User:Etchevers/Notebook/Conference notes/2009/09/14: Difference between revisions
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Group in Houston but also detachment in Bilbao who welcomed Rick over. Collaborates with everyone here practically. | Group in Houston but also detachment in Bilbao who welcomed Rick over. Collaborates with everyone here practically. | ||
''Questions:'' | |||
Heart defects in KO mice? Anticipates yes but had not yet looked. Find a good cohort to study relationship between heart defects and NTDs? | |||
Careful to never say folate deficiency - compliments from RJ Berry. These are folate insufficiency effects. | |||
Type I and II diabetes lumped together one immune the other not - but autoantibody response in pregnant women present before pregnancy and only see the effect on susceptible target? Not proposing that response was correlated | |||
Irene: Placenta really effect on NT closure? not relevant for exencephaly? (too late for that) Vascularization... Andy wonders if toxicity on yolk sac. "Yeah, that's what I meant." | |||
*'''[[User:Etchevers|Heather]] 11:10, 14 September 2009 (EDT)''': | *'''[[User:Etchevers|Heather]] 11:10, 14 September 2009 (EDT)''': | ||
Revision as of 08:15, 14 September 2009
 Conference and seminar notes
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Third day of 6th Intl Neural Tube Defects conferenceMegan Kooistra on Arhgap2 and Cecr2 (McDermid group)Balb/c different susceptibility to NTD on cecr2 hypomorph allele. Whole genome linkage analysis for modifiers suggestive on X, present on Chr19. Dominant or semi- within FBV/N strain. Human susceptibility? Marcy Speer study 2005 with screen chr 7 (largest, from one family) and chr10 (q25.3) - syntenic to mouse chr 19D2. Gene expn analysis using microarray and embryos E8.5 cranial NT closure. Compared wt between two strains and mutants on same BGs. Other genes lipid metabolism also foxd4. Most differential = Arhgap19. One paper showing fetal expn by RT-PCR. Rho GTPase protein acts as a negative regulator of GTPases. Fits into PCP by potential interaction with RhoA to lead to actin cytoskeleton remodeling. qRT-PCR confirm change in two strains (Balb/c very reduced re: FVB/N, C57 and 129S2), then check for sequence changes, and exencephaly penetrance when move cecr2 genetrap onto 129S2 and C57Bl/6 strains. 35 SNPs and 2 insertions, as well as 1 deletion of 132bp in 3' UTR. 3 synonmymous mutations and one nonsense mutation. T insertion after a stretch, end of exon 6 leads to fs and stop codon. Mutation not present in other strains. NMD mediating degradation? If residual mRNA translated, predicted truncation removes everything after the RhoGAP domain, perhaps affecting folding. Bred mice onto different backgrounds to see if change penetrance of exencephalic Cecr2 phenotype. Encouraging preliminary data correlating - in the 129S2 penetrance 43% whereas on BALB/c background penetrance >70%. Screening other strains now, will carry out ISH. Developing new mouse line gene trap fusion Arhgap19-beta-gal. Questions: Muriel - any defects caused by Arhgap19 on cilia, subtle effect on the BALB/c strain? not looked yet. Any back-rescue to increase penetrance by adding back in? Need superovulation but the BALB/c strain doesn't superovulate well. Examining actin cytoskeleton to see if changes in genetrap mutant. Irene asks if have checked on Vangl mice to backcross? Rick comment thinks he has the Arhgap19 gene trap with B-gal reporter and offers it. Cool! This is what conferences should be all about!
