Yield of RASopathy Gene Panel in Karyotype-Normal or Microarray-Normal Fetuses With Increased Nuchal Translucency

Diagnostic yield of the RASopathy gene panel was 3.3% with isolated and 15.1% with non-isolated increased nuchal translucency and normal karyotype or chromosomal microarray.

Abstract

OBJECTIVE:

To characterize the diagnostic yield of RASopathy gene panels performed due to increased nuchal translucency (NT) measurements with normal karyotype or chromosomal microarray (CMA) results or both.

DATA SOURCES:

PubMed, OVID, SCOPUS, CINAHL, and ClinicalTrials.gov databases were searched from inception through January 8, 2025 (PROSPERO CRD 42023353582).

METHODS OF STUDY SELECTION:

Studies were deemed eligible for inclusion if the cohort consisted of fetuses with increased NT with normal karyotype or CMA or both that were tested with a targeted RASopathy gene panel. Quality assessment and critical appraisal of included studies were independently conducted using the Quality Assessment tool for Diagnostic Accuracy Studies. The primary outcome was the diagnostic yield of a RASopathy gene panel in fetuses with increased NT and normal karyotype or CMA or both. Nuchal translucency measurement, associated congenital abnormalities, the presence of cystic hygroma, and the number of genes evaluated in each study were collected.

TABULATION, INTEGRATION, AND RESULTS:

Our systematic review identified 1,800 cases that met inclusion criteria across 14 studies. The RASopathy gene panel identified a diagnostic variant in 114 cases, for an overall yield of 8.1% (0.081, 95% CI, 0.036–0.139). In fetuses with isolated increased NT, the RASopathy gene panel identified a diagnostic variant in 3.3% of cases (0.033, 95% CI, 0.006–0.074). In fetuses with additional ultrasound abnormalities, the RASopathy gene panel identified a diagnostic variant in 15.1% of cases (0.151, 95% CI, 0.011–0.381). Cardiac abnormalities were the most common associated finding. When a cystic hygroma was identified, total diagnostic yield was 18.4% (0.184, 95% CI, 0.110-0.270).

CONCLUSION:

Testing for RASopathies with a targeted gene panel identified a diagnostic variant in 3.3% with isolated increased NT and 15.1% with nonisolated increased NT after normal karyotype or CMA or both.

SYSTEMATIC REVIEW REGISTRATION:

PROSPERO, CRD42023353582.

Noonan syndrome is a rare genetic disorder with an incidence of 1:1,000 to 1:2,500 births.^1,2 Noonan syndrome belongs to the largest known group of malformation syndromes, the RASopathies, which are caused by germline mutations in genes involved in the Ras/mitogen-activated protein kinase pathway.³ Examples of other RASopathies include Costello syndrome, cardiofaciocutaneous syndrome, Noonan syndrome with multiple lentigines, neurofibromatosis type 1, capillary malformation arteriovenous malformation syndrome, Legius syndrome, SYNGAP1, and central conducting lymphatic anomalies. Significant clinical overlap can be observed between these different disorders, as they all result from genetic variation along the same Ras/mitogen-activated protein kinase pathway. This includes craniofacial dysmorphology, cardiac, cutaneous, musculoskeletal, gastrointestinal, and ocular abnormalities, as well as a predisposition to cancer.⁴

When screening for aneuploidy, the American College of Obstetricians and Gynecologists as well as the American Institute of Ultrasound in Medicine recommend evaluation of the nuchal region during the first-trimester ultrasonogram to note any abnormalities, such as an increased nuchal translucency (NT) or cystic hygroma. Nuchal translucency measurement is one of several screening tools for fetal aneuploidy that can be offered between 11 and 14 weeks of gestation, along with select maternal serum screening and cell-free DNA.^5,6 An increased NT measurement or cystic hygroma may be the first indicator of fetal structural abnormalities and genetic aberrations, such as fetal aneuploidy, copy number variants, and single gene disorders, including RASopathies. Given the association of increased NT with fetal genetic aberrations, genetic counseling is recommended for additional assessment with diagnostic genetic testing by karyotype or chromosomal microarray (CMA) analysis or both.⁷

