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OBJECTIVEHydrocephalus occurs in children with congenital toxoplasmosis and can lead to severe disability. In these cases, the decision to intervene is often influenced by the expectation of neurological recovery. In this study, clinical responses to neurosurgical intervention in children with hydrocephalus secondary to congenital toxoplasmosis are characterized.METHODSSixty-five participants with hydrocephalus due to congenital Toxoplasma gondii infection were evaluated as part of the National Collaborative Chicago-based Congenital Toxoplasmosis Study, and their neuroradiographic findings were reviewed. Clinical outcomes were scored on the basis of cognition and motor skills through the use of IQ scores and Gross Motor Function Classification System (GMFCS) level. Outcomes were then analyzed in relation to approach to management, anatomy of hydrocephalus, and time from diagnosis of hydrocephalus to surgical intervention.RESULTSThere was considerable variation in the outcomes of patients whose hydrocephalus was treated in early life, ranging from normal cognitive and motor function to profound developmental delay and functional limitation. Of the 65 participants included in the study, IQ and GMFCS level were available for 46 (70.8%). IQ and motor score were highly correlated (r = −0.82, p < 0.001). There were people with differing patterns of hydrocephalus or thickness of cortical mantle on initial presentation who had favorable outcomes. Time to neurosurgical intervention data were available for 31 patients who underwent ventriculoperitoneal (VP) shunt placement. Delayed shunt placement beyond 25 days after diagnosis of hydrocephalus was associated with greater cognitive impairment (p = 0.02). Motor impairment also appeared to be associated with shunt placement beyond 25 days but the difference did not achieve statistical significance (p = 0.13). Among those with shunt placement within 25 days after diagnosis (n = 19), the mean GMFCS level was 1.9 ± 1.6 (range 1–5). Five (29.4%) of 17 of these patients were too disabled to participate in formal cognitive testing, after excluding 2 patients with visual difficulties or language barriers that precluded IQ testing. Of the patients who had VP shunt placement 25 or more days after diagnosis (n = 12), the mean GMFCS level was 2.7 ± 1.4 (range 1–4). Of these, 1 could not participate in IQ testing due to severe visual difficulties and 8 (72.7%) of the remaining 11 due to cognitive disability.CONCLUSIONSVP shunt placement in patients with hydrocephalus caused by congenital toxoplasmosis can contribute to favorable clinical outcomes, even in cases with severe hydrocephalus on neuroimaging. Shunt placement within 25 days of diagnosis was statistically associated with more favorable cognitive outcomes. Motor function appeared to follow the same pattern although it did not achieve statistical significance.

Symptomatic hydrocephalus is a common condition associated with myelomeningocele (open spina bifida). Traditionally, hydrocephalus was treated with insertion of a ventriculo-peritoneal (VP) shunt. This has been the standard of treatment since the introduction of the Holter shunt valve for the VP shunt in the late 1950s. Now there are other treatments that offer alternatives to VP shunt diversion for hydrocephalus. This article is a review of hydrocephalus associated with myelomeningocele and its treatment options. Treatment in the form of a VP shunt, endoscopic third ventriculostomy (ETV), and conservative management are discussed.

Objective Children with occult spinal dysraphism represent a wide spectrum of patients. Previous studies assessing urologic outcomes have in part been deficient due to the inability to appropriately categorize these patients and gather long-term follow-up data. In this study, a uniform set of patients that had occult spinal dysraphism with magnetic resonance imaging findings of a fatty filum terminale (FF) and/or low-lying cord (LLC) was identified. Utilizing long-term follow-up data, predictors for achieving urinary continence following tethered cord release (TCR) were determined. Methods A retrospective chart review of pediatric patients with a diagnosis of tethered cord who underwent TCR from 1995 to 2005 was performed. Analysis was limited to patients who had primary TCR by one of two neurosurgeons within our multidisciplinary spina bifida clinic, who had greater than 1-year follow-up, and who were old enough to have continence status assessed (age > 6 years unless definitively toilet trained earlier). Patients with other associated forms of spinal dysraphism (lipomyelomeningeocele, spinal lipomas, sacral agenesis), anorectal malformations, and genitourinary anomalies were excluded. Pre- and post-TCR urodynamics, radiographic studies, functional orthopedic status, and urologic outcomes were assessed. Urodynamic results were categorized by three blinded urologists into one of three urodynamic patterns: (1) normal, (2) indeterminate, and (3) high risk. Results A total of 147 patients with FF and/or LLC that underwent TCR were reviewed. 51 patients were excluded because of another associated spinal dysraphism (15/51 patients) or an anorectal/genitourinary anomaly (36/51 patients). Fifty-nine of the remaining 96 patients had adequate long-term follow-up data to be included in the study. 20 patients were asymptomatic at the time of TCR while 39 presented with orthopedic and/or urologic symptoms. The average age at surgery was 59.3 months (range 2–277 months) with an average follow-up of 7.0 years (range 1–16 years). At latest follow-up, 47 (80%) patients were continent while 12 (20%) were either incontinent or utilizing clean intermittent catheterization (CIC). Statistical analysis revealed that age of untethering, type of cutaneous lesion, level of conus, presence of hydronephrosis, and high-grade vesicoureteral reflux (VUR) were not independent predictors of continence. In patients with a cutaneous lesion who were asymptomatic, 19/20 obtained continence post-TCR (* p = 0.036). In patients who were old enough to assess continence pre-TCR, 14/25 patients were continent pre-TCR and 11/25 were incontinent. Of the 14 who were continent pre-TCR, all remained continent post-TCR (* p = 0.002). Of the 11 who were incontinent pre-TCR, five (45%) eventually became continent post-TCR. Assessment of urodynamic data revealed that neither pre- nor post-TCR urodynamics predicted continence status. Conclusion Isolated cutaneous lesions and preoperative continence status are positive predictors for post-TCR continence. While pre- and post-TCR urodynamics do not predict continence status, their utility in preoperative work-up, monitoring for retethering, and long-term urologic follow-up requires further examination.

