Rare copy number variants analysis identifies novel candidate genes in heterotaxy syndrome patients with congenital heart defects.

Background: Heterotaxy (Htx) syndrome comprises a class of congenital disorders resulting from malformations in left-right body patterning. Approximately 90% of patients with heterotaxy have serious congenital heart diseases; as a result, the survival rate and outcomes of Htx patients are not satisfactory. However, the underlying etiology and mechanisms in the majority of Htx cases remain unknown. The aim of this study was to investigate the function of rare copy number variants (CNVs) in the pathogenesis of Htx.

Methods: We collected 63 sporadic Htx patients with congenital heart defects and identified rare CNVs using an Affymetrix CytoScan HD microarray and real-time polymerase chain reaction. Potential candidate genes associated with the rare CNVs were selected by referring to previous literature related to left-right development. The expression patterns and function of candidate genes were further analyzed by whole mount in situ hybridization, morpholino knockdown, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated mutation, and over-expressing methods with zebrafish models.

Results: Nineteen rare CNVs were identified for the first time in patients with Htx. These CNVs include 5 heterozygous genic deletions, 4 internal genic duplications, and 10 complete duplications of at least one gene. Further analyses of the 19 rare CNVs identified six novel potential candidate genes (NUMB, PACRG, TCTN2, DANH10, RNF115, and TTC40) linked to left-right patterning. These candidate genes exhibited early expression patterns in zebrafish embryos. Functional testing revealed that downregulation and over-expression of five candidate genes (numb, pacrg, tctn2, dnah10, and rnf115) in zebrafish resulted in disruption of cardiac looping and abnormal expression of lefty2 or pitx2, molecular markers of left-right patterning.

Conclusions: Our findings show that Htx with congenital heart defects in some sporadic patients may be attributed to rare CNVs. Furthermore, DNAH10 and RNF115 are Htx candidate genes involved in left-right patterning which have not previously been reported in either humans or animals. Our results also advance understanding of the genetic components of Htx.