We further calculated the ratio of the number of mutant alleles to the total number of mutant and wild-type alleles, denoted by P r , present in the plasma samples. The P r is dependent on the fractional fetal DNA concentration in the samples. We calculated the fractional fetal DNA concentration as previously described.
Because there was less than one template molecule per reaction well, the actual number of template molecules distributed to each reaction well followed the Poisson distribution. A count indicates number of wells positive for the A allele; and T count, number of wells positive for the T allele.
Average reference template concentration per PCR well. The reference template referred to the allele with the lesser count in each sample. Two additional well digital PCR sets were performed after which classification could be made.
We used the sequential probability ratio test SPRT to determine whether the dosage imbalance between the mutant and the wild-type alleles in maternal plasma was statistically significant. The 2 hypotheses were 1 the mutant allele was over-represented when compared to the wild-type allele, and 2 the mutant allele was underrepresented when compared to the wild-type allele.
SPRT was performed by constructing a pair of curves, which defined the probabilistic boundaries for accepting or rejecting the hypotheses Figure 2 B.
Hypothesis 1 or 2 was accepted if the experimental P r was above the upper boundary or below the lower boundary, respectively.
The equations for calculating the SPRT boundaries were adapted from El Karoui et al, 23 with the level of statistical confidence adjusted to a threshold likelihood ratio of 8. The cutoff P r values were dependent on the fractional fetal DNA concentration as described above, as well as the average reference template concentration per PCR well m r. We used digital PCR to measure the concentration difference between the total amount maternal- plus fetal-derived of mutant and wild-type alleles in the plasma of heterozygous pregnant women carrying male fetuses.
Because a male fetus possesses a single chromosome X, the relative concentration between the wild-type and the mutant allele is always in dosage imbalance Figure 2 A. An overrepresentation or underrepresentation of the mutant allele represents an affected or nonaffected fetus, respectively.
We used SPRT to test for dosage imbalance. Samples with data points above the upper curve or below the lower curve were classified as affected or nonaffected, respectively. Samples with data points in between the 2 curves were not classified because of insufficient statistical power, and additional digital PCRs were performed. Digital RMD for noninvasive detection of X-linked diseases in maternal plasma.
The male fetus inherits either the mutant allele M or the wild-type allele N from his mother, leading to an overrepresentation of the M or the N allele, respectively, in maternal plasma. B SPRT for hemophilia detection. Samples with mutant allele proportion P r above the upper boundary and below the lower boundary are classified as mutant and wild-type, respectively.
Samples with P r between 2 curves are unclassifiable and require additional digital PCR analysis. The current RMD analysis is relevant to at-risk pregnant cases, ie, pregnant women who are heterozygous for mutations on chromosome X and are carrying male fetuses.
Hence, we studied the plasma samples from 10 pregnant women who were heterozygous for the SNP on chromosome X and were carrying male fetuses. We developed an allele-discriminative digital real-time PCR assay to measure the concentrations of the A- and T-allele in each sample.
The digital RMD result is shown in Table 2. Hence, the result confirmed the feasibility of the digital RMD strategy. We next applied the digital RMD approach for hemophilia mutation detection.
We developed 7 duplex digital real-time PCR assays to detect 3 mutations in the F8 gene, 4 mutations in the F9 gene, and their corresponding wild-type counterparts. We evaluated the performance of the digital PCR assays by constructing artificial DNA mixtures that simulated the composition of maternal plasma samples with a minority male fetal DNA component among a majority maternal DNA background.
The artificial mixtures were constructed to simulate the fetal and maternal DNA compositions in maternal plasma. SNP genotypes were determined by mass spectrometry. Fetal DNA was obtained from the placenta of a normal male fetus. Maternal DNA was obtained from the blood cells of pregnant women heterozygous for the corresponding mutations. Mutant count indicates the number of wells positive for the mutant allele; and wild-type count, the number of wells positive for the wild-type allele.
We tested the digital RMD method for detecting fetal genotypes for the hemophilia mutations through maternal plasma DNA analysis. We performed digital PCR on 12 plasma samples obtained from 7 pregnant women heterozygous for the causative mutations Table 1.
All of the cases involved male fetuses. The digital RMD results are shown in Table 4. The fetal genotypes were correctly classified in all studied cases by the SPRT algorithm supplemental Figure 1. Hence, the degree of quantitative difference between the amount of mutant and the wild-type alleles was too small to be classified with data from one well digital PCR set. Additional well digital PCR sets were therefore performed until classifications could be made.
