Methylome analysis for spina bifida shows SOX18 hypomethylation as a risk factor with evidence for a complex (epi)genetic interplay to affect neural tube development.

BACKGROUND: Neural tube defects (NTDs) are severe congenital malformations that arise from failure of neurulation during early embryonic development. The molecular basis underlying most human NTDs still remains largely unknown. Based on the hypothesis that folic acid prevents NTDs by stimulating methylation reactions, DNA methylation changes could play a role in NTDs. We performed a methylome analysis for patients with myelomeningocele (MMC). Using a candidate CpG analysis for HOX genes, a significant association between HOXB7 hypomethylation and MMC was found. METHODS: In the current study, we analyzed leukocyte methylome data of ten patients with MMC and six controls using Illumina Methylation Analyzer and WateRmelon R-packages and performed validation studies using larger MMC and control cohorts with Sequenom EpiTYPER. RESULTS: The methylome analysis showed 75 CpGs in 45 genes that are significantly differentially methylated in MMC patients. CpG-specific methylation differences were next replicated for the top six candidate genes ABAT, CNTNAP1, SLC1A6, SNED1, SOX18, and TEPP but only for the SOX18 locus a significant overall hypomethylation was observed (P value = 0.0003). Chemically induced DNA demethylation in HEK cells resulted in SOX18 hypomethylation and increased expression. Injection of sox18 mRNA in zebrafish resulted in abnormal neural tube formation. Quantification of DNA methylation for the SOX18 locus was also determined for five families where parents had normal methylation values compared to significant lower values for both the MMC as their non-affected child. SOX18 methylation studies were performed for a MMC patient with a paternally inherited chromosomal deletion that includes BMP4. The patient showed extreme SOX18 hypomethylation similar to his healthy mother while his father had normal methylation values. CONCLUSIONS: This is the first genome-wide methylation study in leukocytes for patients with NTDs. We report SOX18 as a novel MMC risk gene but our findings also suggest that SOX18 hypomethylation must interplay with environmental and (epi)genetic factors to cause NTDs. Further studies are needed that combine methylome data with next-generation sequencing approaches to unravel NTD etiology.

DNA methylation analysis of Homeobox genes implicates HOXB7 hypomethylation as risk factor for neural tube defects.

Neural tube defects (NTDs) are common birth defects of complex etiology. Though family- and population-based studies have confirmed a genetic component, the responsible genes for NTDs are still largely unknown. Based on the hypothesis that folic acid prevents NTDs by stimulating methylation reactions, epigenetic factors, such as DNA methylation, are predicted to be involved in NTDs. Homeobox (HOX) genes play a role in spinal cord development and are tightly regulated in a spatiotemporal and collinear manner, partly by epigenetic modifications. We have quantified DNA methylation for the different HOX genes by subtracting values from a genome-wide methylation analysis using leukocyte DNA from 10 myelomeningocele (MMC) patients and 6 healthy controls. From the 1575 CpGs profiled for the 4 HOX clusters, 26 CpGs were differentially methylated (P-value < 0.05; ß-difference > 0.05) between MMC patients and controls. Seventy-seven percent of these CpGs were located in the HOXA and HOXB clusters, with the most profound difference for 3 CpGs within the HOXB7 gene body. A validation case-control study including 83 MMC patients and 30 unrelated healthy controls confirmed a significant association between MMC and HOXB7 hypomethylation (-14.4%; 95% CI: 11.9-16.9%; P-value < 0.0001) independent of the MTHFR 667C>T genotype. Significant HOXB7 hypomethylation was also present in 12 unaffected siblings, each related to a MMC patient, suggestive of an epigenetic change induced by the mother. The inclusion of a neural tube formation model using zebrafish showed that Hoxb7a overexpression but not depletion resulted in deformed body axes with dysmorphic neural tube formation. Our results implicate HOXB7 hypomethylation as risk factor for NTDs and highlight the importance for future genome-wide DNA methylation analyses without preselecting candidate pathways.

Long-term outcome of intravesical oxybutynin in children with detrusor-sphincter dyssynergia: with special reference to age-dependent parameters.

