Trisomy 21

Trisomy 21, or Down syndrome, is the most frequent autosomal chromosome syndrome with characteristic craniofacial features and congenital anomalies ( Fig. 10-1 ; see also Table 10-1 ). The relationship of Down syndrome to advanced maternal age is well known (see Table 10-2 ). Approximately 95% of cases arise in maternal meiosis, usually meiosis I, and have 47 chromosomes (47,XX,+21 or 47,XY,+21). Mosaicism for chromosome 21 occurs in 2% to 4% of cases of Down syndrome and usually results in a higher IQ (70 to 80). Women with Down syndrome are usually fertile, and although relatively few trisomic mothers have reproduced, about 30% of their offspring are also trisomic. Men are invariably infertile.

An infant with trisomy 21.

(From Simpson JL, Elias S. Genetics in Obstetrics and Gynecology, ed 3. Philadelphia: WB Saunders; 2003:24.)

Translocations (sporadic or familial) most commonly associated with Down syndrome involve chromosomes 14 and 21. One parent may have the same translocation, 45t(14q;21q), referred to as a robertsonian translocation. Empiric risks for having an offspring with Down syndrome are approximately 10% for female robertsonian translocation carriers and 2% for male translocation carriers. A potential concern is that offspring who are diploid (46,XX or 46,XY) actually have uniparental disomy (UPD), a condition in which both chromosomes originate from the same parent. In a study of 65 robertsonian translocation carriers (44t[13q;14q], 11t[14q;21q], 4t[14q;22q], and six others), only one UPD case was observed (0.6%). The authors also surveyed 357 inherited and 102 de novo published cases and concluded that overall risk for UPD 14 or 15 was 3%.

Other structural rearrangements that result in Down syndrome include t(21q;21q) and translocations that involve chromosome 21 and other acrocentric chromosomes (13,15 or 22). In t(21q;21q) carriers, normal gametes do not ordinarily form. Thus only trisomic or monosomic zygotes are produced, and the latter presumably appear as preclinical embryonic losses. Parents who have other translocations have a low empiric risk of having offspring with Down syndrome.

Trisomy 13

Trisomy 13 occurs in about 1/20,000 live births. The clinical features of trisomy 13 are summarized in Table 10-1 . Most cases are caused by nondisjunction (47,XX,+13 or 47,XY,+13) and are maternal in origin. Robertsonian translocations are responsible for fewer than 20% of cases and are invariably associated with two group D (13 to 15) chromosomes joining at their centromeric regions. If neither parent has a rearrangement, the risk for subsequent affected progeny is not increased. If either parent has a balanced 13q;14q translocation, the recurrence risk for an affected offspring is increased but only to 1% to 2%. The exception is a 13q;13q parental translocation in which no normal gametes are formed; this has the same dire prognosis as a 21q;21q translocation. For live births with trisomy 13, survival beyond 3 years is rare.

Trisomy 18

Trisomy 18 occurs in 1 per 8000 live births (see Table 10-1 ). Stillbirth is not uncommon. Fetal movement is feeble, and approximately 50% develop nonreassuring fetal status during labor. For live births, mean survival is measured in months, and pronounced developmental and growth retardation is apparent. Approximately 80% of trisomy 18 cases are caused by primary nondisjunction (47,+18). Errors usually arise in maternal meiosis, frequently meiosis II. Recurrence risk is about 1%.

Other Autosomal Trisomies

All autosomes show trisomies, but usually the trisomies other than those described above end in abortuses. In addition to trisomies 13, 18, and 21, only a few other trisomies are detected in liveborns (8, 9, 14, 16, and 22), often as mosaics in conjunction with a normal cell line (46 chromosomes). All exhibit some degree of mental retardation, various structural anomalies, and intrauterine growth restriction (IUGR).

Monosomy X (45,X)

The incidence of 45,X in liveborn girls is about 1 in 10,000. Monosomy X, or Turner syndrome, accounts for 10% of all first-trimester abortions; therefore it can be calculated that more than 99% of 45,X conceptuses are lost early in pregnancy. The error usually (80%) involves loss of a paternal sex chromosome. Mosaicism is frequent and usually involves a coexisting 45,X cell line.

