Multidisciplinary spina bifida clinic: the Chicago experience.

Open spina bifida (open SB) is the most complex congenital abnormality of the central nervous system compatible with long-term survival. Multidisciplinary care is required to address the effect of this disease on the neurological, musculoskeletal, genitourinary, and gastrointestinal systems, as well as the complex psychosocial impact on the developing child. Individuals with SB benefit from the involvement of neurosurgeons, orthopedic surgeons, urologists, physical medicine and rehabilitation specialists, pediatricians, psychologists, physical/occupational/speech therapists, social workers, nurse coordinators, and other personnel. Multidisciplinary clinics are the gold standard for coordinated, optimal medical and surgical care. Ann and Robert H. Lurie Children's Hospital, formerly known as Children's Memorial Hospital, was one of the first hospitals in the USA to manage patients with this complex disease in a multidisciplinary manner. We describe the longitudinal experience of the multidisciplinary Spina Bifida Center at our institution and highlight the advances that have arisen from this care model over time. This clinic serves as an exemplar of organized, effective, and patient-centered approach to the comprehensive care of people living with open SB.

A critical role of noggin in developing folate-nonresponsive NTD in Fkbp8 -/- embryos.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Folic acid remodels chromatin on Hes1 and Neurog2 promoters during caudal neural tube development.

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.

Key basic helix-loop-helix transcription factor genes Hes1 and Ngn2 are regulated by Pax3 during mouse embryonic development.

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.

The changing incidence of myelomeningocele and its impact on pediatric neurosurgery: a review from the Children's Memorial Hospital.

INCIDENCE: 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. IMPACT OF CHANGES: 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. CLINICAL IMPLICATIONS: One of the greatest challenges facing these young adults is the transitioning of their medical care into an adult medical community.

Linkage to chromosome 2q36.1 in autosomal dominant Dandy-Walker malformation with occipital cephalocele and evidence for genetic heterogeneity.

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.

Current Management Strategies of Hydrocephalus in the Child With Open Spina Bifida.

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.

The anatomy of the ventricular system.

The embryology of the ventricular development of the brain assists in understanding the final relations between structures forming these cavities. An accurate concept of this anatomy allows the endoscopist to maneuver within the ventricular system.

Predictors of urinary continence following tethered cord release in children with occult spinal dysraphism.

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.

Amniotic fluid and serum biomarkers from women with neural tube defect-affected pregnancies: a case study for myelomeningocele and anencephaly: clinical article.

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.

Fetal neural tube stem cells from Pax3 mutant mice proliferate, differentiate, and form synaptic connections when stimulated with folic acid.

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 (P<0.05). 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 (P<0.01). 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.

Nuclear localization of folate receptor alpha: a new role as a transcription factor.

Folic acid (FA) has traditionally been associated with prevention of neural tube defects; more recent work suggests that it may also be involved in in the prevention of adult onset diseases. As the role of FA in human health and disease expands, it also becomes more critical to understand the mechanisms behind FA action. In this work we examined the hypothesis that folate receptor alpha (FRα) acts as a transcription factor. FRα is a GPI-anchored protein and a component of the caveolae fraction. The work described here shows that FRα translocates to the nucleus, where it binds to cis-regulatory elements at promoter regions of Fgfr4 and Hes1, and regulates their expression. The FRα recognition domain mapped to AT rich regions on the promoters. Until this time FRα has only been considered as a folate transporter, these studies describe a novel role for FRα as a transcription factor.

Epigenetic regulation of sensory neurogenesis in the dorsal root ganglion cell line ND7 by folic acid.

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.

Maternal intake of folic acid and neural crest stem cells.

Maternal folic acid (FA) intake has beneficial effects in preventing neural tube defects and may also play a role in the prevention of adult onset diseases such as Alzheimer's disease, dementia, neuropsychiatric disorders, cardiovascular diseases, and cerebral ischemia. This review will focus on the effects of maternal FA intake on neural crest stem cell proliferation and differentiation. Although FA is generally considered beneficial, it has the potential of promoting cell proliferation at the expense of differentiation. In some situations, this may lead to miscarriage or postnatal developmental abnormalities. Therefore, a blind approach such as "FA for everyone" is not necessarily the best course of action. Ultimately, the best approach for FA supplementation, and potentially other nutritional supplements, will include customized patient genomic profiles for determining dose and duration.

Retethering of transected fatty filum terminales.

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.

Role of Pax3 acetylation in the regulation of Hes1 and Neurog2.

Pax3 plays a role in regulating Hes1 and Neurog2 activity and thereby stem cell maintenance and neurogenesis. A mechanism for Pax3 regulation of these two opposing events, during caudal neural tube development, is examined in this study. Pax3 acetylation on C-terminal lysine residues K437 and K475 may be critical for proper regulation of Hes1 and Neurog2. Removal of these lysine residues increased Hes1 but decreased Neurog2 promoter activity. SIRT1 deacetylase may be a key component in regulating Pax3 acetylation. Chromatin immunoprecipitation assays showed that SIRT1 is associated with Hes1 and Neurog2 promoters during murine embryonic caudal neural tube development at E9.5, but not at E12.5. Overexpression of SIRT1 decreased Pax3 acetylation, Neurog2 and Brn3a positive staining. Conversely, siRNA-mediated silencing of SIRT1 increased these factors. These studies suggest that Pax3 acetylation results in decreased Hes1 and increased Neurog2 activity, thereby promoting sensory neuron differentiation.