Utility of chromosomal microarray for diagnosis in cases of nonimmune hydrops fetalis

Abstract

Purpose

Chromosomal microarray (CMA) is recommended in the diagnostic evaluation of cases with fetal structural anomalies when invasive testing is pursued. However, the utility of CMA for nonimmune hydrops fetalis (NIHF) specifically is not well known. Our objective was to describe the overall yield of CMA in the diagnostic evaluation of NIHF, comparing isolated cases to those with concurrent structural anomalies.

Methods

This was a retrospective cohort study of all prenatally diagnosed NIHF cases evaluated at the University of California, San Francisco from 2008 to 2018. NIHF due to twin-twin transfusion syndrome was excluded.

Results

There were 131 cases of prenatally diagnosed NIHF. In 43/44 cases with a CMA performed, results were categorized as normal or likely benign. One case was found on CMA to have a large pathogenic duplication of 21p11.2q22.3, which could have been detected by karyotype and was consistent with a diagnosis of Down syndrome. There was no incremental yield demonstrated for CMA over karyotype. Conclusions: Among a cohort of prenatally diagnosed NIHF cases, CMA did not identify any copy number variants beyond those detectable by karyotype, and the vast majority of CMAs were normal. These results suggest that CMA has low diagnostic utility for NIHF.

1 |. INTRODUCTION

Nonimmune hydrops fetalis (NIHF) is diagnosed in the setting of at least two abnormal fluid collections in fetal soft tissues and serous cavities, including ascites, pleural or pericardial effusions, and skin edema. It portends a poor prognosis for the fetus, with an overall perinatal mortality of 50%–75%, though risks vary significantly by the underlying cause of the NIHF.^1–4 Numerous etiologies of NIHF have been reported, including chromosomal abnormalities, single gene disorders, congenital anomalies, viruses, and others. Despite the heterogeneous group of genetic disorders known to lead to NIHF, the optimal approach to the evaluation for a genetic explanation remains unclear.

The American College of Obstetricians and Gynecologists as well as the Society for Maternal-Fetal Medicine (SMFM)⁵ recommend chromosomal microarray (CMA) as the genetic test of choice for fetuses with structural anomalies when invasive testing is pursued, and both SMFM and the Society of Obstetricians and Gynaecologists of Canada recommend a karyotype and/or CMA when diagnostic testing is pursued for NIHF specifically.^1,6 However, the utility of CMA for NIHF remains poorly understood. A secondary analysis of a large prospective study⁷ suggested an incremental yield of 6% in clinically relevant cytogenetic information when CMA was compared to karyotype among cases with at least one abnormal fetal fluid collection, although this was not designed as the primary outcome of the study.⁸ A substantial proportion of NIHF cases also remain of unknown etiology despite evaluation with karyotype and CMA. ⁹ There are very few case reports of copy number variants (CNVs) detected by CMA in the literature for NIHF cases,^10–13 and many of the reported CNVs could have been detected by karyotype. Examples of CNVs reported with NIHF that would only be detected by CMA include 22q11.2 microdeletion or microduplication syndromes,^14,15 16p13.3 microdeletion with alpha thalassemia,¹⁶ 11p15.4 microdeletion with εγδβ-thalassemia,¹⁷ 20p12.3 microduplication with Wolff-Parkinson-White syndrome,¹² 1p12p21 microdeletion with congenital diaphragmatic hernia,¹¹ and 3q29 microduplication and 15q24.3 microdeletion with cardiac dysfunction.¹⁸

We designed a retrospective cohort study of all NIHF cases undergoing CMA at our institution over the past 10 years, in order to determine the utility of CMA for establishing a diagnosis in cases of isolated NIHF as well as NIHF with concurrent structural anomalies. We hypothesized that CMA would establish a diagnosis in a small proportion of isolated NIHF cases, and that the yield would likely be higher for cases of NIHF with concurrent structural anomalies.

2 |. MATERIALS AND METHODS

This is a retrospective cohort study of cases with prenatally diagnosed with NIHF evaluated at the University of California, San Francisco (UCSF) between 2008 and 2018. Patients were included if they had a prenatal ultrasound report showing at least two of the following: ascites, skin edema, pleural effusions, or pericardial effusions. Included cases were then categorized as isolated NIHF or NIHF with concurrent structural anomaly. Dichorionic twins were still eligible for inclusion if one or both twins were affected by NIHF. Cases of NIHF due to twin-twin transfusion syndrome were excluded though, owing to the different pathophysiology in these cases. Cases of hydrops resulting from alloimmunization were not included, as these are a cause of immune hydrops. The primary outcome was defined as the proportion of the overall cohort with a pathogenic or likely pathogenic CNV on CMA. The secondary outcome was the proportion of cases with a pathogenic or likely pathogenic CNV among cases with isolated NIHF, compared to those with a concurrent structural anomaly. Multiinstitutional review board reliance registry approval was obtained (Institutional Review Board No. 1004093).

