Exome sequencing and CRISPR/Cas genome editing identify mutations of ZAK as a cause of limb defects in humans and mice

The CRISPR/Cas technology enables targeted genome editing and the rapid generation of transgenic animal models for the study of human genetic disorders. Here we describe an autosomal recessive human disease in two unrelated families characterized by a split-foot defect, nail abnormalities of the hands, and hearing loss, due to mutations disrupting the SAM domain of the protein kinase ZAK. ZAK is a member of the MAPKKK family with no known role in limb development. We show that Zak is expressed in the developing limbs and that a CRISPR/Cas-mediated knockout of the two Zak isoforms is embryonically lethal in mice. In contrast, a deletion of the SAM domain induces a complex hindlimb defect associated with down-regulation of Trp63, a known split-hand/split-foot malformation disease gene. Our results identify ZAK as a key player in mammalian limb patterning and demonstrate the rapid utility of CRISPR/Cas genome editing to assign causality to human mutations in the mouse in <10 wk.Split-hand/split-foot malformation (SHFM) is a limb anomaly characterized by median clefts with missing or malformed central rays (Elliott et al. 2005). SHFM is clinically and genetically heterogeneous and represents a paradigmatic genetic disorder displaying different modes of inheritance, variable expressivity, and incomplete penetrance (Birnbaum et al. 2012; Klopocki et al. 2012). Submicroscopic duplications at 10q24 and 17p13.3, TP63 mutations, and deletions of exonic enhancers in DYNC1I1 represent major SHFM-causing mechanisms (Ianakiev et al. 2000; de Mollerat et al. 2003; Birnbaum et al. 2012; Klopocki et al. 2012). Mutations in other genes, including WNT10B in an autosomal recessive form, have been reported. However, in up to two thirds of affected individuals, the causative mutation remains unknown (Ugur and Tolun 2008; Tayebi et al. 2014). One of the key challenges in rare Mendelian disorders is to identify additional disease alleles in unrelated families. CRISPR/Cas genome editing can now be used to create a large number of new alleles in the mouse within a few weeks by creating specific mutations and deletions in a gene of interest (Wang et al. 2013; Kraft et al. 2015). Here we report on the combination of whole-exome sequencing in patients with CRISPR/Cas genome editing in mice to identify and validate a novel disease-causing gene and to assign an unexpected role to the protein kinase ZAK in mammalian limb development.     

The association between second-trimester maternal serum alpha-fetoprotein in 14-22 weeks and adverse pregnancy outcome

Aim of this study is to determine the risk of adverse pregnancy outcome by maternal serum alpha-fetoprotein (MSAFP) level. We followed 295 pregnant women from MSAFP screening in the 14th to 22th week of gestation until the end of pregnancy and information on pregnancy outcome have been recorded in questionnaires. Of 295 pregnant women, 270 had term labor and 25 had preterm labor. The frequencies of pregnancy outcomes were as following: 3 (1.01%) stillbirths, 25 (8.47%) preterm labor, and 10 (3.4%) preterm rupture of membranous (PROM), 15 (5.1%) pre-eclampsia, 23 (7.8%) oligohydramnious, and 1 (0.33%) miscarriage. The mean of preterm labor was significantly associated with the higher level of MSAFP (P = 0.021). The mean was 55.1 ng/cc in preterm labor and 41.1 ng/cc in term labor. Also, second trimester MSAFP levels were higher in women with pre-eclampsia (P < 0.001). The significant association was found between higher level of MSAFP with oligohydramnious (P < 0.001) and low birth weight (P < 0.001). Pregnancies with an elevated MSAFP level are associated with adverse obstetric outcomes and need more prenatal care.     

Noncanonical protein kinase A activation by oligomerization of regulatory subunits as revealed by inherited Carney complex mutations

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function.