Common features include primary ovarian failure, absent pubertal development due to gonadal dysgenesis (streak gonads), and short stature (<150 cm). Structural abnormalities of the X chromosome may also result in premature ovarian failure. Both the long arm and the short arm of the X chro­mosome contain determinants necessary for ovarian differentiation and for normal stature. Various somatic anomalies include renal and cardiac defects, skeletal abnormalities (cubitus valgus and clinodactyly), vertebral anomalies, pigmented nevi, nail hypoplasia, and a low posterior hairline. Performance IQ is lower than verbal IQ, but overall IQ is considered normal. Adult-onset diseases include hypertension, coronary artery disease, hypothyroidism, and type 2 diabetes mellitus.

Low-dose estrogen therapy is needed to induce puberty, and long-term hormone replacement is needed in adulthood. Pregnancy may be achieved with the use of donor eggs but requires careful monitoring of cardiovascular status before and throughout pregnancy and in the postpartum period. Growth hormone treatment increases the final adult height 6 to 8 cm. Comprehensive guidelines for evaluation and clinical management of Turner syndrome are available.

Klinefelter Syndrome

About 1 in 1000 males are born with Klinefelter syndrome, the result of two or more X chromosomes (47,XXY; 48,XXXY; and 49,XXXXY). Characteristic features include small testes, azoospermia, elevated follicle-stimulating hormone (FSH) and luteinizing hormone levels, and decreased testosterone. The most common chromosome complement associated with this phenotype is 47,XXY.

Mental retardation is uncommon in 47,XXY males, but behavioral problems and receptive language difficulties are common. Mental retardation is almost invariably associated with 48,XXXY and 49,XXXXY. Skeletal, trunk, and craniofacial anomalies occur infrequently in 47,XXY but are commonly observed in 48,XXXY and 49,XXXXY. Regardless of the specific chromosome com­plement, patients with Klinefelter syndrome all have male phenotypes. The penis may be hypoplastic, but hypospadias is uncommon. With intracytoplasmic sperm injection and other assisted reproductive technology (ART), siring a pregnancy is now possible. Simpson and colleagues and Graham and colleagues have provided guidelines on evaluation and clinical management.

Polysomy X in Girls (47,XXX; 48,XXXX; 49,XXXX)

About 1 in 800 liveborn girls has a 47, complement. The IQ of such individuals is 10 to 15 points lower than that of their siblings. The absolute risk for mental retardation does not exceed 5% to 10%, and even then, IQ is usually 60 to 80. Most of these individuals have a normal reproductive system. The theoretic risk of women with the 47, complement delivering an infant who also has an abnormal chromosome complement is 50%, given half of the maternal gametes carry 24 chromosomes (24,XX). Empiric risks are much less. Somatic anomalies are uncommon in those with the 47, complement, but anomalies may occur and have been observed in some prenatally detected cases. However, 48,XXXX and 49,XXXXX individuals are invariably retarded and are more likely to have somatic malformations than individuals with the 47, complement.

Polysomy Y in Boys (47,XYY and 48,XXYY)

Presence of more than one Y chromosome is another frequent chromosome abnormality in liveborn boys (1 in 1000). Those born 47,XYY are more likely than 46,XY boys to be tall and are at increased risk for learning disabilities, speech and language delay, and behavioral and emotional difficulties. These individuals have normal male phenotype and sexual development.

First-Trimester Screening

First-trimester screening can be performed between 11 and 14 weeks using a combination of biochemical markers, pregnancy-associated plasma protein A (PAPP-A) and free β–human chorionic gonadotropin (β-hCG), and ultrasound measurement of the nuchal translucency (NT), a sonolucent space present in all fetuses behind the fetal neck. The detection rate for trisomy 21 is greater than 80% with a false-positive rate of 5% compared with a 70% detection rate based on NT measurement alone. In trisomy 21, PAPP-A levels are typically reduced, whereas hCG and the NT measurement are increased. First-trimester screening is comparable or superior to second-trimester screening alone and, most importantly, it provides parents with the option of earlier diagnostic testing in the event the screen indicates that the fetus is at high risk for aneuploidy. However, mandatory training and quality assurance for the NT measurement is a critical necessity.