Mutant count, number of wells positive for the mutant allele. Wild-type count, number of wells positive for the wild-type allele. As controls, we also studied 5 maternal plasma samples obtained from normal pregnant women by using each of the mutation-specific assays.
As shown in supplemental Table 3, no mutant alleles were detected in most of the cases. For 6 of the 35 studied maternal plasma cases, the positive wells containing the mutant alleles constituted less than 0. These positive signals might have resulted from cross hybridizations of the fluorescent probes during PCR. Nonetheless, such low numbers of mutant-positive wells would not skew the allelic ratio between mutant and wild-type alleles to an extent that would alter the RMD classification by SPRT.
Current prenatal diagnosis for hemophilia largely relies on invasive procedures such as chorionic villus sampling, which poses a finite risk to the fetuses. However, invasive diagnostic testing is still required for one-half of the pregnant cases involving male fetuses. In this study, we have developed a noninvasive prenatal diagnostic strategy to directly detect causative mutations carried by male fetuses in at-risk pregnancies. By using the digital RMD approach for genetic loci on chromosome X, we have accurately identified the mutant or the wild-type alleles inherited by the male fetuses in all of the 12 studied maternal plasma samples from 7 pregnant carriers of hemophilia Table 4.
The fetal genotypes could be detected as early as the 11th week of gestation Table 1 , demonstrating the potential for early diagnostic use of the method. It is important to know as soon as possible after birth whether a baby has hemophilia so that special steps can be taken to prevent bleeding complications for the baby. Mothers who carry the hemophilia gene are at risk for serious bleeding after delivery.
This is because the high levels of factor VIII during pregnancy fall back to lower levels after delivery. If the woman has low levels of factor IX, then she can bleed after delivery or surgery, such as Cesarean section. Some women have bleeding from the birth canal that lasts a long time.
This is called postpartum hemorrhage and can require treatment to stop the bleeding. Cord blood can be used to test for clotting proteins. This should be repeated when the baby is 6 months of age to confirm the diagnosis of hemophilia. Learn more about testing and diagnosis. Some parents choose to have their baby boys circumcised removing the foreskin from the penis.
Bleeding from circumcision is the most common cause of bleeding among babies with hemophilia. It can occur days after the procedure is done and, for babies who have not been diagnosed already, often leads to the initial hemophilia diagnosis. In the baby who may have hemophilia, avoid circumcision if possible. However, if circumcision is done, then a pediatric hematologist a doctor who specializes in blood should be consulted before the procedure to ensure that the child receives proper treatment to prevent excessive bleeding.
However, doctors perform prenatal tests for hemophilia, such as amniocentesis or chorionic villus sampling, only after they identify a specific genetic mutation in a parent or a close relative with the condition. NYU Langone genetic counselors can help you understand the results of these tests. Amniocentesis is usually performed early in the second trimester of pregnancy. Chorionic villus sampling can be performed after the 11th week of pregnancy. During this test, the doctor threads a thin tube through the vagina and cervix and into the placenta—a temporary organ that delivers oxygen and nutrients to an unborn child—to remove a small amount of tissue.
During a physical exam, the doctor asks you for a detailed account of any symptoms you may have noticed in your child, such as nosebleeds, unusual bruises, or blood in the urine or stool.
The doctor also examines your child for raised bruises or swelling around the joints, and to see how much movement your child has in the joints. Doctors use blood tests to screen for and confirm a diagnosis of hemophilia. Children with a longer-than-usual clotting time may not have enough clotting factors. The more a child has, the better the blood is able to clot. For children who have family members with unusually heavy menstrual periods, blood tests may also be used to measure levels of von Willebrand factor, another protein involved in clotting.
Doctors can confirm a diagnosis of hemophilia and determine the severity of the condition based on the levels of these factors. The disorder usually is inherited, although it also can be acquired. Severe hemophilia often is diagnosed within the first year of life, while milder forms of the disease may go unnoticed until adulthood.
Expectant parents with a family history of the disorder may request their unborn child to be tested. Amniocentesis and chorionic villus sampling are two prenatal screening tests that can be used to identify genetic mutations associated with hemophilia in the unborn fetus.
Amniocentesis consists of using a needle to remove a small amount of amniotic fluid from the womb. The fluid is examined in the lab for the presence of hemophilia-causing genetic mutations. It typically is performed during the second trimester of pregnancy.
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