A spectrum project: preterm birth and small-for-gestational age among infants with birth defects.

OBJECTIVE: The aim of this study is to investigate the association between birth defects (BDs), prematurity and small-for-gestational age (SGA) in a population-based sample. STUDY DESIGN: Participants were singleton live births enrolled in the National Birth Defects Prevention Study, including 18 737 case infants with one or more BD and 7999 controls. Logistic regression models to evaluate associations between BDs, prematurity and fetal growth were computed while adjusting for covariates. RESULT: Cases were significantly more likely to be born prematurely than controls, particularly at 24 to 28 weeks of gestation. The highest odds ratios for preterm birth were found for intestinal atresia, anencephaly, gastroschisis and esophageal atresia. Infants with BDs were also significantly more likely to be SGA than controls (17.2 and 7.8%). CONCLUSION: Infants with BDs are more likely than controls to be born prematurely and SGA. Findings from this study present additional evidence demonstrating a complex interaction between the development of BDs, prematurity and intrauterine growth.

Assisted reproductive technology and major structural birth defects in the United States.

BACKGROUND: With >1% of US births occurring following use of assisted reproductive technology (ART), it is critical to examine whether ART is associated with birth defects. METHODS: We analyzed data from the National Birth Defects Prevention Study, a population-based, multicenter, case-control study of birth defects. We included mothers of fetuses or live-born infants with a major birth defect (case infants) and mothers who had live-born infants who did not have a major birth defect (control infants), delivered during the period October 1997-December 2003. We compared mothers who reported ART use (IVF or ICSI) with those who had unassisted conceptions. Multiple logistic regression was used to adjust for the following confounders: maternal race/ethnicity, maternal age, smoking and parity; we stratified by plurality. RESULTS: ART was reported by 1.1% of all control mothers, and by 4.5% of control mothers 35 years or older. Among singleton births, ART was associated with septal heart defects (adjusted odds ratio [aOR] = 2.1, 95% confidence intervals [CI] 1.1-4.0), cleft lip with or without cleft palate (aOR = 2.4, 95% CI 1.2-5.1), esophageal atresia (aOR = 4.5, 95% CI 1.9-10.5) and anorectal atresia (aOR = 3.7, 95% CI 1.5-9.1). Among multiple births, ART was not significantly associated with any of the birth defects studied. CONCLUSIONS: These findings suggest that some birth defects occur more often among infants conceived with ART. Although the mechanism is not clear, couples considering ART should be informed of all potential risks and benefits.

Congenital gastrointestinal defects in Down syndrome: a report from the Atlanta and National Down Syndrome Projects.

We report Down syndrome (DS)-associated congenital gastrointestinal (GI) defects identified during a 15 year, population-based study of the etiology and phenotypic consequences of trisomy 21. Between 1989 and 2004, six sites collected DNA, clinical and epidemiological information on live-born infants with standard trisomy 21 and their parents. We used chi-squared test and logistic regression to explore relationships between congenital GI defects and infant sex, race, maternal age, origin of the extra chromosome 21, and presence of a congenital heart defect. Congenital GI defects were present in 6.7% of 1892 eligible infants in this large, ethnically diverse, population-based study of DS. Defects included esophageal atresia/tracheoesophageal fistula (0.4%), pyloric stenosis (0.3%), duodenal stenosis/atresia (3.9%), Hirschsprung disease (0.8%), and anal stenosis/atresia (1.0%). We found no statistically significant associations between these defects and the factors examined. Although not significant, esophageal atresia was observed more often in infants of younger mothers and Hispanics, Hirschsprung disease was more frequent in males and in infants of younger mothers and blacks, and anal stenosis/atresia was found more often among females and Asians.

Congenital heart defects and genetic variants in the methylenetetrahydroflate reductase gene.

