Amniotic fluid homocysteine levels, 5,10-methylenetetrahydrafolate reductase genotypes, and neural tube closure sites

A specific gene mutation leading to altered homocysteine metabolism has been identified in parents and fetuses with neural tube defects (NTDs). In addition, current animal and human data indicate that spine closure occurs simultaneously in five separate sites that then fuse. We sought to determine whether either this mutation or abnormal amniotic fluid homocysteine levels are associated with all five neural tube closure sites. We retrieved stored amniotic fluid from cases of isolated fetal neural tube defect diagnosed from 1988 to 1998 (n = 80) and from normal controls matched for race, month and year of amniocentesis, and maternal age. Cases were categorized according to defect site by using all available medical records. The presence or absence of the 677C-->T mutation of 5, 10-methylenetetrahydrafolate reductase (MTHFR) gene was determined, and homocysteine levels were measured; case and controls were compared. Significantly more cases than controls were heterozygous or homozygous for the 677C-->T MTHFR mutation (44% vs. 17%, P < or = 0. 001). Likewise, cases were significantly more likely than controls to have amniotic fluid homocysteine levels >90th centile (>1.85 micromol/L), 27% vs. 10%, P = 0.02. Most (83%) of control cases had both normal MTHFR alleles and normal amniotic fluid homocysteine levels (normal/normal), whereas only 56% of NTD case were normal/normal (P = 0.001). When evaluated by defect site, only defects involving the cervical-lumbar spine, lumbosacral spine, and occipital encephalocele were significantly less likely to be normal/normal than controls (P = 0.007, 0.0003, and 0.007, respectively), suggesting a strong association with the 677C-->T allele. In contrast, anencephaly, exencephaly, and defects confined to the sacrum included many cases that had both normal MTHFR alleles and normal homocysteine and were not significantly different from controls. The 677C-->T MTHFR mutation and elevated homocysteine levels appear to be disproportionately associated with defects spanning the cervical-lumbar spine, lumbosacral spine, and occipital encephalocele. In contrast, anencephaly, exencephaly, and defects confined to the sacrum may not be related to altered homocysteine metabolism.     

Role of amniotic fluid homocysteine level and of fetal 5,10‐methylenetetrahydrafolate reductase genotype in the etiology of neural tube defects

A mutation in the gene 5,10‐methylenetetrahydrofolate reductase (MTHFR), leading to altered homocysteine metabolism, has been identified in parents and fetuses with fetal neural tube defects. We sought to determine which is of greater importance in fetal neural tube defect formation: the fetal MTHFR mutation or elevated amniotic fluid homocysteine level. We retrieved stored amniotic fluid from cases of isolated fetal neural tube defect diagnosed from 1988 to 1998 (n = 80), and from normal controls matched for race, month and year of amniocentesis, and maternal age. The presence or absence of the 677C→T mutation of MTHFR was determined and homocysteine levels were measured; cases and controls were compared. Significantly more cases than controls were heterozygous or homozygous for the 677C→T MTHFR mutation (44% vs 17%, P ≤ 0.001). Cases were also significantly more likely than controls to have an amniotic fluid homocysteine level above the 90th centile (>1.85 μmol per liter); 27% vs 10%, P = 0.02. Thirty one cases and 12 controls had an abnormal genotype; however, amniotic fluid homocysteine levels were not significantly different between these two groups (6/31, or 19% of cases had an elevated homocysteine compared to 1/12, or 8% of controls; P = 0.65). In contrast, 40 cases and 60 controls had a normal genotype; the neural tube defect cases had significantly higher homocysteine levels (13/40, or 32% of cases had an elevated homocysteine level compared to only 6/60, or 10% of controls; P = 0.008). Although both abnormal fetal MTHFR genotype and abnormal amniotic fluid homocysteine concentration are significantly associated with neural tube defects, the association with amniotic fluid homocysteine concentration is significant regardless of the fetal MTHFR genotype. The relationship between maternal and fetal homocysteine metabolism is complex. Am. J. Med. Genet. 90:12–16, 2000. © 2000 Wiley‐Liss, Inc.     

