Category Archives: DNA


Determining a baby’s gender in the lab

Male Female It's rare that I come across a pregnant woman who doesn't already know the gender of the baby she is carrying.  Ultrasound assessment of a fetus is routinely performed during pregnancy to identify fetal anomalies and, very often, gender identification is offered.  After the 13th week of pregnancy, ultrasound is quite accurate in determining a baby's sex.  There are circumstances, however, where knowing a fetus is a boy or a girl is important.  For example, male fetuses born to women who are carriers of X-linked genetic disorders are at risk of inheriting the disorder.  In such instances, knowledge of a fetus' gender before 13 weeks can be advantageous.

Today, the Journal of the American Medica Association (JAMA) published a systematic review and meta-analysis (a systematic method of evaluating statistical data based on results of several independent studies of the same problem) that sought to determine the performance of DNA tests performed on maternal blood to determine fetal sex.  As a reminder, DNA from a fetus circulates in maternal blood.  This cell-free fetal DNA can be detected and amplified and is the basis of other emerging diagnostic tests.

This report evaluated the results from 57 different studies that tried to determine fetal gender using a test performed on a sample of mother's blood or urine.  The 57 reports contained results from 6,541 women; 54% who delivered boys and 46% who delivered girls.  Because this was a meta-analysis, several different testing techniques were used in the included studies but all of them looked for the presence or absence Y chromosome-specific molecular markers.  If those markers were detected, the fetus predicted to be a male.  If they were not detected, the fetus was predicted to be a female.

Collectively, the tests performed very well.  They correctly identified 95.4% of the male fetuses (test sensitivity) and 98.6% of the female fetuses (test specificity).  The tests did not work well if maternal urine was used as the test sample which is not surprising because cell-free fetal DNA is very difficult to detect in urine.  The report also noted that real-time quantitative PCR (RTQ-PCR) methods had better performance than conventional PCR techniques.

As might be expected, test performance improved along with the gestational age of the fetus.  At less than 7 weeks test sensitivity (see above for definition) was 74.5% but increased to 95% after 7 weeks at was 99.6% at more than 20 weeks.  That makes sense because there is more fetal DNA circulating in the mom's blood as the pregnancy progresses and the fetus increases in size.

Because of the potential clinical use of the test to identify male fetuses of mother's with X-linked genetic diseases, the authors caution that these types of sex-predicting tests should be optimized to achieve high sensitivity (detection of male fetuses) even if it compromises the specificity (detection of female fetuses).  They also cautioned that any tests developed and offered by laboratories should thorough validate that a negative (i.e. female fetus) test result reflects the presence of a female fetus and not simply the inability to detect any fetal DNA (which would also give a negative (female) result).

While there are some direct-to-consumer labs that offer this type of DNA-based testing to determine fetal sex, the test is marketed as one of convenience to satisfy a mother's curiosity regarding her baby's gender.  Currently, I am unaware of any clinical laboratories in the United States that offer such testing for diagnostic purposes although the tests are available for that reason in the European Union.  I predict that this type of test will become more widely available in clinical labs over the next few years.  There is much promise to be realized from tests based on circulating cell-free DNA!

Noninvasive antenatal testing using cell free fetal DNA

Today's post is by a guest author, Joshua Cook, M.D.  Dr. Cook is a pathology resident at the University of Utah.

Fetal cells, largely of placental origin, circulate freely in the mother’s bloodstream during gestation.  In some cases fetal cells are detectible in maternal blood for years post delivery.  Cell free fetal DNA (cffDNA) is also present in the peripheral blood of pregnant women, though it disappears rapidly at delivery.  cffDNA appears early in the first trimester and tends to increase in concentration as gestation progresses.  A number of studies over the last decade have shown this naked fetal DNA provides a noninvasive method for rendering prenatal diagnoses without risk to the fetus or mother.

Consider hemolytic disease of the newborn (HDN).  About 10% of pregnancies in Caucasian RBCs populations involve a rhesus Rh(D)- negative mother carrying an Rh(D)-positive fetus.  Such a mother is at risk of becoming sensitized, or isoimmunized, to Rh(D)-positive red blood cells following exposure to fetal blood. 

Small physiologic fetal-maternal hemorrhages commonly occur at delivery or during miscarriage.  Exposure of the mother’s immune system to fetal red blood cells may result in the generation of maternal antibodies to fetal antigens (sensitization or isoimmunization).  These antibodies may then cross the placenta in subsequent pregnancies and harm a future baby.  In pregnancies with Rh(D) incompatibility [Rh(D)-negative mom with Rh(D)-positive fetus] the mother has historically had about a 10% risk of becoming sensitized. 

Knowledge of the fetal RHD genotype in Rh(D) negative women greatly simplifies antenatal management.  Modern medical practice requires that immunoprophylaxis (anti-D IgG immunoglobulin; aka RhoGAM) routinely be given to all non-sensitized RhD-negative woman in pregnancy to prevent isoimmunization.  This had led to a dramatic decrease in HDN since the 1960’s.  When administered, the anti-Rh(D) antibodies circulate in the mother’s bloodstream and prevent her immune system from reacting to Rh(D)-positive fetal blood.  The standard of care is to provide anti-Rh(D) administration at 26 to 28 weeks of gestation, and after suspected antepartum fetal-maternal hemorrhage (i.e. amniocentesis, chorionic villous sampling, pregnancy loss, ectopic pregnancy).  Anti-D IgG is also given with within 72 hours of delivery if the baby is found to be Rh(D)-positive.

