Abstract
We report a case of two consecutive pregnancies in the same couple presenting with very low pregnancy-associated plasma protein A (PAPP-A), with both pregnancies affected by multiple anomalies of a similar phenotype identified during mid-trimester ultrasound, and eventual diagnosis of Peters-plus syndrome. This case is important in expanding the differential for very low PAPP-A. It also demonstrates the diagnostic value of whole-exome sequencing (WES) after prenatal diagnosis of recurrent fetal ultrasonographic findings. The importance and complexity of providing patient education to enable informed consent for next generation sequencing technologies is discussed.
Keywords
Introduction
Peters-plus syndrome is characterized by postnatal findings of anterior chamber eye abnormalities, short limbs, brachydactyly, developmental/cognitive disabilities, characteristic facial features including an exaggerated Cupid’s bow of the upper lip, and/or cleft lip sometimes accompanied by cleft palate [1–3]. An increased risk for miscarriages and late term fetal loss have been observed in at-risk couples [3]. Prenatal diagnosis has been reported, with ultrasound findings including microphthalmia, short limbs with broad extremities and unilateral multicystic kidney [4]. Mutations have been identified in the B3GLCT (beta-1-3-glucosyltransferase) gene on chromosome 13q12.3 and the inheritance pattern is autosomal recessive [5]. Beta-1,3-glucosyltransferase is involved in the O-glycosylation pathway, which leads to correct localization of many gene products and defines Peters-plus as a disorder of O-glycosylation. The gene plays a role in many cell types and exerts effects on multiple organ systems with variable severity.
Pregnancy-associated plasma protein A (PAPP-A) is a large glycoprotein produced in the placenta that facilitates the release of insulin-like growth factors important for fetal development. Low maternal serum levels of PAPP-A in the first trimester may be used as a serum marker along with B-hCG and ultrasound (and sometimes other markers) to identify pregnancies at increased risk for chromosomal aneuploidy and is a marker for certain pregnancy complications. Extremely low PAPP-A (less than 2 percentile) with normal karyotype may represent an important subset at risk for adverse pregnancy outcome [6].
When obtaining a prenatal diagnosis through amniocentesis or chorionic villus sampling secondary to fetal ultrasound anomalies, laboratory testing has traditionally been done cytogenetically. However, with advances in molecular genetics, targeted chromosomal microarray analysis is now indicated in preference to karyotype analysis. In patients with a structurally normal fetus undergoing invasive prenatal diagnostic testing, either a fetal karyotype or a targeted prenatal chromosomal microarray analysis may be performed [7, 8].
While microarray will detect more clinically significant abnormalities than karyotype analysis it is not always successful, and may reveal variants of unknown significance (VUS) and incidental findings. These findings often lead to patient anxiety and complex genetic counseling. In respone to this concern, targeted prenatal microarray is used to reduce VUS and incidental findings, and genetics specialists provide pre-test genetic counseling and patient education prior to consent and procedure with use of microarray.
With the introduction of next generation sequencing techniques, including whole-exome sequencing (WES), it is now possible to analyze the protein-coding region of the human genome, which represents 2% of the human genome, but contains approximately 85% of disease relevant mutations. WES can detect genetic abnormalities not identified by karyotype or microarray analysis. WES does not evaluate for trisomies or structural chromosomal disorders, imprinting disorders or triplet repeat disorders. WES is currently more costly [9] and would be expected to generate a greater number of VUS and incidental findings than other techniques. Therefore, pretest patient education and genetic counseling are essential to enable informed consent [10]. However, in properly selected cases, WES may identify disease causing mutation(s) that would not have been detected with karyotype or microarray. The use of targeted methods may further improve the efficacy of WES. A definitive prenatal diagnosis via WES can provide valuable prognostic information for the current pregnancy and recurrence risks for future pregnancies, in addition to potentially enabling prenatal or preimplantation genetic diagnosis in future pregnancies.
