Case report of a primary ectopic extradural and extraspinal meningioma of the brachial plexus.

Primary ectopic extradural and extraspinal meningiomas are rare. We present a unique case of this type of meningioma in the brachial plexus. A 25-year-old man consulted us because of neuropathic supraclavicular pain and the appearance of a supraclavicular mass whose volume had increased. Clinical examination found paresis of the deltoid, biceps brachii and brachialis muscles rated as M4 (MRC) and a strong Tinel sign at the supraclavicular fossa, over the palpable mass. There was no sign pointing towards central nervous system involvement or altered general condition. MRI revealed a mass measuring 53 × 24 mm invading the C5-C6 plexus roots and the primary upper trunk, but not the bone or spinal area. This lesion was hyperintense on DWI/ADC, hyperintense on T2 with hypointense spots, and hypointense on T1 with intense heterogeneous gadolinium enhancement. Excisional biopsy was done 6 months after symptoms started. The tumor had developed at the C5 root, which was fibrous and at the C6 root, which was grossly normal. Anatomical pathology confirmed the WHO grade 1 meningioma, meningothelial and psammomatous histological subtypes. At 6 months, a follow-up MRI found no postoperative tumor remnants or recurrence. During the postoperative course, persistent paralysis of the deltoid muscle at 5 months justified a nerve transfer. This is a rare case of ectopic extraspinal and extradural meningioma of the brachial plexus. The diagnosis of an ectopic meningioma must be considered when a patient presents with a brachial plexus tumor causing neurological deficits. The extradural nature is not sufficient to rule out this diagnosis.

Inflammatory markers and BDNF in obstructive sleep apnea (OSA) in Parkinson's disease (PD).

OBJECTIVE: Obstructive sleep apnea (OSA) exacerbates Parkinson's disease (PD) manifestations including cognitive dysfunction. Both OSA and PD have been associated with inflammation. Brain-derived neurotrophic factor (BDNF) has been implicated in cognitive function. We aimed to investigate inflammatory cytokines and BDNF in relation to OSA and PD symptoms. METHODS: In a prospective observational study, patients with PD underwent overnight polysomnography. Morning serum levels of interleukin (IL)-1ß, IL-6, IL-8, TNFα, and BDNF were quantified at baseline (n = 64) and 6 months (n = 38). Outcomes included non-motor and motor standard scores; Montreal Cognitive Assessment (MoCA); and Epworth Sleepiness scale (ESS). Associations were assessed using linear regression, adjusting for age, sex and body mass index. RESULTS: At baseline, IL-6 was associated with the Apnea-Hypopnea Index (ß = 0.013, p = 0.03), and the Oxygen Desaturation Index (ß = 0.028, p = 0.002). No other associations between cytokines and sleep parameters were found. Motor dysfunction was associated with IL-6 (ß = 0.03, p = 0.001). ESS was associated non-significantly with IL-6 (ß = 0.04, p = 0.07) and BDNF (ß = 555, p = 0.06). At follow-up, change in IL-6 was associated with change in non-motor (ß = 0.08, p = 0.007), and motor (ß = 0.03, p = 0.001) symptoms. Change in BDNF was associated with change in ESS (ß = 1450, p = 0.02). INTERPRETATION: We found an association between IL-6 levels and both OSA severity and PD motor dysfunction. At follow-up, increasing IL-6 correlated with deterioration of motor and non-motor PD symptoms. Increasing BDNF correlated with increasing sleepiness. Further work with a larger sample size is needed, but our results support the hypothesis that OSA-related inflammation plays a role in PD manifestations and progression.

Vibrational Coherence Spectroscopy Identifies Ultrafast Branching in an Iron(II) Sensitizer.

The introduction of N-heterocyclic carbene ligands has greatly increased the lifetimes of metal-to-ligand charge transfer states (MLCT) in iron(II) complexes, making them promising candidates for photocatalytic applications. However, the spectrally elusive triplet metal-centered state (3MC) has been suggested to play a decisive role in the relaxation of the MLCT manifold to the ground state, shortening their lifetimes and consequently limiting the application potential. In this work, time-resolved vibrational spectroscopy and quantum chemical calculations are applied to shed light on the 3MCs' involvement in the deactivation of the MLCT manifold of an iron(II) sensitizer. Two distinct symmetric Fe-L breathing vibrations at frequencies below 150 cm-1 are assigned to the 3MC and 3MLCT states by quantum chemical calculations. On the basis of this assignment, an ultrafast branching directly after excitation forms not only the long-lived 3MLCT but also the 3MC as an additional loss channel.

