A targeted sequencing panel identifies rare damaging variants in multiple genes in the cranial neural tube defect, anencephaly.

Neural tube defects (NTDs) affecting the brain (anencephaly) are lethal before or at birth, whereas lower spinal defects (spina bifida) may lead to lifelong neurological handicap. Collectively, NTDs rank among the most common birth defects worldwide. This study focuses on anencephaly, which despite having a similar frequency to spina bifida and being the most common type of NTD observed in mouse models, has had more limited inclusion in genetic studies. A genetic influence is strongly implicated in determining risk of NTDs and a molecular diagnosis is of fundamental importance to families both in terms of understanding the origin of the condition and for managing future pregnancies. Here we used a custom panel of 191 NTD candidate genes to screen 90 patients with cranial NTDs (n = 85 anencephaly and n = 5 craniorachischisis) with a targeted exome sequencing platform. After filtering and comparing to our in-house control exome database (N = 509), we identified 397 rare variants (minor allele frequency, MAF < 1%), 21 of which were previously unreported and predicted damaging. This included 1 frameshift (PDGFRA), 2 stop-gained (MAT1A; NOS2) and 18 missense variations. Together with evidence for oligogenic inheritance, this study provides new information on the possible genetic causation of anencephaly.

Amniotic fluid brain-specific proteins are biomarkers for spinal cord injury in experimental myelomeningocele.

Myelomeningocele (MMC), the most severe form of spina bifida (SB), causes neurological deficit. Injury to the spinal cord is thought to begin in utero. We investigated whether brain-specific proteins (BSPs) would enable us to monitor the development of MMC-related tissue damage during pregnancy in an animal model with naturally occurring SB (curly tail/loop tail mouse; n = 256). Amniotic fluid levels of neurofilament heavy chain (NfH), glial acidic fibrillary protein (GFAP) and S100B were measured by standard ELISA techniques. The amniotic fluid levels of all BSPs were similar in SB and control mice on embryonic day (E) 12.5 and 14.5, whereas a significant increase was observed for GFAP in SB mice on E16.5. Levels of all BSPs were significantly increased in SB mice on E18.5. The rapid increase in GFAP, paralleled by a moderate increase in NfH and S100B, suggests that spinal cord damage starts to accelerate around E16.5. The macroscopic size of the MMC was related to NfH level on E16.5 and E18.5, suggesting that axonal degeneration is most severe in large MMC. Amniotic fluid BSP measurements may provide important information for balancing the risks and benefits to mother and child of in utero surgery for MMC.

Hyperglycaemia potentiates the teratogenicity of retinoic acid in diabetic pregnancy in mice.

AIMS/HYPOTHESIS: We recently showed in mice that maternal diabetes increases embryonic susceptibility to caudal regression induced by vitamin A metabolite retinoic acid. Here we tested whether in the maternal diabetic milieu hyperglycaemia is the critical factor responsible for mediating this increased susceptibility. METHODS: Non-diabetic pregnant mice were made hyperglycaemic by subcutaneous injections of glucose at regular intervals. Conversely, diabetic pregnant mice were treated with phlorizin to induce renal glucosuria and thus reduce blood glucose concentrations. Pregnant mice were treated with retinoic acid and the extent of caudal regression in mouse embryos, measured in terms of the ratio of tail length to crown-rump length was assessed. Embryos were also examined for Wnt-3a expression and cell death. RESULTS: Embryos of mice treated with glucose had a greater extent of caudal regression induced by retinoic acid than saline-treated controls, with enhanced down-regulation of Wnt-3a expression and exacerbated cell death specifically at the caudal end of the embryo. Embryos of diabetic mice treated with phlorizin had a similar extent of caudal regression to embryos of non-diabetic mice after treatment with retinoic acid. CONCLUSIONS/INTERPRETATION: Hyperglycaemia increases embryonic susceptibility to caudal regression induced by retinoic acid, with the underlying cellular and molecular changes closely mimicking those that occur in maternal diabetes. Reduction of blood glucose concentrations in diabetic mice completely abolishes this increased susceptibility to retinoic acid. These results suggest that in maternal diabetes hyperglycaemia is the critical factor responsible for potentiating the teratogenic effect of retinoic acid.

