Non-invasive Prenatal Diagnosis For Single Gene Disorders Research Articles

Applying amplification refractory mutation system technique to detecting cell-free fetal DNA for single-gene disorders purpose.

Non-invasive prenatal diagnosis for single-gene disorders (NIPD) is still in development and deserves further study. The advent of next-generation sequencing technology significantly improved the detection of multiple mutations for non-invasive prenatal diagnosis for single-gene disorder purposes. However, bespoke amplicon-based NGS assays are costly. In this study, we developed a new strategy for non-invasive prenatal screening for single-gene disorders based on a capillary electrophoresis (CE) platform using an amplification refractory mutation system (ARMS)-PCR technique. Allele-specific primers for several disease-correlated mutations were designed, and subsequently, sensitivity and specificity assays were conducted. Assays on simulated two-person DNA mixtures showed that three primers targeting the mutant allele could detect minor DNA components in 1:500 mixtures. All primers showed positive results at 0.01ng of the template DNA. Cell-free fetal DNA was extracted from a pregnant woman's peripheral blood for the detection of paternally inherited mutations. Our results showed that one primer successfully amplified the mutant allele of fetal DNA in maternal plasma, which was confirmed by genotyping the genomic DNA extracted from amniotic fluid. This study suggested that the ARMS-PCR technique, a fast and cost-effective method, might be a promising method used to target de novo or paternally inherited pathogenic mutations in maternal plasma.

Non-invasive prenatal diagnosis (NIPD) of cystic fibrosis: an optimized protocol using MEMO fluorescent PCR to detect the p.Phe508del mutation

BackgroundAnalysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches. MethodsTo make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3′-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence. ResultsWe collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses. ConclusionsThis NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF.

Client Views and Attitudes to Non‐Invasive Prenatal Diagnosis for Sickle Cell Disease, Thalassaemia and Cystic Fibrosis

In the near future the availability of non-invasive prenatal diagnosis (NIPD) for single gene disorders will change the prenatal diagnosis options available to couples who are carriers of conditions such as cystic fibrosis, sickle cell disorder and thalassaemia. Client opinions about NIPD are needed to inform the implementation of NIPD for single gene disorders. This qualitative study used two focus groups (n = 12) and one-to-one interviews (n = 16) with carriers and support group representatives of sickle cell disease, thalassaemia and cystic fibrosis. Discussions were digitally recorded, transcribed verbatim and analysed using thematic analysis. Opinions about NIPD were very positive and participants valued the opportunity to have safe and early testing. Uptake of prenatal testing is likely to increase as women who had previously declined invasive testing expressed interest in having NIPD. Participant concerns about NIPD centred on the need for accuracy to be high to be used for subsequent decision making about termination of pregnancy. Participants also raised concerns that less thought may be given to having a blood test compared to an invasive test and that the perceived ease of a blood test may bring increased pressure to have testing. Participants thought NIPD should be offered through existing specialist services to ensure appropriate genetic counseling and support. Maintaining all testing options is important as some people may prefer invasive testing over NIPD if invasive testing was more accurate or if invasive testing could give information about other conditions such as Down syndrome.

Views and preferences for the implementation of non‐invasive prenatal diagnosis for single gene disorders from health professionals in the united kingdom

Non-invasive prenatal diagnosis (NIPD) will offer new options in prenatal diagnosis for carriers of single gene disorders. This will affect carrier couples and health professionals involved in prenatal care. The aim of this study was to determine health professional opinions on NIPD for single gene disorders to guide development of infrastructure required for implementation. A qualitative approach was adopted using focus groups (N = 17) and one-to-one interviews (N = 30) with health professionals from a range of backgrounds involved in caring for carriers of single gene disorders. Data were digitally recorded, transcribed verbatim and analyzed using thematic analysis. Participants were very positive about the introduction of NIPD, describing benefits arising from no risk of miscarriage, earlier testing and a simple procedure. A number of concerns for implementation were raised. Participants emphasized the need for the new test to be highly accurate and thoroughly validated. There was concern that people may not give as much thought to having a blood test compared to an invasive test or that it may be viewed as routine and as such NIPD may negatively impact on informed consent. In addition there was concern that the simplicity of a blood test may lead to increased pressure to test and terminate. However, participants felt these concerns could be overcome with thorough pre- and post-test counseling. To ensure high quality care, offering NIPD through genetics or other specialist services is essential. Ongoing education and training of health professionals will be important, and guidelines and regulation are needed for effective implementation.