BACKGROUND: Most non-syndromic congenital heart defects (CHD) are caused by a complex interaction between maternal lifestyle factors, environmental exposures, and maternal and fetal genetic variants. Maternal periconceptional intake of folic acid containing vitamin supplements is reported to decrease the risk of CHD. The 677C-->T and 1298A-->C polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene decrease enzyme activity. OBJECTIVE: To examine the relation between CHD and maternal and fetal MTHFR polymorphisms. METHODS: 375 nuclear families were studied. The transmission/disequilibrium test was used to test for transmission distortion in complete triads. A log-linear approach was used to test for associations between CHD and maternal and offspring polymorphisms, and to estimate independently the contributions of maternal and fetal variants to relative risks. Haplotype frequencies were estimated and a haplotype transmission disequilibrium test carried out. RESULTS: The 1298C allele was transmitted less often than expected (p = 0.0013). There was no distortion in the transmission of the 677T allele, neither was there evidence of a parent of origin effect in the transmission of either of the single nucleotide polymorphisms. The 677C-1298C haplotype was also transmitted less often than expected (p = 0.0020). The relative risk associated with inheriting one copy of the 1298C allele was 0.64 (95% confidence interval, 0.48 to 0.87) and the that associated with inheriting two copies of the 1298C allele, 0.38 (0.21 to 0.70). CONCLUSIONS: The apparent protective effect of the MTHFR 1298C allele against CHD could have several explanations and further study is needed.

Collaborative strategies for unraveling the complexity of birth defects.

This article summarizes a presentation by one of the authors (CAH) to the Fifth Annual Meeting of the Diabetes in Pregnancy Study Group of North America. Dr Hobbs, MD, PhD, the Director of the Arkansas Center for Birth Defects Research and Prevention, presented an overview of the collaborative strategies developed for investigating the etiology of birth defects. A multidisciplinary group of scientists and clinicians explores current hypotheses in diabetic embryopathy, drawing upon expertise in experimental biology, genetic epidemiology, genomics, metabolomics and translational research. The prevalence rate of birth defects among infants of diabetic mothers is as high as 11%, despite the knowledge that maternal metabolic control is strongly correlated with the risk of malformations. Specifically, caudal dysgenesis, renal anomalies, heart and neural tube defects and situs abnormalities occur more often among infants of diabetic women than non-diabetic women. Researchers are also working to discover the underlying mechanisms that make some women with diabetes more susceptible than others to having infants with birth defects.

Sources of variability in birth defects prevalence rates.

BACKGROUND: The characteristics and methodologies of state-based birth defect surveillance systems might influence reported prevalence rates, making comparisons among states difficult. Standardizing methods to minimize variability beyond true differences in prevalence will aid national surveillance efforts and birth defects prevention programs. METHODS: Using data provided in the January 2000 Congenital Malformations Surveillance Report from the National Birth Defects Prevention Network, we characterized the surveillance methodologies among all sites. We then identified prevalence rates that are highly varied among systems that use each of our specified methodologies. We also examined the standards used by other collective health registries that exist across geographical boundaries. RESULTS: Large differences in prevalence rates across case ascertainment methods (active, passive, or combination of both) were observed for some conditions, but not for others. We identified additional factors which may influence prevalence rates, including case ascertainment sources, case inclusion criteria, and inclusion of elective terminations and stillbirths. The impact of each of these factors on prevalence rates may be defect-specific. CONCLUSIONS: We conclude that while some variability is expected due to differences in the true prevalence of birth defects, extreme differences among states are more likely due to differences in surveillance practices. The Birth Defects Prevention Act prompted new initiatives to develop birth defect surveillance systems, but there are no nationally agreed upon standards in existence to guide the process. This study was performed in support of developing standards that will influence new and existing state surveillance systems.

Improving estimates of caregiver time cost and family impact associated with birth defects.

BACKGROUND: Birth defects impose substantial costs on both families and society because of medical, developmental, and special education needs. Caring for children with birth defects also may influence caregiver time and impact the family. However, the economic cost of caregiver time and other impacts on the family has received far less attention than traditional healthcare costs. METHODS: This study reviews the literature on measuring caregiver time costs and family impact in an economic framework. The economic framework involves translating caregiver time or difficulties into appropriate units such as cost or quality adjusted life years (QALYs). RESULTS: Despite the potential important contribution of caregiver time costs to the total cost estimate of birth defects, few studies estimate caregiver time costs related specifically to birth defects. Only two studies provide estimates of these costs. Recent work has investigated the impact of chronic illness on caregivers in QALY terms, but birth defects have not been studied. Several issues need to be addressed in both the estimation of caregiver time costs and family impact to improve cost estimates. CONCLUSIONS: Improved estimates of caregiver time costs and impact on the family will assist policy makers in allocating resources for the prevention and treatment of birth defects. Future research should investigate the economic costs of caregiver time and family impact associated with caring for children with birth defects.

The National Birth Defects Prevention Study.

