GENE EXPRESSION SIGNATURES IN PRENATAL DIAGNOSIS OF ANEUPLOIDIES

Kromosomske bolezni predstavljajo pomemben zdravstveni problem, saj so v predrojstnem obdobju pogosto povezane z razvojnimi nepravilnostmi ali s spontano izgubo nosečnosti. Številčne kromosomske nepravilnosti avtosomov niso združljive z življenjem, razen trisomije kromosoma 21, 18 in 13 (T21, T18, T13). Multiomski pristop omogoča sestavljeno raziskovanje genoma in transkriptoma ter posledično prepoznavanje biooznačevalcev za različna patološka stanja, pri čemer nam kromosomski sindromi z dobro opredeljeno klinično sliko lahko služijo kot model patološkega stanja. Transkriptomske raziskave predrojstnih vzorcev s T21, T18 in T13 so pokazale spremembe v genskem izražanju, niso pa raziskovale diagnostičnih možnosti za napoved nosečnosti z visokimi tveganji za trisomijo, niti niso ugotavljale skupnih značilnosti v genskem izražanju pri trisomijah. Postavili smo hipotezo, da obstajajo razlike v genskem izražanju, ki jih lahko uporabimo kot diagnostični biooznačevalec za specifično trisomijo in da je del razlik v genskem izražanju skupen pri različnih trisomijah, kar lahko obravnavamo kot homeostatski odgovor. Opravili smo ekspresijske mikromrežne ekperimente na predrojstnih vzorcih gojenih amniocitov s T21 in T18 ter gojenih celic horionskih resic s T13, ki smo jih primerjali z ujemajočimi vzorci z normalnim kariotipom. V prvem delu smo opravili diferencialno gensko ekspresijo (DGE) za vse trisomije in prepoznali podpise izražanja genov, ki so bili najbolj statistično značilni za T21, 18 in T13. Napovedno zanesljivost izbranih podpisov genskega izražanja smo preverili na dostopnih objavljenih transkriptomskih raziskavah in potrdili, da izbrani biooznačevalci z visoko zanesljivostjo napovedo status trisomije 21, 18 in 13. V drugem delu raziskave smo primerjali podobnosti v DGE med vzorci s T21 in T18 ter ugotovili 6 genov s spremenjenim izražanjem, ki so skupni pri obeh trisomijah. Analiza obogatitve genskih setov in bioloških procesov sta pokazali obogatitev genov povezanih s PI3K/AKT signalno potjo, G2/M kontrolno točko pri DNA okvari in predvideno inhibicijo regulatorja TP53 ter obogatitev genov povezanih s celičnim ciklom, celično smrtjo in preživetjem, preživetjem organizma ter razvojem in delovanjem tkiv. Z raziskavo smo ugotovili specifične podpise izražanja genov, ki so za preiskovane trisomije značilni in jih lahko uporabimo kot bioznačevalec. Zaznali smo tudi določene skupne podobnosti v genskem izražanju pri T21 in T18, za katere menimo, da lahko odražajo genomski homeostatski odgovor na kromosomsko nepravilnost. Naši rezultati predstavljajo temelj za nadaljnje raziskave biooznačevalcev za nosečnosti z visokimi tveganji in za razumevanje mehanizmov s katerimi se trisomična celica upira kromosomski motnji.Chromosome diseases, associated with developmental abnormalities, represent an important health problem and often lead to pregnancy loss. Numerical autosome abnormalities are incompatible with life, with the exception of trisomy of chromosomes 21, 18 and 13 (T21, T18 and T13). A multiomics approach allows for combined genome and transcriptome analysis and the identification of biomarkers for various pathological conditions, where chromosome syndromes with a well-defined clinical presentation can serve as a model of the pathological condition. Reported transcriptomic studies on prenatal samples with T21, T18 and T13 showed changes in gene expression, but they did not investigate the diagnostic potential for predicting high-risk pregnancies, nor did they identify the common features in gene expression among trisomies. We hypothesized that specific gene expression signature can be used as a diagnostic biomarker for certain trisomy and that there are some similarities in gene expression that are common in different trisomies and can be considered as a homeostatic genome response. We performed gene-expression experiments on prenatal samples of cultivated amniocytes with T21 and T18, and cultivated cells of chorionic villi with T13 in comparison to matched prenatal samples with normal karyotype. In the first part of the study, we carried out differential gene expression (DGE) analyses and selected gene expression signatures as biomarkers specific for T21, 18 and T13. The classification performance of the selected gene subsets were verified on independent GEO datasets and confirmed that the selected biomarkers predict the T21, 18 or T13 with high reliability. In the second part of the study, we compared the similarities in DGE for T21 and T18 and found 6 same-directionally dysregulated genes that are common in both data sets. The gene set enrichment and pathway analyses showed enrichment of genes associated with the PI3K / AKT signal pathway, the G2 / M check point in DNA defects and the predicted inhibition of the TP53 regulator and the enrichment of gene-related cell cycle, cell death and survival, the survival of the organism, and the development and function of tissues. Our study found gene expression signature specific for the individual trisomy that can be used as a biomarker. In addition, we detected some similar differences in gene expression, that are common in T21 and T18, which may reflect the genomic homeostatic response to the chromosome change. Our results provide a basis for further investigations of biomarkers in high risk pregnancies and understanding of the mechanisms of trisomic cell resisting chromosomal perturbation

Genomic Testing for Prenatal Clinical Evaluation of Congenital Anomalies

Congenital anomalies occur in about 2–3% of liveborn and 20% of stillborn infants. They constitute a serious public health and epidemiological problem. The etiology of congenital anomalies is complex; they can result from genetic factors, environmental factors, or a combination of both. It is estimated that genetic factors represent an important cause of congenital anomalies and may be due to different genetic mechanisms: aneuploidies, deletions and duplications of DNA segments, and single gene disorders. Due to the genetic complexity, the targeted prenatal genetic diagnostics of congenital anomalies is usually problematic and challenging. In recent years new diagnostic algorithms for prenatal genetic testing are being developed with the advent of new genomic technologies, like molecular karyotyping and next-generation sequencing. These technologies offer testing options that exceed conventional karyotyping and targeted molecular genetic testing with better diagnostic yield. In this chapter, an overview of the conventional genetic diagnostic approach and the use of new genomic technologies in the diagnostic algorithm of prenatally detected congenital anomalies are discussed

Sadašnjost i budućnost prenatalne dijagnostike u Sloveniji

Prenatal genetic testing is under the remit of the National Health Service in Slovenia and has been included in clinical routine since the 1980s. Traditionally, prenatal services have consisted of karyotyping and rapid fetal aneuploidy screening to detect chromosome abnormalities, whereas targeted mutation testing was used for single gene disorders. Development of array comparative genomic hybridization and next generation sequencing allows for genome analysis at better resolution in a single experiment. While technological advances in medicine continue to evolve, increasing diagnostic accuracy and broadening the spectrum of indications, all these innovations require more investment along with more equipment and higher staffi ng rations trained to use it, placing burden upon healthcare funding and expenditure. This prompts us to consider how to implement new techniques into the existing services in order to update genetic services for the 21st century. Our aim is to develop a new approach to prenatal genetic services, which would maximize diagnostic yield at an acceptable cost.Prenatalno genetičko testiranje u nadležnosti je Državne zdravstvene djelatnosti u Sloveniji i uključeno je u kliničku praksu od 1980.-ih godina. Prenatalne usluge tradicionalno obuhvaćaju kariotipiziranje i brz probir na fetalne aneuploidije kako bi se otkrile kromosomne anomalije, dok se za poremećaje jednog gena provodilo ciljano testiranje na mutacije . Razvoj komparativne genomske hibridizacije na mikropostroju i sekvenciranje sljedeće generacije omogućava analizu genoma uz bolju rezoluciju u jednom testu. Dok se tehnološki napredak u medicini nastavlja poboljšavajući tako dijagnostičku točnost i šireći lepezu indikacija, ove inovacije zahtijevaju sve veća ulaganja i sve više opreme te dodatno osposobljeno osoblje koje će raditi s tom opremom, što opterećuje zdravstvene fondove i povećava troškove. To nas potiče da razmotrimo kako uklopiti nove tehnike u postojeću službu kako bismo genetičke usluge prilagodili potrebama 21. stoljeća. Cilj nam je razviti nov pristup prenatalnoj genetici kojim će se postići najučinkovitiji rezultati uz prihvatljive troškove

A critical update on endothelial nitric oxide synthase gene variations in women with idiopathic recurrent spontaneous abortion: genetic association study, systematic review and meta-analyse

A number of case–control studies investigated the association between idiopathic recurrent spontaneous abortion (IRSA) and variations in the gene encoding endothelial nitric oxide synthase (NOS3), but yielded contradictory results. Our aim was to test the association of the NOS3 variable number of tandem repeats (VNTR) in intron 4 and +894 G/T single-nucleotide polymorphism (SNP) with IRSA in Slovenian women (148 IRSA and 149 control women), conduct a systematic review of literature on the association between NOS3 gene variations and IRSA, and perform meta-analyses of studies that met the inclusion criteria, defined by virtue of the European Society for Human Reproduction and Embryology evidence-based guidelines for recurrent spontaneous abortion. Genotyping was performed using PCR and restriction fragment length polymorphism methods. The systematic review of literature (English language) was conducted using PubMed and Scopus databases, to 1 November 2014. We determined no association of IRSA with the VNTR in intron 4 and +894 G/T SNP in Slovenian women. Furthermore, 16 case–control studies were identified on the association between 15 NOS3 gene variations and IRSA. However, significant inconsistencies exist in the selection criteria of patients and controls between studies. The meta-analysis of VNTR in intron 4 was performed on five studies (894 patients, 944 controls), whereas the meta-analysis of +894 G/T SNP included six studies (1111 patients, 1121 controls). The association with IRSA was significant for the +894 G/T SNP under the dominant genetic model (GT+TT versus GG) based on fixed (odds ratio (OR) = 1.54, 95% confidence interval (CI) = 1.28–1.86, P = <0.01) and random effects models (OR = 1.54, 95% CI = 1.03–2.31, P = 0.03). In conclusion, the GT and TT genotypes of the +894 G/T SNP in women might contribute to a predisposition to IRSA. Additional genetic association and functional studies in different populations with larger numbers of participants and a uniformly defined IRSA are needed to clarify the contribution of NOS3 +894 G/T gene variation to IRSA

