Reduction of procarbazine-induced cleft palates by prenatal folic acid supplementation in rats

We investigated the effects of prenatal folic acid supplementation on procarbazine (PCZ)-induced intra-uterine growth retardation (IUGR), cleft palates, and microgenia. Three groups of gravid rats were treated with 200 mg/kg body weight (BW) PCZ on day 13.5 of gestation (GD13.5). Two groups of them were additionally supplemented with 1 and 2.5 mg/kg folic acid, respectively, from GD13.5 through GD16.5. On GD19.5, all fetuses were delivered by caesarian sections and sexed subsequently. Numbers of live and dead fetuses as well as resorptions were counted. Data on fetal BW, crown-rump length, tail length, placental weight, and diameter were collected. Fetal heads were histologically scrutinized for the occurrence of cleft palates and microgenia. Folic acid at 2.5 mg/kg diminished PCZ-induced IUGR. In male fetuses, both folic acid doses significantly reduced the incidence of cleft palates and microgenia, while in females, only the high folic acid dose was capable of lowering the occurrence frequency of cleft palates. We conclude that folic acid supplementation at the used doses confers a substantial protection against PCZ-induced IUGR and incidence of cleft palates and microgenia. However, these effects are gender-related and dose-dependent.     

Epidemiology of orofacial clefts in a Danish county over 35 years – Before and after implementation of a prenatal screening programme for congenital anomalies

In 2004 the Danish National Board of Health changed its screening recommendations. Since 2005 a first trimester screening for Down syndrome and a prenatal ultrasound screening for congenital anomalies in the second trimester of pregnancy has been offered to all pregnant women.The aim of this study was to describe the prevalence of cleft lip with or without cleft palate and cleft palate in a Danish area and to describe associated anomalies and the development in prenatal diagnosis over time. The study was based on data from the EUROCAT Registry for Funen County. The registry is based on multiple data sources and includes information about live births, fetal deaths with a gestational age >20 weeks and terminations of pregnancy after prenatal diagnosis of severe fetal anomaly. The study included all fetuses/infants out of a population of 182,907 births diagnosed with orofacial clefts born between 1980 and 2014. There were 271 cases diagnosed with cleft lip with or without cleft palate and 127 cases diagnosed with cleft palate, giving a prevalence of 14.8 per 10,000 births for cleft lip with or without cleft palate and 6.9 per 10,000 births for cleft palate. There were no significant changes in prevalence over time for the two anomalies, calculated with and without inclusion of genetic and chromosomal cases. Overall 66 cases were diagnosed prenatally (17% of total). For isolated cleft lip with or without cleft palate none of the 157 cases born before 2005 were diagnosed prenatally compared to 34 of 58 cases (59%) born in 2005–2014 (p?<?0.01). The proportion of liveborn infants with multiple congenital anomalies also changed after 2005 with 15% (39/266) of all liveborn infants with orofacial clefts born 1980–2004 having multiple anomalies compared to 7% (7/96) in 2005–2014 (p?<?0.05).The implementation of the new screening programme in 2005 has given a major change in prenatal detection rate and reduced the proportion of liveborn infants with orofacial clefts classified as multiple congenital anomaly cases. The prevalence of cleft lip with or without cleft palate was higher than reported from many other countries.     

Use of folic acid supplements and risk of cleft lip and palate in infants: a population-based cohort study

Background

Orofacial clefts occur when the lips or the roof of the mouth do not fuse properly during the early weeks of pregnancy. There is strong evidence that periconceptional use of folic acid can prevent neural tube defects but its effect on oral clefts has generated debate.

Aim

To identify factors associated with suboptimal periconceptional use of folic acid and its potential effect on oral clefts.

Design and setting

The population-based infant cohort of the national Growing Up in Ireland study, which consists of 11 134 9-month-old infants.

Method

Data collection comprised questionnaires conducted by interviewers with parents in parents’ homes. Characteristics of mothers who did or did not take folic acid before and during pregnancy, as well as the effect of folic acid use on the prevalence of cleft lip and palate were recorded.

