Best Practice & Research Clinical Obstetrics and Gynaecology 28 (2014) 453–466
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Cell-free DNA testing: An aid to prenatal sonographic diagnosis Lyn S. Chitty, PhD MRCOG, Professor of Genetics and Fetal Medicine and Honorary Consultant a, b, * a
Clinical and Molecular Genetics, UCL Institute of Child Health and Great Ormond Street Hospital for Children NHS Trust, London, UK Fetal Medicine Unit, University College London Hospitals NHS Foundation Trust, London, UK
b
Keywords: fetal ultrasound genetic syndromes non-invasive prenatal diagnosis cell-free fetal DNA prenatal diagnosis
Sonographic diagnosis of fetal abnormalities is based on the recognition of sonographic patterns associated with structural abnormalities. Although diagnosis in some situations, such as neural tube defects, gastroschisis, and omphalocoele, can be straightforward, in many situations, the constellation of fetal abnormalities suggest an underlying chromosomal or genetic cause. In these situations, invasive testing is needed to provide the information required to make a definitive diagnosis, and thus accurately counsel parents. Since the identification of cell-free fetal DNA in maternal plasma, the potential for non-invasive prenatal diagnosis is increasingly becoming possible. In this chapter, the current role and future potential of non-invasive prenatal diagnosis, combined with new molecular techniques as an aid to sonographic diagnosis, will be discussed. Ó 2014 Elsevier Ltd. All rights reserved.
Properties of cell-free fetal DNA Cell-free fetal DNA (cffDNA) was first identified in maternal plasma in the late 1990s [1]. It originates from the placenta [2] and comprises fragments of DNA that are shorter on average than maternal cellfree DNA [3], but generally constitutes around 10% of total cell-free DNA (cfDNA) in maternal plasma, the majority being maternal in origin [4]. Cell-free fetal DNA can be detected in the maternal blood
* North East Thames Regional Molecular Genetics Laboratories, Great Ormond Street Hospital, York House, 37 Queen Square, London WC1N 3BH, UK. Tel.: þ44 (0) 207 813 8533. Fax: þ44 (0) 207 813 8578. E-mail address: [email protected]. 1521-6934/$ – see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bpobgyn.2014.01.002
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from 4 weeks’ gestation [5]. As it is rapidly cleared from the maternal circulation, it is undetectable 2 h after delivery [6]. It, therefore, makes a potentially ideal source of fetal genetic material for noninvasive prenatal diagnosis (NIPD). This approach is safer than current invasive prenatal diagnosis, as it is based on a maternal blood sample, and thus avoids the small but significant risk of miscarriage associated with chorionic villus sampling and amniocentesis [7]. Key properties of cffDNA are listed in Table 1. Current clinical applications of cell-free fetal DNA The earliest uses of cffDNA were for the detection of alleles not present in the mother but present in maternal blood because they had been inherited from the father. These include the two most widely used applications, namely fetal sex determination [8] and fetal Rhesus D status in Rhesus D-negative mothers [9]. More recently, some developments have allowed for NIPD of some single gene disorders, mainly those that have arisen de-novo at conception or are inherited from the father [10]. Aneuploidy testing for the major trisomies is also available through commercial providers in the USA, Asia, and much of Europe, but is not yet available in the public sector [11,12]. Analysis of cfDNA in the maternal plasma for fetal sex determination, some single gene disorders, and aneuploidy can aid sonographic diagnosis. These aspects will be discussed in more detail here. Fetal sex determination using cell-free fetal DNA in maternal plasma Fetal sex determination using cffDNA is based on the detection of Y-chromosome sequences, either SRY or DYS14, in maternal plasma. If Y-chromosome sequences are detected, the fetus is predicted to be male and, if they are absent, the fetus is predicted to be female. The exact laboratory methodology used can vary, and a recent meta-analysis of 57 reports detailing more than 6000 pregnancies tested suggested that before 7 weeks’ gestation cffDNA testing is unreliable, with the best results reported after 20 weeks’ gestation [8]. In the UK, an audit of fetal sex determination using cffDNA reported that it was highly accurate when carried out after 7 weeks’ gestation, but that it should be used in conjunction with fetal ultrasound to confirm the gestational age before testing [13]. Ultrasound should also be used to detect multiple pregnancies, and the presence of an empty gestation sac, as it is known that the placenta can continue to shed cffDNA after demise of the fetal pole p2]. Non-invasive prenatal diagnosis for fetal sex determination is now increasingly used to determine fetal sex in pregnancies at increased risk of X-linked genetic disorders and congenital adrenal hyperplasia. In the UK and some other European countries, it is now the standard of care in these situations [14]. Both women and health professionals value this approach to sex determination in these high-risk pregnancies [15,16], as it is a test that can be done early in pregnancy and is highly accurate (>99%) when delivered by accredited molecular genetic laboratories, reducing the rate of invasive testing by around 45%, and thereby avoiding unnecessary exposure to miscarriage risk [13]. Furthermore, in pregnancies at risk of congenital adrenal hyperplasia, it allows early cessation of steroid treatment where the fetus is predicted to be male [13,17]. Indeed, in some European countries, dexamethasone treatment is delayed until NIPD carried out at 7 weeks’ gestation indicates the presence of a female fetus. This allows avoidance of any unnecessary steroid treatment in male-bearing pregnancies. Finally, a detailed health economic analysis has shown that, when used in pregnancies at high risk of serious X-linked conditions (where parents might elect to terminate an affected pregnancy) and those at risk of congenital adrenal
Table 1 Key properties of cell-free fetal DNA. Placental in origin Minority of cell-free DNA in maternal plasma Detectable from 4 weeks gestation Cell-free fetal DNA is shorter on average than maternal cell-free DNA Cleared from the maternal circulation within hours of delivery
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hyperplasia, the savings generated by avoiding invasive testing and unnecessary steroid treatment mean that NIPD for fetal sex determination is no more expensive than invasive diagnostic testing [18]. Fetal sex determination using cell-free fetal DNA in the investigation of genital ambiguity Sonographic detection of an isolated abnormality of the external genitalia occurs infrequently. More common is the identification of a genital anomaly or ambiguity when detailed examination is carried out after detecting another structural abnormality. Whether an isolated finding, or in association with other structural anomalies, determination of genetic sex can be useful in defining the underlying diagnosis and directing parental counselling. In the past, this required invasive testing, but first-line testing in many cases should now be analysis of cffDNA after a maternal blood sample, although invasive testing may ultimately be required for definitive diagnosis. Isolated genital anomalies It can be difficult to differentiate cliteromegaly from hypospadias using ultrasound alone (Fig. 1), and the use of cffDNA testing to confirm the genetic sex is useful in this situation (Fig. 1), as in the absence of other anomalies or intrauterine fetal growth restriction (IUFGR), further testing for chromosomal abnormalities is not indicated. When seen in isolation in a fetus confirmed to be male, the diagnosis is most likely to be hypospadias (Table 2), and the parents should be referred to a paediatric urologist for further discussion of management and prognosis. A small risk of an underlying abnormality of steroid biosynthesis remains (Table 2), but these are rare and difficult to diagnose in cases arising de-novo in the prenatal period as, although molecular testing is available, it has a relatively low yield in the absence of a family history [19]. If cffDNA testing suggests that the fetus is female, there is a greater risk of an underlying genetic abnormality of steroid biosynthesis, and the parents are best referred to the disorders of sexual development team for further investigation and management (Table 2). In this situation, invasive testing may be required for more accurate evaluation of steroid profiles [20,21].
Fig. 1. (A) Ultrasound image of hypospadias; (B) the reverse transcription-polymerase chain reaction showing amplification of the SRY sequences in cffDNA in the maternal plasma; (C) note the similarity of the image of cliteromegaly; and (D) the lack of amplification of SRY sequences in this female fetus. The amplification of control DNA in (B) and (D) indicates that absence of amplification is not due to assay failure.
Other sonographic findings
cffDNA testing
Differential diagnosis
Other aids to prenatal diagnosis
Management
Ambiguous genitalia
None.
Male.
Isolated hypospadias. Inadequate production of testosterone caused by Leydig cell hypoplasia or biosynthetic defects.congenital lipoid adrenal hyperplasia 17a-hydroxylase deficiency 3b-hydroxysteroid dehydrogenase deficiency 17,20-lyase deficiency 17b-hydroxysteroid dehydrogenase deficiency
Steroid profile.a Consider sequencing of the androgen receptor gene.a
Refer to DSD team for investigation and counselling.
Amniotic steroid levels.a Maternal serum androgen levels.a Maternal urinary oestrogen levels. Maternal ovarian scan for multicystic change.
Refer to DSD team for investigation and counselling. Refer to gynaecology or oncology if luteoma.
Fetal biometry. cfDNA or invasive testing to exclude aneuploidy
Serial monitoring by high-risk pregnancy team. Urology team for management of hypospadias counselling.
Ambiguous genitalia
Ambiguous genitalia or hypospadias
None.
Biometry 3rd percentile. Abnormal maternal and fetal Dopplers
Female.
Male.