The International Society for Prenatal Diagnosis (ISPD), the Society for Maternal Fetal Medicine (SMFM) and the Perinatal Quality Foundation (PQF) issued a joint position statement on the use of diagnostic sequencing techniques, including WES, for fetal diagnosis [11]. This statement elucidates the complexity of new sequencing technologies, the importance of a multidisciplinary approach to their use, and the essential nature of patient education and genetic counseling in the process. Among their “points requiring consideration” is the suggestion that WES may be beneficial when the phenotype or family history strongly implicate a genetic etiology but clinical testing for a specific gene is not available, where there is a high degree of genetic heterogeneity, or where previous genetic testing has been uninformative. Our experience with this case supports this point and the previous ACOG Committee Opinion [7] that prenatal exome analysis should be considered in select cases of fetuses with multiple anomalies or recurrent pregnancies affected by anomalies of a similar phenotype with no diagnosis by standard genetic testing.
Case summary
We describe a healthy 37 year old female with a history of 3 unexplained first trimester pregnancy losses followed by two consecutive pregnancies diagnosed prenatally with multiple congenital anomalies. She was diagnosed with hypothyroidism and Synthroid therapy was initiated prior to her fourth pregnancy. The fourth pregnancy presented with an increased risk for Down syndrome and very low PAPP-A (0.5 percentile) on first trimester screening. Detailed ultrasound revealed multiple anomalies at 20 weeks and fetal MRI was performed. Findings included complete agenesis of the corpus callosum, long bone growth lagging 2-3 weeks, mild polyhydramnios, paucity of meconium signal, low termination of the spinal cord (suspected tethered cord), and pulsatile umbilical artery (Table 1). After genetic counseling, amniocentesis was performed. Results indicated a normal karyotype and normal targeted prenatal microarray results. The family elected not to continue the pregnancy based on the severe prognosis. Autopsy was declined.
Summary of ultrasound/MRI findings
Summary of ultrasound/MRI findings
In the fifth pregnancy, the couple presented early for obstetrical care and first trimester screening again revealed very low PAPPA (0.1 percentile). On ultrasound evaluation at 14 weeks, 5 days, short long bones were noted. At 16 weeks, multiple anomalies were seen (Table 1). MRI at 19 weeks confirmed multiple anomalies (Table 1). Given a similar spectrum of abnormalities, recurrence of the same condition was suspected in this fetus. Detailed consultation was provided and informed consent was obtained. The family elected pregnancy termination without autopsy and underwent prenatal diagnosis using amniocentesis with full microarray, which was normal.
The suspicion of possible recurrence of the same condition spurred us to offer WES analysis on cultured amniocytes in a further attempt to elucidate a genetic cause. Single gene testing was also considered, but based on the number of possible syndromic etiologies, WES was expected to be more efficient, cost effective and likely to provide an informative result. The couple returned for a dedicated pretest consult to review the benefits and limitations of WES including that results might not confirm a diagnosis or could reveal unexpected findings that would impact their own health. They opted to proceed with the testing and signed consent. WES analysis was performed and revealed that the fetus was homozygous for a known splice site mutation, c.660 + 1G > A, in the B36ALTL gene [5], consistent with a diagnosis of Peters-plus syndrome. Both parents were identified as carriers of the pathogenic variant which is the most common cause of Peters-plus syndrome. It was presumed that both pregnancies discussed here were affected. In addition, WES revealed that the fetus was mosaic for a de novo variant of uncertain significance (VUS). The couple returned for a post-test genetic consultation to review results and implications for future pregnancies. They ultimately opted to pursue IVF with preimplantation genetic diagnosis (PGD) in the hopes of achieving a successful, unaffected pregnancy (Figs. 1–6).
Coronal MRI of the fetal brain and trunk at 21 weeks 3 days GA, first pregnancy presumably affected by the mutation. There is complete agenesis of the corpus callosum (white arrow indicates absence of fibers crossing midline).
Ultrasound of the femur at 21 weeks 3 days GA. The long bone measurements were diffusely low for gestational age: in this case, below the 2nd percentile. Long bone measurements were similarly affected in the subsequent pregnancy.
Ultrasound of the nose and lips obtained during the second pregnancy at 19 weeks 3 days GA reveals bilateral cleft lip and palate (arrows).
Axial MRI of the fetal brain at 19 weeks 3 days GA. There is colpocephaly and brachycephalic contour of the calvarium.
Sagittal MRI of the fetal brain and trunk at 19 weeks 3 days GA, demonstrating a low lying conus (arrow).
Sagittal MRI of the fetal brain and trunk at 19 weeks 3 days GA, demonstrating abnormal cortical infolding in the occipital region (arrow) indicating likely migrational abnormality.