Protein kinase A (PKA) plays a central role in how cells respond to multiple G protein-coupled receptor (GPCR)-binding hormones, such as thyrotropin or the parathyroid hormone (1, 2). The physiological responses controlled by PKA range from metabolic regulation to cellular differentiation and proliferation (3, 4). PKA includes catalytic and regulatory subunits denoted here as C and R, respectively (4). Under resting conditions, C is inhibited by the R₂ dimer through the formation of a stable holoenzyme complex (R₂C₂). Upon cellular stimulation and activation of adenylyl cyclase by GPCRs, cAMP levels increase sufficiently for cAMP to bind the tandem cAMP-binding domains of R (CBD-A and B; Fig. 1) and release C, which in turn phosphorylates downstream substrates that control a wide array of signaling pathways (4–8).Open in a separate windowFig. 1.The PKA R1α system and disease-related mutations. (A) Domain organization of PKA R1α with non-haploinsufficient CNC and ACRDYS1 mutants within the tandem cAMP-binding domains (CBDs) indicated in green and red, respectively. The blue star in CBD-A denotes Ala-211, which is mutated to Asp in CNC and to Thr in ACRDYS1, and in CBD-B denotes Gly-287, which is mutated to Trp in CNC and to Glu in ACRDYS1. Further details are available in SI Appendix, Fig. S1A. (B) Structure of R1α CBDs in the cAMP-bound state [dark colors; PDB: 1RGS (26)] and C-subunit-bound state [light colors; PDB: 2QCS (5)]. CNC and ACRDYS1 mutation sites are highlighted with green and red spheres, while cAMP is shown with black sticks. The N3A motif (αN-helix, 3₁₀-loop, and αA helix) and phosphate binding cassette (PBC) are highlighted in red and yellow, respectively, while the rest of CBDs is in cyan. The Insets show details of the cAMP-binding sites adjacent to the A211 and G287 mutations, with selected hydrogen bonds and capping interactions highlighted by dashed lines and surfaces.Autosomal dominant inherited mutations in the 1α isoform of PKA R have been associated with type 1 acrodysostosis (ACRDYS1) or the Carney complex (CNC) (1, 9). ACRDYS1 is a severe skeletal dysplasia leading to defects in the development of facial, finger, and toe bones as well as dwarfism (1, 2). CNC refers to a multiple endocrine neoplasia often linked to a generalized predisposition for tumors, including pituitary and breast ductal adenoma, heart and breast myxomas, adrenocortical tumors, pancreatic and liver cancers, as well as skin tumors characterized by spotty pigmentation (9–12). ACRDYS1 arises from hormonal resistance caused by an impaired PKA R1α response to cAMP (1, 2). In fact, most ACRDYS1 PKA R1α mutations are found in the vicinity of the cAMP-binding site of CBD-B. Since CBD-B functions as a “gatekeeper” for binding of cAMP also to CBD-A, these ACRDYS1 mutants exhibit elevated activation constants, which explain the decrease sensitivity to cAMP and lower degree of C activation in ACRDYS1 vs. wild-type (wt) PKA (1, 13, 14).Unlike ACRDYS1, the CNC pathology reflects increased PKA activity arising from losses of the tumor-suppressor function of PKA R1α (11). Most CNC PKA R1α mutations incur nonsense-mediated mRNA decay (NMD), resulting in PKA R1α haploinsufficiency and overactivation of the PKA C kinase (15, 16). However, the most harmful CNC PKA R1α mutants often escape NMD and result in the expression of defective PKA R1α (16, 17). The majority of CNC PKA R1α mutations that are not subject to NMD cluster in CBD-A (Fig. 1A and SI Appendix, Fig. S1A) (15), which is essential for binding and inhibiting PKA C. Some of these CNC mutants, such as R144S and S145G, are located at the protomer–protomer interface of the R1α dimer and have been shown to perturb the allosteric response to cAMP. These mutants result in lowered Hill coefficients and activation constants, thus explaining the hypersensitivity to cAMP and the consequent PKA overactivation in these CNC mutations (18). However, for other CNC mutations, such as A211D and G287W (Fig. 1 and SI Appendix, Fig. S1A), the opposite effect is observed (13). The cAMP activation constants of A211D and G287W are significantly higher than those of wt PKA R, while no significant losses are observed in the ability to inhibit PKA C (13). The A211 and G287 CNC mutant sites are of particular interest because of their unique ability to switch from the CNC to the opposite ACRDYS1 phenotype simply by changing the side chains of these residues. Specifically, A211T and G287E are classified as ACRDYS1 mutations, suggesting that the 211 and 287 sites are critical for understanding PKA-linked pathologies. Taken together, these observations indicate that the molecular mechanisms underlying CNC PKA R1α mutations are currently not fully understood, especially in the context of the classical model of cAMP-dependent PKA activation.Here, we hypothesize that a viable mechanism to explain the CNC phenotype of the A211D and G287W PKA R1α mutations is the activation of PKA C through noncovalent oligomerization-induced losses of the PKA R inhibitory function. Upon partial denaturation, wt PKA R1α tends to aggregate (19). If a CNC mutant is more prone to unfold than wt, either because of reduced cAMP affinity or intrinsic destabilization of R1α (20), it is more likely to oligomerize into open-ended assemblies than wt (21). Bioinformatics analyses indicate that PKA R1α aggregates sequester and shield key loci of the inhibitory R:C interface (19), suggesting that PKA R1α aggregation reduces the inhibitory competency of PKA R, thus explaining the PKA C overactivation typical of CNC mutants. In order to test this hypothesis, here we comparatively analyze the structure, unfolding, aggregation, and inhibition profiles of wt PKA R1α and the CNC mutants A211D and G287W. We also extend our comparative analyses to the cognate ACRDYS1 mutations A211T and G287E, which, unlike the corresponding CNC mutants, result in PKA overinhibition. The ability to compare CNC vs. ACRDYS1 mutations of the same residue eliminates possible positional biases from the comparisons.Structural perturbations were evaluated by combining NMR and X-ray crystallography. Unfolding was investigated at progressive degrees of resolution (i.e., full-length, domain and residue-resolution) by combining urea unfolding monitored by intrinsic fluorescence, bioinformatics, and hydrogen/deuterium exchange (HDX) monitored by NMR. Aggregation was probed by size-exclusion chromatography (SEC), extrinsic fluorescence using the ThT and ANS fluorophores, NMR, dynamic light scattering (DLS), and transmission electron microscopy (TEM), while PKA C activation was gauged through luminescence-based kinase assays. Our results show that the A211D and G287W CNC mutations unfold the tandem CBDs of PKA R1α to a larger extent than wt and the cognate ACRDYS1 mutants, leading to correspondingly higher propensities to oligomerize into open-ended assemblies, which are incompetent to inhibit PKA C. Taken together, our data support the hypothesis that PKA R oligomerization sequesters R from C, thus defining a noncanonical, yet viable, mechanism for PKA C activation that was not previously appreciated but is relevant for CNC mutations.     