Christine Dawe on Cecr2 phenotype (McDermid group)PCP pathway review. She did it all for me! And everyone has their own modification from Kibar 2007! NTDs (craniorachischisis), open eyelids during development, and inner ear defects. KO of Fz3/Fz6 has the same phenotype. Cecr2 chromatin modifier protein expressed many places including limb, neural tube, nose or whisker follicles? ear. Mouse has exencephaly. BBS4 mutant has exencephaly, open eyelids and inner ear pbs. Interaction with Vangl protein in zebrafish. Cecr2 other phenotypes? Gorgeous stain - + in crania and - in caudal NT, and in hindgut - ectopically? Measured inner ear defects as basal more mature than apical eclls of the cochlea, so misalignment more pronounced basally than apically (predicted) - imaged along axis to see inner ear cells. Size of cochlea is already macroscopically different between wt and mutants. (My thoughts: eyelid closure prevention leads to any eye problems? further development of retinal pigment epithelia? Jun and target EGFR reduced in skin keratincytes leads to open eyelids at birth in mutants. Painstaking analysis of cells and their orientation to demonstrate misalignment in Cecr -/-, +/- and +/+ mice. Chi-squared with Bonferroni downward adjustment to compare the measurements. at 50% maturation, wt to mutant comparison of misaligned cells statistically different. The most affected as expected from spatiotemporal maturation. Similar to Cecr1, Ptk1, Fz7 phenotypes. (Not sure I heard gene names right.) Want to cross htz to Vangl htz to see if worsen phenotypes. Question: BBS4 as example not considered PCP gene - link between ciliogenesis and PCP but will extend to other members involved in ciliogenesis? thinks it would be valuable. How will study chromatin remodeling? Answer where Cecr2 is remodeling - using ChIP-Seq? Expression of other PCP genes in mutants to see if affected? Not by in situs. Simon: Correction of orientation of cells over time? Yes, they do. Wnt5a and Vangl expressed in cochlear floor - Cecr2 not directly within cells. Prospective regulation of a signal eg as a Wnt but also not close spatially?
Nicolas Fairbridge on cecr2 phenotype and metanephros (22q11.2) (McDermid group)Kidney defects - loss of glomeruli, duplex kidney disorder, tips of fingers in limbs, also meso/pronephros (segmented). Kidney defects only show up on the FVB/N strain - will look if Arhgap19 involved. Only expressed in outer edge of cortex where glomeruli are. Maturation from least (cortex) to most (medullar) differentiated glomeruli. Convergent-extension defects in collecting ducts. Not in the tubule, but in the outer mesenchyme aggregating into early glomeruli and in S-bodies as they differente into definitive glomeruli and connect to collecting duct. Then turned off. 40% "duplex" kidney - two separate ureters or bifurcation, leads to fused kidney. Is this similar to horseshoe kidney? ALso kidneys are smaller, so developmental progression delayed. Size related to fewer glomeruli. Adult FVB/N Cecer2m/m get L/R size asymmetry, loss of one kidney (12/5%), or hydronephrosis unilaterally. Double ureter increases its risk. PCP: loss of Fat4 leads to cystic and smaller kidneys when double KO with Fjx. Worsening of cysts, which are not observed in Cecr2. Polycystic kidneys in PCP defects (eg Wnt9b) , rather than from the glomerular side. Same microarray assay as above. Cecr2 itself down in mutants, bc mutant is only hypomorph (splice around). Zfp9, Csn3, Alx1, Rgn, Cd36, Hey2, Hoxd1, Tshz3, Wnt5b, Lix1, Frzb, Rab2, Procr, Perp are downregulated when comparing Balb/c and FV. Other 19 other PCP-related genes significant but more minor expression changes. Alx1/Cart1 strain-dependent exencephaly, TF needed for forebrain "mesenchyme" cf Gray et al., 2004. Cecr2 in the retina and ciliary ganglion, forebrain, nose - but Alx1 is not obviously connected to PCP, more so to beta-catenin (canonical pathway). Exon 1 deletion of Cecr2 get 96% penetrance of exencephaly. No limb defects ever observed. Questions: wonder if short list b/c whole embryo and why not dissect affected tissues? Underway. I asked if anyone looked at the histology of the liver for potential effect on biliary ducts - answer, no. Claudia - why expn based on bgal gene trap? ISH never worked well for Cecr2, currently redoing. Andy - Alx1 also a chromatin remodelling agent but not overlapping - how explain? Answer - might Wnt5b be an intermediary, upstream of Alx1?