There is variation in practice regarding genetic evaluation beyond CMA for patients with an increased NT measurement, because normal CMA test results do not rule out the presence of genetic variants due to single gene disorders, such as metabolic syndromes, RASopathies, or skeletal dysplasia. Given the increasing knowledge of an association between increased NT and RASopathies, it is becoming more common for maternal–fetal medicine practitioners to order a prenatal RASopathy gene panel in this setting. The diagnostic yield of prospectively ordering a RASopathy gene panel for cases of increased NT in the setting of normal karyotype or CMA or both remains unknown. It is important to understand the diagnostic yield for this specific cohort so that the decision for additional genetic testing can be evidence based and adhere to the principles of value based care. The objective of our systematic review and meta-analysis is to characterize the diagnostic yield of RASopathy gene panels for fetuses with increased NT measurements or normal karyotype or CMA or both.

SOURCES

This systematic review was conducted according to PRISMA guidelines and registered with PROSPERO (CRD 42023353582, April 22, 2023).⁸ The online electronic databases PubMed, OVID, SCOPUS, CINAHL, and ClinicalTrials.gov were searched from inception to January 8, 2025, using the Boolean combination of: “fetal” or “prenatal” AND “nuchal translucency” or “cystic hygroma,” AND “Noonan Syndrome” or “Costello syndrome” or “cardio-facio-cutaneous syndrome” or “Noonan syndrome with multiple lentigines” or “capillary malformation-arteriovenous malformation syndrome” or “Legius syndrome” or “neurofibromatosis type 1” or “SYNGAP1” or “autosomal dominant intellectual disability type 5” or “central conducting lymphatic anomalies” or “RASopathy.” Furthermore, a variety of related search terms were added as free-text words and MeSH terms. A university-based library specialist assisted in the expansion of the search terms. No specific restriction was applied to select studies by publication date or language. The full search strategy is available (Appendix 1, available online at http://links.lww.com/AOG/E150). Citations of included studies found in the search were evaluated to identify any relevant missed studies.

STUDY SELECTION

After removal of duplicates, studies were independently assessed for eligibility by two authors (J.P.B., M.B.R.) through screening of the title and abstract, followed by evaluation of the full text by a third author (J.E.P., J.P.B., M.B.R.). Discrepancies were resolved by discussion. Studies (observational studies, cohort studies, both prospective and retrospective) were eligible for inclusion when the cohort consisted of fetuses with increased NT, with normal karyotype or CMA or both that were tested with a targeted RASopathy gene panel. Associated ultrasound abnormalities and cystic hygroma data were collected when reported. Increased NT and cystic hygroma were identified as such by the primary study authors. If studies reported data on increased NT separately from cystic hygroma, we collected the data as two separate categories. Some studies combined increased NT and cystic hygroma as one entity. Studies were excluded if the study retrospectively assessed NT in a cohort including only fetuses with confirmed diagnosis of RASopathy. Studies were also excluded if the cohort had an abnormal karyotype or abnormal CMA, or if exome or genome sequencing was performed instead of a targeted RASopathy gene panel. Further exclusion criteria were case series with less than 5 cases, case reports, reviews, textbook chapters, and editorials. Selected manuscripts included retrospective and prospective cohort studies, case series, and one cohort study presented as an abstract.⁹

Additional congenital abnormalities and cystic hygroma data were collected if described as well as NT measurements. The number of genes evaluated in each study was also extracted. The cohort was subdivided based on whether an increased NT was an isolated finding or if additional associated ultrasound abnormalities or cystic hygroma were present for a subgroup analysis. Nonisolated was defined as additional findings at the time of the NT measurement, as reported by primary authors. Genetic variants were considered diagnostic if they were reported as “pathogenic” or “likely pathogenic” by the authors at the time of publication.

The methodologic quality assessment and critical appraisal of the included studies were independently conducted by two authors (J.P.B., M.B.R.) using QUADAS-2 (Quality Assessment tool for Diagnostic Accuracy Studies)¹⁰ (Appendix 2, available online at http://links.lww.com/AOG/E150). The tool was modified to suit the design of this study by evaluating four key domains, including patient selection, reference standard, flow and timing, and inclusion criteria. These domains were assessed to determine the risk of bias, and studies were rated as having low, high, or unclear risk of bias based on the reviewer's evaluation. All included studies were assessed independently by two reviewers (J.P.B., M.B.R.) with discrepancies resolved by discussion.