All CMA results were categorized at the time of report by the UCSF Cytogenetics Laboratory into categories of benign, likely benign, variant of uncertain clinical significance (VUS), likely pathogenic, and pathogenic as defined by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.¹⁹ For the purposes of our analyses, we categorized CNVs into three clinically relevant groups by combining benign and likely benign as well as pathogenic and likely pathogenic. CMA was initially used at our site as a clinical test in 2008, and its use increased in frequency over the study time period. Initially, array comparative genomic hybridization was primarily used; this has been gradually replaced by single-nucleotide polymorphism (SNP) array over time. The CMA build also evolved over time from hg18 to hg19. The overall resolution of the platforms to detect CNVs ranged from approximately 20–75 kb. Due to the referral nature of our patient population, 45.5% of CMAs were performed at our institution. The remainder was performed at outside laboratories, including Kaiser Permanente, Integrated Genetics, Quest Diagnostics, LabCorp, CombiMatrix, University of Washington, and University of California, San Diego.

All cases of NIHF had a detailed obstetrical ultrasound with middle cerebral artery Doppler and fetal echocardiogram. Prenatally detected concurrent structural anomalies were categorized as central nervous system (CNS), face/neck, cardiovascular (CV), pulmonary/thoracic, gastrointestinal (GI)/abdominal, genitourinary (GU), skeletal, sacrococcygeal teratoma (SCT), and placental. Specific anomalies included within each category are listed in Data S1, Appendix A. If the patient did not continue her prenatal care at UCSF following consultation, or did not deliver at UCSF, one of the key personnel (K.G.) in our Fetal Treatment Center contacted the patient’s provider to obtain information about the duration of the pregnancy, as well as any subsequent genetic testing performed. All data were abstracted by Maternal-Fetal Medicine providers and trained research assistants using data from patients’ electronic medical records.

Planned analyses included Fisher’s exact test or Chi square test to compare proportions as appropriate in terms of CNV categories by isolated NIHF vs NIHF with concurrent structural anomaly. The rank sum test compared nonparametric continuous variables after evaluating for normalcy. Statistical significance was defined using a two-sided P value of <.05, and STATA software (version 15.0, College Station, TX) was used for data analysis.

3 |. RESULTS

There were 131 fetuses with prenatally diagnosed with NIHF during the study period. Among these were 44 cases (33.6%) of isolated NIHF and 87 cases (66.4%) of NIHF with at least one associated structural anomaly. Comparing cases that underwent CMA to those that did not, no differences were observed in maternal age, race/ethnicity, history of hydrops, pregnancy outcome, or category of associated structural anomaly (Table 1). However, median gestational age at initial evaluation at our center was later among those who underwent CMA (26 + 0 vs 23 + 0 weeks, P = .01). Additionally, patients who had had noninvasive prenatal testing (NIPT) were more likely to have had CMA (39% vs 5%, P < .001). All of those who underwent CMA had low-risk NIPT results (14/14). Among those who did not have CMA, three patients had low-risk NIPT results, and three were positive for Turner syndrome on NIPT (one confirmed on karyotype, the other had an in utero fetal demise without diagnostic testing). The most common etiology of NIHF was a structural anomaly (40.0%), of which 26.9% underwent a CMA. Aneuploidy was also a common etiology, including Down syndrome and Turner syndrome; 7.7% of these cases had a CMA performed. Others etiologies included arrhythmia (6.2%), lymphatic (5.4%), infectious (2.3%), hematologic (3.9%), and thyrotoxicosis (1.5%). The etiology was unknown in 28.5% of NIHF cases. Specific types of etiologies in each category are listed in Data S1, Appendix B.

TABLE 1.