Several large, multicenter, prospective studies have validated the clinical application of first-trimester screening. When comparing studies, it is important to keep in mind that detection rates vary according to sample characteristics. In particular, sensitivity for noninvasive screening is age dependent, and software is constructed such that the proportion of cases detected at a given age is greater for older women than for younger women. Hence the false-positive rate and the related procedure rate also increase with maternal age. Detection rates also depend not just on the trimester but on the week of gestation and on the arbitrarily set false-positive rate. If more procedures are accepted (i.e., the false-positive rate is higher), detection rates increases. The converse is also true.

In the first U.S. large-scale, prospective study of over 5800 women using both ultrasound (NT) and serum analytes (PAPP-A, free β-hCG), the detection rate for trisomy 21 was 87.5% (7 of 8 cases) in women younger than 35 years of age. In women older than 35 years, the detection rate was 92% (23 of 25), albeit with higher false-positive and invasive procedure rates. For trisomy 18, detection rates were 100% in both age groups. In 2003, an National Institute of Child Health and Human Development (NICHD) multicenter cohort study, the Biochemstry Ultrasound Nuchal Translucency (BUN) Study, reported results in 8514 women screened between 74 and 97 days’ gestation ( Table 10-5 ). Applying the traditional midtrimester screen positive cutoff of 1 in 270, 85.2% of trisomy 21 pregnancies were identified with a false-positive rate of 9.4%. The high false-positive rate was predictable, given the higher mean maternal age of the sample. Stratifying by age, the detection rate for trisomy 21 was 66.7% for patients younger than age 35 years with a 3.7% false-positive rate, and it was 89.8% in patients older than age 35 years with a 15.2% false-positive rate. The detection rate for trisomy 18 was 90.9%. Modeling for the general population (with a lower mean age) and setting a false-positive procedure rate of 5%, sensitivity for trisomy 21 was estimated to be 78.7%; at a false-positive rate of 1%, the sensitivity was estimated to be 63.9%. These findings were consistent with other studies.

Two other large, collaborative studies also have provided results comparable to the NICHD BUN study. The Serum, Urine, and Ultrasound Screening Study (SURUSS) was a 25-center European trial in which 47,000 patients were evaluated in both the first and second trimesters. Using a similar design, Malone and colleagues studied 38,167 women in 15 U.S. centers in the NICHD First- and Second-Trimester Evaluation of Risk (FASTER) trial. Detection rates for Down syndrome were 87% at 11 weeks and 85% at 12 weeks ( Table 10-6 ). In this study, 134 women who had fetuses with a septated cystic hygroma were removed from the cohort; 51% had a chromosome abnormality and 34% had other major abnor­malities. The group later stratified their data and found that NT greater than 4 mm was never associated with a normal noninvasive screen; therefore women with an NT that exceeded this threshold should be offered diagnostic testing without needing to undergo further analyte analysis. In fact, only 8% of pregnancies with NT greater than 3 mm had a screen-negative value.

Nicolaides tabulated that NT, PAPP-A, and hCG detected 87% of 215 trisomy fetuses at a false-positive rate of 5%. Later results of Avgidou and colleagues reported superior results from the same U.K. group. This group screened 30,564 women with NT, PAPP-A, and hCG, providing results the same day and detecting 93% of trisomy 21 cases. The incorporation of the other sonographic markers such as the presence of a nasal bone, reverse ductus venosus flow, and tricuspid regurgitation has been proposed to increase the detection rates further. In general these markers are not utilized except in specialized centers.

When an increased NT measurement is associated with a normal karyotype, fetal loss rates are increased and other fetal anomalies and genetic syndromes are observed, in particular, congenital heart defects. A targeted ultrasound examination during the second trimester and fetal echocardiography are recommended when the NT measurement is 3.5 mm or greater and the fetal karyotype is normal.

