Impact of variants in coding and noncoding regions of disease-causing and modifier genes on phenotype of patients with hyperphenylalaninemia

Hiperfenilalaninemija (HPA) predstavlja najčešći nasledni poremećaj metabolizma aminokiselina (učestalost 1:10 000) koji se karakteriše povišenim nivoom fenilalanina u krvi. Višak fenilalanina ima toksičan efekat na razvoj mozga, i ukoliko se pravovremeno ne uvede odgovarajuća terapija, dolazi do teške i ireverzibilne mentalne retardacije pacijenta. U oko 98% slučajeva, HPA je uzrokovana mutacijama u genu za fenilalanin hidroksilazu (PAH) i tada je poremećaj poznat kao fenilketonurija (PKU). U 1-2% slučajeva HPA je posledica deficijencije esencijalnog kofaktora enzima PAH, tetrahidrobiopterina (BH4), koja nastaje usled mutacija u genima odgovornim za biosintezu (gen za piruvoil tetrahidropterin sintazu – PTS i gen za GTP ciklohidrolazu – GCH1) ili metabolizam BH4 (gen za dihidropteridin reduktazu – QDPR i gen za pterin-4a-karbinolamin dehidratazu, PCBD). S obzirom na ozbiljne i ireverzibilne posledice koje HPA izaziva, od presudne važnosti je precizno postavljanje dijagnoze i što ranija primena odgovarajuće terapije. U Srbiji je molekularno genetička karakterizacija gena PAH uvedena 2004. godine, međutim, neophodno je uspostaviti dodatne metode radi sveobuhvatne identifikacije i funkcionalne karakterizacije varijanti identifikovanih u različitim genima uzročnicima HPA. Ovakav pristup imao bi značajnu primenu za diferencijalnu dijagnostiku pacijenata sa HPA u Srbiji i pravovremeno uvođenje odgovarajuće terapije specifične za genotip pacijenta. U slučaju fenilketonurije, iako mutirani genotip PAH predstavlja glavnu odrednicu PKU fenotipa, primećeno je da nije uvek moguće predvideti fenotip samo na osnovu genotipa. Smatra se da genetički faktori koji doprinose razvoju finalnog PKU fenotipa (posebno kognitivnog PKU fenotipa) još uvek nisu dovoljno istraženi. Stoga bi identifikacija novih intra-genskih (npr., novih elementa uključenih u regulaciju transktipcije) modulatora PKU fenotipa, kao i novih gena modifikatora doprinela njegovom boljem razumevanju..

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

Covid-19 disease severity associated with vitamin d related genetic Variants

COVID-19 pandemic has proved to be an unrelenting health threat for more than a year now. The emerging amount of data indicates that vitamin D could be important for clinical presentation of COVID-19. Here, we investigated association of genetic variants related to the altered level and bioavailability of vitamin D with clinical severity of COVID-19. We analyzed variants in genes significant for the status of vitamin D (DHCR7/NADSYN1 rs12785878, GC rs2282679, and CYP2R1 rs10741657), and vitamin D effect (VDR rs2228570) in 120 Serbian adult and pediatric COVID-19 patients using allelic discrimination. Furthermore, we carried out comparative population genetic analysis among European and other worldwide populations to investigate variation in allelic frequencies of selected variants. The results showed that DHCR7/NADSYN rs12785878 and CYP2R1 rs10741657 variants were associated with severe COVID-19 in adults (p = 0.03, p = 0.017, respectively); carriers of DHCR7/NADSYN TG+GG and CYP2R1 GG genotypes had 0.21 and 5.9 the odds for developing severe disease, OR 0.21 (0.05–0.9) and OR 5.9 (1.4–25.2), respectively. There were no associations between selected genetic variants and disease severity in pediatric patients. Comparative population genetic analysis revealed that Serbian population had the lowest frequency of CYP2R1 rs10741657 G allele compared to other non-Finish Europeans (0.58 compared to 0.69 and 0.66 in Spanish and Italian population, respectively), suggesting that other populations should also investigate the relationship of CYP2R1 variant and the COVID-19 disease course. The results of the study indicated that vitamin D related genetic variants were implicated in severe COVID-19 in adults. This could direct prevention strategies based on population specific nutrigenetic profiles.Book of abstracts: International Conference of Biochemists and Molecular Biologists in Bosnia and Herzegovina - ABMBBIH May, 202

