Obstetrical Outcomes of Amniocentesis or Chorionic Villus Sampling in Dichorionic Twin Pregnancies.

BACKGROUND: Under certain situations, women with twin pregnancies may be counseled to undergo invasive prenatal diagnostic testing. Chorionic villus sampling and amniocentesis are the two generally performed invasive prenatal diagnostic tests. Studies comparing procedure-related fetal loss between first-trimester chorionic villus sampling and second-trimester amniocentesis in twin pregnancies are limited. This study aimed to evaluate the procedure-related fetal loss and the obstetrical outcomes of these two procedures, chorionic villus sampling and amniocentesis in twin pregnancies. METHODS: The data from dichorionic-diamniotic twin pregnancies on which first-trimester chorionic villus sampling (n = 54) or second-trimester amniocentesis (n = 170) was performed between December 2006 and January 2017 in a single center were retrospectively analyzed. The procedure-related fetal loss was classified as loss of one or all fetuses within 4 weeks of procedure, and overall fetal loss was classified as loss of one or all fetuses during the gestation. The groups were compared with respect to the procedure-related and obstetrical outcomes. RESULTS: The difference in proportion of procedure-related fetal loss rate (1.9% for chorionic villus sampling vs. 1.8% for amniocentesis; P = 1.000) and the overall fetal loss rate (7.4% for chorionic villus sampling vs. 4.7% for amniocentesis; P = 0.489) between the two groups was not significant. The mean gestational ages at delivery were not statistically significant. CONCLUSION: Both the overall fetal loss rate and the procedure-related fetal loss rate of chorionic villus sampling and amniocentesis in dichorionic twin pregnancies had no statistical significance. Both procedures can be safely used individually.

Scoring model to predict massive post-partum bleeding in pregnancies with placenta previa: A retrospective cohort study.

AIM: We aimed to identify factors associated with massive post-partum bleeding in pregnancies with placenta previa and to establish a scoring model to predict post-partum severe bleeding. METHODS: A retrospective cohort study was performed in 506 healthy singleton pregnancies with placenta previa from 2006 to 2016. Cases with intraoperative blood loss (≥2000 mL), packed red blood cells transfusion (≥4), uterine artery embolization, or hysterectomy were defined as massive bleeding. After performing multivariable analysis, using the adjusted odds ratios (aOR), we formulated a scoring model. RESULTS: Seventy-three women experienced massive post-partum bleeding (14.4%). After multivariable analysis, seven variables were associated with massive bleeding: maternal old age (≥35 years; aOR 1.79, 95% confidence interval [CI] 1.00-3.20, P = 0.049), antepartum bleeding (aOR 4.76, 95%CI 2.01-11.02, P < 0.001), non-cephalic presentation (aOR 3.41, 95%CI 1.40-8.30, P = 0.007), complete placenta previa (aOR 1.93, 95%CI 1.05-3.54, P = 0.034), anterior placenta (aOR 2.74, 95%CI 1.54-4.89, P = 0.001), multiple lacunae (≥4; aOR 2.77, 95%CI 1.54-4.99, P = 0.001), and uteroplacental hypervascularity (aOR 4.51, 95%CI 2.30-8.83, P < 0.001). We formulated a scoring model including maternal old age (<35: 0, ≥35: 1), antepartum bleeding (no: 0, yes: 2), fetal non-cephalic presentation (no: 0, yes: 2), placenta previa type (incomplete: 0, complete: 1), placenta location (posterior: 0, anterior: 1), uteroplacental hypervascularity (no: 0, yes: 2), and multiple lacunae (no: 0, yes: 1) to predict post-partum massive bleeding. According to our scoring model, a score of 5/10 had a sensitivity of 81% and a specificity of 77% for predicting massive post-partum bleeding. The area under the receiver-operator curve was 0.856 (P < 0.001). The negative predictive value was 95.9%. CONCLUSION: Our scoring model might provide useful information for prediction of massive post-partum bleeding in pregnancies with placenta previa.

Early detection of preeclampsia using inhibin a and other second-trimester serum markers.

