New molecular tools for prenatal diagnosis

Abstract: Prenatal diagnosis enables identification of severe disease in the fetus, and allows for planning and management of future pregnancies if an underlying genetic mechanism is identified. The studies included in this thesis have taken advantage of the dramatic progress in medical genetics, in order to develop and evaluate new procedures to diagnose genetic disorders in fetal life. A correct diagnosis is important not only for the management and counseling of the patient or couple, but may also have an impact on the extended family, as there may be undetected carriers. The incidence of stillbirth in Sweden, i.e. fetal death occurring at gestational week ≥22, has essentially remained constant since the 1980’s, and despite thorough investigation, many cases remain unexplained. In Paper I, clinical chromosome analysis results from 481 stillbirth cases were compiled, and a subgroup of 90 cases were analyzed using chromosomal microarray (CMA) to study the potential benefits of the method. The conventional analysis detected chromosomal aberrations in 7.5% of the cases. CMA additionally identified two known syndromes, one disruption of a known disease gene, and 26 variants of unknown significance. Furthermore, CMA had a significantly higher success rate than cytogenetic analysis (100% vs. 80%, p<0.001). As CMA increased both the success rate and diagnosis rate, we concluded that it is a valuable tool in stillbirth investigation. In Paper II, DNA from 290 stillbirth cases was analyzed by massive parallel sequencing of 79 genes associated with heart disease. Sixty-two cases (21.4%) had putative pathogenic variants in genes associated with channelopathies (53%), cardiomyopathies (24%) or congenital heart defects (23%). Screening for pathogenic variants in genes associated with heart disease might be a valuable complement in cases where the conventional investigation does not reveal the underlying cause of fetal demise. Fetal tissue calcifications are occasionally noted during autopsy or ultrasound, but their biological importance is largely unexplored. In Paper III, a case-control study including 151 calcification cases and 302 matched controls was performed, to identify factors associated with calcifications. Chromosomal abnormalities, detected by conventional chromosome analysis or quantitative fluorescence-polymerase chain reaction, were significantly more common in cases compared with controls; 50% vs. 20% (p<0.001). When comparing cases and controls with chromosomal abnormalities, the cases had a significantly higher prevalence of malformations (96% vs. 77%, p=0.002). We concluded that fetal tissue calcifications are associated with chromosomal abnormalities, especially in combination with malformations. Non-invasive prenatal testing (NIPT) for fetal aneuploidy, based on sequencing analysis of cell-free fetal DNA in maternal plasma, has recently been made possible. Current methods require DNA amplification prior to sequencing, which inevitably leads to a bias because some DNA fragments amplify more efficiently than others. In Paper IV, an amplification-free NIPT protocol was evaluated on 31 samples, of which 15 had confirmed aneuploidies. All aneuploidies were correctly classified, except in one case where cytogenetic testing had confirmed the presence of both trisomy 18 and XXY, but NIPT failed to identify the extra X chromosome. Further analyses revealed that the fetus was mosaic, which probably explains why the extra X chromosome was missed. We concluded that the amplification-free protocol can potentially be used for NIPT, as it could distinguish aberrant samples from healthy controls. Compared to other NIPT protocols, it can be used on smaller quantities of input DNA, reduces GC bias and increases genome coverage. In Paper V, we aimed to gain an increased understanding of pregnant women’s awareness, attitudes, and preferences concerning prenatal testing with emphasis on NIPT, before its introduction into Swedish healthcare. In total, 1,003 pregnant women were recruited to fill in a questionnaire at nine maternity clinics located in different areas of Stockholm. The overwhelming majority of the women (91%) considered examinations aiming to detect fetal abnormalities to be good. Regarding NIPT, 60% stated that they had heard about the method previously, yet 74% would like to use the test if available. The main factor affecting the women’s decision to undergo prenatal chromosomal screening was worry about the baby’s health (83%), followed by the desire to have as much information as possible about their fetuses (55%). The results from the studies have provided support for the value of various genetic analysis methods in the field of prenatal diagnosis, as well as insights to the prevalence of genetic aberrations in fetuses.