From a cohort of 40 BAV adult patients [26] and 20 BAV pediatric cases, we sought to determine the implication of the ROBO-SLIT pathway in patients with no relevant variants in known BAV-related genes. Interestingly, the yield of rare variants in ROBO and SLIT genes was greater in the pediatric cohort (13/20, 65%) compared to only 4 out 40 BAV cases (10%) from the adult cohort (Table 2).

Family segregation was performed for two BAV patients only, both with ROBO4 variants.

The first patient (BAV-PED-10) is a male pediatric case with BAV. His medical records include small aortic insufficiency in the posterior commissure, fusion of the anterior commissure, right anterior leaflet prolapse, aortic annulus dilatation and dilated ascending aorta (z score 3.2 and 3.3). The patient had a positive family history of aortic valve defects. His paternal and maternal grand-mothers underwent aortic valve replacement.

The patient (BAV-PED-10) carried a homozygous ROBO4 variant (p. Arg776Cys) (Table 3). His parents were found heterozygous for the variant (Fig. 1). The MAF of this variant (rs138481093) is 0.004699 in gnomAD with a total number of homozygotes equal to 8. Of note, our patient is of white European non-Finnish ethnic group which represents the highest MAF population (0.007781).

Family segregation of the ROBO4: p.Arg776Cys variant. Darkened left upper quadrant: Affected child with BAV

The second case (BAV-PED-12) is a male pediatric case with BAV. The analysis of his WES data allowed us to identify a splice site heterozygous variant in ROBO4 (c.3001 + 3G > A). The patient’s father was found to have hypoplastic left coronary artery and his brother had VSD. His mother and sister are healthy.

The ROBO4: c.(3001 + 3G > A) variant was found in the patient’s father (I-1) and brother (II-2). The mother (I-2) and sister (II-3) do not carry the variant.

Family pedigree and segregation are shown in Fig. 2.

Family segregation of the ROBO4: c.3001 + 3G > A variant. The index-case (BAV-PED-12) is marked with a star

As for the first patient (BAV-PED-10), this case is European non-Finnish also. The MAF of the ROBO4: c.3001 + 3G > A variant in this population is 0.01448. The highest population MAF of this variant (rs145918924) is 0.02831 in the Ashkenazi Jewish population.

Of note, Gould et al. reported a heterozygous splice site variant ROBO4: c.2056 + 1G > T in a multigenerational BAV-family. Interestingly, seven of eight affected cases were male [10]. These findings underline the intrafamilial variability as well as the phenotypic pleiotropy of ROBO4 variants.

As we mentioned above, the identification of more ROBO and SLIT variants in the pediatric BAV cases than in the adult cohort prompted us to investigate the implication of this pathway in another CHD phenotype. Thus, we analyzed 10 ToF patients and one CoA case. Five out 10 ToF patients carried variants in ROBO and SLIT genes. Three patients carried variants in the ROBO3 gene and strikingly, the two other patients (ToF-PED-9 and ToF-PED11) were carrying the aforementioned ROBO4 variants (p.(Arg776Cys) and c.3001 + 3G > A) at a heterozygous state (Table 2).

The clinical resume of the ToF-patient (ToF-PED-9) carrying the ROBO4: p.Arg776Cys variant is as following: Pregnancy was complicated by gestational diabetes. Pulmonary atresia and VSD as well as partial corpus callosum agenesis were prenatally diagnosed. Amniocentesis was refused by the parents. The anatomy was confirmed after birth. Pulmonary arteries were noted to be extremely hypoplastic (2 mm, z-value -5, birth weight 4 kg). A malformation of the arterial duct was noted, with no signs of spontaneous closure. At the age of 27 months, a total cardiac repair was performed.

The clinical resume of the ToF-patient (ToF-PED-11) carrying the ROBO4: c.3001 + 3G > A splicing variant is as following: Severe ToF and thoraco-abdominal situs inversus was prenatally diagnosed. Birth weight was small for gestational age (2.4 kg, 38W). Anatomy was confirmed after birth. Pulmonary annulus was very hypoplastic (Z value − 3.7) as well as pulmonary arteries (Z value RPA − 2.4, LPA − 2). The complete cardiac repair (closure VSD and patch enlargement of pulmonary valve and artery) was performed 11 months later.

ToF is defined by the presence of four cardiac defects namely; ventricular septal defect (VSD), pulmonary valve stenosis, right ventricular hypertrophy and overriding aorta, which potentially arise from a misalignment of the great arteries [27, 28]. The identification of the same ROBO4 variants in BAV and ToF patients points out the pleiotropic role of this gene with its implication in several CHD entities with different pattern of inheritance. This pleiotropy can be explained by the potential contribution of ROBO4 gene in different cardiac cell populations [8, 13, 29], but also by the difference of the genetic background of each individual and epigenetics mechanisms acting during heart morphogenesis.

Three additional ROBO4 variants were identified in the present study. The ROBO4 variant (p. Arg908Gln) was identified in a BAV patient with aortic stenosis (BAV-PED-1). This patient carried a second missense variant in ROBO2 gene (p. Arg811Trp). The ROBO4: p.(Ala303Asp) has been identified in a pediatric BAV-case (BAV-PED-7) with aneurysm. Similarly, this patient carried a second variant in the SLIT1 gene (p.Glu1340Asp). The third ROBO4: p.(Ala446Asp) variant was found in BAV-PED-8 case (Table 2). No BAV-related complications were noted for this patient.

In regards to BAV adult patients with variants in ROBO1, ROBO2, SLIT1 and SLIT3 genes, the presence of BAV-related complications such as aortic regurgitation, aortic stenosis, and AscAA was checked. Only the patient (BAV-AD-1) with the ROBO1: p.(Val610Ile) variant had AscAA.

Within this study, we report two stop-gain variants in SLIT1 (p.Cys263Ter) and SLIT3 (p.Cys1355Ter) genes. The patient carrying the SLIT3 stop-gain variant had BAV with mitral regurgitation.

Collectively, a total of 24 rare variants were identified including 21 missense, 2 stop-gain, and 1 splice site variants (Table 2). The majority of variants were found in the pediatric cohort. Indeed, 19 pediatric cases carried variants in ROBO and SLIT genes (19/31 CHD-patients; 61%), whereas, only 4 adult patients (10%) had missense variants in ROBO1, ROBO2, SLIT1 and SLIT3 genes.

It should be noted that, all the patients carried heterozygous variants except a BAV- patient (BAV-PED-10) with the homozygous ROBO4 variant (p. Arg776Cys).

Overall, the in-silico predictions of variant pathogenicity are quite consistent among the different software tools, specifically, variants in ROBO1, ROBO3, and SLIT genes, were predicted to have a large decrease of protein stability and high CADD scores (Table 4). Indeed, except for SLIT1 (p.Cys263Ter) and SLIT3 (p.Cys1355Ter) stop-gain variant with a very high CADD-scores (36 and 42, respectively) which is mainly due to the truncating type of the variants, the highest scores (≥ 30) are attributed to variants located in the fibronectin type III-3 domain of ROBO genes. As an example, the ROBO2: p. (Arg811Trp) and the ROBO3: p.(Pro859Gln) variants, with CADD-scores 31 and 32, respectively, are located within the Fibronectin type-III 3 domain of each gene (Additional file 1).

A more detailed description of variant localization and their predicted impact on protein structure, interaction and physicochemical properties is provided in the Additional file 1. Sanger confirmation of the prioritized variants is provided in Additional file 2.