Prompt diagnosis and rapid initiation of medical treatment are critical for the best outcomes in infants with congenital toxoplasmosis. This is important for pregnant women, fetuses, and infants, including those with active retinitis and choroidal neovascular membranes. For hydrocephalus, prompt placement of a ventriculoperitoneal shunt is key for improved outcomes. Pyrimethamine and Sulfadiazine with Leucovorin are first-line medicines. For later recurrences of active retinitis, Azithromycin or Clindamycin are sometimes substituted for Sulfadiazine as second-line treatments, given with Pyramethamine. Following resolution of active retinitis, these medicines may be useful without Pyrimethamine for suppression and avoid the risk of hypersensitivity from Trimethoprim/Sulfamethoxazole. Antibody to VEGF, in conjunction with antimicrobial therapy, results in resolution of choroidal neovascular membranes. Serologic screening of seronegative pregnant women to detect primary infection during gestation, and facilitating medicine administration and thereby preventing or treating fetal infection, is an optimal, apparently cost-effective, means to reduce disease. Definitively curative medicines currently being developed likely will improve future management and outcomes of this disease.

Object The authors sought to identify novel biomarkers for early detection of neural tube defects (NTDs) in human fetuses. Methods Amniotic fluid and serum were drawn from women in the second trimester of pregnancy. The study group included 2 women pregnant with normal fetuses and 4 with fetuses displaying myelomeningocele (n = 1), anencephaly (n = 1), holoprosencephaly (n = 1), or encephalocele (n = 1). Amniotic fluid stem cells (AFSCs) were isolated and cultured. The cells were immunostained for the stem cell markers Oct4, CD133, and Sox2; the epigenetic biomarkers H3K4me2, H3K4me3, H3K27me2, H3K27me3, H3K9Ac, and H3K18Ac; and the histone modifiers KDM6B (a histone H3K27 demethylase) and Gcn5 (a histone acetyltransferase). The levels of 2 markers for neural tube development, bone morphogenetic protein–4 (BMP4) and sonic hedgehog (Shh), were measured in amniotic fluid and serum using an enzyme-linked immunosorbent assay. Results The AFSCs from the woman pregnant with a fetus affected by myelomeningocele had higher levels of H3K4me2, H3K4me3, H3K27me2, and H3K27me3 and lower levels of KDM6B than the AFSCs from the women with healthy fetuses. The levels of H3K9ac, H3K18ac, and Gcn5 were also decreased in the woman with the fetus exhibiting myelomeningocele. In AFSCs from the woman carrying an anencephalic fetus, levels of H3K27me3, along with those of H3K9Ac, H3K18ac, and Gcn5, were increased, while that of KDM6B was decreased. Compared with the normal controls, the levels of BMP4 in amniotic fluid and serum from the woman with a fetus with myelomeningocele were increased, whereas levels of Shh were increased in the woman pregnant with a fetus displaying anencephaly. Conclusions The levels of epigenetic marks, such as H3K4me, H3K27me3, H3K9Ac, and H3K18A, in cultured AFSCs in combination with levels of key developmental proteins, such as BMP4 and Shh, are potential biomarkers for early detection and identification of NTDs in amniotic fluid and maternal serum.

Although maternal intake of folic acid (FA) prevents neural tube defects in 70% of the population, the exact mechanism of prevention has not been elucidated. We hypothesized that FA affects neural stem cell (NSC) proliferation and differentiation. This hypothesis was examined in a folate-responsive spina bifida mouse model, Splotch (Sp(-/-)), which has a homozygous loss-of-function mutation in the Pax3 gene. Neurospheres were generated with NSCs from the lower lumbar neural tube of E10.5 wild-type (WT) and Sp(-/-) embryos, in the presence and absence of FA. In the absence of FA, the number of neurospheres generated from Sp(-/-) embryos compared with WT was minimal (P0.05). Addition of FA to Sp(-/-) cultures increased the expression of a Pax3 downstream target, fgfr4, and rescued NSC proliferative potential, as demonstrated by a significant increase in neurosphere formation (P0.01). To ascertain if FA affected cell differentiation, FA-stimulated Sp(-/-) neurospheres were allowed to differentiate in the continued presence or absence of FA. Neurospheres from both conditions expressed multi-potent stem cell characteristics and the same differentiation potential as WT. Further, multiple neurospheres from both WT and FA-stimulated Sp(-/-) cell cultures formed extensive synaptic connections. On the whole, FA-mediated rescue of neural tube defects in Sp(-/-) embryos promotes NSC proliferation at an early embryonic stage. FA-stimulated Sp(-/-) neurospheres differentiate and form synaptic connections, comparable to WT.

The epigenetic mechanism of folic acid (FA) action on dorsal root ganglion (DRG) cell proliferation and sensory neuron differentiation is not well understood. In this study, the ND7 cell line, derived from DRG cells, was used to elucidate this mechanism. In ND7 cells differentiated with dbcAMP and NGF, Hes1 and Pax3 levels decreased, whereas Neurog2 levels showed a modest increase. Chromatin immunoprecipitation (ChIP) assays examining epigenetic marks at the Hes1 promoter showed that FA favored increased H3K9 and H3K19 acetylation and decreased H3K27 methylation. Hence, FA plays a positive role in cell proliferation. In differentiated ND7 cells, H3K27 methylation decreased, whereas H3K9 and H3K18 acetylation increased at the Neurog2 promoter. FA did not favor this phenotypic outcome. Additionally, in differentiated ND7 Neurog2 associated with the NeuroD1 promoter, FA decreased this association. The results suggest that the switch from proliferation to sensory neuron differentiation in DRG cells is regulated by alterations in epigenetic marks, H3K9/18 acetylation and H3K27 methylation, at Hes1 and Neurog2 promoters, as well as by Neurog2 association with NeuroD1 promoter. FA although positive for proliferation, does not appear to play a role in differentiation.

Object Untethering of a tethered spinal cord (TSC) by transecting or removing a fatty filum terminale is a relatively simple procedure that can prevent or ameliorate neurological symptoms, and the postoperative prognosis is usually good. Progressive neurological deterioration caused by recurrent tethering has been rarely reported. The authors present their experience in cases in which a sectioned fatty filum terminale has become retethered. Methods The authors retrospectively analyzed the surgical results of pediatric patients with fatty filum terminale–TSC treated by transection of the filum. The patients' charts were reviewed for demographic data, clinical presentation, surgical therapy, and follow-up data. Results Of the 225 children who underwent TSC release by sectioning the fatty filum from 1992 to 2005, there were 6 patients (2.7%; 3 males, 3 females) in whom the fatty filum retethered. The mean age at the first diagnosis of TSC was 5.2 years (range 2 months–12.3 years). The mean duration from the first untethering procedure to retethering was 5.4 years. The mean age at the time of retethering was 10.6 years (range 7–17.5 years). Symptoms of retethering were urinary incontinence, low-back pain, difficulty walking, constipation, leg pain, and worsening foot deformity. Patients underwent cystometrography at the time retethering was indicated by increased bladder capacity, large post-void residual volume, decreased bladder capacity, increase in filling pressure, and poor sensation of filling. Magnetic resonance imaging revealed adherence of the rostral stump of the sectioned filum to the midline dorsal dural surface. All patients underwent the second untethering procedure. Four patients improved neurologically and experienced no retethering thereafter (mean follow-up period 5.5 years). Two patients experienced additional retethering after temporary improvement following the second untethering procedure. Conclusions Retethering of the spinal cord is a rare condition occurring after the sectioning of a fatty filum terminale. Awareness of this rare sequela is necessary for appropriate long-term management of TSC caused by a fatty filum terminale. Cystometrography is useful for detecting the lesion and confirming the diagnosis of retethering.

