The trials performed worldwide towards Non-Invasive Prenatal Diagnosis (NIPD) of Down syndrome (or Trisomy 21) have demonstrated the great 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 qPCR to achieve fetal chromosome dosage assessment which can be performed non-invasively through the analysis of fetal-specific DMRs. We achieved non-invasive 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 the above fetal-specific methylation ratio approach provided correct diagnosis of 14 trisomy 21 and 26 normal cases. Down Syndrome (or Trisomy 21) (OMIM190685) is considered to be the most frequent etiology of mental retardation with an incidence of 1 in 700 child births in all populations worldwide 1 . Prenatal genetic diagnosis of trisomy 21 is currently performed using conventional cytogenetic or DNA analyses, which require fetal genetic material to be obtained by amniocentesis, chorionic villus sampling or cordocentesis. However, the above procedures are invasive and are associated with a considerable risk of fetal loss 1 . Therefore, there is a need for the development of Non-Invasive Prenatal Diagnostic (NIPD) strategies.Correspondence should be addressed to P.C.P. (patsalis@cing.ac.cy). Author contributions: E.A.P. and E.T. have carried out the experiments. E.A.P. has written the manuscript. E.A.P. and A.K. performed the statistical analysis. E.T. and V.V. have collected the majority of the samples in this study. P.C.P. was the principal investigator and has supervised the project. All authors reviewed, critiqued and offered comments to the text.Competing financial interest: P.C.P. and E.A.P. declare conflict of interest as they have filed a U.S. provisional patent for the approach (Application No. 61/405,421 We have selected a subset of DMRs on chromosome 21 and we have applied the MeDiP methodology in combination with real-time qPCR in normal and trisomy 21 cases. To provide chromosome dosage information, the ffDNA has to be hypermethylated compared to the maternal DNA. This is essential to achieve fetal-specific methylation enrichment which is the key element in our study. We hypothesize that we would be able to discriminate normal from trisomy 21 cases by comparing the ratio values obtained from normal and trisomy 21 cases using fetal-specific methylated regions located on chromosome 21 (fetalspecific methylation ratio approach) ( Fig. 1). Furthermore, we hypothesize that a combination of DMRs and not a single DMR may be able to give an accurate NIPD of normal and trisomy 21...

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.

Next-generation mate-pair sequencing (MPS) has revealed that many constitutional complex chromosomal rearrangements (CCRs) are associated with local shattering of chromosomal regions (chromothripsis). Although MPS promises to identify the molecular basis of the abnormal phenotypes associated with many CCRs, none of the reported mate-pair sequenced complex rearrangements have been simultaneously studied with state-of-the art molecular cytogenetic techniques. Here, we studied chromothripsis-associated CCR involving chromosomes 2, 5 and 7, associated with global developmental and psychomotor delay and severe speech disorder. We identified three truncated genes: CDH12, DGKB and FOXP2, confirming the role of FOXP2 in severe speech disorder, and suggestive roles of CDH12 and/or DGKB for the global developmental and psychomotor delay. Our study confirmes the power of MPS for detecting breakpoints and truncated genes at near nucleotide resolution in chromothripsis. However, only by combining MPS data with conventional G-banding and extensive fluorescence in situ hybridizations could we delineate the precise structure of the derivative chromosomes.

The trials performed worldwide towards Non-Invasive Prenatal Diagnosis (NIPD) of Down syndrome (or Trisomy 21) have demonstrated the great 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 qPCR to achieve fetal chromosome dosage assessment which can be performed non-invasively through the analysis of fetal-specific DMRs. We achieved non-invasive 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 the above fetal-specific methylation ratio approach provided correct diagnosis of 14 trisomy 21 and 26 normal cases. Down Syndrome (or Trisomy 21) (OMIM190685) is considered to be the most frequent etiology of mental retardation with an incidence of 1 in 700 child births in all populations worldwide 1 . Prenatal genetic diagnosis of trisomy 21 is currently performed using conventional cytogenetic or DNA analyses, which require fetal genetic material to be obtained by amniocentesis, chorionic villus sampling or cordocentesis. However, the above procedures are invasive and are associated with a considerable risk of fetal loss 1 . Therefore, there is a need for the development of Non-Invasive Prenatal Diagnostic (NIPD) strategies.Correspondence should be addressed to P.C.P. (patsalis@cing.ac.cy). Author contributions: E.A.P. and E.T. have carried out the experiments. E.A.P. has written the manuscript. E.A.P. and A.K. performed the statistical analysis. E.T. and V.V. have collected the majority of the samples in this study. P.C.P. was the principal investigator and has supervised the project. All authors reviewed, critiqued and offered comments to the text.Competing financial interest: P.C.P. and E.A.P. declare conflict of interest as they have filed a U.S. provisional patent for the approach (Application No. 61/405,421 We have selected a subset of DMRs on chromosome 21 and we have applied the MeDiP methodology in combination with real-time qPCR in normal and trisomy 21 cases. To provide chromosome dosage information, the ffDNA has to be hypermethylated compared to the maternal DNA. This is essential to achieve fetal-specific methylation enrichment which is the key element in our study. We hypothesize that we would be able to discriminate normal from trisomy 21 cases by comparing the ratio values obtained from normal and trisomy 21 cases using fetal-specific methylated regions located on chromosome 21 (fetalspecific methylation ratio approach) ( Fig. 1). Furthermore, we hypothesize that a combination of DMRs and not a single DMR may be able to give an accurate NIPD of normal and trisomy 21...

