The Frequency and Efficacy of Genetic Testing in Individuals with Scimitar Syndrome

Abstract

Background:

Scimitar syndrome (SS) is a rare congenital heart defect composed of partial anomalous pulmonary venous connection from the right lung, via a scimitar vein, to the inferior vena cava rather than the left atrium. Genetic conditions associated with SS have not been well-investigated at present.

Methods:

Our study included patients with SS diagnosed at Texas Children’s Hospital from January 1987 to July 2020. Medical records were evaluated to determine if genetic testing was performed, including chromosomal microarray analysis (CMA) or whole-exome sequencing (WES). Copy number variants (CNVs) identified as pathogenic/likely pathogenic and variants of unknown significance were collected. Analysis of cardiac and extracardiac findings were performed via chart review.

Results:

Ninety-eight patients were identified with SS, 89 of which met inclusion criteria. A chromosome analysis or CMA was performed in 18 patients (20%). WES was performed in six patients following negative CMA testing. A molecular genetic diagnosis was made in 7 of 18 cases (39% of those tested). Ninety-six percent of the cohort had some type of extra-cardiac finding, with 43% having asthma and 20% having a gastrointestinal pathology. Of the seven patients with positive genetic testing, all had extra-cardiac anomalies with all but one having gastrointestinal findings and 30% having congenital diaphragmatic hernia.

Conclusions:

Genetic testing revealed an underlying diagnosis in roughly 40% of those tested. Given the relatively high prevalence of pathogenic variants, we recommend CMA and WES for patients with SS and extra-cardiac defects.

INTRODUCTION

Scimitar syndrome (SS) is a rare congenital heart defect composed of a partial anomalous pulmonary venous connection from the right lung to the inferior vena cava via a scimitar vein. The birth prevalence of SS is approximately 1 in 50,000 live births¹. SS is often associated with varying degrees of right lung hypoplasia and patients with SS often suffer from chronic lung parenchymal disease, asthma, and recurrent respiratory infections². There is also an increased prevalence of right pulmonary artery hypoplasia, atrial and ventricular septal defects, coarctation of the aorta, tetralogy of Fallot and other congenital heart lesions including single ventricle lesions^1,2. Patients with SS and multiple congenital anomalies can be challenging to manage due to their multiple organ system pathologies.

Although some forms of congenital heart disease have been linked to various genetic syndromes, the mechanisms that contribute to SS remains elusive^3–9. Studies have demonstrated an association between SS and extracardiac anomalies including congenital diaphragmatic hernia, imperforate anus and variants of VACTERL association^10–14.

Although pathogenic variants in specific genes can clearly cause congenital heart defects, the genetic factors contributing to most cases of SS remain unidentified^3–9. In this study we sought to determine the frequency and efficacy of genetic testing in patients with SS from a single institution. We also use the results of clinically based genetic testing to identify genes and pathways whose alteration may lead to SS development. We hypothesized that genetic testing would be performed in a minority of patients with SS but would have a considerable rate of positive findings.

METHODS

This retrospective cohort study included all patients evaluated at Texas Children’s Hospital (TCH) from January 1987 through July 2019 diagnosed with Scimitar syndrome. Patients were first identified from institutional echocardiographic and clinical databases. All patient charts were reviewed to confirm an anatomic diagnosis of SS. A portion of these patients have been used in a prior publication from our institution, focusing on different aspects than our study and not consisting of our entire cohort. Patients were only included if they had been seen by a provider and had sufficient data for chart review. Co-existing diagnoses were also identified and extracardiac defects were classified as: asthma (any diagnosis of asthma, irrespective of medical management), right lung hypoplasia (if documented on imaging), tracheal stenosis, frequent respiratory infections (if indicated as such by the provider), pulmonary sequestration (if indicated on cross-sectional imaging) any gastrointestinal symptoms (poor feeding, failure to thrive), or status post nasogastric feeding requirement or gastrointestinal tube placement, liver dysfunction, omphalocele, pyloric stenosis or imperforate anus. Developmental delay, intellectual disability, and psychiatric diagnosis (attention-deficit hyperactivity disorder, autism spectrum disorder, anxiety disorder, obsessive-compulsive disorder) were also identified. Cardiac abnormalities were identified by review of echocardiograms, magnetic resonance imaging, and computed tomography and reported, with the exclusion of patent foramen ovale (PFO) and small patent ductus arteriosus (PDA).

