Common Types of SCA

There are four relatively common types of SCA, all of which can have consequences although these will often be mild. Combined together, these occur at a birth incidence of approximately 1 in 1000, being somewhat more common in phenotypic males than females. This compares to a birth incidence for trisomy 21 of approximately 1 in 700 live births. One of the genes in the pseudo-autosomal region of the X chromosome is SHOX , which promotes skeletal growth. People with only one sex chromosome—who have Turner syndrome (45,X)—are on average shorter than women who have two X chromosomes, while people with any of the three conditions with an additional chromosome (XXX, XXY, and XYY) tend to be taller. Dosage differences at other loci will contribute to the other effects of the SCAs, only some of which can be fully accounted for in a simple “gene dosage–phenotype” relationship.

People with Turner syndrome are usually infertile and may have one or more congenital anomalies (such as coarctation of the aorta or other cardiovascular defects, renal anomalies or cystic hygroma, that develops in utero but usually resolves by the time of birth to leave webbing of the neck). Girls with Turner syndrome may also be fully affected by sex-linked disorders, as if they were male, as they are hemizygous for all X chromosome genes. It should be remembered that Turner syndrome can also be caused by deletions and other rearrangements affecting the X chromosome. People with Klinefelter syndrome (47, XXY) are also usually infertile; people with XYY and XXX may have subfertility. All four of these conditions are associated with a modest drop in mean IQ but most of the individuals involved have an IQ in the normal range. Subtle neurocognitive problems may also be found in Turner syndrome. There is also a modest association with some behavioral problems in the SCAs, with XYY syndrome associated with autistic spectrum disorder. People with four or more X chromosomes have more serious cognitive and behavioral difficulties.

Benefit of Early Diagnosis

Early diagnosis is very helpful in Turner and Klinefelter syndromes because it enables the prompt recognition and appropriate management of endocrine problems. In Turner syndrome, expert management is important if growth is to be optimized. As well as growth hormone in childhood, the prescription of estradiol and then also progesterone triggers puberty with its growth spurt and enables pseudo-menstrual cycles. In Klinefelter syndrome, prompt treatment with supplementary testosterone permits a normal male puberty with normal male musculature; it may also have helpful behavioral effects.

It is difficult to make objective quality of life assessments of these conditions as there has been a long-standing problem with ascertainment bias. Although some unbiased, population-based cytogenetic studies have been carried out, much clinical experience is skewed to those with more marked difficulties and many affected individuals are probably never diagnosed [ ]. However, experience indicates that an early diagnosis is on balance helpful; it enables the optimization of medical and educational support [ ] and avoids the sudden discovery of a diagnosis as a cause of infertility in adult life [ ]. Putting to one side the question of infertility in most of the people with Turner and Klinefelter syndromes, these conditions are often compatible with “normal,” happy, and fulfilled lives.

Strategies for Detecting the Sex Chromosomes in CELL-FREE DNA Testing

There are three principal molecular strategies to detect the sex chromosomes in cfDNA testing: (i) PCR amplification of sequences from the Y chromosome, as used in the offer of fetal sexing by cfDNA testing. It is essentially a Yes/No test that aims to detect Y chromosome sequences, often the sex-determining SRY gene. It is good at determining whether or not there is a Y chromosome present but is not so effective at counting how many Y chromosomes there are, and it cannot determine how many X chromosomes are present. The other two methods are based on sequencing either (ii) unselected cfDNA in maternal plasma, as performed in whole genome sequencing, or (iii) cfDNA that has been enriched for sequences (i.e., chromosomes) of interest, often by means of a microarray that contains target sequences from the X and Y chromosomes.

The sequencing methods rely on counting the copies of the sequences of interest, to determine their ratio against sequences of reference chromosomes. Sequencing has to be performed to a sufficient depth: enough copies have to be sequenced that one can be confident of the ratio of the placenta-derived sequences in the maternal plasma.

The key is the calibration of the test on pregnancies of known chromosomal constitution, using the sequences that are to be detected and counted when performing the test in a clinical situation. This is technically simpler but demands more sequencing if one sequences unselected cfDNA—method (ii)—than if one enriches the cfDNA for specific chromosomal regions-method (iii). With the latter method one can achieve statistical significance more readily, and much more cheaply [ ]. The disadvantage of the enrichment step is that it could introduce a bias in sequence representation unless great care is taken at the design stage and in each analytical step, hence the need for careful calibration.