Severe neural tube defects in the loop-tail mouse result from mutation of Lpp1, a novel gene involved in floor plate specification.

Neural tube defects (NTD) are clinically important congenital malformations whose molecular mechanisms are poorly understood. The loop-tail (Lp) mutant mouse provides a model for the most severe NTD, craniorachischisis, in which the brain and spinal cord remain open. During a positional cloning approach, we have identified a mutation in a novel gene, Lpp1, in the Lp mouse, providing a strong candidate for the genetic causation of craniorachischisis in LP: Lpp1 encodes a protein of 521 amino acids, with four transmembrane domains related to the Drosophila protein strabismus/van gogh (vang). The human orthologue, LPP1, shares 89% identity with the mouse gene at the nucleotide level and 99% identity at the amino acid level. Lpp1 is expressed in the ventral part of the developing neural tube, but is excluded from the floor plate where Sonic hedgehog (Shh) is expressed. Embryos lacking Shh express Lpp1 throughout the ventral neural tube, suggesting negative regulation of Lpp1 by SHH: Our findings suggest that the mutual interaction between Lpp1 and Shh may define the lateral boundary of floor plate differentiation. Loss of Lpp1 function disrupts neurulation by permitting more extensive floor plate induction by Shh, thereby inhibiting midline bending of the neural plate during initiation of neurulation.

Circletail, a new mouse mutant with severe neural tube defects: chromosomal localization and interaction with the loop-tail mutation.

Circletail (Crc) is a new mouse mutant that exhibits a severe form of neural tube defect, craniorachischisis, in which almost the entire neural tube fails to close. This phenotype is seen in very few other mutants, the best characterized of which is loop-tail (Ltap(Lp), referred to hereafter as Lp). We tested the possibility of allelism between Lp and Crc by intercrossing Lp/+ and Crc/+mice. A proportion of double heterozygotes (Lp/+,Crc/+) exhibit craniorachischisis, revealing failure of complementation. However, genetic analysis shows that Crc is not linked to the markers that flank the Lp locus and cannot, therefore, be an allele of Lp. A genome-wide scan has localized the Crc gene to a region of 8.8 cM on central chromosome 15. Partial penetrance of the craniorachischisis phenotype in Crc/+,Lp/+double heterozygotes suggests the existence of a third, unlinked genetic locus that influences the interaction between Crc and Lp.

Cardiovascular defects associated with abnormalities in midline development in the Loop-tail mouse mutant.

Loop-tail (Lp) is a naturally occurring mouse mutant that develops severe neural tube defects. In this study, we describe complex cardiovascular defects in Lp homozygotes, which include double-outlet right ventricle, with obligatory perimembranous ventricular septal defects, and double-sided aortic arch, with associated abnormalities in the aortic arch arteries. Outflow tract and aortic arch defects are often related to abnormalities in the cardiac neural crest, but using molecular and anatomic markers, we show that neural crest migration is normal in Lp/Lp embryos. On the other hand, the heart fails to loop normally in Lp/Lp embryos, in association with incomplete axial rotation and reduced cervical flexion. As a consequence, the ventricular loop is shifted posteromedially relative to its position in wild-type embryos. This suggests that the observed cardiac alignment defects in the Lp mutant may be secondary to failure of neural tube closure and incomplete axial rotation. Double-sided aortic arch is a rare finding among mouse models. In humans, it is usually an isolated malformation, only rarely occurring in combination with other cardiac defects. We suggest that the double-sided arch arises as a primary defect in the Lp mutant, unrelated to the alignment defects, perhaps reflecting a role for the (as-yet-unknown) Lp gene in maintenance/regression of the aortic arch system.

