Exploring RBFOX2 haploinsufficiency: A New Genetic Link to Hypoplastic Left Heart Syndrome

RNA binding fox-1 homolog 2 (RBFOX2) has been proposed as a candidate gene for left-sided obstructive heart defects, based on mouse models with loss-of-function (LoF) variants leading to left heart hypoplasia¹. These range from isolated left valvular defects to complex forms such as Shone complex and hypoplastic left heart syndrome (HLHS), which result from defective development of left-sided cardiac structures.

We report the first international cohort of congenital heart disease (CHD) individuals with heterozygous LoF variants or deletions involving RBFOX2, recruited through collaboration calls and via Genematcher. In addition, we reviewed previously reported RBFOX2 LoF variants and deletions from databases including ClinVar, DECIPHER, and the literature. The study was conducted under ethics approval number DC-2020-4073 from the French Ministry of Research and Innovation. All subjects provided informed consent. All data are available upon request.

Our cohort includes 19 individuals from 12 unrelated families. Cardiac anomalies predominantly affect the left heart: 10 individuals (52.6%) had HLHS, 10 (52.6%) had ascending aorta abnormalities (coarctation or hypoplasia), 7 (36.8%) had mitral valve defects (atresia or stenosis), and 6 (31.5%) had aortic valve defects (bicuspid valve, stenosis or atresia). Septal defects were also observed: 8 individuals (42.1%) had ventricular septal defect (VSD), and 6 (31.5%) had atrial septal defect (ASD) (Figure A). Substantial phenotypic variability was observed both between and within families (Figure B). Right-sided lesions were also present, such as double outlet right ventricle (DORV, P8-P10, P19) and tetralogy of Fallot (TOF, P18, family F11).

Figure 1.

A. Characteristics of individuals with RBFOX2 variant (n=33). Reference genome is Hg38 and reference transcript is NM_001349999.2 (RBFOX2).

HLHS = Hypoplastic Left Heart syndrome, DORV = Double outlet right ventricle, TOF = Tetralogy of Fallot, VSD = Ventricle Septal Defect, ASD = Atrial Septal Defect, PDA = Patent Ductus Arteriosus, M = male, F = female, NR = not reported, htz = heterozygous. ACMG classification according AnnotSV for CNV and reported for SNV. All variants were ACMG-classified as (likely) pathogenic, except P13 CNV (class 3 in AnnotSV, tool class 5 in french Achropuce tool).

B. Pedigree trees for familial cases. IUFD = in utero fetal demise

C. CNVs encompassing RBFOX2. RBFOX2 MANE transcript in green, OMIM genes with autosomal dominant inheritance (purple) and autosomal recessive inheritance (orange). Genes without associated disease in OMIM database are in blue. Heterozygous deletions from patients of databases (DECIPHER or ClinVar) without CHD are in dark red, heterozygous deletions from patients of our cohort and patients from database (DECIPHER or ClinVar) with CHD are in red.

D. RBFOX2 transcript shown with SNVs and intragenic deletions. Exons included in RBFOX2 cardiac transcript ENST00000405409.6 are marked with green color. RRM protein domain (Recognition RNA motif) is in dark green. Upper, LoF SNVs found in patients without CHD in databases are represented with dark red triangles and deletions from patients in database without CHD are in dark red. Lower, patients in database with CHD and cohort’s missense variants are represented with red circles, splicing variants with red rhombs, truncating variants (frameshift, non-sense and intragenic deletion) with red triangles. Intragenic deletions are shown with horizontal square brackets.

Genetic analyses identified copy number variants (CNVs) in 7 individuals (36.8%) and single nucleotide variants (SNVs) in 12 (63.1%). In 14 of these 19 individuals, molecular analysis (WGS, WES or CHD genes panels) excluded other known genetic causes of CHD. Among the CNVs, five were RBFOX2 heterozygous intragenic deletions and two involved the entire gene (Figure C). All variants were ACMG-classified as (likely) pathogenic, except P13 CNV (class 3 in AnnotSV, class 5 in french Achropuce tool). All deletions shared a minimal overlapping region encompassing the last three exons of RBFOX2; such deletions are absent in control databases (gnomAD, DGV).

