Differential DNA Methylation as a Tool for Noninvasive Prenatal Diagnosis (NIPD) of X Chromosome Aneuploidies
2010; Elsevier BV; Volume: 12; Issue: 6 Linguagem: Inglês
10.2353/jmoldx.2010.090199
ISSN1943-7811
AutoresFloriana Della Ragione, Paola Mastrovito, Ciro Campanile, A. Conti, Elisavet A. Papageorgiou, Maj Hultén, Philippos C. Patsalis, Nigel P. Carter, Maurizio D’Esposito,
Tópico(s)Genetic Syndromes and Imprinting
ResumoThe demographic tendency in industrial countries to delay childbearing, coupled with the maternal age effect in common chromosomal aneuploidies and the risk to the fetus of invasive prenatal diagnosis, are potent drivers for the development of strategies for noninvasive prenatal diagnosis. One breakthrough has been the discovery of differentially methylated cell-free fetal DNA in the maternal circulation. We describe novel bisulfite conversion- and methylation-sensitive enzyme digestion DNA methylation-related approaches that we used to diagnose Turner syndrome from first trimester samples. We used an X-linked marker, EF3, and an autosomal marker, RASSF1A, to discriminate between placental and maternal blood cell DNA using real-time methylation-specific PCR after bisulfite conversion and real-time PCR after methylation-sensitive restriction digestion. By normalizing EF3 amplifications versus RASSF1A outputs, we were able to calculate sex chromosome/autosome ratios in chorionic villus samples, thus permitting us to correctly diagnose Turner syndrome. The identification of this new marker coupled with the strategy outlined here may be instrumental in the development of an efficient, noninvasive method of diagnosis of sex chromosome aneuploidies in plasma samples. The demographic tendency in industrial countries to delay childbearing, coupled with the maternal age effect in common chromosomal aneuploidies and the risk to the fetus of invasive prenatal diagnosis, are potent drivers for the development of strategies for noninvasive prenatal diagnosis. One breakthrough has been the discovery of differentially methylated cell-free fetal DNA in the maternal circulation. We describe novel bisulfite conversion- and methylation-sensitive enzyme digestion DNA methylation-related approaches that we used to diagnose Turner syndrome from first trimester samples. We used an X-linked marker, EF3, and an autosomal marker, RASSF1A, to discriminate between placental and maternal blood cell DNA using real-time methylation-specific PCR after bisulfite conversion and real-time PCR after methylation-sensitive restriction digestion. By normalizing EF3 amplifications versus RASSF1A outputs, we were able to calculate sex chromosome/autosome ratios in chorionic villus samples, thus permitting us to correctly diagnose Turner syndrome. The identification of this new marker coupled with the strategy outlined here may be instrumental in the development of an efficient, noninvasive method of diagnosis of sex chromosome aneuploidies in plasma samples. Aneuploidies, ie, an abnormal number of chromosomes, are responsible for a range of genetic disorders. The most frequent aneuploidies compatible with life are represented by trisomy 21 causing Down syndrome, trisomy 13 causing Patau syndrome, trisomy 18 causing Edwards syndrome, and sex chromosome aneuploidies. Sex chromosome aneuploidies include 45,X causing Turner syndrome (1/2500 living females), 47,XXX associated with triple X syndrome (1/1000 live births), 47,XXY associated with Klinefelter syndrome (prevalence 1/500 live males) (for prevalence and incidence data refer to http://www.orpha.net/consor/cgi-bin/index.php, last accessed on March 9, 2010) and the 47,XYY karyotype (the incidence