Knockdown of Crispld2 in zebrafish identifies a novel network for nonsyndromic cleft lip with or without cleft palate candidate genes.

Orofacial development is a multifaceted process involving tightly regulated genetic signaling networks, that when perturbed, lead to orofacial abnormalities including cleft lip and/or cleft palate. We and others have shown an association between the cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2) gene and nonsyndromic cleft lip with or without cleft palate (NSCLP). Further, we demonstrated that knockdown of Crispld2 in zebrafish alters neural crest cell migration patterns resulting in abnormal jaw and palate development. In this study, we performed RNA profiling in zebrafish embryos and identified 249 differentially expressed genes following knockdown of Crispld2. In silico pathway analysis identified a network of seven genes previously implicated in orofacial development for which differential expression was validated in three of the seven genes (CASP8, FOS, and MMP2). Single nucleotide variant (SNV) genotyping of these three genes revealed significant associations between NSCLP and FOS/rs1046117 (GRCh38 chr14:g.75746690 T > C, p = 0.0005) in our nonHispanic white (NHW) families and MMP2/rs243836 (GRCh38 chr16:g.55534236 G > A; p = 0.002) in our Hispanic families. Nominal association was found between NSCLP and CASP8/rs3769825 (GRCh38 chr2:g.202111380 C > A; p < 0.007). Overtransmission of MMP2 haplotypes were identified in the Hispanic families (p < 0.002). Significant gene-gene interactions were identified for FOS-MMP2 in the NHW families and for CASP8-FOS in the NHW simplex family subgroup (p < 0.004). Additional in silico analysis revealed a novel gene regulatory network including five of these newly identified and 23 previously reported NSCLP genes. Our results demonstrate that animal models of orofacial clefting can be powerful tools to identify novel candidate genes and gene regulatory networks underlying NSCLP.

Accuracy and sampling error of two age estimation techniques using rib histomorphometry on a modern sample.

Most age estimation methods are proven problematic when applied in highly fragmented skeletal remains. Rib histomorphometry is advantageous in such cases; yet it is vital to test and revise existing techniques particularly when used in legal settings (Crowder and Rosella, 2007). This study tested Stout & Paine (1992) and Stout et al. (1994) histological age estimation methods on a Modern Greek sample using different sampling sites. Six left 4th ribs of known age and sex were selected from a modern skeletal collection. Each rib was cut into three equal segments. Two thin sections were acquired from each segment. A total of 36 thin sections were prepared and analysed. Four variables (cortical area, intact and fragmented osteon density and osteon population density) were calculated for each section and age was estimated according to Stout & Paine (1992) and Stout et al. (1994). The results showed that both methods produced a systemic underestimation of the individuals (to a maximum of 43 years) although a general improvement in accuracy levels was observed when applying the Stout et al. (1994) formula. There is an increase of error rates with increasing age with the oldest individual showing extreme differences between real age and estimated age. Comparison of the different sampling sites showed small differences between the estimated ages suggesting that any fragment of the rib could be used without introducing significant error. Yet, a larger sample should be used to confirm these results.