Because the Rh(D) status of the fetus is usually not known prior to birth, about 40% of immunoprophylaxis is given unnecessarily to Rh(D)-negative mothers who are carrying Rh(D)-negative fetuses.  cffDNA testing restricts immuneprophylaxis to the 60% of pregnancies with a true Rh(D)-positive fetus.  Fetal RHD genotyping using maternal plasma is about 95% accurate at 11 -13 weeks gestation and is nearly 99.8% accurate by 26 weeks (when the first dose of immuneprophlyaxis is administered).  Accuracy improves throughout gestation because the amount of cffDNA in maternal peripheral blood increases with gestational age. 

cffDNA testing is currently used in Europe to identify high risk pregnancies [i.e. Rh(D)-positive fetus  in a previously sensitized mother] which benefit from intensive antenatal monitoring.  cffDNA RHD genotyping is used to determine the risk of chorionic villous sampling (CVS) in sensitized women.  CVS is a technique used in the prenatal diagnosis of fetal aneuploidies (such as Down syndrome) and other inherited genetic disorders; however it is relatively contraindicated in isoimmunized women since procedure related fetal-maternal hemorrhage exacerbates pre-existing sensitization.  If the fetus lacks Rh(D) then boosting the maternal amnestic response is not an issue.

Cost and outcome studies for noninvasive cffDNA prenatal screening are in progress, but obstetricians may soon have a powerful new tool in their armamentarium.



A perfect Down syndrome screening test?

In a January post, I wrote about a possible new Down syndrome screening test that detected 100% of affected fetuses and had a very low false-positive rate.  Recently a different group of scientists in Cyprus reported they had developed a perfect test to detect Down syndrome: one that correctly detects all Down sydrome fetuses but had zero false positive results.

While the earlier report utilized a time-consuming and expensive technique called massively parallel sequencing, this new study focused on a process called DNA methylation.  Methyl groups are chemical structures that are naturally attached to regions of DNA.  They function to turn genes on or off.  Because DNA from fetuses have different methylation patterns compared to their mothers', fetal DNA can be distinguished from mom DNA in a blood sample taken from the mother.

After enrichment of the methylated fetal DNA from the mother's blood, quantitative PCR was used to amplify specific methylated regions of chromosome 21.  Fetuses with Down syndrome were identified because they have three, rather than two, copies of chromosome 21, and the increased copies were readily detected by the test.  Out of 40 women who were 11 to 14 weeks pregnant when tested, the method was 100% accurate: all of the 14 Down syndrome and all of the 26 unaffected fetuses were correctly identified.

In contrast to a sequencing approach, the techniques used in this study are rather easy and fast to perform and don't require expensive equipment or software.  It is also a lot cheaper.

This is a promising test but there's still a lot more to be done before it makes the leap into clinical use.  The Cyprus researchers are planning to do larger-scale clinical trials with many more women.  Let's hope that the results from those trials are as exciting as this early report.

New blood test for Down syndrome screening

This is impressive!  A study reported yesterday in the British Medical Journal describes a new blood test that could vastly decrease the number of invasive procedures that are currently used to confirm if a fetus has Down syndrome.

Current blood tests used for Down syndrome screening can identify up to 90-95% of Down syndrome pregnancies but about 5% of normal fetuses are incorrectly identified as having Down syndrome.  That means that lots of women have follow-up tests, like amniocentesis, to definitively determine if their baby is affected.  That's a problem because amniocentesis is an invasive procedure that can lead to the loss of the pregnancy.  Current estimates put the risk of fetal loss due to amniocentesis at about 1 out of 300. 

914335_85510317 This new blood test measures the amount of DNA from chromosome 21 that is present in the mother's blood.  Down syndrome is caused by having an extra copy of chromosome 21.  We know that a pregnant woman's blood contains small amounts of her baby's DNA but that amount is very
low compared to the amount of her own DNA in her blood.  Using a technique called massively parallel sequencing, this test looks for increased amounts of pieces of chromosome 21 in the mother's blood.  In a Down syndrome pregnancy, the mother's blood will have more pieces of chromosome 21 because the fetus has an extra copy of that chromosome.

The study used blood collected from 753 women who were at high-risk of having a baby with Down syndrome.  86 of these pregnancies were carrying a fetus with Down syndrome and the test was able to correctly identify 100% of them.  While that alone is impressive, even more exciting is that only 2.1% of the unaffected fetuses were incorrectly identified as having Down syndrome.  Stated another way, in 98% of the cases Down syndrome could be ruled-out sparing the need for invasive follow-up testing.  Like some of the existing blood tests used to screen for Down syndrome, this new test can be performed in the first trimester of pregnancy.

Although the test is not yet available, there is a company that is working on making it available soon.