Second-Trimester Serum Screening

The most widely used second-trimester aneuploidy screening test is the so-called quad screen, which utilizes four biochemical analytes—alpha-fetoprotein (AFP), hCG, unconjugated estriol (uE ₃ ), and dimeric inhibin A (INHA). Performed between 15 and 22 weeks’ gestation, the detection rate for trisomy 21 is about 75% in women who are less than 35 years of age and over 80% in women 35 years or older, with a false-positive rate of 5%. For trisomy 18, using only the first three markers provides a detection rate of about 70%. Serum screening does not detect other age-related forms of aneuploidy such as Klinefelter syndrome (47,XXY).

Serum hCG and INHA levels are increased in women carrying fetuses with Down syndrome. Levels of AFP and uE ₃ in maternal serum are lower in pregnancies affected with Down syndrome compared with unaffected pregnancies. Typically, levels of AFP, uE ₃ , and hCG are reduced in trisomy 18. A simple approach to detect trisomy 18 is to offer invasive prenatal diagnostic testing whenever serum screening for each of these three markers falls below certain thresholds (maternal serum α-fetoprotein [MSAFP], 0.6 multiples of the median [MoM]; hCG, 0.55 MoM; uE ₃ , 0.5 MoM). Using these thresholds would detect 60% to 80% of trisomy 18 fetuses with a 0.4% amniocentesis rate. Calculating individual risk estimation on the basis of three markers and maternal age, Palomaki and colleagues reported that 60% of trisomy 18 pregnancies can be detected with a low false-positive rate of 0.2%. The value of individual risk estimates is that one in nine pregnancies identified as being at increased risk for trisomy 18 by serum screening would actually be affected.

Confounding factors influence serum screening, and adjustments for gestational age, maternal weight, ethnicity, diabetes, and number of fetuses are necessary. Weight adjustment is needed because without adjustment, dilutional effects would result in heavier women having a spuriously low value, whereas thinner women would have a spuriously elevated value. In women with type 1 diabetes mellitus, a population at increased risk for NTDs, the median levels of MSAFP, uE ₃ and hCG are lower than in nondiabetic women. In black women, who have a lower risk for a fetal NTD, the median MSAFP is higher than in other ethnic groups. Maternal smoking increases MSAFP by 3% but decreases maternal serum uE ₃ and hCG levels by 3% and 23%, respectively. Maternal serum hCG is higher and MSAFP is lower in pregnancies conceived in vitro compared with pregnancies conceived spontaneously. A claim has been made that adjustments should be made for prior aneuploidy; β-hCG is reported to be 10% higher in a pregnancy after aneuploidy, whereas PAPP-A is increased 15% in the first trimester.

First- and Second-Trimester Screening

Several approaches have been proposed using the com­bination of both first- and second-trimester screening to increase the detection rate over that achieved by screening in either trimester alone, with detection rates of 88% to 96% with false-positive rates of 5% reported. A caveat is that independent screening (i.e., using both first- and second-trimester screening tests to assess the risk separately and independently) is not recommended because of the unacceptably high false-positive rates.

Sequential screening begins with first-trimester screening. A woman is informed of the adjusted risk for aneuploidy based on the first-trimester results. If her risk is high (greater than 1 in 50), she is offered genetic counseling and diagnostic testing. If the risk is low or moderate, a second-trimester screening test is performed with results of both the first- and second-trimester screening tests used to generate a final adjusted risk for trisomies 21 and 18. This is called the stepwise approach . With contingency screening, not all women will proceed to second-trimester screening because this occurs only with an intermediate risk; if the risk is low after the first-trimester screening, no further testing is indicated. The detection rates of the contingency approach are about 90% with low positive screening rates (2% to 3%). Malone and colleagues compared several different first- plus second-trimester contingent sequential approaches (see Table 10-5 ). They concluded that the optimal method was contingency screening, in which patients were divided into three groups: (1) women whose calculated (NT, PAPP-A, hCG) first-trimester risk was greater than 1/30 would undergo chorionic villus sampling (CVS); (2) women whose risk was less than 1/1500 would undergo no further testing; and (3) all other women would undergo second-trimester serum testing. Using this approach, only 21.8% of the cohort would need second-trimester testing in order to detect 93% of trisomy 21 cases with a 4.3% false-positive rate; 65% would be detected in the first trimester with only 1.5% of patients having CVS procedures.