The National Birth Defects Prevention Study was designed to identify infants with major birth defects and evaluate genetic and environmental factors associated with the occurrence of birth defects. The ongoing case-control study covers an annual birth population of 482,000 and includes cases identified from birth defect surveillance registries in eight states. Infants used as controls are randomly selected from birth certificates or birth hospital records. Mothers of case and control infants are interviewed and parents are asked to collect buccal cells from themselves and their infants for DNA testing. Information gathered from the interviews and the DNA specimens will be used to study independent genetic and environmental factors and gene-environment interactions for a broad range of birth defects. As of December 2000, 7,470 cases and 3,821 controls had been ascertained in the eight states. Interviews had been completed with 70% of the eligible case and control mothers, buccal cell collection had begun in all of the study sites, and researchers were developing analysis plans for the compiled data. This study is the largest and broadest collaborative effort ever conducted among the nation's leading birth defect researchers. The unprecedented statistical power that will result from this study will enable scientists to study the epidemiology of some rare birth defects for the first time. The compiled interview data and banked DNA of approximately 35 categories of birth defects will facilitate future research as new hypotheses and improved technologies emerge.

Polymorphisms in genes involved in folate metabolism as maternal risk factors for Down syndrome.

Down syndrome is a complex genetic and metabolic disorder attributed to the presence of three copies of chromosome 21. The extra chromosome derives from the mother in 93% of cases and is due to abnormal chromosome segregation during meiosis (nondisjunction). Except for advanced age at conception, maternal risk factors for meiotic nondisjunction are not well established. A recent preliminary study suggested that abnormal folate metabolism and the 677C-->T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene may be maternal risk factors for Down syndrome. The present study was undertaken with a larger sample size to determine whether the MTHFR 677C-->T polymorphism was associated with increased risk of having a child with Down syndrome. Methionine synthase reductase (MTRR) is another enzyme essential for normal folate metabolism. A common polymorphism in this gene was recently associated with increased risk of neural tube defects and might also contribute to increased risk for Down syndrome. The frequencies of the MTHFR 677C-->T and MTRR 66A-->G mutations were evaluated in DNA samples from 157 mothers of children with Down syndrome and 144 control mothers. Odds ratios were calculated for each genotype separately and for potential gene-gene interactions. The results are consistent with the preliminary observation that the MTHFR 677C-->T polymorphism is more prevalent among mothers of children with Down syndrome than among control mothers, with an odds ratio of 1.91 (95% confidence interval [CI] 1.19-3.05). In addition, the homozygous MTRR 66A-->G polymorphism was independently associated with a 2. 57-fold increase in estimated risk (95% CI 1.33-4.99). The combined presence of both polymorphisms was associated with a greater risk of Down syndrome than was the presence of either alone, with an odds ratio of 4.08 (95% CI 1.94-8.56). The two polymorphisms appear to act without a multiplicative interaction.

High levels of intracellular polyamines promote histone acetyltransferase activity resulting in chromatin hyperacetylation.

Polyamines stimulate expression of a variety of genes, including many implicated in cell proliferation. Indeed, aberrant expression of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis, plays a causal role in tumorigenesis. Gene activity is influenced by dynamic changes in acetylation of nucleosomal histones. Although polyamines influence the histone acetyltransferase and deacetylase activities in cell-free systems, their ability to modulate these enzymes in live cells has never been established. To examine the effects of elevated intracellular levels of ODC and polyamines on gene transcription and histone acetylation, cells were infected with a retrovirus containing a cDNA for ODC. ODC overexpression potentiated the stimulatory effects of histone deacetylase inhibitors on reporter gene expression beyond that promoted by ODC or inhibitor treatment alone. Indeed, elevated intracellular levels of ODC promoted hyperacetylation of histones in several epidermal and fibroblast cell types. The ODC-mediated increase in acetylated histones was abrogated when cells were treated with alpha-difluoromethylornithine, a specific inhibitor of ODC activity, implying a distinct role for polyamines. Specifically, polyamines were found to enhance the action of histone acetyltransferases either directly or indirectly. Our studies document effects of elevated intracellular polyamine levels on histone acetylation in proliferating cells, suggesting a mechanism by which altered polyamine biosynthesis contributes to aberrant expression of genes, facilitating tumor growth. In addition, these studies may have implications for the development of drugs designed to regulate enzymes that modify the acetylation status of histones.

Differential regulation of gene expression in vivo by triple helix-forming oligonucleotides as detected by a reporter enzyme.