The primary outcome was the diagnostic yield of RASopathy gene panel for fetuses with increased NT and normal karyotype or CMA or both. A meta-analysis was performed to summarize the diagnostic yield among the included studies. A subgroup analysis also was performed to summarize the diagnostic yield among those with isolated increased NT and nonisolated increased NT, as well as studies that included more than one RASopathy gene. These analyses were performed using Metaprop 8.02.

RESULTS

A total of 695 records were identified in our search, 388 of which were screened after deduplication. In total, 26 studies met inclusion criteria for full text review regarding study design and population studied. Of the 26 studies that underwent full text review, seven were excluded for using exome sequencing rather than a targeted gene panel, three were excluded for including fetuses with abnormal karyotype or CMA, and two were excluded because targeted RASopathy gene panel was not performed. No reports were identified via citation searching. In total, 14 studies describing 1,800 fetuses were included in our review (Fig. 1).

Fig. 1. PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) flow diagram. CMA, chromosomal microarray analysis.

Powel. RASopathy Gene Panel Yield With Increased Nuchal Translucency. O&G Open 2025.

We identified six studies that detailed fetuses with isolated increased NT and five studies that described fetuses with nonisolated increased NT with normal karyotype or CMA or both in which RASopathy gene panel had been completed (Table 1).^1,9,11–22 Three studies did not specify if the increased nuchal translucency was an isolated finding or associated with additional abnormalities (Tables 1 and 2).^16,18,20 Six studies reported karyotype testing alone before targeted RASopathy gene panel testing, and three studies report CMA testing alone. The remaining five studies reported a combination of karyotype and CMA. Eleven studies reported the specific genes included in their RASopathy gene panel (Table 1). Two studies reported only the identified positive RASopathy genes, and one study did not report which genes were tested or positive.^15,17,19 Cutoff values for increased NT measurement ranged from 2.5 to 3.5 mm. Two studies did not specify a cutoff value for increased NT.^11,15 Only one study reported the ethnicities of their patient cohort, with 98% identified as “Caucasian.”¹⁵ A variety of geographic locations are represented in our review, including studies from the United States, Belgium, the Netherlands, Italy, Canada, and China.

Table 1.

RASopathy Gene Panel Results and Diagnostic Yield in the Setting of Increased Nuchal Translucency With and Without Associated Abnormalities