Comparative physical and transcript maps of approximately 1 Mb around loop-tail, a gene for severe neural tube defects on distal mouse chromosome 1 and human chromosome 1q22-q23.

The homozygous loop-tail (Lp) mouse has a severe neural tube closure defect, analogous to the craniorachischisis phenotype seen in humans. Linkage analysis and physical mapping have previously localized the Lp locus to a region on mouse chromosome 1 defined by the markers D1Mit113-Tagln2. Here we report the construction of sequence-ready bacterial clone contigs encompassing the Lp critical region in both mouse and the orthologous human region (1q22-q23). Twenty-two genes, one EST, and one pseudogene have been identified using a combination of EST database screening, exon amplification, and genomic sequence analysis. The preliminary gene map is Cen-Estm33-AA693056-Ly9-Cd48-Slam-Cd84-Kiaa1215-Nhlh1-Kiaa0253-Copa-Pxf-H326-Pea15-Casq1-Atp1a4-Atp1a2-Estm34-Kcnj9-Kcnj10-Kiaa1355-Tagln2-Nesg1-Crp-Tel. The genes between Slam and Kiaa1355 are positional candidates for Lp. The comparative gene content and order are identical between mouse and human, indicating a high degree of conservation between the two species in this region. Together, the physical and transcript maps described here serve as resources for the identification of the Lp mutation and further define the conservation of this genomic region between mouse and human.

Curly tail: a 50-year history of the mouse spina bifida model.

This paper reviews 50 years of progress towards understanding the aetiology and pathogenesis of neural tube defects (NTD) in the curly tail (ct) mutant mouse. More than 45 papers have been published on various aspects of curly tail with the result that it is now the best understood mouse model of NTD pathogenesis. The failure of closure of the spinal neural tube, which leads to spina bifida in this mouse, has been traced back to a tissue-specific defect of cell proliferation in the tail bud of the E9.5 embryo. This cell proliferation defect results in a growth imbalance in the caudal region that generates ventral curvature of the body axis. Neurulation movements are opposed, leading to delayed neuropore closure and spina bifida, or tail defects. It is interesting to reflect that these advances have been achieved in the absence of information on the nature of the ct gene product, which remains unidentified. In addition to the principal ct gene, which maps to distal Chromosome 4, the curly tail phenotype is influenced by several modifier genes and by environmental factors. NTD in curly tail are resistant to folic acid, as is thought to be the case in 30% of human NTD, whereas they can be prevented by myo-inositol. These and other features of NTD in this system bear striking similarities to the situation in humans, making curly tail a model for understanding a sub-type folic acid-resistant human NTD.

Neuronal migration defects in the Dreher (Lmx1a) mutant mouse: role of disorders of the glial limiting membrane.

Dreher (dr(J)) is an autosomal recessive mutation in the newly identified LIM homeobox gene, Lmx1a. The homozygous mutant phenotype includes misplaced neurons (heterotopia) in the cerebral cortex, cerebellum and hippocampus, which mimic the mild end of the spectrum of neuronal migration disorders in humans. Heterotopic neurons are found mainly in the normally cell-sparse layer I within the cerebral hemispheres of dr(J) homozygotes. Neu-N immunostaining confirms the neuronal nature of these heterotopic cells, while bromodeoxyuridine-birthdating shows that the misplaced neurons are generated predominantly during the late stages of corticogenesis (E15-E17), suggesting an over-migration of neurons destined for layer II. Immunohistochemistry for laminin, and staining of reticulin fibres, reveals disruption of the glial limiting membrane specifically overlying the areas of heterotopic neurons. Factor VIII (von Willebrand factor) staining shows an abnormal vascular network in layer I, associated with the fragmented glial limiting membrane. Layer I astrocytes, recognized by immunostaining for glial fibrillary acidic protein, exhibit attachment of their end feet to the fragmented glial limiting membrane. We suggest that disruption of the glial limiting membrane is central to the pathogenesis of heterotopic neurons in dreher, perhaps via defective radial glial-guided neuronal migration.