OBJECTIVE: The purpose of this study was to determine whether second-trimester maternal serum markers including inhibin A are useful for the detection of preeclampsia. METHODS: Between January 2005 and March 2009, we analyzed the data of 4,764 subjects who underwent second-trimester multiple-marker screening for Down syndrome. Serum samples were assayed at 15+0 to 20+6 weeks for maternal serum alpha-fetoprotein (MSAFP), human chorionic gonadotrophin (hCG), unconjugated estriol (uE(3)) and inhibin A. We reviewed all medical records retrospectively, and assessed the relationships of several markers with preeclampsia using logistic regression analysis. RESULTS: The study sample included 41 patients who developed preeclampsia and a control group consisting of the other 4,723 healthy subjects treated between January 2005 and March 2009. There were no significant differences in gestational ages at blood sampling, maternal weights, gravidity and parity between the two groups. However, the mean ages, Apgar scores, gestational age at delivery and neonatal weights were significantly different between the study group and the control group. The levels of markers in the study group were significantly increased compared to the control group, 1.76 ± 2.68 for inhibin A, 1.18 ± 0.69 for MSAFP, and 1.62 ± 1.18 for hCG, but uE(3) did not differ significantly between the two groups. The AUC of inhibin A was 0.715, but the AUC of a three-marker combination model (0.800) was even better. A mid-trimester inhibin A concentration of 1.5 MoM or greater had a sensitivity of 60% and a false-positive rate of 16% for the prediction of preeclampsia. Inhibin A was the best predictor of preeclampsia. Three other markers were reliable predictive markers of preeclampsia. CONCLUSIONS: Inhibin A and other second-trimester serum markers may be useful for early detection of preeclampsia. Inhibin A was in fact the most important predictable marker among the markers we surveyed. The results of this study support those of previous studies, and provide quantified data elucidating the occurrence of preeclampsia.

A preeclampsia risk prediction model based on maternal characteristics and serum markers in twin pregnancy.

AIM: To develop preeclampsia (PE) risk prediction model based on maternal characteristics and serum markers at the first and second trimester in the twin pregnancy. METHODS: Between January 2005 and September 2017, we retrospectively reviewed medical records of 532 twin pregnant women who underwent maternal serum integrated test and gave birth at a Bundang CHA Medical Center. Maternal serum pregnancy-associated plasma protein A (PAPP-A) was determined at 10+0 to 13+6 weeks and the serum alpha-fetoprotein (MSAFP), human chorionic gonadotrophin (hCG), unconjugated estriol (uE3) and inhibin A were assayed at 14+0 to 22+0 weeks. We assessed the relationships of maternal characteristics and serum markers by using multiple logistic regression analysis. RESULTS: The study included 35 patients who diagnosed PE and a control group consisting of the other 497 patients in twin pregnancy. There were no significant differences in the maternal age and body mass index (BMI) between two groups. However, the gestational age and placenta weight at delivery were significantly different between groups (p < .001, p = .005, respectively. Among the maternal serum markers, inhibin A value was significantly higher in women with PE compared to those without preeclampsia (p < .001). In addition, we predicted the PE using maternal age, BMI, uE3, and inhibin A which were achieving an area under the curve of 0.73 overall in twin pregnancy. CONCLUSION: A risk prediction model of PE which combined maternal age, BMI, uE3, and inhibin A was better early predictors than any individual marker twin pregnancy.

First-trimester screening for early preeclampsia risk using maternal characteristics and estimated placental volume.

OBJECTIVE: The purpose of this study was to evaluate the strategy of screening for preeclampsia (PE) in the first trimester based on maternal characteristics, and estimated placental volume (EPV). METHODS: A prospective cohort study was performed with 351 women enrolled, of which 13 women developed PE. This study analyzed the risk of PE according to ultrasonography findings of the placenta, maternal characteristics, and serum markers. The placental ultrasound exam and maternal serum pregnancy-associated plasma protein A (PAPP-A) was determined at 11+0 to 13+6 weeks and the serum alpha-fetoprotein (MSAFP), human chorionic gonadotropin (hCG), unconjugated estriol (uE3), and inhibin A were assayed at 14+0 to 22+0 weeks. We reviewed all antenatal medical screening records and assessed the relationships of EPV of ultrasound examination, maternal characteristics, and serum markers by using multiple logistic regression analysis. RESULTS: Thirteen of the 351 women (3.7%) developed PE in singleton pregnancy. The gestational age at delivery was significantly different between the normal and PE group (p < .001). In the PE group, the placental weight at delivery was not statistically different between the normal and the PE group. The EPV at the first trimester was significantly lower in women with PE compared to those without PE (p = .002). In addition, we predicted PE using combined maternal age, BMI, and EPV, which were achieving an area under the curve of 0.83 overall. CONCLUSION: A risk prediction model of PE, which combined maternal age, BMI, and EPV can be adopted for the screening of PE at the first trimester in singleton pregnancy.

