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...

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...