In contrast, we identified six individuals (R28–R33) from patients’ databases who carried RBFOX2 variants without reported cardiac anomalies (Figure A). Four carried intragenic deletions (R28–R31) affecting exons absent in the cardiac transcript (ENST00000405409.6). Surprisingly, two individuals (R32, R33) carried frameshift SNVs in this cardiac transcript (Figure D), however no phenotypic information, especially regarding cardiac anomalies, was available for these individuals (Figure A). These findings suggest that the pathogenicity of RBFOX2 variants may depend on their location within the cardiac-expressed transcript.

Additional evidence supports RBFOX2 as a gene implicated in HLHS. Glessner et al.² reported a de novo 111 kb CNV including RBFOX2 in a patient with HLHS. Homsy et al.³ identified three more individuals with HLHS carrying de novo LoF variants in RBFOX2. Verma et al.⁴ conducted transcriptomic analysis on these patients hearts, providing supporting evidence for a role of RBFOX2 haploinsufficiency in HLHS pathogenesis.

Our findings further delineate the RBFOX2-associated phenotype. Left heart anomalies predominate, but phenotypic variability is considerable. This may reflect the function of the RBFOX2 protein, which regulates alternative RNA splicing and polyadenylation in cardiomyoblasts, particularly in genes involved in cell regulation and adhesion⁴. Although many RBFOX2 targets have been identified via CLIP-seq studies ^1,4 (e.g., Pnn, Spcs1, Ddx39, Mcm7, Phkb, Abi1, Fn1, Ect2), none are currently linked to CHD in OMIM database. We hypothesize that unidentified cardiac-relevant targets or qualitative splicing changes contributed to phenotypic diversity.

Variant type may also influence the phenotype. CNVs, especially large deletions including RBFOX2 and neighboring genes, are more often associated with syndromic CHD, while SNVs or small intragenic deletions appear more commonly in isolated cardiac defects. Furthermore, variant impact may depend on transcript location. LoF SNVs that do not affect exons of the cardiac transcript are more frequent in controls than in CHD cases. This supports our observation of intragenic deletions in non-CHD individuals (R28–R31) involving exons absent from the cardiac isoform. Four missense variants in our cohort [p.(Arg183Trp), p.(Lys221Arg), p.(Arg249Cys), p.(Gly224Arg)] are located within the RRM (RNA recognition motif) domain, particularly in or near beta-sheet structures crucial for RNA binding. A mouse model carrying a missense variant [p.(Gly224Arg)] in the RRM had abnormal heart development⁵. Further functional studies are required to determine the role of these missense variants in CHD.

In summary, we present an international cohort of 19 individuals with RBFOX2 heterozygous LoF or deletion variants, alongside a comprehensive review of known variants from patients and controls databases. Our results support a role for RBFOX2 haploinsufficiency in HLHS and other left-sided CHDs. We propose that the impact of RBFOX2 variants depends on both their genomic context and their effect on cardiac-specific transcripts and splicing regulation.

Nonstandard Abbreviations and Acronyms

ACMG

American Council of Medical Genetics classification

ASD

atrial septal defects

CHD

congenital heart defects

ClinVar

Clinically relevant Variation database

CLIP-seq

cross-linking immunoprecipitation sequencing

CNV

copy number variant

DECIPHER

DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources

DGV

Database of Genomic Variant

DORV

double outlet right ventricle

gnomAD

Genome Aggregation Database

HLHS

hypoplastic left heart syndrome

Htz

heterozygous

IUFD

in utero fetal demise

LoF

loss-of-function variant

OMIM

Online Mendelian Inheritance in Man database

PDA

patent ductus arteriosus

RBFOX2

RNA binding fox-1 homolog 2

RRM

RNA recognition motif

SNV

single nucleotide variant

TOF

tetralogy of Fallot

VSD

ventricular septal defects

WES

whole exome sequencing

WGS

whole genome sequencing