Role of amniotic fluid homocysteine level and of fetal 5, 10-methylenetetrahydrafolate reductase genotype in the etiology of neural tube defects

A mutation in the gene 5,10-methylenetetrahydrofolate reductase (MTHFR), leading to altered homocysteine metabolism, has been identified in parents and fetuses with fetal neural tube defects. We sought to determine which is of greater importance in fetal neural tube defect formation: the fetal MTHFR mutation or elevated amniotic fluid homocysteine level. We retrieved stored amniotic fluid from cases of isolated fetal neural tube defect diagnosed from 1988 to 1998 (n = 80), and from normal controls matched for race, month and year of amniocentesis, and maternal age. The presence or absence of the 677C-->T mutation of MTHFR was determined and homocysteine levels were measured; cases and controls were compared. Significantly more cases than controls were heterozygous or homozygous for the 677C-->T MTHFR mutation (44% vs 17%, P < or = 0.001). Cases were also significantly more likely than controls to have an amniotic fluid homocysteine level above the 90th centile (>1.85 micromol per liter); 27% vs 10%, P = 0.02. Thirty one cases and 12 controls had an abnormal genotype; however, amniotic fluid homocysteine levels were not significantly different between these two groups (6/31, or 19% of cases had an elevated homocysteine compared to 1/12, or 8% of controls; P = 0.65). In contrast, 40 cases and 60 controls had a normal genotype; the neural tube defect cases had significantly higher homocysteine levels (13/40, or 32% of cases had an elevated homocysteine level compared to only 6/60, or 10% of controls; P = 0.008). Although both abnormal fetal MTHFR genotype and abnormal amniotic fluid homocysteine concentration are significantly associated with neural tube defects, the association with amniotic fluid homocysteine concentration is significant regardless of the fetal MTHFR genotype. The relationship between maternal and fetal homocysteine metabolism is complex.     

A second common variant in the methylenetetrahydrofolate reductase (MTHFR) gene and its relationship to MTHFR enzyme activity, homocysteine, and cardiovascular disease risk

Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinemia, an independent and graded risk factor for cardiovascular disease (CVD). We examined the relationship of two polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, the 677C-->T and 1298A-->C variants, to MTHFR activity, homocysteine concentrations, and risk of CVD in a population of 190 vascular disease patients and 601 apparently healthy controls. The mean specific and residual MTHFR activities were significantly lower in 677CT and 677TT individuals (both P<0.001). The 1298A-->C mutation alone showed no effect on MTHFR activities. However, when the 677C-->T genotype was taken into account, the 1298A-->C mutation also caused a significant decrease in MTHFR activities, which was observed in both the homozygous 1298CC (P<0.001) and the heterozygous 1298AC states (P=0.005). Both the 677TT as the 677CT genotypes were associated with significantly higher fasting and postload homocysteine levels than 677CC (P<0.001 and P=0.003, respectively). The 1298A-->C mutation had no effect on fasting or postload homocysteine levels. Since homocysteine itself is considered to be positively associated with the risk of CVD, these findings indicate that the 1298A-->C mutation cannot be considered a major risk factor for CVD.     

Low blood folates in NTD pregnancies are only partly explained by thermolabile 5,10-methylenetetrahydrofolate reductase: Low folate status alone may be the critical factor