The mechanism(s) behind folate rescue of neural tube closure are not well understood. In this study we show that maternal intake of folate prior to conception reverses the proliferation potential of neural crest stem cells in homozygous Splotch embryos (Sp(-/-)) via epigenetic mechanisms. It is also shown that the pattern of differentiation seen in these cells is similar to wild-type (WT). Cells from open caudal neural tubes of Sp(-/-) embryos exhibit increased H3K27 methylation and decreased expression of KDM6B possibly due to up-regulation of KDM6B targeting micro-RNAs such as miR-138, miR-148a, miR-185, and miR-339-5p. In our model, folate reversed these epigenetic marks in folate-rescued Sp(-/-) embryos. Using tissue from caudal neural tubes of murine embryos we also examined H3K27me2 and KDM6B association with Hes1 and Neurog2 promoters at embryonic day E10.5, the proliferative stage, and E12.5, when neural differentiation begins. In Sp(-/-) embryos compared with WT, levels of H3K27me2 associated with the Hes1 promoter were increased at E10.5, and levels associated with the Neurog2 promoter were increased at E12.5. KDM6B association with Hes1 and Neurog2 promoters was inversely related to H3K27me2 levels. These epigenetic changes were reversed in folate-rescued Sp(-/-) embryos. Thus, one of the mechanisms by which folate may rescue the Sp(-/-) phenotype is by increasing the expression of KDM6B, which in turn decreases H3K27 methylation marks on Hes1 and Neurog2 promoters thereby affecting gene transcription.

Worldwide, the incidence of neural tube defects (NTDs) varies from 0.17 to 6.39 per 1,000 live births. The declining prevalence of myelomeningocele, the most common NTD, is secondary to several factors including folic acid fortification, prenatal diagnosis with termination of affected fetuses, and unknown factors.Of those born with myelomeningocele, survival during infancy and preschool years has improved over the last 25 years (Bowman et al., Pediatr Neurosurg 34:114-120). Fewer newborns today require shunt placement, which will hopefully improve the long-term mortality associated with this disease (Chakraborty et al., J Neurosurg Pediatr 1(5):361-365, unpublished data). Of a cohort born in 1975-1979 and treated at a single US institution, 74% have survived into young adulthood.One of the greatest challenges facing these young adults is the transitioning of their medical care into an adult medical community.

Object All children born with a myelomeningocele at the authors' institution undergo aggressive treatment to maintain or improve functional outcome. Consequently, when any neurological, orthopedic, and/or urological changes are noted, a search for the cause is initiated. The most common cause of decline in a child born with a myelomeningocele is shunt malfunction. The second most common cause is tethering of the distal spinal cord at the site of the original back closure. In this report, the authors review the indicators of symptomatic spinal cord tethering and discuss the surgical interventions and outcomes in the children with myelomeningocele who underwent treatment at Children's Memorial Hospital from 1975 to 2008. Methods Among the 502 children who underwent original closure at Children's Memorial Hospital, a symptomatic tethered spinal cord developed in 114 (23%). Eighty-one patients (71%) have undergone 1 untethering procedure, and 33 patients (29%) have undergone multiple untetherings, for a total of 163 total surgeries. The indicators of symptomatic spinal cord tethering include scoliosis, decline in lower-extremity (LE) motor strength, LE contractures, LE spasticity, gait change, urinary changes, and pain. Results Pain has shown the best response to surgical untethering, with 100% of children experiencing postoperative improvement. The results of long-term follow-up (average 12 years, range 1 month–23.3 years) in this cohort demonstrated scoliosis progression after surgical untethering in 52% of patients, with 28% requiring spinal fusion. On the 3-month postoperative manual muscle test, 70% of patients showed improved LE muscle strength compared to preoperatively. Gait was also similarly improved after untethering as evaluated by an orthopedic surgeon. Spasticity improved in two-thirds of the cohort, and as expected, LE contractures were stable (78%) postoperatively, as assessed by orthopedic and rehabilitation medicine specialists. Urologically, 64% of patients showed improvements on postoperative bladder evaluation. Conclusions Although this is a clinical outcome study with no control group, the authors' experience has been that tethered cord release is beneficial in maintaining neurological, urological, and orthopedic functioning in children born with a myelomeningocele.

Pax3 regulates neural crest cell migration and is critical during neural crest development. TGFbs modify neural crest cell migration and differentiation. TGFbeta2 nullizygous embryos (TGFbeta2(-/-)Pax3(+/+)) display open neural tube and bifid spine, whereas in wild type embryos, the neural tube is closed. In previous work, we have demonstrated that Pax3 regulates TGFbeta2 by directly binding to cis-regulatory elements on its promoter. In this study, we found that the TGFbeta2 nullizygous phenotype can be reversed to the wild type phenotype by down-regulating one allele of Pax3, as in TGFbeta2(-/-)Pax3(+/-) embryos obtained through breeding TGFb2(+/-)Pax3(+/-) mice. The data in this paper suggest that Pax3 and TGFbeta2 interact in a coordinated gene regulatory network, linked by common downstream effector genes, to bring about this phenotypic reversal. Downstream effectors may include Hes1, Ngn2 and Sox9, as well as other genes involved in neuronal differentiation.