Partial androgen insensitivity syndrome. 5a-reductase deficiency. True hermaphrodite. Isolated cliteromegaly. Congenital adrenal hyperplasia 21-OH deficiency 11-OH deficiency 3b-hydroxysteroid dehydrogenase deficiency True hermaphrodite. Maternally derived androgens (e.g. luteoma of pregnancy). Placental aromatase deficiency. Isolated hypospadias with intrauterine growth restriction. Aneuploidy. Confined placental mosaicism.
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Genital anomaly
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Table 2 Causes of sonographically detected anomalies of the external genitalia where determination of genetic sex using cffDNA may be a useful in aiding diagnosis. Results are based on the experience of a tertiary Fetal Medicine Unit over a 10-year period and is adapted from Pajkrt et al. [20] and Chitty et al. [21]. Further details of the steroid investigations can be found in Chitty et al. [21].
No intra-abdomina; bladder. Low cord insertion.
Male.
Bladder exstrophy. Cloacal exstrophy.
Detailed anomaly scan.
Abnormal genitalia
Absent bladder, intra-abdominal cystic mass, dilated bowel, and abnormal spine. Omphalocoele and gastroschisis, abnormal spine, absent bladder, and hydronephrosis
Female.
Detailed anomaly scan. cfDNA snf invasive testing to exclude aneuploidy. cfDNA and invasive testing to exclude aneuploidy.
Abnormal genitalia or micropenis
Echogenic kidneys, with or without polydactyly
Male.
Cloacal exstrophy. Other cloacal abnormality. Aneuploidy. OEIS complex (omphalocoele, bladder exstrophy, imperforate anus, spinal defects). Cloacal abnormality. Aneuploidy. Bardet–Biedl syndrome. Trisomy 13.
Ambiguous or female appearing genitalia
Multiple anomalies, cardiac anomaly, polysyndactyly, intrauterine fetal growth restriction, oedema, cleft lip, central nervous system anomalies, microcephaly, and short limbs.
Male.
Smith–Lemli–Opitz syndrome. Cranio–cerebellar–cardiac syndrome. Short-ribbed polydactyly syndromes. Other genetic syndrome. Aneuploidy.
Ambiguous or female appearing genitalia
Bowing of femora, with or without tibia and fibula. Micrognathia, cardiac anomalies. None.
Male.
Campomelic dysplasia.
cfDNA and invasive testing to exclude aneuploidy. Detailed scan. Maternal urinary steroids levels. Mostly autosomal recessive, take family history for affected members and consanguinity. Detailed scan.
Male.
Laboratory or clerical error. Androgen insensitivity syndrome.
Abnormal genitalia
Genotype discordant with phenotype a
Male or female.
cfDNA and invasive testing to exclude aneuploidy.
cfDNA and invasive testing to confirm discordance between genotype and phenotype.b
Refer to urologists and paediatric surgeons for counselling. Refer to combined fetal-urology team for counselling. Refer to paediatric surgeons and urologists.
Autosomal recessive: family history for affected members and consanguinity. Refer to clinical genetics. Refer to urology and nephrology teams for counselling. Refer to clinical geneticist. Refer to all relevant paediatric teams for discussion of prognosis.
Refer clinical geneticist and skeletal dysplasia clinic. Refer specialist DSD team.
Investigations such as steroid profiling, sequencing of androgen receptor gene, or both, are best managed by a DSD team. In situations where there is an abnormality in the SRY gene (i.e. as androgen insensitivity syndrome), cffDNA using SRY may give misleading results. cffDNA, cell-free fetal DNA; DSD, disorders of sex development. b
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No penis seen, splayed glans and micropenis
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Genital anomalies and intrauterine fetal growth restriction Sonographically detected genital anomalies and ambiguities in a fetus with intrauterine fetal growth restriction (IUFGR), as shown by biometry on or below the third percentile, with or without abnormal maternal and fetal Doppler measurements, is not uncommon [22]. Determination of fetal sex using cffDNA can be a useful aid to diagnosis, and adds weight to the diagnosis of IUFGR. The noninvasive approach can be particularly useful in situations where parents are keen to avoid the risks associated with invasive testing. Uniparental disomy [23] or confined placental mosaicism [24] have been implicated as possible causes, but aneuploidy-related or idiopathic IUFGR are more common. In view of the risk of chromosomal abnormalities, analysis of cfDNA could include testing for aneuploidy (see section on non-invasive prenatal testing for aneuploidy), but currently this will only detect the major trisomies. Therefore, invasive testing and microarray analysis may be more appropriate, as this will detect a wider range of chromosomal rearrangements [25]. Genital anomalies in association with urogenital tract abnormalities Genital abnormalities are most commonly identified in association with other fetal abnormalities [21]. In this situation, the underlying pathology can be broad, with the two main categories being a urogenital tract anomaly or genetic syndrome (Table 2). Probably the most common association is with abnormalities of development of the urogenital tract, including bladder and cloacal exstrophy. Here, knowledge of genetic sex aids prenatal counselling, as the prognosis for these conditions, and hence prenatal counselling, varies for affected males and females [26,27]. Accurate sonographic gender assignment in these conditions is rarely achieved because of the involvement of external genitalia [28]. As an association with aneuploidy is uncommon in the absence of extra-renal anomalies, fetal sex determination using cffDNA is appropriate in these situations, facilitating accurate prenatal counselling without recourse to unnecessary invasive testing. Genital anomalies in association with other fetal abnormalities The association of genital anomalies with genetic syndromes is broad. There are more than 350 syndromes with hypospadias or micro-penis and around 40 with cliteromegaly [29]. In many instances, exclusion of aneuploidy may be the most appropriate first-line investigation in fetuses with multiple abnormalities; however, in some cases, accurate determination of fetal genetic sex using cffDNA can be instrumental in arriving at a definitive diagnosis without recourse to invasive testing (Table 2). Examples include Smith–Lemli–Opitz (SLO) syndrome, Bardet–Biedl syndrome, and campomelic dysplasia. Smith Lemli Opitz syndrome is a genetic syndrome, inherited in an autosomal recessive fashion, which results in an abnormality in cholesterol biosynthesis owing to a mutation in the 7dehydrocholesterol reductase (7DHC) gene. The clinical spectrum of SLO is wide, varying from developmental delay and mild dysmorphic features to those with major structural defects with early or prenatal lethality [30]. The severe form usually presents prenatally with a wide variety of fetal abnormalities [31], including postaxial polydactyly, facial clefts, anomalies of the brain, heart and renal tract, hydrops, and genital anomalies, which are reported to occur in around 90% of affected males [30]. Fetal sex determination in the presence of this spectrum of abnormalities in association with genital ambiguity can increase suspicion of SLO and direct investigations towards analysis of maternal urine for measurement of dehydro-oestriol and dehydropregnanetriol in maternal urine [32] rather than testing for aneuploidy. Another genetic syndrome where fetal sex determination using cffDNA may be of value in coming to a definitive diagnosis is Bardet–Biedl syndrome. This is a rare condition, usually inherited in an autosomal recessive fashion and characterised by obesity, developmental delay, polydactyly, genital anomalies, and renal failure in some cases [33]. In our unit, we have seen cases presenting with large echogenic kidneys, polydactyly, and abnormal external genitalia (Fig. 2), features compatible with trisomy 13, except that fetal growth was normal. Determination of male sex after analysis of cffDNA helped arrive at a probable diagnosis of Bardet–Biedl syndrome. Definitive diagnosis in cases at low
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Fig. 2. Ultrasound images showing the genital ambiguity (*) in (A) large echogenic kidneys; (B) polydactyly; and (C) in a male fetus subsequently found to have Bardet–Biedl syndrome.
prior risk with no family history currently requires molecular genetic analysis. Although it is becoming possible using next-generation sequencing to screen for mutations in the multiple genes responsible for this condition [34], it remains difficult in low-risk cases presenting de-novo in pregnancy. The development of comprehensive gene panels, however, may make rapid, definitive prenatal diagnosis possible in the near future. Furthermore, given the advances in NIPD, this may become possible by analysing cell-free DNA in maternal plasma [10]. Anomalies of the lower leg, excluding isolated talipes, are rare and not usually associated with aneuploidy. The differential diagnosis usually lies between isolated femoral hypoplasia (which can be part of the caudal regression spectrum), femoral hypoplasia, unusual facies syndrome, and campomelic dysplasia. In the latter, bilateral shortening and bowing of the long bones is confined to the legs, all other long bones being normally formed and grown. Other sonographic findings can include talipes, micrognathia, and cardiac abnormalities [35]. A high incidence of genital ambiguity or sex reversal occurs in affected males (Fig. 3). Analysis of cffDNA in maternal plasma to confirm sex reversal in the presence of this constellation of findings is sufficient to reach a definitive diagnosis of campomelic dysplasia and counsel parents accordingly. Non-invasive prenatal diagnosis as an aid to the diagnosis of skeletal abnormalities Skeletal dysplasias are a genetically heterogeneous group of disorders, many of which can be diagnosed using fetal ultrasound [36], with most that are lethal amenable to detection in early pregnancy [37]. Until recently, for most dysplasias presenting in pregnancy in the absence of a family history, definitive diagnosis had to await postnatal investigations, molecular genetic testing, radiology, histopathology, or both. Definitive molecular diagnosis in pregnancy is difficult because of heterogeneity and multiple mutations responsible for these conditions so that definitive prenatal diagnosis was largely confined to pregnancies at high risk because of a relevant family history.