Ретке болести у ери геномике

Tokom poslednjih decenija, istraživanja na polju retkih bolesti napreduju ogromnom brzinom usled sve veće upotrebe sekvenciranja nove generacije (engl. Next-generation sequencing, NGS). Sekvenciranje kompletnog ljuskog genoma (engl. Whole genome sequencing, WGS) osoba koje boluju od retkih bolesti je postalo lako dostupno. Pored pronalaženja novih varijanti i novih gena koji leže u osnovi retkih bolesti, genomika je omogućila i otkriće gena modifikatora koji mogu da objasne uočene nedoslednosti u korelaciji genotipa i fenotipa. Fenilketonurija je urođena metabolička retka bolest koja je uzrokovana varijantama u genu za fenilalanin hidroksilazu (PAH). U ovoj studiji, sproveli smo sekvenciranje kompletnog genoma 4 osobe iz nepovezanih nesrodnih porodica koje su imale patogene varijante u PAH genu, ali nisu razvile fenilketonuriju uprkos tome što nisu bile lečene. Otkrili smo dve nove varijante, p.Pro1591Ala u SHANK1 i p.Asp18Asn u SHANK2 genima, kao i prethodno opisane SHANK2:p.Gly46Ser, SHANK2:p.Pro1388_Phe1389insLeuPro i SHANK3:p.Pro1716Thr varijante. Računarske predikcije su pokazale da identifikovane varijante ne ukidaju funkciju SHANK proteina. Međutim, promene u posttranslacionim modifikacijama SHANK proteina mogu uticati na funkcionisanje glutamatergičnih sinapsi, regulaciju citoskeleta i doprineti održavanju optimalne sinaptičke gustine i broja dendritskih bodlji. Naši rezultati po prvi put povezuju porodicu SHANK gena i osobine neuroloških promena kod osoba sa fenilketonurijom.Током последњих деценија, истраживања на пољу ретких болести напредују огромном брзином услед све веће употребе секвенцирања нове генерације (енгл. Next-generation sequencing, NGS). Секвенцирање комплетног љуског генома (енгл. Whole genome sequencing, WGS) особа које болују од ретких болести је постало лако доступно. Поред проналажења нових варијанти и нових гена који леже у основи ретких болести, геномика је омогућила и откриће гена модификатора који могу да објасне уочене недоследности у корелацији генотипа и фенотипа. Фенилкетонурија је урођена метаболичка ретка болест која је узрокована варијантама у гену за фенилаланин хидроксилазу (PAH). У овој студији, спровели смо секвенцирање комплетног генома 4 особе из неповезаних несродних породица које су имале патогене варијанте у PAH гену, али нису развиле фенилкетонурију упркос томе што нису биле лечене. Открили смо две нове варијанте, p.Pro1591Ala у SHANK1 и p.Asp18Asn у SHANK2 генима, као и претходно описане SHANK2:p.Gly46Ser, SHANK2:p.Pro1388_Phe1389insLeuPro и SHANK3:p.Pro1716Thr варијанте. Рачунарске предикције су показале да идентификоване варијанте не укидају функцију SHANK протеина. Међутим, промене у посттранслационим модификацијама SHANK протеина могу утицати на функционисање глутаматергичних синапси, регулацију цитоскелета и допринети одржавању оптималне синаптичке густине и броја дендритских бодљи. Наши резултати по први пут повезују породицу SHANK гена и особине неуролошких промена код особа са фенилкетонуријом.Knjiga sažetaka: Treći Kongres biologa Srbije, Zlatibor, Srbija 21 - 25. 9. 2022

Functional characterization of novel variants in the dnai1 gene in a patient with primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a rare motor ciliopathy, which predominantly affects the lungs and reproductive organs. PCD has a heterogeneous genetic basis, and it is necessary to analyze more than 40 causative genes in order to establish a precise diagnosis, which is essential for optimal treatment and adequate genetic counseling. Five patients suspected of PCD were analyzed using next-generation sequencing (NGS). The pathogenicity of the genetic variants was tested by in silico, qRT-PCR and Western blot methods. Two newly discovered variants p.N450Lfs*6 and p.D562N in the DNAI1 gene were detected in one patient suspected of PCD. The results of in silico prediction showed that the p.N450Lfs*6 variant affects the structure of the 3D model of the protein, abolishes ligand binding sites and post-translational modifications, thereby disrupting protein-protein interactions (PPI). The p.D562N variant has no effect on the 3D structure of the protein, but affects the ligand binding site and is located in the WD-40 domain, which most likely disrupts PPI. The results of the qRT-PCR method showed a decreased expression level of DNAI1 mRNA by about 50% in the patient compared to the control group, while Western blot analysis showed the presence of two protein products (699 ak and 455 ak). By analyzing the obtained results, it was concluded that the changes p.N450Lfs*6 and p.D562N affect the length and quantity of the DNAI1 protein, leading to the loss of protein function and are responsible for the occurrence of primary ciliary dyskinesia in the analyzed patient