Pregnancy outcomes of elective induction in low-risk term pregnancies: A propensity-score analysis.

We investigated the mode of delivery and perinatal outcomes in low-risk pregnant women whose labor was electively induced or expectantly managed at term.Healthy women with viable, vertex singleton pregnancies at 37 to 40 weeks of gestation were included. Women electively induced (n = 416) in each week (37-37, 38-38, 39-39, 40-40 weeks) were compared with pregnant women with spontaneous labor (n = 487). The primary outcome was mode of delivery. A propensity score (PS) was derived using logistic regression to model the probability of elective induction group as a function of potential confounders. Altogether, 284 women with elective induction were matched with 284 women who underwent expectant management to create a PS-matched population. All analysis was performed using SAS software, version 9.4 (SAS Institute Inc., Cary, NC). All P values reported of the significance level was set at <.05.There are no significant differences of delivery mode, neonatal intensive care unit (NICU) admission, and neonatal complication between PS-matched groups. Incidence of antepartum complications showed higher in the elective induction group compared to the spontaneous labor group (P = .04). When comparing each gestational week, incidence of NICU admission at 38 weeks in the elective induction group [10/74 (13.5%)] was significantly higher than in and the spontaneous labor group [2/74 (2.7%)] (P = .04).Elective induction of labor at term is not associated with increased risk of cesarean delivery. However, overall incidence of NICU admission at 38 gestational weeks seems to be increased in elective induction.

Global gene expression analysis of cell-free RNA in amniotic fluid from women destined to develop preeclampsia.

Preeclampsia (PE) is a disorder specific to pregnancy characterized by new-onset hypertension and proteinuria after 20 weeks of gestation. There is no definite treatment for PE except delivery of the placenta. The purpose of this study was to elucidate the biological pathways involved in the development of PE and to discover a novel biomarker for PE by performing global gene expression analysis of amniotic fluid cell-free RNA.The participants were recruited from the Department of Obstetrics and Gynecology of CHA Gangnam Medical Center (Seoul, Korea) between March 2014 and February 2015. Eight samples were collected from 8 subjects at second trimester who were later diagnosed with PE. From the amniotic fluid samples, cell-free RNA extraction was performed and gene expression was analyzed using the GeneChip PrimeView Array. Transcriptome data previously analyzed by our group from 9 euploid mid-trimester amniotic fluid samples were used as the control for comparative analysis. Functional analysis of the probe sets was performed using the online Database for Annotation, Visualization, and Integrated Discovery (DAVID) toolkit 6.7.We identified 1841 differentially expressed genes (DEGs) between the PE group and the control. Of these, 1557 genes were upregulated in the PE group, while 284 genes were upregulated in the control. The functional annotation of DEGs identified specific enriched functions such as "transport," "signal transduction," and "stress response." Functional annotation clustering with enriched genes in the PE group revealed that translation-related genes, cell-cell adhesion genes, and immune-related genes were enriched. KEGG pathway analysis showed that several biological pathways, including the ribosome pathway and various immune pathways, were dysregulated. Several genes, including RPS29, IGF-2, and UBC, were significantly upregulated in PE, up to tenfold.This study provides the first genome-wide expression analysis of amniotic fluid cell-free RNA in PE. The results showed that gene expression involving the ribosome pathway and immunologic pathways are dysregulated in PE. Our results will aid in understanding the underlying pathogenesis of PE.

Occult hepatitis B virus infection in pregnant woman and its clinical implication.