Integrated screening has the highest theoretic detection rate (93% to 96%), but with this approach, the first-trimester screening results are withheld until the second-trimester screen is completed. The individual receives only a single adjusted risk for trisomy 21 and trisomy 18 based on the results of both the first- and second-trimester screen. The obvious disadvantage with this approach is that the individual does not have an option of early diagnostic testing in the event that the first-trimester screen would have indicated a high risk for trisomy 21 or 18. Another is that patients may not return for their second-trimester screening. Fortunately, Cuckle and colleagues showed that disclosure of first-trimester results could be made with very little loss in sensitivity of integrated screening.

When an NT measurement cannot be obtained, and in communities where NT measurement is not available, serum integrated screening is acceptable. In the FASTER trial, the sensitivity was 88%. With this approach, the first-trimester PAPP-A and the second-trimester serum analytes are used to adjust the risk for trisomy 21, and the individual receives one adjusted risk after the second-trimester screen is completed.

Cell-Free DNA Analysis

The newest screening test for aneuploidy uses maternal cell-free DNA (cfDNA). Maternal plasma contains small fragments of cfDNA (50 to 200 base pairs) derived from the breakdown of both maternal and fetal cells, primarily derived from the placenta. The concept of using cfDNA for prenatal diagnosis is not new; cfDNA has been used successfully to determine fetal sex in pregnancies at risk for X-linked disorders by identifying the Y-chromosome signal. In Europe, noninvasive testing is commonly used to determine fetal Rhesus factor (Rh) status in RhD-negative women using real-time polymerase chain reaction (PCR) amplification. A similar approach can be adapted for the detection of some single-gene disorders. However, to screen for aneuploidy requires a different approach—the use of massively parallel DNA shotgun sequencing (MPSS).

The detection of aneuploidy is more difficult than for single-gene disorders because detecting fetal trisomy must reflect quantitative differences between affected and unaffected pregnancies. With MPSS technology, millions of fragments of maternal and fetal DNA are sequenced simultaneously in a single sample of maternal plasma, which is assigned to a chromosome region and counted. A woman carrying a trisomy 21 fetus will have relatively more chromosome 21 counts than a woman carrying a normal fetus. Alternatively, some laboratories use a targeted approach that sequences specific chromosomes of interest, such as 18 and 21, and adjusts for the proportion of fetal DNA (fetal fraction) to provide a patient-specific risk assessment that takes into account maternal age. An alternative approach is to use single nucleotide polymorphism (SNP)-based sequencing, which allows for the detection of triploidy and some of the common deletion syndromes.

Several studies have demonstrated the ability to detect fetal trisomy 21 using MPSS. A blinded, nested, case-control study of 4664 pregnancies at increased risk for trisomy 21 from 27 prenatal diagnostic centers worldwide validated the use of cfDNA analysis as a screening test for trisomy 21. In this study, 209 of 221 cases of trisomy 21 were detected; sensitivity was 98.6% with a false-positive rate of 0.2%. Subsequently, Palomaki and colleagues reported that all cases of trisomy 18 in this cohort were detected with a false-positive rate of 0.28%, but only 91.7% of the cases of trisomy 13 were detected with a false-positive rate of 0.97%.

Norton and colleagues conducted a multicenter, prospective cohort study—the Noninvasive Chromosomal Evaluation (NICE) study—of over 3200 primarily high-risk women undergoing invasive diagnostic testing. For trisomy 21, the sensitivity was 100% with a false-positive rate of 0.03%, whereas the sensitivity for trisomy 18 was 97.4% and the false-positive rate was 0.07%. Two patients were classified as high risk for trisomy 18 and had a normal karyotype; this highlights the need for confirmatory diagnostic testing when an NIPT is positive or shows high risk for aneuploidy. Further, 29% of the chromosome abnormalities in this cohort were abnormalities other than trisomy 18 and 21 or were sex chromosome abnormalities such as unbalanced translocations, deletions, and duplications.

In a series of 1982 consecutive pregnancies at 12 weeks’ gestation or greater referred for NIPT at a center in Hong Kong, 11 pregnancies screened positive for sex chromosome abnormalities, and 85.7% were confirmed to be fetal in origin. Fetal mosaicism was detected in two cases, emphasizing that it is important to counsel patients about this possibility. Maternal mosaicism for 45,X/XX and confined placental mosaicism also lead to false-positive NIPT results, underscoring the importance of confirmatory invasive diagnostic testing.