Insertion/deletion polymorphism in intron 16 of ACE gene in idiopathic recurrent spontaneous abortion: case-control study, systematic review and meta-analysis

Abstract The insertion/deletion (I/D) polymorphism in intron 16 of the angiotensin I-converting enzyme gene ( ACE ) has been ex- tensively studied as a predisposing factor for idiopathic recurrent spontaneous abortion (IRSA). A case-control study including 149 women with ≥ 3 spontaneous abortions and 149 controls was performed to test the association of ACE I/D polymorphism with IRSA. A systematic review was conducted of previous case-control studies, with strict selection criteria for meta-analyses. We also aimed to evaluate the potential differences in summary estimates between studies defining IRSA as ≥ 2 and ≥ 3 spontaneous abortions. Genotyping was performed by PCR, and systematic review conducted using PubMed and Scopus. There was no association of the polymorphism with IRSA in Slovenian women. Sixteen case-control studies, showing substantial differences regarding IRSA definition and selection criteria for women were identified. Meta-analysis was performed and included four studies defining IRSA as ≥ 2 spontaneous abor- tions and the current study, which defined IRSA as ≥ 3 spontaneous abortions. Based on random effects model, meta-analysis con- ducted on 1192 patients and 736 controls showed no association with IRSA under dominant (DD + IDvsII) and recessive (DDvsID + II) genetic models. Well-designed studies are needed to evaluate the role of ACE I/D polymorphism in IRSA defined as ≥ 3 spontaneous abortion

Y chromosome azoospermia factor region microdeletions are not associated with idiopathic recurrent spontaneous abortion in a Slovenian population: association study and literature review

Objective To investigate the potential association of Y chromosome microdeletions with idiopathic recurrent spontaneous abortion (IRSA) in a Slovenian population and compare our results with those of previously published studies in different populations, with the intention of clarifying the potential impact of Y chromosome microdeletions on IRSA. Design Case–control and association study. Setting Departments of gynecology and obstetrics and university-based research laboratory. Patient(s) Male partners of 148 couples with at least three spontaneous pregnancy losses of unknown etiology, and 148 fertile men. Intervention(s) Multiplex polymerase chain reactions. Main Outcome Measure(s) Azoospermia factor (AZF) regions were tested for Y chromosome microdeletions according to European Academy of Andrology/European Molecular Genetics Quality Network guidelines. The PubMed database was searched to retrieve articles linking Y chromosome microdeletions and susceptibility to IRSA. Result(s) None of the IRSA or control men had microdeletions in the AZFa, AZFb, or AZFc regions. A total of nine previous studies examined the relationship between Y chromosome microdeletions and IRSA, yielding contradictory results, which we discuss in detail. Conclusion(s) On the basis of our comparisons, it is unlikely that Y chromosome microdeletions contribute to IRSA and are therefore currently not recommended for the routine evaluation of IRSA couples

Leberjeva hereditarna optična nevropatija – pregled bolezni z analizo prisotnosti v Sloveniji

Leberjeva hereditarna optična nevropatija (LHON) je redka dedna mitohondrijska bolezen, ki povzroča slepoto najpogosteje pri mladih odraslih. Navadno se izrazi kot subakutna, neboleča izguba vida na eno oko, ki ji sledi poslabšanje vida drugega očesa v nekaj tednih do mesecih. Bolezen večinoma pušča trajne posledice, le pri nekaterih bolnikih lahko v redkih primerih pride do delnega spontanega izboljšanja vida. Razmerje med moškimi in ženskimi bolniki se ocenjuje na 3 : 1. V zadnjih letih je z razvojem zdravilne učinkovine idebenone možno podporno farmakološko zdravljenje, ki lahko prispeva k delnemu izboljšanju vidne funkcije. Bolezen zaradi svoje redkosti velikokrat ostane ne- ali napačno diagnosticirana. V članku predstavljamo štiri klinične primere bolnikov, pri katerih se je po obsežnem in dolgotrajnem diagnosticiranju izkazalo, da imajo LHON. Od leta 1996 se v Sloveniji vodi baza bolnikov z redkimi dednimi očesnimi boleznimi. Na tej podlagi je ocenjena prevalenca LHON 1/72.000. Ob sumu na bolezen so ključni družinska anamneza slabovidnosti po materini strani, genetsko testiranje in napotitev na obravnavo ter zdravljenje v terciarno ustanovo