BACKGROUND/AIMS: The objective of this study was to document the prevalence rate of occult hepatitis B virus (HBV) in healthy pregnant woman and the possibility of transmission to the foetus. METHODS: This study was performed prospectively with 202 healthy pregnant women. HBV-DNA testing was performed using two specific quantitative tests with two independent sets of sera and cord blood. DNA sequencing analysis was carried out to confirm the specificity of polymerase chain reaction (PCR) product of HBV-DNA testing. RESULTS: Eight of 202 (4%) individuals with the TaqMan PCR assay and 23 of 202 (11.4%) with the COBAS Amplicor HBV Monitor test were HBV-DNA positive. Six (3%) individuals were positive with both methods. Sequencing and genotyping analysis of HBV polymerase gene with sera of the 75th subject resulted in genotype C. HBV-DNA testing with four cord blood samples showed that all were HBV-DNA negative. CONCLUSION: Occult HBV infection shows a difference in prevalence rate depending on the test method but the existence has been confirmed by sequencing analysis. Our results also suggest that vertical transmission through the cord blood is not so high as to be clinical problems and warrants further investigation.

Differential impacts of serum vitamin D levels and age at menarche on metabolic syndrome in premenopausal and postmenopausal women: findings from the Korea national cohort.

It is suggested that vitamin D level and age at menarche are related to each other, and the prevalence of low vitamin D status and early menarche in women is increasing worldwide. Moreover, several studies revealed that both of them are associated with metabolic syndrome (MetS). Therefore, we hypothesized that there are significant associations among vitamin D status, age at menarche, and MetS and that the relationships differ according to menstrual state. We assessed whether the association among MetS, vitamin D, and menarche age is different between premenopausal and postmenopausal women and whether there is a change in risk of MetS according to vitamin D level in different age-at-menarche groups. We used data from the Korea National Health and Nutrition Examination Survey, using 1:1 age-matching for this cross-sectional study. Individuals were stratified into 25-hydroxyvitamin D (25[OH]D) levels (deficient, <10 ng/mL; insufficient, 10-19 ng/mL; and sufficient, ≥20 ng/mL) and categorized as having either early, average, or late menarche (<13, 13-16, and ≥17 years). In premenopausal women, early menarche, not vitamin D level, was associated with risk of MetS (odds ratio [95% confidence interval], 1.65 [1.18-2.33]). In contrast, in postmenopausal women, vitamin D deficiency, not age at menarche, was associated with risk of MetS (1.39 [1.03-1.87]). In a stratified analysis regarding interactions of a change in risk of MetS according to vitamin D level in different ages at menarche, vitamin D deficiency was significantly associated with the risk of MetS (1.36 [1.01-1.86]), but this was only in the average-age-at-menarche group. This study suggests that the time of entry into puberty for women may be an important factor in the development of MetS in adulthood, and vitamin D status in women at average menarche age may contribute to the development of MetS.

Immunomodulatory effect of CD200-positive human placenta-derived stem cells in the early phase of stroke.

Human placenta amniotic membrane-derived mesenchymal stem cells (AMSCs) regulate immune responses, and this property can be exploited to treat stroke patients via cell therapy. We investigated the expression profile of AMSCs cultured under hypoxic conditions and observed interesting expression changes in various genes involved in immune regulation. CD200, an anti-inflammatory factor and positive regulator of TGF-ß, was more highly expressed under hypoxic conditions than normoxic conditions. Furthermore, AMSCs exhibited inhibition of pro-inflammatory cytokine expression in co-cultures with LPS-primed BV2 microglia, and this effect was decreased in CD200-silenced AMSCs. The AMSCs transplanted into the ischemic rat model of stroke dramatically inhibited the expression of pro-inflammatory cytokines and up-regulated CD200, as compared with the levels in the sham-treated group. Moreover, decreased microglia activation in the boundary region and improvements in behavior were confirmed in AMSC-treated ischemic rats. The results suggested that the highly expressed CD200 from the AMSCs in a hypoxic environment modulates levels of inflammatory cytokines and microglial activation, thus increasing the therapeutic recovery potential after hypoxic-ischemic brain injury, and further demonstrated the immunomodulatory function of AMSCs in a stroke model.