Fig. 3. Ultrasound image showing (A) genital ambiguity; (B) bowed femora, short lower leg bone and talipes; and (C) male fetus with campomelic dysplasia.
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Achondroplasia is the most common non-lethal skeletal dysplasia and presents with short limbs detected after 25 weeks’ gestation (Fig. 4). It is an autosomal dominant condition, but most cases arise de-novo as a result of a sporadic mutation in the FGFR3 gene. Other sonographic features that can be seen include frontal bossing, short fingers, mild femoral bowing, polyhydramnios, and a slightly small chest (Fig. 5) [38]. Although the sonographic findings can be seen from 25 weeks, presentation is more usual late in the third trimester when scanning is indicated because of polyhydramnios. In this situation, detection of other features may be difficult, and a major differential diagnosis is aneuploidy. As most cases (98%) of achondroplasia are caused by a single mutation in the FGFR3 gene [39], definitive molecular prenatal diagnosis of this condition has been possible after amniocentesis for some years [40]. More recently, NIPD for this condition has been developed [38], and is available in the UK through regional genetics and fetal medicine services (http://ukgtn.nhs.uk/find-a-test/search-by-disordergene/test-service/achondroplasia-nipd-600/). The advantage of the NIPD approach in this situation is that it only requires a maternal blood sample, and so avoids the risk of precipitating preterm labour, while allowing a definitive diagnosis to be made. NIPD can be used for safe prenatal diagnosis of achondroplasia in cases arising de-novo or inherited from the father, but is not appropriate currently for molecular diagnosis of cases inherited from the mother owing to the high background of maternal mutant allele present in cell-free DNA. Thanatophoric dysplasia is the most common lethal skeletal dysplasia. There are two types, TDI and TDII, both of which are caused by mutations in the FGFR3 gene, but rather than one common mutation causing the condition, at least 12 causative mutations occur, although three occur more commonly [41,42]. There are two types of thanatophoric dysplasia, both present with short limbs from early in pregnancy (Fig. 4). Other sonographic findings include a small chest, short ribs, short fingers (trident hands), bowing of the femora (otherwise known as telephone receiver femora) and, in Type II thanatophoric dysplasia, craniosynostosis [43] (Fig. 6). The differential diagnosis for thanatophoric dysplasia includes other skeletal dysplasias, including Jeunes asphyxiating thoracic dystrophy, shortribbed polydactyly syndromes, and achondrogenesis. All are serious and potentially lethal conditions, but making a definitive diagnosis is important as many are autosomal recessive, and thus carry a high recurrence risk in future pregnancies, whereas thanatophoric dysplasia arises de-novo as a lethal dominant mutation and thus has a low, gonadal mosaicism, recurrence risk. Definitive molecular prenatal diagnosis for the common mutations has been available for some time after chorionic villus
Fig. 4. Fetal femur length chart showing normal centiles (solid lines) with femur measurements of fetuses with achondroplasia (open circle) and thanatophoric dysplasia (closed circle). Published with permission [43].
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Fig. 5. Ultrasound image of (A) the profile of a fetus with achondroplasia showing frontal bossing and polyhydramnios; (B) short fingers; and (C) a short, and slightly bowed femur.
sampling or amniocentesis [44], with NIPD for the two most common mutations also possible since 2012 [43]; (http://ukgtn.nhs.uk/find-a-test/search-by-disorder-gene/test-service/thanatophoricdysplasia-nipd-599/). The original NIPD approach was based on a polymerase chain reaction with restriction enzyme digest (PCR-RED) methodology. This approach cannot be developed for all thanatophoric dysplasia mutations, and in any event, testing has to be done for individual mutations, which is both time-consuming and costly. Thus, although it is reasonably straightforward to use NIPD testing for the presence or absence of the common mutations, if these are negative, a diagnosis of thanatophoric dysplasia cannot be completely excluded. The development of desk-top, next generation sequencing platforms has provided the technology to deliver a sensitive and flexible approach to NIPD, as it is possible to design gene panels that can be used to screen for multiple mutations in a single assay [12,45]. In our Regional Genetics Laboratory, we have designed such a panel for all mutations in the FGFR3 gene [45]. By using this approach, we have accurately determined the mutation status in all 15 cases at risk of thanatophoric dysplasia because of relevant sonographic findings (n ¼ 11) or germline mosaicism (n ¼ 4). In addition to identifying the two most common mutations, we also detected rarer mutations in eight of these cases. Non-invasive prenatal diagnosis for thanatophoric dysplasia offers considerable benefits, particularly as the increasing use of early ultrasound for combined Down’s syndrome screening has resulted in early diagnosis of many skeletal dysplasias [37]. Thanatophoric
Fig. 6. (A) Sonographic features of thanatophoric dysplasia showing an axial view of the head in a fetus with thanatophoric dysplasia II and a cloverleaf skull; (B) a longitudinal view of the thorax and abdomen demonstrating the small chest; (C) a sagittal view showing the frontal bossing also demonstrating a small chest; (D) a view of a hand illustrating the short fingers; and (E) a postnatal radiograph showing the bony features.
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dysplasia is readily diagnosed early in pregnancy, as the femurs are short from 12 weeks and other sonographic signs are also present (Figs. 4 and 6). Although definitive diagnosis of thanatophoric dysplasia is possible after chorionic villus sampling, our experience is that many women when given the diagnosis of a lethal condition in their fetus are reluctant to undergo invasive testing. Many will elect to terminate an affected pregnancy and, if diagnosed early enough, would opt for a surgical termination, which precludes a post-mortem examination for definitive diagnosis. The availability of NIPD in this situation allows for definitive diagnosis based on a maternal blood sample. If positive, a surgical termination is a reasonable approach, but if negative other differential diagnoses need to be considered and the medical approach to termination is preferable to allow for a post-mortem, definitive diagnosis and accurate genetic counselling for risks in future pregnancies. The other situation in which NIPD may prove to be useful is in multiple pregnancies discordant for abnormalities compatible with thanatophoric dysplasia. Here, definitive diagnosis based on analysis of cffDNA avoids invasive testing with its associated risks to the normal fetus, and also informs pregnancy management as there is no need to consider feticide for the affected fetus as the condition is lethal. Apert syndrome is a rare autosomal dominant genetic disorder, with 98% of cases caused by one of two mutations in the FGFR2 gene [46]. It is characterised by craniosynostosis, syndactyly (also known as ‘mitten hands’), proptosis, mid-facial hypoplasia, central nervous system anomalies, and varying degrees of development delay, although many have low to normal IQ [47,48]. It is amenable to sonographic diagnosis from early in the second trimester (Fig. 7) [48], but definitive diagnosis requires molecular genetic confirmation. This can readily be done using NIPD for all cases arising de-novo or those at risk of inheriting a paternal mutation [12]. The role of cell-free DNA testing for aneuploidy in the presence of sonographic abnormalities The high background level of maternal chromosome 21 cfDNA in maternal plasma make noninvasive testing for Down’s syndrome and other aneuploidies more challenging than the testing described above, which is designed to test for the presence or absence of an allele not present in the mother. For aneuploidy diagnosis, detection of the relatively small changes in the level of individual chromosome-specific fragments in maternal plasma when the fetus has aneuploidy must be accurately quantified. This has proved to be possible using next-generation sequencing, and several large-scale validation studies have now been conducted, which report detection rates for Down’s syndrome of greater than 99%, with a false positive rate of 0.1–1% [11]. This approach can also detect other
Fig. 7. (A) Two-dimensional ultrasound images of the fetal head showing the unusual skull shape and (B, D) mitten hands. The use of three-dimensional ultrasound (D and E) clearly show the syndactyly of the toes and hands (C, G, H, I) and the proptosis (F).
