Cell-free fetal DNA ( cffDNA ) is the fetal DNA that circulates freely in the mother's blood. Maternal blood is sampled with venipuncture. Analysis of cffDNA is a non-invasive prenatal diagnostic method that is often reserved for expectant mothers. Two hours after delivery, cffDNA is no longer detectable in the mother's blood.
Video Cell-free fetal DNA
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cffDNA comes from the trophoblast of the placenta. Fetal DNA is fragmented when the placental microparticle is passed into the maternal blood circulation.
cffDNA fragments of about 200 base pairs (bp) in length. They are significantly smaller than maternal DNA fragments. The size difference allows cffDNA to be distinguished from maternal DNA fragments.
About 11 to 13.4 percent of the cell-free DNA in the mother's blood is the origin of the fetus. The amount varies from one pregnant woman to another. cffDNA is present after five to seven weeks of pregnancy. The amount of cffDNA increases as pregnancy progresses. The amount of cffDNA in the mother's blood decreases rapidly after delivery. Two hours after delivery, cffDNA is no longer detectable in the mother's blood.
Analysis of cffDNA can provide a diagnosis of fetal conditions earlier than current techniques. Because cffDNA is found in maternal blood, sampling does not carry a risk of spontaneous abortion. The cffDNA analysis has the same ethical and practical problems as other techniques such as amniocentesis and chorionic villus sampling.
Some disadvantages of cffDNA sampling include low cffDNA concentrations in maternal blood; variation in the quantity of cffDNA between individuals; high concentration of maternal-free DNA cells compared with cffDNA in maternal blood.
New evidence suggests that failure rates of cffDNA tests are higher, fetal fractions (fetal DNA versus maternal proportions in maternal blood samples) are lower and PPV for trisomy 18, 13 and SCA decreases in IVF pregnancy compared with those spontaneously conceived.
Maps Cell-free fetal DNA
Laboratory methods
A number of laboratory methods have been developed for cell-free fetal DNA screening for genetic defects have been developed. The main ones are (1) sequenced massive shotgun sequencing (MPSS), (2) large massive sequencing targets (t-MPS) and (3) single nucleotide polymorphism (SNP) approaches.
The mother's peripheral blood sample was taken with venesection in about ten weeks of pregnancy.
Separation of cffDNA
Blood plasma is separated from mother's blood sample using laboratory centrifuge. CffDNA is then isolated and purified. The standard protocol for doing this is written through an evaluation of the scientific literature. The highest results in cffDNA extraction were obtained with "QIAamp DSP Virus Kit".
The addition of formaldehyde to the mother's blood sample increases the cffDNA yield. Formaldehyde stabilizes intact cells, and therefore inhibits further release of maternal DNA. With the addition of formaldehyde, the percentage of cffDNA recovered from maternal blood samples varied between 0.32 percent and 40 percent with an average of 7.7 percent. Without the addition of formaldehyde, the average percentage of cffDNA recovered has been measured at 20.2 percent. However, other numbers vary between 5 and 96 percent.
The recovery of cffDNA may be related to the length of the DNA fragment. Another way to increase fetal DNA is based on the physical length of the DNA fragment. Smaller fragments can represent up to seventy percent of total cell-free DNA in a maternal blood sample.
Analysis of cffDNA
In real-time PCR, fluorescent probes are used to monitor the accumulation of amplicons. The reporter fluorescent signal is proportional to the amount of amplicon produced. The most appropriate real time PCR protocols are designed according to mutations or specific genotypes to be detected. Point mutations were analyzed by qualitative real time PCR by using allele-specific probes. insertion and removal were analyzed by dose measurements using real time quantitative PCR.
cffDNA can be detected by finding DNA sequences that are paternally derived via polymerase chain reaction (PCR).
Real time PCR
genes Y sex-determination (SRY) and short Y tandem chromosomes repeating "DYS14" in cffDNA from 511 pregnancies were analyzed using a quantitative real-time PCR (RT-qPCR). In 401 of 403 pregnancies in which maternal blood is taken at seven weeks or more of pregnancy, both segments of DNA are found.
Nested PCR
The use of nested polymerase chain reactions (PCR nesting) is evaluated to determine sex by detecting a specific chromosome Y signal in cffDNA from maternal plasma. Nested PCR detected 53 of 55 male fetuses. The cffDNA of plasma 3 of 25 women with female fetuses contain a special Y chromosome signal. The PCR sensitivity lodged in this experiment was 96 percent. The specification is 88 percent.