Background Neural tube defects (NTDs), including spina bifida and anencephaly, are the second most common birth defect with an incidence of 1/1000. Genetic factors are believed to contribute to NTD risk and family-based studies can be useful for identifying such risk factors. Methods We ascertained 1066 NTD families (1467 affected patients), including 307 multiplex NTD families. We performed pedigree analysis to describe the inheritance patterns, pregnancy outcomes, and recurrence risks to relatives of various types. Results Myelomeningocele or spina bifida (66.9%) and cranial defects (17.7%) were the most common NTD subtypes observed. The overall male:female ratio for affected individuals was 0.82, and there were even fewer males among individuals with an upper level NTD (0.62). Among twins, 2 of the 5 monozygotic twins and only 3 of 35 dizygotic twins were concordant, while 27% of the same sex twins were concordant, but none of the different sex twins. The estimated 6.3% recurrence risk to siblings (CI 0.04-0.08) is consistent with previous reports. Families with two or more affected individuals show a higher proportion of female transmitters (p = 0.0002). Additionally, the number of affected relatives in maternal compared to paternal lineages was more than double (p = 0.006). There were significantly more miscarriages, infant deaths, and stillborn pregnancies of the maternal aunts and uncles (p Conclusions Our data provide several lines of evidence consistent with a maternal effect, as well as a sex-influenced effect, in the etiology of NTDs.

Pax3 is expressed early during embryonic development in spatially restricted domains including limb muscle, neural crest, and neural tube. Pax3 functions at the nodal point in melanocyte stem cell differentiation, cardiogenesis and neurogenesis. Additionally Pax3 has been implicated in migration and differentiation of precursor cell populations. Currently there are questions about how Pax3 regulates these diverse functions. In this study we found that in the absence of functional Pax3, as in Splotch embryos, the neural crest cells undergo premature neurogenesis, as evidenced by increased Brn3a positive staining in neural tube explants, in comparison with wild-type. Premature neurogenesis in the absence of functional Pax3 may be due to a change in the regulation of basic helix–loop–helix transcription factors implicated in proliferation and differentiation. Using promoter-luciferase activity measurements in transient co-transfection experiments and electro-mobility shift assays, we show that Pax3 regulates Hairy and enhancer of split homolog-1 (Hes1) and Neurogenin2 (Ngn2) by directly binding to their promoters. Chromatin immunoprecipitation assays confirmed that Pax3 bound to cis-regulatory elements within Hes1 and Ngn2 promoters. These observations suggest that Pax3 regulates Hes1 and Ngn2 and imply that it may couple migration with neural stem cell maintenance and neurogenesis.

We previously reported a Vietnamese-American family with isolated autosomal dominant occipital cephalocele. Upon further neuroimaging studies, we have recharacterized this condition as autosomal dominant Dandy-Walker with occipital cephalocele (ADDWOC). A similar ADDWOC family from Brazil was also recently described. To determine the genetic etiology of ADDWOC, we performed genome-wide linkage analysis on members of the Vietnamese-American and Brazilian pedigrees. Linkage analysis of the Vietnamese-American family identified the ADDWOC causative locus on chromosome 2q36.1 with a multipoint parametric LOD score of 3.3, while haplotype analysis refined the locus to 1.1 Mb. Sequencing of the five known genes in this locus did not identify any protein-altering mutations. However, a terminal deletion of chromosome 2 in a patient with an isolated case of Dandy-Walker malformation also encompassed the 2q36.1 chromosomal region. The Brazilian pedigree did not show linkage to this 2q36.1 region. Taken together, these results demonstrate a locus for ADDWOC on 2q36.1 and also suggest locus heterogeneity for ADDWOC.

Prenatal folic acid (FA) supplementation prevents neural tube defects. Folate receptor alpha (FRα) is critical for embryonic development, including neural crest (NC) development. Previously we showed that FRα translocates to the nucleus in response to FA, where it acts as a transcription factor. In this study, we examined if FA through interaction with FRα regulates stem cell characteristics of cranial neural crest cells (CNCCs)-critical for normal development. We hypothesized that FRα upregulates coding genes and simultaneously downregulates non-coding miRNA which targets coding genes in CNCCs. Quantitative RT-PCR and chromatin immunoprecipitation showed that FRα upregulates Oct4, Sox2, and Klf4 by binding to their cis-regulator elements-5′ enhancer/promoters defined by H3K27Ac and p300 occupancy. FA via FRα downregulates miRNAs, miR-138 and miR-let-7, which target Oct4 and Trim71 (an Oct4 downstream effector), respectively. Co-immunoprecipitation data suggests that FRα interacts with the Drosha-DGCR8 complex to affect pre-miRNA processing. Transfecting anti-miR-138 or anti-miR-let-7 into non-proliferating neural crest cells (NCCs) derived from Splotch (Sp−/−), restored their proliferation potential. In summary, these results suggest a novel pleiotropic role of FRα: (a) direct activation of Oct4, Sox2, and Klf4 genes; and (b) repression of biogenesis of miRNAs that target these genes or their effector molecules.

Four anatomical patterns of hydrocephalus secondary to congenital Toxoplasma gondii infection were identified and characterized for infants enrolled in the National Collaborative Chicago-based Congenital Toxoplasmosis Study. Analysis of parasite serotype revealed that different anatomical patterns associate with Type-II vs Not-Exclusively Type-II strains (NE-II) (P = .035).

The neural crest (NC) is a population of cells between the dorsal ectoderm and the neural tube in vertebrate embryos. Regulatory mechanisms ranging from epigenetics to microRNAs (miRNAs) to post-translational protein modifications, work coordinately to fine-tune NC activity. miRNAs affect genes transcriptionally by directly interacting with either their 3′UTR or their 5′UTR. Affected genes include those of chromatin-modifying enzymes such as EZH2 and KDM6B. A lack of optimal levels of chromatin-modifying enzymes may in turn affect miRNA biogenesis as well as the transcriptional regulation of other genes. Overall, noncoding RNA genes and protein-coding genes interact in a network that, when connected to chromatin regulation, affects the expression of both noncoding and coding genes in neural crest development. This chapter highlights that role.