Objective To reevaluate the efficiency of the 12 differentially methylated regions (DMRs) used in the methylated DNA immunoprecipitation (MeDIP) real‐time quantitative polymerase chain reaction (real‐time qPCR) based approach, develop an improved version of the diagnostic formula and perform a larger validation study. Methods Twelve selected DMRs were checked for copy number variants in the Database of Genomic Variants. The DMRs located within copy number variants were excluded from the analysis. One hundred and seventy‐five maternal peripheral blood samples were used to reconstruct and evaluate the new diagnostic formula and for a larger‐scale blinded validation study using MeDIP real‐time qPCR. Results Seven DMRs entered the final model of the prediction equation and a larger blinded validation study demonstrated 100% sensitivity and 99.2% specificity. No significant evidence for association was observed between cell free fetal DNA concentration and D value. Conclusion The MeDIP real‐time qPCR method for noninvasive prenatal diagnosis of trisomy 21 was confirmed and revalidated in 175 samples with satisfactory results demonstrating that it is accurate and reproducible. We are currently working towards simplification of the method to make it more robust and therefore easily, accurately, and rapidly reproduced and adopted by other laboratories. Nevertheless, larger scale validation studies are necessary before the MeDIP real‐time qPCR‐based method could be applied in clinical practice. © 2012 John Wiley & Sons, Ltd.

BackgroundThe purpose of the study was the application and evaluation of array Comparative Genomic Hybridization (array CGH) in selected cases during prenatal diagnosis. Array CGH was applied in 25 fetal samples out of which 15 had normal karyotypes and abnormal ultrasound findings and 10 had apparently balanced structural aberrations with or without abnormal ultrasound findings. DNA was extracted from peripheral blood, chorionic villi samples (CV) and amniotic fluid. Bacterial Artificial Chromosome (BAC) array CGH (Cytochip, BlueGnome Ltd.) of 1 Mb was applied and results were confirmed with either Fluorescence In Situ Hybridization (FISH), Multiplex Ligation-dependant Probe Amplification (MLPA) or Real-Time PCR.ResultsThree out of 25 samples (12%), referred for prenatal array CGH, were found to carry copy number alterations. The number of cases with clinically significant alterations was 2/25 (8%), while one (4%) was of uncertain clinical significance. Two benign Copy Number Variations (CNVs) were also found in 1/25 cases (4%).ConclusionsThe outcome of this study indicates the ability of array CGH to identify chromosomal abnormalities which cannot be detected during routine prenatal cytogenetic analysis, therefore increasing the overall detection rate.

Array Comparative Genomic Hybridization analysis is replacing postnatal chromosomal analysis in cases of intellectual disabilities, and it has been postulated that it might also become the first-tier test in prenatal diagnosis. In this study, array CGH was applied in 64 prenatal samples with whole genome oligonucleotide arrays (BlueGnome, Ltd.) on DNA extracted from chorionic villi, amniotic fluid, foetal blood, and skin samples. Results were confirmed with Fluorescence In Situ Hybridization or Real-Time PCR. Fifty-three cases had normal karyotype and abnormal ultrasound findings, and seven samples had balanced rearrangements, five of which also had ultrasound findings. The value of array CGH in the characterization of previously known aberrations in five samples is also presented. Seventeen out of 64 samples carried copy number alterations giving a detection rate of 26.5%. Ten of these represent benign or variables of unknown significance, giving a diagnostic capacity of the method to be 10.9%. If karyotype is performed the additional diagnostic capacity of the method is 5.1% (3/59). This study indicates the ability of array CGH to identify chromosomal abnormalities which cannot be detected during routine prenatal cytogenetic analysis, therefore increasing the overall detection rate. In addition a thorough review of the literature is presented.

The clinical significance of trisomy 20 mosaicism detected prenatally remains uncertain due to the rarity of liveborn cases with inconsistent clinical findings, and lack of long-term follow-up and outcome. We describe a case of true trisomy 20 mosaicism in a liveborn girl with maternal uniparental isodisomy of chromosome 20 in the diploid blood cells. Trisomy 20 mosaicism was originally detected in amniotic fluid (98%) and was confirmed in the term placenta (100%), as well as in the blood (10%) and urine sediment (100%) of the neonate. There was intrauterine and postnatal growth retardation, but otherwise the newborn manifested no gross abnormalities. At 9 months of age moderate psychomotor retardation, central hypotonia with peripheral hypertonia, numerous minor morphogenetic variants, marked kyphosis, and extensive Mongolian spot were observed. To our knowledge this represents the first case of trisomy 20 mosaicism detected prenatally and confirmed in different tissues of the newborn, where uniparental disomy was demonstrated in the diploid cell line. The clinical and laboratory findings in our patient are compared with those of five previously reported cases of UPD20, suggesting that maternal UPD20 might be associated with a characteristic phenotype.