All clinically obtained genetic testing was reviewed, including chromosome analysis, chromosomal microarray analyses (CMA) and whole exome sequencing (WES). Chromosome analysis and/or single gene or panel sequence testing in isolation were not performed on any patients in this cohort. Positive findings on genetic testing were labeled by the third-party laboratory as “abnormal” or indicative of a pathologic change. All copy number variants (CNV) reported on CMA were recorded and results were described. Variants identified by WES were interpreted according to the American College of Medical Genetics and Genomics guidelines¹⁵. Studies that revealed “pathogenic” or “likely pathogenic” variants in genes associated with the patient’s phenotype were considered positive if their inheritance pattern was also consistent the proposed diagnosis. Variants of unknown significance (VUS) found in genes that associated with the patient’s phenotype were also recorded and results described.

Descriptive analyses included the assessment of categorical variables expressed as counts and percentages. This study was approved by the Baylor College of Medicine Institutional Review Board.

RESULTS

Ninety-eight patients with SS were identified. Eighty-nine of these patients met study criteria (Table 1). The majority of patients were female (60/89, 67.0%). Of these 89 patients, 18 patients (20.2%) had chromosome analysis or CMA testing performed and six (7%) had WES performed in addition to CMA (Table 2). No genetic tests were performed prior to 2000, and the majority of genetic testing was performed since 2010, with 16 of 18 CMAs performed after 2010 and all WES performed after 2010 (supplemental figure 1). Of the individuals for whom CMA was obtained, 4 of 18 (22.2%) revealed clearly pathogenic CNVs (Patients 1, 2, 6 and 7). Three patients had positive genetic findings delineated by WES. Hence, a genetic diagnosis was made in 7 of 18 (38.9%) of patients in whom genetic testing was obtained (Figure 1).

Table 1.

Patient characteristics

Patient characteristics	N (%)
Demographics N = 89
Sex
Female	60 (67.0)
Male	29 (33.0)
Race/ethnicity
Non-Hispanic White	46 (51.7)
Non-Hispanic Black	7 (7.9)
Hispanic	28 (31.4)
Asian	4 (4.5)
Other	4 (4.5)
Coexisting diagnoses/extracardiac defects
Any coexisting diagnosis/extracardiac defect	86 (96.6)
Respiratory/pulmonary pathology	75 (84.3)
Asthma	38 (42.7)
Right lung hypoplasia	69 (77.5)
Frequent respiratory infections	11 (12.4)
Tracheal stenosis	5 (5.6)
Tracheoesophageal fistula	1 (1.1)
Pulmonary sequestration	4 (4.5)
Congenital diaphragmatic hernia	12 (13.2)
Gastrointestinal pathology	21 (23.6)
Poor feeding	19 (21.3)
NG tube	3 (3.4)
Gastrostomy tube	12 (13.2)
Liver dysfunction	2 (2.2)
Omphalocele	1 (1.1)
Pyloric stenosis	1 (1.1)
Imperforate anus	2 (2.2)
Any musculoskeletal pathology	18 (20.2)
Scoliosis	14 (15–7)
Vertebral anomalies	6 (6.7)
Pectus deformity	2 (2.2)
Developmental delay	7 (7.9)
Psychiatric diagnosis	11 (12.4)
Additional cardiovascular abnormalities
Aortopulmonary collateral	50 (56.2)
Status post-occlusion	34 (38.2)
Atrial septal defect	38 (42.7)
Sinus venosus defect	4 (4.5)
Secundum defect	33 (37.1)
Left superior vena cava	19 (21.3)
Ventricular septal defect	7 (7.9)
Arrhythmia	3 (3–4)
Coarctation of the aorta	3 (3.4)
Single ventricle	3 (3.4)
Hypoplastic left heart syndrome	2 (2.2)
DORV, mitral atresia	1 (1.1)
Pulmonary valve stenosis	3 (3.4)
Aberrant right subclavian artery	2 (2.2)
Tetralogy of Fallot	2 (2.2)
Subaortic stenosis	2 (2.2)
Hypertrophic cardiomyopathy	1 (1.1)
Unroofed coronary Sinus	1 (1.1)
Interventions/outcomes
Status post-tracheostomy	3 (3.4)
Status post-Scimitar vein surgery	28 (31.4)
Residual pulmonary vein stenosis	6 (6.7)
Deceased	4 (4.5)

DORV = double-outlet right ventricle; NG = nasogastric.