BACKGROUND: Neural tube defects (NTDs) are considered complex, with both genetic and environmental factors implicated. To date, no major causative genes have been identified in humans despite several investigations. The first genomewide screen in NTDs demonstrated evidence of linkage to chromosomes 7 and 10. This screen included 44 multiplex families and consisted of 402 microsatellite markers spaced ∼10 cM apart. Further investigation of the genomic screen data identified a single large multiplex family, pedigree 8776, as primarily driving the linkage results on chromosome 7. METHODS: To investigate this family more thoroughly, a high-density single nucleotide polymorphism (SNP) screen was performed. Two-point and multipoint linkage analyses were performed using both parametric and nonparametric methods. RESULTS: For both the microsatellite and SNP markers, linkage analysis suggested the involvement of a locus or loci proximal to the telomeric regions of chromosomes 2q and 7p, with both regions generating a LOD* score of ∼3.0 using a nonparametric identity by descent relative sharing method. CONCLUSIONS: The regions with the strongest evidence for linkage map proximal to the telomeres on these two chromosomes. In addition to mutations and/or variants in a major gene, these loci may harbor a microdeletion and/or translocation; potentially, polygenic factors may also be involved. This single family may be promising for narrowing the search for NTD susceptibility genes. Birth Defects Research (Part A) 76:499-505, 2006.

BACKGROUND Vitamin A (retinol), in the form of retinoic acid (RA), is essential for normal development of the human embryo. Studies in the mouse and zebrafish have shown that retinol is metabolized in the developing spinal cord and must be maintained in a precise balance along the anteroposterior axis. Both excess and deficiency of RA can affect morphogenesis, including failures of neural tube closure. METHODS We chose to investigate 5 genes involved in the metabolism or synthesis of RA, ALDH1A2, CYP26A1, CYP26B1, CRABP1, and CRABP2, for their role in the development of human neural tube defects, such as spina bifida. RESULTS An association analysis using both allelic and genotypic single-locus tests revealed a significant association between the risk for spina bifida and 3 polymorphisms in the gene ALDH1A2; however, we found no evidence of a significant multilocus association. CONCLUSIONS These results may suggest that polymorphisms in ALDH1A2 may influence the risk for lumbosacral myelomeningocele in humans. Birth Defects Research (Part A), 2005. © 2005 Wiley-Liss, Inc.

Neural tube defects (NTDs) are the second most common birth defects (1 in 1000 live births) in the world. Periconceptional maternal folate supplementation reduces NTD risk by 50–70%; however, studies of folate related and other developmental genes in humans have failed to definitively identify a major causal gene for NTD. The aetiology of NTDs remains unknown and both genetic and environmental factors are implicated. We present findings from a microsatellite based screen of 44 multiplex pedigrees ascertained through the NTD Collaborative Group. For the linkage analysis, we defined our phenotype narrowly by considering individuals with a lumbosacral level myelomeningocele as affected, then we expanded the phenotype to include all types of NTDs. Two point parametric analyses were performed using VITESSE and HOMOG. Multipoint parametric and nonparametric analyses were performed using ALLEGRO. Initial results identified chromosomes 7 and 10, both with maximum parametric multipoint lod scores (Mlod) >2.0. Chromosome 7 produced the highest score in the 24 cM interval between D7S3056 and D7S3051 (parametric Mlod 2.45; nonparametric Mlod 1.89). Further investigation demonstrated that results on chromosome 7 were being primarily driven by a single large pedigree (parametric Mlod 2.40). When this family was removed from analysis, chromosome 10 was the most interesting region, with a peak Mlod of 2.25 at D10S1731. Based on mouse human synteny, two candidate genes (Meox2, Twist1) were identified on chromosome 7. A review of public databases revealed three biologically plausible candidates (FGFR2, GFRA1, Pax2) on chromosome 10. The results from this screen provide valuable positional data for prioritisation of candidate gene assessment in future studies of NTDs.

An increasing number of children with Chiari I malformations are coming to the attention of neurosurgeons today, although a consensus on the surgical approach to these lesions has yet to be found. We present a retrospective analysis of posterior fossa decompression (PFD) performed at our institution on 96 patients from 1989 to 2001. Statistical analyses based on clinical and radiographic presentation and the types of surgical procedures used formed the basis for our review. Most of the patients with hydromyelia underwent duraplasty procedures with or without tonsillar manipulation. In contrast, most patients without hydromyelia underwent bony decompression with dural scoring and intraoperative ultrasound. PFD with bony decompression and dural scoring showed a 72% success rate, compared with 68% for duraplasty. Dural opening was not more likely to improve or arrest hydromyelia. The group subjected to duraplasty, however, had a significantly higher complication rate. Patients under the age of 8 fared better than their older counterparts. Overall, we favor a tailored posterior fossa craniectomy with dural scoring as the initial surgical procedure in children with Chiari I malformation with or without a syrinx. This less invasive approach minimizes complications associated with dural opening and offers comparable success rates.

It is the Chiari II malformation and its effects that determine the quality of life of the individual born with spina bifida. The cause of this malformation has been a source of debate for many years. Understanding the cause enables strategies for the management of problems created by this malformation to be developed. An open neural tube defect allows fluid to escape from the cranial vesicles, altering the intracranial environment and leads to all of the brain changes seen in the Chiari II malformation. Decompression of the intracranial vesicles causes overcrowding, decrease in the size of the third ventricle, and changes in the fetal skull. It also permanently links the intracranial ventricular system to the spinal cord central canal.

Folate supplementation appears to reduce the risk for neural tube defects (NTDs). Methylenetetrahydrofolate reductase (MTHFR) is a candidate gene in the folate metabolism pathway that has been extensively studied in different human populations. We examined the risk associated with having the thermolabile variant (TT) of MTHFR in a study of 175 American Caucasians with NTDs and their families. We found a significant association in patients compared with 195 unrelated controls [odds ratio (OR) = 2.13, 95% confidence interval (95% CI) = 1.11-4.09)], but not in mothers (OR = 1.29, 95% CI = 0.622-2.67) or in fathers (OR = 1.45, 95% CI = 0.681-3.09). We found no evidence for unequal transmission from parents to an affected child (p > 0.10). We failed to find a previously reported association for a combined haplotype for MTHFR and cystathionine beta-synthase, except in subjects with NTDs compared with 559 pooled controls (OR = 2.87, 95% CI = 1.03-8.03). We found no evidence for an association for a novel CA-repeat polymorphism identified in a gene closely linked to MTHFR (p > 0.10). Our studies continue to suggest that additional candidate genes other than MTHFR may be responsible for an increased risk to NTD in some American Caucasian families.

