Silvia Galbiati

No evidence of fetal DNA persistence in maternal plasma after pregnancy

Short- and long-term persistence of fetal DNA in maternal plasma has been investigated. Short-term persistence at very low concentration was detected in 47 out of 105 women within two days after delivery. Twelve out of 13 samples re-tested within three days scored negative. No long-term persistence was detected in 172 women who had previous sons or abortions. Molecular microchimerism due to circulating fetal DNA persisting from previous pregnancies should not hamper non-invasive plasma-based prenatal testing.

Fetal DNA detection in maternal plasma throughout gestation

The presence of fetal DNA in maternal plasma may represent a source of genetic material which can be obtained noninvasively. We wanted to assess whether fetal DNA is detectable in all pregnant women, to define the range and distribution of fetal DNA concentration at different gestational ages, to identify the optimal period to obtain a maternal blood sample yielding an adequate amount of fetal DNA for prenatal diagnosis, and to evaluate accuracy and predictive values of this approach. This information is crucial to develop safe and reliable non-invasive genetic testing in early pregnancy and monitoring of pregnancy complications in late gestation. Fetal DNA quantification in maternal plasma was carried out by real-time PCR on the SRY gene in male-bearing pregnancies to distinguish between maternal and fetal DNA. A cohort of 1,837 pregnant women was investigated. Fetal DNA could be detected from the sixth week and could be retrieved at any gestational week. No false-positive results were obtained in 163 women with previous embryo loss or previous male babies. Fetal DNA analysis performed blindly on a subset of 464 women displayed 99.4, 97.8 and 100% accuracy in fetal gender determination during the first, second, and third trimester of pregnancy, respectively. No SRY amplification was obtained in seven out of the 246 (2.8%) male-bearing pregnancies. Fetal DNA from maternal plasma seems to be an adequate and reliable source of genetic material for a noninvasive prenatal diagnostic approach.

Full COLD-PCR Protocol for Noninvasive Prenatal Diagnosis of Genetic Diseases

To the Editor: After the discovery of fetal DNA in maternal plasma, investigators reported different strategies for the noninvasive prenatal diagnosis of genetic diseases (1). Despite the advances in improving the analytical sensitivity of methods, distinguishing between fetal and maternal sequences remains very challenging, and the field of noninvasive prenatal diagnosis of genetic diseases has yet to attain a routine application in clinical diagnostics. An innovative strategy that has been reported is based on COLD-PCR (coamplification at a lower denaturation temperature PCR),1 which exploits melting temperature ( T m) differences between variant or mismatched sequences and wild-type sequences. This approach uses a critical denaturation temperature ( T c) lower than the T m to selectively amplify minority mutated alleles (2). We have developed assays for the identification of fetal paternally inherited mutations in maternal plasma. These assays use full COLD-PCR for the detection of IVSI.110 (G>A) and Cd39 (C>T) HBB (globin, beta) gene mutations that cause β-thalassemia. Full COLD-PCR is based on the generation of heteroduplexes between mutant and wild-type sequences. These heteroduplexes melt at lower temperatures than the wild-type homoduplexes. They are selectively denatured at the T c and then subsequently amplified. Given that both the fetal and maternal DNA content in maternal plasma has been demonstrated to vary from pregnancy to …

Peptide-nucleic acid-mediated enriched polymerase chain reaction as a key point for non-invasive prenatal diagnosis of β-thalassemia

This study describes a novel approach to non-invasive pre-natal diagnosis of β-thalassemia based on microchip analysis of fetal DNA extracted from maternal plasma. The presence of fetal DNA in maternal plasma can be exploited to develop new procedures for non-invasive prenatal diagnosis. Tests to detect 7 frequent β-globin gene mutations in people of Mediterranean origin were applied to the analysis of maternal plasma in couples where parents carried different mutations. A mutant enrichment amplification protocol was optimized by using peptide nucleic acids (PNAs) to clamp maternal wild-type alleles. By this approach, 41 prenatal diagnoses were performed by microelectronic microchip analysis, with total concordance of results obtained on fetal DNA extracted from chorionic villi. Among these, 27/28 were also confirmed by direct sequencing and 4 by pyrosequencing.

Feasibility study for a microchip-based approach for noninvasive prenatal diagnosis of genetic diseases

Abstract: Fetal DNA in maternal plasma may represent a source of genetic material for prenatal noninvasive diagnosis of genetic diseases. We evaluated a cohort of physiological pregnancies to determine if fetal DNA can be retrieved at any gestational week in sufficient quantity to be analyzed with advanced mutation detection technologies. We performed fetal DNA quantification by real‐time polymerase chain reaction (PCR) on the SRY gene in 356 women sampled from 6 to 40 gestational weeks. Fetal DNA was retrieved at any week. All female fetuses were correctly identified. In 5 of 188 (2.6%) male‐bearing pregnancies, no amplification was obtained. For noninvasive testing, complete clearance of fetal DNA after delivery is mandatory. Long‐term persistence was not detected in women with previous sons or abortions. These findings confirm that maternal plasma may represent the optimal source of fetal genetic material. For noninvasive diagnosis of genetic diseases, we evaluated microchip technology. The detection limit for a minority allele determined by diluting a mutated DNA into a wild‐type plasma sample was 5 genome equivalents, indicating that the test might be applied to the identification of paternally inherited fetal alleles in maternal plasma. The addition of peptide nucleic acids (PNAs) to either the PCR reaction or the chip hybridization mixture allowed approximately 50% inhibition of wild‐type allele signals.

Scanning mutations of the 5'UTR regulatory sequence of L-ferritin by denaturing high-performance liquid chromatography: identification of new mutations.