Table 2.

Genetic testing results in patients with SS

Test	N (%)
Chromosomal microarray (CMA)	18 (20.2)
CMA with no structural variation	11 (61.1)
CMA with structural variation of unknown significance	3 (16.7)
CMA with pathogenic structural variation	4 (22.2)
Whole-exome sequencing (WES)	6 (6.7)
WES with no concerning variants	3 (50.0)
WES with variants of unknown significance	0 (0.0)
WES with pathogenic/likely pathogenic variants	3 (50.0)

Figure 1.

Pie graph indicating the distribution of chromosomal microarray (CMA) and/or whole-exome sequencing (WES) results on patients with SS. No CMA or WES findings were noted in 8 of 18 patients (44%). CMA revealed copy number variants of unknown significance (VUS) in 3 of 18 (17%). CMA was positive in 4 of 18 (22%) and WES was positive, with negative CMA, in 3 of 18 (17%).

Extra-cardiac defects and coexisting diagnoses are described in Table 1, and at least one of these was identified in a total of 86 of 89 patients (96.6%). Among the entire cohort, 43% of patients were diagnosed with asthma compared to 14% of the genetic-positive subgroup. Four patients were found to have pulmonary sequestration. Congenital diaphragmatic hernia was identified in 13% of the entire cohort and 29% in the genetic-positive subgroup. Any gastrointestinal pathology was identified in only 24% of the entire cohort versus 86% of the genetic-positive subgroup. All patients that had positive genetic findings had some type of extra-cardiac finding (Table 3) and their genetic findings are summarized in table 4.

Table 3.

Cardiac and extracardiac findings in patients with pathogenic/likely pathogenic or VUS on genetic testing

	Pathogenic/likely pathogenic variants N = 7 (%)	Structural variation or sequence VUS N =3 (%)
Coexisting diagnoses/extracardiac defects
Any coexisting diagnosis/extracardiac defect	7 (100)	3 (100)
Respiratory/pulmonary pathology	6 (85.7)	3 (100)
Asthma	1 (14.3)	1 (33.3)
Right lung hypoplasia	6 (85.7)	2 (66.6)
Frequent respiratory infections	2 (28.6)	0
Tracheoesophageal fistula	0	1 (33.3)
Tracheal stenosis	1 (143)	0
Congenital diaphragmatic hernia	2 (28.6)	0
Gastrointestinal pathology	6 (85.7)	1 (33.3)
Poor feeding	4 (57.1)	1 (33.3)
NG tube	1 (14.3)	0
Gastrostomy tube	4 (57.1)	1 (33.3)
Any musculoskeletal pathology	2 (28.6)	2 (66.6)
Scoliosis	1 (14.3)	1 (33.3)
Vertebral anomalies	1 (14.3)	1 (33.3)
Developmental delay	2 (28.6)	1 (33.3)
Psychiatric diagnosis	0	1 (33.3)
Additional cardiovascular abnormalities
Aortopulmonary collateraln	5 (71.4)	1 (33.3)
Status post occlusio	3 (42.9)	1 (33.3)
Atrial septal defect	4 (57.1)	3 (100)
Sinus venosus defect	0	0
Secundum defect	4 (57.1)	3 (100)
Left superior vena cava	2 (28.6)	0
Ventricular septal defect	1 (14.3)	1 (33.3)
Arrhythmia	1 (14.3)	0
Coarctation of the aorta	1 (14.3)	0
Single ventricle	1 (14.3)	0
Hypoplastic left heart syndrome (MA/AS)	1 (14.3)	0
DORV, mitral atresia	0	0
Aberrant right subclavian artery	1 (14.3)	1 (33.3)
Tetralogy of Fallot	1 (14.3)	1 (33.3)
Pulmonary valve stenosis	1 (14.3)	0
Hypertrophic cardiomyopathy	1 (14.3)	0

AS = aortic stenosis; DORV = double-outlet right ventricle; MA = mitral atresia; NG = nasogastric.