We investigated the T locus as a candidate gene in a series of patients and families with lumbosacral myelomeningocele. Single-strand conformation polymorphism (SSCP) analysis was used to identify sequence variation in all 8 exons and in intron 7 of this locus. We found evidence of substantial polymorphism within this locus, as previously reported [Papapetrou et al., 1999, J Med Genet 36:208-213], and moderately significant evidence of linkage disequilibrium with the CacI polymorphism of exon 8. However, when the locus was considered as a whole, with all single nucleotide polymorphisms (SNPs) integrated into a haplotype, there was no evidence for linkage disequilibrium. In addition, we did not identify any new sequence variants. Thus, we conclude that the T locus is not a major locus for human NTDs in this sample.

Objective: Cerebrospinal fluid (CSF) production rates influence shunt design and the care of children with hydrocephalus. Measurement of hourly CSF output through external ventricular drainage (EVD) reflects the CSF production. In the present study, hourly CSF outputs in children with hydrocephalus were measured while they were treated with EVD and correlated with the age, sex and body weight of the patients as well as other clinical parameters. Methods: One hundred children with hydrocephalus due to various causes had EVD treatment. Twenty-six had EVD on two or three separate occasions; thus, the CSF output measurements were observed and analyzed on the basis of 130 EVDs. The most common reason for EVD treatment was shunt infection (n = 75). The duration of EVDs ranged from 25 to 774 h (mean 269 h). The height of the drip chamber from the mid-head position ranged from 0 to 23 cm (mean 9.8 cm). The hourly CSF output was analyzed according to the patient’s age, sex and body weight as well as the presence of CSF infection. Results: The hourly CSF output rapidly increases during the first year of life. By the second year, it reaches 64% of the hourly CSF output of 15-year-old children. The mean hourly output ranged from 0.1 to 26.5 ml/h (mean 8.1 ml/h), with the standard deviation ranging from 0.4 to 10.8 ml/h (mean 5.2 ml/h). A regression analysis indicated that the age and body weight appeared to correlate with the hourly CSF output. Using the natural logarithm of age, body weight and sex, these predictors accounted for 50.9% of the variability in hourly CSF output. The regression equation is as follows: hourly CSF output = 2.78 – 2.23(male = 0, female = 1) + 0.97 log(age in years) + 2.26 log(body weight in kg). R sd = 3.36, R2 = 0.509. The type of infecting organism and the height of EVD did not influence the overall CSF output. Conclusion: The hourly CSF output fluctuates, but the CSF output increases logarithmically with age and body weight. The gender also influences the CSF output, with males having a greater output than females. The data produced by the present study will help us to understand CSF production rates in developing children. They will also help us in the care of children receiving EVD treatment, as well as in selecting and designing shunt systems.

The primary urologic objectives for lipomyelomeningocele (LMM) and myelomeningocele (MM) are preserving renal integrity and achieving continence. Due to this common ground, LMM and MM are urologically treated the same. However, unlike MM, LMM may present with no evident functional concerns. Indications for and timing of tethered cord release (TCR) in LMM are therefore controversial. Long-term urologic outcomes are not well defined.Expectations for continence and potential for intermittent catheterization (CIC) following TCR in LMM are important for realistically counseling families regarding future needs. The present study aimed to identify prognostic factors for continence and need for CIC in LMM.The present study retrospectively identified 143 patients from the multidisciplinary clinic who underwent TCR for LMM between 1995 and 2010. Concomitant anorectal/genitourinary anomalies, filar lipoma, fatty filum, previous TCR, and follow-up1 year were excluded. Analysis was limited to those toilet trained or aged ≥6 years at latest follow-up. Lipomyelomeningocele was classified as dorsal, distal, transitional or chaotic. Pre- and post-TCR urologic status was assessed. Ability to achieve urinary continence, with or without CIC, was the primary outcome, and need for CIC was the secondary outcome of interest.A total of 56 patients met inclusion criteria. Median age at TCR was 4.4 months (range 1.0-224.0) with a median follow-up of 10.7 years (range 1.3-19.1); 68% were asymptomatic at presentation. Clinical symptoms were urologic in 7%. At the latest follow-up, 86% of patients were continent spontaneously or with CIC (Summary Fig.). Of the four patients who presented with urologic symptoms, all were continent, but three required CIC. Overall, 23% of patients required CIC. Median age at CIC initiation was 7.6 years (range 1.6-17.4). Long-term continence was not associated with any demographic, anatomic, surgical or functional variable. Need for CIC at latest follow-up was associated with symptomatic presentation, partial TCR, transitional lipoma, and high-risk pre-operative urodynamics.In this series of primary TCR for LMM, where 93% of patients were urologically asymptomatic before TCR, prospects for continence were excellent. No studied parameter clearly impacted continence; however, need for CIC was associated with multiple variables.Clear predictors for continence after TCR will require additional long-term patient outcomes. Families can anticipate 23% likelihood of CIC, which is considerably less than in MM, but long-term urologic follow-up is still strongly recommended.

Maternal folate intake has reduced the incidence of human neural tube defects by 60-70 %. However, 30-40 % of cases remain nonresponsive to folate intake. The main purpose of this study was to understand the molecular mechanism of folate nonresponsiveness in a mouse model of neural tube defect.We used a folate-nonresponsive Fkbp8 knockout mouse model to elucidate the molecular mechanism(s) of folate nonresponsiveness. Neurospheres were grown from neural stem cells isolated from the lumbar neural tube of E9.5 Fkbp8 (-/-) and wild-type embryos. Immunostaining was used to determine the protein levels of oligodendrocyte transcription factor 2 (Olig2), Nkx6.1, class III beta-tubulin (TuJ1), O4, glial fibrillary acidic protein (GFAP), histone H3 Lys27 trimethylation (H3K27me3), ubiquitously transcribed tetratricopeptide repeat (UTX), and Msx2, and quantitative real-time (RT)-PCR was used to determine the message levels of Olig2, Nkx6.1, Msx2, and noggin in neural stem cells differentiated in the presence and absence of folic acid.Fkbp8 (-/-)-derived neural stem cells showed (i) increased noggin expression; (ii) decreased Msx2 expression; (iii) premature differentiation--neurogenesis, oligodendrogenesis (Olig2 expression), and gliogenesis (GFAP expression); and (iv) increased UTX expression and decreased H3K27me3 polycomb modification. Exogenous folic acid did not reverse these markers.Folate nonresponsiveness could be attributed in part to increased noggin expression in Fkbp8 (-/-) embryos, resulting in decreased Msx2 expression. Folate treatment further increases Olig2 and noggin expression, thereby exacerbating ventralization.