Dual Effects of Human Placenta-Derived Neural Cells on Neuroprotection and the Inhibition of Neuroinflammation in a Rodent Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disease in the elderly and the patients suffer from uncontrolled movement disorders due to loss of dopaminergic (DA) neurons on substantia nigra pars compacta (SNpc). We previously reported that transplantation of human fetal midbrain-derived neural precursor cells restored the functional deficits of a 6-hydroxy dopamine (6-OHDA)-treated rodent model of PD but its low viability and ethical issues still remain to be solved. Albeit immune privilege and neural differentiation potentials suggest mesenchymal stem cells (MSCs) from various tissues including human placenta MSCs (hpMSCs) for an alternative source, our understanding of their therapeutic mechanisms is still limited. To expand our knowledge on the MSC-mediated PD treatment, we here investigated the therapeutic mechanism of hpMSCs and hpMSC-derived neural phenotype cells (hpNPCs) using a PD rat model. Whereas both hpMSCs and hpNPCs protected DA neurons in the SNpc at comparable levels, the hpNPC transplantation into 6-OHDA treated rats exhibited longer lasting recovery in motor deficits than either the saline or the hpMSC treated rats. The injected hpNPCs induced delta-like ligand (DLL)1 and neurotrophic factors, and influenced environments prone to neuroprotection. Compared with hpMSCs, co-cultured hpNPCs more efficiently protected primary neural precursor cells from midbrain against 6-OHDA as well as induced their differentiation into DA neurons. Further experiments with conditioned media from hpNPCs revealed that the secreted factors from hpNPCs modulated immune responses and neural protection. Taken together, both DLL1-mediated contact signals and paracrine factors play critical roles in hpNPC-mediated improvement. First showing here that hpMSCs and their neural derivative hpNPCs were able to restore the PD-associated deficits via dual mechanisms, neuroprotection and immunosuppression, this study expanded our knowledge of therapeutic mechanisms in PD and other age-related diseases.

Risk factors for massive postpartum bleeding in pregnancies in which incomplete placenta previa are located on the posterior uterine wall.

OBJECTIVE: To identify factors associated with massive postpartum bleeding in pregnancies complicated by incomplete placenta previa located on the posterior uterine wall. METHODS: A retrospective case-control study was performed. We identified 210 healthy singleton pregnancies with incomplete placenta previa located on the posterior uterine wall, who underwent elective or emergency cesarean section after 24 weeks of gestation between January 2006 and April 2016. The cases with intraoperative blood loss (≥2,000 mL) or transfusion of packed red blood cells (≥4) or uterine artery embolization or hysterectomy were defined as massive bleeding. RESULTS: Twenty-three women experienced postpartum profuse bleeding (11.0%). After multivariable analysis, 4 variables were associated with massive postpartum hemorrhage (PPH): experience of 2 or more prior uterine curettage (adjusted odds ratio [aOR], 4.47; 95% confidence interval [CI], 1.29 to 15.48; P=0.018), short cervical length before delivery (<2.0 cm) (aOR, 7.13; 95% CI, 1.01 to 50.25; P=0.049), fetal non-cephalic presentation (aOR, 12.48; 95% CI, 1.29 to 121.24; P=0.030), and uteroplacental hypervascularity (aOR, 6.23; 95% CI, 2.30 to 8.83; P=0.001). CONCLUSION: This is the first study of cases with incomplete placenta previa located on the posterior uterine wall, which were complicated by massive PPH. Our findings might be helpful to guide obstetric management and provide useful information for prediction of massive PPH in pregnancies with incomplete placenta previa located on the posterior uterine wall.

Preclinical Analysis of Fetal Human Mesencephalic Neural Progenitor Cell Lines: Characterization and Safety In Vitro and In Vivo.

We have developed a good manufacturing practice for long-term cultivation of fetal human midbrain-derived neural progenitor cells. The generation of human dopaminergic neurons may serve as a tool of either restorative cell therapies or cellular models, particularly as a reference for phenotyping region-specific human neural stem cell lines such as human embryonic stem cells and human inducible pluripotent stem cells. We cultivated 3 different midbrain neural progenitor lines at 10, 12, and 14 weeks of gestation for more than a year and characterized them in great detail, as well as in comparison with Lund mesencephalic cells. The whole cultivation process of tissue preparation, cultivation, and cryopreservation was developed using strict serum-free conditions and standardized operating protocols under clean-room conditions. Long-term-cultivated midbrain-derived neural progenitor cells retained stemness, midbrain fate specificity, and floorplate markers. The potential to differentiate into authentic A9-specific dopaminergic neurons was markedly elevated after prolonged expansion, resulting in large quantities of functional dopaminergic neurons without genetic modification. In restorative cell therapeutic approaches, midbrain-derived neural progenitor cells reversed impaired motor function in rodents, survived well, and did not exhibit tumor formation in immunodeficient nude mice in the short or long term (8 and 30 weeks, respectively). We conclude that midbrain-derived neural progenitor cells are a promising source for human dopaminergic neurons and suitable for long-term expansion under good manufacturing practice, thus opening the avenue for restorative clinical applications or robust cellular models such as high-content or high-throughput screening. Stem Cells Translational Medicine 2017;6:576-588.