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aneuploidies, including trisomy 18 (99% accurate), trisomy 13 (up to 90% accurate) [49–52], and sex chromosome abnormalities. A small, but consistently false–positive rate is reported, which results from the fact that cffDNA arises from the placenta, and that, when screening for aneuploidy, it is the total cfDNA that is analysed, not just the fetal component. Thus, discrepant results have been reported as a result of confined placental mosaicism [53,54], detection of maternal chromosomal rearrangements [54], and even maternal tumours secreting an abnormal chromosome compliment [55]. For these reasons, cfDNA testing for aneuploidy is not fully diagnostic, but rather considered as an advanced screening test (non-invasive prenatal testing [NIPT]), and positive results should be confirmed by invasive testing [56–58]. In the presence of abnormalities consistent with the relevant trisomy (e.g. duodenal atresia and an atrio-ventricular septal defect in Down’s syndrome, holoprosencephaly and IUFGR in trisomy 13 or omphalocoele, talipes, clenched hands in trisomy 18), it might be considered reasonable to consider NIPT diagnostic. A negative NIPT result in the presence of fetal abnormalities does not necessarily mean that there is no chromosomal abnormality present: not only is there a small false negative rate associated with NIPT [11], but currently available tests only screen for the major trisomies and do not detect other chromosomal rearrangements or micro-deletion syndromes, which may be found in a significant proportion of structurally abnormal fetuses [27,59]. Non-invasive prenatal testing for a wider range of chromosomal abnormalities may become possible in the not too distant future as detection of other chromosomal rearrangements using next-generation sequencing has been reported, but the depth of sequencing required is significantly higher than for standard NIPT, making the cost of testing currently too high for routine use [60,61]. Conclusion Non-invasive prenatal diagnosis and testing for fetal sex determination, single disorders, and aneuploidy, can aid diagnosis of the fetus with sonographic abnormalities. In selected circumstances, it can offer safe definitive diagnosis of the underlying pathology based on a maternal blood sample, avoiding the risks associated with invasive diagnostic testing. Sex determination by analysis of cffDNA should be considered in most situations in which genital abnormalities are detected. Non-invasive prenatal diagnosis NIPD for single gene disorders is becoming increasingly possible. It is already the test of choice when faced with sonographic findings associated with the skeletal anomalies associated with mutations in the FGFR2 and 3 genes. With the further development of gene panels for a wide variety of conditions, NIPD will become a useful aid to sonographic diagnosis of many anomalies (e.g. large echogenic kidneys, and a wider variety of skeletal dysplasias). Given the pace of developments in this area, sonographic diagnosis suggestive of an underlying genetic syndrome should prompt referral to a genetic team familiar with NIPD. Cell-free DNA testing for the common trisomies is highly accurate, but currently does not detect other unbalanced rearrangements or micro-deletion syndromes, thus invasive testing for full karyotyping or microarray analysis of chorionic villi or amniocytes may be required in the presence of fetal abnormalities and a normal NIPT result.
Practice points Analysis of cffDNA in maternal plasma is a safe and accurate method for determining genetic sex. It should be considered after sonographic identification of genital anomalies or ambiguity, as it can avoid invasive diagnostic testing and facilitate appropriate parental counselling. Analysis of cffDNA for determining genetic sex should be considered in the presence of female or ambiguous genitalia, and anomalies compatible with SLO or campomelic dysplasia, as identification of sex reversal in genetically male fetuses can facilitate accurate prenatal diagnosis. NIPD based on cffDNA is available for selected genetic conditions; in particular, some skeletal dysplasias (achondroplasia and thanatophoric dysplasia) and syndromes associated with
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craniosynostosis (Apert syndrome). It should be considered as a safe and robust alternative to invasive testing. NIPT for aneuploidy is available privately, and could be considered as an alternative to invasive testing in the presence of sonographic abnormalities suggestive of aneuploidy. As this currently only tests for the common trisomies, however, invasive testing may be more appropriate, as karyotyping detects a wider range of unbalanced rearrangements, and microarray testing can also detect micro-deletion and duplication syndromes. NIPD and NIPT are a rapidly developing technology, and close liaison should take place with clinical geneticists familiar with NIPD and NIPT so that patients can access new tests as they become available if appropriate.
Research agenda Further development of gene panels designed to analyse short fragments of cffDNA to cover a wider range of genetic conditions to enable molecular genetic diagnosis of more fetal abnormalities. Development of cost effective NIPT for sub-chromosome abnormalities, including microdeletion and duplication syndromes.
Conflict of interest None declared. References [1] Lo YMD, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350:485–7. [2] Alberry M, Maddocks D, Jones M, et al. Free fetal DNA in maternal plasma in anembryonic pregnancies: confirmation that the origin is the trophoblast. Prenat Diagn 2007;27:415–8. [3] Chan KCA, Zhang J, Hui ABY, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem 2004;50: 88–92. [4] Lun FMF, Chiu RWK, Allen Chan KC, et al. Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Clin Chem 2008;54:1664–72. [5] Illanes S, Denbow M, Kailasam C, et al. Early detection of cell-free fetal DNA in maternal plasma. Early Hum Dev 2007;83: 563–6. [6] Lo YMD, Zhang J, Leung TN, et al. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 1999;64:218–24. [7] Tabor A, Alfirevic Z. Update on procedure-related risks for prenatal diagnosis techniques. Fetal Diagn Ther 2010;27:1–7. [8] Devaney SA, Palomaki GE, Scott JA, et al. Non-invasive fetal sex determination using cell-free fetal DNA: a systematic review and meta-analysis. JAMA 2011;306:627–36. [9] Daniels G, Finning K, Martin P, et al. Non-invasive prenatal diagnosis of fetal blood group phenotypes: current practice and future prospects. Prenat Diagn 2009;29:101–7. *[10] Lench N, Barrett A, Fielding S, et al. The clinical implementation of non-invasive prenatal diagnosis for single-gene disorders: challenges and progress made. Prenat Diagn 2013;33:555–62. *[11] Boon EM, Faas BH. Benefits and limitations of whole genome versus targeted approaches for non-invasive prenatal testing for fetal aneuploidies. Prenat Diagn 2013;33:563–8. [12] Walsh JM, Goldberg JD. Fetal aneuploidy detection by maternal plasma DNA sequencing: a technology assessment. Prenat Diagn 2013;33:514–20. [13] Hill M, Finning K, Martin P, et al. Non-invasive prenatal determination of fetal sex: translating research into clinical practice. Clin Genet 2011;80:68–75. *[14] Hill M, Lewis C, Jenkins L, et al. Implementing non-invasive prenatal fetal sex determination using cell-free fetal DNA in the UK. Expert Opin Biol Ther 2012;12(Suppl. 1):S119–26. [15] Lewis C, Hill M, Skirton H, et al. Fetal sex determination using cell-free fetal DNA: service users’ experiences of and preferences for service delivery. Prenat Diagn 2012;32:735–41. *[16] Lewis C, Hill M, Skirton H, et al. Non-invasive prenatal diagnosis for fetal sex determination - benefits and disadvantages from the service users’ perspective. Eur J Hum Genet 2012;20:1127–33. [17] Hyett JA, Gardener G, Stojilkovic-Mikic T, et al. Reduction in diagnostic and therapeutic interventions by non-invasive determination of fetal sex in early pregnancy. Prenat Diagn 2005;25:1111–6.
