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Notes for Seckel syndrome presentation and ATR DNA repair pathway seminar
Marjolaine Willems en M2 chez Valerie, chef de clinique après – séminaire de lundi 6 juillet 2009
Voie ATR – syndrome de Seckel. PHRC 2009.
Repair mechanism of DNA damage eg stalled replication forks, UV, etc. ATR more in S-phase during stagnation of replication fork: ATRIP+RAD17+RFC2-5, recruit TOPBP1, kinase activity, but ATM for other types of damage.
CHK1 blocks CDC25A and therefore its activation of CDK1 and Cyclin B, stop cell cycle to repair DNA. ATR acts in the Fanconi pathway.
700 substrates phosphorylated by ATM or ATR. Most ATR targets are also ATM targets, ionizing radiation damage for this test. Seckel syndrome mutations in ATR (?) – inhibitor of DNA polymerase can find fragile sites on chromosome b/c most difficult to replicate.
Detect ATR pathway defects via lack of degradation of CDC25A (regulating entry into mitosis) even after UV treatment, or lack of checkpoint G2/M . Cf. Alderton et al., 2004 for other pathway anomalies in Seckel syndrome even w/o ATR mutations themselves.
Microcephaly vera = -3 to -13SD head with -3SD body growth, lack of neuronal migration. But no pb immunitaire or more cancer or sensitivity to ionizing radiation. MCPH1 is in the ATR pathway.
Centrosomal proteins interacting with gamma-tubulin: microcephalin, pericentrin Nat Genet 2009 Griffith et al., and Rauch et al., 2008 Science. Phenotypes a bit different: functional and morph problems like ATR deficiency, but Rauch et al., anomalies of mitoses in syndrome “MOPH-II” – greater growth defect, major radiological defects, cutis marmorata vascular dysplalis, hyper- and hypopigmented skin lesions, and some CV anomalies and vascular brain problems (aneurysm), but no mental retardation and evolving facial dysmorphy rather than the bird-like face in Seckel syndrome (large, beak-like nose, large incisors). Pb in chromosomal segregation like in centrosomal problems. ATR pathway is less cellularity as an effect, but link between them is now clearer: Tibelius et al., 2009 – MCPH1 recruits pericentrin to centrosome.
Sequencing of PCNT in compatible patients with Seckel or MOPD-II, with 16 families, only 3 non consanguineous, and 8 MOPD-II, 3 non consanguineous. Found 13 mutations all over pericentrin. Arnold asked for what were the diagnostic criteria – Marjolaine answers “diagnosis by a geneticist” – Size >1m10 adult, MR, no severe skeletal defects. Looking back at the pericentrin-mutated patients, facial dysmorphy is present and similar in both groups. Large forehead but not necessarily “fuyant”, nose = hypoplasia nasal alae. Growth retardation and mental retardation or psychomotor is progressive. Bone pbs – variable and less severe in Seckel diagnosed patients – short top of femur, but pelvic pbs in both syndromes. “Coxa vara”. Similar lower leg profiles. Clinodactyly, delayed bone growth. Some endocrine problems, obesity in a few, others – microdontia as opposed to the large incisors; ORL problems sometimes as well. Skin hyperpigmentation/hypo – confusion between two syndromes, but these should orient to MOPD-II and pericentrin.
Two patients have liver problems going from cholestasis, duct proliferation to cirrhosis. Rauch and Valerie both think that PCNT accounts for all MOPD-II with genetic homogeneity. However, phenotypically variable in particular for height, and it’s evolving over time. Seckel is both clinically and genetically heterogeneous.
Asked for PHRC to study of all similar Seckel/MOPD-II patients. Will look for additional genes in Seckel as they progress. (cf. e-mail to Valerie in March 2008).
ATR is hypomorph mutations – acquired mutations in some cancers, but complete KO is lethal.
A few Seckel cases have osteosarcoma and/or leukemia. KO of pericentrin, asked Alexandra? Not known.
Obesity and liver problems – and pancreatic insulinoresistance cf cohort J Hall. Pigmentation problems are evolutive as well, not congenital.
Chr 18 locus paper maybe in fact a MOPD-II patient considering clinical signs – pericentrin perhaps still compatible. Thinks chr 14 locus is badly positioned. What fraction of Seckel not linked to SKL1 or -4? (ATR/PCTN) checked for other effectors of ATR – more than half of them are not – and have not checked more than a few candidate genes. Many of these are still consanguineous. What other genes will test? MW says better to get more families, better clinical homogeneity rather than candidate gene approach.
Tania asks if placental problems. Not known.
Phenotype-genotype correlation for the two with liver cholestasis, asked Yves? Not sure.
Small group meeting on Sophie's return after maternity leave:
Jean-Paul in Alexis’ seminar suggested remove 2ndary neurulation zone from embryo – does this provoke a teratoma?
Injection – if environmental factor, inject at the caudal region. Localization on slides Nanog/Oct3-4 nucleolar vs. Sox9/2 nuclear. Caudal mesenchyme in regular embryos.
Fresh teratomas – one older girl 12 y – first culture. This one injected after dissoc and no teratomas growing, two babies of which one dead, same day. Both in culture currently. St Etienne verolé. 2 other injections 5x10^6 cells. After 2 weeks, can find the mass.
Fibroblasts p5 – Nanog, Oct3/4, Sox2/Sox9. also all positive. Primordial germ cells stay in or localize to caudal pole?
RT sur tissue congele pour confirmer immunomarquage – Isabelle Andre-Schmutz, immunofluo
Proposed SA Bioscience puce Stemness suggested by Isabelle, cf primers – go directly to RT-PCR perhaps.
Find better control.
Late teratoma: all similar.
Early teratomas: look in culture.
SC profile: ES + cells + tumors.
2 mice – 100K cells, 3 weeks, 2 more mice 5x10^6 3 weeks later – this teratoma is pretty quiescent, not growing and maybe no dividing cells. Is this the negative control?
Sabine – retour le 20 juillet, negocier prix des souris de retour.
C12/15/ 17-18 ; voir R1060 en particulier ; voir si on ajoute + tard ? Tumors of tumorotheque – tardifs, Currarino (uniquement endopelvien – known for being mutated) ? en plus des neonataux.
Yacine de Evry – ferait le FACS qu’Isabelle n’a pas pu faire faute de place. Avec quels marqueurs ?
Christelle – review since last March
ISL1 : consensus sequences not in all peaks ; Gregory Nuel – biostats – script BioPerl to look for ISL1/GATA motifs.
35 peaks – remove ISL1- peaks – Sophie asks how many to begin with? I thought a hundred or so, but Chris says fewer. 15 with GATA of which BMP2 is expressed, others not. Most of these 15 have both GATA and ISL1, only a few with ISL1 itself. NKX2-5 and MEF2C regulated by both ISL1/GATA(4).
C15-16 semilunar valves form. Septation of 2 ventricles.
Sites ISL1 prospective to sequence in CARREG patients. M1 Santé Gregoire. TAC, TGV, VDDI.