1st Author, Year	Country	Isolated NT	NT Abnormal Cutoff (mm)	Previous Karyotype or CMA	No. of Eligible Fetuses	No. of Genes on Panel	Genes	Reported Positive Genes With Variants	Diagnostic Yield (%)*
Lee, 200911	United States	Yes	NA†	Karyotype	56	1	PTPN11	PTPN11 T553M	1/56 (1.8)
Pergament, 201112	United States	Yes	3.0	Karyotype	120	5	PTPN11 SOS1 KRAS RAF1 MEK1	PTPN11 A72G PTPN11 I309V PTPN11 S502P PTPN11 T854C PTPN11 Y63C SOS1 P655L SOS1 P655L RAF1 S259F	8/120 (6.7)
Croonen, 201313	The Netherlands	No	3.5	Karyotype	67	4 or 10‡	PTPN11 SOS1 KRAS RAF1 BRAF HRAS MAP2K1 MAP2K2 SHOC2 NRAS	PTPN11 c.174C>G (p.Asn58Lys) dn PTPN11 c.181G>C (p.Asp61His) dn PTPN11 c.182A>G (p.Asp61Gly) dn PTPN11 c.184T>G (p.Tyr62Asp) dn PTPN11 c.205G>C (p.Glu69Gln) dn PTPN11 c.227A>T (p.Glu76Val) dn PTPN11 c.417G>C (p.Glu139Asp) dn PTPN11 c.854T>C (p.Phe285Ser) dn PTPN11 c.1381G>A (p.Ala461Thr) dn KRAS c.173C>T (p.Thr58Ile) dn RAF1 c.770C>T (p.Ser257Leu) dn RAF1 c.770C>T (p.Ser257Leu) dn RAF1 c.775T>C (p.Ser259Pro) dn NR (2 fetuses)	15/67 (22.4)
Ali, 201714	United States	No	3.0	Karyotype	39	9 or 11	PTPN11 SOS1 KRAS RAF1 BRAF HRAS MAP2K1 MAP2K2 SHOC2 ±RIT1 NRAS	PTPN11 (4 fetuses) Variants NR VUS (2): KRAS dn, BRAF inherited variant	4/39 (10.3)
Schreurs, 201815	Belgium	No	NA§	Karyotype or CMA	15	Unknown	Unknown	Variants NR	6/15 (40.0)
Faiola, 20199	Italy	No	3.5	Karyotype	9	14	PTPN11 SOS1 KRAS RAF1 BRAF HRAS MAP2K1 MAP2K2 SHOC2 RIT1 NRAS CBL LZTR1 SOS2	Variants NR	2/9 (22.2)
Stuurman, 201916	The Netherlands	Not reported	3.5	CMA	166	14	PTPN11 SOS1 KRAS RAF1 BRAF HRAS MAP2K1 MAP2K2 SHOC2 RIT1 NRAS CBL SPRED1 A2ML1	PTPN11 c.179G>T (p.Gly60Val) dn PTPN11 c.179G>T (p.Gly60Val) dn PTPN11 c.184T>G (p.Tyr62Asp) dn PTPN11 c.213T>G (p.Phe71Leu) dn PTPN11 c.214G>C (p.Ala72Pro) dn PTPN11 c.417G>C (p.Glu139Asp) dn PTPN11 c.767A>G (p.Gln256Arg) PTPN11 c.854T>C (p.Phe285Ser) dn PTPN11 c.922A>G (p.Asn308Asp) dn PTPN11 c.922A>G (p.Asn308Asp) dn PTPN11 c.1403C>T (p.Thr468Met) PTPN11 c.1504T>G (p.Ser502Ala) PTPN11 c.1507G>C (p.Gly503Arg) dn PTPN11 c.1530G>C (p.Gln510His) dn RAF1 c.770C>T (p.Ser257Leu) dn RAF1 c.770C>T (p.Ser257Leu) dn RAF1 c.770C>T (p.Ser257Leu) dn RAF1 c.778A>C (p.Thr260Pro) dn RAF1 c.782C>G (p.Pro261Arg) dn BRAF c.1391G>T (p.Gly464Val) dn HRAS c.38G>A (p.Gly13Asp) dn RIT1 c.319A>G (p.Met107Val) RIT1 c.319A>G (p.Met107Val) dn	23/166 (13.9)
Bardi, 202017	The Netherlands	No	95th percentile or higher for CRL	Karyotype	771	6‖	PTPN11 SOS1 BRAF MAP2K1 RIT1 LZTR1	Variants NR	20/771 (2.6)
Sinajon, 202018	Canada	Not reported	3.5	Karyotype or CMA	103	9	PTPN11 SOS1 KRAS RAF1 BRAF HRAS MAP2K1 MAP2K2 SHOC2	PTPN11 c.1505C>T (S502L) PTPN11 c.1505C>T (S502L) MAP2K2 c.169T>C (F57L)	3/103 (2.9)
Diderich, 202119	The Netherlands	Yes	3.5	CMA	79	At least 3‖	PTPN11 SOS1 RAF1	Variants NR	3/79 (3.8)
Scott, 202120	Canada, Italy	Not reported	3.0	CMA or karyotype	230	At least 11¶	PTPN11 SOS1 KRAS RAF1 BRAF HRAS SHOC2 RIT1 NRAS LTZR1 SOS2	PTPN11 c.181G>A (p.Asp61Asn) PTPN11 c.218C>T (p.Thr73Ile) PTPN11 c.218C>T (p.Thr73Ile) PTPN11 c.417G>C (p.Glu139Asp) PTPN11 c.844A>G (p.Ile282Val) PTPN11 c.922A>G (p.Asn308Asp) PTPN11 c.922A>G (p.Asn308Asp) PTPN11 c.922A>G (p.Asn308Asp) PTPN11 c.1471C>T (p.Pro491Ser) SOS1 c.806T>C (p.Met269Thr) SOS1 c.2536G>A (p.Glu846Lys) KRAS c.40G>A (p.Val14Ile) KRAS c.466T>A (p.Phe156Ile) RAF1 c.770C>T (p.Ser257Leu) RAF1 c.770C>T (p.Ser257Leu) RAF1 c.788T>C (p.Val263Ala) BRAF c.1785T>G (p.Asp595Glu) HRAS c.35G>C (p.Gly12Ala) HRAS c.38G>A (p.Gly13Asp) SHOC2 c.4A>G (p.Ser2Gly) SHOC2 c.807_808delinsTT (p.Gln269_His270delinsHisTyr) RIT1 c.244T>C (p.Phe82Leu) RIT1 c.245T>G (p.Phe82Cys) RIT1 c.246T>G (p.Phe82Leu) RIT1 c.419A>G (p.Gln140Arg) NRAS c.34G>A (p.Gly12Ser) LZTR1 c.628C>T (p.Arg210*), c.610A>G (p.Thr204Ala) SOS2 c.800T>A (p.Met267Lys)	28/230 (12.2)
Zhen, 202221	China	Yes	3.5	CMA	6	10	PTPN11 SOS1 BRAF HRAS KRAS MAP2K1 MAP2K2 RAF1 CBL NRAS	SOS1 c.508A>G (p.K170E) dn	1/6 (16.7)
Spataro, 202322	Italy	Yes	3.5	Karyotype then CMA	114	14	PTPN11 SOS1 KRAS RAF1 BRAF HRAS MAP2K1 MAP2K2 SHOC2 RIT1 NRAS LZTR1 SOS2 RRAS	Variants NR	3/114 (2.6)
Mastromoro, 20221	Italy	Yes	2.5	Karyotype then CMA	25	16	PTPN11 SOS1 KRAS RAF1 BRAF HRAS MAP2K1 MAP2K2 SHOC2 RIT1 NRAS LZTR1 CBL SOS2 RRAS PPP1CB	SOS1 c.643T>C BRAF c.26G>C LZTR1 c.842del	3/25 (12.0)