Clinical outcome and cost in patients with off-pump vs. on-pump coronary artery bypass surgery

General concept and major emphasis on off-pump coronary artery bypass surgery (OPCAB) is maintaining quality of care and patient safety while reducing cost and resource utilization. OPCAB probably avoids the potential complications of cardiopulmonary bypass. However its acceptance depends on clinical and economic outcome. The aim of this study is to compare clinical and economic outcome of off-pump and on pump coronary artery bypass surgery. This is a report of an analytic cross-sectional study on 304 patients underwent coronary artery bypass surgery that were randomized into conventional on pump and off-pump groups. Variables and costs were obtained for each group and these data were analyzed using parametric methods. There was no difference between the two groups with respect to perioperative and intraoperative patient's variables. OPCAB reduced the need for postoperative transfusion requirement (P<0.05) which was statistically significant and showed a trend towards reduction of morbidity although didn't reach statistical significance (P>0.05). There were no statistically significant differences in surgical re exploration and length of stay between the two groups. The mean cost for an on pump surgery was 8312000 ± 2859 Rials per patient that was significantly higher than an off-pump surgery. Based on the findings of this study, clinical outcome has no statistically significant difference between on pump and off-pump CABG but the costs are significantly higher in the on pump group.     

The effects of face-to-face education for student health ambassadors on the health-promoting lifestyle of adolescent female students: a randomized controlled trial

Journal of Public Health - Adolescence is a critical life stage with significant effects on lifestyle in later life. Promoting adolescents’ lifestyle requires the use of appropriate...