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[18] Hill M, Taffinder S, Chitty LS, et al. Incremental cost of non-invasive prenatal diagnosis versus invasive prenatal diagnosis of fetal sex in England. Prenat Diagn 2011;3:267–73. [19] Adam MP, Fechner PY, Ramsdell LA, et al. Ambiguous genitalia: what prenatal genetic testing is practical? Am J Med Genet A 2012;158A:1337–43. [20] Pajkrt E, Petersen OB, Chitty LS. Fetal genital anomalies: an aid to diagnosis. Prenat Diagn 2008;28:389–98. *[21] Chitty LS, Chatelain P, Wolffenbuttel KP, et al. Prenatal management of disorders of sex development. J Pediatr Urol 2012; 8:576–84. [22] Nemec SF, Nemec U, Brugger PC, et al. Male genital abnormalities in intrauterine growth restriction. Prenat Diagn 2012; 32:427–31. [23] Hansen WF, Bernard LE, Langlois S, et al. Maternal uniparental disomy of chromosome 2 and confined placental mosaicism for trisomy 2 in a fetus with intrauterine growth restriction, hypospadias, and oligohydramnios. Prenat Diagn 1997;17:443–50. [24] Kalousek DK, Howard-Peebles PN, Olson SB, et al. Confirmation of CVS mosaicism in term placentae and high frequency of intrauterine growth retardation association with confined placental mosaicism. Prenat Diagn 1991;11:743–50. [25] Callaway JL, Shaffer LG, Chitty LS, et al. The clinical utility of microarray technologies applied to prenatal cytogenetics in the presence of a normal conventional karyotype: a review of the literature. Prenat Diagn 2013;33:1119–23. [26] Wilcox DT, Chitty LS. Non-visualisations of the fetal bladder: aetiology and management. Prenat Diagn 2001;21:977–83. [27] Bischoff A, Calvo-Garcia MA, Baregamian N, et al. Prenatal counseling for cloaca and cloacal exstrophy-challenges faced by pediatric surgeons. Pediatr Surg Int 2012;28:781–8. [28] Goyal A, Fishwick J, Hurrell R, et al. Antenatal diagnosis of bladder/cloacal exstrophy: challenges and possible solutions. J Pediatr Urol 2012;8:140–4. [29] Winter RM, Baraitser M. The London dysmorphology database. Oxford: Oxford University Press, Electronic Publishing; 2001. [30] Ryan AK, Bartlett K, Clayton P, et al. Smith-Lemli-Opitz syndrome: a variable clinical and biochemical phenotype. J Med Genet 1998;35:558–65. [31] Chitty L, Gardener G, Overton T. Edematous polydactyly in Smith–Lemli–Opitz syndrome Type II. Ultrasound Obstet Gynecol 2004;23:629–30. [32] Shackleton CHL, Roitman E, Kratz LE, et al. Mid-gestational maternal urine steroid markers of fetal Smith-Lemli-Opitz (SLO) syndrome (7-dehydrocholesterol 7-reductase deficiency). Steroids 1999;64:446–52. [33] Forsythe E, Beales PL. Bardet-Biedl syndrome. Eur J Hum Genet 2013;21:8–13. [34] Janssen S, Ramaswami G, Davis EE, et al. Mutation analysis in Bardet-Biedl syndrome by DNA pooling and massively parallel resequencing in 105 individuals. J Hum Genet 2011;129:79–90. [35] Mansour S, Hall CM, Pembrey ME, et al. A clinical and genetic study of campomelic dysplasia. J Med Genet 1995;32:415–20. [36] Krakow D, Lachman RS, Rimoin DL. Guidelines for the prenatal diagnosis of fetal skeletal dysplasias. Genet Med 2009;11: 127–33. [37] Khalil A, Pajkrt E, Chitty LS. Early prenatal diagnosis of skeletal anomalies. Prenat Diagn 2011;31:115–24. *[38] Chitty LS, Griffin DR, Meaney C, et al. New aids for the non-invasive prenatal diagnosis of achondroplasia: dysmorphic features, charts of fetal size and molecular confirmation using cell-free fetal DNA in maternal plasma. Ultrasound Obstet Gynecol 2011;37:283–9. [39] Bellus GA, Hefferon TW, Ortiz de Luna RI, et al. Achondroplasia is defined by recurrent G380R mutations of FGFR3. Am J Hum Genet 1995;56:368–73. [40] Mesoraca A, Pilu G, Perolo A, et al. Ultrasound and molecular mid-trimester prenatal diagnosis of de novo achondroplasia. Prenat Diagn 1996;16:764–8. [41] Tavormina PL, Shiang R, Thompson LM, et al. Thanatophoric dysplasia (types I and II) caused by distinct mutations in fibroblast growth factor receptor 3. Nat Genet 1995;9:321–8. [42] Wilcox WR, Tavormina PL, Krakow D, et al. Molecular, radiologic, and histopathologic correlations in thanatophoric dysplasia. Am J Med Genet 1998;78:274–81. [43] Chitty LS, Khalil A, Barrett AN, et al. Safe, accurate, prenatal diagnosis of thanatophoric dysplasia using ultrasound and free fetal DNA. Prenat Diagn 2013;33:416–23. [44] Sawai H, Komori S, Ida A, et al. Prenatal diagnosis of thanatophoric dysplasia by mutational analysis of the fibroblast growth factor receptor 3 gene and a proposed correction of previously published PCR results. Prenat Diagn 1999;19:21–4. [45] Barrett AN, Fielding S, McKay F, et al. Analysis of cell-free DNA in maternal plasma using next generation sequencing allows for safer, accurate and more comprehensive prenatal diagnosis of monogenic disorders. (Under review). [46] Oldridge M, Zackai EH, McDonald-McGinn DM, et al. De novo alu-element insertions in FGFR2 identify a distinct pathological basis for Apert syndrome. Am J Hum Genet 1991;64:446–61. [47] Cohen MM, Kreiborg Jr S. An updated pediatric perspective on the Apert syndrome. Am J Dis Child 1993;147:989–93. [48] David AL, Turnbull C, Scott R, et al. Diagnosis of Apert syndrome in the second-trimester using 2D and 3D ultrasound. Prenat Diagn 2007;27:629–32. [49] Sparks AB, Struble CA, Wang ET, et al. Non-invasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18. Am J Obstet Gynecol 2012;206:319.e1–9. *[50] Norton ME, Brar H, Weiss J, et al. Non-Invasive Chromosomal Evaluation (NICE) Study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol 2012;207:137.e1–8. *[51] Futch T, Spinosa J, Bhatt S, et al. Initial clinical laboratory experience in non-invasive prenatal testing for fetal aneuploidy from maternal plasma DNA samples. Prenat Diagn 2013;33:569–74. [52] Liang D, Lv W, Wang H, et al. Non-invasive prenatal testing of fetal whole chromosome aneuploidy by massively parallel sequencing. Prenat Diagn 2013;33:409–15. [53] Pan M, Li FT, Li Y, et al. Discordant results between fetal karyotyping and non-invasive prenatal testing by maternal plasma sequencing in a case of uniparental disomy 21 due to trisomic rescue. Prenat Diagn 2013;33:598–601. *[54] Lau TK, Jiang FM, Stevenson RJ, et al. Secondary findings from non-invasive prenatal testing for common fetal aneuploidies by whole genome sequencing as a clinical service. Prenat Diagn 2013;33:602–8.
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[55] Osborne CM, Hardisty E, Devers P, et al. Discordant non-invasive prenatal testing results in a patient subsequently diagnosed with metastatic disease. Prenat Diagn 2013;33:609–11. *[56] ACOG Statement – The American College of Obstetricians and Gynecologists Committee on Genetics; The Society for Maternal-Fetal Medicine Publications Committee. Committee Opinion. Obstet Gynecol 2012;120:1532–4. [57] Benn PA, Borrell A, Cuckle H, et al. Prenatal detection of Down syndrome using massively parallel sequencing (MPS): a rapid response statement from a committee on behalf of the Board of the International Society for Prenatal Diagnosis 24 October 2011. Prenat Diagn 2012;32:1–2. [58] Langlois S, Brock JA, Genetics Committee, et al. Current status in non-invasive prenatal detection of Down syndrome, trisomy 18 and trisomy 13 using cell-free DNA in maternal plasma. J Obstet Gynaecol Can 2013;35:177–81. [59] Ogilvie CM, Lashwood A, Chitty L, et al. The future of prenatal diagnosis: rapid testing or full karyotype? An audit of chromosome abnormalities and pregnancy outcomes for women referred for Down’s Syndrome testing. BJOG 2005;112: 1369–13675. [60] Srinivasan A, Bianchi DW, Huang H, et al. Non-invasive detection of fetal sub-chromosome abnormalities via deep sequencing of maternal plasma. Am J Hum Genet 2013;92:167–76. [61] Chen S, Lau TK, Zhang C, et al. A method for non-invasive detection of fetal large deletions/duplications by low coverage massively parallel sequencing. Prenat Diagn 2013;33:584–90.
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10
Cell-free DNA testing: An aid to prenatal sonographic diagnosis Lyn S. Chitty, PhD MRCOG, Professor of Genetics and Fetal Medicine and Honorary Consultant a, b, * a
Clinical and Molecular Genetics, UCL Institute of Child Health and Great Ormond Street Hospital for Children NHS Trust, London, UK Fetal Medicine Unit, University College London Hospitals NHS Foundation Trust, London, UK
b
Keywords: fetal ultrasound genetic syndromes non-invasive prenatal diagnosis cell-free fetal DNA prenatal diagnosis
Sonographic diagnosis of fetal abnormalities is based on the recognition of sonographic patterns associated with structural abnormalities. Although diagnosis in some situations, such as neural tube defects, gastroschisis, and omphalocoele, can be straightforward, in many situations, the constellation of fetal abnormalities suggest an underlying chromosomal or genetic cause. In these situations, invasive testing is needed to provide the information required to make a definitive diagnosis, and thus accurately counsel parents. Since the identification of cell-free fetal DNA in maternal plasma, the potential for non-invasive prenatal diagnosis is increasingly becoming possible. In this chapter, the current role and future potential of non-invasive prenatal diagnosis, combined with new molecular techniques as an aid to sonographic diagnosis, will be discussed. Ó 2014 Elsevier Ltd. All rights reserved.