generally reported is 1/1000 live births). Compared to trisomy 21, 13, and 18, sex chromosome aneuploidies show less severe clinical phenotypes, but taken together, the incidence of sex chromosome aneuploidies is high and, in the case of 47,XXX, 47,XXY and 47,XYY, this is largely underestimated. Moreover, although the mortality in utero of fetuses with Turner syndrome is high, most fetuses with other sex chromosome aneuploidies survive to term.1Kim YJ Park SY Han JH Kim MY Yang JH Choi KH Kim YM Kim JM Ryu HM Parental decisions of prenatally detected sex chromosome abnormality.J Korean Med Sci. 2002; 17: 53-57PubMed Google Scholar Major malformations may occur in Turner syndrome, but not in the XXY Klinefelter, XXX and XYY syndromes.2Bender BG Linden MG Robinson A Neuropsychological impairment in 42 adolescents with sex chromosome abnormalities.Am J Med Genet. 1993; 48: 169-173Crossref PubMed Scopus (126) Google Scholar To date, conventional prenatal diagnosis of genetic disorders has been based on the analysis of fetal cells obtained using invasive procedures such as amniocentesis and chorionic villus sampling (CVS). These techniques are very reliable, but the downside is that both are associated with a small but significant risk of fetal loss, ie, in the order of 0.5 to 1.0% of cases. For this reason, invasive prenatal diagnosis is offered only if the perceived risk of abnormal pregnancy, estimated by maternal age, ultrasonography and other noninvasive methods, exceeds the miscarriage risk.3Lo YM Noninvasive prenatal detection of fetal chromosomal aneuploidies by maternal plasma nucleic acid analysis: a review of the current state of the art.BJOG. 2009; 116: 152-157Crossref PubMed Scopus (60) Google Scholar Several groups have investigated noninvasive methods of prenatal diagnosis.3Lo YM Noninvasive prenatal detection of fetal chromosomal aneuploidies by maternal plasma nucleic acid analysis: a review of the current state of the art.BJOG. 2009; 116: 152-157Crossref PubMed Scopus (60) Google Scholar Attempts have been made to isolate fetal nucleated cells from maternal blood4Cheung MC Goldberg JD Kan YW Prenatal diagnosis of sickle cell anaemia and thalassaemia by analysis of fetal cells in maternal blood.Nat Genet. 1996; 14: 264-268Crossref PubMed Scopus (244) Google Scholar5Bianchi DW Flint AF Pizzimenti MF Knoll JH Latt SA Isolation of fetal DNA from nucleated erythrocytes in maternal blood.Proc Natl Acad Sci USA. 1990; 87: 3279-3283Crossref PubMed Scopus (388) Google Scholar6Herzenberg LA Bianchi DW Schroder J Cann HM Iverson GM Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting.Proc Natl Acad Sci USA. 1979; 76: 1453-1455Crossref PubMed Scopus (380) Google Scholar but their rarity and the possibility of cells persistent from previous pregnancies have so far made this strategy unreliable. Recent strategies for noninvasive prenatal diagnosis (NIPD) have been based on the observation in 1948 of the presence of cell free circulating nucleic acid in blood plasma7Mandel P Metais P Les acides nucleiques du plasma sanguin chez l'homme.CR Seances Soc Biol Fil. 1948; 142: 241-243PubMed Google Scholar and the increase in this plasma DNA in cancer.8Leon SA Shapiro B Sklaroff DM Yaros MJ Free DNA in the serum of cancer patients and the effect of therapy.Cancer Res. 1977; 37: 646-650PubMed Google Scholar9Sorenson GD Pribish DM Valone FH Memoli VA Bzik DJ Yao SL Soluble normal and mutated DNA sequences from single-copy genes in human blood.Cancer Epidemiol Biomarkers Prev. 1994; 3: 67-71PubMed Google Scholar10Vasioukhin V Anker P Maurice P Lyautey J Lederrey C Stroun M Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia.Br J Haematol. 