NT, nuchal translucency; CMA, chromosomal microarray; NA, not applicable; dn, de novo; NR, not reported; VUS, variant of unknown significance; CRL, crown–rump length.

^*Number of diagnostic variants/number of fetuses.

^†1.9 mm listed as the lowest measurement observed (range 1.9–6.3 mm).

^‡Croonen et al divided cases into two separate cohorts, based on which gene panel was used for RASopathy panel testing.

^§Inclusion based on cystic hygroma and NT measurement taken, not isolated NT measurement.

^‖Genes identified with a diagnostic variant; however, diagnostic RASopathy gene panel did not specify which genes were included.

^¶Most commonly tested genes, but various number of genes used on various panels.

Table 2.

Diagnostic Yield of RASopathy Gene Panel in Isolated and Nonisolated Increased Nuchal Translucency

1st Author, Year	Diagnosed NT Cases (n/N)
	Total	Isolated	Nonisolated
Lee, 200911	1/56	1/56	0/0
Pergament, 201112	8/120	8/120	0/0
Croonen, 201313*	13/50 2/17	0/0	13/50 2/17
Ali, 201714	4/39	0/0	4/39
Schreurs, 201815	6/15	0/0	6/15
Faiola, 20199	2/9	0/0	2/9
Stuurman, 201916†	23/166	NR	NR
Bardi, 202017	20/771	0/0	20/771
Sinajon, 202018†	3/103	NR	NR
Diderich, 202119‡	3/79	3/79	NA
Scott, 202120†	28/230	NR	NR
Zhen, 202221	1/6	1/6	0/0
Spataro, 202322	3/114	3/114	0/0
Mastromoro, 20221	3/25	3/25	0/0
Total	120/1800	19/400	47/901
Diagnostic yield (%)	8.1% (0.081, 95% CI, 0.036–0.139)	3.3% (0.033, 95% CI, 0.006–0.074)	15.1% (0.151, 95% CI, 0.011–0.381)

NT, nuchal translucency; NR, not reported; NA, not applicable.

^*Croonen et al divided cases into two separate cohorts, based on which gene panel was used for testing. The first cohort used a four-gene RASopathy panel. The number of genes tested in each case in this group depended on amount of sample DNA available. The second cohort used a 10-gene RASopathy panel.

^†Studies did not specify whether the increased nuchal translucency was an isolated finding or associated with congenital abnormalities.

^‡Diderich et al did not provide the denominator, the total number of fetuses tested with nonisolated increased nuchal translucency. Because of this, only cases with isolated increased nuchal translucency were extracted from this study due to data availability.

