We identified laterality defects as the leading clinical feature in individuals with loss of function of DNAH9 (Table 1 and Figure 2A). The ventral embryonic node is the left-right organizing center in mammals. Coordinated beating of nodal monocilia plays a crucial role for establishment of correct laterality by establishing a leftward flow essential for activation of the left-right signaling cascade (Figure 2B). We therefore hypothesized that DNAH9 could be involved in node monocilia function and proceeded to perform in situ hybridization analyses of wild-type mouse embryos (0 somite stage) as previously described.39 The exact developmental stage of mouse embryos was determined by morphological landmarks or the number of somites.40 A 529 bp fragment of mouse Dnah9 (GenBank: NM_001099633.1) cDNA was amplified from wild-type mouse fallopian tube cDNA and subsequently ligated into pCRII-TOPO vector by TOPO cloning reaction (Invitrogen, Thermo Fisher Scientific). After hybridization with the antisense probe, we detected a strong signal with the antisense probe located to pit cells of the node, but not in crown cells of the node (Figure 2C). In contrast, no signal was observed using the sense probe as a negative control (Figure 2C). These findings are consistent with DNAH9 involvement in the function of motile node monocilia and that loss of DNAH9 causes an impairment of nodal ciliary beating and subsequently altered nodal flow, to result in laterality defects. Although all identified individuals with loss-of-function mutations in DNAH9 displayed laterality defects, we expect that loss of function of DNAH9 leads to randomization of the left-right body asymmetry, resembling findings observed in individuals carrying recessive DNAH5,28 DNAH11,31, 32 DNAI1,29 and DNAI230 mutations. However, identification of individuals with loss-of-function mutations in DNAH9 without laterality defects is difficult due to the fact that, in contrast to PCD caused by mutations in other genes, respiratory problems are less prominent and may not lead to thorough medical diagnostic work-up.