Thermolabile 5,10-methylenetetrahydrofolate reductase (MTHFR) is the first folate-related variant to be associated with an increased risk of neural tube defects (NTDs). The variant causes high plasma homocysteine levels and reduced red cell folate (RCF) levels, both of which have also been linked to an increased risk of NTDs. We examined the relationship between folate status and presence of the common mutation MTHFR C677T in 82 NTD-affected and 260 control mothers. Homozygosity for the TT genotype was associated with very low folate status among both the cases (n = 13) and the controls (n = 21). However, after exclusion of TT homozygotes, only 10% of the remaining 240 controls had RCF levels less than 200 μg/L compared with 29% of the 69 cases (odds ratio, 3.67; 95% confidence interval, 1.88–7.18; P < 0.001), and those with RCF less than 150 μg/L had eight times higher risk of NTD than subjects with levels over 400 μg/L. Plasma homocysteine levels of non-TT cases were also higher than those of controls (P = 0.047). This study shows that homozygosity for the C677T MTHFR variant cannot account for reduced blood folate levels in many NTD-affected mothers. Thus, a strategy of genetic screening of all childbearing women for this variant would be ineffective as a method of primary prevention of NTDs. The data suggest that low maternal folate status is itself the major determinant of NTD risk, or else that other folate-dependent genetic variants confer risk through the reduction of folate levels. These results emphasize the importance of a food-fortification program as a population strategy for reducing the occurrence of NTDs. Am. J. Med. Genet. 78:155–159, 1998. © 1998 Wiley-Liss, Inc.     

Methylenetetrahydrofolate reductase and spina bifida: evaluation of level of defect and maternal genotypic risk in Hispanics

The C677T and A1298C mutations in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene are each associated with reduced MTHFR activity. The C677T mutation in the heterozygous and homozygous state correlates with increased enzyme thermolability, with homozygous mutant genotypes showing significantly elevated plasma homocysteine levels and decreased plasma folate levels. The A1298C mutation results in decreased MTHFR activity, but changes in neither homocysteine nor folate levels are associated with A1298C variant genotypes. Our study determined the frequencies of the C677T and A1298C MTHFR mutations for spina bifida (SB) cases, mothers and fathers of SB cases, and controls in Hispanics of Mexican-American descent. In addition, our subject population was further categorized as to whether the spina bifida lesion occurred as an upper or lower level defect, according to the Van Allen "multi-site closure" model. Hispanic SB cases with upper level defects and their mothers were homozygous for the C677T variant allele at a higher rate than their respective controls (OR = 1.5 [95% CI 0.8-2.9], P = 0.30; OR = 2.3 [1.1-4.8], P = 0.04, respectively), with statistically significant results seen only for the maternal homozygous genotype. Homozygosity for the A1298C mutation was seen at a higher rate only in Hispanic mothers of both upper and lower level SB cases when compared to controls, but these results were not statistically significant. Our study provides evidence that the maternal C677T MTHFR homozygous mutant genotype is a risk factor for upper level spina bifida defects in Hispanics.     

5,10 Methylenetetrahydrofolate reductase genetic polymorphism as a risk factor for neural tube defects

Persons with a thermolabile form of the enzyme 5,10 methylenetetrahydrofolate reductase (MTHFR) have reduced enzyme activity and increased plasma homocysteine which can be lowered by supplemental folic acid. Thermolability of the enzyme has recently been shown to be caused by a common mutation (677C^→T) in the MTHFR gene. We studied 41 fibroblast cultures from NTD-affected fetuses and compared their genotypes with those of 109 blood specimens from individuals in the general population. 677C^→T homozygosity was associated with a 7.2 fold increased risk for NTDs (95% confidence interval: 1.8–30.3; p value: 0.001). These preliminary data suggest that the 677C^→T polymorphism of the MTHFR gene is a risk factor for spina bifida and anencephaly that may provide a partial biologic explanation for why folic acid prevents these types of NTD.     

Neural tube defects and maternal hyperthermia in early pregnancy: Epidemiology in a human embryo population

A causal relationship between neural tube defects and maternal febrile illness in early pregnancy was investigated retrospectively using human embryo data from Japan. The frequency of febrile illness was compared with corresponding data from normal controls, and holoprosencephalic and polydactylous embryos. Within the group of neural tube defects, the prevalence of maternal hyperthermia was significantly higher than in normal control and holoprosencephaly mothers. The association was constant and highly significant specifically for exencephaly, which is an embryonic forerunner of anencephaly. The correlation with myeloschisis (without exencephaly) was not significant. In eight out of the ten cases in which the timing of the fever was known, it occurred during the clinically determined critical period of neural tube closure in human embryos. These results support the hypothesis that maternal hyperthermia in early pregnancy can cause neural tube defects in man, especially anencephaly.     

MTHFR基因C677T多态性与神经管缺损的相关性研究

目的探讨5,10-甲基四氢叶酸还原酶（5,10-methylenetetrahydrofolate reductase,MTHFR）基因C677T突变与神经管缺损（neuraltube defectes,NTD）发病的相关性。方法应用PCR—RFLP法,对67名正常儿童和48例NTD患儿（NTD组）（其中无脑儿16例,脊柱裂32例）进行MTHFR基因C677T突变分析。结果NTD组胎儿MTHFR基因TT基因型频率（68．7％）和T等位基因频率（0．820）均显著高正常对照组儿童（31．3％和0．54）（x^2=15．71,P〈0．01和x^2=17．18,P〈0．01）。与MTHFR基因CC基因型相比,携带TT基因型的胎儿发生NTD的相对风险增加6．28倍（95％CI：2．01～19．62）。结论MTHFR基因C677T多态性与潍坊地区人胎儿NTD发病有关联。     

MTHFR基因多态性对血管内皮功能指标的影响

目的：研究社区人群中MTHFR 677C→T基因突变对血管紧张素Ⅱ、前列环素和一氧化氮水平的影响。方法：整群抽取社区汉族成年居民1 146人，采用PCR－RFLP鉴定目标人群MTHFR 677位基因型；采用荧光生化技术测定血浆Hcy浓度；用镉还原法检测其血清一氧化氮水平，用放射免疫分析法检测血浆血管紧张素Ⅱ和前列环素的浓度。所有数据用SPSS 13.0 软件进行统计分析。结果：1 146例居民按MTHFR 677基因型自然分组，三组之间PGI₂、AngⅡ没有统计学差异；C/C和T/C基因型组之间血清NO水平无显著差异（P>0.05），T/T基因型组的血清NO水平较C/C和T/C基因型组都低（P＜0.01）。结论:社区人群中MTHFR 677C→T突变对血浆PGI₂、AngⅡ的影响并不明显；血清NO水平下降，可能是MTHFR 677C→T导致缺血性心脑血管病的机制之一。     

Frequency of the thermolabile variant C677T in the MTHFR gene and lack of association with neural tube defects in the State of Yucatan,Mexico

The C677T variant in the MTHFR gene is considered to be an associated risk factor for neural tube defects. However, the association has not been found in some ethnic groups. In order to assess the association between neural tube defects and the C677T variant, we determined the frequency of this variant in the MTHFR gene in the State of Yucatan, Mexico, where neural tube defects are highly prevalent. The study was performed on 65 subjects with spine bifida, 60 of their mothers and 110 control subjects. The presence of the C677T variant was determined by amplification and digestion with HinF1 of each subject's DNA. Genotypic and allelic frequencies were calculated for all groups. We did not observe any statistically significant difference in the genotypic or allelic frequencies between cases and controls for any of the groups studied (p > 0.05), suggesting that the thermolabile variant C677T is not an associated risk factor neither for the development of neural tube defects nor for mothers to have affected offspring in the population from Yucatan. Interestingly, the frequency of the C677T variant (54%) obtained in the Yucatan population is one of the highest reported (p < 0.01) and confirmed the high frequency of this allele throughout Mexico.     

同型半胱氨酸和MTHFR基因多态性与Alzheimer病的关系

目的探讨同型半胱氨酸(Hcy)和5,10 亚甲基四氢叶酸还原酶(MTHFR)基因多态性与Alzheimer病的关系.方法运用多聚酶链反应限制性内切酶片段长度多态性技术(PCR RFLP)和荧光偏振法(FPIA)检测66例阿尔茨海默病及143例正常人MTHFR基因多态性和血浆总Hcy水平.结果 (1)AD病人甲基四氢叶酸还原酶基因中,基因型C/T占56.06%,明显高于对照组的34.97%(P<0.01,RR=0.355),C/C占39.39%,明显低于对照组的62.94%(P<0.01),T/T占4.25%,与对照组2.09%无明显差异(P>0.05). AD病人中甲基四氢叶酸还原酶基因等位基因C的频率为67.43%、相对危险率(RR)为0.594,T的频率为32.57%、与对照组的C为80.42%、T为19.58%有显著性差异(P≤0.05).(2)AD病例组与对照组血浆Hcy分别为14.72±6.2μmol/L和10.9±2.4μmol/L,两者差异有显著差异(P<0.05).AD患者血浆总Hcy水平显著高于正常组.结论 MTHFR基因突变及高同型半胱氨酸血症与Alzheimer病发生有一定关系.     

Influence of combined methionine synthase (MTR 2756A > G) and methylenetetrahydrofolate reductase (MTHFR 677C > T) polymorphisms to plasma homocysteine levels in Korean patients with ischemic stroke

PURPOSE: Methionine synthase (MTR) and 5,10-methylenetetrahydrofolate reductase (MTHFR) are the main regulatory enzymes for homocysteine metabolism. The present case- control study was conducted to determine whether there is an association between the MTR 2756A > G or MTHFR 677C > T polymorphism and plasma homocysteine concentration in Korean subjects with ischemic stroke. MATERIALS AND METHODS: DNA samples of 237 patients who had an ischemic stroke and 223 age and sex-matched controls were studied. MTR 2756A > G and MTHFR 677C > T genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Frequencies of mutant alleles for MTR and MTHFR polymorphisms were not significantly different between the controls and cases. The patient group, however, had significantly higher homocysteine concentrations of the MTR 2756AA and MTHFR 677TT genotypes than the control group (p=0.04 for MTR, p=0.01 for MTHFR). The combined MTR 2756AA and MTHFR 677TT genotype (p= 0.04) and the homocysteine concentrations of the patient group were also higher than those of the controls. In addition, the genotype distribution was significant in the MTHFR 677TT genotype (p=0.008) and combined MTR 2756AA and MTHFR 677TT genotype (p=0.03), which divided the groups into the top 20% and bottom 20% based on their homocysteine levels. CONCLUSION: The results of the present study demonstrate that the MTR 2756A > G and MTHFR 677C > T polymorphisms interact with elevated total homocysteine (tHcy) levels, leading to an increased risk of ischemic stroke.     

Exencephaly in human fetuses

In some anencephalic fetuses exposed neural tissue mass of varied size can be demonstrated. This is known as exencephaly. The authors diagnosed by ultrasound 10 typical exencephalic cases prenatally between 14 and 21 weeks of gestation. Nine singular pregnancies were terminated and in the twin pregnancy a selective feticide of the exencephalic co-twin was carried out. The pregnancy continued to term and a healthy newborn infant and a fetus papyraceus were born. The mummified co-twin was anencephalic and showed only the remnants of the exposed brain. Authors suggest that, as in experiments with animals, the exencephaly in humans, by the degeneration of the exposed neural tissue converts to anencephaly and in this process the macrophages in fetal circulation and in the amniotic fluid may play a significant role. The large number of these actively phagocytic macrophages can be demonstrated in the amniotic fluid samples from exencephalic fetuses.     

Genetic polymorphisms in methylenetetrahydrofolate reductase and methionine synthase,folate levels in red blood cells,and risk of neural tube defects