The QUADAS-2 assessment for risk of bias for all 14 studies were determined to have a low risk of bias for the patient selection and patient flow domains. Two studies were determined to have high risk of bias in one category, either reference standard or inclusion criteria. One study was determined to have high risk of bias in both reference standard and inclusion criteria domains (Appendix 2, http://links.lww.com/AOG/E150).

Across all 14 studies, a total of 1,800 fetuses were included in the final analysis (Table 1). In our combined cohorts, 120 of 1,800 fetuses had a diagnostic variant for a RASopathy identified on the targeted gene panel, for a total diagnostic yield of 8.1% (0.081, 95% CI, 0.036–0.139) Figure 2. Of the 11 studies that specified isolated compared with nonisolated NT, the total number of fetuses for which increased NT was an isolated finding was 400. Nineteen fetuses with isolated increased NT were found to have a diagnostic variant on the RASopathy gene panel for a diagnostic yield of 3.3% (0.033, 95% CI, 0.006–0.074) Figure 3. Diderich et al¹⁹ identified fetuses with both isolated and nonisolated increased NT with a RASopathy diagnosis in their study; however, the total number of fetuses tested with nonisolated increased NT was not provided. Therefore, only cases with isolated NT were extracted from Diderich et al due to data availability. Of the five studies that included cases with nonisolated NT, a total of 901 fetuses were included in our analysis. In the setting of nonisolated NT, 47 fetuses had a diagnostic RASopathy variant, for a diagnostic yield of 15.1% (0.151, 95% CI, 0.011–0.381) Figure 4. Three studies (n=499 fetuses) did not specify whether the identified increased NT was an isolated finding or if it was associated with additional abnormalities (Table 2).

Fig. 2. Diagnostic yield of RASopathy gene panels of included studies.

Powel. RASopathy Gene Panel Yield With Increased Nuchal Translucency. O&G Open 2025.

Fig. 3. RASopathy diagnostic yield among isolated increased nuchal translucency.

Powel. RASopathy Gene Panel Yield With Increased Nuchal Translucency. O&G Open 2025.

Fig. 4. RASopathy diagnostic yield among nonisolated increased nuchal translucency.

Powel. RASopathy Gene Panel Yield With Increased Nuchal Translucency. O&G Open 2025.

Different targeted RASopathy gene panels were used in each study as reported in Table 1. The number of genes on each panel ranged from one to 16 when reported. Only one study included one gene.¹¹ The median number of genes included was 10. The most common gene tested was PTPN11, reported on all RASopathy panels that specified the genes tested. A subgroup analysis that evaluated the 13 studies (n=1,744 fetuses) and included more than one RASopathy gene on their panel found that 119 of 1,744 fetuses had a diagnostic variant identified, for a diagnostic yield of 8.8% (0.088, 95% CI, 0.040–0.150) Figure 5.

Fig. 5. RASopathy diagnostic yield excluding studies with panel testing one gene.

Powel. RASopathy Gene Panel Yield With Increased Nuchal Translucency. O&G Open 2025.

The NT measurement cutoff value was specified in 12 of 14 studies, ranging from 2.5 to 3.5 mm, with the most common NT cutoff value being 3.5 mm (7/12, 58%), followed by 3.0 mm (3/12, 25%) (Table 1). One study defined increased NT as a measurement at or above the 95^th percentile by crown–rump length–adjusted percentiles (crown–rump length range 45–84 mm).¹⁷ Diagnostic yield by NT measurement is presented in Table 3. Increasing NT measurements appeared to trend toward a higher diagnostic yield; however, data were limited by small numbers due to denominator data availability.

Table 3.