In an attempt to assay the capability of various oligonucleotides to inhibit gene transcription in vivo through triplex formation, we developed a cellular system employing transfection of a reporter plasmid and putative triplex-forming oligonucleotides targeted to Sp1-binding sites contained within the SV40 early promoter. Using this approach, we demonstrated that the activity of the reporter enzyme, alkaline phosphatase, was highly dependent on the sequence of the oligonucleotides: oligonucleotides utilizing G:GC triplets, but not C:GC triplets, promoted a dose-dependent decrease in reporter enzyme activity. Evidence of physical interaction between Sp1-binding sites within the SV40 promoter and sequence-specific G-rich oligonucleotides has been demonstrated, suggesting triple-helix formation as the most probable explanation for the inhibitory effect on alkaline phosphatase activity observed for these oligonucleotides. Surprisingly, Southern analysis of isolated nuclear DNA indicates that the differences in alkaline phosphatase activity associated with transfection of the different oligonucleotides appear to correlate with internalized plasmid DNA copy number rather than inhibition of transcription. It is intriguing to postulate the existence of a nuclease that is able to recognize and cleave triple-helical DNA structures. This hypothesis implies the existence of a novel mechanism of gene regulation specific for triplex structures and, presumably, independent of transcription.

Effect of a 5'-phosphate on the stability of triple helix.

An effect of 5'-phosphorylation on the stability of triple helical DNA containing pyrimidine:purine:pyrimidine strands has been demonstrated by both gel electrophoresis and UV melting. A 5'-phosphate on the purine-rich middle strand of a triple helix lowers the stability of triple helix formation by approximately 1 kcal/mol at 25 degrees C. The middle strand is involved in both Watson-Crick and Hoogsteen base pairing. In contrast, a 5'-phosphate on the pyrimidine-rich strands, which are involved in either Watson-Crick or Hoogsteen base pairing, has a smaller effect on the stability of triple helix. The order of stability is: no phosphate on either strand > phosphate on both pyrimidine strands > phosphate on purine strand > phosphate on all three strands. Differential stability of triple helix species is postulated to stem from an increase in rigidity due to steric hindrance from the 5'-phosphate. This result indicates that labelling with 32P affect equilibrium in triplex formation.

Elucidation of the sequence-specific third-strand recognition of four Watson-Crick base pairs in a pyrimidine triple-helix motif: T.AT, C.GC, T.CG, and G.TA.

We report a specific pattern of recognition by third-strand bases for each of the four Watson-Crick base pairs within a pyrimidine triple-helix motif as determined by PAGE: T.AT, C.GC, T.CG, and G.TA. Our recognition scheme for base triplets is in agreement with previous studies. In addition, we identified another triplet, T.CG, under physiological conditions, in which formation of triple helix was observed at equimolar ratios of the third strand and duplex target. Although different nearest-neighbor effects are expected, this finding extends the base-recognition code to all 4 base pairs in double-stranded DNA under physiological conditions. Base-composition analysis of putative triplex species provided independent evidence for the formation of triplex and confirmed the base-recognition code determined by PAGE. Moreover, the formation of triplex, as detected by gel electrophoresis, was seen to be an all-or-none phenomenon, dependent upon a single-base mismatch among 21 nucleotides. This result suggests a high specificity for the recognition of double-stranded DNA by a third strand. In addition, we report the surprising finding that triplex stability depends on the length and sequence of the target duplex DNA.

Very low birth weight: a problematic cohort for epidemiologic studies of very small or immature neonates.

