Genomic profiling of pediatric patients with primary ciliary dyskinesia: genotype-phenotype correlation and functional characterization of novel genetic variants

Primarna cilijarna diskinezija (PCD) predstavlja retku bolest koja se nasleđuje na autozomno recesivan način ili je nasleđivanje vezano za hromozom X, i predominantno utiče na funkcionisanje pluća, reproduktivnih organa i na lateralnost unutrašnjih organa. PCD je klinički i genetički veoma heterogen poremećaj koji se javlja odmah po rođenju, a karakteriše se promenama na motornim cilijama koje su posledica patogenih varijanti u genima koji kodiraju za proteine koji su neophodni za pravilnu strukturu i funkciju ovih organela. Do sada je opisano 38 gena uzročnika bolesti koje su odgovorne za nastanak PCD-a, a smatra se da je taj broj mnogo veći s obzirom da 2500 proteina učestvuje u izgradnji i pravilnom funkcionisanju cilija. Velika raznolikost kliničke slike PCD pacijenata često dovodi do odloženog uspostavljanja precizne dijagnoze ove bolesti, a nije retkost i da pacijenti sa drugim bolestima pluća budu okarakterisani kao PCD pacijenti. Genetički profil ove bolesti kod pacijenata sa teritorije Srbije do sada nije utvrđen, pa je identifikacija gena uzročnika neophodna. Stoga je u okviru ove teze urađeno genomsko profilisanje pacijenata sa primarnom cilijarnom diskinezijom u cilju razvoja genetičkog algoritma koji bi, osim opisanih patogenih genetičkih varijanti i gena uzročnika, obuhvatio i novootkrivene varijante u kodirajućim regionima gena uzročnika PCD-a i gena kandidata za PCD. Sve ovo omogućava razvoj strategije za diferencijalnu dijagnozu PCD-a i drugih bolesti pluća suspektnih na ciliopatije koji se klinički manifestuju kao PCD. Takođe, pristupljeno je funkcionalnoj karakterizaciji novootkrivenih patogenih varijanti u već poznatim genima koji su odgovorni za razvoj PCD-a i/ili u genima koji do sada nisu asocirani sa PCD-om, a koji bi mogli da dovedu do karakterističnog fenotipa kod pacijenta. Jedna od glavnih odlika PCD pacijenata je strukturna i/ili funkcionalna promena na cilijama, pa su one nepokretne, slabo pokretne, promenjen im je obrazac kretanja ili odsustvuju...Primary ciliary dyskinesia (PCD) is a rare disease that is inherited in autosomal recessive manner or the inheritance is X-linked, and predominantly affects the functioning of the lungs, reproductive organs and the laterality of the internal organs. PCD is clinically and genetically very heterogeneous disorder that occurs immediately after birth, and is characterized by alterations in motor cilia due to pathogenic variants in genes encoding proteins that are necessary for the proper structure and function of these organelles. So far, there have been described 38 PCD-causative genes, and this number is considered to be much higher given that 2500 proteins participate in the formation and functioning of the cilia. Clinical heterogeneity of PCD patients often leads to the delayed establishment of a precise diagnosis of the disease, and it is not unusual that patients with other lung diseases are classified as PCD patients. The genetic background of the disease in patients with Serbian descent has not been established so far, so the mutational profile involved in the pathogenesis of PCD is necessary. Therefore, within this thesis, genomic profiling of patients with primary ciliary dyskinesia was performed in order to develop a genetic algorithm that, in addition to the described disease-causing pathogenic genetic variants and genes, would include novel variants in the coding regions of PCD-causative and candidate genes. This approach allows the establishment of a strategy for the differential diagnosis of PCD and other lung diseases suspected to ciliopathies that are clinically manifested as PCD. Functional characterization of a novel potentially pathogenic variant in PCD disease-causing genes and genes that have not been associated with PCD so far, but could be associated with the characteristic phenotype of PCD, was performed. One of the main features of PCD is ultrastructural defects of cilia leading to ciliary immotility, abnormal motility, or their absence..

