Maternal smoking, genetic variation of glutathione s-transferases, and risk for orofacial clefts

BACKGROUND: Maternal smoking is a known risk factor for orofacial clefts. We investigated whether risk is greater among offspring who lack the genetic capacity to produce glutathione S-transferase enzymes relevant to detoxification of chemicals in cigarette smoke. METHODS: Using a population-based case-control design, we genotyped 423 California infants with an isolated cleft and 294 nonmalformed controls for null variants of the glutathione S-transferases GSTT1 and GSTM1. RESULTS: If a mother smoked during pregnancy and her fetus was homozygous null for GSTT1, the risk of isolated cleft lip with or without cleft palate was tripled (odds ratio = 2.9; 95% confidence interval = 1.2-7.2). For fetuses who were homozygous null for GSTM1 and whose mothers smoked >/=20 cigarettes per day, we found nearly a 7-fold increased risk (6.8; 0.82-57). Combined absence of GSTM1 and GSTT1 enzymes among the offspring of smoking mothers was associated with a nearly 6-fold increased risk for cleft lip (6.3; 1.3-42). A similar increased risk for cleft palate was associated with absence of GSTM1, but not for absence of GSTT1. CONCLUSIONS: Maternal smoking during pregnancy increases risks for clefts among fetuses lacking enzymes involved in the detoxification of tobacco-derived chemicals.     

Risks of human conotruncal heart defects associated with 32 single nucleotide polymorphisms of selected cardiovascular disease-related genes

Investigating possible genetic polymorphisms and gene-environment interactions in the etiology of human conotruncal defects is a prudent research approach. In this study we explore gene-only and gene-environment effects of 32 single nucleotide polymorphisms (SNPs) on conotruncal defect risks. The genes bearing these SNPs participate in one of five pathogenetic processes, homocysteine metabolism, coagulation, cell-cell interaction, inflammatory response, and blood pressure regulation. We used DNA samples and data from a California population-based case-control interview study (1987-1988 birth cohort). We employed a multilocus allele-specific hybridization assay. Allelic variants were determined by genotyping 155 infants with conotruncal defects (cases) and 437 infants without malformations (controls). Among the 32 SNPs, four were associated with odds ratios of 2 or more, and two with odds ratios of 0.5 or less. The four SNPs were F2 G20210A (prothrombin) with an odds ratio of 2.5 (95% confidence interval; 0.9-7.0), F7 promoter (-323) 10-bp del/ins with an odds ratio of 2.3 (0.8-6.8), ITGB3 leu33pro (platelet glycoprotein IIIa) with an odds ratio of 2.2 (0.9-5.7), and NPPA T2238C (atrial natriuretic precursor peptide) with an odds ratio of 2.9 (0.8-10.1). Two SNPs were associated with decreased risks: TNF (tumor necrosis factor, G (-376A)) and ADD1 gly460trp (alpha adducin) with odds ratios of 0.5 (0.1-2.3) and 0.5 (0.2-1.9), respectively. Analyses that investigated a potential gene-nutrient interaction between maternal periconceptional vitamin use and MTHFR genotypes did not indicate that the CT or TT genotype contributed to conotruncal defect risk in infants even in the absence of maternal use of multivitamin supplements with folic acid. Analyses that investigated a potential interaction on risk between NOS3 genes and maternal cigarette smoking, revealed some evidence for higher risk of conotruncal defects in infants whose mothers smoked cigarettes periconceptionally and who had one of the variant alleles for NOS3 A(-922G) or NOS3 glu298asp compared to those infants whose mothers did not smoke and whose genotypes were wild-type. Our results provide some support for involvement of genetic variation of biologically relevant candidate genes for some birth defects whose pathogenesis may be related to altered vascular tone or integrity. In particular, NPPA appears to be a good candidate gene for conotruncal defects and warrants further investigation.     

Structural alterations of chromosome 2 in Leishmania major as evidence for diploidy, including spontaneous amplification of the mini-exon array

We have utilized pulsed field electrophoresis to characterize several karyotypic alterations in Leishmania major. Promastigotes of the LT252 line contain three small chromosomes, of 300, 350 and 385 kb. Quantitative densitometry of ethidium bromide-stained gels suggest that these chromosomes are present in equal levels (2:2:2). Two derivatives of this line, one appearing spontaneously (LT252 delta) and one obtained following selection with methotrexate (11-MTXR20), exhibit altered levels of these chromosomes, in the ratio of 2:1:3, respectively. The variant pattern in both lines is due to an increase in size of chromosome 2, yielding a new chromosome similar in size to chromosome 3. The enlarged chromosome 2 of the LT252 delta line is a result of amplification of the mini-exon gene array normally located on this chromosome, which increases from about 93 to 150 copies of the 0.44-kb mini-exon tandem repeat, as shown by quantitative hybridization and sizing of the mini-exon array. In contrast, the increased size of chromosome 2 within the methotrexate-resistant mutant 11-MTXR20 is not due to mini-exon amplification. In both variant lines, there are equal levels of the wild-type and enlarged chromosome 2, and the wild-type chromosome 2 is now present at 50% of the level of chromosome 1. These and other data suggest that Leishmania is diploid for chromosomes bearing housekeeping genes such as the mini-exon locus.     