Results

The prevalence of cleft lip and palate was 1.98 (95% confidence interval [CI] = 1.31 to 2.99) per 1000 9-month-olds. The odds ratio for cleft lip was 4.36-fold higher (95% CI = 1.55 to 12.30, P = 0.005) for infants of mothers who did not take folic acid during the first 3 months of pregnancy, when compared with those who did have a folate intake during the first trimester. Folic acid use was suboptimal in 36.3% (95% CI = 35.4 to 37.2) of the sample.

Conclusion

These findings support the hypothesis that taking folic acid may partially prevent cleft lip and palate. They are particularly relevant for GPs, because they are usually the first port of call for women before and during early pregnancy.     

Folate and one-carbon metabolism gene polymorphisms and their associations with oral facial clefts

Folate metabolism plays a critical role in embryonic development. Prenatal folate supplementation reduces the risk of neural tube defects and probably oral facial clefts. Previous studies of related metabolic genes have associated polymorphisms in cystathionine-beta-synthase (CBS) and 5,10-methylenetetrahydrofolate reductase (MTHFR) with cleft risk. We explored associations between genes related to one-carbon metabolism and clefts in a Norwegian population-based study that included 362 families with cleft lip with or without cleft palate (CL/P) and 191 families with cleft palate only (CPO). We previously showed a 39% reduction in risk of CL/P with folic acid supplementation in this population. In the present study we genotyped 12 polymorphisms in nine genes related to one-carbon metabolism and looked for associations of clefting risk with fetal polymorphisms, maternal polymorphisms, as well as parent-of-origin effects, using combined likelihood-ratio tests (LRT). We also stratified by maternal periconceptional intake of folic acid (>400 microg) to explore gene-exposure interactions. We found a reduced risk of CL/P with mothers who carried the CBS C699T variant (rs234706); relative risk was 0.94 with one copy of the T allele (95% CI 0.63-1.4) and 0.50 (95% CI 0.26-0.96) with two copies (P = 0.008). We found no evidence of interaction of this variant with folate status. We saw no evidence of risk from the MTHFR C677T variant (rs1801133) either overall or after stratifying by maternal folate intake. No associations were found between any of the polymorphisms and CPO. Genetic variations in the nine metabolic genes examined here do not confer a substantial degree of risk for clefts.     

超声常规三切面与特殊切面法筛查唇腭裂胎儿的价值

目的:观察超声常规三切面与特殊切面法筛查唇腭裂胎儿的应用价值。方法:选取佛山市妇幼保健院6 000例孕妇,对2 556例胎儿唇腭部予以单一切面法（对照组）,3 444例胎儿唇腭部予以超声常规三切面+特殊切面法（观察组）,以出生儿颜面部检查为“金标准”,比较对照组与观察组检查结果灵敏度、特异度、准确性、阳性预测值、阴性预测值、漏诊率。结果:共47例（0.78%）唇腭裂;对照组20例（0.78%）唇腭裂,观察组27例（0.78%）唇腭裂,两组唇腭裂率比较无显著差异（P>0.05）;单一切面法灵敏度60.00%,特异度99.88%,准确性99.57%,漏诊率40.00%,阳性预测值80.00%,阴性预测值99.69%,Kappa值0.68;超声常规三切面+特殊切面法灵敏度92.59%,特异度100.00%,准确性99.94%,漏诊率7.41%,阳性预测值100.00%,阴性预测值99.94%,Kappa值0.96。观察组筛查灵敏度、准确性及阴性预测值均明显高于对照组（P<0.05）。结论:超声常规三切面+特殊切面法可有效提高唇腭裂胎儿筛查准确性,具有较高应用价值。     

Disturbances of palatogenesis and their prophylaxis in animal experiments.

The goal of this study using experimental animals was to induce disturbances of palatogenesis which are comparable to human maxillary clefts. Simultaneously, an in vivo method of testing presumed antiteratogenic substances is presented. 13 gravid Wistar rats bearing 98 fetuses received 200 mg/kg of procarbazine on day 14 post conception (p. c.) to induce malformations. 7 of these gravid animals, bearing 48 fetuses, additionally received 200 mg/kg thiamine daily from day 13-19 p. c. to prevent malformations. On day 20 p. c., the fetuses were teratologically screened: all fetuses were externally examined, the skeletons of 1/3 were visualized using cartilage/bone staining methods, and the heads of 2/3 were histologically examined in 24 sequential frontal sections. At birth, the procarbazine-damaged fetuses exhibited a high rate of cleft palate, primarily involving the secondary palate (94%), which was accompanied by retardation and delayed ossification of the viscerocranium. 66% of the fetuses showed pronounced brachygenia. The disturbances of palatogenesis were frequently accompanied by disturbed odontogenesis, which chiefly manifested itself near the cleft of the frontal maxilla as a reduction in size (63%), retardation (38%) or absence (31%) of the tooth germ. In the trial group additionally treated with thiamine, the findings did not differ significantly from these. The animal model presented here proved suitable for studying palatogenesis and localization-specific testing of substances presumed to have antiteratogenic effects. A prophylactic effect of thiamine initially tested as a highly-dosed monotherapy was not verifiable.     

Methylenetetrahydrofolate reductase thermolabile variant and oral clefts

Folic acid can prevent neural tube defects; in some cases the mechanism is probably a correction of a metabolic defect caused by thermolabile methylenetetrahydrofolate reductase (MTHFR) found in increased frequency in cases. It is less clear whether folic acid can prevent oral clefts, in part because it is not known whether thermolabile MTHFR is more common in those with oral clefts. This study examined the prevalence of the mutation (677 C→T) that causes thermolabile MTHFR in subjects with oral clefts from a national Irish support group, and an anonymous control group randomly selected from a neonatal screening program covering all births in Ireland. Eighty-three of 848 control subjects were homozygous (TT) thermolabile MTHFR (9.8%). This defect was almost three times as common in the 27 subjects (25.9%) with isolated cleft palate (odds ratio 3.23, 95% confidence interval 1.32 –7.86, P = 0.02) and somewhat more common in the 66 subjects with cleft lip with or without cleft palate (15.2%, odds ratio 1.65, 95% confidence interval 0.81–3.35, P = 0.20). When the two groups with different etiologies were combined, the overall odds ratio was 2.06 (95% confidence interval 1.16–3.66, P = 0.02). In the Irish population homozygosity for the common folate-related polymorphism associated with thermolabile MTHFR is significantly more frequent in those with isolated cleft palate, and could be etiologically important. Am. J. Med. Genet. 86:71–74, 1999. Published 1999 Wiley-Liss, Inc.     

Methylenetetrahydrofolate reductase thermolabile variant and oral clefts.

Folic acid can prevent neural tube defects; in some cases the mechanism is probably a correction of a metabolic defect caused by thermolabile methylenetetrahydrofolate reductase (MTHFR) found in increased frequency in cases. It is less clear whether folic acid can prevent oral clefts, in part because it is not known whether thermolabile MTHFR is more common in those with oral clefts. This study examined the prevalence of the mutation (677 C-->T) that causes thermolabile MTHFR in subjects with oral clefts from a national Irish support group, and an anonymous control group randomly selected from a neonatal screening program covering all births in Ireland. Eighty-three of 848 control subjects were homozygous (TT) thermolabile MTHFR (9.8%). This defect was almost three times as common in the 27 subjects (25.9%) with isolated cleft palate (odds ratio 3.23, 95% confidence interval 1.32 -7.86, P = 0. 02) and somewhat more common in the 66 subjects with cleft lip with or without cleft palate (15.2%, odds ratio 1.65, 95% confidence interval 0.81-3.35, P = 0.20). When the two groups with different etiologies were combined, the overall odds ratio was 2.06 (95% confidence interval 1.16-3.66, P = 0.02). In the Irish population homozygosity for the common folate-related polymorphism associated with thermolabile MTHFR is significantly more frequent in those with isolated cleft palate, and could be etiologically important. Am. J. Med. Genet. 86:71-74, 1999. Published 1999 Wiley-Liss, Inc.     

X-rayfilm analysis of the sinus paranasales from cleft patients (in comparison with a healthy group) (author's transl)

A report on 301 x-rayfilms from cleft patients and 120 x-rayfilms of a control group to show the behaviour of the pneumatisation of the maxillar sinus is being given. Control group: The sinus maxillares of the left side by the male and female patients are greater than of the right side. The sinus maxillares are greater by female patients than by male patients. Patients with cleft formation: The sinus maxillares by female patients are significant smaller than the sinus maxillares by the control group. No different are the sinus maxillares by the control group and male with cleft formation patients with the right-sided clefts and female patients with right-sided clefts and on bilateral clefts. The sinus maxillares are of the side of localisation smaller than of the opposite side. By clefts of the primary and secondary palate (L-A-P-clefts) are the sinuses maxillares statistically larger than by clefts of the primary palate (L-A-clefts) and by subtotally and totally clefts of the secondary palate (P-clefts).     

5,10-亚甲基四氢叶酸还原酶基因多态性及补充叶酸与非综合征性唇腭裂的相关性

背景：对于5, 10-亚甲基四氢叶酸还原酶(5, 10-methylene tetrahydrofolate reductase, MTHFR)基因C677T位点多态性与唇腭裂相关性的研究国内外结果不一,未见结合干预因素叶酸影响的相关报道。 目的：探讨河南地区汉族人群MTHFR基因C677T位点多态性及补充叶酸与非综合征性唇腭裂的发病关系。 方法：选取2008-09/2010-03在郑州大学第一附属医院及郑州市第一人民医院整形外科就诊的非综合征性唇腭裂患者110例,采用PCR-RFLP法检测外周血中MTHFR基因C677T位点基因型并与40例健康对照比较频数差异。同时结合母孕期是否补充叶酸进行统计学分析。 结果与结论：病例组和对照组C677T基因型及等位基因频率比较差异均具有显著性意义(P < 0.01),且有家族史的患者TT基因型及T等位基因频率高于无家族史患者(P < 0.05)。对母孕期是否补充叶酸进行比较,发现非综合征性唇腭裂与叶酸摄入呈负相关(χ²=4.304,r=-0.169,P  < 0.05)。结果提示MTHFR基因C677T位点突变与河南汉族人群非综合征性唇腭裂的发生相关,母孕期补充叶酸能降低非综合征性唇腭裂的发病风险。     

实时三维超声产前诊断胎儿典型面裂畸形的临床意义

目的探讨实时三维超声产前诊断胎儿面裂畸形的临床意义,寻求提高胎儿唇腭裂诊断准确性的有效方法。方法应用实时三维超声产前对面裂畸形;唇腭裂胎儿和正常胎儿唇腭部位的二维进行对照。结果 10例正常胎儿面部显示率100%,上牙槽突显示率100%,硬腭显示率70%(7/10)。10例唇腭裂胎儿包括7例单纯唇裂,2例上唇裂合并硬腭裂和1例唇裂合并软腭裂。实时三维诊断了所有7例单纯唇裂,诊断率100%;诊断了单侧唇裂合并腭裂1例,有1例单侧唇裂合并腭裂仅诊断了唇裂而漏诊了腭裂,诊断率70%(7/10),另外1例唇裂合并软腭裂仅诊断了唇裂而漏诊了软腭裂。结论实时三维超声产前诊断对胎儿唇裂,尤其是唇裂合并牙槽突及硬腭裂的诊断具有较大的应用价值,但是对胎儿未合并牙槽突裂的软腭裂及部分硬腭裂做出诊断,仍具有很大的难度。     

实时三维超声在产前诊断胎儿唇腭裂中的临床研究

目的探讨三维超声成像在胎儿唇腭裂中的诊断价值。方法应用实时三维超声仪对10164例孕妇、共10236个胎儿面部行二维和实时三维超声检查,统计并比较超声诊断与产后诊断的符合率。结果静态三维超声诊断胎儿唇裂或唇腭裂43例,实时三维超声诊断胎儿唇裂9例,唇腭裂22例,与二维超声诊断相符,漏诊软腭裂1例。结论实时三维超声提供了直观、立体、逼真的胎儿面部图像,优于单纯使用二维超声,两者联合应用可明显提高胎儿唇腭裂的检出率。     

Comparative teratogenicity of triamcinolone acetonide, triamcinolone, and cortisol in the rat

Pregnant rats were injected im with 0.5 mg/kg triamcinolone acetonide (TAC) on day 12, 13, or 14 of gestation and the fetuses were examined for cleft palate on day 20. All three TAC-treated groups showed an increased proportion of fetuses with cleft palate compared to an untreated control group. Only the group treated on day 13 showed a significant increase in the proportion of litters affected. This indicates that day 13 of gestation is the most sensitive day for cleft palate induction by TAC in the rat. Pregnant rats were then treated on day 13 of gestation with either TAC, triamcinolone (TA), or cortisol. TAC was 59 times as potent as TA in inducing cleft palate, with ED50 values of 1.1 mg/kg and 65 mg/kg respectively. Cortisol induced a significant increase in cleft palates at 500 mg/kg, but the efficacy of this compound was too low to calculate an ED50 and relative teratogenic potency value. Other developmental abnormalities including umbilical hernias, resorption, and fetal death resulted from TAC treatment. Fetal growth retardation was produced by all three compounds. The rank order of teratogenic potency was determined to be TAC greater than TA greater than cortisol.     

Morphological variability in unrepaired bilateral clefts with and without cleft palate evaluated with geometric morphometrics

In subjects with orofacial clefts, there is an unresolved controversy on the effect of congenital maxillary growth deficiency vs. the effect of surgical intervention on the outcome of treatment. Intrinsic growth impairment in subjects with orofacial clefts can be studied by comparing facial morphology of subjects with untreated cleft and unaffected individuals of the same ethnic background. Bilateral cleft lip and palate is the most severe and least prevalent form of the orofacial cleft. The aim of this study was to compare facial morphology in subjects with unrepaired complete bilateral clefts and unaffected controls using geometric morphometrics. Lateral cephalograms of 39 Indonesian subjects with unrepaired bilateral complete cleft lip and alveolus (mean age: 24 years), or unrepaired bilateral complete cleft lip, alveolus, and palate (mean age: 20.6 years) and 50 age and ethnically matched controls without a cleft (25 males, 25 females, mean age: 21.2 years) were digitized and traced and shape variability was explored using principal component analysis, while differences between groups and genders were evaluated with canonical variate analysis. Individuals with clefts had a more pronounced premaxilla than controls. Principal component analysis showed that facial variation in subjects with clefts occurred in the anteroposterior direction, whereas in controls it was mostly in the vertical direction. Regression analysis with group, sex, and age as covariates and principal components from 1 to 6 as dependent variables demonstrated a very limited effect of the covariates on the facial shape variability (only 11.6% of the variability was explained by the model). Differences between cleft and non-cleft subjects in the direction of facial variability suggest that individuals with bilateral clefts can have an intrinsic growth impairment affecting facial morphology later in life.     

Are the betaine-homocysteine methyltransferase (BHMT and BHMT2) genes risk factors for spina bifida and orofacial clefts?

Abnormalities in folate and/or homocysteine metabolism may adversely influence embryonic development, leading to the birth of infants with a variety of congenital malformations, including neural tube defects (NTDs) and craniofacial abnormalities. Based upon suggestive evidence that periconceptional folic acid supplementation is effective in preventing a significant proportion of the aforementioned birth defects, genetic variation in the folate biosynthetic pathways may influence the infant's susceptibility to these birth defects. The goal of our study was to investigate sequence variations in the betaine-homocysteine methyltransferase (BHMT) and betaine-homocysteine methyltransferase (BHMT2) genes as modifiers of risk of spina bifida, cleft palate, and cleft lip and palate. The results of this study indicated that individuals homozygous for the single nucleotide polymorphism R239Q in BHMT did not have elevated risks for spina bifida. Genotype frequencies for the BHMT2 rs626105 polymorphism also did not reveal any elevated risks for spina bifida, and only a modest, imprecise elevation of risk for orofacial clefts. The results of these experiments suggest that variants of the BHMT/BHMT2 genes in infants do not substantially contribute to the risk of spina bifida or orofacial clefts in our study population.     

Analysis of Meox-2 mutant mice reveals a novel postfusion-based cleft palate.