Demographics of the overall cohort, by CMA vs no CMA performed

	CMA performed
	Yes (n = 44)	No (n = 87)	P value
Maternal age [median (range)], years	32 (20–41)	30 (16–45)	0.32
Race/ethnicity, n (%)			0.51
White	9 (22)	6 (7)
Asian	7 (17)	8 (10)
Black	3 (7)	1 (1)
Hispanic	4 (10)	8 (10)
Other	1 (2)	2 (2)
Prior pregnancy with hydrops, n (%)	2 (6)	4 (6)	0.68
GA at initial evaluation [median (range)]	26w0d (14w4d-34w5d)	23w0d (13w0d to 35w4d)	0.01
Plurality, n (%)			0.63
Singleton gestation	41 (93)	78 (91)
Twin gestation	3 (7)	8 (9)
NIPT performed, n (%)	14 (37)	4 (6)	<0.001
Karyotype performed, n (%)	27 (61)	47 (54)	0.42
Pregnancy outcome, n (%)			0.83
SAB	0 (0)	1 (1)
Termination or selective reductiona	8 (19)	19 (22)
IUFD	9 (21)	17 (20)
Live birth	17 (40)	28 (33)
Neonatal death	9 (21)	19 (22)
Concurrent anomalies, n (%)			0.10
Isolated NIHF	19 (43)	25 (57)
≥1 concurrent structural anomaly	25 (29)	62 (71)
Category of concurrent anomaly, n (%)			0.63
CV	10 (40)	20 (32)
CNS	2 (8)	5 (8)
Face/neck	1 (4)	0 (0)
Pulmonary/thoracic	7 (28)	21 (24)
GI/abdominal	1 (4)	0 (0)
GU/genitourinary	0 (0)	4 (7)
Skeletal	4 (16)	9 (15)
SCT	0 (0)	2 (3)
Placental	0 (0)	1 (2)
Etiology of NIHF, n (%)			<0.001
Concurrent structural anomaly	14 (32)	38 (44)
Aneuploidy	1 (2)	12 (14)
Other genetic	3 (7)	0 (0)
Arrhythmia	1 (2)	7 (8)
Lymphatic	5 (11)	2 (2)
Hematologic	0 (0)	5 (6)
Infectious	0 (0)	3 (3)
Thyrotoxicosis	2 (5)	0 (0)
Unknown	18 (41)	18 (21)

Note: P values in bold are considered to be statistically significant.

Abbreviations: CMA, chromosomal microarray; CNS, central nervous system; CV, cardiovascular; GA, gestational age; GI, gastrointestinal; GU, genitourinary; IUFD, intrauterine fetal demise; NIHF, nonimmune hydrops fetalis; NIPT, noninvasive prenatal testing; SAB, spontaneous abortion <20 weeks; SCT, sacrococcygeal teratoma.

^aIncludes termination of pregnancy <20 weeks gestation, selective reduction of twins to singleton, or induction termination >20 weeks gestation.

Of the 131 NIHF cases in the cohort, 44 had a CMA performed. Among cases with a CMA, 43.2% (19/44) were done in the setting of isolated NIHF, and 56.8% (25/44) were done in the setting of ≥1 associated structural anomaly. Thirty-one (70.0%) were prenatal CMAs, performed at a median gestational age of 26 + 0 weeks (12 + 0 to 34 + 5 weeks). Overall, 6.8% (3/44) of CMAs were performed following chorionic villus sampling, 54.5% (24/44) from amniocentesis, 6.8% (3/44) from thoracic fluid from thoracentesis, 27.2% (12/44) from neonatal blood, and 4.5% (2/44) from products of conception. The majority of CMAs were SNP arrays (61.4%, 27/44), with the remainder being comparative genomic hybridization (CGH) arrays.

CMA results are outlined in Table 2. Of the 44 cases with CMAs performed, only one (2.3%) identified a pathogenic variant: a 37-Mb duplication of 21p11.2q22.3 in a fetus with Tetralogy of Fallot and an already known diagnosis of Down syndrome based on prenatal karyotype. None of the cases with a CMA performed had a VUS detected, and the large majority (97.7%) had a normal CMA result. There were 26 patients who had both a karyotype and CMA, and no incremental yield of CMA over karyotype was demonstrated.

TABLE 2.

Categorization of CMA results by isolated NIHF vs NIHF with concurrent structural anomalies

			CMA results
	Number	CMA performed	Pathogenic or likely pathogenic	VUS	Normal or likely benign	P value
Isolated NIHF	44	19/44 (43.2%)	0/19 (0.0%)	0 (0.0%)	19/19 (100.0%)	0.38
≥1 associated anomaly	87	25/87 (28.7%)	1/25 (4.0%)	0 (0.0%)	24/25 (96.0%)
All NIHF	131	44/131 (33.6%)	1/44 (2.3%)	0 (0.0%)	43/44 (97.7%)

Abbreviations: CMA, chromosomal microarray; NIHF, nonimmune hydrops fetalis; VUS, variant of uncertain significance.

4 |. DISCUSSION

Among a relatively large cohort of prenatally diagnosed NIHF cases over 10 years, CMA yielded normal results in the large majority, 97.7%. Furthermore, despite 57% of those with a CMA having at least one concurrent structural anomaly, CMA did not identify any CNVs beyond one case of Down syndrome, which was also detected by karyotype. Therefore, in this cohort, we identified no incremental yield of CMA over karyotype. Our findings suggest that CMA has low diagnostic utility in the setting of NIHF, regardless of concurrent structural anomalies.