Properties of cell-free fetal DNA Cell-free fetal DNA (cffDNA) was first identified in maternal plasma in the late 1990s [1]. It originates from the placenta [2] and comprises fragments of DNA that are shorter on average than maternal cellfree DNA [3], but generally constitutes around 10% of total cell-free DNA (cfDNA) in maternal plasma, the majority being maternal in origin [4]. Cell-free fetal DNA can be detected in the maternal blood
* North East Thames Regional Molecular Genetics Laboratories, Great Ormond Street Hospital, York House, 37 Queen Square, London WC1N 3BH, UK. Tel.: þ44 (0) 207 813 8533. Fax: þ44 (0) 207 813 8578. E-mail address: [email protected]. 1521-6934/$ – see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bpobgyn.2014.01.002
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from 4 weeks’ gestation [5]. As it is rapidly cleared from the maternal circulation, it is undetectable 2 h after delivery [6]. It, therefore, makes a potentially ideal source of fetal genetic material for noninvasive prenatal diagnosis (NIPD). This approach is safer than current invasive prenatal diagnosis, as it is based on a maternal blood sample, and thus avoids the small but significant risk of miscarriage associated with chorionic villus sampling and amniocentesis [7]. Key properties of cffDNA are listed in Table 1. Current clinical applications of cell-free fetal DNA The earliest uses of cffDNA were for the detection of alleles not present in the mother but present in maternal blood because they had been inherited from the father. These include the two most widely used applications, namely fetal sex determination [8] and fetal Rhesus D status in Rhesus D-negative mothers [9]. More recently, some developments have allowed for NIPD of some single gene disorders, mainly those that have arisen de-novo at conception or are inherited from the father [10]. Aneuploidy testing for the major trisomies is also available through commercial providers in the USA, Asia, and much of Europe, but is not yet available in the public sector [11,12]. Analysis of cfDNA in the maternal plasma for fetal sex determination, some single gene disorders, and aneuploidy can aid sonographic diagnosis. These aspects will be discussed in more detail here. Fetal sex determination using cell-free fetal DNA in maternal plasma Fetal sex determination using cffDNA is based on the detection of Y-chromosome sequences, either SRY or DYS14, in maternal plasma. If Y-chromosome sequences are detected, the fetus is predicted to be male and, if they are absent, the fetus is predicted to be female. The exact laboratory methodology used can vary, and a recent meta-analysis of 57 reports detailing more than 6000 pregnancies tested suggested that before 7 weeks’ gestation cffDNA testing is unreliable, with the best results reported after 20 weeks’ gestation [8]. In the UK, an audit of fetal sex determination using cffDNA reported that it was highly accurate when carried out after 7 weeks’ gestation, but that it should be used in conjunction with fetal ultrasound to confirm the gestational age before testing [13]. Ultrasound should also be used to detect multiple pregnancies, and the presence of an empty gestation sac, as it is known that the placenta can continue to shed cffDNA after demise of the fetal pole p2]. Non-invasive prenatal diagnosis for fetal sex determination is now increasingly used to determine fetal sex in pregnancies at increased risk of X-linked genetic disorders and congenital adrenal hyperplasia. In the UK and some other European countries, it is now the standard of care in these situations [14]. Both women and health professionals value this approach to sex determination in these high-risk pregnancies [15,16], as it is a test that can be done early in pregnancy and is highly accurate (>99%) when delivered by accredited molecular genetic laboratories, reducing the rate of invasive testing by around 45%, and thereby avoiding unnecessary exposure to miscarriage risk [13]. Furthermore, in pregnancies at risk of congenital adrenal hyperplasia, it allows early cessation of steroid treatment where the fetus is predicted to be male [13,17]. Indeed, in some European countries, dexamethasone treatment is delayed until NIPD carried out at 7 weeks’ gestation indicates the presence of a female fetus. This allows avoidance of any unnecessary steroid treatment in male-bearing pregnancies. Finally, a detailed health economic analysis has shown that, when used in pregnancies at high risk of serious X-linked conditions (where parents might elect to terminate an affected pregnancy) and those at risk of congenital adrenal
Table 1 Key properties of cell-free fetal DNA. Placental in origin Minority of cell-free DNA in maternal plasma Detectable from 4 weeks gestation Cell-free fetal DNA is shorter on average than maternal cell-free DNA Cleared from the maternal circulation within hours of delivery
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hyperplasia, the savings generated by avoiding invasive testing and unnecessary steroid treatment mean that NIPD for fetal sex determination is no more expensive than invasive diagnostic testing [18]. Fetal sex determination using cell-free fetal DNA in the investigation of genital ambiguity Sonographic detection of an isolated abnormality of the external genitalia occurs infrequently. More common is the identification of a genital anomaly or ambiguity when detailed examination is carried out after detecting another structural abnormality. Whether an isolated finding, or in association with other structural anomalies, determination of genetic sex can be useful in defining the underlying diagnosis and directing parental counselling. In the past, this required invasive testing, but first-line testing in many cases should now be analysis of cffDNA after a maternal blood sample, although invasive testing may ultimately be required for definitive diagnosis. Isolated genital anomalies It can be difficult to differentiate cliteromegaly from hypospadias using ultrasound alone (Fig. 1), and the use of cffDNA testing to confirm the genetic sex is useful in this situation (Fig. 1), as in the absence of other anomalies or intrauterine fetal growth restriction (IUFGR), further testing for chromosomal abnormalities is not indicated. When seen in isolation in a fetus confirmed to be male, the diagnosis is most likely to be hypospadias (Table 2), and the parents should be referred to a paediatric urologist for further discussion of management and prognosis. A small risk of an underlying abnormality of steroid biosynthesis remains (Table 2), but these are rare and difficult to diagnose in cases arising de-novo in the prenatal period as, although molecular testing is available, it has a relatively low yield in the absence of a family history [19]. If cffDNA testing suggests that the fetus is female, there is a greater risk of an underlying genetic abnormality of steroid biosynthesis, and the parents are best referred to the disorders of sexual development team for further investigation and management (Table 2). In this situation, invasive testing may be required for more accurate evaluation of steroid profiles [20,21].
Fig. 1. (A) Ultrasound image of hypospadias; (B) the reverse transcription-polymerase chain reaction showing amplification of the SRY sequences in cffDNA in the maternal plasma; (C) note the similarity of the image of cliteromegaly; and (D) the lack of amplification of SRY sequences in this female fetus. The amplification of control DNA in (B) and (D) indicates that absence of amplification is not due to assay failure.
Other sonographic findings
cffDNA testing
Differential diagnosis
Other aids to prenatal diagnosis
Management
Ambiguous genitalia
None.
Male.
Isolated hypospadias. Inadequate production of testosterone caused by Leydig cell hypoplasia or biosynthetic defects.congenital lipoid adrenal hyperplasia 17a-hydroxylase deficiency 3b-hydroxysteroid dehydrogenase deficiency 17,20-lyase deficiency 17b-hydroxysteroid dehydrogenase deficiency
Steroid profile.a Consider sequencing of the androgen receptor gene.a
Refer to DSD team for investigation and counselling.
Amniotic steroid levels.a Maternal serum androgen levels.a Maternal urinary oestrogen levels. Maternal ovarian scan for multicystic change.
Refer to DSD team for investigation and counselling. Refer to gynaecology or oncology if luteoma.
Fetal biometry. cfDNA or invasive testing to exclude aneuploidy
Serial monitoring by high-risk pregnancy team. Urology team for management of hypospadias counselling.
Ambiguous genitalia
Ambiguous genitalia or hypospadias
None.
Biometry 3rd percentile. Abnormal maternal and fetal Dopplers
Female.
Male.
Partial androgen insensitivity syndrome. 5a-reductase deficiency. True hermaphrodite. Isolated cliteromegaly. Congenital adrenal hyperplasia 21-OH deficiency 11-OH deficiency 3b-hydroxysteroid dehydrogenase deficiency True hermaphrodite. Maternally derived androgens (e.g. luteoma of pregnancy). Placental aromatase deficiency. Isolated hypospadias with intrauterine growth restriction. Aneuploidy. Confined placental mosaicism.
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Genital anomaly
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Table 2 Causes of sonographically detected anomalies of the external genitalia where determination of genetic sex using cffDNA may be a useful in aiding diagnosis. Results are based on the experience of a tertiary Fetal Medicine Unit over a 10-year period and is adapted from Pajkrt et al. [20] and Chitty et al. [21]. Further details of the steroid investigations can be found in Chitty et al. [21].
No intra-abdomina; bladder. Low cord insertion.
Male.
Bladder exstrophy. Cloacal exstrophy.
Detailed anomaly scan.
Abnormal genitalia
Absent bladder, intra-abdominal cystic mass, dilated bowel, and abnormal spine. Omphalocoele and gastroschisis, abnormal spine, absent bladder, and hydronephrosis
Female.
Detailed anomaly scan. cfDNA snf invasive testing to exclude aneuploidy. cfDNA and invasive testing to exclude aneuploidy.
Abnormal genitalia or micropenis
Echogenic kidneys, with or without polydactyly
Male.
Cloacal exstrophy. Other cloacal abnormality. Aneuploidy. OEIS complex (omphalocoele, bladder exstrophy, imperforate anus, spinal defects). Cloacal abnormality. Aneuploidy. Bardet–Biedl syndrome. Trisomy 13.
Ambiguous or female appearing genitalia
Multiple anomalies, cardiac anomaly, polysyndactyly, intrauterine fetal growth restriction, oedema, cleft lip, central nervous system anomalies, microcephaly, and short limbs.
Male.
Smith–Lemli–Opitz syndrome. Cranio–cerebellar–cardiac syndrome. Short-ribbed polydactyly syndromes. Other genetic syndrome. Aneuploidy.
Ambiguous or female appearing genitalia
Bowing of femora, with or without tibia and fibula. Micrognathia, cardiac anomalies. None.
Male.
Campomelic dysplasia.
cfDNA and invasive testing to exclude aneuploidy. Detailed scan. Maternal urinary steroids levels. Mostly autosomal recessive, take family history for affected members and consanguinity. Detailed scan.
Male.
Laboratory or clerical error. Androgen insensitivity syndrome.
Abnormal genitalia
Genotype discordant with phenotype a
Male or female.
cfDNA and invasive testing to exclude aneuploidy.
cfDNA and invasive testing to confirm discordance between genotype and phenotype.b
Refer to urologists and paediatric surgeons for counselling. Refer to combined fetal-urology team for counselling. Refer to paediatric surgeons and urologists.
Autosomal recessive: family history for affected members and consanguinity. Refer to clinical genetics. Refer to urology and nephrology teams for counselling. Refer to clinical geneticist. Refer to all relevant paediatric teams for discussion of prognosis.
Refer clinical geneticist and skeletal dysplasia clinic. Refer specialist DSD team.