All values as n (%).

Table 4.

Summary of genetic testing results

Patient	Testing	Genetic finding indicated
1	CMA	~1.6 Mb gain on 22q11.21 (minimum chr22:17,998,078–19,606,540; maximum chr22:17,982,527–19,627,555; hg19. This patient was also found to have a ~0.6 Mb gain on 10q21.1 (minimum chr10:53,155,144–53,788,294, maximum chr10:53,108,516–53,813,645; hg19) involving two genes; a portion of PRKG1 and all of CSTF2T.
2	CMA	Copy number gain of approximately 16.7 Mb on 10q213q23.1 (minimum chr10:69,734,057–86,426,833; maximum chr10:69,678,160–86,472,655; hg19).
3	WES	Maternally inherited, heterozygous c.239_240delAT, p.(H80Rfs*17) frameshift variant in NAA15.
4	WES	Likely pathologic C.3118A > G, p.(R1040G) variant in MYRF consistent with cardiac urogenital syndrome.
5	WES	Likely pathologic C.2660C > T, p.(T8871) missense variant in EP300 consistent with a diagnosis of Rubinstein–Taybi syndrome 2.
6	CMA	Copy number gain on chromosome 16p13.11p12.3 (chr16:15,333,155–18,242,713; hg19) consistent with 16p13.11 microduplication syndrome.
7	Chromosomal analysis	De novo unbalanced translocation between chromosomes X and 2 with a chromosomal designation of 46,X,der(X)t(X;2)(q26;q31.1).
8	CMA	~0.16 Mb loss at16q22.1 (minimum chr16:68,579,360–68,695,101; maximum chr16:68,550,178 – 68,709,888; hg19) which involved two genes, ZFP90 and CDH3.
9	CMA	~ 7 kb loss at 8p11.23 (minimum chr8:38,091,564–38,098,218; maximum chr8:38,090,832–38,099,625; hg19) leading to loss of exons 3–6 of DDHD2. This patient also had an ~0.1 Mb gain on 12q24.31 (minimum chr12:124,090,709–124,111,801; maximum chr12:124,008,592–124,114,750; hg19) and an ~ 0.21 Mb loss on 12q24.31 (minimum chr12:124,155,205–124,156,920; maximum chr12:124,137,166–124,158,087; hg19).
10	CMA	~ 0.026 Mb loss at 8p11.21 (minimum chr8:42,694,713–42706855; maximum chr8:42,685,068–42,710,811; hg19) leading to loss of exons 4–5 of CHRNB

CMA = chromosomal microarray; WES = whole-exome sequencing.

Four patients were deceased, none of which had genetic testing performed. One patient had VACTERL syndrome with a transitional atrioventricular septal defect, subaortic stenosis and coarctation of the aorta. No further information surrounding the death was available. A second patient had hypoplastic left heart syndrome (mitral and aortic atresia) and underwent stage 1 palliation with Norwood/Sano repair and then stage 2 palliation with Glenn repair. The patient had poor PO feeding, intestinal malrotation and pulmonary sequestration but was eventually discharged from the hospital following Glenn palliation. No further information surrounding the death was available. A third patient had a secundum ASD, right-sided congenital diaphragmatic hernia, hemivertebrae and a sacral anomaly. She had severe lung hypoplasia and oxygen requirement and was managed with home hospice care. A fourth patient had a secundum ASD as well as horseshoe lung, tracheal stenosis requiring surgical repair and subsequent tracheal stent, chiari malformation and pulmonary hypertension and died due to pulmonary hypertensive crisis.

Clinical and molecular summaries

Seven patients (Patient 1–7) had a definitive molecular diagnosis made through genetic testing. Three patients (Patients 8–10) had copy number variants identified on CMA and that were of unknown significance (VUS). None of these patients had WES performed. Patient 11 did not have genetic testing but was diagnosed on a clinical basis with Marfan syndrome.