Repression of p53-target gene Bbc3/PUMA by MYSM1 is essential for the survival of hematopoietic multipotent progenitors and contributes to stem cell maintenance.

p53 is a central mediator of cellular stress responses, and its precise regulation is essential for the normal progression of hematopoiesis. MYSM1 is an epigenetic regulator essential for the maintenance of hematopoietic stem cell (HSC) function, hematopoietic progenitor survival, and lymphocyte development. We recently demonstrated that all developmental and hematopoietic phenotypes of Mysm1 deficiency are p53-mediated and rescued in the Mysm1(-/-)p53(-/-) mouse model. However, the mechanisms triggering p53 activation in Mysm1(-/-) HSPCs, and the pathways downstream of p53 driving different aspects of the Mysm1(-/-) phenotype remain unknown. Here we show the transcriptional activation of p53 stress responses in Mysm1(-/-) HSPCs. Mechanistically, we find that the MYSM1 protein associates with p53 and colocalizes to promoters of classical p53-target genes Bbc3/PUMA (p53 upregulated modulator of apoptosis) and Cdkn1a/p21. Furthermore, it antagonizes their p53-driven expression by modulating local histone modifications (H3K27ac and H3K4me3) and p53 recruitment. Using double-knockout mouse models, we establish that PUMA, but not p21, is an important mediator of p53-driven Mysm1(-/-) hematopoietic dysfunction. Specifically, Mysm1(-/-)Puma(-/-) mice show full rescue of multipotent progenitor (MPP) viability, partial rescue of HSC quiescence and function, but persistent lymphopenia. Through transcriptome analysis of Mysm1(-/-)Puma(-/-) MPPs, we demonstrate strong upregulation of other p53-induced mediators of apoptosis and cell-cycle arrest. The full viability of Mysm1(-/-)Puma(-/-) MPPs, despite strong upregulation of many other pro-apoptotic mediators, establishes PUMA as the essential non-redundant effector of p53-induced MPP apoptosis. Furthermore, we identify potential mediators of p53-dependent but PUMA-independent Mysm1(-/-)hematopoietic deficiency phenotypes. Overall, our study provides novel insight into the cell-type-specific roles of p53 and its downstream effectors in hematopoiesis using unique models of p53 hyperactivity induced by endogenous stress. We conclude that MYSM1 is a critical negative regulator of p53 transcriptional programs in hematopoiesis, and that its repression of Bbc3/PUMA expression is essential for MPP survival, and partly contributes to maintaining HSC function.

Contribution of increased ISG15, ISGylation and deregulated type I IFN signaling in Usp18 mutant mice during the course of bacterial infections.

Host genetics has a key role in susceptibility to Salmonella Typhimurium infection. We previously used N-ethyl-N-nitrosourea (ENU) mutagenesis to identify a loss-of-function mutation within the gene ubiquitin-specific peptidase 18 (Usp18(Ity9)), which confers increased susceptibility to Salmonella Typhimurium. USP18 functions to regulate type I interferon (IFN) signaling and as a protease to remove ISG15 from substrate proteins. Usp18(Ity9) mice are susceptible to infection with Salmonella Typhimurium and have increased expression and function of ISG15, but Usp18(Ity9) mice lacking Isg15 do not show improved survival with Salmonella challenge. Type I IFN signaling is increased in Usp18(Ity9) mice and inhibition of type I IFN signaling is associated with improved survival in mutant mice. Hyperactivation of type I IFN signaling leads to increased IL-10, deregulated expression of autophagy markers and elevated interleukin (IL)-1ß and IL-17. Furthermore, Usp18(Ity9) mice are more susceptible to infection with Mycobacterium tuberculosis, have increased bacterial load in the lung and spleen, elevated inflammatory cytokines and more severe lung pathology. These findings demonstrate that regulation of type I IFN signaling is the predominant mechanism affecting the susceptibility of Usp18(Ity9) mice to Salmonella infection and that hyperactivation of signaling leads to increased IL-10, deregulation of autophagic markers and increased proinflammatory cytokine production.

Susceptibility to lethal cerebral malaria is regulated by epistatic interaction between chromosome 4 (Berr6) and chromosome 1 (Berr7) loci in mice.