Background: Open spina bifida is the most complex congenital abnormality compatible with long-term survival. This report outlines the 20- to 25-year outcome for our original cohort of patients with a myelomeningocele treated in a nonselective, prospective manner. Methods: Of the initial 118 children, 71 patients were available for our most recent review. Nineteen patients have been lost to follow-up and 28 patients have died. Data were collected on: motor level, shunt status, education/employment, seizure history, mobility, bladder/bowel continence, tethered cord, scoliosis, latex allergy, posterior cervical decompression, tracheostomy and/or gastrostomy tube. Results: Mortality (24%) continues to climb into young adulthood. Eighty-six percent of the cohort have cerebrospinal fluid diversion, with 95% having undergone at least one shunt revision. Thirty-two percent have undergone a tethered cord release, with 97% having an improvement or stabilization in their preoperative symptoms. Forty-nine percent have scoliosis, with 43% eventually requiring a spinal fusion. Sixteen patients (23%) have had at least one seizure. Eighty-five percent are attending or have graduated from high school and/or college. More than 80% of young adults have social bladder continence. Approximately 1/3 of patients are allergic to latex, with 6 patients having experienced a life-threatening reaction. Conclusion: At least 75% of children born with a myelomeningocele can be expected to reach their early adult years. Late deterioration is common. One of the greatest challenges in medicine today is establishing a network of care for these adults with spina bifida.

Polysialic acid (PSA) is a developmentally regulated carbohydrate found primarily on neural cell adhesion molecules (NCAM) in embryonic tissues. The majority of NCAM in adult tissues lacks this unique carbohydrate, but polysialylated NCAM (PSA-NCAM) is present in adult brain regions where neural regeneration persists and in some pediatric brain tumors such as medulloblastoma, which show greater propensity for leptomeningeal spread. Pax3, a developmentally regulated paired homeodomain transcription factor, is thought to be involved in the regulation of neural cell adhesion molecules. Overexpression of murine Pax3 into a human medulloblastoma cell line (DAOY) resulted in an increase in NCAM polysialylation and a 2-4-fold increase in alpha2, 8-polysialyltransferase type II mRNA levels. No difference was observed in alpha2,8-polysialyltransferase type IV message. The addition of PSA to NCAM changed the adhesive behavior of these Pax3 transfectants. Transfectants expressing high PSA-NCAM show much less NCAM-dependent aggregation than those with less PSA-NCAM. In addition, Pax3 transfectants having high PSA-NCAM show heterophilic adhesion involving polysialic acid to heparan sulfate proteoglycan and agrin. These observations suggest that a developmentally regulated transcription factor, Pax3, could affect NCAM polysialylation and subsequently cell-cell and cell-substratum interaction.

A large Filipino-American family with progressive matrilineal hearing loss, premature graying, depigmented patches, and digital anomalies was ascertained through a survey of a spina bifida clinic for neural crest disorders. Deafness followed a matrilineal pattern of inheritance and was associated with the A1555G mutation in the 12S rRNA gene (MTRNR1) in affected individuals as well as unaffected maternal relatives. Several other malformations were found in carriers of the mutation. The proband had a myelocystocele, Arnold-Chiari type I malformation, cloacal exstrophy, and severe early-onset hearing loss. Several family members had premature graying, white forelock, congenital leukoderma with or without telecanthus, somewhat suggestive of a Waardenburg syndrome variant. In addition to the patient with myelocystocele, two individuals had scoliosis and one had segmentation defects of spinal vertebrae. The syndromic characteristics reported here are novel for the mitochondrial A1555G substitution, and may result from dysfunction of mitochondrial genes during early development. However, the mitochondrial A1555G mutation is only rarely associated with neural tube defects as it was not found in a screen of 218 additional individuals with spina bifida, four of whom had congenital hearing loss.

Neural tube defects (NTD) are one of the most common birth defects and are caused by both environmental and genetic factors. The approach to identifying the genes predisposing to NTD, through linkage analysis and candidate gene analysis, is reviewed along with characteristics of a large, nationally ascertained cohort of families. Results from specific assessments of p53, PAX3 and MTHFR failed to suggest that these genes play a major role in NTD development in these families. Advances in genetic laboratory and statistical techniques have made this a prime opportunity for investigation into the causes of complex disorders, such as NTD. However, traditional approaches may prove to be challenging due to the difficulty of ascertaining samplable multiplex families.

A hereditary contribution to the etiology of neural tube defects (NTDs) has been suggested by clinical studies and animal models. To evaluate the hypothesis that common genes are important for both neural tube defects and neural crest anomalies, we examined children with developmental abnormalities of the spinal cord for anomalies of neural crest-derived structures. Neural crest anomalies, particularly auditory and pigmentary disorders, were identified and classified according to inheritance and type of anomaly. Of the 515 children screened, 44 (8.5%) had neural crest anomalies, 20 (3.9%) of which were apparently familial. Another 19 (3.7%) families had neural crest anomalies in two or more close relations, but the NTD subject was unaffected. Sixteen (3.1%) children with NTDs had a recognizable syndrome, including nine (1.7%) with a subtype of the Waardenburg syndromes. The coincidence of familial neural crest anomaly syndromes in subjects with spina bifida implies that defects in genes underlying neural crest development may contribute to the etiology of neural tube defects in a fraction of cases. The rate of anomalies and familial syndromes of neural crest-derived structures must be assessed in an adequate control sample to evaluate whether or not these abnormalities constitute risk factors for NTDs.

Embryonic development is determined by preset intrinsic programs and extrinsic signals. To explore the possibility that transcription factors are present at the onset of development, preparations of yolk, albumin, and blastoderm from unfertilized and fertilized white Leghorn chicken eggs were screened by a panel of 16 transcription factor antibodies with Western blot techniques. Yolk was positive for 13 transcription factors, whereas blastoderm was positive for 10, and albumin was positive for 5. In yolk, several transcription factors, GATA-2, E2F-1, MyoD, and TFIID, were developmentally regulated. These results indicate that intracellular yolk and extracellular albumin contain transcription factors which presumably influence early chick embryonic development from prefertilization to the late blastoderm stage. Thus, the utility of preset maternal transcription factors within yolk and albumin complement maternally derived mRNA to determine the early development of the zygote.

It has been known for years that the assembly of the nervous system is under genetic control. During the last 10 years, the genes that direct the formation of the brain and spinal cord have begun to be discovered at an amazing pace. Mutations in the fruit fly and advances in molecular genetics have led the way. Gene mutations that cause many of the malformations of the human brain and spinal cord are now known. This has many physician-scientists hoping that an understanding of cause might lead to cure.