Diagnostic Yield by Nuchal Translucency Measurement

NT Range (mm)	No. of Studies	No. of Studies With Denominator Data Available	Diagnostic Yield by NT [No. of Diagnostic Variants/No. of Cases Tested (%)]	Variant-Positive NT (mm)
2.5–2.91	1	1	1/2 (50)	2.6
3.0–3.91,14,16,19,20	5	2	3/41 (7.3)	3.5 3.5 3.6 3.9 3.5–3.9 (n=2)
4.0–4.91,11,20	3	1	1/7 (14.3)	4.0 4.0, 4.0, 4.2, 4.4, 4.5, 4.7, 4.7, 4.8, 4.8 4.7
5.0–5.912,16,17,20	4	1	1/22 (4.5)	5.2, 5.3, 5.5, 5.5, 5.7, 5.7, 5.8, 5.8 5.0–5.9 (n=1) 5.6 mean (n=8) 5.9 median (n=18)
6.0–6.916	1	1	3/9 (33)	6.0–6.9 (n=3)
7.0–7.914,16,20,22	4	1	1/5 (20)	Greater than 7.0 (n=3) 7.0 7.0–7.9 (n=1) 7.7
8.0–8.99,13,16,18–20	6	1	11/21 (52)	Greater than 8.0 (n=11) 8.0 8.0 median (n=15) 8.0, 8.3 8.0, 8.0, 8.7, 8.7 8.7 median (n=2)
Greater than 9.014,17,18,20	4	NA	Denominator data NA for NT measurement	10, 13.1 10, 14 10, 10.9, 11, 12, 13.5 12, 16.7

NT, nuchal translucency; NA, not available.

Diagnostic yield of RASopathy gene panels after normal CMA compared with karyotype as well as associated major ultrasound abnormalities are presented in Appendix 3, available online at http://links.lww.com/AOG/E150. Cardiac abnormality was the most commonly reported concurrent finding. Other associated findings included distended jugular lymphatic sacs and venous system abnormalities; facial, renal, and skeletal abnormalities; and cystic hygroma, hydrops fetalis, and polyhydramnios. Of the studies that included only fetuses with isolated increased NT, Mastromoro et al¹ also reported associated soft markers. Identified soft markers included hypoplastic or absent nasal bone or both, echogenic bowel, single umbilical artery, and echogenic intracardiac foci.¹

Seven studies reported 467 fetuses with cystic hygroma (Appendix 4, available online at http://links.lww.com/AOG/E150).^{1,11,13,15,16,19,20} In this group, 80 fetuses had a diagnostic variant identified on the RASopathy panel, for an overall diagnostic yield of 18.4% (0.184, 95% CI, 0.110-0.270; Appendix 5, available online at http://links.lww.com/AOG/E150). Two studies (n=33) described isolated cystic hygroma: seven fetuses with isolated cystic hygroma had a positive diagnostic variant with an estimated diagnostic yield of 21.2% (0.212, 95% CI, 0.090–0.389).^1,19 Two studies (n=34) described fetuses with cystic hygroma and additional ultrasound abnormalities: 11 had a positive diagnostic variant with a diagnostic yield of 32.4% (0.324, 95% CI, 0.174–0.505).^13,15

DISCUSSION

Our study is a comprehensive systematic review that describes the diagnostic yield of RASopathy gene panels for fetuses with increased NT and normal karyotype or CMA or both. We described an overall diagnostic yield of 8.1% after a normal karyotype or CMA or both. In the setting of isolated increased NT, the diagnostic yield was 3.3%, and in the setting of increased NT with additional ultrasound abnormalities, the yield of the RASopathy gene panel was 15.1%.

Our study describes the additional yield of a RASopathy gene panel for increased NT after normal karyotype or CMA or both for practicing maternal–fetal medicine physicians. Although Noonan syndrome and other RASopathies have an incidence of 1 in 1,000 to 1 in 2,500, current cohort studies evaluating the yield of gene panels for RASopathies in the setting of an increased NT and normal karyotype or CMA or both have had fairly low study population sizes. By performing this meta-analysis, we are able to leverage a larger sample size to better characterize this clinical question than what each individual study can provide.

In the current literature, retrospective studies of diagnosed cases of RASopathies often had an increased NT or cystic hygroma found on first-trimester ultrasonogram. However, the diagnostic yield of prospectively ordering a RASopathy gene panel in cases of increased NT with normal karyotype or CMA or both is not yet clear.²⁰ The diagnostic yield of RASopathy gene panels in previous cohort studies that addressed this question has been quoted to be 1.0–6.7% for isolated increased NT, and ranging from 2.6 to 22% when the increased NT was associated with other ultrasound abnormalities.^9,17,18,20 Our combined cohort of isolated and nonisolated cases with a normal karyotype or microarray or both determined an overall RASopathy gene panel yield of 8.1%. The diagnostic yield for isolated increased NT was 3.3% and the diagnostic yield for nonisolated increased NT was 15.1%.