In the absence of a pregnancy, the woman’s plasma will contain roughly equal proportions of DNA from her two X chromosomes but in a pregnancy there will usually be a slight excess of DNA from one of the mother’s X chromosomes, along with DNA from either a Y chromosome or from the father’s X chromosome. To detect deviations from these two normal scenarios, a 46,XX or a 46,XY fetus, will require quantification of a large number of DNA fragments. Simply examining the ratio of autosomes:X chromosome:Y chromosome will answer some questions. More precise information about the origin of the chromosomal nondisjunction can be obtained if sequences are generated from polymorphic regions where the mother’s two X chromosomes differ from one another and from the father’s X chromosome. Given a fetal fraction of 5%–10%, the ratio expected between sequences derived from the two maternal and one paternal X chromosome in different scenarios will depend upon both the chromosome constitution of the fetus and the precise meiotic error underlying the SCA. In 47,XXY, for example, the additional X chromosome sequences may derive from meiosis I or II of the mother or come from the father.

Let us take Klinefelter syndrome as an example and consider one X-specific sequence and one Y-specific sequence. If we assume that the fetal fraction of the cfDNA in maternal plasma is 12%, so that the maternal fraction is 88%, then one has to distinguish between a ratio of 94:6 (expected X:Y copy ratio if the fetus is 46,XY) and 96:4 (expected X:Y ratio if the fetus is 47,XXY). Many copies of these sequences have to be counted for us to distinguish these ratios with confidence, more sequence information being required if the fetal fraction is less.

Test Quality of CELL-FREE DNA Testing for SCAs

In terms of analytical validity, reliability of laboratory methods for cfDNA prenatal testing is good, and the results are readily reproducible, when the numbers of chromosomes 21, 18, and 13 are calibrated against other autosomes [ ]. In terms of clinical validity, it is a much better test for these classic trisomies than previous testing options that combined biochemical analytes and nuchal translucency measurement. But even with a sensitivity of ~ 99% and a specificity of ~ 99.9% for the classic trisomies [ ], the test is less than fully accurate. Because the DNA tested represents a combination of maternal and fetal cell-free DNA, with the latter deriving from the placenta, a result signaling a suspected aneuploidy may be generated by other events such as placental mosaicism, a vanishing twin, or a maternal tumor [ ]. The impact of this becomes clear if the test is assessed in terms of its predictive value, rather than only its sensitivity and specificity. PPV also takes the low prevalence of the relevant conditions in the target population into account [ ]. The positive predictive value (PPV) is the chance that an increased risk result corresponds to the fetus being affected. In a screening test for common autosomal trisomies applied to an unselected (population-risk) pregnancies, a positive screening test often corresponds to an 80% or so probability of the fetus being affected, while if the pregnancy is already known through conventional screening to have a somewhat increased chance of an affected fetus, the PPV may be greater than 90% [ ]. Both these values demonstrate that a diagnostic procedure should be advised after a positive cfDNA test result for autosomal trisomy, because the chance of a false-positive result is of the order of 5%–15% (depending on the detailed circumstances).

There are limited data on cfDNA test performance for SCAs. These data indicate that cfDNA testing is screen “positive” in up to 1.1% of pregnancies [ ] but has a lower accuracy for SCA than for trisomy 21 and 18, with an overall PPV of 50%, and a much lower PPV for Turner syndrome (20%–30%) [ ]. For determining detection rates one has to be informed about the number of affected cases missed, but since most children born with SCAs do not have clinical features [ ] and healthy newborns are for obvious reasons not genotyped, detection rates cannot be calculated. Also, one would not necessarily expect cfDNA testing to detect mosaic cases or cases associated with more complex chromosome rearrangements. False-positive SCA findings may, in addition to the causes referred to previously, be attributed to maternal X chromosome aneuploidy and maternal X chromosome copy number variations [ ].