From a spina bifida clinic we have identified two patients with a syndrome of myelomeningocele and Waardenburg syndrome type 3 (WS3). The patients each possess a single, de novo, interstitial deletion of chromosome 2 (2q35–36.2), including the PAX3 gene. Deletion of PAX3 was confirmed by fluorescence in situ hybridization (FISH). Analysis with PAX3 and flanking microsatellites shows that the deleted interval of chromosome 2 is of paternal origin and is at least 2 and 6 cM in the two patients. Interstitial deletions in this region result in the Waardenburg syndrome (WS1), but have not been associated with neural tube defects (NTDs). Although other etiologies have not been formally excluded, these patients raise the possibility of a digenic etiology of their NTDs via a genetic interaction of the deleted PAX3 gene with a second unidentified locus. Am. J. Med. Genet. 75:401-408, 1998. © 1998 Wiley-Liss, Inc.

Important decisions are made at the first meeting of the pediatric neurosurgeon and the parents of a child born with a myelomeningocele. It is essential that the neurosurgeon be aware of the most recent outcome data for children born with a myelomeningocele. Only when armed with this information can he or she make it possible for the family to participate in an informed decision. An understanding of the ethical principles involved in deciding for an individual too young to make any decision is a major part of this process.

Background: The cause of congenital glaucoma is unknown. Methods: To determine whether the site of impaired aqueous outflow is the entrance to the trabecular meshwork (TM), within the TM, the aqueous drainage plexus, or a combination thereof, the process of TM development was examined by scanning and transmission electron microscopy on postnatal day 3 and weeks 1, 2, 3, 4, and 6 in New Zealand rabbits homozygous for the buphthalmic (bu/bu) gene compared with age-matched controls. Results: Openings to the entrance of the TM in congenital glaucoma were observed, and there was no evidence of an endothelial membrane occluding aqueous flow to the TM. The morphology of the congenital glaucoma TM was abnormal in all bu/bu rabbits by 2 weeks and was characterized by a smaller entrance to the TM at the iris base, smaller intertrabecular openings within and between the trabecular lamellae, and at 6 weeks, iris pillars with extensive lateral extensions in the angle recess. Most intertrabecular spaces were open, however, the inner intertrabecular spaces adjacent to the aqueous plexus were compressed. Conclusion: These results suggest the development of congenital glaucoma, which involves a mutation in an autosomal recessive gene and leads to loss of function of a gene(s) required for the differentiation of the TM.

If meningomyelocele is indeed a progressive intrauterine process, then early delivery or possibly intrauterine repair of meningomyelocele becomes an issue. Utilizing the delayed splotch (Spd) mouse, a genetically transmitted neural tube defect model, we looked for evidence of abnormalities of neural tissue exposed to amniotic fluid. Affected embryonic and fetal mice were examined with the light microscope, and also with the transmission and scanning electron microscope. Neuronal development and programmed cell death paralleled normal fetal development. No evidence of inflammation on or within the exposed neural tissue was observed. Because the vascular supply to the alar and basilar plate are different, vascular development was also examined and no difference could be found. In conclusion, we found no evidence of deterioration of the exposed neural tube during the gestational period of a mouse, which suggests that exposure of unneurulated spinal cord to amniotic fluid is not a risk factor to the fetus with a neural tube defect.

To compare the functional development of the fourth ventricular outlet in the myeloschisis-Chiari malformation complex with that of a normal brain, the chronological development of the outlet in C57BL/6J non-neural-tube defect mouse embryos was examined as the first step. Then we compared the results with those of homozygotic delayed splotch (Spd) mouse embryos which had neural-tube defects (NTDs). Ferrous chloride (Prussian blue) solution was injected into the lateral or mesencephalic ventricle on gestation days 13-16 in the case of control C57BL/6J mouse embryos and on gestation days 14-16 in the case of homozygotic Spd mouse embryos which had open spinal NTDs and hindbrain anomalies comparable to human Chiari malformation. At 30 min after the injection, acid fixative was infused through the heart to set off the Prussian blue reaction, which makes the dye visible by the precipitation of ferric chloride. According to the present method, more than 75% of C57BL/6J mouse (non-NTD control) embryos showed the evidence of function of the fourth ventricular (4V) outlet from gestation day 15. It was difficult to apply the same method to Spd mouse embryos with NTDs due to the small size of ventricles. Only 4 injections were successful, of which 3 showed the functioning evidence of the 4V outlet. Though the number of mouse embryos with NTDs studied was small, the results suggest that the chronological progress of functional opening of the fourth ventricle in mouse embryos with NTDs is similar to that of control non-NTD embryos.

An overview of MR imaging of the pediatric spine is presented with a systematic approach to the analysis of spinal dysraphic disorders. An introduction to the most commonly used MR imaging techniques, with an emphasis on the newer pulse sequence of cine MR flow of the cerebrospinal fluid and embryogenesis of the spine and spinal cord, provides the orientation for diagnosis of this common pediatric disorder.

Open neural tube defects developed in 12 of 122 alive chick embryos treated with exogenous lectin (concanavalin-A) at stages between 10 or 14 as defined by Hamburger and Hamilton. Embryos treated at stage 10, the time of anterior neuropore closure, developed exencephaly or extensive neural openings from the level of rhombencephalon to the thoracic spinal cord, while embryos treated at stages between 11 and 14, at posterior neuropore closure, developed only small myeloschisis in the thoracolumbar region. The failure of neural tube closure at a critical time is a major cause of neural tube defects.

BACKGROUND Neural tube defects (NTDs) are the second most common birth defects, after congenital heart defects. Telomerase, the reverse transcriptase that maintains telomere DNA, has been shown to be important for neural tube development and bilateral symmetry in the brain. In knockout mice null for the telomerase RNA component (TERC), telomere loss results in the failure of neural tube closure, primarily at the forebrain and midbrain. METHODS We investigated TERC for variants that may predispose to human NTDs in 477 NTD cases with a variety of phenotypic presentations. RESULTS Two novel single nucleotide polymorphisms were identified in the human TERC sequence but showed no association with the NTD phenotype. CONCLUSIONS Variants in TERC are unlikely to be a major risk factor for the most common form of human NTDs, lumbosacral myelomeningocele. Birth Defects Research (Part A), 2004. © 2004 Wiley-Liss, Inc.