In humans, cerebral malaria is a rare but often lethal complication of infection with Plasmodium parasites, the occurrence of which is influenced by complex genetic factors of the host. We used a mouse model of experimental cerebral malaria (ECM) with Plasmodium berghei ANKA to study genetic factors regulating appearance of neurological symptoms and associated lethality. In a genome-wide screen of N-ethyl-N-nitrosourea-mutagenized mice derived from C57BL/6J (B6) and 129S1/SvImJ (129) mouse strains, we detected a strong interaction between the genetic backgrounds of these strains, which modulates ECM resistance. We have mapped a major gene locus to central chromosome 4 (log of the odds (LOD) 6.7; 79.6-97.3 Mb), which we designate Berr8. [corrected]. B6 alleles at Berr6 are associated with resistance, and are inherited in a co-dominant fashion. In mice heterozygous for Berr6 B6/129 alleles, resistance to ECM is strongly modulated by a second locus, Berr7, that maps to the proximal portion of chromosome 1 (LOD 4.03; 41.4 Mb). 129 alleles at Berr7 are associated with ECM resistance in a dosage-dependent fashion. Results are discussed in view of the possible role of this two-locus system in susceptibility to unrelated inflammatory conditions in mice and humans.

Genetic determinants of susceptibility to Mycobacterial infections: IRF8, a new kid on the block.

Genetic and population studies suggest that onset, progression and ultimate outcome of infection with Mycobacteria, including the agent of tuberculosis Mycobacterium tuberculosis, are strongly influenced by genetic factors. Family-based and case-control linkage and association studies have suggested a complex genetic component for susceptibility to tuberculosis. On the other hand, patients with inborn errors in the IL12/IFNγ circuit may develop disseminated mycobacterial infections following perinatal BCG vaccination. The study of such MSMD (Mendelian Susceptibility to Mycobacterial Diseases) patients has provided much insight into innate and acquired immune defenses against mycobacteria. Parallel genetic analyses in mouse models of mycobacterial infections have also indicated complex genetic control, and have provided candidate genes for parallel testing in humans. Recently, mutations in human IRF8 were discovered and shown to cause two distinct forms of a novel primary immunodeficiency and associated susceptibility to mycobacteria. Autosomal recessive IRF8 deficiency is caused by mutation K108E and associated with severe disease with complete depletion of monocytes and dendritic cells. Mutation T80A causes autosomal dominant IRF8 deficiency and a milder form of the disease with selective loss of a subset of dendritic cells. These findings have established that IRF8 is required for ontogeny of the myeloid lineage and for host response to mycobacteria. The ongoing study of the IRF8 transcriptome has shown promise for the identification of IRF8 dependent pathways that play a critical role in host defense against mycobacteria in particular, and against intracellular pathogens in general.

Genetic control of susceptibility to infection with Plasmodium chabaudi chabaudi AS in inbred mouse strains.

To identify genetic effects modulating the blood stage replication of the malarial parasite, we phenotyped a group of 25 inbred mouse strains for susceptibility to Plasmodium chabaudi chabaudi AS infection (peak parasitemia, survival). A broad spectrum of responses was observed, with strains such as C57BL/6J being the most resistant (low parasitemia, 100% survival) and strains such as NZW/LacJ and C3HeB/FeJ being extremely susceptible (very high parasitemia and uniform lethality). A number of strains showed intermediate phenotypes and gender-specific effects, suggestive of rich genetic diversity in response to malaria in inbred strains. An F2 progeny was generated from SM/J (susceptible) and C57BL/6J (resistant) parental strains, and was phenotyped for susceptibility to P. chabaudi chabaudi AS. A whole-genome scan in these animals identified the Char1 locus (LOD=7.40) on chromosome 9 as a key regulator of parasite density and pointed to a conserved 0.4-Mb haplotype at Char1 that segregates with susceptibility/resistance to infection. In addition, a second locus was detected in [SM/J × C57BL/6J] F2 mice on the X chromosome (LOD=4.26), which was given the temporary designation Char11. These studies identify a conserved role of Char1 in regulating response to malaria in inbred mouse strains, and provide a prioritized 0.4-Mb interval for the search of positional candidates.

Genetic diversity in human erythrocyte pyruvate kinase.