Molecular basis of primary ciliary dyskinesia

Primarna cilijarna diskinezija (PCD) predstavlja redak genetički poremećaj motornih cilija koji se najčešće nasleđuje na autozomno recesivan način, a uočeno je i nasleđivanje vezano za hromozom X i autozomno dominantno nasleđivanje ovog oboljenja. Osnovne karakteristike PCD-a su abnormalnosti pokretnih cilija, koje su posledica patogenih varijanti u genima koji kodiraju za proteine koji su neophodni za pravilnu strukturu i funkciju ovih organela. PCD predominantno obuhvata respiratorni trakt i reproduktivne organe, a utiče i na lateralnost unutrašnjih organa. Klinička prezentacija nije specifična, već obuhvata simptome različitih respiratornih oboljenja, što često dovodi do odloženog uspostavljanja precizne dijagnoze, zbog čega je upotreba dijagnostičkih analiza i genetičkog profilisanja neophodna za utvrđivanje PCD-a. Metode sekvenciranja nove generacije omogućile su poslednjih godina detekciju velikog broja novih gena uzročnika i gena kandidata za PCD, pa je do sada opisano 45 gena uzročnika koje su odgovorni za nastanak PCD-a i 200 gena kandidata, a smatra se da je taj broj mnogo veći, s obzirom da 2500 proteina učestvuje u izgradnji i pravilnom funkcionisanju cilija. Nakon detekcije genetičkih varijanti u uzorcima pacijenata suspektnih na PCD, molekularna karakterizacija varijanti, upotrebom in silico i/ili eksperimentalnih metoda, omogućava determinaciju njihove patogenosti i uspostavljanje korelacije između genotipa i fenotipa pacijenata. Terapijski protokoli kojima se leče PCD pacijenti nisu specifični za ovo oboljenje već se ekstrapoliraju iz terapijskih protokola drugih plućnih oboljenja sa sličnom kliničkom prezentacijom bolesti. Stoga su istraživanja terapeutika na animalnim model sistemima od ključnog značaja za razvoj adekvatne terapije za PCD pacijente. ključne reči: Primarna cilijarna diskinezija (PCD), dijagnostički testovi, genomsko profilisanje PCD pacijenata, funkcionalna karakterizacija varijanti, model sistemi za PCD, terapija za PCD.Primary ciliary dyskinesia (PCD) is a rare genetic disorder that is most often inherited in an autosomal recessive manner, nevertheless X-linked and autosomal dominant inheritance of this disease have been reported. The main characteristics of PCD are abnormalities of motile cilia, which are a consequence of pathogenic variants in genes encoding proteins necessary for the proper structure and function of these organelles. PCD predominantly involves the respiratory tract and reproductive organs, and also affects the laterality of internal organs. The clinical presentation is not specific, and most often includes the symptoms of various respiratory diseases, which leads to the delayed establishment of an accurate diagnosis. Consequently, the use of diagnostic and genetic analyses is necessary for the determination of PCD. Next-generation sequencing methods have enabled the detection of a large number of novel PCD disease-causing and candidate genes in recent years, and so far, 45 PCD disease-causing and 200 candidate genes have been described, and this number is thought to be much higher since 2500 proteins participate in cilia formation. After detection of genetic variants in samples of patients suspected of PCD, molecular characterization of variants, using in silico and/or experimental methods, allows determination of their pathogenicity and establishment of genotype-phenotype correlation in patients. Therapeutic protocols for treating PCD patients are not specific for this disease rather, the therapy is extrapolated from other similar lung diseases. Therefore, research on therapeutics on animal model systems is crucial for the development of adequate therapy for PCD patients

Eradikacija Helicobacter pylori kod bolesnika bez gastričkih simptoma koji imaju rekurentni aftozni stomatitis - pilot studija

