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

Molecular karyotyping – a new approach in clinical and laboratory genetics

Metoda molekularne kariotipizacije omogućava analizu genomskih mutacija s visokom rezolucijom na razini cijelog humanog genoma. Manje genomske mutacije (mikrodelecije, mikroduplikacije) koje nije moguće otkriti klasičnom kariotipizacijom prisutne su u čak 15 % pacijenata s mogućom dijagnozom genetičkog poremećaja. Molekularna kariotipizacija je u određenim kliničkim slučajevima već u potpunosti zamijenila klasičnu kariotipizaciju i primjenjuje se u prenatalnoj i postnatalnoj genetičkoj dijagnostici.Molecular karyotyping enables the analysis of the entire human genome on high resolution scale. Smaller genomic mutations (microdeletions, microduplicaitons) that are not detectable using conventional G-banded karyotyping, are present in about 15 % of patients with suspected genetic disease. In certain clinical settings molecular karyotyping has already replaced classical karyotype analysis and is used in postnatal as well as in prenatal genetic diagnosis

Combination of QF‐PCR and aCGH is an efficient diagnostic strategy for the detection of chromosome aberrations in recurrent miscarriage

Abstract BACKGROUND: Our aim was to conduct a comprehensive genetic evaluation using the combination of QF-PCR (quantitative fluorescence polymerase chain reaction) and aCGH (array comparative genomic hybridization) for the detection of the frequency and type of chromosome aberrations in recurrent miscarriage (RM) in the clinical setting. METHODS: This retrospective study was conducted on 73 first-trimester products of conception (POC) between September 2014 and February 2017. The POCs were collected from 73 women with at least one previous miscarriage and analyzed for chromosomal anomalies using QF-PCR and aCGH as part of the routine clinical evaluation. RESULTS: Chromosome aberrations were detected in 52/73 POCs (71.2%), of which 41 (56.2%) were identified by QF-PCR and an additional 11 (15.1%) by aCGH. Numerical aberrations constituted 92.3% of abnormalities, with trisomies as the most common subtype (72.9%). Causative structural aberrations were found in three samples (5.8%). The frequency of chromosome aberrations was not dependent on the number of previous miscarriages, whereas it significantly increased with advanced maternal age. CONCLUSION: Our results confirm that chromosome aberrations are the most common cause of RM and that QF-PCR and aCGH combination should be included in the routine genetic analysis of POCs of couples with miscarriag

The Interleukin-1 Receptor Antagonist Gene and the Inhibitor of Kappa B-Like Protein Gene Polymorphisms Are Not Associated with Myocardial Infarction in Slovene Population with Type 2 Diabetes

In this study we investigated the association of the interleukin-1 receptor antagonist gene variable number tandem repeat (IL1RN VNTR) polymorphism and of the inhibitor of kappa B-like protein (IKBL) gene polymorphism with myocardial infarction (MI) in a group of patients with type 2 diabetes. The IL1RN VNTR and the IKBL+738T>C gene polymorphisms were tested in 374 Caucasians: 151 cases with MI and 223 subjects with no history of coronary artery disease. The IL1RN VNTR polymorphism was not a risk factor for MI in Caucasians with type 2 diabetes (genotype 22 vs. the rest: odds ratio (OR) 1.6; 95% confidence interval (CI) = 0.8–3.5; p=0.2). We also failed to demonstrate that IKBL+ 738T>C gene polymorphism was associated with MI in patients with type 2 diabetes (OR =0.9; 95% CI = 0.3–2.6; p=0.9). We provide evidence that the IL1RN VNTR and the IKBL+738T>C gene polymorphisms are not risk factors for MI in Caucasians with type 2 diabetes

Tumor Necrosis Factor-alpha-308 Gene Polymorphism in Croatian and Slovenian Multiple Sclerosis Patients

Previous findings regarding the role of TNF-a-308 gene polymorphism in multiple sclerosis (MS) are contradictory.This study's aim was to investigate the possible influence of TNF-a-308 polymorphism on MS susceptibility and MS disease process in a Croatian and Slovenian population. Genotyping was performed in 338 patients and 460 healthy controls. The TNF2 allele was present in 123 (26.8%) of healthy controls versus 67 (19.9%) of MS patients (p=0.023, odds ratio=0.68, 95% confidence interval=0.48-0.95), suggesting that carriage of the TNF2 allele might decrease MS risk. The difference of TNF2-allele carrier frequency between patients and controls was identified in relapsing-remitting (RR) MS group.There was no association between TNF2-allele carrier status and age at disease onset or disease progression. Our results suggest that in the study populations the TNF-a-308 polymorphism may play a role in MS susceptibility

Angiotensin-converting enzyme insertion/deletion gene polymorphism and interferon-β treatment response in multiple sclerosis patients: a preliminary report.

