Rare Autosomal Trisomy Mosaicism during Pregnancy and at Birth

Chromosomal mosaicism is the presence of two or more distinct cell lines in an individual . In a prenatal setting, chromosomal mosaicism most commonly affects only the placenta (confined placental mosaicism; CPM), but may occasionally extend to the fetus (true fetal mosaicism; TFM). The clinical consequences of chromosomal mosaicism identified during prenatal diagnosis can be difficult to predict, ranging from no apparent phenotypic effect to early fetal lethality. In the absence of fetal anomalies, the outcome of TFM in a prenatal setting remains uncertain .

Autosomal trisomy is a common cause of early miscarriage. Of the 10%–15% of pregnancies that end in clinical miscarriage, about half will do so because of a chromosome abnormality, and of these, the majority will involve an autosomal trisomy . With very rare exceptions, only trisomy for chromosomes 13, 18, and 21 (the so-called live birth trisomies) is compatible with survival to term, recognizing that even these conditions are associated with a high rate of miscarriage and stillbirth . All other autosomal trisomies (the so-called rare autosomal trisomies) are lethal in nonmosaic form, notwithstanding occasional reports of survival into the second, and very rarely the third trimester of pregnancy; stillbirth or neonatal death is expected. A large study reporting on the prevalence and types of rare chromosome abnormalities notified to 16 European congenital anomaly registers recorded 58 nonmosaic rare trisomies from 2.3 million births (0.25 per 10,000), none of whom survived . All were notified following prenatal testing or late fetal death (≥ 20 weeks of pregnancy). In contrast, 141 mosaic rare trisomies were reported (0.6 per 10,000 births), of which 78% were identified prenatally. Of these, 41% were liveborn, 7% stillborn, and 49% resulted in pregnancy terminations associated with fetal anomalies. Mosaicism involving trisomies 8 and 9 were most commonly notified. These findings show that true mosaicism for rare autosomal trisomies contributes to a small but significant part of pre- and perinatal adverse pregnancy outcomes.

Rare Autosomal Trisomies in Amniotic Fluid

Amniocentesis for cytogenetic prenatal diagnosis has been in widespread use since the early 1970s. The cells isolated from amniotic fluid closely reflect the chromosome constitution of the fetus, being derived from sources such as the fetal skin, nasopharyngeal tract, and urogenital tract , with extraembryonic cells being contributed from the amniotic membrane (amnion) . Historically, amniocentesis samples used for conventional chromosome analysis have been divided and grown across several independent culture dishes. Specimens that exhibit the same mosaic chromosome abnormality in at least two culture dishes are considered to exhibit true (Level III) mosaicism, which is present in approximately 0.1%–0.3% of amniocentesis samples analyzed by conventional karyotyping . True mosaicism most commonly involves the autosomal trisomies (48%), followed by sex chromosome aneuploidies (40%) and extra structurally abnormal chromosomes (12%) . Confirmation of mosaicism in fetal or newborn samples occurs in about 60%–70% of cases and in one US collaborative study, approximately 38% of autosomal trisomy mosaics were reported to be associated with noticeable phenotypic abnormalities .

Hsu et al. have reported phenotypic outcome data for 151 rare autosomal trisomy mosaics (excluding trisomy 20) ascertained following amniocentesis . This series was recently updated by Wallerstein et al., who reported summary outcomes for all mosaic autosomal trisomies , including 506 cases of rare trisomy mosaicism ( Table 1 ). Cases with prior abnormal ultrasound findings were excluded to help remove ascertainment bias. With regard to recorded abnormal outcomes, mosaic trisomy for chromosomes 2, 9, 16, 20⁎ [⁎see below], and 22 were classified as very high risk (> 60% with abnormal outcomes); chromosomes 5, 14, and 15 were classified as high risk (40%–59% abnormal); chromosomes 7, 12, and 17 as moderately high risk (20%–39%); chromosomes 6 and 8 as moderate risk (up to 19%); and no rare mosaic trisomies were classified as low risk (0%). Mosaic trisomy for chromosomes 1 and 10 was not observed. Mosaic trisomy for chromosomes 3, 4, 11, and 19 was not assigned a risk in the previous study by Hsu et al. due to insufficient cases ( n < 5). In the Wallerstein et al. series, abnormal outcomes were recorded in 3/4 cases of trisomy 3 mosaicism, 3/5 trisomy 4, 0/4 trisomy 11, and 0/1 trisomy 19. Therefore true mosaicism for trisomies 3 and 4 suggests a high to very high risk, based on these small numbers. Trisomy 20 mosaicism⁎ appears to have been misclassified as very high risk, rather than moderate risk (11% of cases with abnormal outcome), which is consistent with the lower frequency of abnormal outcomes in an earlier study .