SCAs as Secondary Findings at Invasive Testing: The Option of Targeted Testing

Most SCAs are found as secondary results of invasive testing offered to women at a higher risk of having a child with a common autosomal aneuploidy (trisomies 21, 18, 13). Given the challenges for decision-making and counseling posed by SCAs, it has been suggested that in pregnancies at elevated risk for trisomies 21, 18, or 13 (but without abnormal ultrasound findings), a targeted diagnostic test, fluorescence in situ hybridization (FISH) or quantitative fluorescence polymerase chain reaction (QF-PCR) might be used that would avoid other findings including SCA. This would protect women from being confronted with secondary findings that they may find difficult to handle. The maxim behind this proposal has been summarized as “test what you screen for” [ ]. When targeted follow-up testing is feasible, which has become the case with the availability of molecular tests such as FISH or QF-PCR, forgoing this option would amount to using prenatal diagnosis as a platform for adding opportunistic screening, for which a justification would be needed [ ]. The prevailing view, however, is that “testing for less” than can be found with karyotyping (or nowadays chromosomal microarray), amounts to making suboptimal use of risky invasive testing, and denies prospective parents relevant information about abnormalities with potential clinical significance that they might consider relevant for reproductive decision-making [ ].

An important question is of course what women or couples would want. This question was addressed in a small-scale qualitative interview study among Dutch women who chose to continue the pregnancy after finding themselves unexpectedly confronted with an SCA diagnosis (full and mosaic forms of Turner, Klinefelter, and Triple-X syndromes) after invasive testing for advanced maternal age. Although all respondents reported initial feelings of anguish and fear, they were sufficiently reassured in posttest counseling and expressed that they expected a good quality of life for their child. When asked whether they would want to have testing for the sex chromosomes included when having follow-up invasive testing in a further pregnancy, most couples said they would [ ]. These couples found it important to be given the information and the opportunity to make a personal decision on the basis of it. However, there were also couples stating that they would rather not have been told.

The latter finding is as important as the former. Even if most pregnant women or couples do indeed appreciate the benefits of “testing for more” [ ], it does not follow that individual women need not be asked about their preferences in this regard [ ]. Ideally, women having prenatal diagnosis for Down syndrome should be given the right to indicate that they do not want to receive secondary information about milder conditions such as SCAs [ ].

Prenatal Screening: The “Autonomy Paradigm” and Its Limits

Generally, the potential benefits (or clinical utility) of screening are defined in terms of prevention: the extent to which the screening contributes to a lower burden of disease in the relevant population. But prenatal screening for fetal abnormalities is a special case [ ]. In order to avoid the moral pitfalls connected with presenting selective terminations as the intended result of the offer of prenatal screening for conditions such as Down syndrome, screening authorities and committees concur that the practice should be understood as aimed, not at prevention, but at providing pregnant women with meaningful options for autonomous reproductive decision-making [ ]. In the ethics literature, this is known as the “autonomy paradigm” of prenatal screening. However important this perspective may be when it comes to responding to the critique of “disability rights” advocates, who argue that the practice of prenatal screening sends a discriminatory message about the worth of people living with the relevant conditions [ ], the problem with the autonomy paradigm is that it does not help us answering the increasingly important question of what the scope of prenatal screening should be [ ]. Clearly, there is no reason why prenatal screening for fetal abnormalities should be limited to Down, Edwards, and Patau syndrome, as other conditions can be brought to light that are no less serious than those. But when it comes to milder or highly variable conditions, it is not obvious that anything that anyone would consider a reason for selective abortion should be regarded as a justification for offering screening. The reductivist view that in the ethics of prenatal screening “it is all about choice” [ ] tends to silence all concerns about possible harms, suggesting that whatever these are, they can simply be dealt with as part of informed consent. However, this is at odds with how the responsibility of those offering screening is understood in the international framework as developed on the basis of the Wilson & Jungner principles. This does not only apply to national or regional screening programs, but also to individual practitioners offering screening to their patients and to commercial companies operating on the “screening market” [ ]. Given that inevitably all screening also leads to harms, it is essential only to offer screening when benefits clearly outweigh harms for the persons being tested [ ]. When considering prenatal screening for SCAs, this is at least not obvious.

Concerns With Prenatal Screening for SCAs

First, with screening for SCAs, positive test results indicative of SCAs will confront women with even more difficult decision-making than is the case with SCAs identified as a secondary result of prenatal diagnosis. Here, the question is not only whether to continue or to terminate the pregnancy after a confirmed SCA diagnosis, but also whether or not to take the risk of invasive testing in order to exclude or confirm the cfDNA test result. Although the chances that invasive diagnostic procedures will lead to a miscarriage are now believed to be much smaller than previously thought [ ], this does not make these procedures entirely safe.

