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...

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.

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Measurement of the thickness of glomerular basement membranes is required for the diagnosis of thin membrane nephropathy. Over the years various morphometric methods have been used but some are laborious so there is a need for establishing a simplified method for measuring thickness. In the present study 20 renal biopsies were used to carry out a comparative morphometric analysis between 2 methods. The first method was based on measuring thickness at the maximum number of available points, whereas for the second method, thickness was measured at only 5 points per loop. Since both methods gave mean values that are not statistically different in each patient, the authors recommend that the simplified method be used routinely for diagnosis.

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Apolipoprotein E (APOE) plays an important role in the multifactorial etiology of both cardiovascular disease and Alzheimer's disease. Polymerase chain reaction (PCR) was used to investigate the APOE gene polymorphism in 335 unrelated Greek Cypriots living on the island of Cyprus. For the most common APOE genotypes, the Greek Cypriots followed the general Caucasian European pattern of having higher genotypic frequencies of E3/3, followed by E3/4, and then E2/3. Among the European populations compared, Greek Cypriots exhibited the lowest relative frequency of the E3/4 genotype (12.83%). Also, the relative frequencies of the E2 and E4 alleles in Greek Cypriots were among the lowest around the world (5.4% and 7.0%, respectively). This was also demonstrated by using the complete and the average clustering methods of analysis where the APOE allele relative frequencies in Greek Cypriots were compared to 46 other populations. The Greek Cypriot population in these analyses clustered with populations mainly from south Europe and Japan which have low E2 and E4 allele frequencies. The Greek Cypriot population will be studied further for elucidating the effect(s) and the role of APOE in cardiovascular disease and the APOE4 allele as a possible metabolic factor affecting the rate of expression of both Alzheimer's disease and vascular dementia.