Association of folate metabolic pathway genes with human spina bifida meningomyelocele

Neural tube defects including spina bifida meningomyelocele (SBMM) are common malformations of the brain and spinal cord, and include all abnormalities resulting from lack of closure of the developing neural tube during embryological development.^ The specific aims of this study were to determine if single nucleotide polymorphic variants (SNPs) in the folate/homocysteine metabolic pathway genes confer a risk for NTD susceptibility within this SBMM population.^ In completion of the first specific aim, two novel SNPs were identified in the FOLR1 gene in Chromosome 11of patients including one in non-coding exon 1 with a C → T transition at nucleotide position 71578317 and another in non-coding exon 3 with a T → G transversion at nucleotide position 71579123. It will be important to determine if these variants are present in the respective parents of these individuals. If they are in fact de novo variants, then these SNPs may be more likely to contribute to the birth defect.^ The second project aim was to analyze genotypes associated with SBMM risk by transmission disequilibrium tests (TDT) and association was detected on several SNPs across the folate metabolic pathway genes in this population. SNPs with significant RC-TDT values were found within the DHFR gene (rs1650723), the MTRR gene (rs327592), the FOLR2 gene (rs13908), four tightly linked variants in the FOLR3 gene (rs7925545, rs7926875, rs7926987, rs7926360) and a variant in the SLC19A1 gene (rs1888530). The product of each of these genes performs a vital function in the folate metabolic pathway. It is conceivable, therefore, that if the individual SNP or SNPs can be proven to perturb the function in some way that they may be involved in the disruption of folate metabolism and in the resulting birth defect. Validating the results of this study in other independent populations will further strengthen the evidence that dysfunction of folate enzymes and receptors may confer SBMM risk in humans. ^

Genetic susceptibility, cigarette smoking, and wood dust exposure in lung cancer: Case control analyses

This case control study was conducted to assess the association between lung cancer risk, mutagen sensitivity (a marker of cancer susceptibility), and a putative lung carcinogen, wood dust exposure. There were 165 cases (98 African-Americans, 67 Mexican-Americans) with newly diagnosed, previously untreated lung cancer, and 239 controls, frequency-matched on age, sex, and ethnicity.^ Mutagen sensitivity ($\ge$1 break/cell) was associated with a statistically significant elevated risk for lung cancer (odds ratio (OR) = 4.1, 95% confidence limits (CL) = 2.3,7.2). Wood dust exposure was also a significant predictor of risk (OR = 2.8, 95% CL = 1.2,6.6) after controlling for smoking and mutagen sensitivity. When stratified by ethnicity, wood dust exposure was a significant risk factor for African-Americans (OR = 4.0, 95% CL = 1.4,11.5), but not for Mexican-Americans (OR = 1.5, 95% CL = 0.3,7.1). Stratified analysis suggested a greater than multiplicative interaction between wood dust exposure and both mutagen sensitivity and smoking.^ The cases had significantly more breaks on chromosomes 4 and 5 than the controls did with ORs of 4.9 (95% CL = 2.0, 11.7) and 3.9 (95% CL = 1.6, 9.3), respectively. Breaks at 4p14, 4q27, 4q31, 5q21-22, 5q31, and 5q33 were significantly more common in lung cancer patients than in controls. Lung cancer risk had a dose-response relationship with breaks on chromosomes 4 and 5. Cigarette smoking had a strong interaction with breaks on chromosomes 2, 4, and 5.^ In a molecular cytogenetic study, using chromosome painting and G-banding, we showed that: (1) the proportion of chromosome 5 abnormalities surviving as chromosome-type aberrations remained significantly higher in cells of lung cancer cases (14%) than in controls (5%) (P $<$ 0.001). However, no significant differences were detected in chromosome 4 abnormalities between cases and controls; (2) the proportion of chromosome 5q13-22 abnormalities was 5.3% in the cases and 0.7% in the controls (P $<$ 0.001). 5q13-22 regions represented 40% of all abnormalities on chromosome 5 in the cases and only 14% in the controls.^ This study suggests that mutagen sensitivity, wood dust exposure, and cigarette smoking were independent risk factors for lung cancer, and the susceptibility of particular chromosome loci to mutagenic damage may be a genetic marker for specific types of lung cancer. ^

ETIOLOGIC AND CLINICAL FACTORS WHICH DIFFERENTIATE PATIENTS WITH CHROMOSOMAL ABNORMALITIES FROM DIPLOID PATIENTS IN ACUTE NONLYMPHOCYTIC LEUKEMIA

Tumor-specific chromosomal abnormalities have been demonstrated in bone marrow of approximately 50% of newly diagnosed acute nonlymphocytic (ANLL) patients. This study examined two hypotheses: (1) Aneuploid (AA) patients are diagnosed later in the course of their disease than diploid (NN) patients; and (2) AA patients are more likely to have been exposed to environmental agents. Of 324 patients eligible for study, environmental exposure data were obtained for 236 (73%) of them. No evidence was found to suggest that AA patients had more advanced disease than NN patients. Aneuploid patients were more likely than NN patients to: (a) report treatment with cytotoxic drugs for a prior medical condition (odds ratio, adjusted for age, sex and other exposures (OR) = 4.25, 95% confidence intervals, 1.38 to 13.17); (b) smoke cigarettes, OR = 1.82 (1.02, 3.26) and (c) drink alcoholic beverages, OR = 1.91 (1.05, 3.48). No statistically significant associations between aneuploidy and occupational exposures were present, OR = 3.59 (0.76, 17.13). Problems in interpreting these ORs are discussed. ^

ANALYSIS OF ESTROGEN-INDUCED ANEUPLOIDY AND CHROMOSOME DISPLACEMENT

Diethylstilbestrol (DES) is a known human carcinogen and teratogen whose mechanism of action remains undetermined. As essentially diploid Chinese hamster cell line (Don) was used to test diethylstilbestrol (DES), dienestrol, hexestrol and the naturally occurring estrogens, estradiol and estriol for their ability to cause metaphase arrest and to induce aneuploidy. These compounds arrest mitosis within a narrow range of high concentrations and induce aneuploidy in recovering cell populations. DES was the most effective arrestant on a comparative molar basis. Estradiol and estriol were less potent as arrestants but were effective inducers of aneuploidy. Aneuploidy was induced in a non-random manner. The smallest chromosomes were most frequently recorded in aneuploid cells. Using anti-tubulin antibody and indirect immunofluorescence, it was found that DES inhibits bi-polar spindle assembly and disrupts the cytoplasmic microtubule complex (CMTC). Estradiol arrests mitosis in a manner that allows spindle assembly. Estradiol has no apparent effect on the CMTC. The naturally occurring estrogens caused chromosome displacement during mitotic arrest. Electron microscopy confirmed that the displaced chromosomes appeared at the polar regions of arrested cells. The arresting effect of estradiol, and to some extent DES, was reduced by the addition of dibutyryl cyclic adenosine monophosphate (db-cAMP). Aneuploidy induction by DES and similar compounds may be related to their carcinogenic and/or teratogenic potential. ^

FANCM AND FAAP24 MAINTAIN GENOMIC STABILITY THROUGH COOPERATIVE AND UNIQUE FUNCTIONS

Fanconi anemia (FA) is a rare recessive genetic disease with an array of clinical manifestations including multiple congenital abnormalities, progressive bone marrow failure and profound cancer susceptibility. A hallmark of cells derived from FA patients is hypersensitivity to DNA interstrand crosslinking agents such as mitomycin C (MMC) and cisplatin, suggesting that FA- and FA-associated proteins play important roles in protecting cells from DNA interstrand crosslink (ICL) damage. Two genes involved in the FA pathway, FANCM and FAAP24, are of particular interest because they contain DNA interacting domains. However, there are no definitive patient mutations for these two genes, and the resulting lack of human genetic model system renders their functional studies difficult. In this study, I established isogenic human FANCM- and FAAP24-null mutants through homologous replacement-mediated gene targeting in HCT-116 cells, and systematically investigated the functions of FANCM and FAAP24 inchromosome stability, FA pathway activation, DNA damage checkpoint signaling, and ICL repair. I found that the FANCM-/-/FAAP24-/- double mutant was much more sensitive to DNA crosslinking agents than FANCM-/- and FAAP24-/- single mutants, suggesting that FANCM and FAAP24 possess epistatic as well as unique functions in response to ICL damage. I demonstrated that FANCM and FAAP24 coordinately support the activation of FA pathway by promoting chromatin localization of FA core complex and FANCD2 monoubiqutination. They also cooperatively function to suppress sister chromatid exchange and radial chromosome formation, likely by limiting crossovers in recombination repair. In addition, I defined novel non-overlapping functions of FANCM and FAAP24 in response to ICL damage. FAAP24 plays a major role in activating ICL-induced ATR-dependent checkpoint, which is independent of its interaction with FANCM. On the other hand, FANCM promotes recombination-independent ICL repair independently of FAAP24. Mechanistically, FANCM facilitates recruitment of nucleotide excision repair machinery and lesion bypass factors to ICL damage sites through its translocase activity. Collectively, my studies provide mechanistic insights into how genome integrity is both coordinately and independently protected by FANCM and FAAP24.

ATTITUDES TOWARD AND UTILIZATION OF NON-INVASIVE PRENATAL TESTING FOR CHROMOSOME ANEUPLOIDY AMONG OB/GYNS

Prenatal diagnosis is traditionally made via invasive procedures such as amniocentesis and chorionic villus sampling (CVS). However, both procedures carry a risk of complications, including miscarriage. Many groups have spent years searching for a way to diagnose a chromosome aneuploidy without putting the fetus or the mother at risk for complications. Non-invasive prenatal testing (NIPT) for chromosome aneuploidy became commercially available in the fall of 2011, with detection rates similar to those of invasive procedures for the common autosomal aneuploidies (Palomaki et al., 2011; Ashoor et al. 2012; Bianchi et al. 2012). Eventually NIPT may become the diagnostic standard of care and reduce invasive procedure-related losses (Palomaki et al., 2011). The integration of NIPT into clinical practice has potential to revolutionize prenatal diagnosis; however, it also raises some crucial issues for practitioners. Now that the test is clinically available, no studies have looked at the physicians that will be ordering the testing or referring patients to practitioners who do. This study aimed to evaluate the attitudes of OB/GYN’s and how they are incorporating the test into clinical practice. Our study shows that most physicians are offering this new, non-invasive technology to their patients, and that their practices were congruent with the literature and available professional society opinions. Those physicians who do not offer NIPT to their patients would like more literature on the topic as well as instructive guidelines from their professional societies. Additionally, this study shows that the practices and attitudes of MFMs and OBs differ. Our population feels that the incorporation of NIPT will change their practices by lowering the amount of invasive procedures, possibly replacing maternal serum screening, and that it will simplify prenatal diagnosis. However, those physicians who do not offer NIPT to their patients are not quite sure how the test will affect their clinical practice. From this study we are able to glean how physicians are incorporating this new technology into their practice and how they feel about the addition to their repertoire of tests. This knowledge gives insight as to how to best move forward with the quickly changing field of prenatal diagnosis.