1994; 86: 774-779Crossref PubMed Scopus (323) Google Scholar More recently, Lo et al11Lo YM Corbetta N Chamberlain PF Rai V Sargent IL Redman CW Wainscoat JS Presence of fetal DNA in maternal plasma and serum.Lancet. 1997; 350: 485-487Abstract Full Text Full Text PDF PubMed Scopus (2325) Google Scholar demonstrated the presence of male fetal DNA in maternal plasma, by amplifying Y specific sequences. Moreover, it was found that cell free fetal DNA (cffDNA) in maternal plasma is fragmented12Chan KC Zhang J Hui AB Wong N Lau TK Leung TN Lo KW Huang DW Lo YM Size distributions of maternal and fetal DNA in maternal plasma.Clin Chem. 2004; 50: 88-92Crossref PubMed Scopus (461) Google Scholar and the half-life is in the order of 16 minutes after delivery.13Lo YM Zhang J Leung TN Lau TK Chang AM Hjelm NM Rapid clearance of fetal DNA from maternal plasma.Am J Hum Genet. 1999; 64: 218-224Abstract Full Text Full Text PDF PubMed Scopus (892) Google Scholar The amount of cffDNA in maternal plasma DNA ranges between approximately 3 to 6% with a mean of 25.4 genome copies/ml of maternal plasma during early pregnancy.14Lo YM Tein MS Lau TK Haines CJ Leung TN Poon PM Wainscoat JS Johnson PJ Chang AM Hjelm NM Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis.Am J Hum Genet. 1998; 62: 768-775Abstract Full Text Full Text PDF PubMed Scopus (1389) Google Scholar cffDNA from maternal plasma has been successfully used to determine fetal rhesus D (RhD) blood type,15Lo YM Hjelm NM Fidler C Sargent IL Murphy MF Chamberlain PF Poon PM Redman CW Wainscoat JS Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma.N Engl J Med. 1998; 339: 1734-1738Crossref PubMed Scopus (602) Google Scholar for determination of fetal sex,14Lo YM Tein MS Lau TK Haines CJ Leung TN Poon PM Wainscoat JS Johnson PJ Chang AM Hjelm NM Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis.Am J Hum Genet. 1998; 62: 768-775Abstract Full Text Full Text PDF PubMed Scopus (1389) Google Scholar,16Sekizawa A Kondo T Iwasaki M Watanabe A Jimbo M Saito H Okai T Accuracy of fetal gender determination by analysis of DNA in maternal plasma.Clin Chem. 2001; 47: 1856-1858PubMed Google Scholar,17Costa JM Benachi A Gautier E New strategy for prenatal diagnosis of X-linked disorders.N Engl J Med. 2002; 346: 1502Crossref PubMed Scopus (157) Google Scholar thus limiting the need for invasive diagnosis in cases of sex specific pathologies as well as for the identification of some fetal disorders due to paternal genetic mutations or recessive conditions where parents are compound heterozygotes.18Ding C Chiu RW Lau TK Leung TN Chan LC Chan AY Charoenkwan P Ng IS Law HY Ma ES Xu X Wanapirak C Sanguansermsri T Liao C Ai MA Chui DH Cantor CR Lo YM MS analysis of single-nucleotide differences in circulating nucleic acids: Application to noninvasive prenatal diagnosis.Proc Natl Acad Sci USA. 2004; 101: 10762-10767Crossref PubMed Scopus (174) Google Scholar,19Chiu RW Lau TK Leung TN Chow KC Chui DH Lo YM Prenatal exclusion of beta thalassaemia major by examination of maternal plasma.Lancet. 2002; 360: 998-1000Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar However, the presence of a great excess of free maternal DNA complicates the use of such methods.14Lo YM Tein MS Lau TK Haines CJ Leung TN Poon PM Wainscoat JS Johnson PJ Chang AM Hjelm NM Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis.Am J Hum Genet. 1998; 62: 768-775Abstract Full Text Full Text PDF PubMed Scopus (1389) Google Scholar The most important source of cffDNA released in maternal plasma during pregnancy appears to be the placenta,20Flori E Doray B Gautier E Kohler M Ernault P Flori J Costa JM Circulating cell-free fetal DNA in maternal serum appears to originate from cyto- and syncytio-trophoblastic cells. Case report.Hum Reprod. 