Patient 1

Patient 1 had SS with a congenital diaphragmatic hernia, right lung hypoplasia, right pulmonary artery narrowing, dextroposition of the heart, secundum atrial septal defect (ASD), and aortic arch elongation. CMA demonstrated ~1.6 Mb gain on 22q11.21 (minimum chr22:17,998,078–19,606,540; maximum chr22:17,982,527–19,627,555; hg19). The proximal portion of the DiGeorge critical region was involved in the duplication, but the TBX1 gene is located outside of the duplication. Duplications involving the DiGeorge critical region have a variable phenotype with significant clinical overlap to 22q11.2 deletion syndrome that has been described in association with total anomalous pulmonary venous return^16,17.

This patient was also found to have a ~0.6 Mb gain on 10q21.1 (minimum chr10:53,155,144–53,788,294, maximum chr10:53,108,516–53,813,645; hg19) involving two genes; a portion of PRKG1 and all of CSTF2T. Missense variants in PRKG1 are associated with autosomal dominant aortic aneurysm, familial thoracic 8 through a gain-of-function mechanism ¹⁸. It is possible that PRKG1 may be disrupted by this gain, but data from normal individuals cataloged in the gnomAD database (https://gnomad.broadinstitute.org/) suggest that haploinsufficiency of PRKG1 is unlikely to be associated with a significant phenotype. Similarly, a gain of CSTF2T is not currently associated with a known phenotype. Hence, is it unlikely that this change contributed to the development of SS in this patient.

Patient 2

Patient 2 had SS with right lung hypoplasia as well as secundum ASD, coarctation of the aorta, and pulmonary valve stenosis. This patient also had extracardiac findings including congenital diaphragmatic hernia, osteopenia and rib fractures, failure to thrive with nasogastric feeding tube requirements, and nystagmus. CMA demonstrated a copy number gain of approximately 16.7 Mb on 10q21.3q23.1 (minimum chr10:69,734,057–86,426,833; maximum chr10:69,678,160–86,472,655; hg19) that overlaps the region associated with 10q22.3-q23.2 deletion syndrome. Duplications overlapping that which was seen in this individual have been reported in patients with congenital heart disease, dysmorphisms and delays in speech and motor development^19,20.

Patient 3

Patient 3 had SS with right lung hypoplasia as well as tetralogy of Fallot, an aortopulmonary collateral to the right side requiring device occlusion, a secundum ASD, and hypertrophic cardiomyopathy. Of note, the patient developed progressive severe biventricular hypertrophy and underwent orthotopic heart transplant with reimplantation of the scimitar vein at 22 months of age. The reimplanted scimitar vein developed stenosis requiring transcatheter angioplasty and stenting of the vein. The patient had a normal CMA. Trio WES revealed a maternally inherited, heterozygous c.239_240delAT, p.(H80Rfs*17) frameshift variant in NAA15. This gene encodes an acetyltransferase subunit and has a role in maintaining cell proliferation²¹. Changes in this gene have been identified in separate patients with complex congenital heart defects, hypertrophic cardiomyopathy, and developmental delay²². Variants in this gene have also been described in patients with isolated congenital diaphragmatic hernia²³. No report was available on whether the mother was symptomatic.

Patient 4

Patient 4 had SS and right lung hypoplasia, with hypoplastic left heart syndrome (HLHS) with mitral atresia/aortic stenosis, left ventricular sinusoids, and severe coarctation. The patient underwent single ventricle palliation consisting of a Norwood procedure and, subsequently, a Glenn palliation. Extracardiac findings for this patient included feeding intolerance requiring gastrostomy tube placement, global developmental delay and a para-esophageal hernia. CMA was normal. WES revealed a likely pathologic c.3118A>G, p.(R1040G) variant in MYRF consistent with cardiac urogenital syndrome^24–26. This patient has previously been reported in the literature²⁶.

Patient 5

Patient 5 had SS, an aberrant right subclavian artery, and a left superior vena cava. The patient also had extracardiac findings of failure to thrive, global developmental delay, right lung hypoplasia, congenital diaphragmatic hernia and a laryngeal cleft. His CMA was normal. WES revealed a likely pathologic c.2660C>T, p.(T8871) missense variant in EP300 consistent with a diagnosis of Rubinstein-Taybi syndrome 2, which has a known association with congenital heart defects²⁷.

