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Background: Fetal sex determination is useful for families at risk of X-linked disorders, such as Duchenne muscular dystrophy, adrenal hypoplasia, hemophilia. At first, this could be obtained through invasive procedures such as amniocentesis and chorionic villus sampling, having a 1% risk of miscarriage. Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma, noninvasive prenatal testing permits the early diagnosis of fetal sex through analysis of cffDNA. However, the low amount of cffDNA relative to circulating maternal DNA requires highly sensitive molecular techniques in order to perform noninvasive prenatal diagnosis. In this context we employed droplet digital PCR (ddPCR) in order to evaluate the earliest possible fetal sex determination from circulating DNA extracted from plasma of pregnant women at different gestational ages., Methods: We identified the fetal sex on cffDNA extracted from 29 maternal plasma samples at early gestational ages, several of them not suitable for qPCR determination, using ddPCR designed for SRY gene target., Results: All maternal plasma samples were determined correctly for SRY gene target using ddPCR even at very early gestational age (prior to 7 weeks)., Conclusions: The ddPCR is a robust, efficient and reliable technology for the earliest possible fetal sex determination from maternal plasma.

Non-invasive prenatal diagnosis (NIPD) is based on fetal DNA analysis starting from a simple peripheral blood sample, thus avoiding risks associated with conventional invasive techniques. During pregnancy, the fetal DNA increases to approximately 3–13% of the total circulating free DNA in maternal plasma. The very low amount of circulating cell-free fetal DNA (ccffDNA) in maternal plasma is a crucial issue, and requires specific and optimized techniques for ccffDNA purification from maternal plasma. In addition, highly sensitive detection approaches are required. In recent years, advanced ccffDNA investigation approaches have allowed the application of non-invasive prenatal testing (NIPT) to determine fetal sex, fetal rhesus D (RhD) genotyping, aneuploidies, micro-deletions and the detection of paternally inherited monogenic disorders. Finally, complex and innovative technologies such as digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) (exhibiting higher sensitivity and/or the capability to read the entire fetal genome from maternal plasma DNA) are expected to allow the detection, in the near future, of maternally inherited mutations that cause genetic diseases. The aim of this review is to introduce the principal ccffDNA characteristics and their applications as the basis of current and novel NIPT. [ABSTRACT FROM AUTHOR]

The β-thalassemias are genetic disorder caused by more than 200 mutations in the β-globin gene, resulting in a total (β0) or partial (β+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for β-thalassemia are: β039, β+IVSI-110, β+IVSI-6 and β0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, TaqMan® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human β-globin gene. Four TaqMan® genotyping assays for the most common β-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 β-thalassemia patients. In addition, 15 specimens at late gestation (21–39 gestational weeks) and 11 at early gestation (5–18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations β039, β+IVSI-110, β+IVSI-6, β0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of β-globin gene mutations, starting from the 9th gestational week. [ABSTRACT FROM AUTHOR]

Shwachman–Diamond syndrome (SDS) is a rare inherited bone marrow failure syndrome, resulting in neutropenia and a risk of myeloid neoplasia. A mutation in a ribosome maturation factor accounts for almost all of the cases. Lymphoid involvement in SDS has not been well characterized. We recently reported that lymphocyte subpopulations are reduced in SDS patients. We have also shown that the mTOR-STAT3 pathway is hyper-activated in SDS myeloid cell populations. Here we show that mTOR-STAT3 signaling is markedly upregulated in the lymphoid compartment of SDS patients. Furthermore, our data reveal elevated IL-6 levels in cellular supernatants obtained from lymphoblasts, bone marrow mononuclear and mesenchymal stromal cells, and plasma samples obtained from a cohort of 10 patients. Of note, everolimus-mediated inhibition of mTOR signaling is associated with basal state of phosphorylated STAT3. Finally, inhibition of mTOR-STAT3 pathway activation leads to normalization of IL-6 expression in SDS cells. Altogether, our data strengthen the hypothesis that SDS affects both lymphoid and myeloid blood compartment and suggest everolimus as a potential therapeutic agent to reduce excessive mTOR-STAT3 activation in SDS. [ABSTRACT FROM AUTHOR]

Background: Fetal sex determination is useful for families at risk of X-linked disorders, such as Duchenne muscular dystrophy, adrenal hypoplasia, hemophilia. At first, this could be obtained through invasive procedures such as amniocentesis and chorionic villus sampling, having a 1% risk of miscarriage. Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma, noninvasive prenatal testing permits the early diagnosis of fetal sex through analysis of cffDNA. However, the low amount of cffDNA relative to circulating maternal DNA requires highly sensitive molecular techniques in order to perform noninvasive prenatal diagnosis. In this context we employed droplet digital PCR (ddPCR) in order to evaluate the earliest possible fetal sex determination from circulating DNA extracted from plasma of pregnant women at different gestational ages. Methods: We identified the fetal sex on cffDNA extracted from 29 maternal plasma samples at early gestational ages, several of them not suitable for qPCR determination, using ddPCR designed for SRY gene target. Results: All maternal plasma samples were determined correctly for SRY gene target using ddPCR even at very early gestational age (prior to 7 weeks). Conclusions: The ddPCR is a robust, efficient and reliable technology for the earliest possible fetal sex determination from maternal plasma. [ABSTRACT FROM AUTHOR]