What are NTDs
Neural tube defects are diseases that stem from the failiure of the neural tube to close.
- They are the largest cause of still borns and do carry some genetic risk, with mothers previous having this being more likely to have another.
- Either failing to ever close, or incompletely closing, or failiure to sufficiently close and re-opening following primary nerulation.
- In many cases this results in death (Anencephaly (40%), craniorachischisisis (10%)) or severe deficits (Encephalocele (skull defects) (10%) and open spina bifida(40%))
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-Issues can aslo occur with secondary neurlation resulting in tethered defects, spinal cord is tethered to muscle and so breathing pulls on it and causes damge (surgery fixes). or Closed spinal dysraphism, closed over lesion called lipoma as filled with fatty tissue.
- The is Ultrasound screening avilable to diagnose this whihc has reduced occurence due to increased terminations.
There is prevenatative work being put into place to reduce the risk of these involving folate, inotiol and vitamin b12.
What are the main forms of NTD (4)
Anencephaly- This results from the failiure of the neural tube to close between closure site 1 and the anterial neuropore (closure site 2).
- This results in the rowth of the brain outside the skull in the amniotic sack early on (EXENCEPHALY)
- later on in preganancy when the kidneys begin to wrok the amniotic fluid is filled with urine and waste and thus becomes extremely toxic resulting in the degenration of the brain.
- This always results in still births.
- This accounts for 40% of cases
Open spina bifida- This resutls from a failiure to close at the caudal/posterior neuropore (Closure site 3)
- This results in the exposure of the spinal cord.
- The lesion can be quickly closed in surgery but the downstream nerved remain degenrate.
- Children often suffer long term motor and bowel deficits.
- This accounts for 40% of cases.
Craniorachischisisis- This results from the failiure of the spinal cord to ever close from closure 1 (hind brain)
- This resutls in the exposure of the brain and spine both being degenerate.
- This results in death and still born children.
- This aaacounts for 10% of cases.
Encephalocele
- This results from skull deficits.
- The skull fdoes not fuse properly leaves gaps through which the brain can grow.
- This leads to issues like hydrocephalus in babies and issues like ataxia and cognitive defects are common.
outline 2 types of neurulation
Primary neurulation.
- This is the main form of neurlation involving the conversion of the neuronal late into the neural tube and thus the shift from a apical-basal polarity to a lateral-medial polarity.
- The neural plate undergoes a medio-lateral convergence towards the dorsal midline, These raised arms are called the neural folds.
- The depressed region between the 2 neural folds is the nerual groove.
- This occurs via convergent extension, this involves the central convergence of cells to force a forward potursion.
- Convergent extension is meadited by WNT and signalling in the Polar cell polaratiy (PCP) pathway, hence their assocaition with NTDS.
- Th bending of the neural folds occurs at 2 hinge sites fromed by the notochord signalling and the action of NOGGIN inhibibtiing BMP.
- The first hinge site in the medain hinge at the base of the neural plate, The 2 lateral hinges find by the convergence of actomyosin filaments to induce buckling of the endothelium to cause bending
- The 2 neural folds will meet at the dorsal midline and fuse. This requires the potrusion of finger like projections (filopodia and lamelipodia) and interdigitation for fusion.
- This requires the action of membrane ruffles, These are formed by reorganisation of the actin skeleton via the action of RAC1 as shown by ROLO et al in KO there were no lamelipodia or filopodia and no fusion.
- This fusion and closure occurs at closure site one and extends bidriectionally t closure 2 and 3. This is commonly behind as the body is growing so it as to catch up.
Secondary nerulation- folowing the caudal neuropore is the tail bud developed in the foetus. Secondary neurlation confers the morphogenic change of this into the most cauda end of the neural tube following primary neurualtion.
- This stems from a solif rod of cells, and te cavity is formed via Canalisation.
What are closure 1, 2, an 3 (NTD)
Closure 1- This at the hind brain, closure from here occurs bidriectional towards the caudal and rostral neuropore.
Closure2- This is the anterior nerupor and failiure to close here is associated with anencephaly.
Closure 3- this is the posterior/caudal neurpore failiure to close here is associated with open spina bifida.
What underpin re-opening of the tube?
This is tought to be mediated by excessive cell proliferation.
- This is demonstrated by the ASSP2 KO mce. This is a tumour supressor that interacts with P53.