Philip Lupo on NBDPS studySubdividing anencephaly and SB and use statistics to estimate effects for individual parameters eg. infant sex, maternal BMI, folate supplementation. 613 spina bifida, 282 anencephaly, 5894 controls. Previously excluded maternal anticonvulsants and diabetes to have more homogeneous case group. Do risk factors have different effects cf the particular NTD? Lovely statistical strategy but I can't copy it down, but it seemed justifiable, as do most statistical approaches to me. I'm not capable of critical thought wrt this. Better statistical power if you split, or if you lump? That is the question. Folic acid use, Hispanic ethnicity somewhat similar effect on anencephaly. Multivariable context to vary some factors and constrain others, to build most parsimonious model to get at variability in disease phenotype. Helpful when increasing sample sizes. Questions: For likelihood ration, test model 1 vs model 2? yes, based on estimates for each. Question - does progression offer advantage over stratified models to improve power? Answer - not necessarily, though believed should be b/c more case groups - but comparative models yield similar results. To what extent does model assume etiologies might be different? Have been lumped historically because of recurrence - noticing either anencephaly or SB if proband has one or the other - contrast with CL/P. Assume same mechanism. Is Craniorachischisis on same continuum? Does model deal with this assumption? Answer - no assumption on etiologies of risk factors. Some studies say homogenous, yet maternal high BMI is only risk for SB. This model allows not assumptions by forcing lumping. Dr Fraser - careful of splitting. Answer - will consider but different proportions of exposure in case and control groups, so comparisons are not just between control and anen, control and SB but also anen and SB. Evadnie: Comparable sample sizes effect? Answer not yet known but think that even if sample sizes not necessarily the same size, but need to be large enough. She proposes a method afterward to randomly select and (fill out?) the patient groups. Bill: Viral cause possible, perhpas "threw baby out with bathwater" by splitting. Considers approach wrong - uniform cause at a deeper level. Answer - not agreed that uniform effects for given risk factors. Might obscure associations.
Pedro Rocha on Med12 (Schrewe H laboratory)More transcriptional regulation. Mediator is a 2 MDa, 30 subunit complex! Some generally required - interactions with RNA pol, others are modulable cf TF recruiting the complex. Some known interactions - NFKb with Med17; El;1 and E1A with Med23; PPARs and other nuclear receptors, Med1. Smad 2/3 with Med15. Sox9, Nanog, B-cat, Gli3 with Med12 - transcriptional activation/repression switch. Med12 X linked MR disorders due to missense mutations - Strabismus, hypotonia, partial/complete CC agenesis. Hypomorphs die at E10.5 from heart malformation. All embryos have pbs in closing NT - DLHP not effective, though folds elevate to start along MHP - wonder if pbs b/c less head mesenchyme. 10% no closure, some close, but NT kinked, in posterior hindbrain. Posterior NP not elevated, stays undulating. Body axis not elongating properly. T and Tbx6 show defect presomitic mesoderm - much reduced. Somites are reduced in size and disorganized, caudally hardly formed. Expn of Pax3 0 reduced, irregular somites - subsequent effect on neural plate. Refer in my talk on affecting outside tissues. NP undulation due to somitic problems. No BA formed at all, and Crabp1 marker expressed but cells stay in the neural folds. Increased cell death where usually NCC migrate out, also in neural folds. Med12 needed for specific functions of Mediator complex. Notochord is well-formed and expresses Shh. But axis elongation and neurulation and heart formation affected. Nice explanation of X-linked genetics for flozed gene crossed to universal expressed Cre female mice. Female progeny will be mosaic with excision and others will express the other chromosome. Get gradient in severity of phenotype. Some had open NT only. Others exem amd SB or curly tail, others complete craniorachischisis. Reminds me a lot of the phenotype of the tri 8 human we observed. Beta-catenin effector of Wnt signalling, shown to bind Med12, some phenotypes reminiscent. Treat TOP-flash reporter to see if cells respond to Wnt signalling - Med12-hypo reduced response re to Med12-flozed (but not excised) but standard same response to Gal4-VP16 control. Target = dkk1, in the hypomorphs the caudal end expn is completely abrogated, also a foregut expression (apparently?) above the heart. Not actually expressed in the heart. Questions: Andy: what predict if combine mesoderm or neural plate (Wnt1-Cre probably too late) - nearly no head, but no NTD. From Nick's talk, Sox1-Cre may be helpful. Brachyury-Cre leads to exactly same phenotype. Neural folds open caudally. Helga - random X inactivation. But in humans, skewed. Does that exist in mice? Hypoxic mice reduce (?) Pax3 defects. Problems of heart formation and oxygenation of embryo for NTD? Yes but does not explain T-Cre crosses with no heart phenotype, problem in neural tube closure.
Michelle O'Byrne on an association study of MTHFR and other folate genes (KS AU group)SNPs and contribution to SB meningomyelocoele. 15 candidate genes in pathway, using 208 unrelated simplex families. >77 SNPs.