We investigated the effect of the functional insertion/ deletion (I/D) polymorphism in the angiotensin- converting enzyme (ACE) gene on the response to interferon-β (IFN-β) therapy in Croatian and Slovenian patients with multiple sclerosis (MS). A total of 275 IFN-β treated MS patients [162 responders (Rs) and 113 nonresponders (NRs)] were genotyped by PCR. The ACE I/D genotype distribution and allele frequencies did not differ between female Rs and NRs. However, male NRs tended to have a greater prevalence of the DD genotype (P=0.073 ; odds ratio: 2.64 ; 95% confidence interval: 0.91–7.60) and a significantly higher frequency of the D allele (P=0.022 ; odds ratio: 2.43 ; 95% confidence interval: 1.13–5.20) than male Rs. Multiple forward stepwise regression analysis indicated that the negative response to IFN-β therapy was associated with the ACE-DD genotype in men ( β=0.371 ; multiple R2 change: 0.132 ; P=0.009) and a higher pretreatment relapse rate in both men ( β=−0.438 ; multiple R2 change: 0.135 ; P=0.015) and women ( β=−0.208 ; multiple R2 change: 0.042 ; P=0.034). The ACE I/D polymorphism and pretreatment relapse rate accounted for ∼26.7% of the IFN-β response variability among the men in the sample. Further studies of a larger number of MS patients from different populations are necessary to evaluate these preliminary findings

Angiotensin-converting enzyme insertion/deletion gene polymorphism and interferon-β treatment response in multiple sclerosis patients: a preliminary report.

We investigated the effect of the functional insertion/ deletion (I/D) polymorphism in the angiotensin- converting enzyme (ACE) gene on the response to interferon-β (IFN-β) therapy in Croatian and Slovenian patients with multiple sclerosis (MS). A total of 275 IFN-β treated MS patients [162 responders (Rs) and 113 nonresponders (NRs)] were genotyped by PCR. The ACE I/D genotype distribution and allele frequencies did not differ between female Rs and NRs. However, male NRs tended to have a greater prevalence of the DD genotype (P=0.073 ; odds ratio: 2.64 ; 95% confidence interval: 0.91–7.60) and a significantly higher frequency of the D allele (P=0.022 ; odds ratio: 2.43 ; 95% confidence interval: 1.13–5.20) than male Rs. Multiple forward stepwise regression analysis indicated that the negative response to IFN-β therapy was associated with the ACE-DD genotype in men ( β=0.371 ; multiple R2 change: 0.132 ; P=0.009) and a higher pretreatment relapse rate in both men ( β=−0.438 ; multiple R2 change: 0.135 ; P=0.015) and women ( β=−0.208 ; multiple R2 change: 0.042 ; P=0.034). The ACE I/D polymorphism and pretreatment relapse rate accounted for ∼26.7% of the IFN-β response variability among the men in the sample. Further studies of a larger number of MS patients from different populations are necessary to evaluate these preliminary findings

Tumor Necrosis Factor-alpha-308 Gene Polymorphism in Croatian and Slovenian Multiple Sclerosis Patients

Previous findings regarding the role of TNF-a-308 gene polymorphism in multiple sclerosis (MS) are contradictory.This study's aim was to investigate the possible influence of TNF-a-308 polymorphism on MS susceptibility and MS disease process in a Croatian and Slovenian population. Genotyping was performed in 338 patients and 460 healthy controls. The TNF2 allele was present in 123 (26.8%) of healthy controls versus 67 (19.9%) of MS patients (p=0.023, odds ratio=0.68, 95% confidence interval=0.48-0.95), suggesting that carriage of the TNF2 allele might decrease MS risk. The difference of TNF2-allele carrier frequency between patients and controls was identified in relapsing-remitting (RR) MS group.There was no association between TNF2-allele carrier status and age at disease onset or disease progression. Our results suggest that in the study populations the TNF-a-308 polymorphism may play a role in MS susceptibility