Previously, we have shown that pyruvate kinase, liver and red cell isoform (PKLR) deficiency protects mice in vivo against blood-stage malaria, and observed that reduced PKLR function protects human erythrocytes against Plasmodium falciparum replication ex vivo. Here, we have sequenced the human PKLR gene in 387 individuals from malaria-endemic and other regions in order to assess genetic variability in different geographical regions and ethnic groups. Rich genetic diversity was detected in PKLR, including 59 single-nucleotide polymorphisms and several loss-of-function variants (frequency 1.5%). Haplotype distribution and allele frequency varied considerably with geography. Neutrality testing suggested positive selection of the genein the sub-Saharan African and Pakistan populations. It is possible that such positive selection involves the malarial parasite.

Contribution of VANGL2 mutations to isolated neural tube defects.

Vangl2 was identified as the gene defective in the Looptail (Lp) mouse model for neural tube defects (NTDs). This gene forms part of the planar cell polarity (PCP) pathway, also called the non-canonical Frizzled/Dishevelled pathway, which mediates the morphogenetic process of convergent extension essential for proper gastrulation and neural tube formation in vertebrates. Genetic defects in PCP signaling have strongly been associated with NTDs in mouse models. To assess the role of VANGL2 in the complex etiology of NTDs in humans, we resequenced this gene in a large multi-ethnic cohort of 673 familial and sporadic NTD patients, including 453 open spina bifida and 202 closed spinal NTD cases. Six novel rare missense mutations were identified in seven patients, five of which were affected with closed spinal NTDs. This suggests that VANGL2 mutations may predispose to NTDs in approximately 2.5% of closed spinal NTDs (5 in 202), at a frequency that is significantly different from that of 0.4% (2 in 453) detected in open spina bifida patients (p = 0.027). Our findings strongly implicate VANGL2 in the genetic causation of spinal NTDs in a subset of patients and provide additional evidence for a pathogenic role of PCP signaling in these malformations.

Forward genetic dissection of innate response to infection in inbred mouse strains: selected success stories.

Mouse genetics is a powerful tool for the dissection of genes, proteins, and pathways important in biological processes. Application of this approach to study the host response to infection has been a rich source of discoveries that have increased our understanding of the early innate pathways involved in responding to microbial infections. Here we review some of the key discoveries that have arisen from pinpointing the genetic defect in mouse strains with unusual or extreme response to infection and have led to insights into pathogen sensing pathways and downstream effector functions of the early innate immune response.

Identification of a novel cerebral malaria susceptibility locus (Berr5) on mouse chromosome 19.

Cerebral malaria (CM) is an acute, generally lethal condition characterized by high fever, seizures and coma. The genetic component to CM can be investigated in mouse models that vary in degree of susceptibility to infection with Plasmodium berghei ANKA. Using survival time to measure susceptibility in an informative F2 cross (n=257), we identified linkage to chromosome 19 (Berr5 (Berghei resistance locus 5), LOD=4.69) controlling, in part, the differential response between resistant BALB/c and susceptible C57BL/6 progenitors. BALB/c alleles convey increased survival through the cerebral phase of infection but have no quantitative effect on parasitemia during the later, anemic phase. The Berr5 locus colocalizes with three other immune loci, including Trl-4 (tuberculosis resistance), Tsiq2 (T-cell secretion of IL-4) and Eae19 (experimental allergic encephalitis 19), suggesting the possibility of a common genetic effect underlying these phenotypes. Potential positional candidates include the family of Ifit1-3 (interferon-inducible protein with tetratricopeptide repeats 1-3) and Fas.

Mapping of Char10, a novel malaria susceptibility locus on mouse chromosome 9.

Resistance to blood-stage malaria in AcB55 and AcB61 is caused by a loss of function mutation in pyruvate kinase (Pklr(I90N)). Likewise, pyruvate kinase (PK) deficiency in humans is protective against Plasmodium replication in vitro. We identified a third AcB strain, AcB62 that also carries the Pklr(I90N) mutation. However, AcB62 mice were susceptible to P.chabaudi infection and showed high levels of parasite replication (54-62% peak parasitemia). AcB62 mice showed the hallmarks of PK deficiency-associated anemia similar to AcB55/61 with reticulocytosis, splenic red pulp expansion, tissue iron overload, and increased expression of iron metabolism proteins. This suggests that malaria susceptibility in AcB62 is not because of absence of PK deficiency-associated pathophysiology. To map novel genetic factors affecting malaria susceptibility in AcB62, we generated an informative F2 population using AcB62 (Pklr(I90N)) and CBA-Pk(slc) (Pklr(G338D)) as progenitors and identified a novel locus on chromosome 9 (Char10; LOD=7.24) that controls peak parasitemia. A weaker linkage to the Pklr region of chromosome 3 (LOD=3.7) was also detected, a finding that may reflect the segregation of the two defective Pklr alleles. AcB62 alleles at both loci are associated with higher peak parasitemia. These results identify Char10 as a novel locus modulating severity of malaria in the context of PK deficiency.