Another limitation of cfDNA screening is the reported assay failure rate of up to 5%. One of the reasons is a low fetal fraction; a minimum fetal fraction of 4% is required. The ability to detect the small differences between euploid and triploid fetuses depends on the relative proportion of fetal to maternal cfDNA. The average fetal fraction at 10 to 22 weeks’ gestation is 10% independent of gestational age, maternal age, race/ethnicity, or fetal karyotype. Fetal fraction decreases with maternal weight, and if NIPT fails in an obese patient, it may be necessary to repeat the test on a second sample or to offer serum and/or ultrasound screening as an alternative. Further consideration should be given to offering invasive testing because recent studies have reported a higher frequency of aneuploidy when cfDNA testing fails.

The American College of Obstetricians and Gynecologists (ACOG) and the American College of Medical Genetics (ACMG) recommend that women be offered aneuploidy screening, and both acknowledge that NIPT is one of the screening options for women at increased risk for aneuploidy. This includes women 35 years or older and those with fetal ultrasound markers and structural anomalies associated with aneuploidy, prior pregnancy with trisomy, positive serum screening test, or a parental robertsonian translocation with risk for trisomy 21 or 13. However, as studies of low-risk women are conducted, we can expect that NIPT will become more widely available. In a cohort study of over 2000 women who presented for routine first-trimester screening (11 to 14 weeks) with a mean maternal age of 31.8 years, Nicolaides and colleagues demonstrated that cfDNA analysis using targeted MPSS is feasible in a lower-risk population. The detection rate for trisomy 21 was 100% with a false-positive rate of 0.1%. Norton and colleagues reported similar results in the international, blinded, prospective multicenter Noninvasive Examination of Trisomy (NEXT) study, which compared cfDNA to first-trimester screening at 10 to 14 weeks’ gestation in 15,841 women (mean age 30.7) with a singleton gestation. The positive predictive value was 80.9% (95% confidence interval [CI], 66.7 to 90.9). Although all cases of trisomy 21 and 13 were detected, it is important to note that cfDNA testing did not detect one in 10 cases of trisomy 18, nor would it have detected the other forms of aneuploidy found in this study population. Furthermore, the rate of aneuploidy among patients with no cfDNA results was 2.7%, a prevalence of 1 in 38. Hence, careful consideration should be given to offering diagnostic testing to this group of patients.

In the United States, Bianchi and colleagues conducted a multicenter study to compare the false-positive rates of cfDNA analysis to routine first- and second-trimester screening. In 1914 women (mean age 29.6 years), the false-positive rate for trisomy 21 with cfDNA testing was significantly lower, 0.3% compared with 3.6%. All cases of aneuploidy were detected, but this finding should be interpreted cautiously because of the small sample size. Patients will need to be counseled that a negative cfDNA test does not ensure an unaffected pregnancy and cannot provide the diagnostic accuracy of an invasive prenatal diagnostic test, especially if fetal structural anomalies exist or with a family history of a genetic disorder.

Aneuploidy Screening in Multiple Gestation

In dizygotic pregnancies, each twin has an individual risk for trisomy 21; analysis of the European National Down Syndrome Cytogenetic Registry found that dizygotic pregnancies were one third more likely to have at least one fetus with trisomy 21 than age-adjusted singleton pregnancies. The pregnancy-specific and fetus-specific risks should be the same for monozygotic twins as for singleton pregnancies, although the study found the actual risk to be about a third that of a singleton pregnancy. Unfortunately, Down syndrome screening using multiple serum markers is less sensitive in twin pregnancies than in singleton pregnancies. Using singleton cutoffs, one study showed that 73% of monozygotic twin pregnancies but only 43% of dizygotic twin pregnancies with Down syndrome were detected, given a 5% false-positive rate. Decreased sensitivity in detecting trisomy 21 in dizygotic twins reflects the blunting effect of the concomitant presence of one normal and one aneuploid fetus. Thus patients with twins should be informed that the detection rate by serum screening is less than that in singleton pregnancies. First-trimester screening identifies about 70% of Down syndrome pregnancies; NT measurement alone has been shown to be as effective a screening test for higher-order multiple gestation as it is for twin gestations. The addition of the nasal bone (NB) assessment to the NT measurement increased the detection rate to 87% at a screen-positive rate of 5% and to 89% when serum analytes were included in a retrospective study of 2094 twin pregnancies. First-trimester screening further provides the option of early diagnostic testing if the risk is increased and if selective reduction is undertaken.

