Noninvasive testing for Down's syndrome (trisomy 21) would greatly reduce the risks associated with the more invasive techniques used currently. Earlier identification of differentially methylated regions between fetal DNA and maternal peripheral blood has now enabled Elisavet Papageorgiou and her colleagues to develop a strategy involving methylated DNA immunoprecipitation in combination with real-time quantitative PCR that discriminates normal from trisomy 21 cases in maternal peripheral blood with high sensitivity. The trials performed worldwide toward noninvasive prenatal diagnosis (NIPD) of Down's syndrome (or trisomy 21) have shown the commercial and medical potential of NIPD compared to the currently used invasive prenatal diagnostic procedures. Extensive investigation of methylation differences between the mother and the fetus has led to the identification of differentially methylated regions (DMRs). In this study, we present a strategy using the methylated DNA immunoprecipitation (MeDiP) methodology in combination with real-time quantitative PCR (qPCR) to achieve fetal chromosome dosage assessment, which can be performed noninvasively through the analysis of fetal-specific DMRs. We achieved noninvasive prenatal detection of trisomy 21 by determining the methylation ratio of normal and trisomy 21 cases for each tested fetal-specific DMR present in maternal peripheral blood, followed by further statistical analysis. The application of this fetal-specific methylation ratio approach provided correct diagnosis of 14 trisomy 21 and 26 normal cases.

Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.

Background: Carriers of apparently balanced translocations are usually phenotypically normal; however in about 6% of de novo cases, an abnormal phenotype is present. In the current study we investigated 12 patients, six de novo and six familial, with apparently balanced translocations and mental retardation and/or congenital malformations by applying 1 Mb resolution array-CGH. In all de novo cases, only the patient was a carrier of the translocation and had abnormal phenotype. In five out of the six familial cases, the phenotype of the patient was abnormal, although the karyotype appeared identical to other phenotypically normal carriers of the family. In the sixth familial case, all carriers of the translocations had an abnormal phenotype. Results: Chromosomal and FISH analyses suggested that the rearrangements were "truly balanced" in all patients. However, array-CGH, revealed cryptic imbalances in three cases (3/12, 25%), two de novo (2/12, 33.3%) and one familial (1/12, 16.6%). The nature and type of abnormalities differed among the cases. In the first case, what was identified as a de novo t(9;15)(q31;q26.1), a complex rearrangement was revealed involving a ~6.1 Mb duplication on the long arm of chromosome 9, an ~10 Mb deletion and an inversion both on the long arm of chromosome 15. These imbalances were located near the translocation breakpoints. In the second case of a de novo t(4;9)(q25;q21.2), an ~6.6 Mb deletion was identified on the short arm of chromosome 7 which is unrelated to the translocation. In the third case, of a familial, t(4;7)(q13.3;p15.3), two deletions of ~4.3 Mb and ~2.3 Mb were found, each at one of the two translocation breakpoints. In the remaining cases the translocations appeared balanced at 1 Mb resolution. Conclusion: This study investigated both de novo and familial apparently balanced translocations unlike other relatively large studies which are mainly focused on de novo cases. This study provides additional evidence that cryptic genomic imbalances are common in patients with abnormal phenotype and "apparently balanced" translocations not only in de novo but can also occur in familial cases. The use of microarrays with higher resolution such as oligo-arrays may reveal that the frequency of cryptic genomic imbalances among these patients is higher.

Background: There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT). Methods: We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing. Results: Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%–100%) cases of trisomy 21, 16/16 (95% CI, 79.4%–100%) cases of trisomy 18, 5/5 (95% CI, 47.8%–100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%–100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%–100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction. Conclusions: The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT.