PAX genes and human neural tube defects: an amino acid substitution in PAX1 in a patient with spina bifida.

From studies in the mouse and from the clinical and molecular analysis of patients with type 1 Waardenburg syndrome, particular members of the PAX gene family are suspected factors in the aetiology of human neural tube defects (NTD). To investigate the role of PAX1, PAX3, PAX7, and PAX9, allelic association studies were performed in 79 sporadic and 38 familial NTD patients from the Dutch population. Sequence variation was studied by SSC analysis of the paired domain regions of the PAX1, PAX7, and PAX9 genes and of the complete PAX3 gene. In one patient with spina bifida, a mutation in the PAX1 gene was detected changing the conserved amino acid Gln to His at position 42 in the paired domain of the protein. The mutation was inherited through the maternal line from the unaffected grandmother and was not detected in 300 controls. In the PAX3 gene, variation was detected at several sites including a Thr/Lys amino acid substitution in exon 6. All alleles were present among patients and controls in about the same frequencies. However, an increased frequency of the rare allele of a silent polymorphism in exon 2 was found in NTD patients, but no significant association was observed (p = 0.06). No sequence variation was observed in the paired domain of the PAX7 and PAX9 genes. Our findings so far do not support a major role of the PAX genes examined in the aetiology of NTD. However, the detection of a mutation in PAX1 suggests that, in principle, this gene can act as a risk factor for human NTD.     

Loss of RAD9B impairs early neural development and contributes to the risk for human spina bifida

DNA damage response (DDR) genes orchestrating the network of DNA repair, cell cycle control, are essential for the rapid proliferation of neural progenitor cells. To date, the potential association between specific DDR genes and the risk of human neural tube defects (NTDs) has not been investigated. Using whole‐genome sequencing and targeted sequencing, we identified significant enrichment of rare deleterious RAD9B variants in spina bifida cases compared to controls (8/409 vs. 0/298; p = .0241). Among the eight identified variants, the two frameshift mutants and p.Gln146Glu affected RAD9B nuclear localization. The two frameshift mutants also decreased the protein level of RAD9B. p.Ser354Gly, as well as the two frameshifts, affected the cell proliferation rate. Finally, p.Ser354Gly, p.Ser10Gly, p.Ile112Met, p.Gln146Glu, and the two frameshift variants showed a decreased ability for activating JNK phosphorylation. RAD9B knockdowns in human embryonic stem cells profoundly affected early differentiation through impairing PAX6 and OCT4 expression. RAD9B deficiency impeded in vitro formation of neural organoids, a 3D cell culture model for human neural development. Furthermore, the RNA‐seq data revealed that loss of RAD9B dysregulates cell adhesion genes during organoid formation. These results represent the first demonstration of a DDR gene as an NTD risk factor in humans.     

Testing for genetic associations in a spina bifida population: analysis of the HOX gene family and human candidate gene regions implicated by mouse models of neural tube defects

Neural tube defects (NTDs) are among the most common severely disabling birth defects in the United States, affecting approximately 1-2 of every 1,000 live births. The etiology of NTDs is multifactorial, involving the combined action of both genetic and environmental factors. HOX genes play a central role in establishing the initial body plan by providing positional information along the anterior-posterior body and limb axis and have been implicated in neural tube closure. There are many mouse models that exhibit both naturally occurring NTDs in various mouse strains as well as NTDs that have been created by "knocking out" various genes. A nonparametric linkage method, the transmission disequilibrium test (TDT), was utilized to test the HOX gene family and human equivalents of genes (when known) or the syntenic region in humans to those in mouse models which could play a role in the formation of NTDs. DNA from 459 spina bifida (SB) affected individuals and their parents was tested for linkage and association utilizing polymorphic markers from within or very close to the HOXA, HOXB, HOXC, and HOXD genes as well as from within the genes/gene regions of eight mouse models that exhibit NTDs. No significant findings were obtained for the tested markers.     