Experience using cfDNA analysis in multiple gestations is limited, but a few studies suggest that cfDNA has similar sensitivity and specificity rates as in singleton pregnancies. For dizygotic twins, a higher fetal fraction (minimum of 8%) may be necessary to detect a quantitative difference. NIPT should be interpreted cautiously in pregnancies with a vanishing twin; the results may be discordant because the vanishing twin may continue to release cfDNA into the maternal circulation. SNP-based NIPT may be helpful in identifying this phenomenon.

Ultrasound Screening for Aneuploidy

Second-trimester ultrasonography may detect anomalies associated with aneuploidy, such as cardiac defects or duodenal atresia (see Chapter 9 ). In 1985, Benacerraf and colleagues showed a significant association between the thickness of the fetal nuchal skin fold and the presence of trisomy 21. For the first time, a “marker,” as opposed to an actual birth defect, could be used to assess the likelihood that Down syndrome was present. Other markers now commonly used in the genetic sonogram include the NB length, short femur or humerus, echogenic intracardiac focus, echogenic bowel, and pyelectasis. The use of these markers has gradually become common clinical practice. As markers were studied in larger numbers of women, it became possible to assign likelihood ratios to each marker and to do a formal risk adjustment from the a priori risk to an ultrasound-adjusted risk. However, most of the markers perform poorly as individual predictors of Down syndrome and have a very low sensitivity and a high rate of false-positive results. Further, the test performance depends on the skill of the examiner and the subjective assessment required for several markers, particularly echogenic intracardiac focus and echogenic bowel. Most women who undergo a genetic sonogram have no markers, but women should be informed that the absence of markers does not rule out the possibility of Down syndrome or other chromosome abnormalities.

The incidental finding of “soft markers,” minor markers such as echogenic intracardiac focus or pyelectasis, in otherwise low-risk pregnancies can cause a great deal of anxiety in the expectant parents. Opinions vary as to what to do when these soft markers are noted in a low-risk patient. A reasonable approach is to refer such a patient for consultation with an expert to evaluate the presence of other markers in the context of other screening tests (i.e., multiple marker screening or cfDNA).

Indications for Prenatal Cytogenetic Testing

In January 2007, ACOG published a new recommendation that all women, regardless of age, should have the option of invasive testing without first having been screened. Prenatal cytogenetic testing can be as basic as a routine G-banded karyotype, or it might include a whole genome microarray. In some cases, a more targeted approach may be desirable and could include either fluorescence in situ hybridization (FISH) for a specific chromosome region of interest or a targeted chromosome microarray.

Chromosome Microarrays

Chromosome MAs allow comprehensive analysis of the entire genome at a finer resolution than a routine karyotype and are capable of detecting trisomies and submicroscopic deletions and duplications of the genome (CNVs). CNVs can be detected using either comparative genomic hybridization (CGH) or SNP arrays ( Fig. 10-2 ). For both, the principle is based on single-stranded DNA annealing (hybridizing) with a complementary single-stranded DNA. To look for CNVs, a sample of test DNA is labeled with a fluorochrome (e.g., red), denatured to single-stranded DNA, and then hybridized to single-stranded copies of DNA of known sequence differentially labeled (e.g., green) and embedded on a platform (array) in an ordered fashion. If equal amounts of control and test DNA are present, the color of the hybridized mixture could be expected to be yellow. If the test DNA were in excess (e.g., trisomy or duplication), the mixture for the relevant chromosome region would be relatively more of the color used to connote test (patient) DNA. This would be red in the previous example. SNP arrays can also detect homozygosity or heterozygosity for regions of DNA and can detect triploidy, UPD, and consanguinity.

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