Despite widespread acceptance of the concept of very low birth weight (VLBW), i.e., birth weight of less than or equal to 1,500 g, VLBW infants represent an extremely heterogeneous group of newborns, including those with very immature gestational age and those who are more mature but extremely growth retarded. To demonstrate how use of the VLBW rubric can lead to confounding bias that is not only large in magnitude but impossible to control satisfactorily, the authors divided 640 consecutive live neonates born in the Royal Victoria Hospital, Montreal, Canada, from 1978 to 1987 into two overlapping groups: a VLBW cohort (birth weight, 500-1500 g; n = 573) and a gestational age cohort (gestational age, 23-30 completed weeks; n = 466). Variation in growth status by gestational age was much more uniform in the 23- to 30-week cohort. Thus, although mean birth weight was similar in the 500- to 1,500-g and 23- to 30-week cohorts (1,055 vs. 1,064 g), the 500- to 1,500-g cohort was more mature (mean gestational age, 28.8 vs. 27.8 weeks; upper range, 39.7 vs. 30.9 weeks) and had twice the rate of intrauterine growth retardation (25.7 vs. 11.5%). These differences in maturity and growth resulted in a misleading protective effect of intrauterine growth retardation against in-hospital death in the 500- to 1,500-g cohort (crude odds ratio = 0.55 (95% confidence interval 0.36-0.83] and a greater discrepancy in maturity between cesarean- and vaginally delivered infants (3.1 vs. 1.5 weeks) in the 500- to 1,500-g vs. 23- to 30-week cohorts. These differences arise from inextricable confounding of growth status and maturity in the 500- to 1,500-g cohort, the most mature infants also being the most growth retarded. The removal of well-grown infants with birth weights of greater than 1,500 g from the VLBW cohort leads to a progressively distorted spectrum of growth with advancing gestational age and an artifactual blunting of the beneficial effects of increasing maturity. The authors suggest that whenever fetal growth is an important exposure, outcome, or confounding variable, epidemiologic studies of extremely small or immature newborns should be based on gestational age rather than the VLBW criterion.

Towards an understanding of position effect variegation.

Most variegating position effects are a consequence of placing a euchromatic gene adjacent to alpha-heterochromatin. In such rearrangements, the affected locus is inactivated in some cells, but not others, thereby giving rise to a mosaic tissue of mutant and wild-type cells. A detailed examination of the molecular structure of three variegating white mottled mutations of Drosophila melanogaster, all of which are inversions of the X chromosome, reveals that their euchromatic breakpoints are clustered and located approximately 25 kb downstream of the white promoter and that the heterochromatic sequences to which the white locus is adjoined are transposons. An analysis of three revertants of the wm4 mutation, created by relocating white to another euchromatic site, demonstrates that they also carry some heterochromatically derived sequences with them upon restoration of the wild-type phenotype. This suggests that variegation is not controlled from a heterochromatic sequence immediately adjacent to the variegating gene but rather from some site more internal to the heterochromatic domain itself. As a consequence of this observation we have proposed a boundary model for understanding how heterochromatic domains may be formed. It has been recognized for many years that the phenotype of variegating position effects may be altered by the presence of trans-acting dominant mutations that act to either enhance or suppress variegation. Using P-element mutagenesis, we have induced and examined 12 dominant enhancers of variegation that represent four loci on the second and third chromosomes. Most of these mutations are cytologically visible duplications or deficiencies. They exert their dominant effects through changes in the copy number of wild-type genes and can be divided into two reciprocally acting classes. Class I modifiers are genes that act as enhancers of variegation when duplicated and as suppressors when mutated or deficient. Conversely, class II modifiers are genes that enhance when mutated or deleted and suppress when duplicated. The available data indicate that, in Drosophila, there are 20-30 loci capable of dominantly modifying variegation. Of these, most appear to be of the class I type whereas only two class II modifiers have been identified so far.(ABSTRACT TRUNCATED AT 400 WORDS)

New cloning vectors and techniques for easy and rapid restriction mapping.

We have modified plasmid, phage lambda and cosmid cloning vectors to be of general use for easily and unambiguously determining restriction maps of recombinant DNA molecules. Each vector is constructed so that it contains the rarely found NotI restriction site joined to a short synthetic linker sequence that is followed by a multiple cloning site. DNA cloned into these vectors may be restriction-mapped by either of two methods. In one technique, the cloned DNA is completely digested with NotI, followed by partial digestion with any other restriction enzyme. After electrophoresis and transfer to a nylon membrane, the fragments are hybridized to a labeled probe complementary to the NotI linker. In the second technique, referred to as recession hybridization detection, cloned DNA is digested with NotI and then briefly treated with exonuclease III to recess the 3' ends. After hybridizing a labeled complementary oligodeoxynucleotide to the single-stranded 5' end containing the linker sequence, the DNA is partially digested with another restriction enzyme, electrophoresed and the gel is exposed to x-ray film. With either method the size of each labeled fragment corresponds directly to the distance that a restriction site is located from the NotI linker terminus. Methods for obtaining partial restriction enzyme digests have been devised so that as many as 20 different enzymes may be conveniently mapped on a single gel in little more than a day. The vectors and techniques described may also be adapted to automated or semi-automated devices that read fragment lengths and calculate the resulting restriction map.(ABSTRACT TRUNCATED AT 250 WORDS)