2004; 19: 723-724Crossref PubMed Scopus (133) Google Scholar,21Alberry M Maddocks D Jones M Abdel Hadi M Abdel-Fattah S Avent N Soothill PW Free fetal DNA in maternal plasma in anembryonic pregnancies: confirmation that the origin is the trophoblast.Prenat Diagn. 2007; 27: 415-418Crossref PubMed Scopus (286) Google Scholar whereas it has been suggested that the cell free maternal DNA (cfmDNA) originates from hematopoietic cells.22Lui YY Chik KW Chiu RW Ho CY Lam CW Lo YM Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation.Clin Chem. 2002; 48: 421-427PubMed Google Scholar On the basis of the placental origin of free fetal nucleic acids (cffDNA and cffRNA23Ng EK Tsui NB Lau TK Leung TN Chiu RW Panesar NS Lit LC Chan KW Lo YM mRNA of placental origin is readily detectable in maternal plasma.Proc Natl Acad Sci USA. 2003; 100: 4748-4753Crossref PubMed Scopus (324) Google Scholar) and the finding of a chromosome 21 placenta-specific mRNA marker in maternal plasma,24Lo YM Tsui NB Chiu RW Lau TK Leung TN Heung MM Gerovassili A Jin Y Nicolaides KH Cantor CR Ding C Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection.Nat Med. 2007; 13: 218-223Crossref PubMed Scopus (317) Google Scholar an approach based on RNA-SNP allelic ratio has been reported, to detect aneuploidies of this chromosome.24Lo YM Tsui NB Chiu RW Lau TK Leung TN Heung MM Gerovassili A Jin Y Nicolaides KH Cantor CR Ding C Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection.Nat Med. 2007; 13: 218-223Crossref PubMed Scopus (317) Google Scholar Recently, Lo and co-workers25Lo YM Lun FM Chan KC Tsui NB Chong KC Lau TK Leung TY Zee BC Cantor CR Chiu RW Digital PCR for the molecular detection of fetal chromosomal aneuploidy.Proc Natl Acad Sci USA. 2007; 104: 13116-13121Crossref PubMed Scopus (366) Google Scholar reported the use of digital PCR to determine the over-representation of chromosome 21 in trisomy 21 samples in mixtures of placental and maternal blood cell DNA, using samples containing at least 25% of fetal DNA, a concentration many fold higher than that present in a first trimester maternal plasma sample. A similar strategy, based on microfluidic digital PCR platform has been applied by Fan and co-workers26Fan HC Blumenfeld YJ El-Sayed YY Chueh J Quake SR Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy.Am J Obstet Gynecol. 2009; 200: e541-e547Abstract Full Text Full Text PDF Scopus (87) Google Scholar in the set-up of diagnosis of chromosome number abnormalities, on amniotic fluid and CVS samples. Recently, high throughput technologies, such as those based on parallel DNA-sequencing27Mardis ER Next-generation DNA sequencing methods.Annu Rev Genomics Hum Genet. 2008; 9: 387-402Crossref PubMed Scopus (1551) Google Scholar are being applied to NIPD strategies. Read depth analysis was used to successfully identify chromosome 13, 18, and 21 aneuploidies of the fetus.28Fan HC Blumenfeld YJ Chitkara U Hudgins L Quake SR Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood.Proc Natl Acad Sci USA. 2008; 105: 16266-16271Crossref PubMed Scopus (839) Google Scholar Chiu and co-workers29Chiu RW Chan KC Gao Y Lau VY Zheng W Leung TY Foo CH Xie B Tsui NB Lun FM Zee BC Lau TK Cantor CR Lo YM Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma.Proc Natl Acad Sci USA. 2008; 105: 20458-20463Crossref PubMed Scopus (699) Google Scholar also applied high throughput DNA-sequencing to quantify the amount of unique chromosome 21 sequences from plasma (maternal and fetal), revealing a potential trisomy. However, high throughput DNA-sequencing