Genomic spectrum of congenital heart disease combined with kidney and urinary tract anomalies uncovered by exome sequencing and array-CGH

Abstract

Congenital heart disease (CHD) combined with anomalies of the kidney and urinary tract (CAKUT) belongs to severe clinical conditions that represent isolated cases or can be part of complex inherited syndromes. To date, knowledge of the genetic basis of cardio-renal birth defects is very limited. Using chromosomal microarray analysis and exome sequencing, we investigated the spectrum of copy number variations (CNVs) and point genetic variants in a pediatric cohort of 30 patients presenting with combined CHD and CAKUT. Two patients possessed a pathogenic deletion in the 22q11.2 genomic region, well-known as the DiGeorge/velocardiofacial syndrome locus. Four other patients harbored rare copy number gains, which included one of the dosage-sensitive genes: DHFR (5q14.1), NPHP1 (2q13), NNT (5p12), and MCTP2 (15q26.2). In six children, novel or previously described causative variants were detected in the genes associated with orphan monogenic disorders such as Kabuki (KMT2D), Noonan (PTPN11), Adams-Oliver (NOTCH1), and Alagille (JAG1) syndromes, as well as SALL4- and MED13-related syndromes. Furthermore, potentially disease-contributing variants were identified in the genes that primarily code for cardiac transcription factors, chromatin remodeling proteins, Notch- or BMP-signaling molecules, and components of cilia. Notably, two children had loss-of-function variants in ROBO1, encoding one of the receptors of the SLIT/ROBO signaling pathway. In vitro studies on cardiac mesenchymal cells demonstrated a dramatically diminished expression level of ROBO1 in a patient with novel compound heterozygous variants c.541_542del (p.Val181CysfsTer18) and c.657+4A>G and a moderately reduced ROBO1 expression in a patient with a heterozygous stop-gain variant c.2758C>T (p.Arg920Ter), which suggests a haploinsufficiency mechanism. Together, our findings underlined the utility of early cytogenetic screening for the 22q11.2 deletion among children with cardio-renal malformations and confirmed the importance of rare single-nucleotide variants in the genes involved in cardiogenesis and kidney development. In particular, ROBO1 can be regarded as a candidate gene associated with combined heart and kidney structural defects and should be included in appropriate diagnostic gene-sequencing panels. The results support the usefulness of WES and array-based comparative genomic hybridization (array-CGH) analysis for new candidate gene identification, more accurate diagnosis, and family genetic counseling.