Given the low PPV of a positive screening test for SCAs, this means a probably more than 50% chance of a false alarm. It is true that this is still better than the ~ 5% PPV of traditional screening for Down syndrome [ ], but given the relatively mild phenotype of SCAs, the question remains whether providing (or confronting) women with this choice is a matter of helping or harming them. There are a few papers reporting on women’s actual choices in the context of screening for SCAs, based on small numbers [ ]. These suggest that upon a cfDNA result positive for SCAs most (but far from all) women accept the offer of diagnostic testing. However, the authors of one of these papers also state that several women expressed regret that they had learned this information prenatally [ ]. Their experience of being caught in a “screening trap” suggests layers of complexity in the dynamics of women’s decision-making that need to be further explored before the routine offer of cfDNA testing for SCAs can be considered to be a responsible policy. In this matter, the specifics of different SCAs will have to be taken into account, for instance, the fact that > 99% of nonmosaic Turner fetuses miscarry, and that those who survive often also have abnormalities that are detected by ultrasound [ ].

Second, given the much lower PPV of cfDNA testing for SCAs, adding testing for these conditions to prenatal screening for common autosomal trisomies will have the potential of reversing the large reduction in invasive testing that is widely considered to be the main benefit of this new screening test. Again, it is true that the PPV for SCAs is higher than was regarded as acceptable for traditional first trimester prenatal screening for Down syndrome. However, the question remains whether the potential higher loss of wanted pregnancies would be outweighed by the benefits of added screening for SCAs.

Benefits for the Child

The recommendation by the European and American Societies of Human Genetics not to offer screening for SCAs at this time [ ] has met with criticism. Not only was this recommendation ethically troubling because it restricts patient choice, but it would also deny future children the benefit of early diagnosis [ ]. The latter argument has also been used to question the policy of offering women the option of targeted testing as to avoid SCA as secondary findings from prenatal diagnosis [ ]. Interestingly, the argument that screening for SCAs might benefit the future child reintroduces “prevention” as a (secondary) aim of prenatal screening for fetal abnormalities, though without raising the moral problems of connecting prevention with selective termination. What is meant here is prevention in the sense of improving long-term health outcomes for children born after a timely diagnosis enabled by prenatal screening. It is expected that prevention in this sense will become more important as an aim of prenatal screening with improved options for fetal therapy [ ]. Even for conditions such as Down syndrome, a timely prenatal diagnosis may in the future open up options for in utero therapeutic intervention with better long-term results than postnatal treatment [ ]. This may in the future lead to possible tensions with the autonomy paradigm, especially with regard to pregnant women’s “right not to know” [ ].

But as long as there is no prenatal or perinatal treatment for SCAs, there is also no benefit for the future child of having a SCA diagnosis during pregnancy rather than early in postnatal life. This means that if an early diagnosis is on balance beneficial, this would be an argument for considering newborn screening for SCAs [ ], rather than trying to convince women that they should have prenatal SCA screening or testing in the interest of any affected child that they would allow to be born.

The professional consensus that an early diagnosis of SCA is beneficial specifically pertains to Turner and Klinefelter syndrome as it allows the timely management of endocrine problems. Whether this conclusion also holds for other SCAs is less clear. Given concerns about potential psychosocial harm (effect on self-esteem, parent–child interaction, and stigmatization) as a consequence of being born with a diagnosis that otherwise might have been made only much later or never at all, the balance of possible benefits and harms for the future child depends on the precise SCA.

Direct-to-Consumer CELL-FREE DNA Testing

Direct-to-consumer (DTC) offers of cfDNA testing for fetal abnormalities in average population-risk pregnancies are likely to add screening for SCAs and indeed microdeletion syndromes as a marketing ploy to increase their attractiveness to naïve customers, who will often fail to appreciate whether or not these tests have met test performance criteria and will eventually greatly increase the chance of an inappropriate follow-on invasive test procedure that puts the pregnancy at risk for no clear purpose. Such unvalidated cfDNA testing will also impose costs of follow-up invasive investigations on either the patient, their health insurer, or the national health care system. In the UK, the Nuffield Council of Bioethics has recommended that such additional costs should be met by the cfDNA testing provider from the initial fee for performing the test. This may discourage inappropriate marketing [ ].