Patient 6

Patient 6 had SS with an ASD and a history of supraventricular tachycardia along with pyloric stenosis. CMA revealed a gain on chromosome 16p13.11p12.3 (chr16:15,333,155–18,242,713; hg19) consistent with 16p13.11 microduplication syndrome. Individuals with this syndrome can have congenital heart defects^28,29 as well as aortopathy^30,31. Developmental delay, seizure disorder, autism spectrum disorder and speech and learning disorders have also been seen in patients with 16q13.11 microduplications.

Patient 7

Patient 7 had SS with perimembranous and muscular VSDs, a history of poor feeding, developmental delay and intellectual disability. A chromosome analysis revealed a de novo unbalanced translocation between chromosomes X and 2 with a chromosomal designation of 46,X,der(X)t(X;2)(q26;q31.1). Additional studies demonstrated preferential inactivation of the abnormal X chromosome, with no inactivation of the translocated material from chromosome 2. This results in the presence of three active copies of the distal long arm of chromosome 2 in most cells.

Patient 8

Patient 8 had SS with tetralogy of Fallot as well as ectrodactyly, right lung hypoplasia, scoliosis, thoracic vertebrae anomalies and developmental delay. CMA revealed a ~0.16 Mb loss at 16q22.1 (minimum chr16:68,579,360–68,695,101; maximum chr16:68,550,178 – 68,709,888; hg19) which involved two genes, ZFP90 and CDH3, neither of which have been associated with congenital heart defects.

Patient 9

Patient 9 had SS with a large ASD, aberrant right subclavian artery and pulmonary hypertension requiring extra-corporeal membrane oxygenation. This patient also had a tracheoesophageal fistula requiring surgical repair, right lung hypoplasia, necrotizing enterocolitis requiring medical management, intraventricular hemorrhage noted at birth, and adrenal insufficiency. CMA revealed a ~ 7 kb loss at 8p11.23 (minimum chr8:38,091,564–38,098,218; maximum chr8:38,090,832–38,099,625; hg19) leading to loss of exons 3–6 of DDHD2, which is not known to be associated with congenital heart defects. This patient also had an ~0.1 Mb gain on 12q24.31 (minimum chr12:124,090,709–124,111,801; maximum chr12:124,008,592–124,114,750; hg19) and an ~ 0.21 Mb loss on 12q24.31 (minimum chr12:124,155,205–124,156,920; maximum chr12:124,137,166–124,158,087; hg19), both of which carry no association with congenital heart defects.

Patient 10

Patient 10 had SS, a perimembranous ventricular septal defect (VSD), a secundum ASD, scoliosis, asthma, and ADHD. This patient also had a tracheoesophageal fistula requiring surgical repair, necrotizing enterocolitis requiring medical management, intraventricular hemorrhage noted at birth and adrenal insufficiency. CMA revealed an ~ 0.026 Mb loss at 8p11.21 (minimum chr8:42,694,713–42706855; maximum chr8:42,685,068–42,710,811; hg19) leading to loss of exons 4–5 of CHRNB3, which has not been associated with a human disorder and carries no association with congenital heart disease.

Additionally, Patient 11 was clinically diagnosed with Marfan syndrome. The patient has a family history of clinical Marfan syndrome in multiple siblings, aunts/uncles and cousins that all carry the clinical diagnosis of Marfan syndrome. The patient had the following findings on evaluation: aortic root dilation with most recent measurement of 4.0 cm (z-score for age + 5.0), ectopia lentis, positive thumb and wrist sign. Of note, no genetic testing has been performed on this patient and specifically FBN1 sequencing has not yet been performed. The patient is being medically managed for Marfan syndrome with atenolol and losartan. She has an extracardiac history of spontaneous pneumothorax requiring apical bleb resection. She has not required surgical intervention on her aortic root.

DISCUSSION

We present a cohort of 89 patients with SS and describe the frequency and findings of genetic testing, describing the genetic variants as well as the extra-cardiac findings seen in these patients. This is largest single-center cohort of patients with SS described to date. Our findings correlate with similar literature, describing a 2:1 female-to-male preponderance¹. The percentage of patients with SS who had genetic testing was relatively low at only 20% of patients in the cohort (18 of 89). However, genetic testing provided a molecular diagnosis in 39% of the individuals tested (7 of 18). Variants of unknown significance were identified in another 3 individuals that had genetic testing performed and one individual was clinically diagnosed with Marfan syndrome.