Summary. Hereditary hyperferritinaemia cataract syndrome is an autosomal dominant disorder caused by heterogeneous mutations of the iron regulatory element (IRE) in the ferritin l‐chain mRNA. The mutations are rare and fast DNA scanning would facilitate diagnosis. The aim of the study was to compare the analytical performances of two fast DNA scanning techniques: denaturing high‐performance liquid chromatography (DHPLC) and double‐gradient denaturing gradient gel electrophoresis (DG‐DGGE). We analysed the sequence encoding the 5′ untranslated flanking region of ferritin l‐chain mRNA, which includes an IRE stem loop structure. The two systems unambiguously identified all the 12 accessible mutations in a single run, including the difficult C–G transversions. DHPLC and DG‐DGGE identified seven abnormal patterns in DNA samples from 47 subjects with unexplained hyperferritinaemia; all had mutations in the IRE sequence, including two not reported before: C36G and A37G. The scanning of 250 DNA samples from subjects genotyped for HFE led to the identification of four new mutations, all outside the IRE structure: C10T, C16T, C90T and del‐T156. We conclude that DHPLC, similar to DG‐DGGE, detects all the mutations in the l‐ferritin 5‘UTR sequence in a single run, and that various mutations occur outside the IRE structure.

Fetal DNA in maternal plasma in twin pregnancies

Twin pregnancies have increased in recent years as assisted reproductive technologies have been adapted. The maternal age of women undergoing assisted reproduction is generally higher than that of women with spontaneous pregnancies. This also implies a higher risk of fetal aneuploidies. Furthermore, twin pregnancies are known to have a higher risk of developing complications such as preeclampsia, intrauterine growth retardation, and preterm labor [for a review, see Ref. (1)]. Of note is that women who have been trying for years to become pregnant and have finally undergone cumbersome therapeutic interventions could be particularly reluctant to expose their pregnancy to any invasive prenatal diagnostic procedure. Thus, noninvasive screening or diagnostic tests could be particularly welcome in this subset of pregnant women. Noncellular fetal DNA circulating in maternal plasma may represent a suitable source of fetal genetic material that can be obtained noninvasively. Several studies have shown a significantly higher concentration of fetal DNA in maternal plasma in some fetal aneuploidies and placental pathologies (2)(3)(4)(5)(6). Nevertheless, existing data about fetal DNA concentrations in maternal blood under physiologic and pathologic conditions refer only to singleton pregnancies, and reference values for twins are still missing. The origin of the fetal DNA released into maternal plasma is still unclear. Although there is evidence that some of the cell-free fetal DNA in the maternal plasma is derived from blood elements, several authors favor the hypothesis that the majority is derived from the placenta (7)(8). Interestingly, twin pregnancies may present with one or two placentas, and although dizygotic twins must have two placentas, monozygotic twins can be either mono- or bichorionic. It has been reported that monozygotic twins exhibit smaller uteroplacental junctional areas than do …

Droplet digital polymerase chain reaction for DNMT3A and IDH1/2 mutations to improve early detection of acute myeloid leukemia relapse after allogeneic hematopoietic stem cell transplantation.

Over the last decades, allogeneic hematopoietic stem cell transplantation (allo-HSCT) has considerably improved the outcome of acute myeloid leukemia (AML). Unfortunately, disease relapse remains a frequent occurrence, and a major cause of post-transplant mortality.[1][1] Most salvage treatments do

Evaluation of a panel of circulating DNA, RNA and protein potential markers for pathologies of pregnancy.

Abstract Background: Among markers of pregnancy complications, corticotropin-releasing hormone (CRH) mRNA, long pentraxin 3 (PTX3) protein and fetal and total DNA had been reported to be increased in the plasma of women with overt preeclampsia (PE). We developed an optimized protocol to evaluate whether concentrations of CRH mRNA, PTX3 mRNA and protein, fetal and/or total DNA are increased in fetal growth restriction (FGR), and whether they predict complications of pregnancy. Methods: The protocol included a preamplification step to enrich rare mRNA species. CRH and PTX3 mRNA, DNA and PTX3 protein were measured in the plasma of women with PE or FGR, in women at risk of developing these pathologies and in healthy women matched for gestational age. Results: CRH mRNA, fetal and/or total DNA and PTX3 protein were significantly increased in women with overt PE when compared to controls. Pregnant women who later developed PE or FGR during pregnancy showed total DNA levels that were significantly increased before the onset of both pathologies, while RNA markers were increased only in women who later developed PE. Conclusions: Our protocol for plasma RNA quantification may allow for the extension of a panel of predictive markers to be investigated in larger patient cohorts. Clin Chem Lab Med 2010;48:791–4.

Different approaches for noninvasive prenatal diagnosis of genetic diseases based on PNA-mediated enriched PCR

Abstract:  The aim of this work was to develop advanced and accessible protocols for noninvasive prenatal diagnosis of genetic diseases. We are evaluating different technologies for mutation detection, based on fluorescent probe hybridization of the amplified product and pyrosequencing, a technique that relies on the incorporation of nucleotides in a primer‐directed polymerase extension reaction. In a previous investigation, we have already proven that these approaches are sufficiently sensitive to detect a few copies of a minority‐mutated allele in the presence of an excess of wild‐type DNA, In this work, in order to further enhance the sensitivity, we have employed a mutant enrichment amplification strategy based on the use of peptide nucleic acids (PNAs). These DNA analogues bind wild‐type DNA, thus interfering with its amplification while still allowing the mutant DNA to become detectable. We have synthesized different PNAs, which are highly effective in clamping wild‐type DNA in the beta‐globin gene region, where four beta‐thalassemia mutations are located (IVSI.110, CD39, IVSI.1, IVSI.6) plus HbS. The fluorescence microchip readout allows us to monitor the extent of wild‐type allele inhibition, thus facilitating the assessment of the optimal PNA concentration.