RhoB is expressed in migrating neural crest and endocardial cushions of the developing mouse embryo.

RhoB mRNA expression was examined in the developing mouse embryo between E8.5 and E11.5. Specific expression was found in migrating neural crest (NC) cells, from the first stages of their migration at E9.5, throughout the migration period. Expression is maintained in NC derivatives for at least one embryonic day after they reach their final destinations, but is then down-regulated. RhoB is also expressed in non NC-derived neural tissues, including motor neurones and the floor plate of the neural tube. RhoB mRNA expression is also found in the developing endocardial cushions of the atrioventricular and outflow regions of the developing heart.

Retinal axon misrouting at the optic chiasm in mice with neural tube closure defects.

In a new mouse mutant, circletail (Crc), failure of neural tube closure (embryonic day [E] 8-9) is associated with errors in retinal axon projection at the optic chiasm (E12-18), such that many axons normally projecting contralaterally instead grow to ipsilateral targets. Although the architecture of the chiasmatic region is altered, neurons and glia containing putative cues for axon guidance are present. The aberrant ipsilateral-projecting cells originate from a nonrandom expansion of the wild-type uncrossed retinal region. These axon pathway defects are found in two other mutants with cephalic neural tube defects (NTD), loop-tail (Lp) and Pax3 (splotch; Sp(2H)). Crc is phenotypically similar to Lp, exhibiting an open neural tube from midbrain to tail (craniorachischisis), while splotch has spina bifida with or without a cranial NTD. The retinal axon abnormalities occur only in the presence of NTD and not in homozygous mutants lacking cranial NTD. Thus, failure of neural tube closure is associated with failure of many retinal axons to cross the ventral midline. This study therefore reveals an unexpected connection between closure of the neural tube at the dorsal midline and development of ventral axon tracts. genesis 27:32-47, 2000.

A genetic risk factor for mouse neural tube defects: defining the embryonic basis.

Genetic polymorphisms are thought to play an important role in determining susceptibility to neural tube defects (NTDs), for example between different ethnic groups, but the embryonic manifestation of these polymorphic genetic influences is unclear. We have used a mouse model to test experimentally whether polymorphic variations in the pattern of cranial neural tube closure can influence susceptibility to NTDs. The site at which cranial neural tube closure begins (so-called closure 2) is polymorphic between inbred mice. Strains with a caudal location of closure 2 (e.g. DBA/2) are relatively resistant to NTDs, whereas strains with a rostrally positioned closure 2 (e.g. NZW) exhibit increased susceptibility to NTDs. We tested experimentally whether altering the position of closure 2 can affect susceptibility to cranial NTDs, by back- crossing the splotch ( Sp (2H) ) mutant gene onto the DBA/2 background. As a control, Sp (2H) was transferred onto the NZW background, which resembles splotch mice in its closure pattern. Approximately 80% of Sp (2H) homozygotes develop NTDs, both cranial (exencephaly) and spinal (spina bifida). After transfer to the DBA/2 background, the frequency of cranial NTDs was reduced significantly in Sp (2H) homozygotes, confirming a protective effect of caudal closure 2. In contrast, Sp (2H) homozygotes on the NZW background had a persistently high frequency of cranial NTDs. The frequency of spina bifida was not altered in either backcross, emphasizing the specificity of this genetic effect for cranial neurulation. These findings demonstrate that variation in the pattern of cranial neural tube closure is a genetically determined factor influencing susceptibility to cranial NTDs.

Neural tube defects: prevention by folic acid and other vitamins.