Among the genes implicated in this study, EP300 and NAA15 may lead to involved changes related to different phases of cell growth and proliferation, including transcription coactivators and post-translational acetylation processing^32,33. MYRF has been shown to play a role in oligodendrocyte cell proliferation³⁴, and we can assume that MYRF plays an important developmental role outside of the central nervous system as well.

Patient 1 had duplication of the 22q11.21 region, previously reported to be associated with total anomalous pulmonary venous return. However, duplications of this region have not been previously reported in individuals with SS³⁵. Altered dosage of the TBX1 gene has been implicated in the commonly noted outflow tract abnormalities in both 22q11.2 deletion syndrome and 22q11.2 duplication syndrome^16,17. However, it is important to note that the TBX1 gene was not involved in our patient’s duplication. This suggests that increased copy number of genes other than TBX1 are responsible for the heart defects seen in this patient.

Patient 7 had an unbalanced chromosomal translocation between chromosome X and 2, that effectively resulted in trisomy of the terminal portion of the q arm of chromosome 2 extending to band 2q31.1. This large region is likely to harbor several genes that play a role in cardiac development. Although congenital heart defect can be seen in Turner syndrome, losses of material distal to Xq25 rarely give rise to phenotypes associated with Turner syndrome beyond secondary amenorrhea or premature menopause³⁶. In keeping with this observation, this patient had no stigmata or findings consistent with Turner syndrome. Of note, the patient population was predominantly female, calling into question if other sex chromosome variants could be implicated.

Patient 6 had a 16p13.11 microduplication, which has been associated with congenital heart disease as well as multiple neuropsychiatric anomalies and developmental delay, as well as hypermobility and various other musculoskeletal changes^28–30,37. At present, there is no clear mechanistic link between the genetic findings described and cardiac embryogenesis or the development of SS.

The copy number variants classified as VUS involved several different cellular pathways. For example, Patient 8 had a loss at 16q22.1 that involved CDH3. Autosomal recessive variants in CDH3, located within the 16q22.1 region, are associated with ectodermal dysplasia, ectrodactyly, and macular dystrophy (OMIM# 225280) and hypotrichosis, congenital, with juvenile macular dystrophy (OMIM# 601553). This gene has not been associated with congenital heart disease in the literature to date.

Extra-cardiac findings were seen in a large majority of our cohort, and in all of the patients with positive genetic findings and copy number VUS. The most common extra-cardiac findings throughout the cohort were asthma and right lung hypoplasia, gastrointestinal and skeletal pathologies. Four patients were found to have pulmonary sequestration, a rare but known finding amongst SS patients^38,39

Our study must be considered in the light of certain limitations. First, this is a retrospective study, which limits the applicability of our results and introduces the risk of selection bias. This was a single center study, and the decision to perform genetic testing was likely altered by institutional practices as well as intrinsic sampling bias towards those patients with extracardiac findings. We relied on chart review for description of extra-cardiac findings, which places great emphasis on accurate documentation as the sole source for description of extra-cardiac findings and instances may have been missed due to this limitation. Right lung hypoplasia was seen in a large percentage of patients, but this and other extracardiac findings may be susceptible to reporting bias. Given the retrospective nature of the study, we also cannot describe what individual aspects drove each clinician to obtain genetic testing on some patients with SS and not others. It is possible that providers performed more genetic tests on patients with SS and other extra-cardiac abnormalities, as compared to those without additional findings. We also note that genetic testing was performed on a relatively small percentage of our cohort. Many of these limitations could be addressed in future studies performed on a prospective basis. Future studies may also consider analysis of the 2:1 female preponderance, which to date has been described but it’s mechanism remains unknown.

In conclusion, our study highlights the ability of genetic testing to identify a molecular diagnosis in a significant percentage of SS patients (7/18, 39%). Since all of these individuals had extra-cardiac defects, we conclude that genetic testing should be performed in all individuals with SS who have extra-cardiac defects. If a specific genetic syndrome is not suspected, CMA analysis should be performed as a first-tier test. If a molecular diagnosis is not identified on CMA, WES should then be performed. This testing will allow the clinician to better counsel families going forward and prepare the provider for extra-cardiac pathologies.