The most likely explanation for the low yield of CMA in cases of NIHF is that most genetic causes of NIHF are due to single gene disorders, rather than CNV. Despite the fact that CMA provides information about microdeletions and microduplications at a much higher resolution than karyotype,⁵ this category of genetic disorders appears to explain few cases of NIHF.

Despite the relatively high prevalence of aneuploidy in fetuses with NIHF, we found only one case of Down syndrome and two likely cases of Turner syndrome. Although this is less than expected based on other published series of NIHF, in which aneuploidy represents between approximately 7% and 16% of cases,¹ this likely reflects the referral nature of our patient population. We suspect that pregnancies with NIHF due to aneuploidy were less likely to be referred to our center for aggressive fetal care and likely many were terminated prior to referral. In addition, the number of cases with a pregnancy loss prior to referral is unknown.

A wide spectrum of genetic disorders has been reported in association with NIHF, including a large number of single gene disorders. These disorders are not detected by either CMA or karyotype, and are likely to explain a significant portion of unexplained cases. For example, CMA cannot detect missense and other pathogenic variants involving single base pair changes, such as might be found in Noonan syndrome, lysosomal storage disorders (LSDs), Pena-Shokeir syndrome, and many other genetic disorders associated with NIHF.^20,21 Prior studies have suggested that 19% of all NIHF cases result from rare genetic diseases, and 29% of unexplained cases may be attributable to LSDs.^21,22 The utility of NIHF gene panels and other sequencing technologies deserve further exploration, in order to capture more of these rare genetic disorders. However, given that so many of the genetic etiologies of NIHF are rare single gene disorders, many of which are not included on NIHF gene panels, it may be reasonable to consider exome sequencing as the next step when standard genetic tests do not yield a diagnosis. Further research on the yield of exome sequencing for NIHF is needed. Additionally, a broad approach to evaluation of NIHF including infectious or environmental pathogens is merited, as it is possible that there are many infectious causes of NIHF beyond those that are currently recommended for testing (parvovirus, cytomegalovirus, and toxoplasmosis).¹

There are several strengths of our study. We did not identify other series that report specifically on the yield of CMA in NIHF cases as the primary outcome. We had a relatively large number of cases given the rarity of this disease process, and our cohort spanned a decade at a major tertiary care center. However, there are still multiple limitations to acknowledge. Despite our cohort being relatively large for this rare disorder, the numbers are still small overall, which may have limited some of our comparisons. CMA and karyotype were not performed for all cases in our cohort, which may limit our ability to accurately determine incremental yield of CMA over karyotype. As is the case with other similar cohorts, CMA was not performed for all NIHF cases, or even for all NIHF cases with concurrent structural anomalies, which may have led us to miss underlying CNVs. However, it is standard of care to recommend CMA and among all fetuses with structural anomalies at our institution, and 21% have abnormal CMA results.¹⁸ Finally, given that our institution is a referral center, NIHF cases with abnormal results of diagnostic testing that was done at outside institutions may not have come to our attention, decreasing the overall proportion of cases with abnormal CMA and/or karyotype results. In addition, approximately one third of patients in our cohort delivered elsewhere and therefore we did not have results of postnatal genetic testing that may have been done. However, this avoids bias introduced in situations where postnatal findings may have led to greater likelihood of having CMA performed.

In conclusion, in this relatively large cohort of NIHF cases, CMA did not confer a clinically significant yield of cytogenetic information for establishing an underlying diagnosis, regardless of the presence of concurrent structural anomalies. This likely stems from the nature of the genetic etiologies that are known to lead to NIHF, including the multitude of single gene disorders that are not detected by CMA. To address this, further research should focus on the incremental yield of other genetic testing modalities for NIHF, such as gene panels, exome sequencing, and potentially eventually genome sequencing.

What’s already known about this topic?

• A chromosomal microarray (CMA) is recommended in the diagnostic evaluation in cases of fetal structural anomalies when invasive testing is pursued. This includes cases of nonimmune hydrops fetalis (NIHF).

• There are very few case reports of copy number variants detected by CMA in the literature for NIHF cases.

What does this study add?

• This study investigates the actual utility of a CMA for establishing a diagnosis in cases of NIHF, with or without concurrent structural anomalies.

• We found that there was no additional yield of microarray over karyotype for cases of hydrops and conclude that further investigation of other diagnostic genetic tests for hydrops should be pursued.