000 ljudi definiÅ”e se kao retka bolest. Iz tog razloga, broj retkih
bolesti je veliki. Do sada je opisano preko 6000 razliÄitih retkih bolesti. Preko 80% retkih bolesti ima genetiÄku
osnovu i to je razlog zaÅ”to su znanja iz molekularne biologije od neprocenjivog znaÄaja za
istraživanje molekularne osnove retkih bolesti, postavljanje taÄne dijagnoze i razvoj inovativnih terapeutika.
Cilj ovog rada je da objasni važnost otkrivanja molekularno-genetiÄke osnove retkih bolesti i da prikaže
desetogodiŔnje iskustvo primene sekvenciranja nove generacije u Srbiji (2014.-2023.) u tu svrhu. U
prethodnom periodu za istraživanje retkih bolesti koriÅ”Äeni su sekvenciranje kliniÄkog egzoma, sekvenciranje
kompletnog egzoma i sekvenciranje kompletnog genoma. TakoÄe, date su i perspektive za buduÄnost
gde Äe genomika biti kompletirana tehnologijom sekvenciranja dugaÄkih fragmenata i
komplementirana upotrebom transkriptomike, proteomike, metabolomike i drugih āomikaā.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 nauÄne aktivnosti Centra za genetiÄku dijagnostiku
retkih bolesti IMGGI
The molecular basis of monogenic diabetes
Monogenski dijabetes je heterogen oblik dijabetesa u Äiji nastanak je ukljuÄen veliki broj gena. Promene u
genima uzroÄnicima monogenskog dijabetesa naruÅ”avaju funkciju Ī²-Äelije pankreasa dovodeÄi do smanjene
ili oÅ”teÄene sekrecije insulina i, poslediÄno, hiperglikemije kod pacijenata. Produkte ovih gena uglavnom
Äine transkripcioni faktori, zatim membranski kanali i proteini sa specifiÄnom funkcijom u Äeliji. Mnogi
transkripcioni faktori monogenskog dijabetesa imaju plejotropno dejstvo, te se kod nosioca promena u
ovim genima mogu uoÄiti ne samo poremeÄaji u funkcionisanju Ī²-Äelija, veÄ i defekti u drugim organima ili
multisistemski poremeÄaji. Dva glavna oblika monogenskog dijabetesa su neonatalni dijabetes, koji se javlja
u prvih 6 meseci života, i mnogo ÄeÅ”Äi, MODY dijabetes koji nastaje u mlaÄem odraslom dobu. NajÄeÅ”Äi geni
uzroÄnici neonatalnog dijabetesa su geni KCNJ11, ABCC8 i INS kao i lokus 6q24, dok se u sluÄaju MODY
dijabetesa izdvajaju dva dominantna gena HNF1A i GCK. Iako je monogenski dijabetes veoma redak, njegovo
prepoznavanje meÄu rasprostranjenijim oblicima dijabetesa je od izuzetnog znaÄaja, s obzirom na to da su
terapija, kliniÄka prezentacija, subklasifikacija i prognoza toka bolesti specifiÄni prema genu uzroÄniku.
Savremene tehnologije sekvenciranja (NGS) su pronaŔle svoje mesto u dijagnozi monogenskog dijabetesa,
buduÄi da je ova metoda nezamenjiva kada je u pitanju analiza velikog broja gena i heterogen fenotip koji
se sreÄe kod ovog tipa dijabetesa. RastuÄa saznanja o genima uzroÄnicima monogenskog dijabetesa su
izdvojila ovaj oblik dijabetesa kao dobrog kandidata za implementaciju relativno novog koncepta, precizne
medicine dijabetesa, Äiji 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 Ī²-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 Ī²-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 patientās 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 poremeÄajima, ukljuÄujuÄi i respiratorne. Primarna cilijarna diskinezija (PCD) najÄeÅ”Äa je ciliopatija, koja nastaje kao posledica poremeÄaja u motornim cilijama. Promenjena struktura i/ ili funkcija motornih cilija dovodi do neonatalnog respiratornog distresa, hroniÄnog vlažnog kaÅ”lja, simptoma nazalne sekrecije, bronhoektazija, hroniÄne upale sinusa i uha, a 50% bolesnika ima i situs inversus. Ovi simptomi su priliÄno uobiÄajeni kod male dece i u drugim stanjima; stoga je uspostavljanje precizne dijagnoze otežano. Cilj ovog istraživanja je ukazivanje na znaÄaj genomskog profilisanja bolesnika i dizajniranje strategije za genetiÄku analizu podataka kod bolesnika suspektnih na ciliopatije sa kliniÄkom slikom sliÄnom drugim bolestima pluÄa. Metode Sproveli smo bioinformatiÄku analizu podataka dobijenih metodom sekvenciranja nove generacije 21 bolesnika sa potvrÄenom ili suspektnom dijagnozom PCD-a. Analizirano je 93 gena: 29 PCD gena, 45 gena asociranih sa pojedinaÄnim simptomima pluÄnih bolesti i 19 gena asociranih sa senzornim ciliopatijama. Rezultati Dizajnirani algoritam za genetiÄku analizu NAM je omoguÄio da potvrdimo kliniÄku i uspostavimo genetiÄku dijagnozu kod 17/21 (80,95%) bolesnika, meÄu 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 pluÄna oboljenja, dok je kod 1/21 (4,76%) bolesnika genetiÄka osnovna bolesti ostala nerazjaÅ”njena. ZakljuÄak Dizajniranje strategije za lakÅ”e i brže uspostavljanje konaÄne dijagnoze ciliopatija je obavezno i ukljuÄuje i kliniÄku i genetiÄku 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