Genetic polymorphisms and cerebral palsy in very preterm infants

In the present study, we examine whether selected genetic polymorphisms contribute to the development of cerebral palsy (CP) in very preterm infants. Subjects were 96 singleton infants with later-diagnosed CP and 119 control children, white non-Hispanic (n for CP=74, controls=88) or white Hispanic (CP=22, controls=31), born <32 wk gestation. Presence of CP was identified through state service agencies, with review of medical records. DNA extracted from archived neonatal blood was genotyped using multi-locus polymerase chain reaction amplification and immobilized sequence-specific oligonucleotide probes. Single nucleotide polymorphisms (SNPs) showing evidence of association with development of CP were endothelial nitric oxide synthase (eNOS) A(-922)G, factor 7 (F7) arg353gln and del(-323)10bp-ins, and lymphotoxin A (LTA) thr26asn. In white non-Hispanic children, beta-2 adrenergic receptor gln27glu was associated with CP risk; in Hispanic children, plasminogen activator inhibitor-1 (PAI-1) 4G(-675)5G and G11053T were associated with risk of CP. In a logistic regression considering these SNPs simultaneously in non-Hispanics, an association with CP was observed for heterozygotes of eNOS -922 (OR 3.0, CI 1.4-6.4), F7 (OR 2.7, CI 1.1-6.5), LTA (OR 2.1, CI 1.0-4.6), and PAI-1 (OR 3.2, CI 1.2-8.7). Factor 5, Factor 2, methylene tetrahydrofolate reductase, tumor necrosis factor-alpha, and other SNPs tested were not significantly associated with CP risk. We conclude that further study of genetic factors that may influence susceptibility to CP in very preterm infants is warranted.     

NAT2 variation and idiopathic talipes equinovarus (clubfoot)

Idiopathic talipes equinovarus (ITEV), or isolated clubfoot, is a common developmental anomaly that is characterized by a rigid foot, adducted forefoot, cavus midfoot, equinovarus of the hindfoot, and hypoplastic calf musculature. The etiology of this common birth defect is largely unknown, but genetic factors have been implicated in population and family studies and maternal smoking during pregnancy has been identified as an environmental risk factor. The biotransformation of exogenous substances, such as tobacco smoke, is modulated by numerous genes including N-acetylation genes, NAT1 and NAT2. Functional variants of these genes exist and can be distinguished by genotyping. We hypothesized that variation in NAT1 and NAT2 genes might be associated with ITEV. To test this hypothesis, NAT1 and NAT2 were genotyped in a sample of 56 multiplex ITEV families, 57 trios with a positive family history and 160 simplex trios with ITEV. The results detected a slight decrease in the expected number of homozygotes for the NAT2 normal allele in the Hispanic simplex trios. In addition, in a pilot case-control study of ITEV, there were significantly more slow NAT2 acetylators among the cases. This suggests that slow acetylation may be a risk factor for ITEV. This study is the first to find evidence suggesting a role for a biotransformation candidate gene in the etiology of ITEV and provides a scientific foundation to further explore the contributions of other tobacco metabolism genes in the etiology of clubfoot.     

Evolution of nuclear DNA and the occurrence of sequences related to new small chromosomal DNAs in the trypanosomatid genus Endotrypanum

Comparisons of nuclear DNA restriction fragment patterns were used to examine the evolutionary relatedness among 17 strains previously identified as Endotrypanum, a trypanosomatid parasite of sloths. Fragments were obtained with 6 restriction enzymes and analyzed by Southern blotting with hybridization probes from three loci. An estimate of the percent nucleotide sequence divergence among strains, Δ, was calculated and used to construct molecular evolutionary trees. The 17 isolates fell into four distinct groups, one of which (group D) showed no more relationship to groups A–C than it did to other genera (Leishmania, Crithidia, Leptomonas, Trypanosoma), being too distant to be resolved with this method. These and other data suggest that group D may not actually be Endotrypanum. Molecular karyotype analysis revealed considerable variation among the chromosomes of these strains. One strain (LV88, group B) contained a linear 70-kb chromosome not evident in other isolates. Hybridization probes specific for this chromosome (LV88-70) were developed and revealed that related sequences were present at high levels in group B isolates and low levels in group A isolates, although a complex hybridization pattern was evident. Sequences related to LV88-70 were not present in groups C and D, nor in Leishmania major, showing that this DNA has a disjunct distribution which curiously parallels that of virus-like particles present in these isolates.     

Maternal occupational chemical exposures and biotransformation genotypes as risk factors for selected congenital anomalies

In a case-control study using an assessment of occupational tasks by an industrial hygienist, the authors investigated whether women's occupational exposures increased risks of delivering infants with cleft palate (CP), cleft lip with or without cleft palate (CLP), conotruncal defects, or limb deficiencies. For CP and CLP, exposures were further considered in the presence/absence of infant genetic variants for glutathione-S-transferase M1, glutathione-S-transferase T1, and N-acetyltransferases 1 and 2. The study included 1987-1989 California stillbirths and livebirths. Telephone interviews were conducted with mothers of 662 CLP and CP cases, 207 conotruncal defect cases, 165 limb deficiency cases, and 734 nonmalformed controls. Occupational tasks were assigned to a priori-defined exposure categories: 74 chemical groups and nine "end-use" chemical groups. Odds ratios of 1.5 or greater were observed for a small number of exposure-defect comparisons. Risks associated with end-use groups revealed odds ratios of 1.5 or greater for exposures to dyes and pigments (conotruncal and CP), propellants (CP), and insecticides (conotruncal and CP). Numerous odds ratios of 2.5 or greater were observed for combined effects of exposures and homozygous mutant genotypes, particularly for CP. Although potential associations were observed, most results suggested that maternal occupational chemical exposures did not contribute substantially to the occurrence of these anomalies in this California population.