Abstract 4367934: Comprehensive Genetic Analyses Combined with Animal Studies Provide New Insights into Genetic Modes of Outflow Tract Defects: A Large-Scale Japanese Investigation

Background: Persistent truncus arteriosus (PTA), pulmonary atresia with ventricular septal defect (PA-VSD) and tetralogy of Fallot (ToF) give a developmental spectrum of outflow tract (OFT) defects and share genetic etiologies such as 22q11.2 deletion. We recently identified a frameshift variant of TMEM260 (c.1617del), named “Keio-Tohoku variant (KTv)” as the most frequent single gene variant of PTA in Japanese patients. FLT4 and NOTCH1 have been reported as the first and second frequent gene responsible for ToF. Objective: To explore the genetic background of OFT defects through comprehensive genetic analyses using numbers of patients’ DNA, combined with animal models. Methods: A total of 433 Japanese patients with OFT defects (PTA: n=37; PA-VSD: n=161; ToF: n=235) subjected to genetic testing including G-banding, FISH, and exome sequencing. Mouse models with genetic manipulation of Tmem260 , Flt4 and Notch1 were established and morphologically analyzed. Results: Genetic causes were identified in 211 of 433 cases (49%; PTA 51%, PA-VSD 56%, ToF 43%), including 178 with 22q11.2 deletion. Biallelic TMEM260 KTv were found in 6 (5 in PTA) cases. Interestingly, compound heterozygous for TMEM260 KTv and a ~7kb deletion involving exons 6–7 in TMEM260 were identified in 2 PTA patients (5.4%). This deletion was also enriched in Japanese population (0.17% allele freq.). NOTCH1 and FLT4 variants were found in 12 patients with ToF or PA-VSD. Notably, two PTA cases harbored compound heterozygous variants in both NOTCH1 and FLT4 . Variants in GATA6 (0.7%), GATA4 (0.5%), NKX2-5 (0.2%), and MEF2C (0.2%) were also detected. Initial mouse analyses showed that Tmem260 homozygous KO mice exhibited perinatal lethality and OFT defects while normal phenotype of heterozygous mice. Compound heterozygous Flt4 / Notch1 KO mice had more frequent and severe OFT defects than each single heterozygote. Conclusion: Genetic causes were identified in nearly 50% of our patients with OFT defect. TMEM260 is responsible for PTA with not only homozygous variants, but also compound heterozygous variant/deletion in an autosomal recessive fashion that was confirmed by mouse models. Moreover, our genetic analyses suggest that compound heterozygous variants in NOTCH1 and FLT4 may play a combinatorial contribution to OFT defects that was also supported by the findings from mouse models. This result showed new evidence for oligogenic/multifactorial origin in OFT defects.