PCR Digital
The microfluidic device allows the quantification of cffDNA segments in maternal plasma with accuracy beyond that of real-time PCR. Point mutations, loss of heterozygosity and aneuploidy can be detected in one PCR step. Digital PCR can differentiate between maternal blood plasma and multiplex DNA of the fetus.
Shotgun sequencing
High absorption shotgun rifles using tools such as Solexa or Illumina, produce about 5 million sequence tags per maternal serum sample. Aneuploid pregnancies such as trisomy are identified when testing during the fourteenth week of pregnancy. The entire genome mapping of the fetus by parental haplotip analysis was solved using cffDNA sequencing of maternal serum. Pregnant women were studied using maternal DNA sequences of 2-step parallel DNA and trisomy were diagnosed with a z-score of more than 3. The sequencing gave 100 percent sensitivity, 97.9 percent specificity, 96.6 percent positive predictive value and 100 percent positive predictive value.
Bulk Spectrometry
Matrix assisted matrix/ionization-time-of-flight matrix (MALDI-TOF MS) combined with single base expansion after PCR allows cffDNA detection with single base specificity and single-molecule DNA sensitivity. DNA is amplified by PCR. Then, linear amplification with a basic extension reaction (with a third primer) is designed for anneal to the upstream region of the mutation site. One or two bases are added to the extension primer to produce two extension products of wild-type DNA and mutant DNA. The specificity of a single base provides advantages over hybridization-based techniques using the TaqMan hydrolysis probe. When assessing the technique, no false or negative positives were found when looking for cffDNA to determine fetal sex in sixteen samples of maternal plasma. The gender of ninety-ninety-one male fetuses was detected correctly using MALDI-TOF mass spectrometry. This technique has accuracy, sensitivity and specificity of more than 99 percent.
Epigenetic modifications
Differences in gene activation between maternal and fetal DNA can be exploited. epigenetic modification (a heritable modification that alters gene function without altering DNA sequence) can be used to detect cffDNA. Hypermethylated RASSF1A promoter is a universal fetal marker used to confirm the presence of cffDNA. A technique described where cffDNA is extracted from maternal plasma and then digested with restriction enzymes is sensitized and insensitive. Then, real-time PCR analysis from RASSF1A, SRY, and DYS14 was performed. The procedure detects 79 of the 90 (88 percent) maternal blood samples in which RASSF1A hypermethylation is present.
mRNA
Transcripts of mRNA from genes expressed in the placenta are detected in maternal plasma. In this procedure, the plasma is centrifuged so that an aqueous layer appears. This layer is transferred and from it RNA is extracted. RT-PCR is used to detect selected RNA expression. For example, human placental lactogen (hPL) and beta-hCG mRNA are stable in maternal plasma and can be detected. (Ng et al., 2002). This may help to confirm the presence of cffDNA in the mother's plasma.
Apps
Prenatal sex acuity
The cffDNA analysis of maternal plasma samples allows for prenatal sex assertion. Prenatal sex contraceptive applications include:
- Disease test : Whether the sex of a male or female fetus allows the determination of the risk of X-linked recessive genetic disorders in certain pregnancies, especially where the mother is a carrier of genetic disorders.
- Preparation , for all aspects of childcare depending on gender.
- Sex selection , which after preimplantation genetic diagnosis can be performed by selecting only the embryo of the desired sex, or, after the post-implantation method by sex- selective abortion depends on the test results and personal preference.
Compared with unreliable obstetric ultrasonography for first-trimester gender determination and amniocentesis that carries a small risk of miscarriage, maternal plasma sampling for riskless cffDNA analysis. The primary target in the cffDNA analysis is the gene responsible for the Y protein that determines the sex (SRY) on the Y chromosome and the order of DYS14.
Congenital adrenal hyperplasia
In congenital adrenal hyperplasia, the adrenal cortex does not have the correct corticosteroid synthesis, causing excess androgen to adrenal and affecting the female fetus. There is an external masculinization of the genitals in the female fetus. The mother of the fetus is at risk for dexamethasone at 6 weeks' gestation to suppress the release of androgen glands in the pituitary.
If the cffDNA analysis obtained from maternal plasma samples does not have a genetic marker found only on the Y chromosome, then this indicates a female fetus. However, it may also indicate the failure of the analysis itself (false negative results). Paternal genetic polymorphisms and independent-sex markers can be used to detect cffDNA. A high degree of heterozygosity from this marker must exist for this application.
Daddy line test
Prenatal DNA paternity testing is commercially available. The test can be done at nine weeks of pregnancy.