Folic acid has been demonstrated in clinical trials to reduce significantly the recurrence (and probably occurrence) of neural tube defects (NTD). In the U.K., there has been no decline in prevalence of NTD since the publication of the findings with folic acid. This article examines a series of questions relating to the action of folic acid, with emphasis on the use of mouse models as a source of experimental information which cannot easily be obtained by direct study of humans. Several mouse genetic NTD models exhibit sensitivity to prevention by folic acid, whereas other mice which develop morphologically similar NTD are resistant. Folic acid normalises neurulation in the sensitive mouse strains, providing evidence for a direct effect on the developing embryo, not on the pregnant female: Mouse studies do not support the proposed action of folic acid in encouraging the in utero demise of affected fetuses (i.e. terathanasia). Polymorphic variants of several folate-related enzymes have been shown to influence risk of NTD in humans and an inherited abnormality of folate metabolism has been demonstrated in one mouse NTD model. However, the biochemical basis of the action of folic acid in preventing NTD remains to be determined in detail. NTD in one folate-resistant mouse strain can be prevented by myo-inositol, both in utero and in vitro, raising the possibility of a therapeutic role also in humans. Gene-gene interactions seem likely to underlie the majority of NTD, suggesting that poly-therapy involving folic acid and other agents, such as myo-inositol, may prove more effective in preventing NTD than folic acid treatment alone.

Neuronal migration disorders in humans and in mouse models--an overview.

The spectrum of neuronal migration disorders (NMD) in humans encompasses developmental brain defects with a range of clinical and pathological features. A simple classification distinguishes agyria/pachygyria, heterotopia, polymicrogyria and cortical dysplasia as distinct clinico-pathological entities. Many of these conditions are associated with intractable epilepsy. When considering the pathogenesis of NMD, a critical developmental process is the migration of neuroblasts along the processes of radial glia during the formation of the layered structure of the cerebral cortex. In addition, faulty cytodifferentiation and programmed cell death play important roles in the generation of dysplasias and heterotopias respectively. A number of genes have been identified that participate in the regulation of neuronal migration. Mouse models, in which these genes are mutated, provide insight into the developmental pathways that underlie normal and abnormal neuronal migration.

Retinoic acid induces down-regulation of Wnt-3a, apoptosis and diversion of tail bud cells to a neural fate in the mouse embryo.

The tail bud comprises the caudal extremity of the vertebrate embryo, containing a pool of pluripotent mesenchymal stem cells that gives rise to almost all the tissues of the sacro-caudal region. Treatment of pregnant mice with 100 mg/kg all-trans retinoic acid at 9.5 days post coitum induces severe truncation of the body axis, providing a model system for studying the mechanisms underlying development of caudal agenesis. In the present study, we find that retinoic acid treatment causes extensive apoptosis of tail bud cells 24 h after treatment. Once the apoptotic cells have been removed, the remaining mesenchymal cells differentiate into an extensive network of ectopic tubules, radially arranged around the notochord. These tubules express Pax-3 and Pax-6 in a regionally-restricted pattern that closely resembles expression in the definitive neural tube. Neurofilament-positive neurons subsequently grow out from the ectopic tubules. Thus, the tail bud cells remaining after retinoic acid-induced apoptosis appear to adopt a neural fate. Wnt-3a, a gene that has been shown to be essential for tail bud formation, is specifically down-regulated in the tail bud of retinoic acid-treated embryos, as early as 2 h after retinoic acid treatment and Wnt-3a transcripts become undetectable by 10 h. In contrast, Wnt-5a and RAR-gamma are still detectable in the tail bud at that time. Extensive cell death also occurs in the tail bud of embryos homozygous for the vestigial tail mutation, in which there is a marked reduction in Wnt-3a expression. These embryos go on to develop multiple neural tubes in their truncated caudal region. These results suggest that retinoic acid induces down-regulation of Wnt-3a which may play an important role in the pathogenesis of axial truncation, involving induction of widespread apoptosis, followed by an alteration of tail bud cell fate to form multiple ectopic neural tubes.