is still costly and difficult to manage in a routine laboratory, due to the large bioinformatic and computer resources required for analysis. For the foreseeable future, it may be difficult to translate this approach for widespread noninvasive diagnosis of aneuploidies. Strategies exploiting differential DNA methylation may also be used to discriminate tissues/cells of different origin. DNA methylation, the major post-biosynthetic modification found throughout mammalian genomes, is involved in many important biological phenomena, such as X chromosome inactivation and genomic imprinting, and in controlling tissue-specific expression in adult somatic tissues.30Robertson KD Wolffe AP DNA methylation in health and disease.Nat Rev Genet. 2000; 1: 11-19Crossref PubMed Scopus (874) Google Scholar31Scarano MI Strazzullo M Matarazzo MR D'Esposito M DNA methylation 40 years later: its role in human health and disease.J Cell Physiol. 2005; 204: 21-35Crossref PubMed Scopus (99) Google Scholar32Matarazzo MR De Bonis ML Gregory RI Vacca M Hansen RS Mercadante G D'Urso M Feil R D'Esposito M Allelic inactivation of the pseudoautosomal gene SYBL1 is controlled by epigenetic mechanisms common to the X and Y chromosomes.Hum Mol Genet. 2002; 11: 3191-3198Crossref PubMed Scopus (43) Google Scholar Differential DNA methylation between maternal and fetal DNA has been investigated for use in NIPD. Differential methylation between fetal CVS and maternal blood cell DNA was first reported in 200233Poon LL Leung TN Lau TK Chow KC Lo YM Differential DNA methylation between fetus and mother as a strategy for detecting fetal DNA in maternal plasma.Clin Chem. 2002; 48: 35-41PubMed Google Scholar and three years later the first universal epigenetic marker of fetal DNA in maternal plasma, SERPINB5 was described. The promoter region of SERPINB5 is hypomethylated in placenta and hypermethylated in maternal blood cells. Fetal SERPINB5 was distinguished from maternal SERPINB5 in maternal plasma DNA34Chim SS Tong YK Chiu RW Lau TK Leung TN Chan LY Oudejans CB Ding C Lo YM Detection of the placental epigenetic signature of the maspin gene in maternal plasma.Proc Natl Acad Sci USA. 2005; 102: 14753-14758Crossref PubMed Scopus (300) Google Scholar after bisulfite modification35Frommer M McDonald LE Millar DS Collis CM Watt F Grigg GW Molloy PL Paul CL A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.Proc Natl Acad Sci USA. 1992; 89: 1827-1831Crossref PubMed Scopus (2515) Google Scholar and methylation specific PCR.36Herman JG Jen J Merlo A Baylin SB Hypermethylation-associated inactivation indicates a tumor suppressor role for p15INK4B.Cancer Res. 1996; 56: 722-727PubMed Google Scholar Subsequently, the application of bisulfite-independent methods were also reported.37Chan KC Ding C Gerovassili A Yeung SW Chiu RW Leung TN Lau TK Chim SS Chung GT Nicolaides KH Lo YM Hypermethylated RASSF1A in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis.Clin Chem. 2006; 52: 2211-2218Crossref PubMed Scopus (295) Google Scholar,38Old RW Crea F Puszyk W Hulten MA Candidate epigenetic biomarkers for non-invasive prenatal diagnosis of Down syndrome.Reprod Biomed Online. 2007; 15: 227-235Abstract Full Text PDF PubMed Scopus (75) Google Scholar Despite the appeal of using epigenetic differences between maternal and fetal DNA to develop NIPD for aneuploidies, the search for chromosome specific markers is challenging: this is particularly true for X linked sequences. An alternative high-throughput approach for identifying chromosome specific methylated markers is based on immunoprecipitation of methylated DNA (MeDiP39Weber M Davies JJ Wittig D Oakeley EJ Haase M Lam WL Schubeler D Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells.Nat Genet. 