AIMS: Intravesical instillation of oxybutynin is an accepted and effective treatment in children with neuropathic bladder-sphincter dysfunction, when oral oxybutynin results in inadequate suppression of detrusor overactivity or intolerable side effects. However, as yet no data are available on long-term use and outcome. METHODS: A patient cohort with detrusor-sphincter dyssynergia that started oral oxybutynin between 1995 and 1997 was re-evaluated 15 ± 1 years after the switch from oral to intravesical (n = 10), with urodynamic investigations, renal ultrasounds, DMSA-scintigraphy, (51)Cr-EDTA-clearance, and validated questionnaires on incontinence and quality of life. RESULTS: At follow-up, cystometric bladder capacity (CBC) had increased to the 25-50% percentiles for age, from the 5% percentile; mean end-filling pressure, 24.5 ± 14.4 cm H2O, had returned to the safe zone; bladder compliance expressed as a fraction of normal compliance for age (Wahl units) showed a statistically significant increase. At follow-up, the prevalence of renal scars was 30% (95% CI: 6-65%). Kidney lengths correlated with scarring at DMSA-scintigraphy, (51)Cr-EDTA-clearance did not. In 2 years of oral oxybutynin we documented 10 pyelonephritic episodes, in 15 years of intravesical oxybutynin only three. Urinary continence was reported as satisfying, its impact on quality of life as acceptable. CONCLUSION: Percentile charts for cystometric bladder capacity and individual kidney lengths, age-dependent parameters, were invaluable in estimating long-term outcome, and the same goes for bladder compliance in Wahl units. We can conclude that intravesical oxybutynin provided more than adequate suppression of detrusor activity, without side effects, over a period of 15 years.

Home screening for bacteriuria in children with spina bifida and clean intermittent catheterization.

BACKGROUND: Significant bacteriuria (SBU) and urinary tract infections (UTIs) are common in patients with spina bifida and neuropathic detrusor sphincter dysfunction. Laboratory agar plated culture is the gold standard to establish SBU. It has the disadvantage of diagnostic and subsequent therapeutic delay. Leukocyte esterase tests (LETs) and dip slides proved to be useful in the general populations to exclude SBU and UTI. The aim of this study was to evaluate the reliability of LET and dip slide in children with spina bifida without symptoms of UTI. The reliability in children with asymptomatic SBU was not studied before. METHODS: In one hundred and twelve children with spina bifida on clean intermittent catheterization LETs and dip slides were compared with laboratory cultures. Both tests and agar plated cultures were performed on catheterized urine samples. The hypothesis was that the home tests are as accurate as laboratory cultures. RESULTS: A SBU was found in 45 (40%) of the 112 laboratory cultures. A negative LET excluded SBU (negative predictive value 96%), while a positive LET had a positive predictive value of 72%. The false positive rate was 28%. Dip slide determination of bacterial growth had no added value, other than serving as transport medium. CONCLUSIONS: In spina bifida children, leukocyte esterase testing can be used to exclude significant bacteriuria at home, while dip slide tests have no added value to diagnose or exclude significant bacteriuria.

Antibiotic prophylaxis for urinary tract infections in children with spina bifida on intermittent catheterization.