Characterization of a major colon cancer susceptibility locus (Ccs3) on mouse chromosome 3.

Treatment of mice with the carcinogen azoxymethane (AOM) induces a number of lesions in the colon, including hyperplastic lesions, as well adenomas and carcinomas in situ. Inbred strains of mice show different responses to AOM-induced carcinogenesis. A/J mice are highly susceptible and develop a greater number of hyperplastic lesions and tumors (15-70 tumors per mouse) than resistant C57BL/6J mice (0-6 tumors per mouse). Susceptibility to AOM-induced tumors segregates as a co-dominant trait in (A x B6)F1 hybrids. Using a set of 23 AcB and BcA recombinant congenic mouse strains derived from A/J (susceptible) and B6 (resistant) parents, we observed that the number of hyperplastic lesions and tumors induced by AOM was under different genetic controls in AcB/BcA strains. The multiplicity of AOM-induced tumors is controlled by a major locus that we have mapped on the distal portion of chromosome 3, to which we have given the temporary designation colon cancer susceptibility locus 3 (Ccs3). B6 and A/J alleles at Ccs3 are associated with resistance and susceptibility, respectively. Haplotype analysis in key informative AcB/BcA strains restricts the size of the Ccs3 locus to a 14 Mb segment that contains 94 annotated genes. The expression level of all these genes in normal colon has been established by transcript profiling with microarrays, and has led to the identification of a subset of positional candidates that are expressed at high levels in this tissue. The 4q and 1p human chromosomal segments sharing syntenic homology with the mouse Ccs3 segment are known to be associated with inflammatory bowel diseases and colorectal tumors in humans, suggesting that the study of the mouse Ccs3 locus may help further the pathogenesis of these human conditions.

Genetic analysis of resistance to infections in mice: A/J meets C57BL/6J.

Susceptibility to infectious diseases has long been known to have a genetic component in human populations. This genetic effect is often complex and difficult to study as it is further modified by environmental factors including the disease-causing pathogen itself. The laboratory mouse has proved a useful alternative to implement a genetic approach to study host defenses against infections. Our laboratory has used genetic analysis and positional cloning to characterize single and multi-gene effects regulating inter-strain differences in the susceptibility of A/J and C57BL/6J mice to infection with several bacterial and parasitic pathogens. This has led to the identification of several proteins including Nrampl (Slc11a1), Birc1e, Icsbp, C5a, and others that play critical roles in the antimicrobial defenses of macrophages against intracellular pathogens. The use of AcB/BcA recombinant congenic strains has further facilitated the characterization of single gene effects in complex traits such as susceptibility to malaria. The genetic identification of erythrocyte pyruvate kinase (Pklr) and myeloid pantetheinase enzymes (Vnn1/3) as key regulators of blood-stage parasitemia has suggested that cellular redox potential may be a key biochemical determinant of Plasmodium parasite replication. Expanding these types of studies to additional inbred strains and to emerging stocks of mutagenized mice will undoubtedly continue to unravel the molecular basis of host defense against infections.

Forward genetic dissection of immunity to infection in the mouse.

Forward genetics is an experimental approach in which gene mapping and positional cloning are used to elucidate the molecular mechanisms underlying phenotypic differences between two individuals for a given trait. This strategy has been highly successful for the study of inbred mouse strains that show differences in innate susceptibility to bacterial, parasitic, fungal, and viral infections. Over the past 20 years, these studies have led to the identification of a number of cell populations and critical biochemical pathways and proteins that are essential for the early detection of and response to invading pathogens. Strikingly, the macrophage is the point of convergence for many of these genetic studies. This has led to the identification of diverse pathways involved in extracellular and intracellular pathogen recognition, modification of the properties and content of phagosomes, transcriptional response, and signal transduction for activation of adaptive immune mechanisms. In models of viral infections, elegant genetic studies highlighted the pivotal role of natural killer cells in the detection and destruction of infected cells.