Investigations such as steroid profiling, sequencing of androgen receptor gene, or both, are best managed by a DSD team. In situations where there is an abnormality in the SRY gene (i.e. as androgen insensitivity syndrome), cffDNA using SRY may give misleading results. cffDNA, cell-free fetal DNA; DSD, disorders of sex development. b
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No penis seen, splayed glans and micropenis
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Genital anomalies and intrauterine fetal growth restriction Sonographically detected genital anomalies and ambiguities in a fetus with intrauterine fetal growth restriction (IUFGR), as shown by biometry on or below the third percentile, with or without abnormal maternal and fetal Doppler measurements, is not uncommon [22]. Determination of fetal sex using cffDNA can be a useful aid to diagnosis, and adds weight to the diagnosis of IUFGR. The noninvasive approach can be particularly useful in situations where parents are keen to avoid the risks associated with invasive testing. Uniparental disomy [23] or confined placental mosaicism [24] have been implicated as possible causes, but aneuploidy-related or idiopathic IUFGR are more common. In view of the risk of chromosomal abnormalities, analysis of cfDNA could include testing for aneuploidy (see section on non-invasive prenatal testing for aneuploidy), but currently this will only detect the major trisomies. Therefore, invasive testing and microarray analysis may be more appropriate, as this will detect a wider range of chromosomal rearrangements [25]. Genital anomalies in association with urogenital tract abnormalities Genital abnormalities are most commonly identified in association with other fetal abnormalities [21]. In this situation, the underlying pathology can be broad, with the two main categories being a urogenital tract anomaly or genetic syndrome (Table 2). Probably the most common association is with abnormalities of development of the urogenital tract, including bladder and cloacal exstrophy. Here, knowledge of genetic sex aids prenatal counselling, as the prognosis for these conditions, and hence prenatal counselling, varies for affected males and females [26,27]. Accurate sonographic gender assignment in these conditions is rarely achieved because of the involvement of external genitalia [28]. As an association with aneuploidy is uncommon in the absence of extra-renal anomalies, fetal sex determination using cffDNA is appropriate in these situations, facilitating accurate prenatal counselling without recourse to unnecessary invasive testing. Genital anomalies in association with other fetal abnormalities The association of genital anomalies with genetic syndromes is broad. There are more than 350 syndromes with hypospadias or micro-penis and around 40 with cliteromegaly [29]. In many instances, exclusion of aneuploidy may be the most appropriate first-line investigation in fetuses with multiple abnormalities; however, in some cases, accurate determination of fetal genetic sex using cffDNA can be instrumental in arriving at a definitive diagnosis without recourse to invasive testing (Table 2). Examples include Smith–Lemli–Opitz (SLO) syndrome, Bardet–Biedl syndrome, and campomelic dysplasia. Smith Lemli Opitz syndrome is a genetic syndrome, inherited in an autosomal recessive fashion, which results in an abnormality in cholesterol biosynthesis owing to a mutation in the 7dehydrocholesterol reductase (7DHC) gene. The clinical spectrum of SLO is wide, varying from developmental delay and mild dysmorphic features to those with major structural defects with early or prenatal lethality [30]. The severe form usually presents prenatally with a wide variety of fetal abnormalities [31], including postaxial polydactyly, facial clefts, anomalies of the brain, heart and renal tract, hydrops, and genital anomalies, which are reported to occur in around 90% of affected males [30]. Fetal sex determination in the presence of this spectrum of abnormalities in association with genital ambiguity can increase suspicion of SLO and direct investigations towards analysis of maternal urine for measurement of dehydro-oestriol and dehydropregnanetriol in maternal urine [32] rather than testing for aneuploidy. Another genetic syndrome where fetal sex determination using cffDNA may be of value in coming to a definitive diagnosis is Bardet–Biedl syndrome. This is a rare condition, usually inherited in an autosomal recessive fashion and characterised by obesity, developmental delay, polydactyly, genital anomalies, and renal failure in some cases [33]. In our unit, we have seen cases presenting with large echogenic kidneys, polydactyly, and abnormal external genitalia (Fig. 2), features compatible with trisomy 13, except that fetal growth was normal. Determination of male sex after analysis of cffDNA helped arrive at a probable diagnosis of Bardet–Biedl syndrome. Definitive diagnosis in cases at low
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Fig. 2. Ultrasound images showing the genital ambiguity (*) in (A) large echogenic kidneys; (B) polydactyly; and (C) in a male fetus subsequently found to have Bardet–Biedl syndrome.
prior risk with no family history currently requires molecular genetic analysis. Although it is becoming possible using next-generation sequencing to screen for mutations in the multiple genes responsible for this condition [34], it remains difficult in low-risk cases presenting de-novo in pregnancy. The development of comprehensive gene panels, however, may make rapid, definitive prenatal diagnosis possible in the near future. Furthermore, given the advances in NIPD, this may become possible by analysing cell-free DNA in maternal plasma [10]. Anomalies of the lower leg, excluding isolated talipes, are rare and not usually associated with aneuploidy. The differential diagnosis usually lies between isolated femoral hypoplasia (which can be part of the caudal regression spectrum), femoral hypoplasia, unusual facies syndrome, and campomelic dysplasia. In the latter, bilateral shortening and bowing of the long bones is confined to the legs, all other long bones being normally formed and grown. Other sonographic findings can include talipes, micrognathia, and cardiac abnormalities [35]. A high incidence of genital ambiguity or sex reversal occurs in affected males (Fig. 3). Analysis of cffDNA in maternal plasma to confirm sex reversal in the presence of this constellation of findings is sufficient to reach a definitive diagnosis of campomelic dysplasia and counsel parents accordingly. Non-invasive prenatal diagnosis as an aid to the diagnosis of skeletal abnormalities Skeletal dysplasias are a genetically heterogeneous group of disorders, many of which can be diagnosed using fetal ultrasound [36], with most that are lethal amenable to detection in early pregnancy [37]. Until recently, for most dysplasias presenting in pregnancy in the absence of a family history, definitive diagnosis had to await postnatal investigations, molecular genetic testing, radiology, histopathology, or both. Definitive molecular diagnosis in pregnancy is difficult because of heterogeneity and multiple mutations responsible for these conditions so that definitive prenatal diagnosis was largely confined to pregnancies at high risk because of a relevant family history.
Fig. 3. Ultrasound image showing (A) genital ambiguity; (B) bowed femora, short lower leg bone and talipes; and (C) male fetus with campomelic dysplasia.
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Achondroplasia is the most common non-lethal skeletal dysplasia and presents with short limbs detected after 25 weeks’ gestation (Fig. 4). It is an autosomal dominant condition, but most cases arise de-novo as a result of a sporadic mutation in the FGFR3 gene. Other sonographic features that can be seen include frontal bossing, short fingers, mild femoral bowing, polyhydramnios, and a slightly small chest (Fig. 5) [38]. Although the sonographic findings can be seen from 25 weeks, presentation is more usual late in the third trimester when scanning is indicated because of polyhydramnios. In this situation, detection of other features may be difficult, and a major differential diagnosis is aneuploidy. As most cases (98%) of achondroplasia are caused by a single mutation in the FGFR3 gene [39], definitive molecular prenatal diagnosis of this condition has been possible after amniocentesis for some years [40]. More recently, NIPD for this condition has been developed [38], and is available in the UK through regional genetics and fetal medicine services (http://ukgtn.nhs.uk/find-a-test/search-by-disordergene/test-service/achondroplasia-nipd-600/). The advantage of the NIPD approach in this situation is that it only requires a maternal blood sample, and so avoids the risk of precipitating preterm labour, while allowing a definitive diagnosis to be made. NIPD can be used for safe prenatal diagnosis of achondroplasia in cases arising de-novo or inherited from the father, but is not appropriate currently for molecular diagnosis of cases inherited from the mother owing to the high background of maternal mutant allele present in cell-free DNA. Thanatophoric dysplasia is the most common lethal skeletal dysplasia. There are two types, TDI and TDII, both of which are caused by mutations in the FGFR3 gene, but rather than one common mutation causing the condition, at least 12 causative mutations occur, although three occur more commonly [41,42]. There are two types of thanatophoric dysplasia, both present with short limbs from early in pregnancy (Fig. 4). Other sonographic findings include a small chest, short ribs, short fingers (trident hands), bowing of the femora (otherwise known as telephone receiver femora) and, in Type II thanatophoric dysplasia, craniosynostosis [43] (Fig. 6). The differential diagnosis for thanatophoric dysplasia includes other skeletal dysplasias, including Jeunes asphyxiating thoracic dystrophy, shortribbed polydactyly syndromes, and achondrogenesis. All are serious and potentially lethal conditions, but making a definitive diagnosis is important as many are autosomal recessive, and thus carry a high recurrence risk in future pregnancies, whereas thanatophoric dysplasia arises de-novo as a lethal dominant mutation and thus has a low, gonadal mosaicism, recurrence risk. Definitive molecular prenatal diagnosis for the common mutations has been available for some time after chorionic villus
Fig. 4. Fetal femur length chart showing normal centiles (solid lines) with femur measurements of fetuses with achondroplasia (open circle) and thanatophoric dysplasia (closed circle). Published with permission [43].
