Neural tube defects (NTDs) are among the most common complex congenital malformations observed in newborns. family of birth defects. Neural tube defects (NTDs) are characterized by a failure of neural PF-04971729 tube closure during early embryonic development. The most frequent types of NTDs are spina bifida which are defects of low spinal closure below the level of T12 and anencephaly which results from incomplete closure of cranial neural tube. Failure of the neural folds to elevate results in the entire neural tube remaining open is referred to as craniorachischisis. The worldwide prevalence of NTDs is 0.5–1 per 1000 newborns with variations among different populations [1]. The etiology of NTDs is complex including both genetic and environment factors. In mice so far there are more than 300 genes were linked to NTDs [2]. However no causative mutations have been identified in humans to date. One possible reason is that there are very few large multigenerational families that could be used to identify causative PF-04971729 NTD genes using linkage mapping. Other obstacles to identifying NTD causative genes using mouse models is that most of these gene knockout models do not express an NTD phenotype as heterozygotes yet the homozygous embryos most often suffer from lethality. In thinking about the genetic basis of NTDs many investigators consider PF-04971729 the notion that multiple combined heterozygous variants in same gene same pathway genetic or physical interaction partner work together to produce the NTD phenotype in humans. These combined functional variants could be inherited or result from germline and/or somatic mutations. However it has been very difficult to directly test this hypothesis due to the limitations of our existing genome editing technologies. Recently the development of next generation sequencing (NGS) techniques [3] and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genetic editing technique [4] provide an excellent opportunity to test this hypothesis. Next generation sequencing (NGS) also called (high throughput) massive parallel sequencing including NGS-whole exome sequencing (NGS-WES) NGS-whole genome sequencing (NGS-WGS) and NGS-target enrichment sequencing. Compared with first generation (Sanger) sequencing the newer approaches can generate large amounts of sequencing data in a short time at a reasonably low cost. For example the human genome sequencing project took 13 years and cost over $3 COLL6 billion dollars. Using the latest NGS equipment (eg. Illumina HiSeq 4000) sequencing a whole human genome can be completed in a week at a cost approaching $1 0 Since more than 300 genes have been reported to be involved in murine neural tube closure it is highly likely that even more genes that contribute to the expression of human NTDs will be discovered. We believe that one approach to identifying new candidate NTDs genes in humans is to appropriate the NGS-WES and NGS-WGS methodologies/strategies that are currently being successfully used for identifying risk PF-04971729 genes in autism spectrum disorder [5] a multifactorial disease similar to NTDs. For human NTDs it is also assumed that multiple related (eg. in a pathway) functional variants combined can be the underlying genetic etiology of some cases. Thus far millions of genetic variants have been identified; therefore the potential combinations PF-04971729 of multiple variants could be in the billions or trillions. To test whether combined rare variants in a pathway are human NTD genetic risk factors scientists need to sequence thousands of NTD cases for all the known candidate PF-04971729 pathway genes. The NGS-target enrichment sequencing technique is perfect for this purpose. Currently there are three types of target capture/enrichment methods: multiple- PCR based method capture hybrid capture (on-array or in-solution) and molecular inversion probes (MIP) capture [6]. Each method has its advantages and disadvantages. MIP has been successfully used for autism risk genes validation in a large sample size due to its low cost ease of use and template saving advantages [7]. We believe that this technique has the potential successfully enhance our understanding of NTDs risk genes/pathways by performing validation studies on large NTD cohorts. The functional characterization of identified variants is important for.