2005; 37: 853-862Crossref PubMed Scopus (1412) Google Scholar) coupled with high resolution tiling oligonucleotide array analysis. More than 2000 differentially methylated regions between placenta and maternal blood cells, on respectively chromosome 13, 18, 21, X and Y, both in non-genic regions and in the CpG islands have been recently reported using this approach.40Papageorgiou EA Fiegler H Rakyan V Beck S Hulten M Lamnissou K Carter NP Patsalis PC Sites of differential DNA methylation between placenta and peripheral blood: molecular markers for noninvasive prenatal diagnosis of aneuploidies.Am J Pathol. 2009; 174: 1609-1618Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Taking advantage of these latter data, we selected three putative X-linked markers that are differentially methylated between placenta and maternal blood cell DNA, and after further validation we developed a NIPD strategy to determine copy number of fetal sex chromosomes. Pregnant women with singleton pregnancies were recruited from the Laboratory of Cytogenetics and Prenatal Diagnosis at the University Federico II, Naples, Italy. After informed consent was obtained, placenta and peripheral maternal blood samples were collected from first trimester subjects, recruited among women undergoing chorionic villus sampling for prenatal diagnosis, or third trimester subjects undergoing elective caesarean delivery. Maternal peripheral blood samples were collected just before the obstetric procedures. One to two hours after collection, blood samples were centrifuged at 1000 × g for 10 minutes at 4°C. The plasma portion was recovered and recentrifuged at 14,700 × g for 10 minutes at 4°C. DNA from peripheral blood cells was extracted using the Wizard Genomic DNA purification Kit (Promega Corporation, Madison, WI). DNA from third trimester placental tissues was extracted incubating the tissue in lysis solution (100 mmol/L Tris-Hcl pH 8.5, 5 mmol/L EDTA pH 8, 0.2% SDS, 200 mmol/L NaCl, and 0.1 mg/ml proteinase K) at 64°C overnight. After a centrifugation for 1 minute at 12,000 × g the supernatant was precipitated with 1 volume of isopropanol, centrifuged for 1 minute at 12,000 × g and the DNA pellet washed with 70% ethanol, dried and resuspended in TE 1× (10 mmol/L Tris-HCl, 1 mmol/L EDTA ph 8). Ten μg of DNA were incubated with 30 μg of RNase A (Roche, Mannheim, Germany) in a final volume of 30 μl for 1 hour at 37°C, then purified using phenol/chloroform extraction. DNA from first trimester placenta (CVS) was extracted using the Wizard Genomic DNA purification Kit (Promega Corporation). DNA from maternal plasma was extracted with the QIAamp DSP Virus Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Starting from 0.5 ml of plasma, the final elution was carried out in 25 μl of elution buffer. One μg of maternal blood cell and placental DNA and 10 μl of plasma DNA were bisulfite-converted using the EpiTect Bisulfite Kit (Qiagen). Samples were eluted in a final volume of 20 μl. One μl of bisulfite-converted first and third trimester maternal blood cell and placenta DNA was amplified using nested PCR carried out on Gradient PCR Express (Hybaid, Middlesex, UK). EF3 marker was amplified using EF3F1 and EF3R1 primers for the first step and EF3F2 and EF3R2 for the second step (Table 1).Table 1The Primers Used for Bisulfite Sequencing, RT-MSP, Control RT, PCR, and RT after Methylation-Sensitive DigestionsPrimer nameNucleotidic sequenceProduct lengthEF3F15′-TTGGTTTGGTTTTTTGGTGAG-3′694 bpEF3R15′-CAAAACAAAAACAAACAAAAATC-3′EF3F25′-GATTTAGGGTTGTTATTATGTT-3′498 bpEF3R25′-CTATTATCAACTTTTAAACAA-3′mMSP-EF3F1C5′-AAGTCGTATGGGTAAAGTTTTTGTCTC-3′179 