PURPOSE: Antibiotic prophylaxis (low dose chemoprophylaxis) has been prescribed since the introduction of clean intermittent catheterization in children with spina bifida. We hypothesized that stopping low dose chemoprophylaxis does not increase the number of urinary tract infections in these patients. MATERIALS AND METHODS: A total of 176 patients with spina bifida participated in a randomized controlled trial (ISRCTN trial number 56278131) of either continuation or discontinuation of low dose chemoprophylaxis. During the 18-month study period biweekly urine samples were evaluated for leukocyturia and bacteriuria with dipsticks and cultures. Asymptomatic significant bacteriuria (positive culture results without clinical symptoms) and urinary tract infections (significant bacteriuria with clinical symptoms and leukocyturia) were analyzed. RESULTS: Discontinuation of low dose chemoprophylaxis resulted in higher rates of asymptomatic significant bacteriuria (incidence rate ratio 1.23, 95% CI 1.08-1.40, p = 0.002) and urinary tract infection (IRR 1.44, 95% CI 1.13-1.83, p = 0.003). For urinary tract infection the number needed to harm was 2.2, that is if 2 patients discontinued low dose chemoprophylaxis for a year, 1 extra urinary tract infection would result. Febrile urinary tract infection occurred once in every 30 patient-years and slightly more often in the discontinuation group (relative risk 2.0, 95% CI 0.38-10.6, p = 0.4). Of 88 patients allocated to discontinuation of low dose chemoprophylaxis 38 (43%) switched back to chemoprophylaxis. The urinary tract infection rate was nonsignificantly higher in the presence of vesicoureteral reflux. Male gender and a low pre-study rate of urinary tract infection predicted successful discontinuation. CONCLUSIONS: Patients with spina bifida on clean intermittent catheterization and antibiotic prophylaxis for urinary tract infections can safely discontinue this prophylaxis, in particular males, patients with low urinary tract infection rates and patients without vesicoureteral reflux.

Metformin therapy to reduce weight gain and visceral adiposity in children and adolescents with neurogenic or myogenic motor deficit.

The aim of this randomized, placebo-controlled study was to explore the effect of metformin in children with a neurogenic or myogenic motor deficit, who are therefore prone to develop overweight, adiposity, and insulin resistance. Study participants (n = 42) had a mean age of 15.5 yr, a short stature (height -2.4 SD), a relatively high BMI (+1.7 SD), and a high body fat fraction (41.9% or +2.8 SD). Abdominal CT confirmed the high fat mass and disclosed a high fraction of visceral fat. As expected, insulin resistance was increased. As compared to placebo, metformin intake for 6 months exerted an insulin sensitizing effect and lowered weight (mean difference of 2 kg within 6 months, p = 0.007) and BMI (p = 0.016). Weight loss appeared to be primarily due to loss of visceral fat ( approximately 20% vs. placebo; p < 0.0001). Results were similar across diagnostic subgroups. In conclusion, metformin treatment for 6 months was associated with a rise in insulin sensitivity and with a reduction of visceral adiposity in children and adolescents with a primary muscle disorder or with a neural tube defect. These findings suggest that insulin resistance underpins, at least partly, the overweight and visceral adiposity of these patients, who are not necessarily obese.

The neurogenic bladder: medical treatment.

Neurogenic bladder sphincter dysfunction (NBSD) can cause severe and irreversible renal damage and bladder-wall destruction years before incontinence becomes an issue. Therefore, the first step in adequate management is to recognize early the bladder at risk for upper- and lower-tract deterioration and to start adequate medical treatment proactively. Clean intermittent catheterization combined with anticholinergics (oral or intravesical) is the standard therapy for NBSD. Early institution of such treatment can prevent both renal damage and secondary bladder-wall changes, thereby potentially improving long-term outcomes. In children with severe side effects or with insufficient suppression of detrusor overactivity despite maximal dosage of oral oxybutynin, intravesical instillation is an effective alternative. Intravesical instillation eliminates systemic side effects by reducing the first-pass metabolism and, compared with oral oxybutynin, intravesical oxybutynin is a more potent and long-acting detrusor suppressor. There is growing evidence that with early adequate treatment, kidneys are saved and normal bladder growth can be achieved in children so they will no longer need surgical bladder augmentation to achieve safe urinary continence in adolescence and adulthood.

Segmental spinal cord hypoplasia and meningocele with preservation of medullary function: case report.

BACKGROUND: In very rare dysraphic cases, it is not clear whether the primary abnormalities are in the neural elements, or in the bony elements. CASE DESCRIPTION: We describe a case of segmental hypoplasia of the spinal cord, with absent nerve roots in the afflicted segments, and associated meningocele and vertebral abnormalities. We illustrate the arguments for and against the classification of this lesion either as an atypical case of myelomeningocele (MMC) or as a mild case of segmental spinal dysgenesis (SSD). CONCLUSIONS: Possibly, in this exceptional case, the primary defect is in the neural tissue like in more usual cases of MMC and not in the spine, like in segmental spinal dysgenesis.