Background/Aim. Helicobacter (H.) pylori is a widespread bacterium and its involvement in pathogenesis of gastric diseases is well-known. However, H. pylori role in etiology of other histologically similar conditions, especially recurrent aphthous stomatitis (RAS) is still controversial. Research regarding H. pylori and its association with RAS, as well as the treatment options were always conducted on patients with diagnosed gastric problems. The aim of this study was to determine whether H. pylori is present in the oral cavity of patients suffering from RAS but without any symptoms or medical history of gastric disease. Methods. A total of 15 patients with RAS participated in the study. None of the participants suffered from any gastrointestinal disorders. Two dental plaque samples from each participant were collected. The first was analyzed using rapid urease test and the second one was put in transport medium and sent for cultivation. The sensitivity of H. pylori to antibiotics was established using disk diffusion method of sensitivity testing for every patient individually and adequate therapy was prescribed. Results. Before the treatment the mean annual recurrence rate of RAS was 8.1 ± 2.1, with the average number of lesions being 3.9 ± 1.9. During the 12-month observation period after the eradication therapy, none of the patients reported recurrence of aphthous lesions. The treatment was successful in all cases. Conclusion. This study shows that RAS can be effectively treated by successful eradication of oral H. pylori, and that RAS could be possibly considered as an early warning sign of potential gastric infection by H. pilory.Uvod/Cilj. Helicobacter (H.) pylori je široko rasprostanjena bakterija i njen uticaj na nastanak gastričkih oboljenja vrlo dobro je dokumentovan. Međutim, uloga H. pylori u patogenezi histološki sličnih oboljenja, posebno rekurentnog aftoznog stomatitisa (RAS), nije dovoljno istražena. Dosadašnje studije, u kojima je ispitivana veza između H. pylori i RAS, kao i moguće terapijske opcije, bile su usmerene ka bolesnicima sa prethodno dijagnostikovanim gastričkim smetnjama. Cilj ovog istraživanja bio je da se utvrdi da li je H. pylori prisutan u usnoj duplji i kod bolesnika bez simptoma i istorije gastričkih oboljenja koji pate od RAS. Metode. U studiji je učestvovalo 15 bolesnika koji pate od RAS. Bolesnici nisu imali smetnje vezane za gornji deo digestivnog trakta. Po dva uzorka dentalnog plaka prikupljena su od svakog bolesnika. Jedan plak je ispitivan uz pomoć brzog ureaza testa, dok je drugi stavljen u transportni medijum i poslat na kultivaciju. Osetljivost H. pylori na antibiotike određivana je uz pomoć antibiograma za svakog bolesnika posebno i, u skladu sa rezultatima, prepisivana je odgovarajuća terapija. Rezultati. Pre lečenja prosečan broj epizoda RAS tokom godine iznosio je 8,1 ± 2,1, sa prosečno 3,9 ± 1,9 aftoznih lezija. Tokom 12-mesečnog perioda nakon eradikacione terapije, ni kod jednog bolesnika nije došlo do ponovne pojave afti. Terapija je bila uspešna kod svih bolesnika. Zaključak. Rezultati ovog istraživanja pokazuju da se RAS može uspešno lečiti eradikacijom H. pylori i da se sama pojava RAS može posmatrati kao rano upozorenje na moguću gastričku infekciju

Hybrid dysgenesis in two natural populations of Drosophila melanogaster

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Antigenotoxic effects of royal jelly in the sex linked recessive lethal test with Drosophila melanogaster

The antitoxic and antimutagenic effect of royal jelly was assayed by a standard testing procedure on Drosophila melanogaster. The similarity of metabolic pathways between Drosophila and mammals makes the test widely applicable for detecting the impact of various potential promutagens and antimutagenic effects could be recognized accordingly. The flies were treated with the potent mutagen MMS, alone and combined with royal jelly. The frequency of sterile males and sex linked recessive lethal mutations increased significantly after MMS treatment and decreased after combined treatment. The results strongly indicate that, in addition to its well documented action on development, life-span and reproductive ability, royal jelly has an antimutagenic potential as well.Antigenotoksični i antimutageni efekat pčelinjeg mleča ispitivan je korišćenjem standardne procedure testiranja pomoću Drosophilae melanogaster. Sličnost metaboličkih puteva između Drosophila i sisara čini ovaj test široko primenljivim u otkrivanju uticaja različitih potencijalnih mutagena i promutagena, a analogno tome se može ispitivati i mogući antimutageni potencijal substanci. Mušice odgovarajućeg genotipa tretirane su jakim mutagenom, metil-metan-sulfonatom (MMS), samostalno i u kombinaciji sa mlečom. Učestalost sterilnih mužjaka i polno vezanih recesivnih letalnih mutacija rasla je značajno nakon tretmana sa MMS i opadala nakon kombinovanog tretmana. Dobijeni rezultati značajno ukazuju da uz već potvrđeni uticaj na razviće, dužinu života i reproduktivnu sposobnost, mleč ima i antimutageni potencijal.Projekat ministarstva br. 152

Inversion polymorphism in populations of Drosophila subobscura from urban and non-urban environments

Populations of Drosophila subobscura from the urban area of Belgrade and from the locality, Deliblato, which is not under strong anthropogenic influence, were studied with the aim to characterize and compare their genetic structure by examining chromosomal inversion polymorphism. Additional analysis and comparison of this type of polymorphism with several other populations from different habitats in the central Balkans, was done. The obtained results indicate higher heterozygosity in the population from Belgrade. Despite being ecologically marginal and under strong and complex influences, this population did not show a decline in the number of inversions and it is not highly differentiated compared to the referent populations.