The assessment of abnormal outcomes was made mostly by postmortem examination following termination or after birth. The authors note that subtle anomalies may not have been recognized and that neurodevelopmental follow-up after birth was rare. Nonetheless, these summary data are invaluable for helping evaluate possible outcomes following a diagnosis of rare trisomy mosaicism with normal ultrasound and to help guide patient counseling. Genetic counseling in the setting of normal fetal ultrasound remains problematic, but the presence of ultrasound anomalies indicates a very high risk for developmental and physical disabilities following the detection of rare trisomy mosaicism .

One last consideration is the introduction of chromosome microarray (CMA) into prenatal diagnosis. Few data currently exist on the interpretation of chromosomal mosaicism found in uncultured amniotic fluid samples ascertained using CMA. In the State of Victoria, Australia, > 85% of all samples received for cytogenetic prenatal diagnosis are now analyzed using CMA ; the majority of which use DNA extracted directly from uncultured cells. In my own laboratory, using a single-nucleotide polymorphism (SNP) CMA (Illumina Inc.), which has a lower limit of detection of 7%–12% for trisomy mosaicism , we sometimes observe discrepancies between the results of CMA on uncultured cells, and the results of conventional karyotyping on cultured cells. In some, but not all cases of discrepancy, the SNP CMA will exhibit mosaicism, while the cultured cells are karyotypically normal, or perhaps show only a single colony of abnormal cells. Insufficient data currently exist to determine which method more accurately reflects the true fetal karyotype or provides a better prediction of phenotype.

Confined Placental Mosaicism in Chorionic Villi and its Relationship to CELL-FREE DNA-Based NIPT

Chorionic villus sampling (CVS) for cytogenetic prenatal diagnosis emerged in the mid-1980s . The procedure enables prenatal testing from the first trimester of pregnancy, using samples of chorionic villi biopsied from the placenta at around 10 to 12 weeks of gestational age. The basis of CVS is that the karyotype of the placental chorionic villi represents the karyotype of the fetus .

Samples of chorionic villi for conventional chromosome analysis can be prepared using two methods: (i) a direct or short-term (24–48 h) culture method (STC) that analyzes rapidly dividing cells from an outer villi layer of cytotrophoblast, and (ii) a long-term culture method (LTC) that analyzes cells grown from the mesodermal core of the chorionic villi. Over the past two decades, some laboratories have substituted STC for other rapid methods of analysis, using techniques such as fluorescence in situ hybridization (FISH) or quantitative fluorescence polymerase chain reaction (QF-PCR) . Whereas STC provides a low-resolution G-banded karyotype, FISH and QF-PCR typically only target aneuploidy for chromosomes 13, 18, 21, X, and Y. More recently, CMA using DNA extracted from whole chorionic villi has replaced LTC in some laboratories .

Reports of discrepancies between the karyotype of cells from STC and/or LTC, and the chromosome constitution of fetus emerged shortly after CVS was implemented into clinical practice . These discrepancies usually involved chromosomal mosaicism that was present in the chorionic villi but not in the fetus—a phenomenon known as confined placental mosaicism (CPM) , which affects up to 2% of CVS samples . This frequency of mosaicism is at least 10 times higher than the rate of TFM seen after amniocentesis.