Maternal serum PAPP-A of less than the first percentile was a feature in both affected pregnancies and may suggest that extremely low PAPP-A represents a useful clinical marker in identifying pregnancies at risk for rare but significant fetal conditions, perhaps including Peters-plus syndrome. The fact that Peters-plus syndrome is now known to be a disorder of O-glycosylation and PAPP-A is a glycoprotein appears to support this hypothesis.
WES may be considered for patients with recurrence of multiple congenital anomalies suspected to have been caused by a genetic condition for which microarray analysis has previously failed to identify an abnormality. Such appropriate use of next-generation sequencing technologies, including WES, may have a significant impact on medical care. Congenital anomalies remain the leading cause of infant mortality [12]. While formal genetics evaluation with chromosome analysis and other specialized testing has been shown to identify the cause for many, almost 50% of these anomalies could not be linked to a genetic or environmental etiology. The introduction of microarray analysis in the early 2000s allowed for a specific diagnosis in thousands more (reportedly 8 to 15%) previously undiagnosed infants and children [13]. Next-generation sequencing techniques stand to expand medical genetic diagnostic capabilities to an even greater degree.
In this prenatal case, both targeted and full chromosome microarray analysis failed to detect causative changes. While microarray analysis has been shown to detect some of the mutations associated with Peters-plus syndrome [5], WES was needed to provide the diagnosis. Counseling involved multiple visits at two different institutions. There were no secondary findings to discuss, however a VUS was identified in addition to the confirmed pathogenetic variants. We cannot exclude that the VUS contributed to the abnormalities seen in the fetus. Counseling was not completely straightforward, but fortunately the couple was extremely capable and WES results provided information for the couple to use for future pregnancies. This further illustrates the potential complexity of WES results and demonstrates the need for thorough pre and post test counseling with a genetic professional.
There have been no standard guidelines or systematically approved methods to determine when to use WES in the prenatal setting of multiple congenital anomalies. However, the joint position statement of the ISPD, SMFM and PQF supports its use in selected situations [11]. Studies have shown the utility of testing, including a large cohort of diverse clinical manifestations that concluded WES provided a potential molecular diagnosis in 25% of patients [14]. Pangalos, et al. used a targeted exome sequencing technique with bioinformatics to test 14 euploid fetuses with prenatally diagnosed malformations. They reported a definitive or highly likely diagnosis in 6 of 14 cases (43%) [15]. Future studies are certain to elucidate the value, efficacy and optimal application of these sequencing techniques.
Because of its complexity and potential to detect findings of uncertain or unintended nature, pretest education for WES presents a challenge in providing fully informed consent. Adequate time and consideration should be dedicated to this counseling. ACMG recommends that pretest counseling include a thorough review of risks, benefits, and limitations of testing with an emphasis on the possibility of incidental/secondary findings that could have clinical significance beyond the initial intent for the testing [16]. This recommendation is reiterated in the ACMG Choosing Wisely statement in 2015: “Don’t order exome or genome sequencing before obtaining informed consent that includes the possibility of secondary findings” [17].
Conclusions
Whole-exome sequencing expands our ability to provide a diagnosis in fetuses with multiple anomalies. This case demonstrates the potential value of WES with fully informed consent in the setting of a recurrent fetal phenotype which remained unexplained by standard testing methods. WES may also be considered in other select prenatal cases, such as when no specific syndrome or too many possible syndromes makes single gene testing impractical and/or more expensive.
The history of very low PAPP-A in two pregnancies that likely both had Peters-plus syndrome (confirmed in one, presumed in the other) is remarkable and may be secondary to the underlying etiology of Peters-plus syndrome. Therefore, very low PAPP-A may be considered a diagnostic feature requiring further evaluation in cases where Peters-plus syndrome is suspected.
Next-generation sequencing techniques for genetic diagnostic testing present a challenge for genetic counseling and fully informed consent, particularly in the prenatal setting. When fetal abnormalities are detected, the focus of the discussion is typically on the associated risks, expected outcome and pregnancy management options. Diagnostic testing options are an integral part of this discussion. In our experience, having the couple return separately at a less stressful time for a dedicated pretest consultation allowed for a comprehensive discussion with better patient comprehension and fully informed consent. As exemplified by the implementation of previous technologies for prenatal diagnosis, with appropriate counseling by a genetics professional, WES has the potential to provide invaluable information for additional families in the settting of fetal abnormalities.