Folic acid administration to women in the periconceptional period reduces the occurrence of neural tube defects (NTDs) in their offspring. A polymorphism in the gene encoding methylenetetrahydrofolate reductase (MTHFR), 677C→12;T, is the first genetic risk factor for NTDs in man identified at the molecular level. The gene encoding another folate-dependent enzyme, methionine synthase (MTR), has recently been cloned and a common variant, 2756A→12;G, has been identified. We assessed genotypes and folate status in 56 patients with spina bifida, 62 mothers of patients, 97 children without NTDs (controls), and 90 mothers of controls, to determine the impact of these factors on NTD risk. Twenty percent of cases and 18% of case mothers were homozygous for the MTHFR polymorphism, compared to 11% of controls and 11% of control mothers, indicating that the mutant genotype conferred an increased risk for NTDs. The risk was further increased if both mother and child had this genotype. The MTR polymorphism was associated with a decreased O.R. (O.R.); none of the cases and only 10% of controls were homozygous for this variant. Red blood cell (RBC) folate was lower in cases and in case mothers, compared to their respective controls. Having a RBC folate in the lowest quartile of the control distribution was associated with an O.R. of 2.56 (95% CI 1.28–5.13) for being a case and of 3.05 (95% CI 1.54–6.03) for being a case mother. The combination of homozygous mutant MTHFR genotype and RBC folate in the lowest quartile conferred an O.R. for being a NTD case of 13.43 (CI 2.49–72.33) and an O.R. for having a child with NTD of 3.28 (CI 0.84–12.85). We propose that the genetic-nutrient interaction—MTHFR polymorphism and low folate status—is associated with a greater risk for NTDs than either variable alone. Am. J. Med. Genet. 84:151–157, 1999. © 1999 Wiley-Liss, Inc.     

糖尿病视网膜病MTHFR基因多态性及其与血浆同型半胱氨酸水平的关系

目的：研究亚甲基四氢叶酸还原酶（methylenetetrahydrofolate reductase,MTHFR）基因多态性及血浆同型半胱氨酸水平与2型糖尿病视网膜病的关系。方法：应用聚合酶链反应－限制性内切酶片段长度多态性技术检测208例2型糖尿病患者（其中110例伴视网膜病）及57名正常对照的MTHFR C677T基因型，采用高效液相色谱法测定血浆同型半胱氨酸水平。结果：糖尿病视网膜病组MTHFR基因TT纯合基因型、CT杂合基因型及T等位基因频率（分别为28．18％、41．82％、49．09％）均明显高于糖尿病不伴视网膜病组（分别为18．37％、29．59％、33．16％）及正常对照组（分别为17．54％、28．07％、31．58％），基因型和等位基因频率分布差异均有显著性（P＜0．01），而MTHFR基因多态性在糖尿病不伴视网膜病组与正常对照组之间差异无显著性（P＞0．05），T等位基因与糖尿病视网膜病的发生密切相关（OR＝1．94，95％CI；1．31－2．88）。糖尿病视网膜病组、糖尿病不伴视网膜病组及正常对照组中，MTHFR基因有C677T突变者血浆同型半胱氨酸水平均显著高于无基因突变者。结论：MTHFR基因C677T位碱基突变致血浆同型半胱氨酸水平升高可能是糖尿病视网膜病发病的重要遗传因素。     

Methylenetetrahydrofolate reductase polymorphism and pre-eclampsia.

A common missense mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, a C to T substitution at nucleotide 677, is responsible for reduced MTHFR activity and associated with modestly increased plasma homocysteine concentrations. Since underlying maternal vascular disease increases the risk of pre-eclampsia, we had the working hypothesis that pre-eclampsia patients would have an increased T677 allele frequency compared with controls. The MTHFR genotypes were determined in 67 pre-eclampsia patients, 98 normal pregnant women, and 260 healthy adults by the PCR/RFLP method. The T677 allele and the genotype homozygous for the T677 allele were significantly increased in the pre-eclamptic group compared with the controls (p < 0.02 and p < 0.004, respectively). The data indicate that the T677 variant of the MTHFR gene is one of the genetic risk factors for pre-eclampsia.     

Linkage disequilibrium of MTHFR genotypes 677C/T-1298A/C in the German population and association studies in probands with neural tube defects(NTD)