CPM can be present in STC only (CPM I), in LTC only (CPM II), or in both (CPM III) . Trisomy for CPM types I and II usually has a mitotic origin, where postzygotic gain of the trisomic chromosome is confined to the cytotrophoblast or the mesenchyme, respectively. CPM type III is more likely to have a meiotic origin and involve a trisomic conception that has lost one of the trisomic chromosomes in the first few cell divisions after fertilization—so-called trisomy rescue . Rarely, a false-negative result may be reported. Here, the chorionic villi have a normal karyotype, but the fetus has a chromosome abnormality, either as a full trisomy or with TFM. False-negative results for the common autosomal trisomies occur almost exclusively during analysis of STC cytotrophoblast cells . From a developmental view point, the cytotrophoblast cells are more distantly related to the embryo proper than are cells from the LTC mesenchyme; the mesenchyme cells being known to more accurately reflect the fetal karyotype . This is because the LTC mesenchyme cells derive from the hypoblast of the inner cell mass (ICM); the ICM also giving rise to the epiblast and embryo proper .

Discrepancies involving mosaicism in chorionic villi are critically important to our understanding and management of cfDNA test results, as the origin of “fetal” cfDNA is apoptotic cytotrophoblast cells. Thus cfDNA-based prenatal testing is analogous to CVS STC and is essentially a liquid biopsy of these placental cells. Several groups have used this knowledge to review large databases of CVS test results to predict the frequency of false-positive cases that will occur during cfDNA analysis and to help guide the choice of follow-up prenatal procedure. In particular, these large reviews are helpful for the interpretation and management of rare autosomal trisomy cases identified during cfDNA-based NIPT . A caveat here is that the cfDNA result is a proxy for the CVS STC result only. No information is provided on the LTC mesenchyme cells, which are available to aid interpretation during the analysis of a diagnostic CVS sample (see Table 2 ). Professional societies governing standards in cytogenetic testing recommend against the analysis of CVS STC alone, because of the increased chance of both false-positive and false-negative results . This recommendation is a salient reminder that cfDNA-based NIPT should always be regarded as a screening test.

Rare Autosomal Trisomies in Chorionic Villi and Fetal Compromise

The clinical implications of CPM involving the rare autosomal trisomies are well documented, and pregnancy outcomes may vary greatly, even for the same chromosome . Reported pregnancy complications include spontaneous miscarriage, IUGR, intrauterine fetal demise (IUFD), preterm birth and stillbirth, but many pregnancies also proceed to term uneventfully . Pregnancy-induced hypertension and preeclampsia are frequently reported complications of trisomy 16 mosaicism , while uniparental disomy [the inheritance of two homologous chromosomes from one parent without a contribution of that chromosome from the other parent] may lead to imprinting disorders for those chromosomes known to harbor imprinted genes (chromosomes 6, 7, 11, 14, 15, and 20), following trisomy rescue . Residual TFM has been reported more commonly in pregnancies associated with trisomies 9, 16, and 22, but in practice mosaicism can involve almost any rare trisomy . Cryptic mosaicism, where fetal malformations are seen in association with CPM, but where the rare trisomy mosaicism cannot be demonstrated in the fetus or newborn, may be present in up to 10% of cases , and has been frequently suspected in trisomy 16 CPM .

The likelihood of an adverse pregnancy outcome after the detection of rare trisomy mosaicism following CVS appears to be influenced by 3 key variables. These are: (i) the distribution of trisomic cells in the chorionic villi [CPM types I, II, or III], (ii) the actual trisomy involved, and (iii) the frequency of abnormal cells.

Early studies of CVS mosaicism reported an association between very high frequencies of trisomic cells in the cytotrophoblast cell lineage (CPM I), or both the cytotrophoblast and mesenchyme cell lineages (CPM III), and serious pregnancy complications . UPD was also more commonly ascertained when the frequency of trisomic cells in both cell lineages was high. Both Robinson et al. and Wolstenholme et al. correlated CPM III with a meiotic origin of the trisomy and an increase in propensity for pregnancy complications. CPM types I and II were more commonly benign, these being associated with a somatic, postzygotic origin of the trisomy, particularly when the frequency of trisomic cells was low.

Toutain et al. reported a high frequency of pregnancy complications involving CPM III in a review of 13,809 CVS samples with either CPM II (37 cases) or CPM III (20 cases) . The authors found no difference in the frequency of low birth weight, prematurity, and other adverse pregnancy outcomes for CPM II, when compared with a control population. Nor were there any confirmed cases of UPD reported (0/6 cases investigated). In contrast, the mean birth weight was lower, and the incidence of prematurity, IUGR, and pregnancy loss higher for CPM III, compared with controls. UPD was reported in 4/13 (30.8%) CPM III cases, which is consistent with the 1 in 3 theoretical prevalence expected following trisomy rescue.