- The theory was that this caused proliferation to burst through the neral tube, causes the abnormal location of openings seen.
- Support for this comes via the temporally graded severity. Th condition worstened with gestational age of onset, suggesting that it is re-opening and thus worst if occuring at later stages.
what are the core targets of gentic mutations implicated in NTD?
- PCP 25 mutations identified
- glucose metabolism 10 indetified
- 45 identified for folate metabolism.
Discuss the role of environment in NTD.
There is a wide range of environemntal facotrs that have been shown to increase the risk of NTD.
basic
- ALCOHOL
-CAFFEINE
COMPLEX
- Valproate- the anti-convulsant was shown to increase the risk of NTD by 10 fold if given within the first trimester of pregancy. Studies have postulated that this is a result of its role in HDAC inhibition and thus the dirsuption of epigeneitc regualtion of expression in develkopemnt preventing NTC.
- Folate, Inostiol and vitamin B12 defficiencies increase risk hence why they are targetted in preventative treamtent.
- Fumonism- This is produced by a fungus that contamnated flour and was responisile for an outbreak of NTDs in mexico as it was used to make tortillas. This acted as a folate antagonist.
Discuss content and research pertaining to the prevention of NTD. first state other treaments
palliative
- surgery in open spina bifids
preventative diagnosis
- ultrasound diagnosis 85% aborted.
preventative=
Pregnavite FORTE F
- This is folate supplementation with viamin solution (riboflavin, vit A and D e.t.c)
- Wald ea al carried out a trial in which they took mothers with previous issues (1/25 risk) and placed then into groups
- They found those not on it has a 3.5% and those on it had a 1% (21/602 vs 6/593)
- issue with this is it is a voluntary preventative in the UK otherwsie is only recmended once they have their first meeting. You are not told to go for this until 2 weeks after missing a period.
- ovulation occurs around 2 weeks after the period and hence if trying for a child this is when fertilisation is most likely to occur. 2 weeks later if the period is missed they would wait 2 weeks until thye appear pregnant. This means that they would have been 4 weeks without folate supplement and this is when neurulation usually occurs and so its too late.
- The advic is to go on folate if trying. 400 micro grams is normal, 4-5 miligrams is a previous issue.
FOOD FORTIFICATION
- Dues to the issues countries like the US use folate in general foods like bread to supplement the diet and prvent these issus.
- 100 micro grams of folic acid is added in many cases.
- these countries have significant decreases in occurence.
- A case study for this is NEWFOUNDLAND where the average rates where above avergae and are now at average.
INOSITOL supplementation
Inostiol is the only nutrient deffeicny shown to cause NTDs in mice models
- Curly tail mutants- scribble muants were found to be resitsnat to folate supplementation in 30% of cases in humans though to eb this case. . and so inositol was decided to be used. (Greene et al showed insotiol worked in 70% of SB cases.)
- This was beneficial and so has led to in PONTI trial (Prevention Of Neural Tube defects by Inositol)
*risk females split into folate only or folate and insitol groups.
- vast majority in new drug trial as refused basc.
-out of all patients tested, 3 had NTDs. and they all came from the folate only trial.
-this was a smal trial ) however and larger ones are now in planning.
DISCUSS evidence of the PCPs link to NTDS
Polar cell polarity has been linked to a funcion of WNT down a non-canonical path.
several muations causing disticny NTD phenotypes in mice embryos have nw been identified as part of this pathway.
Priamrily asscoiated with craniorachischisiss but human mutaytions cause a range of conditions rangind from craniorachischisis to spina bifida.
- loop tail (VANGL2- TM that interacts with DVL)
- circle tail (Scribble (SCRB)
- Crash (Celsr)
- DVL double ko
- prk7 ko
- frizzled 3/6 KO (WNT receptor)
Studies using GFP fluorescence showed that in muations the expression of these proteins which is usally along the whole midline is shortened and restricted to smaller location.
Further study has linked this pathway to the regulation of cell polarity neccesary for apical cnstrcition an convergent extention. thus they are key to neural tube closure. implicating their muation in NTDs like open spina bifida.
- This pathway regulates the exprssion of RAC 1 downstream. ROLO et al showed RAC1 KO goes to no lamelipodia or filopodia formation needed for membrane ruffle formation and potrusion for fusion of Neural Tube at dorsal midline.
- Celsr KO prevented the formation of the median hinge point.
- VANGL2 investgation by YANG et al- showed
- low expression led to failed NTC and NTD.