A number of studies have demonstrated that the common polymorphism 677C→T in the gene encoding 5, 10-methylenetetrahydrofolate reductase (MTHFR) leads to a thermolabile variant with decreased enzyme activity and to mildly elevated plasma homocysteine. 677TT homozygosity was shown to be more frequent in NTD probands compared with controls in some studies. Recently, another polymorphism, 1298A→C, in the MTHFR gene was described and combined heterozygosity 677CT/1298AC was suggested to be an additional risk factor for NTD. The present study examines the genotype and haplotype distribution of the two polymorphisms in the German population and evaluates the impact on NTD individuals and their relatives. To determine the haplotype of all individuals tested, we developed an easy-to-perform ARMS-RFLP test. Our data show that the two polymorphisms are in linkage disequilibrium in the general population and in NTD individuals. There was no statistically significant difference in allele and genotype frequency between probands (patients, fetuses) and controls (P > 0.10) and between observed and expected values for mother–child pairs (P > 0.80). Taking into account gender, an increased rate of 677CT heterozygotes was found in affected and unaffected males compared to affected and unaffected females. A family-based association study using a multiallelic transmission disequilibrium test (TDT) also shows that transmission rates do not deviate significantly from equilibrium (P > 0.50). Thus, our data provide no evidence for an association between NTD phenotype and MTHFR 677C/T-1298A/C genotypes and haplotypes. Am. J. Med. Genet. 87:23–29, 1999. © 1999 Wiley-Liss, Inc.     

Differential staining techniques in amniotic fluid cytology diagnosis of neural tube defects: a prospective study of 129 pregnancies

In order to assess the utility of amniotic fluid cytology in the diagnosis of neural tube defects (NTDs), we performed a prospective study of 129 pregnancies selected for maternal age or history of prior congenital defects or multiple stillbirths. Amniotic fluid obtained between the 16th and 22nd weeks of gestation was cytocentrifuged and stained with hematoxylineosin (H&E), Papanicolaou, and Diff-Quik. Chromosomal analysis, amniotic fluid alpha fetoprotein (AFP) level, and eventual pregnancy outcome were recorded for each case. The AFP levels were normal in 128 cases, including two closed NTDs (meningomyelocele and encephalocele). Three patients had abnormal chromosomal studies: two with 47,XXX and one with trisomy 21. Of these 128 cases, 16 had inadequate cytologic preparations; the remainder, including the five aforementioned patients, showed predominantly squamous cells, amnion cells, and minicells. Other cell types, including genito-urinary cells, umbilical cord cells, respiratory cells, and macrophages, were also present in smaller numbers. One case exhibited a large population of small, darkly staining neural cells and large macrophages. The AFP in this case was markedly elevated, and the fetus was noted to be anencephalic. We found the H&E to be the preferred stain for both cytologic detail and minicell preservation; however papanicolaou was better for bloody specimens. Amniotic fluid cytology may provide a rapid and inexpensive method of confirming the diagnosis of open NTDs.     

Homocysteine and atheromatous renal artery stenosis

Hyperhomocysteinemia is an independent risk factor for vascular disease, frequently observed in patients with severe renal impairment. Hyperhomocysteinemia has never been considered as a possible risk factor in renal artery stenosis. We investigated plasma folate and vitamin B₁₂, methylenetetrahydrofolate reductase (MTHFR) C577T and cystathionine β-synthase (CBS) 844ins68 polymorphisms, and homocysteine levels before and after methionine (100 mg/kg) loading in 58 patients with angiographically documented renal artery stenosis and mildly impaired renal function. One hundred and two normotensive subjects with angiographically normal coronary arteries and no history or clinical or angiographic evidence of atherosclerosis in other vascular districts, were considered as a control group. Mean total homocysteine levels were significantly higher in patients than in controls (P<0.01), as was the prevalence of hyperhomocysteinemia (51.7% vs. 32.3%, P<0.05). However, MTHFR alleles and genotypes as well as CBS 844ins68 mutation frequencies were similar in the two groups, whereas a lower folate level was observed in the patients. Moreover, patients with MTHFR A/A genotype showed a poorer folate status than control subjects, suggesting that a subclinical folate deficiency may be very frequent in renal artery stenosis, regardless of C677T mutation. In conclusions, hyperhomocysteinemia is common in patients with atheromatous renal artery stenosis; a subclinical folate deficiency seems to be involved, regardless of MTHFR thermolabile or CBS insertion genotypes. Folate supplementation might be useful in the management of overall vascular risk of these patients. Received: 13 November 2001 / Accepted: 13 December 2001