From these and other studies it is clear that trisomy 16 mosaicism is the most frequent rare trisomy associated with pregnancy complications. Almost all cases have their origins in an error of maternal meiosis . A placenta containing a high proportion of trisomy 16 cells appears to be particularly vulnerable to placental insufficiency leading to fetal growth restriction and other complications. Many pregnancies with CPM for trisomy 16 are associated with very low pregnancy-associated plasma protein A (PAPP-A) levels, with a median MoM of 0.13 reported, equivalent to the 0.2th percentile . Pregnancies with a PAPP-A level below the 5th percentile have a higher likelihood for IUGR, premature birth, preeclampsia, and stillbirth , consistent with outcomes reported for trisomy 16 CPM. Other rare trisomies might also have a predisposition toward abnormal first trimester screening analytes, with a large Danish study reporting 77% of confirmed rare trisomies as being screen positive by CFTS, usually in association with low PAPP-A measurements .

The fact that CPM types I and II are more likely to have a somatic origin of trisomy also explains their much lower association with UPD. This is particularly true for trisomy 3 (CPM 1) and trisomy 2 (CPM II) mosaicisms, which are commonly reported as being confined to the cytotrophoblast and mesenchymal core of the chorionic villi, respectively. Based on the distribution of rare trisomies confined to these cell lineages, Wolstenholme et al. estimated a postzygotic origin for trisomies 2, 3, 7, and 8 of 81%, 95%, 87%, and > 95%, respectively . Therefore the risk of UPD for these chromosomes after a finding of CPM should be low. A large Italian study on mosaicism in CVS provides evidence for this, with 0/65 and 0/74 cases with UPD2 and UPD7, respectively . If a meiotic origin was common for these trisomies, up to one-third of all cases would be expected to have UPD following trisomy rescue.

Interest in pregnancy outcomes for the rare autosomal trisomies has existed since their increased prevalence was first noted after the introduction of CVS for prenatal diagnosis. A renewed interest has emerged with the arrival of genome-wide cfDNA-based NIPT. While diagnostic CVS examines only a small, localized region of the placenta, cfDNA screening provides information on the entire placental cytotrophoblast. Therefore bioinformatics algorithms can be employed to identify pregnancies with very high proportions of trisomic cells in the placenta . It is often these pregnancies that are conceived with trisomy. Several decades of CVS outcome data indicate these pregnancies are those most at risk for complications that include fetal growth restriction, premature delivery, residual trisomy mosaicism, and UPD.

Rare Autosomal Trisomies as a Cause of Unusual, False, or Failed CELL-FREE DNA Results

Bioinformatics algorithms used for cfDNA-based NIPT are designed to detect aneuploidies in the test autosomes by comparing and normalizing their sequence counts to nontest reference chromosomes (see Chapter 3 ). A potential disadvantage of this approach is that a true aneuploidy involving a reference chromosome may cause a false aneuploidy call on a test chromosome. If the reference chromosome is trisomic, a false monosomy call can occur for the test chromosome when the higher sequence counts are used for normalization. The opposite is true if the reference chromosome involves a monosomy; a false trisomy call may occur on the test chromosome.

A recent cfDNA-based NIPT study reporting on two large patient cohorts used a common quality metric (NCD; normalized chromosome denominator) to monitor for unusual sequence counts on reference and other nontest chromosomes . The larger of the two cohorts recorded 246 rare trisomies of nontarget chromosomes in approximately 73,000 pregnancies. Of these, 172 involved a reference chromosome that produced 21 cases with one or more putative false monosomy calls on the test chromosomes. Pregnancy outcomes were not available except in a small number of cases previously reported in association with maternal malignancy; these cases were associated with multiple false aneuploidy calls on the test chromosomes. The second, smaller patient cohort was analyzed using the same quality metric but in this instance the algorithm was designed to cancel the analysis rather than generate a false call on the test chromosome. Of 60 trisomies involving a reference or nontest chromosome in 16,885 pregnancies, 24 resulted in test cancellations. Cytogenetic or pregnancy outcome data in these cases indicated the fetus and/or placenta was affected by a rare trisomy that was often associated with miscarriage or other complications including IUGR, TFM, and UPD. However, normal pregnancy outcomes were also recorded. One test cancellation was found to be caused by a low-grade maternal mosaicism for trisomy 8.