Single gene disorder
A single dominant and recessive autosomal dominant gene disorder has been diagnosed prenatally by analyzing paternally inherited DNA including cystic fibrosis, beta thalassemia, sickle cell anemia, spinal muscular atrophy, and myotonic dystrophy. A prenatal diagnosis of a single gene abnormality caused by an autosomal recessive mutation, a maternal inherited autosomal dominant mutation or a large sequence mutation that includes duplication, expansion or insertion of DNA sequences is more difficult.
In cffDNA, fragments along the 200 - 300 bp involved in single gene disorders are more difficult to detect.
For example, an autosomal dominant condition, achondroplasia is caused by a mutation of the FGFR3 gene. In two pregnancies with a fetus with achondroplasia there was found a mutated G1138A mutation by solder from cffDNA from a maternal plasma sample in one and a de novo G1138A mutation from the other.
In Huntington's chorea genetic studies using qRT-PCR cffDNA from maternal plasma samples, CAG repetitions have been detected at normal levels (17, 20 and 24).
cffDNA can also be used to diagnose single gene disorders. Development in a laboratory process using cffDNA allows prenatal diagnosis of aneuploidies such as trisomy 21 (Down syndrome) in the fetus.
Hemolytic disease of the fetus and newborn
The incompatibility of fetal and maternal RhD antigens is a major cause of hemolytic disease in newborns. About 15 percent of Caucasian women, 3 to 5 percent of black African women and less than 3 percent of Asian women are RhD negative.
An accurate prenatal diagnosis is important because the disease can be fatal for the newborn and because treatment including intramuscular immunoglobulin (Anti-D) or intravenous immunoglobulin may be given to the mother at risk.
PCR to detect RHD (gene) gene exons 5 and 7 of cffDNA obtained from maternal plasma between 9 and 13 weeks of pregnancy provides a high level of specificity, diagnostic sensitivity and accuracy (& gt; 90 percent) when compared with RhD determination of cord blood newborn center. serum. Similar results were obtained by targeting exons 7 and 10. Dropplet digital PCR in the determination of fetal RhD was comparable to the real-time PCR routine technique.
Regular determination of fetal RhD status of cffDNA in maternal serum allows early management of risky pregnancies while reducing unnecessary anti-D use by up to 25 percent.
Aneuploidy
- Sex chromosomes
Maternal serum cffDNA serum analysis with high-throughput sequences can detect common fetal sex chromosomal anusuploids such as Turner syndrome, Klinefelter syndrome and triple X syndrome but a positive predictive value of this procedure is low.
- Trisomy 21
The trisomy of the chromosome 21 fetus is the cause of Down's syndrome. This trisomy can be detected by cffDNA analysis of maternal blood by sequencing massive shotgun sequencing (MPSS). Another technique is the digital analysis of the selected area (DANSR). However, the tests show an inconsistent degree of sensitivity and specificity and therefore may be best used to confirm positive maternal screening tests such as ultrasound markers of the condition.
- Trisomies 13 and 18
Analysis of cffDNA from maternal plasma with MPSS looking for trisomy 13 or 18 is possible
Factors that limit sensitivity and specificity include cffDNA levels in maternal plasma; maternal chromosomes may have mosaicism.
A number of fetal nucleic acid molecule derived from aneuploid chromosome can be detected include SERPINEB2 mRNA, clad B, hypomethylated SERPINB5 of chromosome 18, placenta-specific 4 (PLAC4), hypermethylated holocarboxylase synthetase (HLCS) and c21orf105 mRNA of complete trisomy of chromosome 12. With, allelic mRNA in maternal plasma is not a ratio of 1: 1 is normal, but it is actually 2: 1 ratio of allelic determined by epigenetic markers may also be used to detect trisomy complete. Massive parallel sequences and digital PCR for fetal aneuploidy detection can be used without limitation of fetal specific nucleic acid molecules. (MPSS) is estimated to have a sensitivity between 96 â € <â €
Preeclampsia is a complex condition of pregnancy involving hypertension and proteinuria usually after 20 weeks of pregnancy. This is associated with a poor cytotrophoblastic invasion of the myometrium. The onset of conditions between 20 and 34 weeks of pregnancy, is considered "early". Maternal plasma samples in pregnancy complicated by preeclampsia had significantly higher cffDNA levels than those in normal pregnancies. This applies to early-onset preeclampsia.
Future perspective
New generation sequencing can be used to generate a whole genome sequence of cffDNA. This raises ethical questions. However, the usefulness of this procedure can be increased when a clear association between specific genetic variants and disease status is found.
See also
- Trial three times
- Quad Test
References
Source of the article : Wikipedia
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