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Fig. 5. Ultrasound image of (A) the profile of a fetus with achondroplasia showing frontal bossing and polyhydramnios; (B) short fingers; and (C) a short, and slightly bowed femur.
sampling or amniocentesis [44], with NIPD for the two most common mutations also possible since 2012 [43]; (http://ukgtn.nhs.uk/find-a-test/search-by-disorder-gene/test-service/thanatophoricdysplasia-nipd-599/). The original NIPD approach was based on a polymerase chain reaction with restriction enzyme digest (PCR-RED) methodology. This approach cannot be developed for all thanatophoric dysplasia mutations, and in any event, testing has to be done for individual mutations, which is both time-consuming and costly. Thus, although it is reasonably straightforward to use NIPD testing for the presence or absence of the common mutations, if these are negative, a diagnosis of thanatophoric dysplasia cannot be completely excluded. The development of desk-top, next generation sequencing platforms has provided the technology to deliver a sensitive and flexible approach to NIPD, as it is possible to design gene panels that can be used to screen for multiple mutations in a single assay [12,45]. In our Regional Genetics Laboratory, we have designed such a panel for all mutations in the FGFR3 gene [45]. By using this approach, we have accurately determined the mutation status in all 15 cases at risk of thanatophoric dysplasia because of relevant sonographic findings (n ¼ 11) or germline mosaicism (n ¼ 4). In addition to identifying the two most common mutations, we also detected rarer mutations in eight of these cases. Non-invasive prenatal diagnosis for thanatophoric dysplasia offers considerable benefits, particularly as the increasing use of early ultrasound for combined Down’s syndrome screening has resulted in early diagnosis of many skeletal dysplasias [37]. Thanatophoric
Fig. 6. (A) Sonographic features of thanatophoric dysplasia showing an axial view of the head in a fetus with thanatophoric dysplasia II and a cloverleaf skull; (B) a longitudinal view of the thorax and abdomen demonstrating the small chest; (C) a sagittal view showing the frontal bossing also demonstrating a small chest; (D) a view of a hand illustrating the short fingers; and (E) a postnatal radiograph showing the bony features.
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dysplasia is readily diagnosed early in pregnancy, as the femurs are short from 12 weeks and other sonographic signs are also present (Figs. 4 and 6). Although definitive diagnosis of thanatophoric dysplasia is possible after chorionic villus sampling, our experience is that many women when given the diagnosis of a lethal condition in their fetus are reluctant to undergo invasive testing. Many will elect to terminate an affected pregnancy and, if diagnosed early enough, would opt for a surgical termination, which precludes a post-mortem examination for definitive diagnosis. The availability of NIPD in this situation allows for definitive diagnosis based on a maternal blood sample. If positive, a surgical termination is a reasonable approach, but if negative other differential diagnoses need to be considered and the medical approach to termination is preferable to allow for a post-mortem, definitive diagnosis and accurate genetic counselling for risks in future pregnancies. The other situation in which NIPD may prove to be useful is in multiple pregnancies discordant for abnormalities compatible with thanatophoric dysplasia. Here, definitive diagnosis based on analysis of cffDNA avoids invasive testing with its associated risks to the normal fetus, and also informs pregnancy management as there is no need to consider feticide for the affected fetus as the condition is lethal. Apert syndrome is a rare autosomal dominant genetic disorder, with 98% of cases caused by one of two mutations in the FGFR2 gene [46]. It is characterised by craniosynostosis, syndactyly (also known as ‘mitten hands’), proptosis, mid-facial hypoplasia, central nervous system anomalies, and varying degrees of development delay, although many have low to normal IQ [47,48]. It is amenable to sonographic diagnosis from early in the second trimester (Fig. 7) [48], but definitive diagnosis requires molecular genetic confirmation. This can readily be done using NIPD for all cases arising de-novo or those at risk of inheriting a paternal mutation [12]. The role of cell-free DNA testing for aneuploidy in the presence of sonographic abnormalities The high background level of maternal chromosome 21 cfDNA in maternal plasma make noninvasive testing for Down’s syndrome and other aneuploidies more challenging than the testing described above, which is designed to test for the presence or absence of an allele not present in the mother. For aneuploidy diagnosis, detection of the relatively small changes in the level of individual chromosome-specific fragments in maternal plasma when the fetus has aneuploidy must be accurately quantified. This has proved to be possible using next-generation sequencing, and several large-scale validation studies have now been conducted, which report detection rates for Down’s syndrome of greater than 99%, with a false positive rate of 0.1–1% [11]. This approach can also detect other
Fig. 7. (A) Two-dimensional ultrasound images of the fetal head showing the unusual skull shape and (B, D) mitten hands. The use of three-dimensional ultrasound (D and E) clearly show the syndactyly of the toes and hands (C, G, H, I) and the proptosis (F).
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aneuploidies, including trisomy 18 (99% accurate), trisomy 13 (up to 90% accurate) [49–52], and sex chromosome abnormalities. A small, but consistently false–positive rate is reported, which results from the fact that cffDNA arises from the placenta, and that, when screening for aneuploidy, it is the total cfDNA that is analysed, not just the fetal component. Thus, discrepant results have been reported as a result of confined placental mosaicism [53,54], detection of maternal chromosomal rearrangements [54], and even maternal tumours secreting an abnormal chromosome compliment [55]. For these reasons, cfDNA testing for aneuploidy is not fully diagnostic, but rather considered as an advanced screening test (non-invasive prenatal testing [NIPT]), and positive results should be confirmed by invasive testing [56–58]. In the presence of abnormalities consistent with the relevant trisomy (e.g. duodenal atresia and an atrio-ventricular septal defect in Down’s syndrome, holoprosencephaly and IUFGR in trisomy 13 or omphalocoele, talipes, clenched hands in trisomy 18), it might be considered reasonable to consider NIPT diagnostic. A negative NIPT result in the presence of fetal abnormalities does not necessarily mean that there is no chromosomal abnormality present: not only is there a small false negative rate associated with NIPT [11], but currently available tests only screen for the major trisomies and do not detect other chromosomal rearrangements or micro-deletion syndromes, which may be found in a significant proportion of structurally abnormal fetuses [27,59]. Non-invasive prenatal testing for a wider range of chromosomal abnormalities may become possible in the not too distant future as detection of other chromosomal rearrangements using next-generation sequencing has been reported, but the depth of sequencing required is significantly higher than for standard NIPT, making the cost of testing currently too high for routine use [60,61]. Conclusion Non-invasive prenatal diagnosis and testing for fetal sex determination, single disorders, and aneuploidy, can aid diagnosis of the fetus with sonographic abnormalities. In selected circumstances, it can offer safe definitive diagnosis of the underlying pathology based on a maternal blood sample, avoiding the risks associated with invasive diagnostic testing. Sex determination by analysis of cffDNA should be considered in most situations in which genital abnormalities are detected. Non-invasive prenatal diagnosis NIPD for single gene disorders is becoming increasingly possible. It is already the test of choice when faced with sonographic findings associated with the skeletal anomalies associated with mutations in the FGFR2 and 3 genes. With the further development of gene panels for a wide variety of conditions, NIPD will become a useful aid to sonographic diagnosis of many anomalies (e.g. large echogenic kidneys, and a wider variety of skeletal dysplasias). Given the pace of developments in this area, sonographic diagnosis suggestive of an underlying genetic syndrome should prompt referral to a genetic team familiar with NIPD. Cell-free DNA testing for the common trisomies is highly accurate, but currently does not detect other unbalanced rearrangements or micro-deletion syndromes, thus invasive testing for full karyotyping or microarray analysis of chorionic villi or amniocytes may be required in the presence of fetal abnormalities and a normal NIPT result.
Practice points Analysis of cffDNA in maternal plasma is a safe and accurate method for determining genetic sex. It should be considered after sonographic identification of genital anomalies or ambiguity, as it can avoid invasive diagnostic testing and facilitate appropriate parental counselling. Analysis of cffDNA for determining genetic sex should be considered in the presence of female or ambiguous genitalia, and anomalies compatible with SLO or campomelic dysplasia, as identification of sex reversal in genetically male fetuses can facilitate accurate prenatal diagnosis. NIPD based on cffDNA is available for selected genetic conditions; in particular, some skeletal dysplasias (achondroplasia and thanatophoric dysplasia) and syndromes associated with
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craniosynostosis (Apert syndrome). It should be considered as a safe and robust alternative to invasive testing. NIPT for aneuploidy is available privately, and could be considered as an alternative to invasive testing in the presence of sonographic abnormalities suggestive of aneuploidy. As this currently only tests for the common trisomies, however, invasive testing may be more appropriate, as karyotyping detects a wider range of unbalanced rearrangements, and microarray testing can also detect micro-deletion and duplication syndromes. NIPD and NIPT are a rapidly developing technology, and close liaison should take place with clinical geneticists familiar with NIPD and NIPT so that patients can access new tests as they become available if appropriate.
Research agenda Further development of gene panels designed to analyse short fragments of cffDNA to cover a wider range of genetic conditions to enable molecular genetic diagnosis of more fetal abnormalities. Development of cost effective NIPT for sub-chromosome abnormalities, including microdeletion and duplication syndromes.
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