Snyder et al. reported on 113,415 cfDNA cases, of which 138 cases (0.12%) were associated with autosomal monosomy ( n = 65), aneuploidy for both a common trisomy and a sex chromosome ( n = 36), or multiple autosomal aneuploidies ( n = 37) . Not surprisingly, fetal involvement was most frequent in the group involving a common trisomy and sex chromosome aneuploidy (these being true fetal abnormalities) and was least frequent when an autosomal monosomy was involved (false calls as described previously). Several maternal malignancies were confirmed in the multiple aneuploidy group. It is now well recognized that genome-wide copy number imbalances can lead to multiple aneuploidy calls for the test chromosomes and a subsequent suspicion of maternal malignancy , see also Chapter 10 .

Dharajiya et al. reported 55 cases with widespread copy number abnormalities from about 450,000 cfDNA-based NIPT . Of cases with follow-up, 18 confirmed maternal malignancies, 20 benign uterine fibroids (uterine leiomyoma), and 3 cases without evidence of disease were recorded. Of the 18 confirmed malignancies, 7 cases were known prior to NIPT (but not to the testing laboratory) and 11 cases were unknown. Thus at least 11 unknown malignancies were documented among 450,000 cfDNA-based prenatal screening tests. These findings account for a small but important cause of rare and unusual cfDNA screening results. An important advantage of a genome-wide cfDNA analysis approach is the ability to help differentiate false or misleading results involving the common aneuploidies from other genuine genome-wide copy number aberrations that may involve whole chromosomes and/or segmental aneuploidies.

Rare Trisomies Identified During CELL-FREE DNA-Based NIPT and Pregnancy Outcomes

Several investigators have documented their experience of reporting rare autosomal trisomies using genome-wide cfDNA screening . From these studies it is clear that many pregnancies with rare trisomies represent CPM and that these pregnancies may proceed to term uneventfully ( Box 1 ). However, other pregnancies may be complicated by miscarriage, poor fetal growth associated with placental insufficiency, TFM ( Box 2 ), and pathogenic UPD if an imprinted chromosome is involved ( Box 3 ). Samples where the trisomic (affected) fraction of cfDNA is similar to the fetal fraction appear most at risk for adverse outcomes . This is because the placental cytotrophoblast in these pregnancies is expected to be almost uniformly (100%) trisomic, in keeping with a meiotic origin for the trisomy. In these circumstances the consequences for the pregnancy can be catastrophic if the trisomy extends to the embryo proper.

Box 3

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  Indication: Age 36 years, NIPT as primary screening test at 11 wks 0 days

  
  
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  Genome-wide NIPT: Increased risk T15

  
  
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  Trisomic fraction (TF) = 6.6% Fetal Fraction (FF) = 6.8%

  
  
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  Ratio TF/FF: 0.97 (97% T15)

  
  
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  Follow-up ultrasound at 12 wks: viable fetus, no abnormality on scan

  
  
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  Ultrasound at 16 wks: viable fetus, no abnormality on scan.

  
  
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  Amniocentesis at 16 wks: SNP microarray normal male arr(1-22)x2,(XY)x1; comparative analysis of parental and fetal SNPs consistent with maternal UPD15 causing Prader-Willi syndrome (PWS).

  
  
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  TOP elected.

  
  
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  Placental biopsy after TOP: SNP microarray on whole chorionic villi—12%–15% T15 (meiosis I error, no recombination). CPM for trisomy 15 mosaicism confirmed.

  
  
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  Summary: T15 conception with high-grade T15 (100%) on cfDNA analysis. Trisomy rescue associated with maternal UPD15 causing PWS. PWS is characterized by hypotonia and feeding difficulties at birth, poor growth, developmental delay, and mild to moderate intellectual disability. Hyperphagia during childhood leads to obesity.

Abbreviations: PWS , Prader-Willi syndrome; SNP , single nucleotide polymorphism; TOP , termination of pregnancy; T15 , trisomy 15; wks , weeks.

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