Adaptive significance of amylase polymorphism in drosophila, XV: Examination of genotype-by-environment interactions on the viability, developmental time and stability of drosophila subobscura homozygous for Amy during exposure to nutritional changes

Due to the direct interaction between enzyme and substrate, the amylase system can provide valuable information on the relationship between homozygosity and developmental homeostasis under a changing environment in several Drosophila species, The adaptive significance of the relationship between genetic variability and environmental change manifests through the well-known polymorphism of the amylase locus (Amy). We examined the effect of gradual and abrupt changes in starch concentration in the nutritional substrate, on the developmental time, egg-to-adult viability and phenotypic plasticity in the progeny of Drosophila subobscura that was homozygous for 'fast' (AmyF/AmyF) and 'slow' (AmyS/AmyS) Amy alleles. Our findings show that gradual and abrupt nutritional changes exert a significant effect on developmental time and viability. A high heterogeneity among genotypes in fluctuating asymmetry (FA) and no direct association between FA and fitness components under the two experimental regimes of environmental change were observed.Projekat ministarstva br. 17301

The effect of lead on the developmental stability of Drosophila subobscura through selection in laboratory conditions

Fluctuating asymmetry (FA), the increased variation of bilateral symmetry in a sample of individuals, can indicate disturbance in developmental stability caused by environmental and/or genomic stress. This developmental instability was analyzed in Drosophila subobscura maintained for seven generations on two different concentrations of lead in laboratory conditions. The FA4 index showed that the genotypes reared on the higher lead concentration were in developmental homeostasis, except for males in the F7 generation, for both wing size parameters. The results show that different degrees of lead pollution cause different responses to selection of the exposed population in laboratory conditions

Variability of fluctuating asymmetry in ovariole number of Drosophila subobscura caused by microclimatic difference

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Does inbreeding affects developmental stability in Drosophila subobscura populations?

In the present paper, we focused on the coadaptive aspect of genetic variability at population level and its relation to genomic stress such as inbreeding. The paper evaluates the effects of an experimental reduction of average heterozygosity after fourteen generations of systematic inbreeding in laboratory conditions, on developmental stability in Drosophila subobscura populations from two ecologically and topologically distinct habitats, knowing that they possess a certain degree of genetic differences due to their different evolutionary histories. The aims were to analyze: (i) the variability change of wing size (length and width) among the inbred lines from both populations; (ii) the relations between homozigosity and level of fluctuating asymmetry as a potential measure of developmental instability, in inbred lines originating from two populations. Results for the wing size showed similar between line variability pattern across generations of systematic inbreeding in both populations. The obtained results suggest that variability of fluctuating asymmetry as a measure of developmental instability can not be related to homozygosity due to inbreeding per se, in both experimental populations.Rad je fokusiran na ko adaptivni aspekt genetičke varijabilnosti na nivou populacije i u odnosu na genomski stres kao što je inbriding. Analizirani su efekti eksperimentalnog smanjenja prosečne heterozigotnosti nakon 14 generacija sistematskog inbridinga u laboratorijskim uslovima na razvojnu stabilnost Drosophila subobscura populacija sa dva ekološki i topološki odvojena staništa, znajući da one poseduju odredjeni stepen genetičke diferencijacije usled različitih evolutivnih istorija. Ciljevi rada su bili da se analizira: (i) varijabilnost u promeni veličine krila (dužine i širine) medju inbidingovanim linijama i populacijama; (ii) odnosi izmedju homozigotizacije i nivoua fluktuirajuće asimetrije kao potencijalne mere razvojne nestabilnosti u inbridingovanim linijama obe populacije. Rezultati veličine krila pokazuju sličnu varijabilnost medju linijama obe populacije kroz generacije inbridinga. Dobijeni rezultati sugerišu da varijabilnost fluktuirajuće asimetrije kao mere razvojne nestabilnosti ne mogu biti povezani sa homozigotizacijom usled inbridinga per se, u obe eksperimentalne populacije.Projekat ministarstva br. 17301