Structural birth defects (SBD) affect 3-6% of live births and are a leading cause of infant morbidity and mortality worldwide. Most SBD are isolated, occur in the first 10 weeks of human development, and are thought to be driven by genetic, epigenetic, and environmental factors. Technical advances in DNA sequencing have propelled our understanding of the genetics of SBD. Array based technologies identified copy number variants that contribute to SBD and enabled genome wide association studies. Massively parallel ?Next Generation? (NGS) methods sequence large numbers of short (50-300 base pairs) pieces of DNA simultaneously, permitting sequencing of all coding regions (the exome) or the entire genome at once. Exome and genome sequencing of blood-derived DNA identifies the molecular etiology of ~50% of children multisystemic, syndromic SBD. However, the etiology of most SBD- both isolated and syndromic- remains unknown. A potential reason for this is that short-read based sequencing of blood-derived DNA does not provide a comprehensive view of an individual?s genome. The goal of this proposal is to use novel technologies in prospective cohorts of children with SBD to identify genetic variation not identified by current methods. These ?hidden? variants include duplications, inversions, repeat expansions/contractions, and epigenetic modifications that standard short-read based methods cannot identify (Aim 1). Hidden variants also include DNA mutations that arise post-zygotically (?mosaic?) and are not present in blood-derived DNA (Aim 2). We will use long-read based DNA and RNA sequencing methods (PacBio and Oxford Nanopore) on patients with syndromic SBD whose clinical workup (including exome sequencing) has been non-diagnostic. In patients with isolated SBD whose clinical workup has been non-diagnostic, we will apply deep short-read based DNA sequencing of multiple, non-blood derived tissues to identify mosaic variants. This proposal aligns with the stated goals of the NICHD, as our findings will form the basis of new strategies for the diagnosis, treatment, and prevention of human structural birth defects. Our work furthers that goal by identifying previously undetected genetic variation in children with SBD. This is a key first step towards establishing a framework for utilizing these novel technologies in the clinical diagnostic arena.