PRKCI基因单核苷酸多态性与神经管畸形的相关性研究

目的探讨PRKCI基因单核苷酸多态性（SNP）与中国山西省神经管畸形（NTDs）发生的相关性。方法采用病例对照研究,利用MassARRAY分子量阵列分析平台,检测133例NTDs标本和135例非病理性胎儿标本PRKCI基因中17个标签SNPs的基因分型,分析其与NTDs发生的相关性。结果 17个SNPs位点中,16个的微效等位基因频率（MAF）与HapMap或dbSNP数据库的结果基本一致。除rs9876082外,其余SNPs位点的基因型分布和等位基因频率在病例组和对照组均无明显差异。rs9876082位点为纯合的微效等位基因A时,NTDs的发病率增加（P=0.035,比值比=2.135,95%可信区间=1.846-2.471）,但是调整其它变量后进行logistic回归分析,这种相关性不再明显（P=0.057）。结论 PRKCI基因rs9876082位点与NTDs的发生有弱的相关性,这种相关性需进一步加大样本进行验证,PRKCI基因可能不是通过其SNPs影响NTDs的易感性。     

Parental sex effect in spina bifida: a role for genomic imprinting?

Fifty families (491 individuals in 137 sibships) with more than one living case of isolated, nonsyndromic spina bifida (SB) were analyzed genetically. There were twice as many gene-carrier females (56) as gene-carrier males (28) (P < 0.005). This was not an artifact of ascertainment bias because the sex ratio of gene-carriers was the same whether the pedigree was obtained through the proband's father or mother. Also, this effect was not observed in other disorders analyzed by the same method. Neither was the effect due to differential fertility because the number and sex of affected and unaffected children per gene-carrier parent were not different for male or female gene-carrier parents. There was no evidence that the missing male gene-carriers were lost by selective spontaneous abortion. There was no deficit of male-to-male or male-to-female transmission, excluding simple X-linked or simple mitochondrial inheritance. If genomic imprinting plays a role in the unequal female and male carrier frequencies in SB, penetrance should differ with parental sex. Penetrance was higher for offspring of female parents than of male parents, but the difference was not statistically significant. In addition, both male and female gene-carriers were frequently found in the same pedigree. Thus, the present data suggest a possible role for imprinting in SB.     

Nutritional and genetic determinants of vitamin B and homocysteine metabolisms in neural tube defects: a multicenter case-control study

Neural tube defects (NTDs) are severe congenital malformations due to failure of neural tube formation in early pregnancy. The proof that folic acid prevents NTDs raises the question of whether other parts of homocysteine (Hcy) metabolism may affect rates of NTDs. This French case-control study covered: 77 women aged 17-42 years sampled prior to elective abortion for a severe NTDs (cases) and 61 women aged 20-43 years with a normal pregnancy. Plasma and erythrocyte folate, plasma B6, B12 and Hcy were tested as five polymorphisms MTHFR 677 C --> T, MTHFR 1298 A --> C, MTR 2756 A --> G, MTTR 66 A --> G and TCN2 776 C --> G. Cases had significantly lower erythrocyte folate, plasma folate, B12 and B6 concentrations than the controls, and higher Hcy concentration. The odds ratio was 2.15 (95% CI: 1.00-4.59) for women with the MTRR 66 A --> G allele and it was decreased for mothers carrying the MTHFR 1298 A --> C allele. In multivariate analysis, only the erythrocyte folate concentration (P = 0.005) and plasma B6 concentration (P = 0.020) were predictors. Red cell folate is the main determinant of NTDs in France. Folic acid supplement or flour fortification would prevent most cases. Increased consumption of vitamins B12 and B6 could contribute to the prevention of NTDs. Genetic polymorphisms played only a small role. Until folic acid fortification becomes mandatory, all women of reproductive age should consume folic acid in a multivitamin that also contains B12 and B6.     

Predisposition to spina bifida: Search for a relation to maternal gastric acid secretion.