SNP selection on prediction for biological function, submitted to applied biosystems to choose 48 particular SNPs in SNPlex genotyping system. Minor allele frequencies comparing Mexican-Americans cf other Caucasians show more, consistently in former, in certain SNPs in 6 folate metabolic pathway genes. Family based TDT method, larger sample size. 60% Mexican-Americans. Platform is a limitation, less than 90% call rate. Recently published, cf. Hope Northrup and Carla Martinez, in press. Question - were positive SNPs near cut-off for call rate? Do still remaining concerns about some of the SNPs used because not 95%? will go back for the few, but these they are confident. Would revalidate using other platform. Subgroup analyses? Used TDT on combined group. Parent-of-origin effect? Not examined.
Break! Rick Finnell on maternal immune factors and NTDsStarts out with this delightful Dilbert cartoon on front. <html> <a href="http://dilbert.com/strips/comic/1999-03-04/" title="Dilbert.com"><img src="http://dilbert.com/dyn/str_strip/000000000/00000000/0000000/000000/00000/7000/000/7016/7016.strip.gif" border="0" alt="Dilbert.com" /></a></html> Maternal lifestyle choices and genetic factors - remind that mother = environment from POV of developing embryo/foetus. Prevalence estimates of impaired glucose tolerance - actually worse in other countries (eg Russia) than in US, but obesity is an inflammatory disease as well, here, middle east and N Africa up to 45% women are overweight. US data not shown. ABout 30% in US ten years ago, spioking to 45% in non-hispanic black population. Other risk factors - cooking generated PAHs, arsenic and heavy metals in coal, nutritional deficiencies. Enviromental polluants - arsenic, fine particulate air pollution (< 2.5 um), lead, cadmium - induction of autoimmune disease. Associated also with regions with high prevalence NTDs. Antiphospholipid antibodies bind placenta - C5a binds C5aR on neutrophils, upreg expn TF, interacts with PAR2 to activate neurophil, release of NOS. Steven Whitehead showing lots of immune responses modulated by folate status, impinge on NT closure. Cf. Rothenberg, Quadros, Sequeira, Mallow - NEJM 2004 to 2009 not quite clear if autoantibodies or not? (gentle ribbing of controversy). Teratogens. Hypothesis: FR autoantibodies prevent uptake of folate into fetal tissues, may lead to placental inflammation compromising normal development. Thiolactone = homocysteine, reacts with lysine residues of proteins, leading to homocysteinylation (Hcy) which creates neoantigens, resulting in production of autoantibodies. FRs have lots of lysines and also may lead to dysfunction. C5a receptor knockout mouse analysis. Drug developed PMX53 blocks the C5aRs specifically. E8.5 to 10.5 see C9, MCP1 and C5a and its receptor. within the neural folds as well as for Ca in the lip of Sessel's pouch. Add in Hcy to human monocyte receptor, get induction of C5a. Folate rich diet, WT BG or C5aR KO mutants, folate-depleted WT BG nearly no NTDs, but on C5aR get 55%, or WT with the PMX53 drug also get near 40% NTDs. Anophthalmia, microcephaly, clefts, gastroschisis. Not just deprivation of folic acid that affects development - environmental factors likely to be involved in other direct ways in causing NTDs. Maternal autoantibodies to FR may modulate NTD risk, and induction of inflammatory cytokines may compromise placental vascularization, impacting normal embryogenesis. Group in Houston but also detachment in Bilbao who welcomed Rick over. Collaborates with everyone here practically. Questions: Heart defects in KO mice? Anticipates yes but had not yet looked. Find a good cohort to study relationship between heart defects and NTDs? Careful to never say folate deficiency - compliments from RJ Berry. These are folate insufficiency effects. Type I and II diabetes lumped together one immune the other not - but autoantibody response in pregnant women present before pregnancy and only see the effect on susceptible target? Not proposing that response was correlated Irene: Placenta really effect on NT closure? not relevant for exencephaly? (too late for that) Vascularization... Andy wonders if toxicity on yolk sac. "Yeah, that's what I meant."
Bermans Iskandar on folates and repair of the CNSSteve Whitehead on folates and MCP-1My talk which is available upon requestRobert Cabrera on autoantibodies to folate receptor alpha in Norwegian cohortLeah Burke on genetic counseling for NTDs, including Melissa Baker and Karen Lawliss, parents, and Denise Boyun, genetic counselor | |