- VANgle phopshoryaltion was neccesary fro its assymetric expression in the Neural plate which was neccesaray fro NTC.
- DVL was shown to be neccesary fro phosphoryaltion.
-Vangl2 KO led to the widening of medain hinge points which meant that even though the folds converged they never met.
Scrb- In mice muation to the protein leads to the formation of a Truncated protein dna LOF, These wher found for Craniorachischisis.
- Strut et al (2009) used GFP staining to mark the protein finding that it fromed a complex that localised to the membrane and was key to PCP function.
- Looking at the effects of mutants he found some prevented this translocation likely underpinning the LOF of the PCP explaining its contribution to NTDs.
Muations in those primarily associated with apical constriction can be more closely lonked to sina biida. A large on being muations in SHROOM encoding a Actin Binding Protein.
oultine evidence that folate defficiency is a risk in predisposed individuals discuss FOCM.
This is related to the connecttions of NTDs and deffciency in FOLATE ONE CARBON METABOLISM (FOCM).
- Mammals cannot produce folate and so have t get it from there food.
- Folate deffiencincy alone did not cause NTDs in mice
*Instead a prediposed model SPLOTCH in which a mutation in neural developemntal homeodomain protein PAX 3 is carried. They saw the folate defficeny did cause NTD. so seems that supplementing folate in diet is importnat t stop issues in prediposed individauls.
- - Mutations in FOCM found in humans are in Methyl tetrahydroflorate reductase (MHTFR) and Glycine decarboxylase (GLDC).
*MHTFR muations prevent the dividion of 1 carbon units from folate for supply into the methionine pathway. - This did not cause NTDS even in homozugous null mice
(Note this is associated with increased risk in some populations) - GLDC however, whic prevent 1 carbon unit supply into the folate cycle did induce NTDS. (PAI et al 2015)
- To do this studies using a gene trap allele that cause a 90% reduction. This correlated with NTDs in 20% of cases. (1 being spina bifida)
- Using a more potent model that led to unrecordable levels increases this penetrance to 57%
- Maternal supplemntation wit fomate was able t prevent this (again suggests we should supplement)
- Note GLDC is part of the mitchondrial cycle (Glycine cleavage system) in which the output back into the folate cycle is formate.
Spina bifida extra paper-
This stems from te fialiure of the neural tube to close at the caudal neuropore.
- This charcaterisable by the open lesion of the spinal segement.
- This varies in size. for exmaple mice expressing muation in th ZIC2 (zinc finger protein) which is related to delayed neurulation have a failiure to close ealry on reuslting in large lesions.
- VS GRHL3 which is a transcription facotr in primary neurlation causing curly tail form. This causes smaller lumbosacral lesions.
symptoms
-large spinal lesion
-severe motor defects stemming from neurodegeneration as a result of in utero exposure
- Hydroiocephalus- this stretches white matter and is a seen by the thin corpus callosum. This tracts well with the sevrity of cogntive defcets.
- Intelectual disabiltity is very common
- usually have an enlarged putamen and smalle HPC.
Diagnosis
- Can be diagnosed with a ultasound. several give away featres apart from spinal lesion.
- Lemon side, th forward protusion of the frontal skull.
- Banana sign, the shape of the cerrbelelum though to be linked to tethering to the spine.
Treatment
- Besides rpior mentioned preventative treat,ment like folate an inositol supplementation.
- Post natal closure of the lesion. This does not have good outcomes.
- Now they is support for in utero surgery following the MANAGEMENT OF MYELOMININGE;LOCELE STUDY that ended in 2010. This showed that it was beneficial conmapared to psot natal surger with 42% walking vs 21%
Curly tail mutant GRHL3
- these are largely resistant to folate.
- SB forms have small lesions in the lower thoracic regions.
- COPP et al injectig dyes into the close end of the neural tube to report leakage at the neural pore, associating this with an issue of the neural pore.
- Defects where shown to be issues of primary neurulation by tracking the final point of Caudal neuropore closure to the 32=34 somite level. this confers to the sacral regions and Circle tail SB was localised to the lower thoracic levels sshowing this is an issu of primery neurlation.
Studies by PETERS et 1998- Suggests this is an issue of dysfunction proliferation. as glucose is importnat to nuerulation they used uptake as a marker reporting a decline at the caudal nerupore and 25% dcerease in proliferation.