The peak acid output of the stomach in a group of 71 mothers of spina bifida children and in 71 matched controls was estimated indirectly by the serum level of group I pepsinogens. The mean levels did not differ significantly, suggesting that the conjectured teratogen is not specially acid-labile. The variance was significantly higher in index subjects than in controls, but the interpretation of this finding is not clear.     

The development of the human brain from a closed neural tube at stage 13

Summary Twenty-five embryos of stage 13 (28 days) were studied in detail and graphic reconstructions of seven of them were prepared. Thirty or more somitic paris are present, and the maximum is possibly 39. The notochord is almost entirely separated from the neural tube and the alimentary epithelium, and its rostral tip is closely related to the adenohypophysial pocket. Caudal to the cloacal membrane, the caudal eminence is the site of secondary neurulation. The eminence, which usualy contains isolated somites, in the area where new notochord, hindgut, and neural tube are forming. The neural cord develops into neural tube without the intermediate phase of a neural plate (secondary neurulation). Canalization is regular and the lumen is continuous with the central canal. The neural tube is now a closed system, filled with that may be termed ependymal fluid. The brain is widening in a dorsoventral direction. Neuromeres are still detectable. The following features are distinguishable: infundibular area of D 2, chiasmatic plate of D 1, adult lamina terminalis, and commissural plate (at levels of nasal plates). The beginning of the synencephalon of D 2 can be discerned. The retinal and lens discs are being defined. The mesencephalic flexure continnues to diminish. The midbrain possesses a sulcus limitans, and the tegmentum may show the medial longitudinal fasciculus. The isthmic segment is clearly separated from rhombomere 1. Lateral and ventral longitudinal fasciculi are usually present in the hindbrain, and the common afferent tract is beginning. Somatic and visceral efferent fibres are seen in certain nerves: 6, 12, 5, 7, 9–11. The first indication of the cerebellum may be visible in the alar lamina of rhombomere 1. The terminal-vomeronasal crest appears. Various cranial ganglia (e.g., vestibular, superior ganglia of 9, 10) are forming. The trigeminal ganglion may show its three major divisions. Epipharyngeal placodes of pharyngeal arches 2 to 5 contribute to cranial ganglia 7, 9, and 10. The spinal neural crest is becoming segregated, and the spinal ganglia are in series with the somites. Ventral spinal roots are beginning to develop.Supported by research grant No. HD-16702, Institute of Child Health and Human Development, National Institutes of Health (USA)Herrn Professor Dr. Johannes Lang zu seinem 65. Geburtstag gewidmet in Anerkennung seiner wertvollen Beiträge zur klinischen Anatomie von Kopf und Gehirn     

FZD6 is a novel gene for human neural tube defects

Neural tube defects (NTDs) are severe malformations of the central nervous system, affecting 1 of 1,000 live births. Mouse models were instrumental in defining the signaling pathways defective in NTDs, including the planar cell polarity (PCP), also called noncanonical Frizzled/Disheveled pathway. Based on the highly penetrant occurrence of NTDs in double Fzd3/Fzd6(-/-) mutant mice, we investigated the role of the human orthologues, FZD3 and FZD6, by resequencing a cohort of 473 NTDs patients and 639 ethnically matched controls. While we could not demonstrate a significant contribution of FZD3 gene, we identified five rare FZD6 variants that were absent in all controls and predicted to have a functional effect by computational analysis: one de novo frameshift mutation (c.1843_1844insA), three missense changes (p.Arg405Gln, p.Arg511Cys p.Arg511His), and one substitution (c.*20C>T) affecting the 3'-untranslated region (UTR) of the gene. The overall rate of predicted deleterious variants of FZD6 was 5.1-fold higher in cases compared to controls, resulting in a significantly increased NTDs mutation burden. This study demonstrates that rare nonsynonymous variants in FZD6 may contribute to NTDs in humans and enlarges the spectrum of mutations that link PCP pathway to NTDs.