An increasing number of studies continue to assess the diagnostic yield of fetal exome sequencing in this patient population. In 2021, Mellis et al²³ combined cohorts from two large referral tertiary care centers and demonstrated that the diagnostic yield of fetal exome for an isolated increased NT is 1.8% (2/111). Nonisolated cases with at least one structural anomaly or hydrops at the time of the increased NT had an exome sequencing diagnostic yield of 22.2% (12/54). The diagnostic yield in cases with apparently isolated increased NT followed by additional anomalies visualized after more than 14 weeks of gestation was 32.4% (12/37).²³ RASopathies accounted for 33.3% (4/12) of the diagnoses made in cases with initially isolated NT, and six additional fetuses were found to have a “potentially clinically relevant” variant, of which two were found in a RASopathy gene.²³ Though fetal exome is a promising diagnostic tool in cases of an increased NT, until access to such testing and payer policy shift, RASopathy panel remains a reasonable genetic test to order.²⁴ Given the relatively high rate of diagnosis in fetuses with an increased NT, a targeted RASopathy gene panel may be of value as a first-line test built on top of an exome platform when RASopathy gene panel is negative.

Strengths of our review include its comprehensive analysis of studies detailing the RASopathy gene panel yield in the setting of increased NT with a normal karyotype or CMA or both. We describe a large combined cohort of 1,800 fetuses with increased NT and normal karyotype or CMA or both. Our study brings attention to the consideration of a RASopathy gene panel as the next logical step in the prenatal genetic workup for increased NT measurement after a normal CMA, especially for practices in lower resource settings or in the setting of an isolated increased NT measurement. Another strength is the varied geographic locations of included studies, which may enhance generalizability of our findings.

Limitations of this study include a high degree of variance in the number of included genes among the RASopathy gene panels in each of the cohort studies. A RASopathy diagnosis was considered if it was reported as such by the authors at the time of the publication. The rate of diagnosis may be even higher in this patient population as targeted RASopathy gene panels have become more comprehensive over the past decade, with the more recent studies often including more expansive gene panels. We did not include data for exome sequencing in our systematic review. We acknowledge that more genetic diagnoses may be made with exome sequencing that may not be demonstrated by a RASopathy panel in the setting of increased NT.

The variance in the definition for increased NT measurement across studies is also a limitation. Two studies defined increased NT as inclusive of cystic hygroma, which limits our ability to differentiate between the two entities when calculating RASopathy panel diagnostic yield.^15,17 Studies with a higher cutoff value may have a higher index of suspicion for RASopathies and, therefore, a higher yield when a RASopathy gene panel is performed; a lower NT cutoff value (eg, less than 3.0 mm) may be associated with a lower diagnostic yield. Additionally, although increasing NT measurements may appear to demonstrate a trend, conclusions are limited by small numbers due to data availability. Larger studies with increased data availability are needed. We defined nonisolated increased NT as “additional findings at the time of NT measurement,” as reported by primary authors. It is possible that associated abnormalities could have been diagnosed later in pregnancy after the isolated NT measurement in the first trimester, resulting in overestimation of the yield of a RASopathy gene panel for isolated NT. Another limitation is the lack of self-reported ethnicity in the majority of studies. Lastly, the QUADAS-2 bias assessment demonstrated that four studies were determined to have high risk of bias in the reference standard or inclusion criteria or both, though all studies were determined to have low risk of bias in the patient selection and patient study flow domains.

As further genetic evaluation after the finding of an increased NT measurement and normal karyotype or CMA or both has become common in maternal–fetal medicine clinical practice, it is of utmost importance that we equip clinicians with information regarding the diagnostic yield of the genetic tests they order. Our study supports the current practice of RASopathy gene panel testing in the setting of increased NT. This is especially true until fetal exome sequencing can be more feasibly integrated into the work-up of increased NT in terms of cost, insurance coverage, and accessibility. When microarray is normal in the setting of an increased NT associated with fetal abnormalities, exome sequencing should be the first genetic evaluation offered and not a gene panel. However, with the current insurance coverage limitations, that may not be possible for many patients, and a stepwise approach may be necessary. The diagnostic yield is anticipated to increase over time as payer practices shift to permit more broad genetic testing.