bpmMSP-EF3R2G5′-CAAAAAACCCACCCCAACCCGCG-3′MSP-C+-EF3F25′-GGGTTGGGTTGAGATATAATATATGTT-3′242 bpMSP-C+-EF3R15′-ACTCAACCAAATACACTATTATCAACTT-3′mMSP-RASSF1AF5′-TTATCGTTTTTAGTTCGCGGGGTTC-3′99 bpmMSP-RASSF1AR35′-CGCGCGCACTACAAACCTTTACG-3′C+RASSF1AF1bis5′-GTGATAGAATGTAAAGAATGAATAAGGGGT-3′113 bpC+RASSF1AR1bis5′-AATCTTAAACTCCTAACCTCAAATAATCCA-3′EF3-AciI-digest F15′-CCCACCTTGCAAAGAAGAAA-3′162 bpEF3-AciI-digest R15′-CCGTCCACAATGACAAACAT-3′EF3-AciI-digest R35′-TGCAACCGCTTTCAGACTCA-3′RassF1A AciI F5′-GACCTCTGTGGCGACTTCAT-3′233 bpRassF1A AciI R5′-GGAGTGCGACAAGGGATAAA-3′RassF1A AciI R45′-ACCATTTTCGCGCACTCTTC-3′GAPDH F5′-ACATGTTCCAATATGATTCCA-3′291 bp and 162 bpGAPDH R5′-TGGACTCCACGACGTACTCAG-3′The sequence of each primer and the lengths of the PCR products are indicated. The primer EF3-AciI-digest F1 coupled with the primer EF3-AciI-digest R3 provides a 190-bp PCR product. The primer RassF1A AciI F coupled with the primer RassF1A AciI R4 gives a 214-bp PCR product. Open table in a new tab The sequence of each primer and the lengths of the PCR products are indicated. The primer EF3-AciI-digest F1 coupled with the primer EF3-AciI-digest R3 provides a 190-bp PCR product. The primer RassF1A AciI F coupled with the primer RassF1A AciI R4 gives a 214-bp PCR product. Reactions were performed in presence of 0.2 mmol/L each dNTP, 0.15 μmol/L (first step) or 0.3 μmol/L (second step) of each primer, 0.5 units of Amplitaq DNA polymerase (Applied Biosystem, Nieuwerkerk, The Netherlands) using the buffer recommended by the manufacturer. The second step was carried out using 1 μl of the first step product as template. The thermal cycling conditions were: 95°C for 3 minutes, followed by 95°C for 30 seconds, 52°C for 45 seconds, and 72°C for 45 seconds for 25 (first step) or 35 (second step) cycles. PCR products were run on agarose gels and recovered using QIAquick Gel Extraction Kit (Qiagen), following the manufacturer's instructions. Purified PCR products were cloned in the pCR2.1 cloning vector using TA Cloning Kit (Invitrogen Corporation, Carlsbad, CA) and sequenced by using the dye terminator method (PRIMM facility, Naples, Italy). Real-time methylation-specific PCR (RT-MSP) of EF3 and RASSF1A markers was performed on 10 ng of CVS and maternal blood cell bisulfite converted DNA, in a final volume of 20 μl in presence of 0.15 μmol/L of each primer and 1× iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) using DNA Engine Opticon 2 System (Bio-Rad) with mMSP-EF3F1C and mMSP-EF3R2G primer pair (EF3) or mMSP-RASSF1AF and mMSP-RASSF1AR3 primers (RASSF1A) (Table 1). The control primers were MSP-C+-EF3F2 and MSP-C+-EF3R1 (EF3) and C+RASSF1AF1bis and C+RASSF1AR1bis (RASSF1A) (Table 1). The real-time PCR protocol for EF3 marker was 4 minutes at 95°C followed by 15 seconds at 95°C, 20 seconds at 60°C (for RT-MSP) or 58°C (for control RT) and 20 seconds at 72°C for 32 cycles and for RASSF1A marker was 4 minutes at 95°C followed by 15 seconds at 95°C, 20 seconds at 60°C (for RT- MSP) or 62°C (for control RT) and 20 seconds at 72°C for 35 cycles. RT-MSP of EF3 and RASSF1A on plasma samples were performed with the same primers previously described, using 8 μl of 1:4 dilution of converted plasma DNA, extracted from first trimester pregnancies, containing approximately 16 pg of fetal DNA and 309 pg of maternal DNA. For this reason, RT-MSP was carried out, separately, also on 16 pg of CVS converted DNA and 309 pg of converted maternal blood DNA, respectively as positive and negative controls. Reactions were performed in the same conditions previously illustrated except for the cycles number (47 for EF3 and 48 for RASSF1A). At the end