Ground-state conditions promote robust Prdm14 reactivation and maintain an active Dlk1-Dio3 region during reprogramming.

Induced pluripotent stem cells (iPSCs) are capable of unlimited self-renewal and can give rise to all three germ layers, thereby providing a new platform with which to study mammalian development and epigenetic reprogramming. However, iPSC generation may result in subtle epigenetic variations, such as the aberrant methylation of the Dlk1-Dio3 locus, among the clones, and this heterogeneity constitutes a major drawback to harnessing the full potential of iPSCs. Vitamin C has recently emerged as a safeguard to ensure the normal imprinting of the Dlk1-Dio3 locus during reprogramming. Here, we show that vitamin C exerts its effect in a manner that is independent of the reprogramming kinetics. Moreover, we demonstrate that reprogramming cells under 2i conditions leads to the early upregulation of Prdm14, which in turn results in a highly homogeneous population of authentic pluripotent colonies and prevents the abnormal silencing of the Dlk1-Dio3 locus.

Wound healing properties of a 3-D scaffold comprising soluble silkworm gland hydrolysate and human collagen.

Biomaterials that serve as scaffolds for cell proliferation and differentiation are increasingly being used in wound repair. In this study, the potential regenerative properties of a 3-D scaffold containing soluble silkworm gland hydrolysate (SSGH) and human collagen were evaluated. The scaffold was generated by solid-liquid phase separation and a freeze-drying method using a homogeneous aqueous solution. The porosity, swelling behavior, protein release, cytotoxicity, and antioxidative properties of scaffolds containing various ratios of SSGH and collagen were evaluated. SSGH/collagen scaffolds had a high porosity of 61-81% and swelling behavior studies demonstrated a 50-75% increase in swelling, along with complete protein release in the presence of phosphate-buffered saline. Cytocompatibility of the SSGH/collagen scaffold was demonstrated using mesenchymal stem cells from human umbilical cord. Furthermore, SSGH/collagen efficiently attenuated oxidative stress-induced cell damage. In an in vivo mouse model of wound healing, the SSGH/collagen scaffold accelerated wound re-epithelialization over a 15-day period. Overall, the microporous SSGH/collagen 3-D scaffold maintained optimal hydration of the exposed tissues and decreased wound healing time. These results contribute to the generation of advanced wound healing materials and may have future therapeutic implications.

Interleukin-1beta -511T>C genetic variant contributes to recurrent pregnancy loss risk and peripheral natural killer cell proportion.

OBJECTIVE: To identify whether interleukin gene polymorphisms are risk factors for idiopathic recurrent pregnancy loss (RPL) in Korean women. DESIGN: Case-control study. SETTING: Hospital-based study. PATIENT(S): A cohort of 385 idiopathic RPL patients and 232 controls with Korean ethnicity. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Genotyping was assessed with a polymerase chain reaction-restriction fragment length polymorphism assay. We examined polymorphisms in three interleukin (IL) genes: IL-1ß, IL-4, and IL-10. RESULT(S): The IL-1ß -511T>C polymorphism was associated with RPL (-511TT vs. -511CC: adjusted odds ratio 1.826; 95% confidence interval 1.130-2.953). Allelic gene-gene interaction analysis revealed that the T/B2/G (IL-1ß/IL-4/IL-10) allele combination was only detected in the RPL group (adjusted odds ratio 20.046; 95% confidence interval 1.188-338.204). The proportion of peripheral natural killer cells was higher in patients with the IL-1ß -511C allele compared with the -511T allele. CONCLUSION(S): According to these results, IL-1ß -511T>C may be a predisposing factor to RPL susceptibility. However, the mechanism underlying the function of IL-1ß -511T>C in RPL remains to be determined, and further studies are needed to improve understanding of the roles of IL-1ß -511T>C, using a larger and more heterogeneous cohort.