Molecular basis of thalassemia syndromes in Serbia: an update

Introduction: Thalassemia syndromes are heterogeneous group of hereditary anemias characterized by defects in the synthesis of hemoglobin (Hb) polypeptide chains. These disorders comprise thalassemias and thalassemic hemoglobin variants which are predominantly caused by mutationsin a- and b-globin genes (HBA and HBB genes). Clinical manifestations of thalassemia syndromes range from asymptomatic thalassemia minor to severe anemia in thalassemia major cases. The aim of thisstudy was to update our previous findings on frequency of thalassemia mutations which result from a 13-year-old systematic survey in Serbia. Methods: Two hundred and fourteen patients from 149 unrelated families presented with hematological parameters indicative of thalassemia syndromes were studied. Detection of α- and β-globin gene mutations was performed using PCR and direct sequencing. Results: Two Hb variants and twelve different β-thalassemia mutations, including two mutations previously not reported in Serbian population, were detected. Hb variant Lepore Boston-Washington wasthe most common cause of thalassemia, with frequency of 24.3%, followed by HBB:c.316-106C>G mutation detected in 18.1% of families. The third most frequent cause of β-thalassemia were HBB:c.118C>T and HBB:c.93-21G>A mutations with 16.6% incidence each. Together, these four variants account for over 75% of all mutated β-globin alleles. In addition, five families affected with α-thalassemia were detected. Conclusion: Despite the increase in cohort size by 50% between this and our previous studies, the frequency of mutations affecting HBB gene remained unchanged. Results presented in this study will update Serbian national mutation database and contribute to better understanding of geographic history of South European and Balkan populations

The role of MIR-34 family members on the mucociliary process in the cellular respiratory model system

Introduction: Primary ciliary dyskinesia is a rare and heterogeneous disorder primarily affecting the respiratory organs, with impaired mucociliary clearance being a common characteristic. Recently, the importance of the miR34/449 family in ciliogenesisin animal models has been described. Thisstudy aimed to establish a modelsystem to study respiratory diseases and assessfor the first time the role of the miR34 family on the mucociliary process in humans. Methods: We cultured the primary Normal Human Bronchial Epithelial (NHBE) cells in the air-liquid interface system, enabling the differentiation of multiciliated cells(MCCs) and goblet cells(GCs). During the differentiation process, transient overexpression of miR-34a/b/c members was conducted. The model system and treatments were validated through confocal microscopy (β-tubulin, MUC5B, MUC5AC antibodies) and qRT-PCR of miRNAs,specifically ciliogenesis markers(NOTCH1, MCIDAS, GEMC1, CCNO, RFX3), and differentiated cell markers (FOXJ1 and TFF3). Results: Expression levels of ciliogenesis and differentiated cells markers and detection of cilia and mucins at confocal microscopy confirmed the successful establishment of cellular modelsystem. During the initial differentiation stage, an overexpression of miR34a/b/c changed the expression profile of ciliogenesis and differentiated cell markers. Conclusion: The established model system provides a valuable platform for exploring innovative treatment approaches for lung diseases. These findings suggest that overexpression of miR34a/b/c has impact on mucociliary process by reducing the duration required for the process of ciliogenesis. Furthermore, the expression levels of differentiated cell markerssuggest increased number of MCCs and decreased number of GCs, indicating the role of miR34a/b/c in enhancing mucociliary clearance

High-risk population screening for fabry disease in patients with chronic renal failure of unknown etiology

Fabry disease (FD) is a rare X-linked disorder caused by variants in the GLA gene leading to the deficiency of lysosomal α-galactosidase-A and progressive accumulation of globotriaosylceramide affecting the heart, nervous system, and kidneys. FD has overlapping phenotypes and often remains undiagnosed. Therefore, a precise molecular-genetic diagnosis and the earliest possible treatment are essential to avoid significant disease progression. The study aimed to determine the strategy for establishing routine molecular genetic diagnostics of FD in Serbia to provide an early application of appropriate therapy and genetic advice to families with a high risk for the birth of a child with FD. We analyzed 95 (34 female and 61 male) hemodialysis patients with clinical suspicion of FD using Sanger sequencing of all coding exons (7) and flanking intron regions of the GLA gene and measured the relative expression of the GLA gene in available samples. The genetic analysis revealed 3 patients with a missense variant (p.Asp313Tyr), and 10 patients with combinations of non-coding variants, described as complex intronic haplotypes (CIHs). CIH1 (c.-10C>T, c.370-81_370-77delCAGCC, c.640-16A>G, c.1000-22C>T), the most frequent haplotype, was detected in 7 (7.4%) patients. Lyso-Gb3 biomarker levels were within the normal range in each tested patient. However, RT-qPCR analysis revealed decreased relative expression of the GLA gene in PBMC of 2 female patients with CIH1 and one female patient carrying only c.-10C>T variant by 9,1%, 7,4%, 46,3%, respectively, pointing out that further analyses are needed to confirm/exclude FD in these patients. Because the effects of CIHs are not yet fully understood, our work highlights the importance of analyzing intronic regions of the GLA gene as genetic modifiers and the need to include expression analysis in the diagnostic algorithm.Book of abstracts: International Conference of Biochemists and Molecular Biologists in Bosnia and Herzegovina - ABMBBIH May, 202