Genetic control of susceptibility to pulmonary infection with Chlamydia pneumoniae in the mouse.

A mouse model was used to study the genetic control of differential host response to pulmonary infection with Chlamydia pneumoniae. The A/J and C57BL/6 strains show differential response to intranasal infection with respect to their ability to clear pulmonary bacterial load and the extent of lung pathology developed by 2 weeks post infection. The genetic basis of this interstrain difference was studied by whole-genome scan in an informative [A/J x C57BL/6J] F2 cross using the pulmonary microbial load as a phenotypic readout of host response. We detected a highly significant linkage (LOD score=11.5) on chromosome 17 that overlaps with the major histocompatibility (MHC) locus. This quantitative trait locus (QTL) accounts for approximately 30% of the phenotypic variance with B6 alleles conferring susceptibility and inherited in a recessive fashion. Significant linkage was also detected to chromosome 5 in female mice, while chromosome 6 showed suggestive linkage in male mice, pointing to additional complexity in the genetic control of the difference in susceptibility observed in A/J and C57BL/6J.

Reduced in vitro functional activity of human NRAMP1 (SLC11A1) allele that predisposes to increased risk of pediatric tuberculosis disease.

Polymorphic variants within the human natural resistance-associated macrophage protein-1 (NRAMP1, also known as SLC11A1) gene have been shown to impact on susceptibility to tuberculosis in different human populations. In the mouse, Nramp1 is expressed at the macrophage phagosomal membrane and its activity can be assayed by the relative acquisition of mannose 6-phosphate receptor (M6PR) in Salmonella-containing vacuoles. Based on this M6PR recruitment assay, we have now developed an assay in primary human macrophages to test the function of human NRAMP1 gene variants. First, we established that M6PR acquisition was significantly higher (P = 0.002) in human U-937 monocytic cell lines transfected with NRAMP1 as compared to untransfected U-937 cells. Second, the M6PR assay was shown to be highly reproducible for NRAMP1 activity in monocyte-derived macrophages (MDM) from healthy volunteers. Finally, the assay was investigated in MDM from pediatric tuberculosis patients and significantly lower NRAMP1 activity was detected in MDM from individuals homozygous for the NRAMP1-274 high-risk allele (CC genotype) in comparison to heterozygous individuals (CT genotype; P=0.013). The present study describes both an assay for human NRAMP1 functional activity and concomitant evidence for reduced NRAMP1 function in the common genetic variant shown to be associated with tuberculosis susceptibility in pediatric patients.

Host genetics of mycobacterial diseases in mice and men: forward genetic studies of BCG-osis and tuberculosis.

In humans, genetic factors have long been suspected to contribute to the onset and outcome of tuberculosis. Such effects are difficult to identify owing to their complex inheritance, and to the confounding impact of environmental factors, notably pathogen-associated virulence determinants. Recently, forward genetic approaches in mouse models and in human populations have been used to elucidate a molecular basis for predisposition to mycobacterial diseases. The genetic dissection of host predisposition to infection with Mycobacterium bovis BCG and M. tuberculosis will help to define the key molecules involved in host antituberculous immunity and should provide new insights into this important infectious disease.

Toward understanding the genetic basis of neural tube defects.

Neural tube defects (NTDs) represent a common group of severe congenital malformations that result from failure of neural tube closure during early development. Their etiology is quite complex involving environmental and genetic factors and their underlying molecular and cellular pathogenic mechanisms remain poorly understood. Animal studies have recently demonstrated an essential role for the planar cell polarity pathway (PCP) in mediating a morphogenetic process called convergent extension during neural tube formation. Alterations in members of this pathway lead to NTDs in vertebrate models, representing novel and exciting candidates for human NTDs. Genetic studies in NTDs have focused mainly on folate-related genes based on the finding that perinatal folic acid supplementation reduces the risk of NTDs by 60-70%. A few variants in these genes have been found to be significantly associated with an increased risk for NTDs. The candidate gene approach investigating genes involved in neurulation has failed to identify major causative genes in the etiology of NTDs. Despite this history of generally negative findings, we are achieving a rapid and impressive progress in understanding the genetic basis of NTDs, based mainly on the powerful tool of animal models.