of each RT-MSP experiment, the melting curve of each PCR product was checked to confirm the purity and all products were analyzed by agarose gel electrophoresis to verify the amplicon size. The sex chromosomes/autosomes (SC/A) ratio was calculated as the concentration fold change following RT-MSP of EF3 and RASSF1A markers. Ten ng of bisulfite converted DNA extracted from CVS of five Turner cases (45,X; samples T1, T2, T3, T4, and T5) and four normal euploid individuals as controls (N6, N7, N8 and N9) were amplified using methylation specific primers in the same conditions before described, but using the CFX96 Real-Time System (Bio-Rad). The thermal cycling protocol was 4 minutes at 95°C followed by 15 seconds at 95°C, 20 seconds at 62°C or 64°C (for EF3 or RASSF1A, respectively), and 20 seconds at 72°C for 37 cycles. In all RT-MSP experiments, samples were present in triplicate and each experiment was performed three times (technical replicates). SC/A ratio was calculated using the 2−ΔΔCt method,41Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2−Delta Delta C(T) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123336) Google Scholar where N8 has been considered as reference sample (fold change = 1, omitted in the graphic representation) to whom compare all of the other samples. One μg of CVS and maternal blood cell DNA was digested with 10 U of AciI enzyme (New England Biolabs, Ipswich, MA) for 4 hours at 37°C, then adding 10 U of enzyme and incubating for a further 4 hours, and finally adding another 20 U and incubating overnight. Fifty ng of each digested DNA was subject to PCR to amplify EF3 and RASSF1A markers and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genomic region, using EF3-AciI-digestF1 and EF3-AciI-digestR1 primers (EF3), RassF1A AciI F and RassF1A AciI R primers (RASSF1A), and GAPDH F and GAPDH R primers (GAPDH) (Table 1). The reactions were performed in presence of 0.2 mmol/L each dNTP, 0.3 μmol/L (EF3 and RASSF1A) or 0.2 μmol/L (GAPDH) of each primer, 1.5 mmol/L MgCl2, 0.5 U of EuroTaq DNA Polymerase (Euroclone, UK), and 1× reaction buffer in a final volume of 10 μl. The thermal cycling protocol was: 4 minutes at 95°C, followed by 30 seconds at 95°C, 45 seconds at 60°C and 45 seconds at 72°C for 27 (EF3), 33 (RASSF1A), or 35 (GAPDH) cycles and 5 minutes of final extension at 72°C. PCR products were analyzed by agarose gel electrophoresis. The same methylation-sensitive assay was performed on maternal plasma by digesting 10 μl of plasma DNA obtained from a first trimester pregnancy. Two hundred fifty pg of CVS DNA and 4750 pg of maternal blood cell DNA were also digested as positive and negative controls, respectively. DNA samples were incubated with 10 U of Aci I enzyme (New England Biolabs) in a final volume of 20 μl for 6 hours at 37°C, then 10 U of Aci I were added and the samples were incubated overnight. Subsequently, DNAs were precipitated with ethanol, sodium acetate, and magnesium chloride and digested with 10 U of HpaII for 6 hours at 37°C in a volume of 20 μl, then 10 U of enzyme were added and the digestion was continued overnight. To amplify the EF3 and RASSF1A markers and GAPDH genomic region PCR reactions were carried out using 1 μl (EF3 and GAPDH) and 2 μl (RASSF1A) of plasma, CVS and blood digested DNAs using the same conditions as described above, except for 37 (EF3), 45 (GAPDH), or 50 cycles (RASSF1A). All PCR reactions were carried out using Gradient PCR Express (Hybaid, UK). SC/A ratio was obtained by real time and calculated, with the 2−ΔΔCt method.41Livak KJ Schmittgen TD Analysis of relative gene expression data us
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