Cleft palate represents a common human congential disease involving defects in the development of the secondary palate. Major steps in mammalian palatogenesis include vertical growth, elevation, and fusion of the palate shelves. Our current study with the homeobox gene Meox-2 during mouse secondary palate development reveals a novel postfusion-based mechanism for cleft palate. Meox-1 and Meox-2 are two functionally related homeobox genes playing important roles in somitogenesis and limb muscle differentiation. We found that the expression of Meox-2, not Meox-1, marks the specification of early mouse palatal mesenchymal cells in the maxillary processes at embryonic day 11.5 (E11.5). From E12.5 to E15.5, the expression of Meox-2 occupies only the posterior part of the palate, providing an early molecular marker for the anterior-posterior polarity in mouse secondary palate formation. A total of 35.3% of Meox-2-/- (n = 17) and 25.5% of Meox-2+/- (n = 55) mouse embryos display a cleft palate phenotype at E15.5, indicating that the reduction of Meox-2 function is associated with susceptibility to cleft palate. Unlike previously reported clefts, none of the clefts found in Meox-2 mutants contain any epithelial sheets in the medial edge areas, and detailed examination revealed that the clefts resulted from the breakdown of newly fused palates. This article is the first report of a gene required to maintain adherence of the palatal shelves after fusion.     

Variable contribution of the MTHFR C677T polymorphism to non-syndromic cleft lip and palate risk in China

Non-syndromic cleft lip with or without cleft palate (nsCL/P) is one of the most common craniofacial malformations among newborn infants. It has been demonstrated that periconceptional folic acid supplementation may reduce the occurrence of offspring with clefts, particularly in the North China; however, the mechanism remains unknown. Our study of a thermolabile polymorphism (C677T) of methylenetetrahydrofolate reductase (MTHFR) gene in 170 Chinese case-parent triads revealed a moderate association between this MTHFR polymorphism and nsCL/P in a population from North China, but not in a population from South China. Moreover, the study revealed that the heterozygous parents in the North were about twice as likely to transmit the high-risk T allele to affected cases, as that observed in the South (OR = 2.24, 95% CI: 1.08-4.65). Thus, the MTHFR polymorphism is a significant risk factor for nsCL/P in this Northern Chinese population. Our study suggested possible genetic heterogeneity in the development of nsCL/P among Northern and Southern populations in China.     

早孕期唇腭裂的超声诊断初探

目的探讨二维颜面部正交叉三切面联合扫查以及三维新技术在早孕期唇腭裂诊断中的应用价值。方法选取2018年6月至2019年7月于宁夏医科大学总医院接受早孕期产前超声筛查的胎儿599例,头臀径(CRL)50~84 mm。首先应用二维超声评估胎儿腭部的3个重要标志,即腭线(正中矢状切面)、上颌骨牙槽突(横切面)和鼻后三角底部(冠状切面)。然后适当放大可疑胎儿以及30例正常胎儿面部图像,采集正中矢状切面三维容积数据,应用TUI、OmniView等技术观察腭部,并给予脱机分析。所有胎儿均进行中孕期超声畸形筛查,并在产后或引产后给予追踪随访。结果本研究599例胎儿共发现7例不同类型的裂缺,其中单侧唇腭裂3例,双侧唇腭裂2例,中央型唇腭裂2例,其余胎儿3个超声标志均没有回声缺失或连续性中断,敏感度为87.5%,特异性为100%,假阳性率为0,假阴性率为12.5%。产前诊断结果均经产后或引产后证实。结论二维颜面部三切面联合扫查可用于筛查早孕期唇腭裂,三维容积数据分析有利于唇腭裂类型的精确诊断。     

Prenatal diagnosis of an isolated incomplete V-shaped cleft palate using a new three-dimensional ultrasound technique investigation

Objective The objective of this case report is to evaluate the faculty of a recently described original three-dimensional ultrasound technique to detect pathological processes of the fetal palate. Method The palate of the fetus of a patient with a personal history of isolated incomplete cleft palate is evaluated by three-dimensional ultrasound at 34 weeks of gestation. The postnatal findings are compared to the prenatal investigation. Result The used three-dimensional ultrasound technique provides selective visualization of the total hard palate which permits the prenatal diagnosis and multidisciplinary approach of an incomplete cleft palate. Conclusion These prenatal findings might encourage further studies to confirm the value of this recently described innovative technique in the diagnosis and surgical prenatal counselling of fetal cleft palate.